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Molecular cytogenetics in Picea Brown, Garth Robert 1995

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MOLECULAR CYTOGENETICS IN PICEAbyGARTH ROBERT BROWNB.Sc., The University of British Columbia, 1986A THESIS SUBMITTED [N PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of Forest SciencesWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1995© Garth Robert Brown, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Forest SciencesThe University of British ColumbiaVancouver, CanadaOctober 11, 1995Date________________________________DE-6 (2/88)ABSTRACTA fundamental characteristic of a species is its karyotype, a description of eachchromosome. In spruce (Picea) and other conifer genera, chromosome identification ishindered by similarities in chromosome size and morphology within a species. Homeologouschromosomes of related species are also generally indistinguishable. The focus of this Ph.Dthesis is the development of a new technology in conifer cytogenetics, in situ hybridization, toaddress these inherent difficulties and to establish procedures for the physical mapping of thespruce genome.In situ hybridization augments conventional cytogenetics with the methods ofmo1ecuar biology and allows the visualization of defined DNA sequences along metaphasechromosomes and interphase chromatin. The chromosomal locations of three tandemrepeated DNA sequences, including the genes encoding the 18S-5.8S-26S ribosomal RNA,those encoding the 5S ribosomal RNA, and a centromeric satellite DNA (SGR-31), weredetermined in white spruce (Picea glauca (Moench) Voss) and Sitka spruce (P. sitchensis(Bong.) Carr.). A molecular description of the 5S ribosomal RNA genes and SGR-3 1 inwhite spruce, comprising their nuclear organization, nucleotide sequence and genomic copynumber, was also performed.Combining in situ hybridization data from the 18S-5.8S-26S ribosomal RNA and the5S ribosomal rRNA genes permitted the first unequivocal identification of each somaticchromosome of a conifer species. All three repeated DNAs were subsequently mapped ontothe same metaphase spreads, producing the first cytogenetic maps of white and Sitka spruces.The white spruce map consists of twelve loci, seven corresponding to 18S-5.8S-26SHribosomal RNA loci, one to the 5S ribosomal DNA, and four to SGR-31 sites. Comparison ofthis map to that of Sitka spruce (five 18S-5.8S-26S ribosomal RNA loci, one 5S ribosomalRNA site, and five SGR-3 loci) revealed that despite the overall uniformity in appearanceamong homeologous chromosomes, the repeated DNA complement of these closely relatedspruce genomes is in flux.The prospects for the use of in situ hybridization in conifer genome analysis areimmense. It’s greatest contribution, which hinges on improvements in detection sensitivity,will be in the integration of genetically defined linkage groups with the chromosomes onwhich they reside. Investigating the relationship between genetic linkage, recombination andchromosome structure or the co-linearity of gene sequences and chromosomal synteny amongspruce species and other conifer genera, and the eventual cloning of genes based on mapposition, are now within the possibilities of conifer genetics.111TABLE OF CONTENTSAbstractTable of Contents ivList ofAbbreviations ViiList of Tables viiiList ofFigures ixList of Common and Scientific Names of Gymnosperms xList of Common and Scientific Names of Angiosperms XiAcknowledgement xiiForeword xiiiGENERAL INTRODUCTION 1Literature Cited 5Chapter One Literature Review 8White Spruce and Sitka Spruce 8The Nuclear Genome of Spruce 10Organization and Evolution of Repeated DNA 11Tandem Arrayed Repeated DNA 12Dispersed Repeated DNA 15Low Copy DNA Sequences in Conifer Genomes 17In Situ Hybridization 18Literature Cited 22Chapter Two Development of In Situ Hybridization in Spruce:the Chromosomal Location of Genes Encodingthe 18S-5.8S-26S Ribosomal RNA 30Introduction 30Materials and Methods 33Plant Material and Chromosome Preparation 33Probe Labeling 34In Situ, Hybridization 35Microscopy 37Karyotype Analysis 37Results 38ivDiscussion 45Literature Cited 49Chapter Three Characterization of the 5 S Ribosomal RNA Genes in Spruce 51Introduction 51Materials and Methods 52DNA Isolation 52PCR Amplification and Cloning of the 5S rRNA Genes 53Genomic Digestion and Southern Hybridization 54DNA Sequencing 55Estimation of 5 S rDNA Copy Number in White Spruce 55Chromosome Preparation and In Situ Hybridization 55Results 56Characterization of 5S rDNA in White Spruce 56Physical mapping of the 5S rDNA 64Discussion 65Literature Cited 68Chapter Four Characterization of a Centromeric Satellite DNA in WhiteSpruce 70Introduction 70Materials and Methods 71Plant Material and DNA Isolation 71Screening of a Partial White Spruce Library 72DNA Sequencing 74Estimation of the SGR-3 1 Copy Number in White Spruce 74Chromosome Preparation and In S/ti, Hybridization 79Results 79Characterization of SGR-3 1 in White Spruce 79SGR-31 inPicea 80Physical Mapping of SGR-3 1 81Discussion 82SGR-3 1, a Centromeric Satellite DNA 82Evolution of SGR-3 I in Picea 84SGR-3 1 and Spruce Centromere Function 85Literature Cited 87Chapter Five The First Cytogenetic Maps ofWhite and Sitka Spruces 89Introduction 89Materials and Methods 90In Situ Hybridization 90Image Analysis 91Results 91VDiscussion 96Cytogenetic Maps ofWhite and Sitka Spruce 96Repeated DNAs of the Spruce Genome 97Literature Cited 99Chapter Six The Prospects for Non-Isotopic In Situ Hybridizationin Conifer Genome Analysis 101The Sensitivity of In Situ Hybridization 101Applications 104Continued Mapping ofRepeated DNAs 104Low Copy Sequence and Gene Mapping 105Literature Cited 109Appendix Common fluorochromes and their excitation andand emission maxima 113viLIST OF ABBREVIATIONSISH in situ hybridization18S-26S rDNA 18S-5.8S-26S ribosomal DNADAPI 4’ -6-diamidino-2-phenylindoleFITC fluorescein isothiocyanateCMA3 chromomycin A3PCR polymerase chain reactionA.R. arm ratioCI. centromeric indexR.L. relative lengthFLpter fractional length from terminus of the short arm (p)IGS intergenic spacer of the 18S-5.8S-26S ribosomal DNANTS nontranscribed spacer of the 5S rDNAviiLIST OF TABLESI. Picea species included in Southern hybridization experiments with SGR-3 1. 73viiiLIST OF FIGURES2.1 Molecular structure of a typical 18S-5.8S-26S ribosomal RNA genein higher eukaryotes. 312.2 Localization of 18S-26S rDNA in white spruce. 392.3 18S-26S rDNA polymorphism in white spruce. 402.4 Localization of 18S-26S rDNA in Sitka spruce. 412.5 Ideograms of the mitotic chromosomes ofwhite and Sitka spruces. 443.1 Origin of the primer pairs P1/P2 and P3/P4 used in the PCRamplification of the 5S rDNA. 573.2 Consensus sequence of 5S rRNA genes in white spruce. 583.3 Southern hybridization of 5S rDNA to partially digestedwhite spruce genomic DNA. 593.4 Estimation of 5S rDNA copy number in white spruce. 603.5 Localization of 5S rDNA in white spruce. 613.6 Localization of 5S rDNA in Sitka spruce. 624.1 Southern hybridization of SGR-3 1 to partially digested whitespruce genomic DNA. 754.2 Nucleotide sequence of SGR-3 1. 764.3 Southern hybridization of SGR-3 1 to Sau3A digested genomicDNA from 18 species ofPicea. 774.4 Localization of SGR-3 I in white and Sitka spruces. 785.1 Co-localization of the 5S and 18S-26S rDNA, and SGR-31in white and Sitka spruces. 925.2 The first cytogenetic maps of white and Sitka spruces. 93ixLIST OF COMMON AND SCIENTIFIC NAMES OF GYMNOSPERMSCycas Cycas rei’oluta Thunb.Ephedra Ephedra kokanica Regel.Gingko Gingko biloba L.Gnetum Gnetiirn itla Brongn.Douglas-fir Psendotsuga rnenziesii (Mirb.) FrancoWestern redcedar Thujaplicata Donn ex D. DonPine, Loblolly Finns taeda L.Radiata Finns radiata D. DonScots Finns sylvestris L.Slash Finns elliott/i Engeim.Spruce, Black Picea mariana (Mill) B. S .PBlue Ficea pungens Engeim.Candalabra Picea niontigena MastersChihuahua Picea chihuahuana MartinezEngelmann Picea engelnianni (Parry)Koyama Picea koyarnai Shiras.Mexican Picea mexicana MartinezNorway Picea abies (L.) KarstOriental Picea orientalis (L.) LinkRed Picea rubens Sarg.Sargent Ficea brachytyla (Franch.) Pritz.Schrenk’ s Ficea schrenkiana (Fisch and Meyer)Serbian Ficea omorika (Pancic) Purkyne.Siberian Picea obovata Ledeb.Sitka Ficea sitchensis (Bong.) Carr.Tigertail Ficeapolita (Sieb. and Zucc.) Carr.White Picea glanca (Moench) VossYeddo Piceajezoensis (Sieb. and Zucc.)(unavailable) Ficea purpurea MastersxLIST OF COMMON AND SCIENTIFIC NAMES OF ANGIOSPERMSArabidopsis Arabidopsis thaliana (L.) Heynh.barley Hordeum vulgare L.broad bean Viciafaba L.Chinese cabbage Brassica canipestris L.garden pea Pisum salivvni L.lily Li/him speciosum Thumb.kale Brassica oleracea L.maize Zea mays L.mung bean Vigna radiata (L.) Wilczekonion All/nm cepa L.rapeseed Brassica napus L.rice Oryza saliva L.rye Secale cereale L.soybean Glycine max (L.) Merr.tomato Lycopersicon escu/enlum Mill.wheat Triticum aestivum L. em Thell.xiACKNOWLEDGEMENTMy gratitude is extended to the members of my supervisory committee: Dr. John E.Carison, for the opportunity and guidance, Dr. F. Brian Ho!!, for his continua! support andencouragement, and Dr. Yousry El-Kassaby for his attitudes and views, research materials andcollaborative support on behalf of Pacific Forest Products, Ltd.I would like to thank Gyula Kiss of the Kalamalka Research Station, Vernon, B.C. forthe collection and steady supply of seeds and additional research materials from the sprucearboretum. I am also indebted to Michael Weis for his microscopy and computer skills and hiswillingness to share this knowledge, and to Vindhya Amarasinghe for her constructivecriticism and reviews of all aspects of this research. I am grateful for the efforts of CraigNewton and for the friendship of Andree, Ann, and Jeff. My thanks go to all other membersof Dr. Carison’s lab, past and present, for an enjoyable and stimulating work environment, toJim and Scott for showing me the straightest path, and to Paul Bauman for planting the seedof my interest in cytogenetics.xliFOREWORDA portion of the research described in Chapter 2 has been previously published by Mr.Garth R. Brown, Dr. Vindhya Amarasinghe, Mr. Gyula Kiss and Dr. John E. Carison underthe title “Preliminary karyotype and chromosomal localization of the ribosomal DNA sites inwhite spruce using fluorescence in situ hybridization” in the journal Genorne, volume 36, pp.310-316, 1993. The written permission of the copyright holder to include this material in thethesis has been obtained.The thesis author, under the supervision of Dr. Carlson, conducted all experimentalprocedures detailed in the publication with the exception of the operation of the confocal laserscanning microscope (performed by Dr. Amarasinghe). The necessary spruce materials werecollected and supplied by Mr. Kiss.First authorSenior’ AuthorxliiGENERAL INTRODUCTIONMolecular biology provides powerful new tools for the study and manipulation ofplant genomes. Variations in the DNA sequence between homologous loci can be used asgenetic markers in conventional linkage analysis (Botstein et a!., 1980) and genetic linkagemaps are in progress to supplement research programs oriented towards the improvement ofmany agricultural crops. Advances in DNA marker technology, in particular the application ofthe polymerase chain reaction (Williams et a!., 1990), have simplified and reduced the timeand cost associated with mapping. Genetic maps can now be rapidly constructed for virtuallyany sexually reproducing species. Among the species attracting the interest of molecularbiologists are several economically important members of the Pinaceae family of conifers,including white spruce, slash pine, loblolly pine, and Douglas-fir (Tulsieram et at., 1992;Nelson et al., 1993; Devey et al., 1994; Jermstad et a!., 1994). Genetic mapping of conifergenomes may allow the molecular dissection of both simple and complex traits and willprovide a much broader insight into the structure, organization, and evolution of the nucleargenome than previously possible using markers for morphological or biochemical traits.Additionally, if associations between genetic markers and quantitative trait characters inspecific crosses can be consistently detected, genetic mapping may provide the means ofshortening the long generation time particular to conifer breeding programs through markerassisted selection.In studying the genome organization of any species it is prudent to augment geneticlinkage analysis with the characterization of repeated DNAs and the underlying physical1relationship of both repeated and low copy DNA sequences along the chromosomes. A largeportion of the genomes of higher plants, in fact more than 75% of total DNA in thoseexceeding 2 picograms per nucleus, is composed of repeated DNA sequences (Flavell 1980;Thompson and Murray 1981). Some repeated DNAs, such as the genes encoding thecomponents of ribosomes, have critical cellular roles while others serve no discernible protein-encoding function. In its preponderance, repeated DNA is clearly a major determinant ofchromosome size, structure and functioning. It is, however, less amenable then low copyDNA to linkage analysis due to the occurrence of repeating units of a single family found atmultiple loci and difficulties in identifying segregating alleles.A further incentive to characterize the repeated DNA component of any genome inparallel to genetic mapping arises from the unequal distribution of recombination eventsobserved across both animal and plant genomes (Leitch and Heslop-Harrison 1993; Gustafsonand Dille 1992; Lukaszewski 1992; Groover et al., 1995). Since genetic distance is definedby the recombination frequency between markers, chromosomal regions which suppressrecombination, like heterochromatic areas composed of tandem arrayed repeated DNAfamilies, will lack genetic markers (Flavell et al., 1985). The primary constriction, orcentromere, is one such chromosomal domain and can result in tightly linked markers that inreality lie in the distal portion of opposite chromosome arms separated by hundreds ofmegabases of DNA. Additional recombination “cold spots” such as inversions andtranslocations have been described (Lucchesi and Suzuki 1968) as have “hot spots” whichresult in markers appearing unlinked genetically even though they are physically very near toone another (Steinmetz ci a!., 1987). Therefore, the genetic distance between markers canfrequently be a poor indicator of the true physical distance involved.2In situ hybridization (ISH), one of several methods available to generate a physicalmap, combines conventional cytology and methods of molecular biology, enabling thelocalization of defined DNA sequences along metaphase chromosomes or interphasechromatin. The technique involves the hybridization of labeled DNA or RNA to cytologicalpreparations of denatured metaphase chromosomes and chromatin which have been fixed on amicroscope slide. The sites of probe hybridization can be observed microscopically viaradioactive or fluorescent emissions, or by the enzymatic production of a colored precipitate.ISH has been widely applied to the physical mapping of genes and repeated DNAsequences in animal species and, in particular, is a central component of the Human GenomeProject. The presence of a cell wall in plants and the available methods of plant chromosomepreparation present difficulties not experienced in human cytogenetics. While these maycurrently limit the routine detection of gene loci in plants, the physical mapping of repeatedDNAs has been achieved in a wide variety of angiosperms. These studies, along withobservations that repeated DNAs are a dynamic, rapidly evolving part of the genome notsubject to the evolutionary constraints imposed on transcribed sequences and those thatregulate them, have illustrated their value as cytological markers not only to investigategenome organization but also in the identification of somatic chromosomes and in the study ofchromosome evolution, phylogenetic relationships, introgression and the spatial arrangementof chromatin in the interphase nucleus (Lapitan et a!., 1989; Lapitan et a!., 1987; Zhang andDvorak 1989; Bauwens et a!., 1991).By necessity, ISH will play a central role in the physical mapping of conifer genomes,serving as the primary tool in chromosome identification and in integrating genetic andphysical maps. Most conifers, including spruce species, have symmetrical karyotypes3composed of predominantly metacentric chromosomes (Khoshoo 1961) which pose seriousdifficulties in identifying each chromosome pair based solely on morphological criteria.Chromosome identification is, however, an important step in genome analysis.A variety of chromosome banding techniques has been developed to facilitatechromosome identification in many animals and some angiosperm plant species. Thesemethods produce either transverse bands spanning the length of all metaphase chromosomesor darkly staining regions in particular sites of some or all chromosomes. As many as 2000 Gbands can be produced along midprophase chromosomes of humans, greatly simplifyingkaryotyping. However, the method is generally not applicable to plants (Bickmore andSumner 1989). Plant cytogenetics has traditionally relied on techniques which produce muchfewer bands of the second type. For example, Giemsa C- banding, which originated as amodification of ISH (Pardue and Gall 1970), reveals heterochromatic areas associated withcentromeres, telomeres and some interstitial sites on one to all chromosomes of acomplement. Certain fluorescent dyes which have specificity for GC or AT base pairs, such asChromomycin A3 and 4’-6-diamidino-2-phenylindole (DAPI), respectively, revealchromosomal regions with sequence compositions distinct from adjacent areas (Schweizer1980). Fluorescent bands usually correspond to a subset of the heterochromatic regionsrevealed by C-banding. Lastly, N-bands denote the sites of actively expressed ribosomal RNAgenes through the detection of a complex of acidic nonhistone argentophilic proteins boundwith the ribosomal genes (Goessens 1984). Unfortunately, none of these chromosomebanding methods have been applied to spruce cytogenetics. Currently, the best approach tokaryotyping spruce and other conifers is the isolation and physical mapping by ISH of one orseveral repeated DNA sequences having genomic distribution(s) specific to each chromosome.4In many agricultural crops the integration of genetically mapped DNA markers withthe physical map is readily achieved by filter hybridization to genomic DNA from a variety ofwell characterized chromosome addition or substitution lines (Chinoy et at., 1991). Thesecytogenetic stocks have not been produced for any conifer species and, at present, mapintegration in conifers will likely only proceed by determining the chromosomal location oflinkage groups using ISH methods.At the onset of this research, ISH procedures had been demonstrated for only a singleconifer species (Cullis et al., 1988). Repeated DNAs had also not been fully characterized orexploited in any gymnosperm. To correct this situation and to access the potential of ISH foranalysing the spruce genome the specific aims of this thesis were: 1) the development of amethod to localize repeated DNA sequences to the mitotic chromosomes of spruce species byISH and 2) the cloning and characterization of repeated DNA sequences yielding easilydistinguished chromosome-specific hybridization patterns of white spruce (Picea glauca(Moench) Voss) and Sitka spruce (P. sitchensis (Bong.) Can.).LITERATURE CITEDBauwens, S., Van Oostveldt, P., Engler, G. and Van Montagu, M. 1991. Distribution of therDNA and three classes of highly repetitive DNA in the chromatin of interphase nucleiofArabidopsis thaliana. Chromosoma, 101: 4 1-48.Bickmore, W.A. and Sumner, AT. 1989. Mammalian chromosome banding - an expression ofgenome organization. Trends in Genet. 5: 144-148.Botstein, D., White, R.L., Skolnick, M. and Davis, R.W. 1980. Construction of a geneticlinkage map in man using restriction fragment length polymorphisms. Am. J. Hum.Genet. 32: 3 14-331.5Chinoy, C.N., Devos, K.M., Bringloe, D., Gray, J.C., Gale, M.D. and Dyer, T.A. 1991.Chromosomal location of the genes for ferredoxin in wheat, barley and rye. Theor.Appi. Genet. 82: 1-2.Cullis, C.A., Creisson, G.P., Gorman, S.W. and Teasdale, R.D. 1988. The 25S, 18S, and 5Sribosomal RNA genes from Pinus radiata. hi Proceedings of the Second Workshop ofthe IUFRO Working Party on Molecular Genetics, Chalk River, Ontario, June 15-18,1987. Cheliak, W.D. and Yapa, AC.(eds). pp. 34-40.Devey, M.E., Fiddler,T.A., Liu, B.-H., Knapp, S.J. and Neale, D.B. 1994. An RFLP linkagemap for loblolly pine based on a three-generation outbred pedigree. Theor. Appl.Genet. 88: 273-278.Flavell, R.B., ODell, M., Smith, D.B. and Thompson, W.F. 1985. Chromosome architecture:the distribution of recombination sites, the structure of ribosomal DNA loci and themultiplicity of sequences containing inverted repeats. In Molecular form and functionof the plant genome. NATO ASI, 83. van Volten-Doting, L., Groot, G.S.S.P. andHall, T.C. (eds). Plenum Press, New York. pp. 1-14.Flavell, R.B. 1980. The molecular characterization and organization of plant chromosomalDNA sequences. Ann. Rev. Plant. Physiol. 31: 569-5 96.Goessens, G. 1984. Nucleolar structure. mt. Rev. Cytol. 87: 107-158.Groover, A.T., Williams, C.G., Devey, M.E., Lee, J.M. and Neale, D.B. 1995. Sex-relateddifferences in meiotic recombination frequency in Piiii,s taeda. J. Hered. 86: 157-158.Gustafson, J.P. and Dille, J.E. 1992. Chromosome location of Oryza sativa recombinationlinkage groups. Proc. NatI. Acad. Sd. USA 89: 8646-8650.Jermstad, K.D., Reem, A.M., Henifin, J.R., Wheeler, N.C. and Neale, D.B. 1994. Inheritanceof restriction fragment length polymorphisms and random amplified polymorphicDNAs in coastal Douglas-fir. Theor. Appl. Genet. 89: 758-766.Khoshoo, T.N. 1961. Chromosome numbers in gymnosperms. Silvae Genet. 10: 1-9.Lapitan, N.L.V., Ganal, M.W. and Tanksley, S.D. 1989. Somatic chromosome karyotype oftomato based on in situ hybridization of the TGR1 satellite repeat. Genome, 32: 992-998.Lapitan, N.L.V., Gill, B.S. and Sears, R.G. 1987. Genomic and phylogenetic relationshipsamong rye and perennial species in the Triticeae. Crop Sd. 27: 682-686.6Leitch, I.J. and Heslop-Harrison, J.S. 1993. Physical mapping of four sites of the 5S rDNAsequences and one site of the alpha-amylase-2 gene in barley (Hordezim vulgare).Genome, 36: 5 17-523.Lucchesi, J.C. and Suzuki, D.T. 1968. The interchromosomal control of recombination. Ann.Rev. Genet. 2: 53-86.Lukaszewski, A.J. 1992. A comparison of physical distribution of recombination inchromosome 1R in diploid rye and in hexaploid triticale. Theor. Appl. Genet. 83:1048-1053.Nelson, C.D., Nance, W.L. and Doudrick, R.L. 1993. A partial genetic linkage map of slashpine (Finus el/joWl Engelm. var. el/joWl) based on random amplified polymorphicDNAs. Theor. App!. Genet. 87: 145-151.Pardue, M.L. and Gall, 3. 1970. Chromosomal localization of mouse satellite DNA. Science,168: 1356-1358.Schweizer, D. 1980. Fluorescent chromosome banding in plants: applications, mechanismsand implications for chromosome structure. In Proceddings of the Fourth John InnesSymposium, Davies, D.R. and Hopwood, R.A. (eds.), Norwich 1979. The PlantGenome: John Innes Charity, Norwich, pp. 6 1-72.Steinmetz, M., Uematsu, Y. and Lindahi, K.F. 1987. Hotspots of homologous recombinationin mammalian genomes. Trends in Genet. 3: 7-10.Thompson, W.F. and Murray, M.G. 1981. The nuclear genome: structure and function. InBiochemistry of Plants, Strumpf, P.K. and Conn, E.E. (eds.). Academic Press, NewYork. pp. 10-8 1.Tulsieram, L.K., Glaubitz, J.C., Kiss, G. and Carlson, J.E. 1992. Single tree genetic linkagemapping in conifers using haploid DNA from megagametophytes. Bio/Technology,10(6): 686-690.Williams, J.G.K, Kubelik, A.R., Livak, K.J., Rafaiski, J.A. and Tingey, S.V. 1990. DNApolymorphisms amplified by arbitrary primers are useful as genetic markers. Nuc.Acids Res. 18: 6531-6535.Zhang, H.-B. and Dvorak, J. 1990. Isolation of repeated DNA sequences from Lophophyrurnelongatuni for detection of Lophophyrurn chromatin in wheat genomes. Genome, 33:283-294.7CHAPTER 1Literature ReviewChapter 1 summarizes the pertinent literature concerning the genus Picea, repeatedDNAs and the development and use of ISH in genome analysis. This chapter is not intendedas a comprehensive review, in particular of tandem repeated DNA organization and evolutionwhich are discussed throughout, but rather for sufficient background information for thereading of this thesis.WHITE SPRUCE A1\D SITKA SPRUCEThe species of primary interest to this research are the closely related spruces whitespruce (Picea glauca (Moench) Voss) and Sitka spruce (P. sitchensis (Bong.) Carr.), two ofthe approximately 40 species of spruce recognized (Mikkola 1969). White spruce is one ofthe most widely distributed spruce species, ranging across boreal America from coastal Alaskato Newfoundland and south into Montana, Wisconsin, Michigan and the New England states.In British Columbia it is found throughout most of the interior on the eastern side of theCoastal Mountains at elevations below 1000 meters. Throughout much of the B.C. interior,the natural range of white spruce overlaps with that of Engelmann spruce (P. engelnianni(Parry)), which occupies the higher elevations from 1000 metres up to timberline in the centraland southern Rocky Mountains (Owens 1982). Effective barriers to interspecific8hybridization do not exist between these two species resulting in complex hybrid “swarms” inareas where the species are sympatric (Roche 1969), a common occurrence among manydifferent species of spruce (Wright 1955). From an operational perspective, the B.C. Ministryof Forests makes no distinction between white spruce, Engelmann spruce and their hybrids.The species complex is denoted simply as “interior spruce” because of the considerableinterspecific hybridization and similarities in the species’ cultural regimes in seedling nurseriesand in planting sites.Sitka spruce ranges from Kodiak Island, Alaska to northern California, confinedpredominantly to a coastal belt below 700 metres in elevation and seldom wider than 80kilometers. Along several drainage systems, notably the Nass, Bulkley and Skeena Rivers inthe Coastal Mountains and the Skagit River in the Cascades, Sitka spruce may be found as faras 150 kilometers inland where its range also frequently overlaps with that of either whitespruce to the north or Engelmann spruce to the south (Hosie 1990). Zones of introgressivehybridization occur between Sitka spruce and both white and Engelmann spruces where theirranges are sympatric (Fowler 1987; Hosie 1990).Interior spruce is extensively harvested in B.C. owing to its predominance and value aspulpwood, lumber, and as a source of specialty wood products (Owens 1982). It is animportant component of reforestation programs in the province with an estimated 100 millionseedlings being required for artificial regeneration by the year 2000 (Kiss and Yeh 1988).Sitka spruce is also extensively harvested although its value in reforestation in B.C. ispresently limited, in particular owing to a high incidence of plantation failure due tosusceptibility to the white pine terminal weevil (Pissodes strobi Peck) (Heppner and Wood1984).9THE NUCLEAR GENOME OF SPRUCEWith the exception of chromosome numbers and information provided by the kineticsofDNA fragment reassociation in solution it is not known how the nuclear genome of spruce,and indeed any conifer, is organized. Early cytological investigations of the genus noted thatall spruce species are diploids with the nuclear genome packaged into 12 chromosome pairs,i.e., 2n = 24 (Sax and Sax 1933; Santamour 1960). Only the smallest 4 chromosome pairs cangenerally be distinguished in metaphase preparations using conventional stains, the largest 8pairs being metacentric and of similar sizes.These preliminary surveys revealed a remarkable conservation of karyotypemorphology among spruce species as well as other conifer genera (e.g. in Pinus, Pederick1970). This fact, and the difficulties in identifying each mitotic chromosome of a species, hasseverely impacted the field of conifer cytogenetics and limited cytological contributions tophylogenetic studies in spruce and other gymnosperms.Spruce chromosomes are large in comparison to many angiosperm species and thissize is reflected in estimates of the DNA content of the nuclear genome. Using Feulgenmicrospectrophotometry, the diploid nucleus of white spruce contains 17 picograms of DNA(Dhillon 1987) which translates into approximately 8.5 X 10 base pairs per haploid genome.In comparison, the genome of spruce is 100 times the size of the Arabidopsis genome, thesmallest genome among flowering plants, 20 times that of rice and 3-4 times that of maize(Arumuganathan and Earle 1991). Among gymnosperms, nuclear DNA amounts varyapproximately 12-fold (Ohri and Khoshoo 1986). Excluding Gnetum with clearly the smallestgymnosperm genome (4.5 pg) the range is reduced to approximately 4-fold. This is in10contrast to the 100-fold variation among diploid angiosperms and reflects the similaritiesbetween related species and many genera of gymnosperms observed in cytological studies.The difference in genome sizes between Arabidopsis and species with large genomes isunlikely to arise from a need for a battery of new gene products. In fact, as has been shownby reassociation kinetics, increasing genome size is positively correlated with an increasingamount of repeated DNA (Lapitan 1992). In Arabidopsis, only about 15% of the genome isrepeated (Leutwiler et at., 1984) whereas in species with greater than 2 pg of DNA pernucleus the repeated DNA fraction typically exceeds 75% of the genome, much of which hasno apparent cellular function (Flavell 1980). Studies on the reassociation kinetics of conifergenomes confirm this generality: approximately 68% of the white spruce genome reannealswith repeated DNA kinetics (Rake et at., 1980), a value which likely underestimates the truerepeated DNA content of the spruce genome since the technique fails to include repeatedsequences which are too short or have diverged to such an extent as to prevent stable DNAduplexes forming in solution.ORGANIZATION AND EVOLUTION OF REPEATED DNADNA reassociation kinetics describes repeated DNA sequences solely in terms of theirgenomic copy number. Only since the development of techniques in molecular biology has thecomplete characterization of repeated sequences become possible. While no attempts hadbeen made prior to the beginning of this research to study repeated DNA families in conifersor other gymnosperms, a considerable volume of information concerning their organizationand evolution in animals and angiosperm plants has been amassed. These studies have11revealed that repeated DNA sequences are found in two distinct classes, either arranged astandem arrays or interspersed with unrelated repeated or unique DNA sequences (Flavell1986).Tandem repeated DNA sequencesTandem arrays are composed of closely related repeating units arranged in a “head-to-tail” manner and are typically the most highly represented DNA sequence families in complexgenomes. Many distinct families can coexist within a genome, differing in sequence andcomplexity, and in genetic activity. Among these are the least complex repeats of themicrosatellites (repeating units of 6 bp or less) and the minisatellites (with repeating units of11-60 bp), the satellite DNAs composed of tandemly repeated units of 150-500 bp lengths andoriginally named for their distinct banding position away from the main band DNA on cesiumchloride density gradients, the genes encoding the 18S, 5.8 S and 26S ribosomal RNAs andthe 5S ribosomal RNA, and the specialized, highly conserved tandem repeats with theconsensus sequence of (T/A)1G8at the telomeres of each eukaryotic chromosome (Zakian1989; Ganal et a!., 1991). The organizational features of relevant repeated DNA families arediscussed in detail in the introductions of Chapters 2, 3 and 4.The origin of tandem repeated DNA sequences in a genome undoubtedly involvessome form of amplification event at specific chromosomal sites, such as centromeres andtelomeres, or wherever it is tolerated. Although the precise mechanisms are not understood,several possibilities have been proposed, including unequal crossing over between twosequences (Smith 1976), excision followed by a rolling circle type of DNA replication andreintegration (Hourcade et a!., 1973), slippage replication (Tautz and Renz 1984) and12aberrant in situ replication (Schimke 1982).Tandem arrays of a given sequence family are frequently found in similar positions onmore than one, if not all chromosomes of a complement. Thus, arrays can be divided orduplicated and transposed to both homologous and non-homologous chromosomes. Again,although the details of intragenomic movement of tandem repeated sequences are sketchy,Flavell (1985) suggests it may result from double crossovers between interacting chromosomesegments or the excision of a circular segment of arrays and its reintegration elsewhere. Inthis regard, the association of chromosomes in intermitotic nuclei (“Rabl polarization”; Rabi1885) described in some (but not all) plant and animal cell nuclei (Comings 1980) isintriguing. Rabi polarization creates a unique ordering of chromosomes within a cell such thatnon-homologous chromosomes of most similar length are adjacent to one another.Centromeres are then closely associated as are telomeres on chromosome arms of similarlength. Such a physical environment could facilitate the recombination necessary fortransposing members of a tandem array onto homologous and non-homologous chromosomesleading to the fixation of an array in a species.Since most tandem arrays, with the exception of ribosomal RNA genes and thetelomeric repeat structure, appear not to have a strictly sequence-dependant function, theyaccumulate base substitutions, deletions and insertions at higher rates than protein-codinggenes. However, both the repeats within a single tandem array and all members of a sequencefamily in a given genome do not evolve independently. Instead, DNA sequence analysisreveals very high homology among repeats within a species (Lapitan 1992). They thereforeappear to evolve “in concert” (Arnheim et a!., 1980; Dover 1982) through mechanisms suchas unequal crossing over and/or gene conversion events (Flavell 1985). It is precisely because13of comparatively rapid divergence between species and concerted evolution within a speciesthat tandem repeated DNA sequences have attracted considerable attention as phylogenetictools.The debate over the role(s) of tandem repeated DNA (or heterochromatin) continues.How does one ascribe function(s) to DNA sequence families which make up a highly variablecomponent of the genome in terms of copy number, length, sequence and organization?Proponents of the “junk11 DNA hypothesis maintain that tandem repeated DNA, and repeatedDNA in general, have no functional significance, simply accumulating and evolving due toselective neutrality (Orgel and Crick 1980; Doolittle and Sapienza 1980). As Macgregor andSessions (1986) note: “in no case is there any obvious causal relation between amounts ofsatellite DNA and morphological change within a group of related animals. It is possible forsatellite sequences to be lost from both tissue culture cells and in vivo without obvioussomatic effects”. Others hold the view that tandem repeated DNA is selected for itsinvolvement in chromosome pairing, recombination or organization of chromosomes in thenucleus (Flavell 1982). At least in Drosophila, meiotic pairing seems to requireheterochromatic homology (Trick 1994). Martinez-Zapater et al. (1986) propose theinvolvement of satellite DNA in the organization of DNA into nucleosomes based on thecorrespondence of satellite DNA lengths in many species and the distance betweennucleosomes. Some satellites are transcribed (although a cellular function cannot be assignedor the transcriptional event is due to read-through from adjacent structural genes: Macgregorand Sessions 1986). The most recent addition to the debate is the enticing postulate of Vogt(1992) in which tandem repeated DNAs permit the establishment and stabilization of specificchromatin folding structures in distinct chromosome regions such as centromeres and14telomeres.Dispersed repeated DNA sequencesSpecies with very large genomes have not nearly enough tandem repeated DNAsequence families to account for the amount of DNA in excess of protein coding requirements(Smyth 1991). Much of these large genomes, for example more than 50% of the genome ofcereals (Flavell et a!., 1981), is composed of repeated DNA sequences found interspersedwith other repeated DNAs or unique sequences. Dispersed repeats of 5-2000 bp may befound in large genomes on average every 200-4000 bp of unique sequence DNA (Flavell1980). By contrast, in small genomes, the length of unique sequence DNA between dispersedrepeats may exceed 130,000 bp (Ganal et a!., 1988). Dispersed repeats are more difficult tocharacterize due to their association with diverse neighboring sequences and lower copynumber than most tandem arrayed repeated DNAs. However, several have now been studiedin a number of angiosperms including rye, tomato, rice, maize and lily (Rogowsky et a!.,1990; Ganal eta!., 1988; Mochizuki eta!., 1992; Schwarz-Sommer eta!., 1987; Smyth et at.,1989) revealing considerably more size and structural heterogeneity among different copies ina genome than tandem arrayed sequences.The genomic organization of dispersed repeats is consistent with the discovery thatmany have the properties of mobile genetic elements. Several dispersed elements in plantgenomes have been identified as transposable elements, mobile via a DNA intermediate andencoding a transposase gene and sequences necessary for recognition and transposition. Anexample of this type is the Activator transposon of maize (McClintock 1948). However, sincetheir copy numbers generally do not exceed several hundred per genome at the most, this class15of dispersed repeat appears to contribute little to genome size (Smyth 1991). Other mobileelements have structural similarities to retrotransposons, transposing via an RNAintermediate. Among these, two distinct classes have been identified based on whether or notlong terminal repeats are produced during amplification (Smyth 1991). In contrast totransposable elements, some retrotransposon types of dispersed repeats have been found inhigh copy number in several genomes, including the IFG element present in approximately10,000 copies in the sugar pine genome (C.S. Kinlaw, personal communication) and the del2element at 240,000 copies in lily (Leeton and Smyth 1993). Although not all dispersedrepeats share features in common with transposons or retrotransposons, most contain invertedor direct repeats (Flavell 1982) which suggest that they may be the remnants of once activemobile elements. The potential for mobile elements to have contributed significantly to thelarge genome size of plants is further illustrated by electron microscopic observations that asmuch as 10% of wheat DNA can form hairpin structures under high stringency conditions(Flavell 1984).Clearly the frequent amplification and transposition of mobile elements could be aselective disadvantage which might explain why few active elements in plants have beendiscovered. At least in the case of retrotransposons, subsequent activity may be precluded bythe error prone nature of reverse transcriptase, such that inserted elements may already bediverged in sequence and unable to move further. Although gene conversion processes canact to maintain the sequence homogeneity of a dispersed repeat family, Scherer and Davis(1980) noted that this mechanism operates at lower frequency than with tandem arrayedrepeats. Thus over time the individual members of a dispersed repeated family may divergeand eventual become part of the single copy sequence component of a genome (Smyth 1991).16LOW COPY DNA SEQUENCES IN CONIFER GENOMESNo discussion on genome organization is complete without mention of the distributionof protein-coding and other single- or low-copy sequences. As noted previously, 25-30% ofthe genome of white spruce and other conifers reassociates with single- or low-copy kinetics.One might question why in large genomes like those of conifers the repeated DNA fractiondoes not exceed 95%, relegating the low- and single-copy fraction to one up to severalpercent. Although the percentage of repeated DNA is positively correlated with increasingDNA content in plant species, single copy sequences are also found to increase in largegenomes (Hutchinson el a!., 1980). Therefore, conifer genome size is not simply a function ofan elevated repeated DNA content.Prior to RFLP mapping, isozyme linkage analysis was used to investigate theorganization of a limited portion of the low copy component of conifer genomes.Conservation of isozyme linkage groups among pine species and other Pinaceae, includingwhite spruce, was used to infer that no major chromosome rearrangements had occured inthese regions during conifer evolution (Conkle 1981).RFLP mapping vastly improves upon the number of available markers for geneticanalysis and consequently yields a much more detailed view of the organization of low-copysequences. Only in loblolly pine, however, are RFLP maps sufficiently advanced to allowmeaningful insights. Of 65 cDNAs mapped by Devey et a!.,, (1994), 24% detected morethan one segregating locus that could be mapped. According to the authors, many othercDNAs detected multiple loci but the complexity of the hybridization pattern prevented theirmapping. This is in contrast to the genome of Arabidopsis in which 98% of RFLPs map to17single loci (Chang et al., 1988).While polyploidy in the course of evolution can be responsible for multiple loci of agiven gene, as in maize (Helentjaris et al., 1988), this phenomenon has not been a regularfeature of conifer evolution (Khoshoo 1959). Thus, numerous multigene families have arisenby other means in the loblolly pine genome, conceivably by transposition, and in part accountfor the large fraction of single- and low-copy DNA. The possibility that much of thiscomponent is actually composed of ancient families of dispersed repeats which have divergedenough to prevent their inclusion in a repeated DNA family has yet to be investigated.IN SITUHYBRIDIZATIONThe basic principles of ISH to cytological preparations were independantly establishedmore than 25 years ago by Gall and Pardue (1969) and John et a!. (1969). These pioneeringefforts used tritium-labeled RNA probes to visualize ribosomal RNA in Xenopus oocytes. Inthe following years, isotopic ISH was adapted to agricultural plants and used to determine thechromosomal location of both repeated DNA sequences (Bedbrook e. a!., 1980; Hutchinsonet a!., 1981) and single copy DNA sequences (Shen et a!., 1987). Isotopic ISH is verysensitive particularly in human cytogenetics where unique sequences as small as 500 base pairshave been detected on metaphase chromosomes (Jhanwar et a!., 1983). The technique,however, suffers from several drawbacks relating to the use of radioactive labels, includinglong exposure times, limited resolution due to scattering of radioactive emissions and theircapture in an emulsion overlay, a high degree of background signal necessitating statisticalanalysis of hybridization data, and safety and disposal concerns.The major advances and widespread application of ISH techniques have arisen withthe development of non-radioactive labeling methods. The most common method is indirectlabeling in which hapten-like reporter molecules (typically biotin) are incorporated intohybridization probes and subsequently detected by the appropriate enzymatic or fluorescentaffinity reagents. While labeling methods have been developed in which nucleotides labeledwith fluorescent molecules are incorporated directly into hybridization probes, they currentlyprovide lower sensitivity than indirect methods in most cases.Langer et a!. (1981) described the synthesis and use of deoxyuracil-5’-triphosphate(dUTP) to which biotin, a member of the vitamin B complex, had been attached to thepyrimidine ring. Analogs of dUTP, dCTP and dATP are now commercially available and eachbiotin-labeled nucleotide can be readily incorporated into hybridization probes by nicktranslation (Rigby eta!., 1977), random primer labeling (Feinberg and Vogelstein 1983) or thepolymerase chain reaction (Saiki et a!., 1985; Mullis et a!., 1986). Additional reportermolecules include digoxigenin and dinitrophenol which may be incorporated into probes insimilar fashion, and aminoacetylfluorene, mercury and sulfonate which are attached to DNAthrough chemical reactions (Trask 1991).The detection of biotin-labeled probes after hybridization to chromosomes is achievedthrough enzymatic or fluorescent conjugates of either anti-biotin antibodies or avidin, aglycoprotein extracted from egg white. The affinity of avidin (and related molecules such asstreptavidin and ExtrAvidin) for biotin is higher than immunological methods making it themethod of choice for biotin detection. Horseradish peroxidase which catalyses thepolymerization of 1,2-diaminobenzidine in the presence of hydrogen peroxide, producing abrown precipitate at the hybridization site, has been used as an avidin-enzyme conjugate19(Rayburn and Gill 1985). Amplification of the hybridization signal by prolonging the reactioncan improve the sensitivity of this method for the detection of less abundant DNA sequences(Jiang and Gill 1994).Fluorescent tags are increasing in popularity since first reported (Langer-Safer et at.,1982). A variety of fluorochromes, such as fluorescein isothiocyanate, rhodamine, Texas Red,amino methyl coumarin acetic acid, Cy3, and Cy5, each with distinct spectral properties, havebeen conjugated to either antibodies or avidin, enabling the simultaneous hybridization andindependent localization of different DNA probes (Leitch et a!., 1991). (The excitation andemission characteristics of commonly used fluorochromes are presented in Appendix I).Signal amplification can also be achieved using anti-avidin or anti-biotin antibodies (Pinkel etat., 1986). The most impressive use of fluorescent ISH to date has undoubtedly been inhuman cytogenetics where as many as 7 cosmid probes have been physically mapped in asingle experiment by combinatorial labeling (Ried et a!., 1992).Another important advantage of fluorescence ISH in relation to other reporter systemsis the development of sophisticated digital imaging instruments including the confocal laserscanning microscope and the cooled charge coupled device camera. Not only can differentfluorochromes be detected separately using specific filters, but digital images are generatedwhich can be analysed by computer software providing an added degree of precision tophysical mapping.The simplicity and benefits of non-radioactive ISH have led to its broad use in theanalysis of plant genomes. Rayburn and Gill (1985) were the first to report the use of biotinlabeled probes in plants, determining the location of a tandem repeated DNA family on thechromosomes of wheat. In the past decade, additional tandem repeated sequences including20the multigene families of the ribosomal RNAs, the 5S rRNA and specific cloned sequenceshave been mapped in many angiosperms, such as wheat, rye, barley, rice, soybean, tomato,rapeseed, onion and Arabidopsis (Appels et a!., 1980; Bedbrook et al., 1980; Leitch andHeslop-Harrison 1992; Song and Gustafson 1993; Skompska ci a!., 1989; Ganal eta!., 1988;Xia eta!., 1993; Ricroch et a!., 1992; Bauwens ci a!., 1991). Recently ISH in several speciesof pine has also been reported (Cullis ci a!., 1988; Karvonen ci a!., 1993; Doudrick et a!.,1995). Among the useful contributions to the understanding of plant genome structureprovided by these studies have been the detection of additional ribosomal RNA sites notdetectable by Southern hybridization (Mukai et a!., 1991) and the molecular characterizationof telomeric heterochromatin in cereals (Bedbrook et a!., 1980), heterochromatic knobs inArabidopsis (Maluszynska and Heslop-Harrison 1991) and Giemsa C-bands in rye (Mukai eta!., 1992). As a karyotyping tool, the physical mapping of tandem repeats has enabled theidentification of somatic chromosomes in tomato (Lapitan ci a!., 1989), and in combinationwith fluorescent dye banding patterns, the chromosomes of slash pine (Doudrick ci a!., 1995).The chromosomal distribution of dispersed repeated sequences has also been demonstrated inseveral angiosperm genomes (Ganal eta!., 1988; Moore ci a!., 1991).Genomic ISH, an extension of the basic procedure, is an important development in theanalysis of polyploid species and hybrids (Le ci a!., 1989; Schwarzacher ci a!., 1989). Bycombining labeled total DNA from one parental species with unlabeled total DNA from theother parental genome(s) in the hybridization mixture, only species-specific DNA sequences ofthe labeled genome, which in all likelihood are repeated sequences, remain single stranded andcapable of producing hybridization signals. Providing that the genomes contributing to thepolyploid or hybrid species are sufficiently diverged with respect to their repeated DNA21compositions, the chromosomes or chromosomal segments from the different genomes areclearly differentiated after ISH. This approach, therefore, does not require the molecularcloning of species-specific repeated DNA sequences. Genomic ISH has been used todetermine genome origin, relatedness and evolution in allopolyploids such as hexaploid wheat(Mukai et at., 1993). From a breeding perspective, the method has been used to monitor theintrogression of alien chromatin from rye into wheat breeding lines (Schwarzacher et at.,1992). Wide crossing strategies provide valuable sources of genetic variability in agriculturalbreeding programs. Genomic ISH can efficiently and accurately reveal the location oftranslocation breakpoints and the amount of alien chromatin remaining in the crop species insubsequent backcross generations.Non-radioactive ISH of low copy or unique DNA sequences to plant chromosomeshas proven difficult, although not impossible. A number of factors inherent in plantcytogenetics likely account for the limited success, including the presence of the plant cellwall, the low mitotic index of root tips and the methods of plant chromosome preparation.These factors and the methods available to circumvent the difficulties they present in thephysical mapping of low copy DNA sequences are discussed in detail in Chapter 6.LITERATURE CITEDAppels R., Gerlach W.L., Dennis E.S., Swift H. and Peacock, W.J. 1980. Molecular andchromosomal organization of DNA sequences coding for the ribosomal RNAs incereals. 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Recombination of dispersed repeated DNA sequences inyeast. Science, 209: 1380-1384.Schimke, R. 1982. In Gene amplification. Schimke, R. (ed.), pp. 317-333. New York. ColdSpring Harbor Press.Schwarz-Sommer, Z. Leclercq, L., Gobel, E. and Saedler, H. 1987. Cin4, an insert alteringthe structure of the Al gene in Zea mays, exhibits properties of nonviralretrotransposons. EMBO J. 6: 3873-3 880.Schwarzacher, T., Anamthwat-Jonsson, K., Harrison, G.E., Islam, A.K.M.R., Zia, 3. Z., King,I.P., Leitch, A.R., Miller, T.E., Reader, S.M., Rogers, SM., Shi, M. and Heslop28Harrrison, J.S. 1992. Genomic in situ hybridization to identify alien chromosomes andchromosome segments in wheat. Theor. Appi. Genet. 84: 778-786.Shen, D. and Wu, M. 1987. Gene mapping on maize pachytene chromosomes by in situhybridization. Chromosoma, 95: 311-314.Skorupska, H., Albertseen, M.C., Langholz, K.D. and Palmer, R.G. 1989. Detection ofribosomal RNA genes in soybean, Glycine max (L.) Merr., by in situ hybridization.Genome, 32: 1091-1095.Smith, G.P. 1976. Evolution of repeated DNA sequences by unequal crossing over. Science,191: 528-535.Smyth, D.R. 1991. Dispersed repeats in plant genomes. Chromosoma, 100: 355-359.Smyth, D.R., Kalitsis, P., Joseph, J.L. and Sentry, J.W. 1989. Plant retrotransposon fromLiliun2 henryi is related to Ty3 of yeast and the gypsy group of Drosophila. Proc.Nati. Acad. Sci. USA 86: 5015-5019.Song, Y.C. and Gustafson, J.P. 1993. Physical mapping of the 5S rDNA gene complex in rice(Oryza sativa). Genome, 36: 658-661.Swarzacher, T., Leitch, A. R., Bennett, M. D. and Heslop-Harrison, J. S. 1989. In situlocalization of parental genomes in a wide hybrid. Ann. Bot. (London), 64: 3 15-324.Tautz, D. and Renz, M. 1984. Simple sequences are ubiquitous repetitive components ofeukaryotic genomes. Nucl, Acids. Res. 12: 4127-4138.Trask, B.J. 1991. Fluorescence in situ hybridization. Trends in Genet. 7(5): 149-154.Vogt, P. 1992. Code domains in tandem repetitive DNA sequence structures. Chromosoma,101:585-589.Wright, J.W. 1955. Species crossabililty in spruce in relation to distribution and taxonomy.Forest Sciences, 1(4): 3 19-349.Xia, X., Selvaraj, G. and Bertrand, H. 1993. Structure and evolution of a highly repetitiveDNA sequence from Brassica napus. Plant Mol. Biol. 21: 2 13-224.Zakian, V.A. 1989. Structure and function of telomeres. Ann. Rev. Genet. 23: 579-604.29CHAPTER 2Development of In Slit, Hybridization in Spmce: the Chromosomal Location of GenesEncoding the I 8S-5.8S-26S Ribosomal RNAINTRODUCTIONThe genes encoding the 18S-5.8S-26S ribosomal RNA (18S-26S rDNA) were amongthe first isolated and have been studied at the molecular and cytological levels in a widevariety of organisms. From 500 to 40,000 copies of the 18S-26S rDNA, in some casescomprising up to 10% of the total DNA, are found in tandem arrays at one to severalchromosomal sites in plant genomes (Rogers and Bendich 1987). Each repeating unit istypically from 7-12 kb in length in angiosperm plants and consists of the coding region for theRNA products and associated spacers (Fig. 2.1).Within the nontranscribed or intergenic spacer (IGS), sequence repetition is alsoobserved in all organisms. In some lower eukaryotes, such as yeast and slime mold, the 5SrDNA is located here (Appels and Honeycutt 1986). In higher eukaryotes, the 5S rDNA isnot linked to the 1 8S-26S rDNA, and instead, a small tandem subrepeat is found. Forexample, there are 11 copies of a 13 1-133 bp element in wheat (Appels and Dvorak 1982). InDrosophila and Xenopus, the subrepeats share homology with the site of RNA polymerase Itranscription initiation and, while this feature is not observed in all species analysed, it has ledto the postulation that the subrepeats serve as sites for the loading ofRNA polymerase I.3018S 5.8S 26S 18STranscription UnitRepeating UnitFig. 2.1. Molecular structure of a typical 18S-5.8S-26S ribosomal RNA gene in highereukaryotes. The precursor rRNA consists of an external transcribed spacer (ETS)preceding the 1 8S rRNA gene at the 5’ end of the transcript, the 5. 8S rRNA separatedfrom both the 18S and 26S rRNA by intervening spacers (ITS-i and ITS-2), and the26S rRNA at the 3’ end of the transcript. The vertical lines within the intergenic spacerdenote sequence repetition observed in all organisms (Appels and Honeycutt 1986). Inwhite spruce, the restriction enzyme Bg/ll has a single recognition site within thissubrepeat. Several subrepeats have been cloned and sequenced, revealing a 131 bprepeating element (Brown, Newton and Carlson, unpublished).ETS ITS-I ITS-2 ETS31Transcription of the rDNA can be observed cytologically both in interphase and duringcell division. During interphase, the nucleolus forms around the 18S-26S rDNA arrays withinwhich the chromatin is dispersed and transcribed. For this reason, the 18S-26S rDNA loci arefrequently referred to as the nucleolus organizer regions (NOR). The rRNA moleculesassociate with proteins and accumulate within the nucleolus before being exported to thecytoplasm. As the cell cycle continues, rRNA synthesis decreases in prophase and thenucleolus disintegrates. Although the chromatin which was actively transcribed in thenucleolus condenses along with the remainder of the metaphase chromosome, it remainsassociated with nucleolar proteins and stains poorly in comparison to adjacent chromatin,giving rise to a visible gap or secondary constriction (Goessens and Lepoint 1974). It is nowgenerally accepted that secondary constrictions are the sites of rRNA genes that were activelytranscribed in the preceding interphase. Because of the high GC content of the 18S-26SrDNA, secondary constrictions are also easily revealed by fluorescent dyes whichpreferentially bind GC or AT base pairs, such as Chromomycin A3 and DAPI, respectively. Inthe former case they appear as brightly staining regions and, in the latter, as negatively stainingregions.Since the 18S-26S rDNA has both a large physical size and a visible site on metaphasechromosomes, it is an ideal DNA sequence to use in developing ISH methodology.Additionally, although the IGS exhibits both sequence and length variation in many plantspecies (Rogers and Bendich 1987), the coding sequences are highly conserved across a wideevolutionary spectrum obviating the need to screen genomic libraries or to isolatehybridization probes by other means. In this chapter, a heterologous probe from soybean(Zimmer et a!., 1988) was used to determine the chromosomal location of 18S-26S rRNA32genes in white spruce and Sitka spruce. The findings described herein have been published bythe author in the journal Genonie (Brown et al., 1993).MATERIALS AND METHODSPlant material and chromosome preparationOpen pollinated seeds from one white spruce plus tree (Parent #5548 of the BritishColumbia Ministry of Forests Tree Improvement Program) originally located northeast ofPrince George, B.C. were provided by Gyula Kiss of the Kalamalka Research Station. Sitkaspruce seeds, collected from a Sitka spruce seed production orchard were provided by Dr.Yousry El-Kassaby of Pacific Forest Products, Ltd. Both species are diploids with 2n 24and 1 or more B chromosomes occasionally present. For uniform germination, Sitka spruceseeds were imbibed in distilled water overnight and stratified for 3 weeks at 4 OC. Thistreatment was not necessary with the white spruce seedlot used. Seeds of both species weregerminated on water saturated filter papers in a growth chamber for 6 days in the dark. Toaccumulate root tip cells in metaphase, the 1-2 cm long germinants were treated with 0.2%(w/v) aqueous colchicine for 6 hours. Following overnight fixation in 3:1 ethanol:acetic acid,root tips were stored in 70% ethanol at 4 0C until used.Microscope slides were cleaned thoroughly by washing in 2 M chromic acid for threehours followed by several rinses in distilled water and storage in ethanol. Prior to squashing,root tips were rinsed several times in 0.OIM sodium citrate/citric acid buffer (enzyme dilutionbuffer). A lox stock solution of this buffer was prepared by mixing 40 ml of 0.1 M citric acidand 60 ml of 0. 1 M trisodium citrate. The cell walls were digested to facilitate chromosome33spreading and the penetration of hybridization probes by incubating root tips in a solution of2% cellulase (Calbiochem: 11,300 U/g), 1% macerase (Calbiochem: 3100 U/g) and 2% liquidpectinase (Sigma: 9 U/g) in lx enzyme dilution buffer for 1 hour at 37 °C. Meristematicregions were then excised, teased apart in 45% acetic acid, and squashed under a coverslip.Slides were scanned for quality metaphase spreads on a Zeiss Axiophot microscope underphase contrast. The coverslip was removed from high quality slides with a razor blade afterfreezing on dry ice. Slides were then immersed briefly in 100% ethanol and air dried. Thesecould be stored at room temperature for several weeks without noticeable effect onhybridization.Chromosomes were stained prior to ISH in 0.2 .tg/ml 4’,6-diamidino-2-phenylindole(DAPI) in phosphate buffered saline (PBS, 0.13 M NaCl, 7 mMNa2HPO4,pH 7.4). DAPIstaining was visualized using a Zeiss UV filter block and well spread metaphases werephotographed on TMAX 400 film at ASA 1600. A micrometer scale bar was alsophotographed at the same magnification (100 X). Chromosomes were then destained in 3:1methanol:acetic acid for 30 minutes at room temperature, rinsed three times in methanol andair dried.Probe labelingpGmRl, supplied by E. Zimmer (Zimmer et al., 1988), contains a 7.9 kb EcoRlfragment in pBR325 encoding the 18S-5.8S-26S rDNA and the intergenic spacer sequencesof soybean (Glycine max (L.) Merr.). Plasmid DNA was purified from 250 ml bacterialcultures by alkaline lysis and cesium chloride ultracentrifugation (Sambrook et a!., 1989). Theentire plasmid molecule was labeled with biotin-14-dATP using a commercially available nick34translation kit according to the supplier’s recommendations (BRL Life Technologies). The 50pJ labeling reaction consisted of 1 .tg pGmRl, 20 M each of dCTP, dGTP, dTTP and biotin14-dATP, 50 mM Tris-HC1 (pH 7.8), 5 mM MgC12, 10 mM 2-mercaptoethanol, 2 U ofE. coilDNA Polymerase I and 200 pg of DNase I. Labeling was performed at 16 °C for 1.5-2 hoursand the reaction was then terminated by adding EDTA to 30 mM and SDS to 1.25%.Unincorporated nucleotides were removed on Sephadex G-50 (Pharmacia) spun columns orby ethanol precipitation. Lastly, an aliquot (50 ng) of the purified probe was run on a 2%agarose minigel in IX TAE (0.4 M Tris-acetate, 0.001 M EDTA, pH 8) to verify that theaverage fragment size was less than 500 bp.hybridizationSlides were incubated with 20 j.tg/ml DNase-free RNAase A (Boehringer-Mannheim:500 .tg/ml) in 2X SSC (0.6 M NaCI, 0.06 M sodium citrate, pH 7) at 37 °C for 1 hour.Following three washes in 2X SSC at room temperature for 5 minutes each, slides wereimmersed in 0.01 M HC1 for 2 minutes and then incubated in a solution of pepsin (Sigma:3700 U/mg of protein used at 5 p.g/ml in 0.01 M HCI) for 8 minutes at 37 °C. The reactionwas stopped by rinsing slides in distilled water for 2 minutes. Slides were then washed 3 timesin 2X SSC for 5 minutes at room temperature and dehydrated in a 70, 90, and 100% ethanolseries.Chromosomal DNA was denatured in one of two ways. Initial experiments performedthis step by incubating slides in 70% formamide/2X SSC, pH 7.0, for 2 minutes at 72 0C.Formamide used in the denaturing solution and hybridization mixture was de-ionized withBlO-RAD AG 501-X8 ion exchange resin for 4 hours and filtered. Slides were then35immediately transferred through an ice-cold ethanol series and air dried. The hybridizationmixture, consisting of 50-150 ng of labeled pGmRl, 7.5 i.g of sheared salmon sperm DNA,50% formamide, 10% dextran sulfate, 2X SSC, and 0.1% SDS, was then denatured at 80 °Cfor 10 minutes, chilled on ice, and applied to the slide. Hybridization proceeded at 37 °C for12-16 hours in a humidity chamber. Although this method was effective, the development ofequipment to automate chromosome denaturation (Heslop-Harrison et a!., 1991) simplifiedthe ISH protocol and improved consistency between experiments. Chromosomal DNAdenaturation was subsequently performed using the Omnigene Hybaid Temperature Cycler.In this situation, the probe hybridization mixture was denatured as described above, chilled onice, and applied to air dried slides following pepsin treatment. Once a coverslip was put inplace, slides were incubated at 80 °C for 10 minutes in the temperature cycler to denature thechromosomal DNA and then slowly cooled to 37 °C according to Heslop-Harrison et a!.(1991). Slides remained in the temperature cycler at 37 °C for 12-16 hours.After hybridization, slides were washed twice in 50% formamide/2X SSC for 5minutes at 42 °C, 2X SSC for 5 minutes at 42 °C, and 2X SSC for 5 minutes at roomtemperature.For the detection of biotin-labeled probes, slides were washed in 0.2% (v/v) Tween-20in 4X SSC (detection buffer) for 5 minutes at room temperature. They were then incubated in5% BSA in detection buffer at room temperature for 5 minutes to block non-specific avidinbinding. Slides were treated with 100 il of ExtrAvidin conjugated to fluoresceinisothiocyanate (FITC) (Sigma: diluted 1:100 in 5% BSA in detection buffer) at 37 oC for 1hour and then washed 3 times in detection buffer at 37 °C for 8 minutes. Chromosomal DNAwas counterstained for 3-5 seconds in 0.1 .Lg/ml propidium iodide, rinsed in 2X SSC and36mounted in 50% glycerol in PBS.MicroscopyIn situ hybridized chromosomes were imaged using a BlO-RAD MRC 600 confocallaser scanning system equipped with an argonlkrypton laser. FITC and propidium iodide wereexcited simultaneously using 488 nm and 538 nm laser lines. The resulting green and redemissions were separated to the two photomultipliers using Ki and K2 filter blocks.Fluorescein images were obtained in the photon counting mode (with signal accumulation topeak) and the propidium iodide images were taken in the direct mode with Kalman averaging.Images of hybridization sites and counterstained chromosomes were merged using AdobePhotoshop 3.0 software on a Macintosh Quadra 840 personal computer.Kaiyotype analysisPhotographs of DAPI stained metaphases (and micrometer scales printed at the sameenlargement) were digitized using an AGFA Studioscan II flatbed scanner. Chromosomeswere randomly numbered 1-24 and measured using the public domain NIH Image program(written by W. Rasband at the U.S. National Institutes of Health and available from theInternet by anonymous ftp from zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 PortRoyal Rd., Springfield, VA 22161, part number PB93-504868). After calibrating the softwareusing the known distance on the micrometer scale, total chromosome length was obtained bycomputer measurement of a line drawn down the middle of each chromosome. The positionsof the centromere and secondary constriction were marked and, using the Plot Profilecommand, values for the length of the short arm (p), long arm (q) and the fractional length of37the secondary constriction from the terminus of the short arm (FLpter) were derived. Thearm ratio (q/p), and centromeric index (p/p+q x 100) were then calculated. Homologous pairswere identified based on the measurements and hybridization patterns of 5 well spreadmetaphases and validated by the hybridization patterns from an additional 20 cells.Chromosome pairs were ordered according to convention from longest to shortest based onthe relative length of each chromosome pair expressed as a percentage of the diploid cellcomplement.RESULTSOf critical importance to the successful application of ISH to plant chromosomes is theability to generate many well spread metaphases free of the cell wall and cytoplasmic debris.Conventional methods of obtaining squash preparations from plant root tips entail the use ofHC1 hydrolysis to soften the middle lamella and improve chromosome spreading. However,cell wall material remains and, in addition, acid hydrolysis depurinates nucleic acids potentiallyresulting in the loss of chromosomal DNA target sequences. Cell walls and cytoplasmiccomponents will also inhibit the penetration of probe molecules and interact with elements ofthe detection system causing high levels of background signal.Three factors designed to ameliorate these difficulties were optimized for thepreparation of acceptable spruce root tip metaphases. Because spruce chromosomes arecomparatively long, an extended treatment with a colchicine solution considerably strongerthan used with most angiosperm germinants was required to bring about an adequate degreeof chromosome condensation and to allow satisfactory spreading. The cell wall was38Fig. 2.2. Localization of the 18S-26S rDNA in white spruce. a) Fluorescence ISH ofbiotin-labeled pGmRl (green) to the mitotic chromosomes of white spruce (2n = 24).Chromosomal DNA was counterstained with propidium iodide (red). b) The samemetaphase stained with DAPI. Asterisk, an unpaired B chromosome. Bar represents10 tm.39Fig. 2.3. 18S-26S rDNA polymorphism in whitespruce. a) Fluoresence ISH localization of pGmRl andb) DAPI staining reveal that only 13 rDNA loci arepresent in this metaphase. Arrows, chromosome pairshowing rDNA polymorphism. Asterisk, an unpaired Bchromosome. Bar represents 10 Rm.40Fig. 2.4. Localization of the 18S-26S rDNA loci in Sitka spruce.a) Fluorescence ISH ofbiotin-labeled pGmRi (green) to the mitoticchromosomes of white spruce (2n = 24). Chromosomal DNA wascounterstained with propidium iodide (red). b) The samemetaphase stained with DAPI. Bar represents 10 .tm.41effectively removed from most cells by incubating the root tips in a mixture of cellulase,macerase and pectinase prior to squashing. Lastly, most of the cytoplasm which still enclosedmany metaphases in the squash preparations was degraded by a pepsin incubation prior tochromosome denaturation and ISH.Hybridization sites of the biotin-labeled 18S-26S rDNA probe were detected as greenfluorescence from the FITC conjugated ExtrAvidin. Non-hybridizing chromosomal sequencesfluoresced red due to the propidium iodide counterstain. Images were acquired using aconfocal laser scanning microscope and, while the optical sectioning capability of this systemis of little added benefit on thin squash preparations, the ability to record the FITC signalindependently from that of the propidium iodide counterstain signal provides higher sensitivityin detecting hybridization signals compared to epifluorescent microscopy. Over the course ofdeveloping the ISH technique, more than 50 intact metaphases of both white spruce and Sitkaspruce were analysed. The number of pGmRl hybridization sites observed in white sprucevaried from 9 to 14 which was likely due to the quality of metaphase spreads given that locion overlapping chromosomes can be masked from probe or detection reagents. Qualityimproved with experience and the 10 best spreads in which one or no chromosomesoverlapped were selected for further analysis. In 6 metaphases (one shown in Fig. 2.2), 14pGmRl hybridization sites were counted. The same chromosome spread stained with DAPI,in which secondary constrictions appear as negative staining regions, shows that eachhybridization site is associated with a secondary constriction, indicating that all loci weretranscriptionally active in the previous interphase. The probe did not hybridize to the Bchromosome. Therefore, the 18S-26S rDNA in white spruce are located on 7 of the 12chromosome pairs. In the remaining 4 cells, only 13 hybridization sites and secondary42constrictions were observed (Fig. 2.3) suggesting that one of the white spruce chromosomepairs is polymorphic for this character. Of additional note in these metaphases was a veryelongated secondary constriction found on one of the large metacentric chromosomes.ISH and DAPI staining results in Sitka spruce are shown in Fig. 2.4. In all metaphasesobserved, 10 pGmRl sites were evident, each with a corresponding secondary constriction.Although not present in the metaphase pictured, pGmRl did not hybridize to the Bchromosome(s) of Sitka spruce.The mitotic chromosomes of white and Sitka spruce are represented by the ideogramsin Fig. 2.5 arranged from longest to shortest according to the relative length of each pairaveraged over 5 well spread metaphases. Pairing of homologous chromosomes was basedprimarily on the length, arm ratio, and presence and location of the secondary constriction orpGmRl hybridization signal (given as the FLpter value). In most cases a simple visualassessment was sufficient to identify and pair 20 of the 24 chromosomes in each species, theexceptions being chromosomes 2 and 5 in Fig. 2.5. With careful measurements of high qualitymetaphases these can be distinguished by total length differences. Somewhat fortuitously,chromosome 5 also contains the site of the 5S rRNA genes in both species as described inChapter 3.Although in situ hybridization is not an entirely quantitative procedure, in white sprucethe FITC signal on chromosome 6 was usually the most intense while that on chromosome 10was the faintest, sometimes appearing as a discrete dot on each chromatid. Therefore it islikely that the 18S-26S rDNA arrays on chromosome 6 and 10 correspond to the longest andshortest, respectively, in the white spruce genome. Additionally, karyotyping spreads showingthe possible rDNA polymorphism indicated that the potentially polymorphic locus in white4300H o123 U5678 9 1112A.R. 1.08 (0.05) 1.18 (0.03) 1.03 (0.02) 1.21 (0.05) 1.05 (0.04) 1.09 (0.05) 1.04 (0.03) 1.29 (0.06) 1.72(0.07) 1.33 (0.10) 1.17(0.08) 2.02(0.17C.L 0.49(0.01) 0.46(0.00) 0.50(0.01) 0.45(0.01) 0.49(0.01) 0.48(0.01) 049(0.01) 0.44(0.01) 0.37(0.01) 0.43(0.02) 0.46(0.02) 0.33(0.02RL. 10.01 (0.11) 9.28 (0.10) 9.25 (0.23) 9.24 (0.36) 8.88 (0.17) 8.50(0.32) 8.37(0.18) 8.33 (0.22) 7.78 (0.28) 7.46(0.18) 6.81 (0.18) 6.10(0.09FLpter 0.74(0.01) 0.32 (0.01) 0.19(0.01) 0.23 (0.01) 0.13(0.02) 0.72 (0.01) 0.19(0.13)H HH H H H H H H1 2 3 4 5 6 7 8 9 10 1112A.R. 1.07(0.04) 1.05(0,02) 1.12(0.06) 1.17(0.10) 1.05(004) 108(0.06) 1.05(0.03) 1.27(0.08) 1.82(0.11) 1.51(0.13) 1.20(0.10) 1.81(0.10)C.L 0.48(0.01) 0.49(0.01) 0.47(0.01) 0.46(0.02) 0.48(0.01) 0.48(0.01) 0.49(0.01) 0.44(0.02) 0.36(0.01) 0.40(0.02) 0.45(0.02) 0.36(0.01)R.L. 10.35(0.64) 9.57(0.24) 9.40(0.34) 9.37(0.31) 9.07(0.38) 8.87(0.24) 8.75(0.26) 8.42(0.19) 7.65(0.22) 6.93(0.44) 6.58(0.27) 6.01(0.34)FLpter 0.25(0.01) 0.66(0.01) 0.20(0.01) 0.22(0.01) 0,13(0.01)Fig. 2.5. Ideograms of the mitotic chromosomes of white spruce (top) and Sitka spruce(bottom), both 2n = 24. The upper arm is the short arm (p) by convention. A.R. = arm ratio,C.I. centromeric index, R.L. = relative length, FLpter = fractional length of the 18S-26SrDNA locus from the terminus ofthe p arm, as described in the Materials and Methods.44spruce was found on chromosome 2. Only slight variation in hybridization signal intensity wasobserved among the five Sitka spruce loci.As expected among closely related species with a high degree of sexual compatibility,many of the chromosomes share morphological features in white and Sitka spruces, includingranking, arm ratios, and the location of the 1 8S-26S rDNA. Notable discrepancies betweenthe species include the absence of the 18S-26S rDNA arrays on chromosome 8 and 10 inSitka spruce, different positions of the 1 8S-26S rDNA of chromosomes 2 and 3, and slightmorphogical differences between the smaller three chromosome pairs.DISCUSSIONA fluorescence ISH procedure for the detection of highly repeated DNA sequences onmetaphase chromosomes of spruce was developed and used to map the location of the genesencoding the 18S-26S rDNA in white and Sitka spruce. Cross-hybridization of the highlyconserved coding regions of the ribosomal DNA repeat from soybean revealed that the 18S-26S rDNA in white spruce are distributed at one site on seven chromosome pairs. In Sitkaspruce these loci are found on five pairs. All loci correspond to the positions of secondaryconstrictions at metaphase implying that each is actively transcribed during interphase in thespruce root tip meristem.ISH of rDNA probes has recently been reported in a number of pine species. Tenmajor sites and several minor sites were observed in radiata pine by Cullis et a!. (1988), and16 sites were observed in both Scots pine and slash pine (Karvonen et a!., 1993a; Doudrick etat., 1995). Considered with the data presented here on white and Sitka spruce, the genes45encoding the 18S-26S rRNA are more extensively distributed in these conifer genomes incomparison to angiosperms.The tandem organization of a repeated DNA family confers a susceptibility tomolecular mechanisms capable of altering the number of repeats in a given array. Unequalcrossing over, whereby improper meiotic pairing and recombination results in a deletion in onechromatid or chromosome and a corresponding duplication in the other, is one of severalsuggested mechanisms and has been reported to occur in 18S-26S rDNA arrays in yeast(Szostak and Wu 1980). Since a hierarchy of repeat organization exists within the 18S-26SrRNA array, consisting of an IGS subrepeat embedded within the repeating unit itself, boththe size of the 18S-26S rDNA repeat itself and the number of repeats at a given locus aresubject to change.Variation in the length of the 18S-26S rDNA repeat can arise by unequal crossing overbetween subrepeats within the IGS. Among angiosperms, heterogeneity has been found at alllevels, for example, between species related to maize (Zimmer et a!., 1988), betweenindividuals ofwheat (Appels and Dvorak 1982), and within individuals of a number of species,the extreme example being broad bean in which as many as 20 different length variants withina single plant have been observed (Rogers et al., 1986). Although a molecularcharacterization of the 18S-26S rRNA repeating unit of white and Sitka spruce was notundertaken in this study, Bobola et al. (1992) investigated the gene family in two other NorthAmerican spruces, black spruce (Picea niariana) and red spruce (P. rubens). In both species,the coding regions and intergenic spacer, comprising as much as 4% of the genome, rangedfrom 32 kb to greater than 40 kb in length, considerably larger than that found in angiospermplants. Restriction mapping revealed that as many as five different repeat sizes are found46within any individual. Up to four different 1 8S-26S rDNA repeat lengths within an individualtree of Scots pine have also been reported (Karvonen eta!., 1993b).Although the individual members of a tandemly repeated DNA family can showconsiderable sequence or length variation in a single genome, repeats within any one array arefrequently more homogeneous than would be expected if each was evolving independently(Arnheim et a!., 1980). The concerted evolution of 18S-26S rDNA repeating units within thetwo nucleolus organizers of pea was suggested by Polans et a!. (1988) who noted that two18S-26S rDNA length variants segregated in a Mendelian manner, indicating that thecomposition of rDNA repeats at each NOR is distinct. Although linkage analysis of rDNAloci in conifers is hampered by the multitude of sites and the difficulty in identifyingsegregating alleles, the number of length variants in black spruce corresponds with the numberof secondary constrictions observed byNkongolo and Klimaszewska (1994).Unequal crossing over between repeating units outside the IGS subrepeat will give riseto variation in the total number of 1 8S-26S rRNA genes among individuals as well as the sizeof array at a particular chromosomal site. Extensive variability in rDNA copy number hasbeen reported in many populations of angiosperms (Rogers and Bendich 1987). In conifers,Strauss and Tsai (1988) reported a 5-fold variance among 54 individuals sampled from therange of Douglas-fir and a 28-fold variance within individuals of radiata pine was found byCullis and Teasdale (1985). Such variation is presumably tolerated because only a smallfraction of the 18 S-26S rDNA, perhaps as little as 10%, is transcribed (Rogers and Bendich1987).The observed variance in rDNA copy number among individuals predicts that inoutcrossing species, such as spruce, heterozygosity in copy number at a particular locus47should arise. While more difficult to document, Miller et at. (1980) clearly demonstrated itsoccurrence in wheat using radioactive ISH in which grain counts provided a direct measure ofrDNA copy number. The detection of only 13 of the 14 sites in well spread metaphases ofwhite spruce could likewise be ascribed to great differences in rDNA copy number on thehomologs of chromosome 2 as opposed to the complete absence of an array. Since thealternate homozygous state (i.e., 12 pGmR1 hybridization sites due to the absence of a rDNAlocus on both homologs) was not observed among the well spread metaphases analysed, thematernal parent from which the open pollinated seeds were collected may be heterozygous fora rare 18S-26S rDNA deletion. That the secondary constriction found on one homolog ofchromosome 2 in these metaphases was very elongated suggests that unequal recombinationand/or the decondensation and transcription of greater numbers of rDNA genes has occurred.Alternatively, if Engelmann spruce differs from white spruce in its rDNA distribution,specifically lacking the locus on chromosome 2, than a white X Engelmann hybrid would carryonly 13 hybridization sites. While the possibility of contaminating Engelmann spruce pollencannot be ruled out, preliminary DAPI staining and ISH with pGmRl in Engelmann sprucesuggest that the two species have identical 18S-26S rDNA chromosome locations (data notshown). Therefore, interspecific hybridization is unlikely to account for the 13 18S-26SrDNA loci observed in some metaphases.Mapping additional repeated DNA sequences in spruce suggests that the ISHprocedure described here is at least capable of detecting 100 kb of DNA on a metaphasechromosome. Therefore, given the large physical size of the 1 8S-26S rDNA repeat inspruce, one to several copies of the gene are expected to reside at the undetected site onchromosome 2. This situation exemplifies the current difficulty in interpreting ISH results in48plants for which the lower limit of detection is seldom known, that is, that in the absence ofreproducible methods to localize single copy DNA sequences one cannot be certain that allgenomic locations of a given sequence have been identified.LITERATURE CITEDAppels, R. and Dvorak, J. 1982. The wheat ribosomal DNA spacer region: its structure andvariation in populations and among species. Theor. Appl. Genet. 63: 337-348.Appels, R. and Honeycuttt, R.L. 1986. rDNA: evolution over a billion years. In DNASystematics, vol. 2: Plants. S.K. Dutta (ed). CRC, Boca Raton, Fla., pp 81-135.Arnheim, N.D., Krystal, M., Schmickel, R., Wilson, G., Ryder, 0. and Zimmer, E. 1980.Molecular evidence for genetic exchanges among ribosomal genes on non-homologouschromosomes in man and apes. Proc. Nati. Acad. Sci. USA 77: 7323-7327.Bobola, M.S., Smith, D.E. and Klein, A.S. 1992. Five major nuclear ribosomal repeatsrepresent a large and variable fraction of the genomic DNA of Picea rubens and Pmariana. Mol. Biol. Evol. 9(1): 125-137.Brown, G.R., Amarasinghe, V., Kiss, G. and Carlson, I.E. 1993. Preliminary karyotype andchromosomal localization of ribosomal DNA sites in white spruce using fluoresence insitu hybridization. Genome, 36: 310-316.Cullis, C.A. and Teasdale, R.D. 1985. The 25, 18 and 5S ribosomal DNAs from pine. InProceedings of the First International Congress of Plant Molecular Biology, Center forContinuing Education, University of Georgia, Athens. Galau, G.A. (ed.). Abstr. No.P0-1-079.Cullis, C.A., Creisson, G.P., Gorman, S.W. and Teasdale, R.D. 1988. The 25S, 18S, and 5Sribosomal RNA genes from Pinus radiata. In Proceedings of the Second workshop ofthe IUFRO Working Party on Molecular Genetics, Chalk River, Ontario, June 15-18,1987. Cheliak, W.D. and Yapa, A.C. (eds.) pp 34-40.Doudrick, R.L., Heslop-Harrison, J.S., Nelson, C.D., Schmidt, T., Nance, W.L. andSchwarzacher, T. 1995. Karyotyping slash pine (Pinus elliottii var. eiliottii) usingpatterns of fluoresence in situ hybridization and fluorochrome banding. J. Hered. (inpress).49Goessens, G. and Lepoint, A. 1974. The fine structure of the nucleolus during interphase andmitosis in Ehrlich tumor cells cultivated in vitro. Exp. Cell Res. 87: 63-72.Heslop-Harrison, J.S., Schwarzacher, T., Anamthawat-Jonsson, K., Leitch, A.R., Shi, M. andLeitch, I.J. 1991. In situ hybridization with automated chromosome denaturation.Technique, 3: 109-115.Karvonen, P., Karjalainen, M. and Savolainen, 0. 1993a. Ribosomal RNA genes in Scots pine(Pinus syivestris L.): chromosomal organization and structure. Genetica, 88: 59-68.Karvonen, P. and Savolainen, 0. 1993b. Variation and inheritance of ribosomal DNA in Piiizissylvestris L. (Scots pine). Heredity, 71: 6 14-622.Miller, T.E., Gerlach, W.L. and Flavell, RB. 1980. Nucleolus organizer variation in wheatand rye revealed by in situ hybridization. Heredity, 45(3): 377-3 82.Nkongolo, K.K. and Klimaszewska, K. 1994. Karyotype analysis and optimization of mitoticindex in Picea mariana (black spruce) preparations from seedling root tips andembryogenic cultures. Heredity, 73: 11-17.Polans, NO., Weeden, N.F. and Thompson, W.F. 1988. Distribution, inheritance and linkagerelationships of ribosomal DNA spacer length variants in pea. Theor. Appi. Genet. 72:289-295.Rogers, S.0. and Bendich, A.J. 1987. Ribosomal RNA genes in plants: variability in copynumber and in the intergenic spacer. Plant Mol. Biol. 6: 509-520.Rogers, S.0., Honda, S. and Bendich, A.J. 1986. Variation in the ribosomal RNA genesamong individuals of V/ciafaba. Plant Mol. Biol. 6: 339-345.Sambrook, J., Fitch, E.F. and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual.2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Strauss, S.H. and Tsai, C.H. 1988. Ribosomal gene number variability in Douglas-fir. J.Hered. 79(6): 453-458.Szostak, J.W. and Wu, R. 1980. Unequal crossing over in the ribosomal DNA ofSaccharomyces cerevisiae. Nature (Lond.), 284: 426-430.Zimmer, E.A., Jupe, E.R. and Walbot, V. 1988. Ribosomal gene structure, variation, andinheritance in maize and its ancestors. Genetics, 120: 1125-1136.50CHAPTER 3Characterization of the 5S Ribosomal RNA Genes in SpruceINTRODUCTIONRibosomal 5S RNA (5S rRNA) is a component of all ribosomes except in somemitochondria. Like the 18S-26S rDNA, the structure and organization of genes encoding theSS rRNA have been extensively studied. Nucleotide sequences of at least 30 5S rRNA and 425S rDNA from higher plants are known (Barciszewska et at., 1994). In a typical eukaryoticgenome, hundreds to thousands of genes are maintained as long tandem arrays of repeatingunits composed of a highly conserved 120 nucleotide coding sequence and a nontranscribedspacer (NTS). In plants, spacer lengths ranging from 95 bp to 730 bp in mung bean andradiata pine, respectively, have been reported (Hembleden and Werts 1988; Moran et at.,1992). Sequence and length variability within the NTS is commonly observed both within andamong species. In genomes containing more than one spacer length, the variant classes aregenerally restricted to discrete chromosomal sites, as in rye (Reddy and Appels 1989), inconcurrence with the concerted evolution of tandem repeated DNA families.ISH has been used to identify the chromosomal location of 5S rRNA genes in anumber of plant species, including wheat, rye, barley, maize, pea, tomato, and rice (Appels etat., 1980; Leitch and Heslop-Harrison 1993; Mascia et at., 1981; Ellis et al., 1988; Lapitan etat., 1991; Song and Gustafson 1993). In general, one to three chromosomes carry the 5S51rDNA. In all higher eukaryotes examined to date, these genes are not associated with thegenes encoding the 1 8S-26S rRNA, although in wheat and rye the two arrays are situated nearone another (Appels eta!., 1980).Most analyses of plant 5S rRNA genes have involved angiosperm species to date.Consequently, little is known of its organization and chromosome location in the genome ofgymnosperm plants. Cullis et a!. (1988) and Moran et al. (1992) characterized the molecularstructure of the 5S rDNA in radiata pine while Doudrick et a!. (1995) determined itschromosomal locations in slash pine. The present study was intiated not only to developadditional cytological markers for spruce chromosomes but to extend the knowledge of the 5SrRNA gene family in gymnosperms. As a rapid alternative to isolating 5S rDNA onactinomycin-D/CsCI density gradients (Lawrence and Appels 1986), the polymerase chainreaction was employed to amplify the 5S rDNA from the genome of white spruce usingprimers derived from known evolutionarily conserved sequences. The 5S rDNA wassequenced and its organization and copy number in white spruce determined. Additionally,fluorescence ISH was used to physically map the 5S rDNA in white and Sitka spruce.MATERIALS AND METHODSDNA iso!ationGenomic DNA was isolated from white spruce needle samples collected from theKalamalka Research Station of the British Columbia Ministry of Forests, Vernon, B.C. Fivegrams of needles were ground to a fine powder in liquid nitrogen using a mortar and pestleand stirred into 75 mIs of ice cold extraction buffer. The extraction buffer consisted of 5052mM Tris-HC1, pH 8, 5 mM EDTA, 0.35 M sorbitol, 0.1% BSA, 10% PEG 4000, 0.1%spermine and 0.1% spermidine. 2-mercaptoethanol was added to 0.1% just prior to use. Thehomogenate was filtered through four layers of cheesecloth and one layer of miracloth.Nonfiltered plant material was re-extracted with 25 mIs ice cold extraction buffer and the twofiltrates combined and centrifuged at 9000 xg for 15 minutes at 4 °C. The resulting pellet wasresuspended gently in 5 mls of wash buffer (50 mlvi Tris-HC1, 25 mM EDTA, 0.35 M sorbitoland 0.1% 2-mercaptoethanol), transferred to a 15 ml Falcon tube, and 1/4 volume of 5%sarkosyl added. The solution was gently mixed and left at room temperature for 20 minutes.For each ml of solution, one gram of CsCI was then added. This solution was then transferredto an Oakridge tube containing 300 d of 10 mg/mI ethidium bromide and ultracentrifuged at45,000 xg for 16 hours at 20 OC. The band of genomic DNA was removed and placed in aquick-seal tube which was then filled with 1 g/ml CsCl and ultracentrifuged for a second time.Genomic DNA was again removed and the ethidium bromide extracted with water saturatedn-butanol. Finally, the DNA was ethanol precipitated, washed twice with 70% ethanol,vacuum dried and resuspended in 50-150 il of TE (10 mlvi Tris-HC1, 1 mMEDTA, pH 8).PCR anipl/ication and cloning of the 5S rRNA genesPCR amplification (Saiki et a!., 1985; Mullis et a!., 1986) was carried out in a PerkinElmer Cetus DNA Thermal Cycler. The reaction mixture consisted of 5 ng white sprucetemplate DNA, 10 mM Tris-CI, pH 8.3, 50 mM KCI, 0.2 mM dNTPs, I tM primer pair, 2m]V1 MgCI2 and 1.25 U AmpltiTaq DNA Polymerase in a 50 l volume. The primer pairs usedwere synthesized based on the sequences of seven angiosperms reported in Goldsbrough etal., (1982) and included (P1:5-GGGTGCGATCATACCAGCGT-Y and P2: 5’-53GGGTGCAACACTAGGACTTC-3) and (P3 5’-GAGTTCTGATGGGATCCGGTG-3’ andP4 5’-CGCTTGGGCTAGAGCAGTAC-3’). Reactions were initially denatured at 94 °C for 3minutes and subsequently subjected to 20 cycles of 94 °C for 1 minute and 55 °C for 10seconds. Amplifications were completed by a 72 °C final extension for 10 minutes. Reactionproducts were resolved on 2% agarose gels in lx TAE and PCR products to be cloned wereexcised and gel purified. Following reamplification under the identical cycling conditions, thereaction mixture was cloned into the EcoRV site of pBluescript KS using the dideoxy-tailingmethod described by Holton and Graham (1991). Approximately 10 ng of the ligationmixture was used to transform competent DH5 alpha E. coil cells (BRL).Genomic DNA digestion and Southern hybridizationGenomic DNA (1 tg) was partially digested with 10 units of BamHJ or ScaT at 37 oCfor varying amounts of time (from 0-120 minutes). Reactions were stopped by adding EDTAto 25 mM and the restriction fragments resolved in 1.6% agarose gels in lx TAE. Followinga 5 minute depurination in 0.25 M HC1, DNA was blotted to Hybond N (Amersham)membranes by capillary action using 0.4 M NaOH as the transfer buffer. The insert frompWS1 1, a PCR product from amplification with primers P3 and P4, was excised frompBluescript by EcoRI/XhoI digestion, gel purified and labeled with 32P-dCTP by randompriming using a commercially available kit (Boehringer-Mannheim). Following a 1 hourprehybridization at 65 0C in 6X SSC, 20 ig/ml sheared, denatured salmon sperm DNA, 5XDenhardt’s and 0.5% SDS, the labeled probe was denatured at 100 0C for 5 minutes, chilledon ice, and incubated with the membrane at 65 °C overnight. (1X Denhardt’s is 0.02% Ficoll,0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin). Membranes were subsequently54washed in 2X SSC for 15 minutes, 0.2X SSC at 65 °C for 30 minutes and twice in 0.1X SSCat 65 °C for 30 minutes before autoradiography.DNA sequencingSequencing of both strands of each PCR clone was performed by the dideoxy chaintermination method (Sanger et a!., 1977) with the T7 Sequencing Kit (Pharmacia) using theT3 and T7 promotor primers.Estimation of5S rDNA copy number in white spruceThe copy number of the 5S rDNA in white spruce was estimated by reconstructionexperiments. Various dilutions ofpWSll, corresponding to the expected weight of 100, 500,1000, 2000, 5000, 7500, 10,000 and 20,000 copies, were prepared in sterile distilled water.NaOH and EDTA were added to to final concentrations of 0.4 M and 0.01 M, respectively.The DNA was denatured at 80 °C for 10 minutes, chilled on ice, and immobilized on a nylonmembrane using a slot blotting apparatus (BlO-RAD). Equivalent molar amounts ofpBluescript were slotted as controls for cross-hybridization to vector sequences. Aliquots ofwhite spruce genomic DNA (0.5 and 1.0 g) were also applied. Two replicates of each filterset were included. After probing with the insert of pWS 11, membranes were washed at highstringency (as above) and the resulting autoradiograms analyzed by densitometry.C’hromosome preparations and in situ hybridizationPlant materials and procedures performed were as described in Chapter 2 (pp. 33-37).Fifteen well spread metaphases from both white spruce and Sitka spruce were analysed.55RESULTSCharacterization of5S rDNA in white spruceThe 5S rDNA was amplified from white spruce genomic DNA by the polymerasechain reaction. Primers P1 and P2 were designed to amplify the 120 bp coding region onlyand primers P3 and P4 to amplify the entire repeating unit (with the exception of a 12 bpregion between the 5’ ends of P3 and P4, Fig. 3.1). PCR amplification with P1 and P2 gavethe expected 120 bp product. Primers P3 and P4 yielded a prominent amplification product ofapproximately 210 bp and lesser products of approximately 430 and 650 bp (data not shown).Reamplification of either the 430 or 650 bp product produced the 210 bp product, consistentwith these minor PCR products being dimers and trimers, respectively, of a 5S rDNAmonomer.To obtain the sequence of the repeating unit, both the 120 and 210 bp PCR productswere cloned and four representatives of each analyzed. Alignment of the sequences from eachprimer pair revealed that the amplification products represented a 221 bp 5 S rRNA geneconsisting of the 120 bp coding region, as determined by sequence comparison with otherplant 5S rRNA genes, and a 101 bp NTS. Sequence variation among clones was observedcorresponding on average to less than one nucleotide substitution for coding region sequencesand seven nucleotide substitutions for NTS sequences.A consensus sequence derived from the eight PCR products is presented in Fig. 3.2.The coding region shows complete sequence identity with the 5S rRNA of Scots pine(Mashkova et at., 1990), and the 5S rDNA from radiata pine (Moran et a!., 1992) andDouglas-fir (Amarasinghe and Carison, unpublished). Upstream of the transcription start site,56P1 P4 P1 P4ci100 bpFig. 3.1. Origin of primer pairs P1/P2 and P3/P4 used in the PCR amplification of 5SrDNA. The sequences of primer pairs P1/P2 and P3/P4 are given in the Materials andMethods. Line, nontranscribed spacer. Open box, coding region. Shaded region, 12 bpof coding region sequence not amplified using primers P3/P4.5710 20 30 40GGGTGCGATC ATACCAGCGT TAATGCACCG GATCCCATCA50 60 70 80GAACTCCGCA GTTAAGCGCG CTTGGGCTAG AGTAGTACTG90 100 110 120GGATGGGTGA CCTCCCGGGA AGTCCTAGTG TTGCACCCTT130 140 150 160CCCCCCTTTT GCATGGCTCC GCGATGGATC GGGGCGCTTT170 180 190 200TAAGCCCTCC CCCGCGGCTC GGTGATGTCC ATTCAAGAGG210 220GGGAGGGGGC CTGATCCTTG CFig. 3.2. Consensus sequence of the 5S rRNA genes in white spruce.Nucleotides in bold represent the coding region. The nontranscribed spacerbegins at position 121. Regulatory elements referred to in the text (dottedlines) are found in the 3’ region of the nontranscribed spacer. The BarnHI(single line) and ScaT (double line) restriction sites are noted.580.32 —0.1Fig. 3.3. Southern hybridization ofpWSH, a P3/P4 primer pair amplification product. GenomicDNA of white spruce was digested for varying amounts of time with BarnHl or ScaT. Arrows,hybridizing fragments not corresponding to multimers of the 221 bp repeating unit. Molecularweights shown are in kilobases.B a iii HI Seal(I 1 2 5 10 20 30 45 60 90 120 0 1 2 5 10 20 30 .45 60 90 1202.l)4u:1.0 ._ .. ....0.50.459-ye pWS11 WhiteControl Spruce5001000 0.5 ug2000 1RII1i_ IfiJil 1.0 ug5000750010,000Fig.3.4. Estimation of the copy number of 5S rDNA. Dilutions ofwhite spruce genomic DNA, pWS 11, and pBluescript KScorresponding to the expected number of copies shown on the leftwere denatured, slotted onto nylon membranes and hybridized athigh stringency with the 32p labeled insert of pWS 11. Hybridizationto the plasmid vector pBluescript KS was not detected. Copynumber calculations were based on densitometric readings and theassumption that the 1C DNA content of white spruce is 8.5 x 1Obp.60Fig. 3.5. Localization of the 5S rDNA (green) in white spruce. a)Fluorescence ISH of biotin-labeled pWS 11 (green). Chromosomal DNA wascounterstained with propidium iodide (red). b) The same metaphase stainedwith DAPI. Arrowhead, secondary constriction adjacent to 5S rDNA site.Asterisk, B chromosome. Bar represents 10 .Lm.61Fig. 3.6. Localization of the 5S rDNA (green) in Sitka spruce. a)Fluorescence ISH of biotin-labeled pWS11 (green). Chromosomal DNA wascounterstained with propidium iodide (red). b) The same metaphase stainedwith DAPL Arrowhead, secondary constriction adjacent to 5S rDNA site.Asterisk, B chromosome. Bar represents 10 Lm.62from nucleotide -ito -33, the 5S rDNA sequences of radiata pine and Douglas-fir also show80% identity to that of white spruce. Within this region are elements suggested to regulatetranscription (Venkateswarlu et al., 1991), including a cytosine at -1, a GC-rich regionbetween -ii and -17, and an AT-rich region between -24 and -30. Lastly, a stretch of fourthymidines immediately downstream from the 3’ end of the coding region likely represents thesignal for transcription termination.The genes encoding the 5S rRNA molecule are typically arranged in a genome as longtandem arrays. To verify this organization in white spruce, Southern blots of genomic DNApartially digested with either BanzHI or Seal, which have only one restriction site within therepeating unit, were probed with pWS11, a clone with a white spruce insert from the P3 andP4 primer pair amplification (Fig. 3.3). The rationale for this experiment is that partialdigestion will randomly restrict only a limited number of all available sites. Therefore, for atandemly repeated DNA, a group of restriction fragments based on multimers of the repeatingunit will be generated which, when resolved in an agarose gel and analysed by Southernhybridization, appears as a “ladder” of hybridizing fragments. When either partial BarnHI orScaT digests were probed with pWS 11, a ladder of bands based on an approximately 220 bpmonomer was apparent. With increasing digestion time, fragments of approximately 600,1000 and 1200 bp which do not correspond exactly to multiples of the repeating unit wereapparent. These latter fragments, more evident in the completed Seal digests (which is notmethylation sensitive) shown in lanes 5-10 in Fig. 3.3, likely represent minor length variants ofthe 5 S rDNA genes in the white spruce genome (with the 1200 bp fragment possibly being adimer of the 600 bp fragment). Although various PCR reaction and cycling conditions weretested, amplification products corresponding to these length variants were not seen.63The copy number of the 5S rDNA genes in white spruce was calculated fromdensitometry readings of slot blots probed with pWS 11 (Fig. 3.4). A value of approximately1170 +1- 82 copies per haploid genome was derived. Given that chioroplast andmitochondrial DNA sequences can account for a considerable portion of total DNA, this valueis likely an underestimate of the copy number in white spruce.Physical mapping of/he 5S rDNAUsing fluorescence ISH, the 5S rRNA genes were assigned to a single metacentricchromosome pair in white spruce very near to a secondary constriction (Fig. 3.5). There wasvery little difference in the length of the long and short arm of this chromosome and,therefore, fractional lengths were calculated from the end of the arm to which the probehybridized. From 10 chromosomes, the FLpter for the 5S rDNA and the NOR were estimatedas 0.28 +1- 0.02 and 0.23 +1- 0.01, respectively. The average relative length of the hybridizingchromosome pair indicates that it should be designated chromosome 5.Different size classes of 5S rDNAs are often found sequestered at discretechromosomal sites (Dvorak et a!., 1989; Reddy and Appels 1989). However, even withamplification of the ISH signal using overlays of biotin-labeled anti-avidin antibodies andFITC conjugated ExtrAvidin, no additional hybridization sites were detected (data notshown). These results suggest that all 5S rDNA size classes in white spruce are found at thisone chromosome site, either in adjacent tandem arrays or with some degree of interspersion.A single chromosomal site for the 5S rDNA was observed in all metaphases of Sitkaspruce as well (Fig. 3.6), on what appears to be the homeologous chromosome. Fractionallengths calculated from the end of the chromosome arm to which the 5S rDNA and pGmRl64hybridized were determined to be 0.28 +1- 0.02 and 0.22 +1- 0.01, respectively.DISCUSSIONCharacterization of the 5S rDNA amplification products by DNA sequencing,Southern hybridization and fluorescence ISH have shown that the white spruce 5S rDNA istypical of that found in higher plants. The primary form of 5S rDNA observed was a 221 bprepeating unit composed of the 120 bp coding sequence and a 101 bp nontranscribed spacer.Among the clones analysed, sequence variation observed within the NTS was 7-fold greaterthan in the coding region and likely reflects natural variation rather than misincorporation byTaq DNA polymerase. A comparison of the 5S rDNA sequences among several genera in thePinaceae family showed a similar degree of conservation as that observed among families ofangiosperm plants, both in the coding region and in sequences immediately upstream.The 120 bp coding sequence was arranged into the generalized secondary structuremodel proposed for plant 5S rRNAs (Barciszewska ci a!., 1994). An irregularity in thedouble helical region of Stem II was noted where a thymidine at nucleotide 20 must base pairwith a guanine at nucleotide 58. In a recent review of 5S rRNA sequences at the RNA andDNA levels, Barciszewska eta!. (1994) noted that sequence data from 5S rDNA are often notcolinear with the sequence of the mature 5S rRNA molecule. Possible explanations includethe sequencing of non-transcribed pseudogenes or the potential existence of a mechanism toedit the 5S rRNA. Although in this study a number of PCR products were sequenced tominimize errors inherent in PCR, it may still be that sequence comparisons among organismsmay be suspect unless performed at the RNA level. With this in mind, it is interesting to note65that the irregularity in Stem II base pairing found in white spruce is not found in thoseangiosperms analyzed to date, but has been reported in 6 of 7 gymnosperms studied, namelyGingko, Cycas, Ephedra, and Scots pine at the RNA level (see Barciszewska et at., 1994 forreferences) and in radiata pine and Douglas-fir at the DNA level (Cullis ci a!., 1988; Moran etat., 1992; Amarasinghe and Carlson, unpublished).The tandem organization of the 5S rDNA genes in white spruce was confirmed bySouthern hybridization. The presence of additional size classes of hybridizing fragmentssuggests that spacer length variation exists but that the PCR conditions used were ineffectivein amplifiing all size classes. While PCR amplification may be a suitable means of generatinghomologous probes for ISH, established methods of cloning rDNAs may be more appropriatefor the complete characterization of these gene families.One major 5S rDNA locus was identified on the metaphase chromosomes of bothwhite spruce and Sitka spruce, adjacent to a secondary constriction. Hybridizingchromosomes also showed morphological similarities between the two species, suggestingtheir homeologous nature. Assuming that all 5S rDNA repeats are found at this site, the arrayis approximately 260 kb in length. While minor sites were not detected even withamplification of the hybridization signal, it is difficult to rule out their existence given theSouthern hybridization evidence for length variants in white spruce and the preferredclustering of such variants at discrete loci characterisitic of most plants analysed. As in rye(Reddy and Appels 1989), minor 5S rDNA sites may be revealed as the sensitivity of ISHimproves.In most plants and animals, the 5S rDNA repeats outnumber the 18S-26S rDNA genes(Appels et al., 1980). Although the copy number of the 18S-26S rDNA in white spruce has66not been reported, more than 12,000 genes are present per haploid genome in Sitka spruce(Ingle 1975). This is more than 10-fold higher than the 1170 copies of the 5S rDNAestimated here in white spruce. Additionally, the chromosomal distribution of the 5S rDNAarrays has not paralleled the multilocus nature of the 18S-26S rDNA in these spruces.The primary goal in developing the ISH procedure at this stage was to establish ameans of identifying spruce chromosomes quickly and without reliance on chromosomemeasurements. In the exceptional metaphase spread in which squashing had not appreciablydistorted any of the chromosomes, 10 of the 12 chromosome pairs could be distinguished bymorphology and the location of the 1 8S-26S rDNA (revealed by either DAPI staining or ISHusing pGmRl). Length measurements were needed to discern chromosomes 2 and 5 inChapter 2. The 5S rDNA locus in both white spruce and Sitka spruce mapped tochromosome 5 which allowed all the chromosomes to be reliably identified.In both species, the 5S rDNA bearing chromosomes are conserved in size,morphology and the location of two repeated DNA families to date. The single 5S rDNAlocus could serve as the link to integrate the physical chromosome with genetically mappedmolecular markers through segregation analysis of polymorphic restriction fragments withinthe array. Pulsed field gel electrophoresis could also be used in the genetic mapping of verylarge fragments or the entire 5S rDNA array assuming that parents heterozygous for the totalarray length could be identified. Such a study would provide the first look at the synteny of aspecific chromosome between conifer species and reveal insights into chromosome evolutionwithin the genus.67LITERATURE CITEDAppels, R., Gerlach, W.L., Dennis, E.S., Swift, H. and Peacock, W.J. 1980. Molecular andchromosomal organization of DNA sequences coding for the ribosomal RNAs incereals. Chromosoma, 78: 293-311.Barciszewska, M.Z., Szymanski, M., Specht, T., Erdmann, V.A. and Barciszewski, J. 1994.Compilation of plant 5S ribosomal RNA sequences on RNA and DNA levels. PlantSci. 100: 117-128.Cullis, C.A., Creisson, G.P., Gorman, S.W. and Teasdale, R.D. 1988. The 25S, 18S, and 5Sribosomal RNA genes from Finns radiata. In Proceedings of the Second workshop ofthe JUFRO Working Party on Molecular Genetics, Chalk River, Ontario, June 15-18,1987. Cheliak, W.D. and Yapa, A.C. (eds.). pp 34-40.Doudrick, R.L., Heslop-Harrison, IS., Nelson, C.D., Schmidt, T., Nance, W.L. andSchwarzacher, T. 1995. Karyotyping slash pine (Finns e/liottii var. elliottii) usingpatterns of fluoresence in situ hybridization and fluorochrome banding. J. Hered. (inpress).Ellis, T.H.N., Kee, D. and Thomas, C.M. 1988. 5S rRNA genes in Pisuni: Sequence, longrange and chromosomal organization. Mol. Gen. Genet. 214: 333-342.Goldsbrough, F.B., Ellis, T.H.N. and Lomonossoff G.P. 1982. Sequence variation andmethylation of the flax 5S rRNA genes. Nuc. Acids Res. 10(15): 4501-4515.Hembleden, V. and Werts, D. 1988. Sequence organization and putative regulatory elementsin the 5S rRNA genes of two higher plants (Vigna radiata and Maithila incana).Gene, 62: 165-169.Holton, T.A. and Graham, M.W. 1991. A simple and efficient method for direct cloning ofPCR products using ddT-tailed vectors. Nuc. Acids Res. 19: 1156.Ingle, J., Timmis, J.N. and Sinclair, J. 1975. The relationship between satellitedeoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy, and genome sizein plants. Plant Physiol. 55: 496-501.Lapitan, N.L.V., Ganal, M.W. and Tanksley, S.D. 1991. Organization of the 5S ribosomalRNA genes in the genome of tomato. Genome, 34: 509-5 14.Lawrence, G.J. and Appels, R. 1986. Mapping the nucleolus organiser region, seed proteinloci and isozyme loci on chromosome IR in rye. Theor. Appi. Genet. 71: 742-749.68Leitch, I.J. and Heslop-Harrison, J.S. 1993. Physical mapping of four sites of the 5S rDNAsequences and one site of the alpha-amylase-2 gene in barley (Hordeurn vulgare).Genome, 36: 517-523.Mascia, P.N., Rubenstein, I., Philips, R.L., Wang, A.S. and Xiang, L.Z. 1981. Localization ofthe 5S rRNA genes and evidence for diversity in the 5S rDNA region of maize. Gene,15: 7-20.Mashkova, T., Barciszewska, M., Joachimiak, A., Nalaskowska, M. and Barciszewski, J.1990. Molecular evolution of plants as deduced from changes in free energy of 5Sribosomal RNAs. mt. J. Bio. Macromol. 12: 247-250.Moran, G.F., Smith, D., Bell, J.C. and Appels, R. 1992. The 5S RNA genes in Pinus radiataand the spacer region as a probe for relationships between Pinus species. Plant Syst.Evol. 183: 209-221.Mullis, K.B., Faloona, F.A., Scharf S.J., Saiki, R.K., Horn, G.T. and Erlich, H.A. 1986.Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. ColdSpring Harbor Symp. Quant. Biol. 51: 263-273.Reddy, P. and Appels, R. 1989. A second locus for the 5S multigene family in Secale L.:sequence divergence in two lineages of the family. Genome, 32: 456-467.Saiki, R.S., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N.1985. Enzymatic amplification of beta-globin genomic sequences and restriction siteanalysis for diagnosis of sickle cell anemia. Science, 230: 1350-1354.Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminatinginhibitors. Proc. Natl. Acad. Sci. USA 85: 2444-2448.Song, Y.C. and Gustafson, J.P. 1993. Physical mapping of the 5S rDNA complex in rice(Oryza sativa). Genome, 36: 658-661.Southern, E.M. 1977. Detecting of specific sequences among DNA fragments separated bygel electrophoresis. J. Mo!. Biol. 98: 503-5 17.Venkateswarlu, K., Lee, S.-W. and Nazar, R.N. 1991. Conserved upstream sequenceelements in plant 5S ribosoma! RNA-encoding genes. Gene, 105: 249-253.69CHAPTER 4Characterization of a Centromeric Satellite DNA in White SpruceINTRODUCTIONA significant portion of genomic DNA from many plant and animal species can beseparated from the main band of DNA using buoyant density ultracentrifugation. This“satellite” DNA is composed of tandem arrays of one or more highly repeated DNA family.Other tandem repeated sequences, described as cryptic, have densities similar to the bulk ofgenomic DNA and consequently band at the same position. Many distinct satellites andcryptic satellites (herein referred to as satellites) of differing complexities can exist in a plant’sgenome. These include the small tandem repeated microsatellites and minisatellites, and thosewith larger repeating units of typically 150-500 bp (Lapitan 1992). Satellite DNAs aretypically the most highly repeated DNA sequences in plant genomes. For example, inArabidopsis, there are 6000 copies of a 185 bp repeat comprising 1.5% of the genome(Simoens et al., 1988) and, in rye, 10% of the genome is made up of four satellite sequenceslocated near the telomeres of all chromosomes (Bedbrook et al., 1980).While several tandem repeated DNA families are conserved across a broadevolutionary spectrum and have a known or hypothetical function, the majority of tandemrepeats are a dynamic, rapidly changing component of genomes. The distribution of aparticular sequence within a taxonomic family may range from its presence in many or all70species to its confinement to a single genus or species. Many tandem repeated DNAs havebeen classified as “junk” DNA as a reflection of their rapid divergence, high degree ofmethylation and lack of protein coding capacity. Consistent with this apparent geneticinertness, satellite DNA is generally confined to constitutively heterochromatic regions ofchromosomes. These areas, denoted by Giemsa C-bands, include subtelomeric regions andcentromeres as well as some interstitial sites of some or all chromosomes of a complement.The correlation of C-bands with the location of characterized satellite DNAs has beenobserved by ISH in a number of plants (and animals), including rye (Mukai et at., 1992) andonion (Irifbne eta!., 1995).Changes in satellite DNA may represent the primary form of variation in genome sizeamong related species (Flavell et a!., 1977). As such, they have been used to investigate theevolution of species and chromosomes and to support established taxonomic relations(Lapitan et aL, 1987; Harrison and Heslop-Harrison 1995). Since they are a majorcomponent of the nuclear genome, studies on genome organization are lacking withoutknowledge of the types, numbers and distribution of satellite DNAs. To date, however,satellite DNA in conifers has not been investigated or exploited. This chapter details theisolation and characterization of a white spruce satellite DNA, SGR-3 1 (Spruce GenomicRepeat), and investigates its chromosomal location and presence in the genus.MATERIALS AND METHODSPlant material and DNA isolationPlant material used in Southern hybridizations is listed in Table I. Spruce samples71were collected from a spruce arboretum at the Kalmalka Research Station and provided byGyula Kiss. Douglas-fir and western redcedar samples were collected from trees on theUniversity of British Columbia campus. Radiata pine DNA was provided by a post-doctoralfellow in Dr. Carison’s Lab, Dr. Yong-Pyo Hong. Genomic DNA was isolated from needlesamples as described on pp. 52-53 of Chapter 3. Plant materials used for cytogenetic analysisare described on pp. 33 of Chapter 2.Screening ofa partial white spruce libraryCloning and preliminary screening were performed by Dr. Craig H. Newton of B.C.Research, Inc. as part of an unrelated research project. Genomic DNA was fractionated byCsClflToechst 33258 equilibrium ultracentrifugation (Douglas 1988). The gradient fractionfrom which ribosomal DNA had been mostly subtracted was identified, digested to completionwith Sau3A according to the manufacturer’s recommendations and ligated into the BarnHI siteof dephosphorylated pUC8. Approximately 10 ng of the ligation mixture was used totransform competent DH5 alpha E. coil cells (BRL). Two thousand white colonies werescreened for those containing repeated DNA inserts by colony hybridization (Sambrook et a!.,1989). White spruce genomic DNA was labeled with 32P-dCTP by random priming using acommercially available kit (Boehringer-Mannheim) and hybridized to colony lifts under theconditions described on pp. 53 of Chapter 3. Twenty five colonies representing a range ofsignal intensities were picked and supplied to the author for further analysis.Selected clones were screened by Southern hybridization. Genomic DNA (2 .tg) wasdigested with 4-10 units of Dral, HaeIII, HindIII, Hinff, MvaI, RsaI, Sau3A or TaqI for 16hours at 37 °C and resolved in 1% agarose gels using IX TAE as the running buffer.72Table I Picea species included in Southern hybridization experiments with SGR-3 1.Species Species RangePicea glauca Canada, N. United StatesPicea sitchensis W. Canada, W. United StatesPiceapungens W. United StatesPicea niariana Canada, N. United StatesPicea rubens E. Canada, N.E. United StatesPicea chihuahuana MexicoPicea mexicana MexicoPiceajezoensis E. AsiaPicea polita JapanPicea koyamal E. China, JapanPicea asperata central ChinaPicea purpurea central ChinaPicea montigena central ChinaPicea obovata Scandinavia, N. Europe, AsiaPicea schrenkiana E. ChinaPicea oniorika SerbiaPicea orienta/is S. Russia, N. TurkeyPicea abies W. Europe, Scandinavia, W. Russia73Southern blotting was performed as described on pp. 54. The insert from each clone wasexcised from the plasmid by double digestion with EcoRT and HindIII, gel purified in 0.8%low melting agarose in lx TAE, and labeled with 32P-dCTP by random priming. Thehybridization and post-hybridization wash schedule were carried out as described on pp. 54-55 of Chapter 3.For the partial digestion of genomic DNA, 1 g aliquots were digested with 4 units ofSau3A at 37 °C for varying amounts of time. Reactions were stopped by adding EDTA to 25mM. Genomic DNA (1 tg) from the spruce and other Pinaceae species listed in Table I wasdigested overnight with 4 units of Sau3Aat 37 oC These digests were resolved on 1.6%agarose in lx TAE. Southern blotting and hybridizations were performed as described above.DNA sequencingPrior to sequencing, the EcoRl/HindII fragment, representing the white spruce insertand the multiple cloning site of pUC8, was subcloned into pT7T3 (Pharmacia) to make use ofthis vectors T3 and T7 bacteriophage promotor sites. Sequencing of both strands of SGR-3 1was performed by the dideoxy chain termination method (Sanger et a!., 1977) with the T7Sequencing Kit (Pharmacia).Estimation ofSGR-31 copy tiumber in white spruceThe copy number of SGR-3 1 in white spruce was estimated by reconstructionexperiments. Various amounts of SGR-3 1, corresponding to the expected weight of 100,500, 1000, 2000, 5000, 7500, 10,000 and 20,000 copies were applied to a nylon membraneusing a slot blotting apparatus (BlO-RAD). Equivalent molar amounts of pT7T3 were slotted74I) 1)3) 0.06 0 125 L250 0.500 1 2U/ig2000 : - . -1000 —900• - -700600500400 -300.!)J0I0flFig. 4 1 Southern hybridization of 32P1abe1ed SGR31. One microgram aliquots of white spruce genomicDA were digested for 1 hour with varying amounts ofSau3A (indicated at the top of each lane). Molecularweights are in base pairs.7510 20 30 40GATCTATTAA CCCGCATCTG TATTCGGGTT GCTAAGTAAA50 60 70 80TACTCTCTGG TTCCGAATTT GGGGATTTCA GCACTGCAGC90 100 110 120ATTCTTCAGA CAAAAGAATA ACCCCGCAGC TGTAAAAAAA130 140CAAAACAAAA AATGGTGAGA TCFig. 4.2. Nucleotide sequence of SGR-3 1. The Sau3A sites defining therepeating unit are underlined.7623.1 -9.46.6 -)4. .$% // Q QO —‘) 0 01. .1:. .1. 1 1. SL 1 1 i SL 1 S I. S._ i. 1. i. &4.42.32.0 -0.6 -1I. . , , -Fig. 4.3. Southern hybridization of SGR-31 to Sazi3A digested genomic DNA from 18 species ofPicea. Douglas-fir (Pseudotsuga rnenziesii), western red cedar (Thuja plicata) and radiata pine(Pinus radiata) were included to represent three other conifer genera. Molecular weights givenare kilobases.77Fig. 4.4. Localization of SGR-3 I in a) white spruce and b) Sitkaspruce by fluorescence ISH. SGR-3 I (green) hybridized to thecentromeres of four chromosome pairs in a) and five in b).Chromosomal DNA was counterstained with propidium iodide(red). Several interspecific differences SGR-3 l’s chromosomaldistribution were apparent including one observed on chromosome12 (arrowheads). Bar represents 10 m.78separately and served to control for cross-hybridization to vector sequences. Aliquots ofwhite spruce genomic DNA (0.5 and 1.0 .tg) were also applied. After hybridizing with the32P-labeled insert from SGR-3 1, membranes were washed at high stringency and the resultingautoradiograph analyzed by densitometry. Two replicates for each clone were included.Chromosome preparation and in situ hybridizationThe procedures performed were as described on pp. 33-3 7 of Chapter 2.RESULTSCharacterization ofSGR-31 in i’hite spruceThe monomer unit of satellite DNAs in plants (and animals) with small genomes cangenerally be observed and cloned directly from ethidium bromide stained gels of genomicDNA digested with the appropriate restriction enzyme. In contrast, the much higher numberof restriction fragments generated by digestion of large genomes, like those of conifers,prevents the direct observation of satellite DNA monomers and necessitates the cloning ofrestriction digests and screening of recombinants.Clones containing repeated DNA sequences were isolated from a partial white sprucelibrary by comparing relative signal intensities when probed with total genomic DNA. Toinvestigate the genome organization of selected clones, each was hybridized to Southern blotsof white spruce DNA digested with the restriction enzymes DraI, HaeIII, HindIII, HinJI,MvaI, RsaI, Sau3A and TaqI (data not shown). Of the 25 clones screened, 3 clones revealeda prominent ladder of hybridizing fragments in Sau3A digests indicative of a tandem arrayed79repeated sequence. Dot blot hybridizations showed that these clones represented the samesequence family (data not shown) and, therefore, one clone designated SGR-3 1 was selectedfor further analysis.To confirm its genomic organization, SGR-3 1 was hybridized to Southern blots ofwhite spruce DNA partially digested with Sau3A (Fig. 4.1). These results clearly indicatedthe presence of an approximately 140 bp repeated DNA element tandemly arrayed in thegenome. The size of the monomer corresponded to the estimated insert size of SGR-3 1indicating that the entire repeating unit had been cloned. Faint hybridization to approximately0.8 and 2 kb fragments is also evident suggesting a different organization of some SGR-3 1related sequences in the genome.The nucleotide sequence of SGR-3 1 was determined to be 138 bp in length with anA+T content of 60% (Fig. 4.2). No significant internal subrepeats were observed althoughsmall (6 bp or less) direct and inverted repeats were found. A search of the EMBL andGENBANK databases revealed no sequences with greater than 57% homology to SGR-31.Approximately 10,000 copies of this repeated DNA family are found per haploid genome ofwhite spruce as calculated from densitometric analysis of slot blots (data not shown). Thiscorresponds to 0.02% of the genome based on a haploid genome size of 8.5 x 10 bp (Dhillon1987) and is likely to be an underestimate given that considerable amounts of the total DNAare chioroplast or mitochondrial sequences.SGR-3] in PiceaSeventeen additional species of spruce were assessed for the presence and genomicorganization of SGR-3 1 by Southern hybridization to Sau3A digested genomic DNA.80Representative species from three other genera of Pinaceae were also included on the filters.Fragments homologous to SGR-3 1 were observed in all spruces as tandem repeats with theexception of Chihuahua spruce (P. chihuahuana) (Fig. 4.3). The size of the monomer unitwas identical to that of white spruce in all species to the limit of gel resolution. In Chihuahuaspruce, faint hybridization was seen to only the higher molecular weight fragments of 0.8 and2 kb common to all spruce species examined. Differences in SGR-3 1 copy number among thespruces is suggested by relative hybridization intensities between lanes although thisobservation should be treated cautiously since the genome size of most of the species, andhence the number of genome copies per lane, has not been determined. SGR-3 1 appears to bespecific to spruce species since no hybridization to the DNA of radiata pine, Douglas-fir orwestern redcedar was observed.Physical mapping ofSGR-31The SGR-3 1 tandem repeated sequence was found within the primary constriction offour of the twelve chromosome pairs in white spruce by fluorescence ISH (Fig. 4.4a).Hybridization sites appeared to lie within the primary constriction and not in paracentromericregions of the metaphase chromosomes examined although the condensed state of thechromosomes limited a higher resolution analysis. SGR-3 1 is not evenly distributed amongthese chromosome pairs as is evident by variation in signal intensity consistently observed.The highest copy number is found on chromosome 12, easily identified by its size andsubmedian centromere position, where the hybridization signal was usually seen as a broadband encompassing the entire region of the primary constriction. The other threechromosome pairs, identified as chromosomes 4, 6 and 8, showed much smaller, discrete81signals on each chromatid.In Sitka spruce, SGR-3 1 hybridization sites were fairly uniformly distributed over theprimary constriction of five of the twelve chromosome pairs including chromosomes 1, 4, 6, 7,and 8. (Fig. 4.4b). Distinct differences in the distribution of SGR-3 1 were apparent betweenhomeologous chromosomes of the two species. The SGR-3 1 sites on chromosome 1 and 7 ofSitka spruce are absent in white spruce and the prominent SGR-3 I site on chromosome 12 inwhite spruce is not found in Sitka spruce.DISCUSSIONSGR-31, a centromeric satellite DNASGR-3 1, a 138 bp satellite DNA, was isolated from a white spruce genomic library.Fluorescence ISH analysis localized SGR-3 1 sequences to the centromeres of four whitespruce chromosome pairs and five Sitka spruce pairs. Hybridization sites clearly fell withinthe boundaries of the primary constriction. Despite the abundance of repeated sequencescharacterized from a wide variety of plant genomes, only in Arabidopsis, barley, wheat, andseveral Brassica species have centromeric tandem arrays been isolated (Murata et a!., 1994;Dennis et at., 1979; Xia et a!., 1993; Harrison and Heslop-Harrison 1995). No significanthomology between these centromeric repeats and SGR-3 I was found.SGR-3 1 is typical of most characterized satellite DNAs in that its simple sequenceoffers little insight into its origin and role, if any, in the spruce genome. Benslimane et at.(1986) maintain that some satellite DNAs arise from a tRNA gene ancestor although nohomology of SGR-3 1 with any of the tRNA genes was observed. It has also been suggested82that plant satellite DNAs originate from a small 30 bp unit, first duplicated or triplicated, andthen amplified as an entire block (Ingham et a!., 1993). No internal subrepeats of greater than6 bp were observed to support this argument. A potential role for SGR-3 1 in the sprucegenome is no less clear. The folding of centromeric DNA into constitutive heterochromatinhas been suggested by Vogt (1992) to be mediated by a protein binding capacity of tandemrepeated structures involving a particular sequence domain and/or a stable curvature of theDNA. The sequence unit (GGAAT) has been implicated as a component of this interactionbased on its extreme evolutionary conservation (from yeast to sea urchins, maize, chickensand humans), centromeric location, unusual hydrogen bonding properties, high affinity forspecific nuclear proteins and similarities to the fhnctional centromeres isolated from yeast(Grady et al., 1992). However, no such sequence motifs are found in the primary sequence ofSGR-31.The most intriguing feature of SGR-3 1 is its confinement to only a subset of sprucechromosomes. Other centromeric satellite sequences isolated from plants, with the exceptionof pBcKB4 and pBoKB 1 from Chinese cabbage and kale, respectively (Harrison and HeslopHarrison 1995), were physically mapped to all somatic chromosomes of the particular species.While SGR-3 1 sequences may be completely absent from non-hybridizing centromeres, it isconceivable that SGR-3 I copy numbers on these chromosomes present target sequencesbelow the sensitivity of the ISH technique used. Alternatively, non-hybridizing chromosomesmay contain SGR-3 1 related sequences that have diverged sufficiently to precludehybridization under high stringency conditions. Low stringency hybridization of pBcKB4 andpBoKB1 detected additional centromeric locations with divergent sequences. In thechromosomes of humans and other primates, most centromeres have one or more diverged83alpha satellite subsets specific to that particular chromosome type (Willard 1990). Lowstringency ISH might reveal that diverged SGR-3 1 related sequences are organized in asimilar chromosome-specific manner as pBcKB4 and pBoKB1 and the alpha satellite.Evolution ofSGR-31 in PiceaThe evolution of SGR-3 1 was investigated by Southern hybridization to 18 species ofspruce and by comparative ISH to the metaphase chromosomes of Sitka spruce. Thetaxonomic relationship of spruce species is not fully elucidated because of a lack of fossilrecords for many species, relatively few changes to the basic spruce form having occurredduring differentiation into the present day array of species, and geographic, as opposed togenetic, isolation mechanisms having been predominantly involved in speciation (Wright1955). It was hoped that SGR-31 might contribute to current taxonomic views, however,Southern hybridizations provided no additional information. Both the size and organization ofSGR-3 1 sequences are well conserved within all but one of the species assayed. Since only 4of the 138 bp of SGR-3 1 are sampled for sequence variation by digestion with Sau3A a morethorough analysis involving sampling numerous restriction enzymes or DNA sequencing ofSGR-3 1 in other species may contribute more to a taxonomic study.SGR-3 1 should be considered an ancestral tandem repeated DNA family amplifiedearly in the evolution of the genus since it is present and organized as such in all but onespecies assayed. These included P. koyarnai, considered to be the most primitive andgeneralized spruce, and Yeddo spruce (P. jezoensis), the probable link between the olderAsiatic species and those found in North America (Wright 1955). In Chihuahua spruce, anisolated Mexican species with limited range, SGR-3 1 is not organized in tandem arrays. SGR8431 related sequences do exist in the Chihuahua spruce genome as the 0.8 and 2 kb Sau3Afragments present in all spruces but the molecular nature of these has not been investigated.In contrast to Southern hybridization results, comparative ISH mapping of SGR-3 1revealed differences between its chromosomal distribution in white and Sitka spruce. At somepoint since the divergence of these species intragenomic movement of SGR-3 1 has resulted inthe species-specific sites on chromosomes 1, 7 and 12. Experimental observations in plantsand animals that tandem arrays of a given sequence family are usually found on many, if notall, chromosomes of a species (after presumably originating from an amplification event at asingle site) suggest that satellite DNAs can be transposed between homologous and non-homologous chromosomes. Flavell (1986) suggests several mechanisms of transpositionincluding a double crossover between homologous chromosomes or the excision andintegration ofDNA fragments into non-homologous chromosomes. Alternatively, some arraysmay be prone to transposition due to the sequence itself.By whatever means it has occurred, the deletion and transposition of SGR-3 1 has hadlittle effect on chromosome pairing during meiosis since fertile hybrids are invariably producedby interspecific hybridization. It would be interesting to determine the chromosomaldistribution of SGR-3 I in other spruces, and in particular Yeddo spruce, perhaps leading to abetter understanding of taxonomic relations within the genus and the dynamic nature of thespruce genome.SGR-31 and spruce centrornere functionThe kinetochore is the trilaminar structure at the outer surface of the centromere towhich microtubules attach in mediating the movement of chromosomes during mitotis and85meiosis. Its assembly must involve the interaction of specific centromeric DNA sequenceswith kinetochore components (Willard 1990). It has been postulated from studies onmammalian centromere organization that the process might rely on the regular spacing of aparticular DNA curvature or sequence motif embedded in a larger repeated DNA11superstructure” (Vogt 1990; 1992). Two putative motifs have been characterized includingthe (GGAAT) repeat (Grady el al., 1992) described earlier and the CENP-B box, a 17 bpmotif found in a subset of alpha satellite sequences of humans and the mouse minor satellite.CENP-B, a major protein component of human centromeres, binds the CENP-B box both invitro and in vivo (Masumoto et at., 1989; Haafet a!., 1992) and has been immunolocalized tothe central domain of human metaphase chromosomes underlying the kinetochore (Cooke etat., 1990). Vogt’s concept is appealing since only the short motifs and their regular spacingare selected for as the critical requirement for kinetochore assembly. Within an array oftandemly repeated satellite DNA many copies of the sequence or curvature will be present. Ifthe interaction with kinetochore proteins requires the correct spacing of only some of thesemotifs, then amplification, deletion and rearrangement of the surrounding repeated sequences(the spacing domain; Vogt 1992) can result in the chromosome-specific satellite DNAcompositions characteristic of mammalian centromere organization.Little is known about the specific sequence organization of plant centromeres otherthan the few repeated DNAs listed previously. While it appears that SGR-3 1 is not a requiredcomponent of the spruce centromere, Vogts postulate suggests that some so-called junkDNAs might have a cellular role despite chromosome specificity and variation in copy numberbetween chromosomes. Whether SGR-3 1 plays a structural role in centromere function or issimply tolerated in the spruce genome is presently unknown.86LITERATURE CITEDBedbrook, J.R., Jones, J., O’Dell, M., Thompson, R.D. and Flavell, R.B. 1980. A moleculardescription of telomeric heterochromatin in Secale species. Cell, 19: 545-560.Benslimane, A.A., Dron, M., Hartmann, C. and Rode, A. 1986. Small tandemly repeatedDNA sequences of higher plants likely originate from a tRNA gene ancestor. Nuc.Acids Res. 14: 811-819.Cooke, C.A., Bernat, R.L., Earnshaw, W.C. 1990. CENP-B: a major human centromereprotein located beneath the kinetochore. J. Cell. Biol. 110: 1475-1488.Dennis, E.S., Gerlach, W.L. and Peacock, W.L. 1979. Identical polypyrimidine-polypurinesatellite DNAs in wheat and barley. Heredity, 44: 349-3 66.Dhillon, S.S. 1987. DNA in tree species. In Cell and tissue culture in forestry. Vol. 1. Generalprinciples and biotechnology. J.M. Bonga and D.J. Durzan (eds.). 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Genet. 90: 157-165.Ingham, L.D., Hanna, W.W., Baier, J.W. and Hannah, L.C. 1995. Origin of the main class ofrepetitive DNA within selected Pennisetum species. Mo!. Gen. Genet. 238: 350-356.87Irifline, K., Hirai, K., Zheng, J., Tanaka, R. and Merikawa, H. 1995. Nucleotide sequence of ahighly repeated DNA sequence and its chromosomal localization in All/urn Jistulosum.Theor. App!. Genet. 90: 312-316.Lapitan, N.L.V., Gill, B.S. and Sears, R.G. 1987. Genomic and phylogenetic relationshipsamong rye and perennial species in the Triticeae. Crop Sci. 27: 682-687.Lapitan, N.L.V. 1992. Organization and evolution of higher plant nuclear genomes. Genome,35: 171-181.Masumoto, H., Masukata, H., Muro, Y., Nozaki, N. and Okazaki, T. 1989. A humancentromere antigen (CENP-B) interacts with a short specific sequence in alphoidDNA, a human centromeric satellite. J. Cell Biol. 109: 1963-1973.Mukai, Y., Friebe, B. and Gill, B.R. 1992. Comparison of C-banding patterns and in situhybridization sites using highly repetitive and total genomic rye DNA probes of‘Imperial’ rye chromosomes added to ‘Chinese Spring’ wheat. Jpn. J. Genet. 67: 71-83.Murata, M., Ogura, Y. and Motoyoshi, F. 1994. Centromeric repetitive sequences inArabidopsis thaliana. Jpn. J. Genet. 69: 361-370.Sambrook, J., Fitch, E.F. and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual.2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminatinginhibitors. Proc. Nati. Acad. Sd. USA 85: 2444-2448.Simoens, C.R., Gielen, J., Van Mantagu, M. and Inze, D. 1988. Characterization of highlyrepetitive sequences ofArabidopsis thal/ana. Nuc. Acids Res. 16(14): 6753-6766.Singer, M.F. 1982. Highly repeated sequences in mammalian genomes. Tnt. Rev. Cytol. 76:67-112.Vogt, P. 1992. Code domains in tandem repetitive DNA sequence structures. Chromosoma,101: 585-589.Willard, H.F. 1990. Centromeres of mammalian chromosomes. Trends in Genet. 6:410-415.Wright, J.W. 1955. Species crossabililty in spruce in relation to distribution and taxonomy.Forest Sci. 1(4): 3 19-349.Xia, X., Selveraj, G. and Bertrand, H. 1993. Structure and evolution of a highly repetitiveDNA sequence from Brass/ca napus. Plant Mol. Biol. 21: 2 13-224.88CHAPTER 5The First Cytogenetic Maps ofWhite and Sitka SprucesINTRODUCTIONProviding a location on the emerging ISH map of spruce for a new DNA clonenecessitates that its hybridization site can be assigned to a recognized chromosome and relatedto other markers in its proximity. In mammalian cytogenetics, new markers can be quicklyassigned not only to a chromosome but to a well defined region by G-banding after ISH(Bhatt eta!., 1988; Lemieux et a!., 1992). Although G-banding in plants is uncommon, Jiangand Gill (1993) successfully applied modified N- or C-banding protocols following ISH toidentify wheat chromosomes hybridizing repeated DNAs. Similarly, ISH of the 18S-26SrDNA and the 5S rDNA in combination with fluorescent banding patterns produced by DAPIand Chromomycin A3 distinguished most of the chromosomes of slash pine (Doudrick et a!.,1995).In spruce, C- or N- banding procedures have not been defined and fluorescent bandingwith DAPI and Chromomycin A3 are less informative than in slash pine (Brown and Carison,unpublished data; Hizume et aL, 1991). However, the wide distribution of 18S-26S rRNAgenes is sufficient to provide a chromosomal designation for the majority of probes. In mostcases, all that it is needed is a DAPI stained image of the metaphase spread to determine to89which chromosome a probe has hybridized. In others, particularly involving chromosomes 2and 5, additional information such as the chromosome-specific 5S rDNA locus is needed. Inthis case, multiple probes must be physically mapped on the same metaphase spread.DNA clones can be co-localized on the same metaphase spread by eithersimultaneously hybridizing distinctly labeled probes (Leitch et a!., 1991) or by multipleprobings of the same slide (Heslop-Harrison et a!., 1992) in a manner analogous to therepeated hybridizations afforded by nitrocellulose or nylon membranes in Southern analysis.However, wide differences in target sequence copy numbers can make analysis ofsimultaneously hybridized probes difficult and the extra detection reagents required can leadto unacceptably high levels of background signal. In this chapter, the method of HeslopHarrison eta!. (1992) was employed using slides previously hybridized with SGR-3 I (Chapter4). By stripping these of old reagents and reprobing them, first with the 5S rDNA and thenthe 18S-26S rDNA clones, all three repeated DNAs were co-localized on the same metaphasespreads. This approach should be sufficient to designate specific chromosomal locations foradditional repeated and low copy DNA sequences in spruce.MATERIALS AND METHODSIn situ hybridizationAfter acquiring SGR-3 I hybridization images (Chapter 4), slides could be immediatelystripped of old detection reagents, hybridized probe sequences and mounting medium orstored in the dark at 4 0C for at least six months. Stripping was accomplished by washingslides 3 times in 0.1% Tween-20 (v/v) in 4X SSC for 1 hour each and twice in 2X SSC for 590minutes each. Slides were then dehydrated through an ethanol series and air dried. Biotinlabeled pWS1 1 (white spruce 5S rDNA clone) was then denatured at 80 °C, chilled on ice,and applied to the slide. Chromosomal DNA denaturation, ISH and detection were performedas described on pp. 33-36 of Chapter 2. This procedure was repeated a third time for theISH of pGmRl (18S-26S rDNA).Image analysisTo generate the images in Fig. 5.1 of propidium iodide stained chromosomeshybridized with the three repeated DNA sequences, the pWS Li image was first merged withthe propidium iodide image using Adobe Photoshop 3.0 software and converted to an 8-bitgrey scale image. This image was then merged with the 8-bit images of SGR-3 1 and pGmRlhybridization, producing a 24-bit RGB color image in which the images of pWS1 1/propidiumiodide, SGR-3 1 and pGmRl were arbitrarily assigned to the red, green and blue channels,respectively. The two hybridization sites of pWS 11 were then each selected andpseudocolored to more clearly distinguish them from the counterstain.RESULTSThe three repeated DNA sequences described in this research were co-localized on thesame metaphase spreads in white spruce and Sitka spruce (Fig. 5.1). Chromosomemorphology was not affected by the repeated stripping and hybridization. Of the twentyspreads observed after each hybridization only one suffered any chromosome loss. Althoughsome amount of DNA loss is inevitable, these results suggests the potential for further rounds91Fig. 5.1. Fluorescence TSR colocalization of the 5S and 18S-26SrDNA, and SGR-3 1 on the mitotic chromosomes of a) whitespruce and b) Sitka spruce (2n = 24). Fluorescent images ofhybridization sites for the 18S-26S rDNA (purple), the 5S rDNA(pale blue) and SGR-3 1 (green) were merged with the propidiumiodide counterstained image (red). The distinguishing features ofeach chromosome are listed in the text. Bar represents 10 Jim.92Chromosome Number1 2 3 4 5 6 7 8 9 10 11 120.32 019R02313 U flo.19 flH H0.74H U H flo.72H H HH H}0.25 0.20[H H0.66 H H H HFig. 5.2 The first cytogenetic maps ofwhite spruce and Sitka spruce. Map positionsof the 1 8S-26S rDNA (small gaps) and the 5S rDNA (open circles) are given fromthe terminus ofthe short arm. SGR-3 1 sites at the centromeres are denoted by filledcircles.93of hybridization with other repeated DNAs.Chromosome pairs of white and Sitka spruce were readily identified by morphology orISH pattern and are shown diagramatically in the ideograms in Fig. 5.2. Distinguishingfeatures of each white spruce chromosome are described below:Chromosome 1 - Longest chromosome. No hybridization sites.Chromosome 2 - Metacentric. Distinguished from #3 by position of I 8S-26S rDNA in themiddle of long arm. The extended secondary constriction in one homolog frequently observedin this seedlot may account for different arm designations and ratios between white and Sitkaspruces.Chromosome 3 - Metacentric. Easily distinguished from all other chromosomes by the 18S-26S rDNA locus proximal to the centromere on the long arm.Oiromosome 4- 18S-26S rDNA site on short arm and SGR-31 site at centromere.Chromosome 5 - Metacentric. 18S-26S rDNA and 5S rDNA sites on the same arm. This armhas been designated the short arm since arm length measurements were variable.Chromosome 6 - Easily distinguished from all chromosomes by the subtelomeric 18S-26SrDNA locus on the short arm. SGR-3 1 locus also at the centromere.Chromosome 7 - Metacentric. Smaller than #1. No hybridization sites of SGR-3 1, 5S rDNAor 18S-26S rDNA.Chromosome 8 - 1 8S-26S rDNA locus on the long arm and SGR-3 I site at centromere.Chromosome 9 - Easily identified from all other chromosomes by centromere position andarm ratio. No hybridization sites of SGR-3 I, 5S rDNA or 18S-26S rDNA.Chromosome 10 - Smallest chromosome with 18S-26S rDNA site.Chromosome 11 - Smallest metacentric chromosome. No hybridization sites.94Chromosome 12 - Submetacentric. Smallest chromosome. Prominent SGR-3 1 site atcentromere.The unique features of each Sitka spruce chromosome are listed below, withdifferences between their probable white spruce homeologues noted. Chromosomes 2-6, 9and 11 cannot be distinguished between white spruce and Sitka spruce based on morphologyor ISH patterns of the repeated DNAs used.Chromosome 1 - Longest chromosome. Centromeric site of SGR-3 1 in Sitka spruce which isabsent on #1 in white spruce.Chromosome 2 - Similar 18S-26S rDNA locus in middle of chromosome arm as on #2 ofwhite spruce but chromosome measurements place it on the short arm in Sitka spruce. 1 8S-26S rDNA site polymorphism not observed.Chromosome 3 - Similar proximal 18S-26S rDNA locus as on #3 of white spruce althoughchromosome measurements place the NOR in Sitka spruce on the long arm.Chromosome 4 - Similar to #4 ofwhite spruce.chromosome 5 - Similar to #5 ofwhite spruce.Chromosome 6 - Similar to #6 ofwhite spruce.Chromosome 7 - Similar morphologically to #7 of white spruce but with a centromeric SGR31 site in Sitka spruce.Chromosome 8- Medium sized metacentric with a centromeric SGR-3 I hybridization site.Although the arm ratio, centromeric index and relative length correspond to #8 of whitespruce, no 18S-26S rDNA locus is found in Sitka spruce.Chromosome 9- Similar to #9 ofwhite spruce. Easily identified by its size and arm ratio.95Chromosome 10 - Submetacentric. Third smallest in the complement. No hybridization sites.Lacks the 18S-26S rDNA loci on the long arm of #10 in white spruce.Chromosome 11 - Second smallest chromosome. Distinguished from #10 and #12 in Sitkaspruce by both length and arm ratio.Chromosome 12 - Smallest chromosome of the complement. Lacks the prominent site ofSGR-3 1 hybridization on #12 ofwhite spruce.DISCUSSIONytogenetic maps ofwhite and Sitka spruceThe three repeated DNA families used in this research have allowed the placing of thefirst 12 loci onto the cytogenetic map of white spruce, seven correspondingto sites of the18S-26S rDNA, one to the 5S rDNA, and four to SGR-3 1 loci. Elevenloci (five 18S-26SrDNA, one 5S rDNA and five SGR-31 loci) have likewise been mappedin Sitka spruce.While morphology and the locations of most repeated loci mapped here arecommon amongthe two species several clear differences are apparent. As mentioned previously,chromosomes 8 and 10 in Sitka spruce lack the 18S-26S rDNA loci found in white spruce.Chromosomes 1, 7 and 12 have different SGR-31 distributions in each species. Differenceswere also observed in the positioning of the 18S-26S rDNA on chromosomes3 and 4.Despite earlier cytologic observations that karyotype morphology amongspecies ofspruce is highly conserved, the differences in molecular cytogenetic mapsbetween white andSitka spruce indicate that the molecular forces responsible for repeated DNA turnover (e.g.,deletion, amplification and transposition) are clearly active. The tools ofgenetic mapping,96DNA sequencing and 1ST-I are now available to conifer geneticists to approach the question ofwhether or not karytoype conservation reflects gene order colinearity within and potentiallybetween genera. Meiotic chromosome pairing studies in white X Sitka or other interspecifichybrids would also assist in studying chromosome synteny and genome rearrangementscoincident with speciation in the genus.Additional repeated or low copy sequences mapped by ISH can now be easily assignedto a spruce chromosome and in most cases the chromosome arm by reprobing the slide withrepeated DNAs of known location. Currently the 5S and 1 8S-26S rDNA are satisfactoryreferences and could be supplemented with new DNA sequences mapped in the thture.Reprobing the same metaphase spread also ensures that the linear relation of sequences inproximity can be determined with certainty, in contrast to comparing hybridization sitesbetween spreads. This is a very practical contribution to spruce cytogenetics since highquality root tip preparations and metaphase spreads, generally in limited supply, can be reusedseveral times with minimal loss of chromosomal material.RepeatedDNAs of the spruce genonieWhile this thesis focussed on the ISH localization of tandem repeated DNA sequences,two lines of evidence support the notion that families of dispersed repeated DNAs may be thelarger component of the spruce repeated DNA fraction and, therefore, be responsible for thelarge genome size. First, while developing ISH probes at the beginning of this research, ablack spruce genomic library in a lambda bacteriophage vector (constructed and provided byLinda De Verno of Petawawa National Forestry Institute) was screened with white sprucegenomic DNA to identify recombinants with highly repeated DNA inserts. Of approximately9710,000 clones screened, 30 corresponding to those with the most intense hybridization signalwere selected. The inserts, on average 17 kb in length, were digested with Sail producing 52restriction fragments. Twenty six of these were then assigned to six distinct repeated DNAfamilies by dot blot hybridization. Four of the six families were determined to be dispersednuclear repeats based on the “smear” of fragments observed following Southern hybridization(data not shown). The remaining two families were tandem repeated sequences, one encodingthe 18S-265 rDNA as inferred by homology to pGmRl, and the other corresponding totandem repeated sequences found in the large intergenic spacer of the I 8S-26S rDNArepeating unit (Brown, Newton and Carison, unpublished). While this survey represents onlya limited examination of the spruce genome, it does suggest that the most highly repeatedsequences, apart from the I8S-26S rDNA, are dispersed as opposed to tandemly arranged.Secondly, chromosome staining with the fluorescent dyes DAPI and CMA3 revealedfew AT- or GC-rich genomic regions. Fluorescent bands reflect the reiteration of satelliteDNAs with a base composition distinct from the surrounding chromatin. DAPI stains thechromosomes of white, Engelmann and Sitka spruces and Picea brachylyla homogenously,with the exception of centromeres and secondary constrictions which appear as negativelystaining regions (Brown and Carlson, unpublished; Hizume et al. 1991). The centromerictandem arrays of SGR-3 I do not induce a positive DAPI band probably because the sequencecomposition differs little from the genome average of 63% (Miksche and Hotta 1973). In thesame species, CMA3 stains all secondary constrictions intensely, consistent with the high GCcontent of the 185-26S rDNA. In white, Engelmann and Sitka spruces, additional CMA3positive bands are found at several centromeric sites. Most of these are associated with theISH site of the GC-rich tandem repeat found in the IGS of the 18S-26S rDNA and cloned98from the black spruce library described above (data not shown). In summary, it appears thatneither GC- nor AT- rich tandem repeats, apart from the 1 8S-26S rDNA, have been amplifiedto any appreciable extent in the spruce genome. It would be instructive to optimize GiemsaC-banding protocols, which do not rely on differential base composition to revealheterochromatic regions, for spruce chromosomes. This would then enable the fhll extent oftandem repeated DNA sequences found in telomeric, centromeric and interstitialheterochromatin to be determined, and provide a clearer view of their contribution to thespruce genome.LITERATURE CITEDBhatt, B., Burns, J., Flannery, D. and McGee. 3.0. 1988. Direct visualization of single copygenes on banded metaphase chromosomes by nonisotopic in situ hybridization. Nuc.Acids Res. 16(9): 3951-3961.Doudrick, R.L., Heslop-Harrison, J.S., Nelson, C.D., Schmidt, T., Nance, W.L. andSchwarzacher, T. 1995. Karyotyping slash pine (Finns eli/oil/i var. elliottii) usingpatterns of fluoresence in situ hybridization and fluorochrome banding. J. Hered. (inpress).Heslop-Harrison, J. S., Harrison, G.E. and Leitch, 1.3. 1992. Reprobing of DNA:DNA in situhybridization preparations. Trends in Genet. 8: 372-3 73.Hizume, M., Kitazawa, N., Gu Z. and Kondo, K. 1991. Variation of fluorescent chromosomebands in Picea brachytyla var. comp/anata collected in Yunnan, China. LaKromosomo II, 63-64: 2149-2158.Jiang, 3. and Gill, B.S. 1993. Sequential chromosome banding and in situ hybridizationanalysis. Genome, 36: 792-795.Jiang, 3. and Gill, B.S. 1994. Nonisotopic in situ hybridization and plant genome mapping: thefirst 10 years. Genome, 37: 7 17-725.99Leitch, I.J., Leitch, A.R. and Heslop-Harrison, J.S. 1991. Physical mapping of plant DNAsequences by simultaneous in situ hybridization of two differently labeled fluorescentprobes. Genome, 34: 329-333.Lemieux, N., Dutrillaux, B. and Viegas-Pequignot, E. 1992. A simple method forsimultaneous R- or G-banding and fluorescence in situ hybridization of small singlecopy genes. Cytogenet. Cell. Genet. 59: 311-312.Miksche, J.P. and Hotta, Y. 1973. DNA base composition and repetitious DNA in severalconifers. Chromosoma, 41: 29-3 6.100CHAPTER 6The Prospects for Non-Isotopic In Situ Hybridizationin Conifer Genome AnalysisTHE SENSIVITY OF IN SITUHYBRIDIZATIONTechnical advances directed towards improving the detection sensitivity of nonisotopicISH are central to its future applications in the analysis of conifer and other plant genomes.Sensitivity defines the lower size limit of chromosomal target sequences that can be visualized.While in human cytogenetics, sensitivities as low as 0.5 kb allow the routine detection ofcDNAs, low copy DNA sequence mapping in plants has generally required chromosomaltargets of 10 kb or more (Ambros e. a!., 1986; Simpson et a!., 1988; Schaff et at., 1990;Leitch and Heslop-Harrison 1993). Only in rice does ISH sensitivity appear to rival that ofhuman ISH (Gustafson and Dille 1992; Song and Gustafson 1995). Gustafson and Dille(1992) successfully mapped 23 genomic DNA clones ranging in size from 0.7-3.4 kb on themitotic chromosomes of rice, albeit hybridization sites were detected in only 6% ofmetaphases analysed and were usually observed on only a single chromatid. Dong and Quick(1995) also physically mapped a 2.6 kb low copy sequence in wheat and rye but failed toreport the percentage of cells showing hybridization signals.The discrepancy in sensitivity between ISH to human and plant chromosomes arisesprimarily from the presence of the plant cell wall, the source of dividing material and the101methods of metaphase chromosome preparation. The preparation of human chromosomesrelies exclusively on well established tissue culture methods which yield synchronized cellpopulations with a high mitotic index. Chromosome spreads are typically obtained bydropping a hypotonic cell suspension onto microscope slides resulting in many well spreadmetaphases with little or no cytoplasmic debris. In contrast, plant chromosomes prepared forISH are usually produced by the squash technique following enzymatic digestion of the cellwall, as used in this research. This approach is effective in producing greater numbers of wellspread metaphases in comparison to conventional squashes which omit the degradation of thecell wall. However, cell wall and cytoplasmic debris invariably remain and appear to hinderthe hybridization and detection of low copy sequences and increase the non-specific binding oflabeled probes and detection reagents. These factors, coupled with the generally low mitoticindex of root tip meristems and the frequent detection of hybridization signals on only a singlechromatid in a very low percentage of metaphases analysed, may raise questions concerningthe authenticity of hybridization sites of small, low copy DNA probes in plants.Most successes in low copy DNA sequence mapping in plants have been attributed tothe use of protoplasts derived from suspension cultures or root tips, and to droppingprotoplast suspensions onto microscope slides to spread the chromosomes. Tissue culturemethods for conifers, including white spruce and several pine species (Attree et al., 1989;Gupta and Durzan 1987) are well established. Of particular relevance to genome analysis inspruce is the recent demonstration that embryogenic cultures of black spruce can besynchronized by treatment with hydroxyurea and arrested effectively in metaphase usingolchicine. Mitotic indices in these cultures approached 40% (Nkongolo and Klimaszewska1994). Adapting these culture treatments to other conifers and developing protoplast102methods for chromosome preparation (Dille et al., 1990) will significantly improve thesensitivity of ISH.Raap ci a!. (1995) described a new detection principle for fluorescence ISH in humancytogenetics. To detect biotin-labeled probes, a layer of avidin conjugated to horseradishperoxidase is first applied, as in many non-isotopic ISH procedures. The method is based onthe subsequent use of the peroxidase substrate tyramine conjugated to fluorochromes orbiotin. The action of peroxidase on the tyramine derivatives produces many highly reactiveintermediates that effectively immobilize fluorochrome or biotin molecules at or near theperoxidase molecule. Fluorochromes can then be visualized by fluorescence microscopy,while biotin molecules require an additional avidin conjugate layer. This detection method,described as “ultra-sensitive” by the authors should be easily adapted to plants and may alsohelp alleviate low ISH sensitivities.Even using the squash technique, good hybridization signals from low copy DNAsequences could be generated using DNA probes with large inserts, such as yeast or bacterialartificial chromosomes. If the typical conifer genome is composed of 75% repeated DNAthen inserts of 200 kb should contain approximately 50 kb of low copy sequence. Evencosmids or P1 bacteriophage clones (with more easily managed insert sizes of 50 and 100 kb,respectively) may contain enough low copy sequence to produce readily detected signals. It isnecessary to suppress the hybridization of repeated sequences within these clones althoughthis is routinely achieved in the ISH of yeast artificial chromosomes in human cytogenetics bycompetition with Cot-i DNA (Landegent ci a!., 1987). Genomic DNA in the appropriatemolar ratio might also serve as a competitor. Problems with the hybridization of repeatedsequences in large clones may also be circumvented by using clones from unrelated species1I(although this approach might require the verification of linkage relationships between thespecies).APPLICATIONSContinued mapping ofrepeatedDNAsThe value of physically mapping tandem arrayed repeated DNAs in spruce has beenclearly demonstrated in this research as a means of identif’ing the morphologically similarchromosomes of conifers and in comparing the distribution and evolution of repeated DNAsbetween homeologous chromosomes. Even in the absence of technical advances to increasethe sensitivity of ISH, this method and the ribosoma RNA probes described can be likewiseapplied to other conifers as a basis for comparing their genome organization and evolutionwith other plant forms.The chromosomal mapping of dispersed repeated DNAs will also be a useful tool forthose foresters and geneticists in British Columbia interested in the presence and extent ofintrogression among natural populations of Sitka spruce and interior spruce (white,Engelmann and their hybrids). While the tandem repeated DNA families studied here wouldbe sufficient to distinguish parental species and F1 white X Sitka spruce hybrids, meioticrecombination in the hybrids and the limited genome coverage of the repeated DNA familiesavailable restricts their ability to assess species composition in later generations ofintrogressive hybridization. The efforts of Sutton et al. (1991a; 1991b), using species-specificrestriction fragment length polymorphisms (RFLP’s) of the organelle genomes and morerecently RFLP’s of the intergenic spacer of the nuclear-encoded 18S-26S rDNA (Sutton eta!.,041994), are presently more reliable and simpler approaches to investigating introgression thanISH.Some form of ISH with dispersed repeated DNA probes would be a valuablecytological complement to RFLP analysis of introgression in spruce. Genomic ISH, wherebylabeled white spruce genomic DNA and an excess of unlabeled Sitka spruce DNA (or viceversa) are hybridized to chromosome preparations of individuals from putative hybrid zonesor seedlots, is one possibility. However, since the ease of detecting differences betweenspecies by genomic ISH decreases with increasing overall sequence homology between thespecies (Jiang and Gill 1994), the apparently high degree of genome relatedness of white andSitka spruces may preclude this approach. Alternatively, species-specific dispersed repeatspresent throughout only one of the two introgressing genomes could be used as hybridiziationprobes. Either of these ISH approaches provides broader coverage of the genome thanRFLP’s, giving a more direct, easily interpreted analysis of the contribution of each parentalspecies.Low copy DNA sequence and gene mappingA future goal of genome mapping in conifers, and one in which ISH plays a centralrole, is the assignment of genetic linkage groups and individual genes or other low copy DNAloci to specific chromosomal sites. This accomplishment will serve both academic andpractical purposes. Correlating the genetic and physical maps of a species will allow thestatus of the genetic map to be assessed. It will also serve to unite different linkage groups onthe same chromosome and verify that genetic markers at the end of linkage groups actually lievery near to the telomere. Integrated maps will augment future comparative genetic mapping105studies in conifers based on RFLP linkages by providing a chromosomal context in which geneorder co-linearity is assessed. The results could provide insight into the extent andconsequence of chromosomal rearrangements undergone during evolution and the potentialapplication of one species map to another species.It is becoming increasingly clear that the physical distance between genes is oftenmarkedly different than the observed genetic distance (Dvorak et a!., 1984; Song andGustafson 1995). Recombination in the distal region of cereal chromosomes is significantlymore frequent than in regions proximal to the centromere (Lukaszewski 1992). Relating thegenetic map to underlying chromosome structure will enable recombination rates across theconifer genome to be evaluated. The effect of reduced recombination in some genomicregions on the usable genetic variation available to tree breeders and on the value of geneticdiversity estimates derived from biochemical and molecular markers may need to be addressedin the future.Gene transfer methods for white spruce and other conifers have received considerableattention as avenues to integrate new traits into existing populations quickly. This approachto tree improvement would also benefit from successes in gene mapping by ISH as thelocation of the integrated transgene and the number of copies in the genome may affect geneexpresssion.From a practical perspective, low copy DNA sequence mapping by ISH will facilitateconstructing higher density maps of conifer genomes. The current focus of genome analysis intree improvement is the construction of genetic linkage maps and the search for associationsbetween molecular markers and desirable economic traits. To use this information in mapbased cloning or marker-assisted selection the subsequent step in defining the genomic region106involved is to isolate more tightly linked and flanking markers. This requires constructing ahigher resolution map.In genetic mapping, the smaller the map distance between loci, the larger thesegregating population that is needed to ensure, at an acceptable level of probability, that lociare properly ordered. For conifer species, the construction of higher resolution genetic mapsmay be difficult since large numbers of segregating individuals from controlled crosses maynot be available and obtaining progeny from specific genotypes or crosses can take manyyears. Physical mapping by ISH is a particularly valuable complement to linkage analysis insuch cases since it requires seed from only one individual. The locus need not be polymorphicand segregation analysis is not required. Additionally, Lichter et al. (1990) demonstrated thatDNA clones 1.1 Mb apart can be resolved across the width of each chromatid. The resolutionof ISH to human metaphase chromosomes is generally 1-3 Mb (Joos et at., 1994), equivalentto roughly 1-3 centiMorgans in humans. Although it has been suggested that plant metaphasechromosomes are more condensed than human chromosomes (Greilhuber 1977; Jiang and Gill1994), resolution of metaphase chromosome mapping in plants would still likely be less than10Mb.ISH provides a quick and efficient tool in constructing higher resolution maps. Torandomly add markers to a physical map, several probes can be distinctly labeled, hybridizedand independently localized to chromosomes in a single experiment. Clones which map to thesame position on low resolution genetic maps may also be quickly ordered. Once thechromosomal location of a marker linked to a desirable trait is known, then clones mapping inproximity to that marker can be readily identified. To find the most tightly linked flankingmarkers would still require segregation analysis. However, if large insert clones were used as107ISH probes in generating the higher resolution map, the proximal markers could be screenedfor highly informative microsatellite or minisatellite polymorphisms to maximize the possibilityof detecting recombination between the markers and the trait locus.Generating a high resolution map of an entire conifer genome is an enormous task.The random addition of molecular markers to a genetic map is both time and resourceconsuming and could be simplifed if approaches to target directly a specific chromosome orchromosomal region of interest were developed. The construction of chromosome-specificlibraries using flow sorting methods permits this (Arumuganathan et a?., 1991; Wang et a!.,1992), although it may not be possible given the overall uniformity in conifer chromosomesize. Alternatively, microdissection methods (Jung et a?., 1992) or the sorting of individualchromosomes by fluorescence ISH in conjunction with flow sorting or avidin-conjugatedmagnetic particles are possible (Dudin eta?., 1988; Gray eta?., 1986). The chromatin isolatedcould then be cloned into cosmids or other large insert vectors and individual clones rapidlyordered along the chromosome of interest.To what extent high resolution maps of conifer genomes will be required is uncertain.For dissecting quantitative traits or marker-assisted selection, the resolution offered by ISHmapping on metaphase chromosomes is likely sufficient. Cloning genes from a conifergenome based on their map position would require the generation of a much higher resolutionphysical map and the development of techniques to manipulate and sequence large tracts ofDNA. Conifer genomes are large and primarily repetitive which hampers map-based cloningstrategies. Unless a particular gene is specific to a conifer species, it will be more practical toclone it from a well characterized plant with a small genome such as Arabidopsis (or poplar)and then use this to probe a conifer genomic or cDNA library. If the situation arises in which108very high resolution maps are required, ISH methods developed in human cytogenetics boast50 kb - 1 Mb resolution on interphase chromatin (Lawrence et a!., 1988; Trask 1991) and 1 -400 kb resolution using extended chromatin fibers (Parra and Windle 1993; Florijn et al.,1995). Both techniques rely on the decondensed state of chromatin either at interphase orusing extended single fibers ofDNA released from nuclei fixed on slides.Prior to the broad use of molecular cytogenetics, plant and animal genomes could onlybe studied at the DNA sequence level, by genetic recombination, and at the chromosomallevel. Only by knowing the physical position of genes along a chromosome can geneticlinkage, and gene and sequence interpendence, be understood (Heslop-Harrison 1991). ISHtechniques bridge the gap between the resolutions of these methods allowing thechromosomal location of linkage groups to be determined, the relationship between geneticand physical distance to be understood, and the eventual cloning of genes based on mapposition.LITERATURE CITEDAmbros, P.F., Matzke, M.A. and Matzke, A.J.M. 1986. Detection of a 17 kb unique sequence(T-DNA) in plant chromosomes by in sill! hybridization. Chromosoma, 94: 11-18.Arumuganathan, K., Slattery, J.P., Tanksley, S.D. and Earle, E.D. 1991. Preparation and flowcytometric analysis of metaphase chromosomes of tomato. Theor. Appi. Genet. 82:101—111.Attree, S.M., Dunstan, D.I. and Fowke, L.C. 1989. Plantlet regeneration from embryogenicprotoplasts of white spruce (Picea glanca). Bio/Technology, 7: 1060-1063.Dille, J.E., Bittel, D.C., Ross, K. and Gustafson, G.P. 1990. Preparing plant chromosomes forscanning electron microscopy. Genome, 33: 333-339.109Dong, H. and Quick, J.S. 1995. Detection of a 2.6 kb single/low copy DNA sequence onchromosomes of wheat (Triticurn aestivum) and rye (Secale cereale) by fluorescencein situ hybridization. Genome, 38: 246-249.Dudin, G., Steegmayer, E.W., Vogt, P., Schnitzer, H., Diza, E., Howell, K.E., Cremer, T. andCremer, C. 1988. Sorting of chromosomes by magnetic separation. Hum. Genet. 80:111—116.Dvorak, J. and Chen, K.-C. 1984. Distribution of nonstructural variation between wheatcultivars along chromosome 6Bp: evidence from the linkage map and physical map ofthe arm. Genetics, 113: 325-333.Florijn, R.J., Bonden, L.A.J., Vrolijk, H., Wiegant, J., Vaandrager, J.-W., Baas, F., denDunnen, J.T., Tanke, H.J., van Ommen, G.-J. B. and Raap, A.K. 1995. High-resolution DNA Fiber-FISH for genomic DNA mapping and colour bar-coding oflarge genes. Hum. Mol. Genet. 4: 831-836.Gray, J.W., Lucas, J., Peter, D., Pinkel, D., Trask, B., van den Engh, G. and VanDilla, M.1986. Flow karyotyping and sorting of human chromosomes. Cold Spring HarborSymp. Quant. Biol. 51: 141-149.Greilhuber, J. 1977. Why plant chromosomes do not show G-bands. Theor. Appl. Genet. 50:121- 124.Gupta, P.K. and Durzan, D.J. 1987. Biotechnology of somatic polyembryogenesis and plantletregeneration in loblolly pine. Bio/Technology, 5: 147-15 1.Gustafson, J.P. and Dille, J.E. 1992. Chromosome location of Oiyza saliva recombinationlinkage groups. Proc Nail Acad. Sci. USA 89: 8646-865 0.Gustafson, J.P., Butler, E. and McIntyre, C.L. 1990. Physical mapping of a low-copy DNAsequence in rye (Seca/e cereale L.). Proc. Nati. Acad. Sci. USA 87: 1899-1902.Heslop-Harrison, J.S. 1991. The molecular cytogenetics of plants. J. Cell Sci. 100: 15-21.Jiang, J. and Gill, B.S. 1994. Nonisotopic in s/ti, hybridization and plant genome mapping: thefirst 10 years. Genome, 37: 7 17-725.Joos, S., Fink, T.M., Ratsch, A. and Lichter, P. 1994. Mapping and chromosome analysis: thepotential of fluorescence in si/it hybridization. J. BioTech. 35: 135-153.Jung, C., Claussen, U., Horstemke, B., Fisher, F. and Herrmann, R.G. 1992. A DNA libraryfrom an individual Bela pate//ar/s chromosome conferring nematode resistance110obtained by microdissection of meiotic metaphase chromosomes. Plant Mol. Biol. 20:503-5 11.Landegent, J.E., Jansen in de Wa!, N., Dirks, R.H., Baas, F. and van der Ploeg, M. 1987. Useof whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370.Lawrence, J.B., Singer, RH. and McNeil, J.A. 1990. Interphase and metaphase resolution ofdifferent distances within the human dystrophin gene. Science, 249: 928-932.Lawrence, J.B., Villnace, C.A. and Singer, R.H. 1988. Sensitive, high resolution chromatinand chromosome mapping in situ: presence and orientation of two closely integratedcopies of EBV in a lymphoma line. Cell, 52: 5 1-61.Leitch, I.J. and Heslop-Harrison, J.S. 1993. Physical mapping of four sites of the 5S rDNAsequences and one site of the alpha-amylase-2 gene in barley (Hordeurn vulgare).Genome, 36: 5 17-523.Lichter, P., Tang, C.C., Call, K., Hermanson, G., Evans,. G.A., Housman, D. and Ward, D.C.1990. High resolution mapping of human chromosome 11 by in situ hybridization withcosmid clones. Science, 247: 64-69.Lukaszewski, A.J. 1992. A comparison of physical distribution of recombination inchromsome 1R in diploid rye and in hexaploid triticale. Theor. Appl. Genet. 83: 1048-1053.Nkongolo, K.K. and Klimaszewska, K. 1994. Karyotype analysis and optimization of mitoticindex in Picea mariana (black spruce) preparations from seedling root tips andembryogenic cultures. Heredity, 73: 11-17.Parra, I. and Windle, B. 1993. High resolution visual mapping of stretched DNA byfluorescent hybridization. Genetics (Nature), 5: 17-21.Raap, A.K., van de Corput, M.P.C., Vervenne, R.A.W., van Gijlswijk, R.P.M., Tanke, H.J.and Wiegant, J. 1995. Ukra-senstitive FISH using peroxidase-mediated deposition ofbiotin- or fluorochrome tyramides. Hum. Mol. Genet. 4: 529-534.Schaff, D.A., Koehier, S.M., Matthews, B.F. and Bauchan, G.R. 1990. In situ hybridization ofbeta-tubulin to alfalfa chromosomes. J. Hered. 81: 480-483.Simpson, P.R., Newman, M.A. and Davies, D.R. 1988. Detection of legumin gene DNAsequences in pea by in situ hybridization. Chromosoma, 96: 454-458.111Song, Y.C. and Gustafson, J.P. 1995. The physical location of fourteen RFLP markers in rice(OryzasativaL.). Theor. Appl. Genet. 90: 113-119.Sutton, B.C.S., Pritchard, S.C., Gawley, JR., Newton, C.H. and Kiss, G.K. 1994. Analysis ofSitka spruce-interior spruce introgression in British Columbia using cytoplasmic andnuclear DNA probes. Can. J. For. Res. 24: 278-28 5.Sutton, B.C.S., Flanagan, D.J. and El-Kassaby, Y.A. 1991a. A simple and rapid method forspecies determination of spruce seedlots using restriction fragment lengthpolymorphism. Silvae Genet. 40: 119-123.Sutton, B.C.S., Flanagan, D.J., Gawley, R., Newton, C.H., Lester, D. and El-Kassaby, Y.A.199 lb. Inheritance of chloroplast and mitochondrial DNA in Picea and composition ofhybrids from introgression zones. Theor. Appl. Genet. 82: 242-248.Trask, B.J. 1991. Fluorescence in situ hybridization. Trends in Genet. 7: 149-154.Wang, M.L., Leitch, A.R., Schwarzacher, T., Heslop-Harrison, J.S. and Moore, G. 1992.Construction of a chromosome-enriched HpaII library from flow-sorted wheatchromosomes. Nuc. Acids Res. 20(8): 1897-1901.112APPENDIX ICommmon fluorochromes and their excitation and emission maxima.Fluorochrome Excitation Emission FluorescentMaximum (nm) Maximum (nm) ColorNucleotide ConjugatesCoumarin (AMCA) 350 600 blueFluorescein (FITCy’ 494 520 greenCyanine Cy 3 550 565 greenRhodamine600 (TRITC)c 575 600 redTexas Red 596 615 redCyanine Cy 5 650 670 redDNA Stains359 461DAPI bluePropidium iodide 340, 530 617 redChromomycin A3 458_____________590 yellowa 7-amino-4-methly-coumarin-3-acetic acid; b fluorescein isothiocyanate; tetramethylrhodamine isothiocyanate; d 4’ ,6-diamidino-2-phenylindole.113

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