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The genetic control of petal morphology in the sweet pea (Lathyrus odoratus) Woollacott, Christine Marie 2010

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THE GENETIC CONTROL OF PETAL MORPHOLOGY IN THE SWEET PEA (Lathyrus odoratus)  by  Christine Marie Woollacott  B.Sc., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2010  © Christine Marie Woollacott, 2010  Abstract The hooded mutant of sweet pea (Lathyrus odoratus) was characterized in order to better understand the genetic control of petal morphology in papilionoid legumes. The TCP identity gene CYCLOIDEA2 (CYC2) was shown to have different amplification patterns in the standard petals of hooded and wild-type sweet peas in RT-PCR experiments. Lathyrus CYC1 was also examined, but no differences were found between expression patterns in wild-type and hooded plants. Genome walking and additional sequencing revealed that the hooded phenotype was associated with a mutant allele of CYC2. To determine if the mutant allele of CYC2 is responsible for the hooded phenotype, a F2 segregation experiment was conducted. In a population of 118 plants, the hooded phenotype segregated with the mutant allele of CYC2 100% of the time, suggesting that the mutation in CYC2 is likely responsible for the hooded floral character. Scanning Electron Microscopy was conducted to determine if epidermal cell types could be used as micromorphological markers for petal identity. Cells on the adaxial surface of sweet pea petals can be used to distinguish petal identity. When  mutant and wild-type flowers were compared, clear differences in cell type were observed. The standard petal in hooded flowers has taken on wing petal characteristics, indicating that the mutation is caused by a shift in petal identity. To examine localized growth patterns in wild-type and hooded flowers, microscopic grids were printed on the surfaces of sweet pea buds using a specialized inkjet apparatus. The deformation of the printed grid during growth allowed localized patterns of growth to be visualized. Wild-type standard petals have a more uniform rate of growth, whereas hooded standard petals show increased growth at their margins which may account for the overall differences in curvature between wild-type and hooded petals. The results of these analyses suggest that the hooded mutant of sweet pea is caused by a mutation in the CYC2 gene. CYC2 genes have been shown to play a role in establishing standard petal identity in the sweet pea and in a number of other legumes (Feng et al., 2006; Wang et al., 2008a). ii  Preface The data presented in the sixth chapter of this document, ‘Localized Growth Patterns in the Standard Petal of Lathyrus odoratus’, is a result of a collaboration between myself, Lisheng Wang and Simon Beyer who are members of Konrad Walus’ lab in Department of Electrical and Computer Engineering at the University of British Columbia. Simon Beyer and Lisheng Wang operated the specialized inkjet printing apparatus that was used to print microscopic grids on sweet pea standard petals. Lisheng Wang was also responsible for generating the figures that illustrate the localized growth patterns that were observed for each petal examined. I grew and maintained the sweet pea plants that were required for the analyses and interpreted the figures in a biological context. Quentin Cronk and Konrad Walus oversaw the project. I conducted all other experiments (RT-PCR, PCR, genome walking, F2 segregation and SEM) described in this document unless otherwise noted. I have written the entire body of this document including all text found within the sixth chapter.  iii  Table of Contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii Acknowledgements ........................................................................................................................ ix Dedication ....................................................................................................................................... x 1 – Introduction ............................................................................................................................... 1 1.1 – The Sweet Pea .................................................................................................................... 1 1.2 – Lathyrus.............................................................................................................................. 1 1.3 – Sweet Pea Flowers ............................................................................................................. 3 1.4 – TCP-box Genes .................................................................................................................. 6 1.5 – CYCLOIDEA and Flower Development ............................................................................ 7 2 – RT-PCR Analysis of CYCLOIDEA in Lathyrus odoratus ........................................................ 9 2.1 – Aim ..................................................................................................................................... 9 2.2 – Introduction ........................................................................................................................ 9 2.2.1 – The hooded sweet pea ................................................................................................. 9 2.2.2 – CYCLOIDEA genes ................................................................................................... 10 2.2.3 – Similar mutants in other legumes ............................................................................. 11 2.2.4 – The hooded mutation only affects the standard petal ............................................... 11 2.3 – Methods ............................................................................................................................ 12 2.3.1 – Tissue sampling and RNA extraction ....................................................................... 12 2.3.2 – RT-PCR..................................................................................................................... 13 2.4 – Results .............................................................................................................................. 13 2.5 – Discussion ........................................................................................................................ 14 2.5.1 – A mutation affecting LoCYC2 is implicated in the hooded phenotype .................... 14 2.5.2 – Possible roles of LoCYC genes in petal identity ....................................................... 15 2.5.3 – The hooded mutation is homeotic in nature .............................................................. 17 3 – Analysis of CYCLOIDEA Sequence in Lathyrus odoratus .................................................... 19 3.1 – Aim ................................................................................................................................... 19 3.2 – Methods ............................................................................................................................ 19 iv  3.2.1 – Isolating CYCLOIDEA sequence from wild-type and hooded sweet peas ............... 19 3.2.2 – Genome walking ....................................................................................................... 20 3.3 – Results .............................................................................................................................. 21 3.3.1 – Wild-type sequence ................................................................................................... 21 3.3.2 – Mutant CYC2 sequence from hooded plants ............................................................. 22 3.4 - Discussion ......................................................................................................................... 23 4 – F2 Segregation ........................................................................................................................ 25 4.1 – Aim ................................................................................................................................... 25 4.2 – Introduction ...................................................................................................................... 25 4.21 – Phenotypes in crossing experiments .......................................................................... 25 4.2.2 – Predicted phenotypes ................................................................................................ 26 4.3 – Methods ............................................................................................................................ 27 4.3.1 – Crossing .................................................................................................................... 27 4.3.2 – Determination of alleles ............................................................................................ 29 4.4 – Results .............................................................................................................................. 31 4.4.1 – Observed phenotypes ................................................................................................ 31 4.4.2 – Inheritance of the mutant allele of LoCYC2 ............................................................. 33 4.4.3 – χ2 Analysis ................................................................................................................ 34 4.5 - Discussion ......................................................................................................................... 35 5 – Petal Micromorphology in Lathyrus odoratus ........................................................................ 37 5.1 – Aim ................................................................................................................................... 37 5.2 – Introduction ...................................................................................................................... 37 5.2.1 – The role of cell shape in pollination ......................................................................... 37 5.2.2 – Petal-specific cell types in Fabaceae......................................................................... 38 5.3 – Methods ............................................................................................................................ 39 5.3.1 – SEM and tissue sampling .......................................................................................... 39 5.4 – Results .............................................................................................................................. 40 5.4.1 – Scanning electron micrographs ................................................................................. 40 5.4.2 – Cell types present in wild-type sweet peas ............................................................... 41 5.4.3 – Cell types present in hooded sweet peas ................................................................... 42 5.5 – Discussion ........................................................................................................................ 42 5.5.1 – Cell shape as a micromorphological marker ............................................................. 42 5.5.2 – The hooded mutation is homeotic in nature .............................................................. 43 v  5.5.3 – CYCLOIDEA-like genes and epidermal cell shape ................................................... 43 6 – Localized Growth Patterns in the Standard Petal of Lathyrus odoratus ................................. 45 6.1 – Aims ................................................................................................................................. 45 6.2 - Introduction ....................................................................................................................... 45 6.2.1 – Methods of landmark analyses.................................................................................. 45 6.2.2 – Effect of genes on growth patterns and curvature .................................................... 46 6.3 – Methods ........................................................................................................................... 47 6.3.1 – Printing of landmarks ................................................................................................ 47 6.3.2 – Analysis of landmarks............................................................................................... 47 6.4 – Results .............................................................................................................................. 48 6.5 – Discussion ........................................................................................................................ 50 6.5.1 – Growth and curvature in the sweet pea ..................................................................... 50 7 - Conclusion ............................................................................................................................... 52 Works Cited .................................................................................................................................. 55 Appendix – Supplemental Data and Calculations ........................................................................ 58 Sweet Pea Standard Petal Measurements .................................................................................. 58 Relationships Between Standard Petal Length and Width Measurements ............................... 62 Statistical Formulas ................................................................................................................... 62 Sweet Pea Primers ..................................................................................................................... 63 Sweet Pea Sequences ................................................................................................................ 64 Genome walking results: Hooded1L/2L primers .................................................................. 64 The F2 Segregation Ratio .......................................................................................................... 69 F2 PCR Amplification Results .................................................................................................. 70 F2 gels ................................................................................................................................... 76 F2 hooded Sequence.................................................................................................................. 77 Primers: LoHoodedGene4L/4R ............................................................................................ 77 Primers: LoHoodedGene3L/2R ............................................................................................ 80 Localized Growth Patterns in the Standard Petal of Lathyrus odoratus ................................... 86 Petal Micromorphology in Lathyrus odoratus – scanning electron micrographs ..................... 96  vi  List of Tables Table 2.2 – Wild-type and hooded varieties of Lathyrus odoratus...............................................14 Table 3.1 – List of genome walking primers.................................................................................21 Table 4.1 – Expected F2 phenotypes.............................................................................................26 Table 4.9 – χ2 calculations............................................................................................................35  vii  List of Figures Figure 1.1 – Sweet pea flowers........................................................................................................4 Figure 1.2 – Sweet pea standard petals............................................................................................5 Figure 2.1 – Expression of CYC-like homologs in Lathyrus odoratus..........................................13 Figure 3.2 – Gene map of Lathyrus odoratus CYC2.....................................................................22 Figure 3.3 – Lathyrus odoratus CYC2 Alleles...............................................................................24 Figure 3.4 – FASTA protein sequence of the wild-type and hooded alleles of LoCYC2..............24 Figure 4.2 – Insect visitation..........................................................................................................28 Figure 4.3 – F2 segregation...........................................................................................................29 Figure 4.4 – CYC2GENE primers.................................................................................................30 Figure 4.5 – LoHoodedGene primers............................................................................................30 Figure 4.6 – Sweet pea phenotypes................................................................................................32 Figure 4.7 – CYC2GENE4/9 amplification...................................................................................33 Figure 4.8 – Sequence from hooded sweet peas............................................................................34 Figure 5.1 – Epidermal cell types present in wild-type sweet peas (Lathyrus odoratus)..............40 Figure 5.2 – Epidermal cell types present in hooded sweet peas (Lathyrus odoratus).................41 Figure 6.1 – The growth model generated through the petal printing landmark analysis.............48 Figure 6.2 – The growth model generated through the petal printing landmark analysis.............49  viii  Acknowledgements I’d like to thank my supervisor, Quentin Cronk, for his continual support and guidance throughout my research. I would also like to thank the members of my examination committee, Keith Adams (co-supervisor), and Andrew Riseman for their suggestions and advice. I would like to thank the supplemental examiner for my defense, Jeannette Whitton, and the chair for my defense, Patrick Martone. I would like to thank Quentin Cronk and UBC for the grant support that allowed me to conduct my research. I would like to acknowledge Dorothy Cheng, Nyssa Temmel, Isidro Ojeda Alayon and other members of the Cronk lab for training me in the common lab techniques and protocols that were required in my research. I would like to thank Lisheng Wang, Simon Beyer and Konrad Walus for their help gathering data and producing figures for Chapter 6 – Localized Growth Patterns in the Standard Petal of Lathyrus odoratus. Without their assistance and skill, Chapter 6 would not be possible. I would like to thank Farnaz Pournia and my mother, Evelyn Carpenter, for their help tending to the F2 population of sweet pea plants both in the field and in the green house. They were both instrumental in collecting tissue samples for the F2 segregation analysis outlined in Chapter 4. I would like to thank my father, James Woollacott, for his encouragement and support throughout my research. And lastly, I would like to thank Bella Shum for her assistance formatting this document.  ix  Dedication I’d like to dedicate this work to my parents, James Woollacott and Evelyn Carpenter.  x  1 – Introduction 1.1 – The Sweet Pea Although the sweet pea (Lathyrus odoratus L.) is no longer a common model organism, it has a long history of being used in genetic research. The sweet pea was one of the first organisms that was used to verify Mendel’s work with the garden pea (Pisum sativum L.) in the early 1900s (Bateson, 1902). The genetical phenomenon of linkage was first demonstrated in the sweet pea, although these observations did not directly lead to the development of modern chromosomal theory (Bateson et al., 1906; Carlson, 2004). The sweet pea has long been a favourite English garden plant, and commercial varieties have been available on the market since 1724 (Rice, 2002). Since then, hundreds of horticultural varieties displaying both vegetative and floral mutations have been unveiled. These pure-breeding horticultural lines are a valuable resource for studying the genetics behind vegetative and floral traits. Because of its large, colourful and fragrant flowers, the sweet pea is especially suited as a model organism with which to study flower development.  1.2 – Lathyrus The genus Lathyrus, placed in the order Fabales, and the family Fabaceae (Leguminosae), is most closely related to the genera Vicia and Pisum (Kenicer et al., 2005). Approximately 160 species world-wide belong to Lathyrus (Asmussen and Liston, 1998). In the past, Lathyrus has been broken down into a number of sections because of its great diversity and large size (Asmussen and Liston, 1998; Kenicer et al., 2005). Members of Lathyrus generally grow in temperate regions and have been found on all continents except Antarctica (Kenicer et 1  al., 2009; Kenicer et al., 2005). Many species have a chromosome number of 2n = 2x = 14, although some species such as L. pratensis and L. palustris have chromosome numbers of 2n = 4x = 28 or 2n = 6x = 42 respectively (Gutiérrez et al., 1994; Senn, 1938). Members of Lathyrus can be either annual or perennial (Allen and Allen, 1981). Lathyrus leaves are paripinnate and usually possess two pairs of entire leaflets (Allen and Allen, 1981). The petiole can be quite wide and phyllode-like in some species (Allen and Allen, 1981). The plants often exhibit a climbing habit that is facilitated by branched tendrils that terminate the tips of their compound leaves (Allen and Allen, 1981). The leaves can also be terminated by bristles (Allen and Allen, 1981). Plants often possess leaf-like, semisagittate stipules that may be entire or divided at their base (Allen and Allen, 1981). Flowers can be yellow, blue, purple, red, pink or white in colour and are borne in racemes, or as solitary flowers (Allen and Allen, 1981). The flowers are papilionoid and have a reflexed standard petal that may be oval, round or obcordate (Allen and Allen, 1981). The clawed wing petals can be falcate-obovate or oblong, and are longer than the clawed, obtuse keel petals (Allen and Allen, 1981). The flowers have ten stamens, nine of which are connate and one which is free (Allen and Allen, 1981). The style is inflexed, flattened dorsally, and can be either glabrous or pubescent along its dorsal side (Allen and Allen, 1981). Lathyrus seeds are produced in multiples of two in pods that are oblong and compressed (Allen and Allen, 1981). The seeds are often rounded or angular, but can be flattened in some species (Allen and Allen, 1981). A handful of other general morphological characteristics is currently used to distinguish members of Lathyrus from other closely related groups such as Vicia. Many members of Lathyrus possess truncated or squared off staminal tubes, although in some plants the staminal tubes are more oblique (Kenicer et al., 2009). Species of Lathyrus show supervolute vernation in  2  their leaflets during development (leaflets are rolled together in bud) (Kenicer et al., 2009). Some Lathyrus species have leaflets with parallel veins (Kenicer et al., 2009). In reticulate-veined species, the veins do not loop back from the leaflet edges like they do in Vicia (Kenicer et al., 2009).  1.3 – Sweet Pea Flowers The sweet pea (Lathyrus odoratus) has papilionoid flowers, which means they have strong dorsiventral asymmetry. The style is twisted in the sweet pea, as in many other Lathyrus species, so bilateral symmetry is not complete (Senn, 1938). The flowers consist of three specialized petal types (Figure 1.1). A papilionoid flower contains one large standard petal, two wing petals and two keel petals. The keel petals are often fused together and encase the carpel (pistil) and stamens. Sweet pea flowers often occur in bicolour forms in which the standard petal is a different colour from the wing and keel petals. The sweet pea is native to Sicily and occurs in a purple bicolour form where the standard petal has a purple pigmentation and the wings and keel are blue (Punnett, 1923). A red and white bicolour flower similar to the cultivar, ‘Painted Lady,’ is also said to occur in the wild (Bateson, 1905).  3  Figure 1.1 – Sweet pea flowers The wild-type variety ‘Cupani’ and the hooded variety ‘Annie B Gilroy’ are shown from left to right. Specific petal types are indicated with arrows. Besides mutations in colour, some sweet pea flowers display mutations in the shape of their petals. One such floral type called hooded was examined in great detail by Punnett, Bateson and Saunders in the early 1900s. Flowers of hooded sweet peas possess several shape and colour traits that distinguish them from wild-type flowers. The standard of hooded sweet peas is cupped and hood-shaped, rather than erect and banner-like (Figure 1.1). Strongly hooded sweet peas often possess two deep, symmetrical lobes on the edges of the standard petal (Figure 1.2). The hooded standard petal often has a central notch along the mid-vein that is much less pronounced than it is in wild-type standard petals.  4  Figure 1.2 – Sweet pea standard petals Sweet pea standard petals have been flattened to illustrate the differences in shape between hooded ‘Annie B Gilroy’ and wild-type ‘Cupani’ varieties.  In order to accurately describe the differences in shape between wild-type and hooded petals I took measurements of petal length and width. For wild-type flowers I measured the length of a petal down its mid-vein and the width of a petal at its widest portion. To generate a single number that described the relationship between the two values, I divided the width by the length. Wild-type flowers have an average value of 1.17 with a standard deviation of 0.145 and a 95% confidence interval of 1.15-1.20. These values suggest that the width of wild-type petals is consistently greater than their length as measured down the mid-vein. Because strongly hooded standard petals have deep lobes, petal width was measured twice – both at the wide petal base and at the narrower portion of the petal. I calculated the value for the wider lobed portion of hooded standard petals to be 1.05 with a standard deviation of 0.082 and a 95% confidence interval of 1.02-1.08. The average value for the narrower body of the petal was 0.96 with a standard deviation of 0.152 and a 95% confidence interval of 0.89-1.01. These numbers indicate that the standard petal length is either greater than, or almost equal to, the petal width in hooded sweet peas (illustrated in Figure 1.2). The wild-type 95% confidence interval of 1.15-1.20 does  5  not overlap with either of the confidence intervals generated using hooded petal measurements. For calculations and other additional information see Appendix – Supplemental Data and Calculations.  1.4 – TCP-box Genes TCP-box genes are transcription factors that help regulate cell division and growth (Cubas et al., 1999). TCP-box genes get their name from the genes TEOSINTE BRANCHED 1 (TB1) in Maize, CYCLOIDEA (CYC) in Antirrhinum, and PROLIFERATING CELL FACTOR 1 and 2 (PCF1, PCF2) in Oryza (Cubas et al., 1999). TB1 plays a role in establishing the plant’s branching pattern as well as influencing apical dominance (Doebley et al., 1995). CYC plays a vital role in floral organ identity and establishing floral zygomorphy along the dorsoventral plane (Luo et al., 1996). The proteins PCF1 and PCF2 bind to the promoter region of PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA), a gene that functions in a number of processes that occur in dividing cells such as DNA replication and cell cycle control (Jónsson et al., 1998; Kosugi and Ohash, 1997; Warbrick, 2000). As a group, TCP-box genes share two evolutionarily conserved domains which likely function in DNA binding as well as in proteinprotein interactions: the basic-Helix-Loop-Helix structure and the arginine (R) rich ‘R domain’(Cubas et al., 1999). TCP class genes are involved in many fundamental processes in plants. TCP-genes are known to play roles in cell cycle control, plant architecture, lateral organ development and floral development (Martín-Trillo and Cubas, 2009). Expression of TCP genes is regulated both spatially and temporally and thus they are able to influence growth and development at a fine scale (Cubas, 2002). In Antirrhinum, CYC may either reduce or increase the rate of growth 6  depending on the location or timing of expression during development (Luo et al., 1996; Preston and Hileman, 2009). For instance, in early flower development growth is reduced in order to establish proper organ number whereas in late development expression increases growth in petals (Luo et al., 1996). Interestingly, late expression in the staminal whorl leads to the abortion of stamen in Antirrhinum and in other related species (Hileman et al., 2003; Luo et al., 1996).  1.5 – CYCLOIDEA and Flower Development In Antirrhinum, CYCLOIDEA (CYC) plays a large role in flower development, particularly in the establishment of floral organ identity and flower symmetry (Corley et al., 2005; Luo et al., 1996). Antirrhinum flowers have strong bilateral symmetry and are made up of two large dorsal petals, two lateral petals and one ventral petal. CYC interacts with another closely related TCP-box gene called DICHOTOMA (DICH) as well as two MYB-class genes DIVARICATA (DIV) and RADIALIS (RAD) (Corley et al., 2005). CYC and DICH control adaxial (dorsal) petal identity and development and their mutants show at least a partial reversion to an actinomorphic flower morphology (Luo et al., 1996). CYC expression has also been tied to the abortion of stamen development in Antirrhinum (Luo et al., 1996). The expression of CYC and DICH in the adaxial petal leads to the expression of RAD which in turn inhibits the expression of DIV (Corley et al., 2005). RAD expression contributes to lateral petal identity and the expression of DIV contributes to abaxial (ventral) petal identity (Corley et al., 2005). CYCLOIDEA-like genes have also been shown to play a large role in the control of floral zygomorphy in plants other than the snapdragon. Arabidopsis, Lotus japonicus and Gerbera hybrida are just a few species that possess CYCLOIDEA-like TCP genes (Broholm et al., 2008; Cubas et al., 1999; Feng et al., 2006). In Lotus japonicus, five CYCLOIDEA-like homologs, three 7  of which are orthologous to CYC and DICH in Antirrhinum, have been isolated, indicating that CYC-like genes have undergone a duplication event at some point in this lineage (Citerne et al., 2003; Feng et al., 2006). The CYC homolog LjCYC2 was found to control adaxial petal development and petal identity (Feng et al., 2006). Over expression of LjCYC2 caused the papilionoid Lotus flowers to become much more radial in character and caused the wing and keel petals to gain standard-like characters (Feng et al., 2006). The squared standard mutant of Lotus is caused by a mutation in LjCYC2 and results in an oddly shaped standard that shares many wing petal characters (Feng et al., 2006). Mutations similar to the squared standard mutant of Lotus can be found in both Pisum sativum (pea) and Lathyrus odoratus (sweet pea). The mutants of pea and sweet pea are named lobed standard 1 and hooded respectively.  8  2 – RT-PCR Analysis of CYCLOIDEA in Lathyrus odoratus 2.1 – Aim Reverse Transcriptase PCR reactions (RT-PCR) were conducted to determine the petal specific expression patterns of CYCLOIDEA (CYC) homologs in hooded and wild-type sweet peas. This was done in order to see if a change in expression of a CYC homolog in Lathyrus may be responsible for causing the hooded floral phenotype.  2.2 – Introduction 2.2.1 – The hooded sweet pea Flowers of hooded sweet peas possess several shape and colour traits that distinguish them from wild-type flowers. The standard of hooded sweet peas is cupped and hood-shaped, rather than erect and banner-like. The hooded standard petal is more narrow than the wild-type standard petal, and is often more comparable in shape to wing petals because it is longer than it is wide. Strongly hooded sweet peas often possess two deep, symmetrical lobes on the edges of the standard petal. Wild-type standard petals are not lobed. Wild-type sweet peas often display a bicolour phenotype wherein the standard petal displays a contrasting colour when compared to the wing and keel petals. Wild-type sweet pea cultivars such as ‘Cupani’, ‘Matucana’ and ‘Wild Italian’ have a purplish standard petal and darker blue wing and keel petals. Significantly, a bicolour hooded sweet pea has never been described, and this will be discussed in a later section.  9  2.2.2 – CYCLOIDEA genes The purpose of these Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) experiments is to determine if hooded sweet peas show differential expression of a gene in the CYCLOIDEA (CYC) gene family when compared to wild-type sweet peas. CYC and CYC-like genes are TCP-box genes which function primarily in floral development and floral organ identity in a variety of plant groups, including legumes (Citerne et al., 2003; Cubas et al., 1999; Luo et al., 1996). CYC and CYC-like genes which function in floral symmetry possess two evolutionarily conserved regions: an Arginine-rich R domain and a TCP box domain which is present in all members of the TCP group (Cubas et al., 1999). CYC-like genes are likely candidate genes for causing the hooded phenotype for several reasons: 1) Legume CYC2 (or CYC1B) controls dorsal standard petal identity in papilionoid flowers such as Lotus, Lupinus and Pisum and the hooded floral phenotype shows a visible change in the standard petal (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). 2) The lack of a bicolour hooded sweet pea indicates that the standard petal may have undergone a shift in petal identity and is no longer distinct from the wing petals. As legume CYC2 functions in establishing dorsal petal identity, it is a likely candidate gene (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). 3) Legume CYC2 expression is limited to the standard petal during flower development in papilionoid legumes (Feng et al., 2006). In the CYC2 loss of function mutants squared standard 1(squ1) in Lotus and lobed standard 1(lst1) in Pisum, the standard petal is the only petal that displays a visible phenotype (Feng et al., 2006; Wang et al., 2008a). This is important as the only trait that sets hooded sweet peas apart from the wild-type is a change in the shape of the standard petal. 4) The hooded phenotype looks very similar to the squared standard 1(squ1) and lobed standard 1(lst1) mutants in Lotus and Pisum, which are  10  caused by changes in CYC2 expression and influence the shape and curvature of the standard petal (Feng et al., 2006; Wang et al., 2008a).  2.2.3 – Similar mutants in other legumes The squared standard 1 (squ1) mutant of Lotus is caused by a reduction in LjCYC2 expression and results in an oddly shaped standard petal that shares wing petal characters such as epidermal cell shape (Feng et al., 2006). The hooded standard phenotype of the sweet pea is very similar to the squared standard both in size, when compared to wing and keel petals, and in overall shape. The standard petal of squ1 mutants also displays lobed edges much like those present in some hooded varieties. A second mutant similar in appearance to the hooded phenotype is the lobed standard 1 (lst1) mutant in the pea (Pisum sativum). This mutant, much like the squ1 mutant in Lotus, possesses deep lobes on the edges of the standard petal and reduced standard size in comparison to the wings and keel (Wang et al., 2008a). Lst1 lines exhibit variable lobing in their petals much like what is seen in hooded sweet peas (Ambrose, 2003). This mutant was also found to be caused by a mutation in a CYC2 homolog.  2.2.4 – The hooded mutation only affects the standard petal The hooded mutation only has a visible effect on the standard petal. CYC-like genes, specifically CYC2 homologs, are known to have a very strong effect on standard petal development (Feng et al., 2006; Wang et al., 2008a). While other CYC-like genes, for example CYC1 in Lotus, have been found to be expressed in the adaxial petal of the sweet pea, CYC2 is  11  the only CYCLOIDEA-like gene that is expressed exclusively in the standard petal during floral development (Feng et al., 2006).  2.3 – Methods 2.3.1 – Tissue sampling and RNA extraction Sweet pea floral tissue for RNA extraction was collected from developing, unopened buds. Unlike some other genes that function in floral development, CYCLOIDEA is expressed in both early and late stages of flower bud development, and relatively late-stage buds were used for these experiments. The buds collected were approximately one to one-and-a-half centimetres in length and all floral organs had been formed, but pollen had not yet been released. Sweet pea buds were dissected and separated into standard, wing and keel petal types prior to immersion in liquid nitrogen. Tissue samples were stored at -80°C to prevent RNA degradation. Wild-type sweet pea cultivars ‘Cupani’, ‘America’ and hooded cultivars ‘Annie B Gilroy’, ‘Miss Ellen Willmott’ and ‘Lady’s Bonnet’ were used. For each variety, one pooled tissue sample containing 3-5 petals was taken for each petal type. I extracted RNA from the tissue using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, USA), following manufacturer’s protocol. I then purified the extracted RNA by removing the residual DNA contamination using the TURBO DNA-freeTM kit produced by Ambion (Carlsbad, USA). The purified RNA was then used to create cDNA using the RevertAid H minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, CA), following manufacturer’s instructions.  12  2.3.2 – RT-PCR Reverse Transcriptase Polymerase Chain Reactions (RT-PCRs) were carried out using floral cDNA and primers designed to amplify CYCLOIDEA sequences in the closely related species Pisum sativum. Primers specific to Pisum CYC2 (SL0868/SL0970) and CYC1 (SL0932/SL0933) published in Wang et al. (2008) were used to examine expression patterns of homologs in Lathyrus odoratus. General Histone 4 primers SL1815/SL0363 were used as a control (Wang et al., 2008a). To test for genomic contamination in Lathyrus cDNA samples, RTPCR reactions using RNA and Histone 4 primers were conducted. If genomic DNA was present in the RNA sample it would be amplified and subsequently visualized on a 1% agarose gel. If the RNA tested positive for DNA contamination, an additional DNase treatment was performed. Reactions were carried out in an Eppendorf Mastercycler Gradient thermocycler with the following conditions: 94°C for 3 min for initial denaturation, and then 34 cycles of 1 minute at 94°C for denaturation, 1 minute at 50°C for annealing, 90 seconds at 72°C for extension. Once the reaction program was complete, the samples were held at room temperature until they could be placed into a -20°C freezer. 2.4 – Results  Figure 2.1 – Expression of CYC-like homologs in Lathyrus odoratus Gel photos were taken to visualize the expression patterns of genes in individual petals of hooded and wild-type sweet peas. Standard petals, wing petals and keel petals have been abbreviated to S, W, and K respectively. Lathyrus homologs of CYC1, and CYC2 were examined and Histone4 was used as a positive control. One pooled tissue sample was used for each variety and each experiment was replicated between 3 and 5 times, with identical results. 13  Sweet Pea Variety  Wild-type or hooded  Cupani America Annie B Gilroy Lady’s Bonnet Miss Ellen Willmott  Wild-type Wild-type hooded hooded hooded  LoCYC 2 Amplification in Standard Petal Yes Yes No No No  Table 2.2 – Wild-type and hooded varieties of Lathyrus odoratus Five varieties of sweet pea were used in the RT-PCR analysis, three hooded and two wild-type. There was an association between the lack of LoCYC2 amplification and the hooded floral phenotype. One pooled tissue sample was used for each variety and each experiment was replicated between 3 and 5 times, with identical results. In the sweet pea, LoCYC1 is strongly expressed in the standard petals of both hooded and non-hooded varieties. In addition to being expressed in the standard petal, LoCYC1 is also weakly expressed in wing petals. LoCYC2 is strongly expressed in the standard petal of wild-type sweet peas, but is absent in wing and keel petals. In hooded sweet peas LoCYC2 is not amplified in any petal type.  2.5 – Discussion 2.5.1 – A mutation affecting LoCYC2 is implicated in the hooded phenotype The absence of LoCYC2 amplification in the standard petals of Lathyrus is associated with the hooded mutation, which supports the hypothesis that the abnormal floral form is caused by a mutation in LoCYC2. The squ1 mutant in Lotus is caused by a point mutation near the 3’ end of a very short intron that causes the RNA to be incorrectly spliced (Feng et al., 2006). In Wang et al.’s 2008 study, lst mutants were also found to carry mutations in CYC2. It is possible that a similar mutation has occurred in the sweet pea homolog of CYC2 to cause the hooded floral phenotype, although further testing is required to determine the nature of the mutation and 14  whether it is necessary and sufficient to cause the hooded floral phenotype. The expression of the other CYC homolog LoCYC1 did not change across petal types, which suggests that although LoCYC1 and LoCYC2 are closely related genes, LoCYC1 is no longer a candidate for explaining this phenotype. Although the lack of amplification of LoCYC2 in the standard petals of hooded sweet peas suggests the gene may play a role in establishing the phenotype, it is not necessarily the cause of the floral change. It is possible that another regulatory gene is influencing the expression of CYC2 and is directly responsible for the formation of a hooded phenotype. In order to further test whether or not LoCYC2 is the cause of the hooded phenotype a F2 segregation experiment was carried out (Chapter 4).  2.5.2 – Possible roles of LoCYC genes in petal identity Although LoCYC2 is not amplified in the dorsal petal of hooded sweet pea mutants, the standard petal remains identifiable even though it has undergone a distinct change in shape. This suggests that although legume CYC2 has been shown to function in establishing dorsal petal identity, its lack of expression may not be sufficient to completely abolish the presence of a recognizable standard petal. It has been suggested that CYC1 and CYC2 may have a partial redundancy of function (Citerne et al., 2003). Even though there may be a partial redundancy of function, the RT-PCR results suggest that LoCYC2 expression may be necessary for normal standard petal development in sweet peas. The distinct changes in shape that are present in hooded mutants such as the formation of lobed edges and the curving of the once erect petal are likely due to the absence of LoCYC2 expression which has caused the petal to become almost wing-like. 15  Although CYC3 has been shown to be a lateralizing factor in both Pisum and Lotus (Feng et al., 2006; Wang et al., 2008a), it is unlikely that it is the cause of the hooded floral phenotype. Mutants of CYC3 in Lotus and Pisum called keeled wings in Lotus 1 (kew1) and keeled wings (k) have phenotypes where lateral petal identity is lost and the wing petals become keel-like (Feng et al., 2006; Wang et al., 2008a). In Pisum, PsCYC3 is expressed in both the standard and wing petals of wild-type flowers (Wang et al., 2008a). While it is unlikely that a mutation in LoCYC3 is the cause of the hooded phenotype, in the absence of LoCYC2 the gene may cause the standard petal to take on wing petal characters that would not normally be seen, so LoCYC3 may be involved in the development of the phenotype. Unfortunately due to time constraints, the expression pattern of LoCYC3 was not examined in this study. The expression pattern of LoCYC3 would be an interesting subject for future work. Legume homologs of CYC2-like genes have been shown to contribute to dorsalized as well as lateralized floral forms. In Cadia, an unusual member of the Papilionoideae, flowers have become dorsalized and actinomorphic (Citerne et al., 2006). While CYC2-like homologs are expressed in the standard petals of Lotus and Pisum, the gene (referred to as CYC1B in Citerne et al.’s 2006 paper) is strongly expressed in every petal. The actinomorphic flowers observed in Cadia can be compared to the GFP tagged LjCYC2 line SH0578 that displays LjCYC2 expression in every petal and has a completely dorsalized phenotype (Feng et al., 2006). The overexpression of legume CYC2 leads to expansion of dorsal petal identity, whereas mutations in the gene lead to flowers where dorsal identity is incomplete or has been lost (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). This suggests that LegCYC2 is an identity gene that is vital for standard petal development and floral symmetry.  16  2.5.3 – The hooded mutation is homeotic in nature Homeosis is defined as the change of one organ into another based on an alteration of developmental fate (Cronk et al., 2002). In Antirrhinum, CYC is an identity gene that is directly involved in establishing the identity of floral organs by sending developmental signals that help to establish the overall architecture of a flower (Corley et al., 2005; Luo et al., 1996). In cyc/dich double mutants, the identity of the dorsal petal is lost because the identity genes that signal the organ’s developmental fate are no longer expressed (Corley et al., 2005; Luo et al., 1996). The cyc/dich double-mutant is homeotic in nature because the organ in the dorsal position now expresses genes that establish ventral petal identity (Corley et al., 2005). In legumes, CYC-like genes also play a role in establishing petal identity and flower symmetry (Citerne et al., 2006). In Cadia, the expression of CYC2 (referred to as CYC1B) in the wing and keel positions has been suggested to have transformed the other organs and to have conferred dorsal petal identity to all the petals in the actinomorphic flower (Citerne et al., 2006). The loss of function mutants squ1 in Lotus and lst1 in Pisum are also homeotic mutations, as the dorsal standard petal of both mutants has taken on wing petal characteristics (Feng et al., 2006; Wang et al., 2008a). The hooded sweet pea has never been found to occur in a bicolour form. Wild-type sweet peas are bicolour by default, which indicates that the hooded mutant is unique in some fashion. In other sweet pea lines, bicolouration has been lost through changes in gene pathways that control pigmentation. The lack of a bicoloured hooded sweet pea could therefore be due to close linkage between the hooded gene and a gene controlling the bicolour trait, as Bateson thought (Bateson, 1913). However, in over one-hundred years of breeding and cultivation, even the most closely linked genes would have likely undergone a recombination event, but a bicoloured  17  hooded sweet pea has never been described. The conspicuous lack of a bicolour sweet pea indicates that the hooded mutation may be homeotic in nature. If the standard petal has taken on a wing petal identity by default (having lost the gene specifying standard petal identity) a bicolour sweet pea would never been seen because the same genes controlling pigmentation would be activated in both petals.  18  3 – Analysis of CYCLOIDEA Sequence in Lathyrus odoratus 3.1 – Aim To determine the cause of the differential amplification patterns that were observed in the RT-PCR analysis (Chapter 2), gene sequence from wild-type and hooded sweet peas was examined. The change in RT-PCR amplification suggests that there is a difference between wildtype and hooded alleles of the CYCLOIDEA2 homolog in Lathyrus. Failure of RT-PCR primers to amplify cDNA of CYC2 suggests that either the sequence that the specific primers bind to has been altered, that the gene is no longer expressed, or both. Changes in genetic sequence such as deletions, insertions, or single nucleotide polymorphisms may be responsible for changes in expression or RT-PCR amplification patterns.  3.2 – Methods 3.2.1 – Isolating CYCLOIDEA sequence from wild-type and hooded sweet peas I acquired partial sequence of LoCYC2 through direct sequencing using published degenerate LEGCYC/F1 and LEGCYC/R1 primers (Citerne et al., 2003). These primers bound to highly conserved regions of TCP box genes, and amplified fragments of the Lathyrus CYC homologs. All PCR reactions were carried out in an Eppendorf Mastercycler Gradient thermocycler. The LEGCYC/F1 and LEGCYC/R1 primers were used to amplify CYC sequence using the conditions outlined in Citerne et al. 2003. Because CYCLOIDEA-like homologs in the Fabaceae have undergone duplication events in the past (Citerne et al., 2003; Fukuda et al., 2003), when I used the degenerate LEGCYC/F1  19  and LEGCYC/R1 primers, fragments from a number of Lathyrus CYC-like genes were amplified. In order to extract individual sequences from the mixture of amplified DNA fragments I used a TOPO® TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, USA) following the protocol provided by the manufacturer. I also used the Pisum specific primers SL0868/SL0970, LjSSR1084/LjSSR1085, and SL0773/SL0970 (Wang et al., 2008b, Supporting Information) to amplify sequence from LoCYC2. Reactions were carried out in an Eppendorf Mastercycler Gradient thermocycler at the following conditions: 94°C for 3 min for initial denaturation, and then 34 cycles of 1 minute at 94°C for denaturation, 1 minute at 55°C for annealing, 90 seconds at 72°C for extension. Once the reaction program was complete, the samples were held at room temperature until they could be placed into a -20°C freezer. Using these techniques I was able to sequence small fragments of LoCYC1, LoCYC2 and LoCYC3. Once I had partial sequence from LoCYC2, I was able to use the genome walking technique to extend the known sequence.  3.2.2 – Genome walking To extend the length of known sequence, the GenomeWalker Universal Kit (Clontech, Mountain View, CA) was used. This kit requires the construction of a DNA library as well as nested PCR and cloning to isolate the successfully amplified gene fragments. Using a DNA library I constructed from wild-type (‘Cupani’) DNA, I sequenced the full LoCYC2 homolog as well as approximately 300 base pairs of the promoter region. I also sequenced over 1000 base pairs of LoCYC2 sequence from the hooded variety ‘Annie B Gilroy’ using this method. Figure 3.1 shows the sequences of the Lathyrus odoratus specific genome walking primers I used. 20  Primer Name LoCYC2Walk1L LoCYC2Walk2L LoCYC2Walk3L LoCYC2Walk4L Hooded1L Hooded2L Hooded3L Hooded4L Hooded5L  Primer Sequence CAAGAGAAAAAGCAAGAGCAAGAGCAA CAAAAGCTTCACATCTTCTTCGGATTG TCCCTAAGGAAGCAACAAGTTTCAACA TCGGATTGTGAAGATTCAGAAGTTGCT TCCATTCCTTACATTCCAACTCACCATC CACCATCATAGTTCTCATCCTCATCATCA GCCGAAACAAGATCCTTTAACTCTTGGT CTCTTGGTGGTGGTTCTCACTATGGAA GCTTGAGAGATCGTAGGGTTAGGCTTTC  Table 3.1 – List of genome walking primers This table gives the sequence for all LoCYC2 specific primers used with the GenomeWalker Universal Kit (Clontech, Mountain View, CA). All primers were designed based on Lathyrus sequence using the computer program Primer 3 (Rozen and Skaletsky, 2000).  3.3 – Results 3.3.1 – Wild-type sequence CYC2 sequence from wild-type sweet peas showed high homology to the sequence found in the closely related species Pisum sativum and differed only in a handful of locations (Wang et al., 2008a). On closer examination of the protein sequence, these changes appear to be homopolymer repeats (Figure 3.2). The TCP-box domain of LoCYC2 occurs at the 380th base pair and extends to the 521st base pair. The R domain begins at the 758th base pair and extends to the 803rd base pair. The open reading frame of LoCYC2 is 1274 base pairs in length.  21  Figure 3.2 – Gene map of Lathyrus odoratus CYCLOIDEA2 This figure illustrates the open reading frame of the Lathyrus odoratus homolog of CYC2. Bold arrows indicate the locations of homopolymer repeats that distinguish the sequence from that found in Pisum sativum. Important functional domains such as the TCP-box domain and the R domain are also labeled.  3.3.2 – Mutant CYC2 sequence from hooded plants The sequence that was generated from genome walking using ‘Annie B Gilroy’ DNA shows some significant differences when compared to wild-type LoCYC2 sequence. The first portion of the 5’ sequence is identical to that found in the wild-type, however the sequences diverge after the 417th base pair (Figures 3.3, 3.4). Certain CYC2GENE primers designed specifically to bind to portions of the Lathyrus odoratus homolog of CYC2 showed differential PCR amplification patterns that were used to identity mutant sequence in the F2 segregation (Chapter 4). CYC2GENE11/12 primers which amplify a region of DNA between the TCP box and the R domain (Figure 4.4) amplified sequence in both hooded and wild-type plants which suggests that the change in DNA sequence observed in hooded sweet peas is caused by an 22  insertion, rather than a deletion. In an attempt to sequence the entirety of the insertion, over 900 nucleotides were sequenced following the change, however, the 3’ portion of the LoCYC2 gene, including the functionally relevant R domain, was not located.  3.4 - Discussion The change in LoCYC2 sequence that is observed in hooded sweet peas is undoubtedly the cause of the differential amplification patterns that were observed in the RT-PCR analysis (Chapter 2). The change in sequence disrupts the TCP-box domain and would likely render LoCYC2 non-functional. It is possible that because of this change in sequence, the hooded allele of LoCYC2 has been silenced. If the hooded allele of LoCYC2 is actually transcribed and translated into protein, it would produce a prematurely-truncated product because the aberrant sequence is littered with stop codons, the first of which occurs only 63 base pairs after the change in sequence (Figure 3.3). Because the hooded allele of LoCYC2 alters the functional domains of the protein, it is unlikely that the gene could retain its activity as a transcription factor. CYC-like homologs in papilionoid legumes have been shown to function in the determination of petal identity, and are especially important in delineating the identity of the standard petal (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). The changes in gene sequence observed in the hooded allele of LoCYC2 support the results of the RT-PCR analysis and further strengthen the hypothesis that the hooded mutant of sweet pea is a homeotic mutation caused by a change in a CYC2-like gene.  23  Figure 3.3 – Lathyrus odoratus CYC2 alleles This figure shows gene maps of the wild-type and hooded alleles of CYC2. Important functional domains such as the TCP-box domain and the R domain are also labeled  >Lathyrus_odoratusCYC2 MFPYSSNPYPSFLPSSSSSSSSLFPFPFLNPENASSNSNNNTLFRDPFSIPYIPTHHHSSHPHHHLQNISNIPETLA NLSVSQENHNNSNNNINNIAVPMPKQDPLTLGGGSHYGISCFLTKKPAKKDRHSKIYTSQGRDRRVRLSIEI ARKFFDLQEMLGFDKASNTLDWLFTKSKKAIKDLTKSKQRDNNSEVGGDAKSFTSSSDCEDSEVATNDSL NLQKEGAGSKTEDKKLKRVQIKEPACVRASKMKESREKARARARERTSNKISNNNIRGVQEVELKKKYEN SNNNNNNNLQAFHQSRSSSQDHQIVSSNEAQRDNFNVIEESIVIKRKLKPSLMMHHHHHHQQNHVIPKEAT SFNSNGDYNFLPNSSPNWDANNGSSSTNRSTFCAIASMNLSTGLQIFGKSWEECTNPRLH >Hooded Lathyrus_odoratusCYC2 MFPYSSNPYPSFLPSSSSSSSSLFPFPFLNPENASSNSNNNTLFRDPFSIPYIPTHHHSSHPHHHLQNISNIPETLA NLSVSQENHNNSNNNINNIAVPMPKQDPLTLGGGSHYGISCFLTKKPAKKDRHSKIYTSQGCLNHSYKLHLI LGYKLMANQF-KMKIILYTISSSHPKLSLNFF-FI-PNYTNMYKTKSNMPSSLMNFPPLAQTIKAYIDNNHSSP LTPPPPIQILLSKPKDFFRQRRGHVSNILYDENKKHIPAHED-ILLL-LPNSI-SEVYPI-VFNQKVSSLSVNGSTC ANNEYNSSN-SFTNRVFNVPKFHCQFLSSHMPELLTVLYL-EEAAYIYQNLVCKSDHSNQKACQNETVALSP SLNTNCFINPSSSFNSTMRIISLHHNQTRSYVYATSATSQSFKCIGPPKIKEIYSVRFI-YNIIRFCMI  Figure 3.4 – FASTA protein sequence of the wild-type and hooded alleles of LoCYC2 The protein sequences of CYC2 homologs in wild-type and hooded sweet peas. The yellow text indicates conserved sequence. The red dashes indicate stop codons.  24  4 – F2 Segregation 4.1 – Aim The results of the sequence analysis (Chapter 3) show that there is an association between the hooded phenotype and a change in sequence in the LoCYC2 gene. Although it is likely that the change in the DNA sequence is responsible for the hooded floral phenotype, it is possible that the true cause of the mutant is another gene and the change in sequence observed in LoCYC2 in hooded plants is incidental and does not directly influence the phenotype. A F2 cosegregation analysis was therefore used to expand the sample size, and determine if the hooded phenotype was consistently inherited with the mutant allele of LoCYC2. This analysis will confirm or refute the hypothesis that hooded is caused by a change in LoCYC2.  4.2 – Introduction 4.21 – Phenotypes in crossing experiments The pink hooded variety ‘Annie B Gilroy’ was initially crossed with the wild-type blue and purple bicolour variety ‘Cupani’. These varieties were chosen because their phenotypes were well characterized by Punnett in the early 1900s. ‘Annie B Gilroy’ differs from wild-type ‘Cupani’ in a number of ways: 1) ‘Annie B Gilroy’ carries a recessive allele of ‘B’, a ‘purple factor’ that is normally responsible for the purple-blue colour that is present in the wing petals of wild-type sweet peas (Punnett, 1919). ‘B’ is known to be closely linked to ‘E’, the ‘Erect standard’ locus that is responsible for hooded (Punnett, 1919). 2) ‘Annie B Gilroy’ carries a recessive ‘E’ allele, responsible for the hooded character of the standard petal (Punnett, 1919). 3) ‘Annie B Gilroy’ carries a recessive allele of ‘L’, a ‘light wing factor’ that is responsible for  25  reducing the red colour present in wing petals (Punnett, 1919). In ‘Annie B Gilroy’, the recessive ‘l’ causes the wing petals to be more intensely red (Punnett, 1919).  4.2.2 – Predicted phenotypes Self pollination of the F1 plants allowed predictions to be made about segregation patterns. The homozygous wild-type ‘Cupani’ sweet pea has a genotype of BBEE / LL, where B and E are tightly linked, and ‘Annie B Gilroy’ has a genotype of bbee / ll. The F1 generation had a genotype of BbEe / Ll. Punnett’s work in the early 1900s states that these loci have simple Mendelian inheritance patterns. Following self-fertilization, this F2 segregation ratio is expected: 9 ‘Cupani’-type phenotype (B-E- / L-), 3 ‘Deep Purple’ phenotype (B-E- / ll), 3 ‘Hooded Painted Lady’ phenotype (bbee / L-), 1 ‘Annie B Gilroy’ phenotype (bbee / ll) (Figure 4.1). Because ‘B’ and ‘E’ are distinct, but tightly linked loci, some genetic recombination is likely to occur, however it will be rare. The hypothesized ratios were tested against the collected data using Chisquared Goodness of Fit test (χ2).  Gametes BE / L BE / l be / L  be / l  BE / L Cupani BBEE / LL Cupani BBEE / Ll Cupani BbEe / LL  BE / l Cupani BBEE / Ll Deep Purple BBEE / ll Cupani BbEe / Ll  Cupani BbEe / Ll  Deep Purple BbEe / ll  be / L Cupani BbEe / LL Cupani BbEe / Ll Hooded Painted Lady bbee / LL Hooded Painted Lady bbee / Ll  be / l Cupani BbEe / Ll Deep Purple BbEe / ll Hooded Painted Lady bbee / Ll Annie B Gilroy (hooded) bbee / ll  Table 4.1 – Expected F2 phenotypes This Punnett square diagram illustrates the phenotypes that are predicted to arise from the selfpollination of the heterozygous F1 sweet peas. The hooded phenotypes are indicated in bold. Figure 4.6 on page 31 illustrates the appearance of each predicted flower variety. 26  ‘Painted Lady’ is an old name for a bicolour sweet pea that has a red standard petal and very light pink wing and keel petals (it represents a wild-type sweet pea lacking Punnett’s B (blue) factor and is therefore red and pale-pink rather than purple and blue) (Punnett, 1919). If hooded is in fact a homeotic mutation where the standard petal has taken on wing petal identity, the Painted Lady phenotype will no longer have a red standard petal. The flower would no longer have a petal that possesses standard petal identity. If the standard petal and wing petals in a hooded flower share the same petal identity, the entire flower of the ‘Hooded Painted Lady’ phenotype will be light pink. A bicolour sweet pea cannot exist if the dorsal standard petal no longer has a distinct identity apart from the wing petals. 4.3 – Methods 4.3.1 – Crossing In the wild, native species of Lathyrus are legitimately pollinated by leaf cutter bees which are strong enough to work the large, papilionoid flower (Westerkamp, 1993). Although Lathyrus odoratus is capable of producing seed either by outcrossing or by selfing (Brahim et al., 2001) legitimate pollination by insect visitation has not been observed in the field (Figure 4.2). In order to ensure the F1 seeds were not the result of accidental selfing, the recessive hooded ‘Annie B Gilroy´ variety was chosen as the maternal plant. Selfed plants will then be recognized as having the maternal phenotype rather than the expected paternal phenotype. No accidental selfs were observed in the F1.  27  Figure 4.2 – Insect visitation The bumblebee (Bombus sp.) in this photograph is stealing nectar from the sweet pea flower without pollinating it. Bumblebees and honeybees do not appear to be able to extract pollen and cross-pollinate the flowers. The F1 plants were grown and allowed to self-pollinate to produce the F2 generation. All F1 plants displayed a wild-type floral phenotype rather than the recessive ‘Annie B Gilroy’ hooded phenotype ensuring that the F1 plants were the result of cross fertilization rather than accidental selfing. Figure 4.3 illustrates the expected ratios of phenotypes in the F2 generation. F2 seeds were planted in vermiculite and placed in a greenhouse seed germination bed to sprout. Following germination the seedlings were transplanted into pots filled with potting soil. In order to ensure that a large number of F2 plants survived to flowering, approximately half of the population was planted outside in a plot in Totem Field. The other half of the F2 population was retained in the Horticulture greenhouse on UBC campus. The F2 plants in the greenhouse were watered daily, however many died due to the unusually warm weather that was experienced in the summer of 2009. The F2 plants in Totem Field were watered every three to five days. A total of 223 F2 tissue samples were collected from plants grown both outside and under glass.  28  Figure 4.3 – F2 segregation Phenotypes expected from the F2 segregation experiment. The F2 phenotypes shown from left to right are Cupani, Dark Cupani, Hooded Painted Lady and Annie B Gilroy.  4.3.2 – Determination of alleles In order to determine if the hooded floral phenotype is inherited with the mutant allele of LoCYC2, eighteen wild-type-specific and eight mutant-specific PCR primers (Figures 4.4, 4.5) were designed using the computer program Primer 3 (Rozen and Skaletsky, 2000). Leaf tissue samples were collected from F2 plants and stored briefly in silica gel before genomic DNA was extracted. Due to the large F2 sample size, the dried tissue was pulverized using a drill apparatus that could be fitted with sterile acrylic grinding bits. The use of the drill greatly decreased the time required to grind the tissue samples during DNA extraction. Genomic DNA was extracted  29  from the dried leaf tissue using the Plant DNAzol Reagent protocol provided by the manufacturer (Invitrogen, Carlsbad, USA).  Figure 4.4 – CYC2GENE primers This gene map of the wild-type allele of LoCYC2 shows the array of forward and reverse CYC2GENE primers that were designed for the F2 analysis. The arrows denote forward or reverse directionality. The bold numbers indicate the primer name and the numbers below indicate the specific base pair the primer binds to. For primer sequences, see Appendix.  Figure 4.5 – LoHoodedGene primers This gene map of the hooded allele of LoCYC2 shows the array of forward and reverse LoHoodedGene primers that were designed for the F2 analysis. The arrows denote forward or reverse directionality. The bold numbers indicate the primer name and the numbers below indicate the specific base pair the primer binds to. For primer sequences, see Appendix. The concentration of the newly extracted genomic DNA was analyzed using a Nanodrop Spectrophotometer. PCR analyses using ITS primers were then conducted to ensure that the  30  DNA samples were capable of undergoing PCR amplification. Primer tests using the wild-typespecific primers were conducted using DNA from ‘Cupani’ and ‘Annie B Gilroy’ varieties. Differential amplification between wild-type and hooded varieties occurred in a number of the primer pairs. Using one such set of primers, CYC2GENE4/9, I analyzed DNA samples from the F2 population. The CYC2GENE4/9 set of primers amplifies a DNA fragment approximately 100bp in length in wild-type sweet peas but does not amplify strongly in plants with a hooded phenotype. This is expected as the CYC2GENE4 primer binds to the later portion of the TCPbox domain which has been disrupted in the mutant allele of LoCYC2. Mutant-specific primers were then used to verify the results. This was done to ensure that the lack of PCR amplification seen when using wild-type primers was caused by a change in DNA sequence that influenced the primer binding site. Two sets of primers, LoHoodedGene4L/4R and LoHoodedGene3L/2R, were used to amplify sequence around the 417th base pair of LoCYC2 to ensure that the change in sequence that was observed during genome walking was correct.  4.4 – Results 4.4.1 – Observed phenotypes All phenotypes that were expected were observed, in addition to two individuals that I have classified as Burgundy Hooded sweet peas which displayed phenotypes that were likely a result of recombination between the linked ‘B’ and ‘E’ loci. Other unusual phenotypes such as Hooded Violet, Red Flake, Hooded Purple Flake, Hooded Pink Flake and Coral Cerise-bicolour varieties were also seen and may be virus-induced phenotypes (in the case of the flakes), or the result of the segregation of loci that were not predicted. The term ‘flake’ applies to the breakage of colour in a sweet pea flower leading to a striped phenotype where the darker pigment is 31  streaked across a white background. In this case, the flake phenotypes may or may not be genetic in origin. Common viruses that are spread by aphid vectors such as the Bean Yellow Mosaic and Common Pea Mosaic Viruses may cause flaking or colour breakage in sweet pea flowers (Hull, 1965). Aphids were common in the field of F2 plants and it is quite possible that the observed flaking phenotypes were viral in origin, although this was not tested. The Hooded Painted Lady sweet pea did indeed possess light pink standard petals which lends support to the hypothesis that the dorsal standard petal has taken on lateral wing petal identity. Each F2 individual was identified upon flowering. Figure 4.6 shows the four major phenotypes in the F2 segregation.  Figure 4.6 – Sweet pea phenotypes The numbers in bold indicate the expected ratios based on simple Mendelian inheritance. The numbers in parentheses indicate the observed number of individuals in the initial population of 223. Due to lab-wide contamination issues only 118 plants were used in the final F2 analysis.  32  4.4.2 – Inheritance of the mutant allele of LoCYC2 Of the initial F2 population of 223 plants, only 118 individuals were able to be correctly identified and genotyped due to lab-wide DNA contamination issues. The first 118 individuals that were tested using the PCR-based method were successfully genotyped prior to the beginning of the contamination problem. Unfortunately the other samples were contaminated with DNA that was shed from communal-use pipettes and could not be analyzed with confidence. The CYC2GENE4/9 primers successfully amplified LoCYC2 sequence from wild-type individuals, and did not amplify sequence from plants exhibiting the hooded phenotype (Figure 4.7). Mutant-specific primers LoHoodedGene4L/4R and LoHoodedGene3L/2R successfully amplified DNA from hooded individuals and provided additional support for the genome walking results discussed in Chapter 2 and show that the sequence of LoCYC2 diverges from that of the wild-type around the 417th base pair (Figure 4.8). All but three plants were double-checked in this fashion: one plant was not tested with LoHoodedGene4L/4R and LoHoodedGene3L/2R primers due to an oversight on my part and two plants failed to show DNA amplification with all three sets of primers. Sweet Pea Variety  Cupani  Dark Cupani  Wild-type  Wild-type  Hooded Painted Lady hooded  Annie B Gilroy hooded  CYC2GENE4/9 primers Figure 4.7 – CYC2GENE4/9 amplification These primers show differential amplification between wild-type and hooded varieties. The DNA fragment amplified in wild-type plants is approximately 100bp long. The grey smudge seen in the Annie B Gilroy sample is approximately 30bp, and therefore is likely caused by the formation of primer-dimers rather than true amplification of the target sequence.  33  > LoHoodedGene4L/4R_Primers_Row2-5_Annie_B_Gilroy TCACTATGGAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTACAC TTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAAATTAATGGCTAACCA ATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTCACATCCGAAATTGTCATTAAATTTT > LoHoodedGene3L/2R _Primers_Row1-13_Burgundy_Hooded TATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTACACTTC TCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAAATTAATGGCTAACCAATT CTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTG ATTCATATGACCCAACTACACCAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTT CCCACCACTTGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCC ACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTTTCAAATAT TTTATATGATGAAAACAAAAAACACATACCAGCTCAGAGGATTAAATATTACTCCTCTGACTTCCAAA T  Figure 4.8 – Sequence from hooded sweet peas This figure shows the sequence of DNA that was amplified using hooded specific primers. The yellow text indicates conserved portion of DNA retained from the wild-type LoCYC2 gene.  4.4.3 – χ2 Analysis To analyze whether or not the F2 population segregates in the predicted ratio, a Chisquared Goodness of Fit test using the formula  was used. In this  formula, Observedi is the number of plants that are observed in the ith category, and Expectedi is the number of individuals that are predicted in that category under the null hypothesis of a 9:3:3:1 phenotypic ratio (Whitlock and Schluter, 2008). The plants were divided into four groups based on their phenotype. Unexpected types were placed into the closest category, for instance Burgundy Hooded sweet peas were grouped with the Hooded Painted Lady type because they are hooded and distinct from the parental Annie B Gilroy type. The critical χ2 value at α = 0.05 for 3 degrees of freedom is 7.81(Whitlock and Schluter, 2008). Since the observed χ2 value of 5.45 is less than the critical value of 7.81 at α = 0.05 for 3 degrees of freedom, I fail to reject the null hypothesis. For more detailed calculations see Figure 4.9.  34  Sweet Pea Type  Cupani  Dark Cupani  Number of Plants Observed Number of Plants Expected  62 66.375 0.288370998  31 22.125 3.560028249  Hooded Painted Lady 21 22.125 0.05720339  Hooded Painted Lady 4 7.375 1.544491525  = 5.45 Degrees of Freedom: 4-1 = 3 Critical χ2 value at α=0.05 for 3 degrees of freedom = 7.81 (Whitlock and Schluter, 2008) Table 4.9 - χ2 Calculations The χ2 Goodness of Fit test was used to determine if the 118 plants used in the F2 segregation analysis exhibited the predicted 9:3:3:1 ratio.  4.5 - Discussion Because the observed χ2 value was less than the critical value at three degrees of freedom, I failed to reject the null hypothesis of the χ2 analysis. This means that the data are consistent with the predicted 9:3:3:1 ratio. In the 118 individuals tested, the mutant allele of LoCYC2 segregated with the hooded floral phenotype 100% of the time. Wild-type sweet peas always possessed an intact copy of LoCYC2, although on occasion DNA could be amplified from wild-type flowers using LoHoodedGene4L/4R and LoHoodedGene3L/2R primers. This is not surprising because hooded has been characterized as a recessive trait and heterozygous individuals are predicted to be common in the segregating F2 population. Three of the hooded individuals were only tested using CYC2GENE4/9 primers. One of these plants was simply overlooked accidentally, whereas the other two did not show any amplification while using the LoHoodedGene4L/4R and LoHoodedGene3L/2R primers. Because the result for hooded plants relies on a negative result (a lack of amplification) using 35  CYC2GENE4/9 primers, ITS primers were initially used to prove that the genomic DNA was capable of undergoing PCR amplification. It is possible that the two plants that did not show amplification using the hooded specific primers have additional mutations in the binding sites of the hooded specific primers.  36  5 – Petal Micromorphology in Lathyrus odoratus 5.1 – Aim I used Scanning Electron Microscopy (SEM) to examine cell types present on the adaxial and abaxial surfaces of standard, wing and keel petals in the sweet pea (Lathyrus odoratus). Although not all papilionoid legumes in the Inverted Repeat Loss (IRL) clade have discernable differences in petal-specific cell types (Ojeda et al., 2009), cell shape and size can be used as a tool to determine petal identity in the sweet pea. Cell shape and cell type were examined in all petals of the hooded floral mutant of sweet pea. The standard petal of hooded sweet peas has taken on cell characteristics found in wing petals of wild-type flowers, suggesting that the mutation is homeotic in nature.  5.2 – Introduction 5.2.1 – The role of cell shape in pollination In many plants, epidermal cell type can have a great influence on the way a surface is perceived. Qualities such as cell shape and curvature can change the focus and absorption of both visible and ultraviolet light (Gorton and Vogelmann, 1996). In addition to light focus and absorption, epidermal cell type can modify the intensity at which pigment is perceived (Gorton and Vogelmann, 1996; Kay et al., 1981; Noda et al., 1994). Pigment type and UV-absorption both play large roles in the attractiveness of flowers to pollinators. UV-absorption and UVreflection patterns, although invisible to the human eye, often provide visual cues for insects and are of particular importance in bee-pollinated flowers (Kevan, 1983).  37  In Antirrhinum, a MYB transcription factor called MIXTA is involved in the formation of conical epidermal cells in flower petals (Noda et al., 1994). In mixta mutant lines, flowers appear lighter in colour and the adaxial surface of the petals have flattened epidermal cells rather than the conical-papillate cells that are see in the wild-type (Noda et al., 1994). While the mixta phenotype is clearly distinguishable from wild-type by the human eye, the change did not influence the behaviour of the bumblebee (Bombus terrestris) in an observable fashion (Dyer et al., 2007). In Lotus, petal epidermal cell shape varies greatly between insect and bird pollinated species leading to the hypothesis that MIXTA may play a role in establishing pollination syndrome (Cronk and Ojeda, 2008).  5.2.2 – Petal-specific cell types in Fabaceae The Leguminosae is a large and diverse group. Within the Papilionoideae petal-specific cell types can vary between lineages even though the overall floral form is conserved (Ojeda et al., 2009). In Ojeda et al.’s 2009 survey of petal micromorphology, six major cell types were commonly found in the petals of legume flowers. In this paper, the cell types were identified both by the cell’s overall shape and by the appearance of its cell wall surface, as is the custom with similar studies in angiosperms (Barthlott, 1990; Kay et al., 1981; Ojeda et al., 2009). Tabular Rugose Striate (TRS) and Tabular Rugose Granular (TRG) cells are both puzzle-shaped and differ in the texture of the cell’s surface. Tabular Flat Striate (TFS) cells are brick-shaped. Papillose Knobby Rugose (PKR) cells are more round in character and have a raised lens-like quality. Papillose Conical Striate (PCS) cells are also more round in character, but have a conical raised surface that is absent in PKR cells. Papillose Lobular Striate (PLS) cells have a three  38  dimensional lobed appearance. For the sake of simplicity, epidermal cell shape will be classified in Lathyrus odoratus using these customary cell categories. While six cell-types were identified in legume flowers, Ojeda et al. 2009 describe some general trends that link a certain cell type to a particular petal. In general, papillose conical cells are characteristics of dorsal and lateral petals, tabular rugose cells are usually associated with lateral petals and tabular flat cells are commonly restricted to ventral petals (Ojeda et al., 2009). In some species, such as Lotus japonicus, epidermal cell types can be used as micromorphological markers for petal identity (Feng et al., 2006; Ojeda et al., 2009). In the IRL clade of legumes (which includes Lathyrus), some of the diversity of epidermal cell types that is common in other lineages is not present (Ojeda et al., 2009). Although generally members of this group only possess TRS cells, differences in size and shape can be used as micromorphological markers to identify specific petal types (Ojeda et al., 2009).  5.3 – Methods 5.3.1 – SEM and tissue sampling All SEM data were collected using a Hitachi S-2600N scanning electron microscope at an acceleration voltage of 20kV. Petals were extracted from blooming flowers and mounted onto scanning electron microscope stubs using double-sided adhesive tape. Fresh tissue was used under low-vacuum conditions (20 Pa) due to the ready availability of flowers and time constraints. Both the adaxial and abaxial petal surfaces were examined to determine if there were visible differences in cell type that could be used as micromorphological markers to determine petal identity. Seven different cultivated varieties of sweet pea were examined to verify that any trends in cell shape that were observed were conserved traits. Three varieties (Annie B Gilroy, 39  Lady’s Bonnet and Lady Grisel Hamilton) that displayed hooded standard petals and four varieties (Cupani, Miss Wilmott, NAB Scarlet, Painted Lady) that displayed wild-type standard petals were examined. One to five samples of each flower petal type were used to generate the results.  5.4 – Results 5.4.1 – Scanning electron micrographs  Figure 5.1 – Epidermal cell types present in wild-type sweet peas (Lathyrus odoratus) The adaxial surface of the three petal types showed significant variation in cell shape and size. The white scale bars represent 100μm.  40  Figure 5.2 – Epidermal cell types present in hooded sweet peas (Lathyrus odoratus) In the hooded floral mutant of sweet pea, the adaxial surface of the standard petal has taken on wing petal characters. The hooded cultivated variety ‘Annie B Gilroy’ is shown, however this pattern was seen for all hooded mutants examined. The wild-type variety ‘Cupani’ is shown for the purposes of comparison. The white scale bars represent 100μm.  5.4.2 – Cell types present in wild-type sweet peas The adaxial surface of the standard petal of wild-type sweet peas is composed primarily of elongate TRS cells. The adaxial surface of wild-type wing petals is composed primarily of PKR cells. It could be argued that the cells may have a character that is similar to TRS (as suggested by Ojeda et al 2009), although the tabular quality is much less pronounced. The epidermal cells found in the standard and wing petals can easily be used to distinguish petal type. The cells found in the wing petals are nearly isodiametric and have a lens-like surface which is a quality seen in PKR cells. The adaxial surface of wing petals of wild-type sweet peas is composed of brick-shaped, flat TFS cells.  41  5.4.3 – Cell types present in hooded sweet peas The adaxial surface of hooded standard petals is made up of PKR cells that are indistinguishable from those found in the wing petals of both hooded and wild-type sweet pea petals. The adaxial surface of hooded wing and keel petals is unchanged in comparison to wildtype petals.  5.5 – Discussion 5.5.1 – Cell shape as a micromorphological marker In the sweet pea, cell size and shape differ significantly on the adaxial surface of petals of different identities, and can therefore be used as an indicator of petal identity. Cell size and shape has been used successfully as a marker of identity in Lotus japonicus (Feng et al., 2006). Ojeda’s 2009 survey of legume flower micromorphology showed that cell shape and size are easily visible under SEM and are qualities that can be used to distinguish the difference between specific petal types in many papilionoid groups. Interestingly, although many of the IRLC legumes display only TRS cells, sweet pea petals possess PKR cells and TFS cells on the wing and keel petals respectively in addition to the TRS cells found on the adaxial surface of the standard petal. The presence of lens-shaped PKR cells on the adaxial surface of wing petals likely plays a role in the attraction of pollinators, as this cell type has previously been found to increase colour depth and alter light absorption (Gorton and Vogelmann, 1996; Noda et al., 1994).  42  5.5.2 – The hooded mutation is homeotic in nature In hooded sweet peas, the epidermal cells found on the adaxial surface of the standard petal have been replaced by cells that are very similar in shape and size to those characteristically found in the wing petals. In wild-type sweet peas the elongate TRS cells found on the adaxial surface of the standard petal are easily distinguishable from the PKR cells found on the wing petals. This observation lends support to the hypothesis that hooded is a homeotic mutation where the standard petal has taken on wing petal identity (Chapters 2 and 4). If the hooded mutation is homeotic in nature it would also explain why a bicolour hooded sweet pea has never been observed. If the standard took on wing petal identity it would not be able to express the different pigmentation patterns that are seen in wild-type bicolour sweet peas because wingspecific pigment biosynthetic pathways would be active instead of the standard-specific ones.  5.5.3 – CYCLOIDEA-like genes and epidermal cell shape CYC-like genes have been found to play a role in establishing petal identity in papilionoid legumes (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). Mutations in Legume CYC3 homologs have been shown to cause wing petals to take on keel petal identity in Lotus and Pisum (Feng et al., 2006; Wang et al., 2008a). Mutations in Legume CYC2 have also been shown to cause changes in standard petal identity and even influence floral symmetry as in Cadia (Citerne et al., 2006; Feng et al., 2006; Wang et al., 2008a). Besides playing roles in establishing floral organ number and identity, these master regulator genes are likely responsible for controlling the genes that determine epidermal cell types as CYC2 mutants in Lotus and Pisum show petalspecific changes in epidermal cell types (Feng et al., 2006; Wang et al., 2008a). In Antirrhinum, CYC and DICH interact with two MYB-class genes, DIV and RAD to establish floral symmetry 43  and petal identity (Corley et al., 2005). In Legumes it is possible that CYC2 homologs interact with MYB genes that are similar to MIXTA. MIXTA has been shown to influence epidermal cell type and may also play a role in pigmentation (Noda et al., 1994).  44  6 – Localized Growth Patterns in the Standard Petal of Lathyrus odoratus 6.1 – Aims While many cultivated varieties of sweet pea possess hooded standard petals, very little has been done to properly characterize the phenotype in a developmental context. The standard petal of the hooded sweet pea differs both in shape (lobes) and curvature (hood) in comparison to the erect wild-type standard petal (Chapter 1, Figures 1.3). A landmark analysis was conducted in order to determine the localized differences in growth that separate hooded standard petals from those found in wild-type flowers.  6.2 - Introduction 6.2.1 – Methods of landmark analyses While the hooded mutant has been characterized for over a hundred years, very little has been done to describe how the hooded standard petal differs from the wild-type during growth. The final result is a petal quite obviously altered in both shape and curvature, which raises the question of how the characteristic curvature of the petal arises. Some studies have been done using internal landmarks in growth, such as the branching patterns created by developing vascular tissue, to determine local growth patterns (Maksymowych, 1973). Natural landmarks are often very difficult to identify and therefore this method of describing growth can be quite challenging. Alternatively, artificial landmarks can be applied to the surface of an expanding organ such as a petal or a leaf which would then allow for greater ease in describing localized growth patterns. Unfortunately, by using artificial landmarks the landmarks themselves no longer  45  hold any biological significance apart from allowing patterns in growth to be observed, whereas natural landmarks can be helpful in studying the overall ontogeny of a structure. One method of using artificial landmarks is to apply a grid to the surface of a growing organ using India ink (Avery, 1933). A similar method is to apply graphite particles randomly to the surface of an organ and to track the changes in the overall pattern (Basu et al., 2007). One difficulty that is often encountered in landmark studies that use either natural or artificial landmarks is that tracing the movement of the landmarks can be quite difficult and labour intensive depending on the scale and level of detail that is desired. Despite the technical challenges that may arise, landmark analysis remains an excellent method of characterizing the patterns of growth and surface curvature that occur in a particular organ.  6.2.2 – Effect of genes on growth patterns and curvature TCP transcription factors often play important roles in establishing organ identity, plant architecture, and patterns of cell growth and development (Martín-Trillo and Cubas, 2009). The TCP transcription factor CINCINNATA (CIN) in Antirrhinum has been shown to influence patterns of growth and curvature in leaves (Nath et al., 2003). Where wild-type leaves are oval in shape, the leaves of CIN mutants are more rounded and often have curled margins (Nath et al., 2003). The obvious change in shape and curvature is caused by longer periods of growth along the marginal regions of the leaf (Nath et al., 2003). Interestingly, CIN mutations also influence epidermal cell shape (Crawford et al., 2004).  46  6.3 – Methods 6.3.1 – Printing of landmarks In order to determine the localized growth patterns that occur during petal development, F2 sweet pea buds approximately 1 cm long were stripped of their sepals to reveal the folded outer surface of the standard petal. The immature standard petal sheathes the wing and keel petals and is folded in half along the plane of bilateral symmetry in the flower bud. The flower bud was gently fixed to a glass slide using tape. Special care was taken not to damage the newly exposed floral tissue because any injury sustained at this stage of flower development would influence the growth observed during maturation and anthesis. Artificial landmarks were printed on one side of the exposed standard petal using a specialized inkjet printing apparatus that is described in detail in Wang et al. 2010. A microscopic grid was printed on the abaxial side of half of a standard petal using the printing parameters outlined in Wang et al. 2010. Blue food colouring was used instead of violet in order to increase the contrast between the printed landmarks and the natural purple pigmentation that is present in wild-type standard petals. The ink was used to print latitudinal and longitudinal lines that consisted of 40m dots 87m apart. These lines were used to generate a fine-scale grid consisting of squares that measured 435m in each direction. 6.3.2 – Analysis of landmarks Following the printing steps, photographs were of the petal’s surface using the methods and instruments described in Wang et al. 2010. The sweet pea plant was then returned to the greenhouse. Flower buds were allowed to develop naturally and the standard petal was harvested once the bud had bloomed, approximately three to five days after the initial printing step. The mature standard petal was photographed and the deformation of the grid was analyzed using 47  specialized computer programs (Wang et al., 2010). Five hooded and five wild-type flowers from the F2 generation were analyzed using this method.  6.4 – Results  Figure 6.1 – The growth model generated through the petal printing landmark analysis A graph was printed on one half of a developing wild-type standard petal. The scale diagram shows the rates of growth that were observed during development. Cool colours show areas of less growth whereas warm colours show areas of rapid growth.  48  Figure 6.2 – The growth model generated through the petal printing landmark analysis A graph was printed on one half of a developing hooded standard petal. The scale diagram shows the rates of growth that were observed during development. Cool colours show areas of less growth whereas warm colours show areas of rapid growth.  While both hooded and wild-type standard petals show increased growth rates along their margins, the character is much more pronounced in hooded standard petals. Mutant hooded standard petals also display a greater range of growth rates on their surfaces in comparison to wild-type petals as can be seen by the greater variation in colour present in Figure 6.2 when compared to Figure 6.1.  49  6.5 – Discussion 6.5.1 – Growth and curvature in the sweet pea Growth can be used to describe the curvature seen in expanding tissue and can be expressed in terms of Gaussian curvature (Dumais and Kwiatkowska, 2002; Nath et al., 2003; Todd, 1985). A circle of cells that is expanding at a uniform rate will have a Gaussian curvature of zero and will be flat (Nath et al., 2003). If the growth rate increases at the margins, the surface will be ruffled at its surface and will have negative Gaussian curvature (Nath et al., 2003). Alternatively, if the growth rate is higher in the center, the surface will be domed and will have positive Gaussian curvature (Nath et al., 2003). While both wild-type and hooded standard petals have a negative Gaussian curvature, this trait is much more pronounced in hooded petals. This is unsurprising because while the standard petals of wild-type sweet peas are generally erect and banner-like, they often possess a slight frilly quality near their margins which can be explained by negative curvature. Both hooded and wild-type plants show areas of increased growth near the base of the standard petal. This is likely what allows the wild-type sweet pea to create its distinctive reflexed form. The area of increased growth would also contribute to the formation of the lower lobes that are seen in a notched hooded standard petal. It is counter-intuitive that the standard petal of hooded sweet peas displays negative Gaussian curvature, however. Often hooded standard petals have areas of differing growth rates along their margins whereas wild-type standard petals usually show less variation towards the tip of the petal. It is possible that distinct areas with different growth rates allow hooded standard petals to fold and form the distinctive hooded phenotype instead of forming a saddle-shaped petal that would be characteristic of negative curvature. It is also possible that the base of the 50  standard petal plays a role in the orientation of the standard petal but this could not be accurately visualized using petal printing method without the risk of damaging delicate floral tissue.  51  7 - Conclusion CYCLOIDEA and CYC-like genes play important roles in establishing petal identity and floral symmetry in a number of groups and species. In the Fabaceae, CYC2 genes are responsible for the determination of standard petal identity (Feng et al., 2006; Wang et al., 2008a). In the unusual actinomorphic species Cadia purpurea, CYC2 has been shown to greatly influence floral symmetry (Citerne et al., 2006). The goal of my research was to determine the genetic cause of the hooded mutant of sweet pea and to expand the current data on the genetic control of petal morphology in legumes to include another papilionoid species. The results from the RT-PCR experiments supported the hypothesis that the hooded mutation is caused by a change of sequence or expression in LoCYC2. While no change in expression was observed in LoCYC1, the primers specific for CYC2, showed differential amplification patterns that may have been caused by a change in gene expression. At this point I can not be certain whether this change in amplification is due to a change in gene expression, or due to a change in DNA sequence at the binding site of one of the CYC2-specific primers that was used in the analysis. In any case, the result was enough to warrant an in depth analysis of the Lathyrus CYC2 gene. Unfortunately I was unable to examine CYC3 expression in wild-type and hooded plants, though the information would have made my research more complete. Since CYC3 has been shown to influence lateral petal identity (Feng et al., 2006; Wang et al., 2008a) and the hooded standard seems to have taken on wing petal identity, it would be very interesting to see if the gene is expressed in the standard petal of hooded sweet peas. The results from the analysis of CYC2 genes in Lathyrus provided further support for the results of the RT-PCR experiments. It is quite clear that there are two alleles of CYC2 present in sweet peas. Through using the technique of genome walking I was able to get the full gene  52  sequence of the wild-type allele of CYC2, but I was unable to fully sequence the mutant allele. I expect that the mutant allele of CYC2 is caused by an insertion because I was able to sequence DNA fragments from the 3’ region of the gene. These data suggest that if I had continued sequencing the mutant allele I may have eventually re-discovered the 3’ portion of the gene, including the functionally relevant R domain. While the F2 segregation results were clear for 118 individuals, a larger sample size would have increased the confidence of the results. In every individual examined, the mutant allele of CYC2 segregates with the hooded phenotype, lending support for the hypothesis that the floral mutant is caused by a change in CYC2. Bateson (1913) suspected that there could be close linkage between hooded and the gene that controls the bicolour trait; however, in the time since this prediction was made even the most closely linked genes would have undergone a recombination event. The conspicuous lack of a bicoloured sweet pea in the F2 population lends support for the hypothesis that the hooded mutant is homeotic in nature. The patterns of petal micromorphology that were observed in the sweet pea also support this hypothesis and show that the standard petal of hooded sweet peas has taken on wing-like characters. Also, it is interesting to note that while not all members of the IRL clade of Fabaceae have discernable differences in petal-specific cell types (Ojeda, FranciscoOrtega et al. 2009), the standard, wing and keel petals of sweet peas can be easily identified by epidermal cell type. Examination of the localized growth patterns in the standard petal of the sweet pea show that there are observable differences in growth rate that lead to the differences in shape and curvature that are seen in the hooded mutant. Although a developmental series was not examined, it would be interesting to determine if the shape of the hooded standard petal can be  53  attributed to a process such as neoteny, where an early petal organ form is retained in a mature flower. Future research can be done using the Lathyrus-specific CYC2 primers I have created. Marker-assisted plant breeding could be conducted using the primers designed for the F2 segregation experiment. These primers could be used to determine if a sweet pea plant is hooded or wild-type prior to blooming. Currently Sæmundur Sveinsson is using the CYC2GENE primers to generate a phylogenetic tree to show the relationships between species in Lathyrus (Personal communication, April 11, 2010). Because the CYC2 gene has both conserved functional domains and variable regions, it is ideal for generating phylogenetic information. Sæmundur Sveinsson has also informed me that the CYC2GENE primers are capable of amplifying sequence from the closely related group Vicia (Personal communication, August 15, 2010). While the hooded mutant of sweet pea has been characterized for nearly a century, its genetic basis has now been illuminated. The mutants squ1 in Lotus and lst1 in Pisum share much more in common with hooded sweet peas than just the shape of the dorsal standard petal. The Lathyrus CYC2 homolog functions in establishing local growth patterns and dorsal petal identity.  54  Works Cited  Allen, O. N., Allen, E. K., 1981. The Leguminosae, A Source Book of Characteristics, Uses, and Nodulation. The University of Wisconsin Press, Madison, Wisconsin. Ambrose, M. J., 2003. 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Roberts & Company Publishers, Greenwood Village, Colorado.  57  Appendix – Supplemental Data and Calculations Sweet Pea Standard Petal Measurements Row  Flower Type  Standard length down mid-vein (cm)  Standard width at widest part (wild-type and hooded) (cm)  Row 4-12 Row 1-7 GH 7 Row 1-11 Row 1-10 Row 1-24  Shell Pink Cupani Cupani Dark Cupani Dark Cupani Hooded Painted Lady Annie B Gilroy Cupani Cupani Hooded Painted Lady Cupani Dark Cupani Cupani Hooded Painted Lady Annie B Gilroy Cupani Cupani Purple Flake Cupani Cupani Cupani Cupani Hooded Painted Lady Cupani Hooded Painted Lady Cupani Cupani Cupani Cupani Dark Cupani Cupani  2.6 2.5 2.1 2.8 2.4 2.8  3.6 3.4 2.8 3.3 3.4 3  3  3.2  1.066667  2.5 2.7 2.3  3.1 3.6 2.4  1.24 1.333333 1.043478  2.3 2.7 2.8 2.8  3 3.4 3.6 2.7  1.7  1.304348 1.259259 1.285714 0.964286  0.607143  3.7  3.6  3  0.972973  0.810811  2 2.3 3.1 2.7 2.7 2.5 2.1 2.9  2.2 2.5 3.6 3.6 3.3 3.1 2.9 2.9  2.3  1.1 1.086957 1.16129 1.333333 1.222222 1.24 1.380952 1  0.793103  2.4 2.9  3.1 2.9  2.2  1.291667 1  0.758621  2.5 2.7 2.5 2.2 2.5 2.4  3.2 3.7 2.7 2.9 2.9 3.1  Row 1-23 GH 16 Row 1-12 GH 53 GH 54 Row 1-21 Row 1-6 Row 1-8 Row 1-9 GH 15 GH 12 Row 2-14 Row 2-15 Row 4-1 Row 4-2 Row 4-3 Row 4-19 Row 4-4 Row 4-20 Row 4-5 Row 3-22 Row 3-23 Row 3-24 Row 3-28 Row 3-25  Standard width at narrow part (notched hooded only) (cm)  2  Standard width at widest part (cm) / Standard length down mid-vein (cm) 1.384615 1.36 1.333333 1.178571 1.416667 1.071429  Standard width at narrow part (cm) / Standard length down mid-vein (cm)  0.714286  1.28 1.37037 1.08 1.318182 1.16 1.291667  58  Row  Flower Type  Standard length down mid-vein (cm)  Standard width at widest part (wild-type and hooded) (cm)  Row 3-30 Row 3-29 Row 4-24  Dark Cupani Cupani Hooded Painted Lady Dark Cupani Hooded Painted Lady Cupani Cupani Cupani Annie B Gilroy Cupani  2.7 2.6 2.7  3.5 3 2.6  2.8 3  3.3 3.1  1.178571 1.033333  2.3 2.6 2.5 2.3  3.3 3 3.2 2.1  1.434783 1.153846 1.28 0.913043  2.1  2.1  1  Burgundy Hooded Coral Cerise Hooded Painted Lady Cupani Hooded Painted Lady Cupani Cupani Hooded Painted Lady Cupani Cupani Cupani Cupani Pink Flake Hooded Cupani Cupani Cupani Dark Cupani Cupani Dark Cupani Cupani Cupani Cupani Dark Cupani Cupani  3.2  3.7  1.15625  1.9 2.8  2.5 3  1.315789 1.071429  2.3 3.2  2.8 3  2.1 2.6 3  3.1 3.3 3.1  1.47619 1.269231 1.033333  2.5 2.5 2.3 2.6 3.5  2.9 3.2 3 3.2 4.3  1.16 1.28 1.304348 1.230769 1.228571  2.2 2.4 2.2 2.6 2.7 2.6 2.5 2.9 2 2.8 2.6  3.3 3.1 3.1 3.7 3.4 3.6 3.4 4.1 2.3 3.4 3.2  1.5 1.291667 1.409091 1.423077 1.259259 1.384615 1.36 1.413793 1.15 1.214286 1.230769  Row 4-7 Row 4-30 Row 4-8 Row 4-10 Row 4-21 Row 4-23 GH 16 Row 3-1 Row 3-2 Row 3-17 Row 3-3 Row 3-26 Row 3-4 Row 4-6 Row 4-22 Row 3-20 Row 3-7 Row 3-5 Row 3-6 Row 3-27 Row 2-7 Row 2-4 Row 2-3 Row 4-17 Row 4-16 Row 4-15 Row 4-13 Row 4-14 Row 2-26 Row 2-27 Row 2-25  Standard width at narrow part (notched hooded only) (cm)  2.1  1.8  2.3  Standard width at widest part (cm) / Standard length down mid-vein (cm) 1.296296 1.153846 0.962963  1.217391 0.9375  Standard width at narrow part (cm) / Standard length down mid-vein (cm)  0.777778  0.782609 *Very Large Bud  0.71875  59  Row  Flower Type  Standard length down mid-vein (cm)  Standard width at widest part (wild-type and hooded) (cm)  Row 2-28 Row 2-24 Row 4-18 Row 3-21 Row 3-18 Row 3-18 Row 3-16 Row 3-15 Row 3-13 Row 3-14 Row 3-12 Row 3-10 Row 3-11 GH 45 GH 48 GH 46 GH 50 GH 55  Cupani Dark Cupani Dark Cupani Dark Cupani Cupani Dark Cupani Dark Cupani Cupani Cupani Cupani Dark Cupani Dark Cupani Dark Cupani Cupani Cupani Dark Cupani Cupani Annie B Gilroy Hooded Painted Lady Cupani Cupani Burgundy Hooded Cupani Cupani Cupani Dark Cupani Hooded Painted Lady Dark Cupani Cupani Dark Cupani Dark Cupani Hooded Painted Lady Dark Cupani Cupani Dark Cupani Cupani Dark Cupani Cupani  2.3 2.5 2.4 2.2 2.3 2.5 2.3 2.3 2.9 2.5 2.6 2.5 2.8 2.4 3 2.5 2.5 2.2  3.2 3 3 3.4 3 3.4 3.1 2.9 3.5 3.4 3.5 3.2 3.7 3 4.1 3 3.1 2.2  2.9  3  1.034483  2.5 2.6 3.6  3.4 3.2 4.4  1.36 1.230769 1.222222  2.7 2.1 2.7 2 3.2  3.2 2.9 3.2 2.8 3.6  1.185185 1.380952 1.185185 1.4 1.125  2.7 2.6 2.4 2.8 3.2  3.6 3.4 3.4 3.6 3.5  1.333333 1.307692 1.416667 1.285714 1.09375  2.6 2.5 1.8 2.6 2.3 2.6  2.9 3.3 2.2 3.5 3.1 3.6  1.115385 1.32 1.222222 1.346154 1.347826 1.384615  Row 1-30 Row 3-9 GH 14 Row 1-13 Row 1-14 Row 1-16 Row 1-15 GH 51 Row 1-18 Row 1-29 Row 1-19 Row 1-17 Row 1-20 Row 1-5 Row 1-4 Row 1-3 GH 49 Row 2-6 Row 2-8 Row 2-9  Standard width at narrow part (notched hooded only) (cm)  1.7  Standard width at widest part (cm) / Standard length down mid-vein (cm) 1.391304 1.2 1.25 1.545455 1.304348 1.36 1.347826 1.26087 1.206897 1.36 1.346154 1.28 1.321429 1.25 1.366667 1.2 1.24 1  Standard width at narrow part (cm) / Standard length down mid-vein (cm)  0.772727  60  Row  Flower Type  Standard length down mid-vein (cm)  Standard width at widest part (wild-type and hooded) (cm)  Row 2-11 Row 4-25 Row 4-26 Row 4-29 Row 4-28 Row 2-29 Row 2-23 Row 2-13 Row 2-17 Row 2-18 Row 2-19 Row 2-20 Row 2-21 Row 2-22 Row 2-30 Row 1-27  Cupani Dark Cupani Cupani Cupani Dark Cupani Cupani Cupani Red Flake Dark Cupani Cupani Cupani Cupani Dark Cupani Cupani Cupani Violet Speckle Dark Cupani Hooded Painted Lady Cupani Cupani Cupani Cupani Dark Cupani Cupani Cupani Cupani Cupani Hooded Painted Lady Coral Cerise Cupani Cupani Cupani Hooded Painted Lady Cupani  2.3 2.5 2.3 2.2 2.3 2.5 2.1 2.4 2.5 2.8 2.2 2.7 2.6 2.2 2.5 3  3 3.1 2.8 3.3 2.6 3.3 2 2.9 3.3 3.6 2.6 3.5 3.2 3 3.1 3.4  2.1 3  2.7 3  2.7 2.3 2.6 2.4 2.2 2 2.4 2.6 2.1 3.1  3.9 3.2 3.1 2.9 2.6 2 2.7 3.2 3.3 3.4  1.444444 1.391304 1.192308 1.208333 1.181818 1 1.125 1.230769 1.571429 1.096774  2.7 2.6 2.5 2.5 3.4  3.1 3.3 2.8 3 3.7  1.148148 1.269231 1.12 1.2 1.088235  2.5  3.1  1.24  Row 1-28 Row 1-26 Row 1-22 Row 1-2 Row 1-1 GH 52 Row 1-25 GH 56 GH 42 GH 43 Row 3-8 Row 2-15 Row 4-11 Row 4-9 GH 44 Row 2-2 Row 2-12 Row 2-1  Standard width at narrow part (notched hooded only) (cm)  2.4  Standard width at widest part (cm) / Standard length down mid-vein (cm) 1.304348 1.24 1.217391 1.5 1.130435 1.32 0.952381 1.208333 1.32 1.285714 1.181818 1.296296 1.230769 1.363636 1.24 1.133333  Standard width at narrow part (cm) / Standard length down mid-vein (cm)  1.285714 1  0.8  61  Relationships Between Standard Petal Length and Width Measurements Mean value of (Width / Length) Standard width at narrow part (cm) / Standard length down midvein (cm) (hooded and notched hooded) Standard width at widest part (cm) / Standard length down midvein (cm) (hooded) Standard width at widest part (cm) / Standard length down midvein (cm) (wild-type)  Sample size (N)  Standard Deviation  Standard Error  Lower 95% Confidence Interval  Upper 95% Confidence Interval  0.956778611  24  0.152591  0.031148  0.895729  1.017828  1.052043879  24  0.082138  0.016766  1.019182  1.084906  1.172783504  123  0.145023  0.013076  1.147154  1.198413  Statistical Formulas  62  Sweet Pea Primers Primer Name CYC2GENE1 CYC2GENE2 CYC2GENE3 CYC2GENE4 CYC2GENE5 CYC2GENE6 CYC2GENE7 CYC2GENE8 CYC2GENE9 CYC2GENE10 CYC2GENE11 CYC2GENE12 CYC2GENE13 CYC2GENE14 CYC2GENE15 CYC2GENE16 CYC2GENE17 CYC2GENE18 LoHoodedGene1L LoHoodedGene1R LoHoodedGene2L LoHoodedGene2R LoHoodedGene3L LoHoodedGene3R LoHoodedGene4L LoHoodedGene4R  Primer Sequence AATAACATTGCGGTTCCGATGCCG GCCAATCAAGTGTGTTGCTGGCTT TCTTTCCCTTGCTCTTGCTCTTGC GCCAGCAACACACTTGATTGGCTT CGTGGATTGGTGCATTCTTCCCAT ATGGGAAGAATGCACCAATCCACG AGCAAGAGCAAGAGCAAGGGAAAG CGGCATCGGAACCGCAATGTTATT GGCATCGCCACCAACTTCACTATT AAGCCAATCAAGTGTGTTGCTGGC TCTTCAAGAGATGTTAGGGTTTGAC CCTCCTTTTGTAGGTTTAAGGAATC TCCCTTATAGTTCAAACCCTTATCC GAGAACTATGATGGTGAGTTGGAAT GCAATTAAGGATCTAACAAAAAGCA CTTGACTCCTTCATCTTACTTGCTC CTCTTGACTCCTTCATCTTACTTGC CAAGCAAAGAGACAACAATAGTGAA CCGAGTTGCTAACAGTTTTGTATTTAT GGAGAGATATTATACGCATTGTTGAAT TAAAGCATATATTGACAACAACCACTC GACTATATTGAATTTGGAAGTCAGAGG GGTTCTCACTATGGAATTTCTTGTTT TTTTAGAATTGGTTAGCCATTAATTTG TCACTATGGAATTTCTTGTTTCCTTAC AAAAATTTAATGACAATTTCGGATGT  Primer Target Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 Wild-type LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2 hooded LoCYC2  63  Sweet Pea Sequences Genome walking results: Hooded1L/2L primers >1AnnieBGilroyGenomeWalkingHooded1L/2L TCACCATCATAGTTCTCATCCTCATCATCATCTTCAAAACATTTCAAATATTCCAGAA ACCCTAACAAATTTGTCTGTTTCACAAGAGAATCACAATAATAGCAACAACAATATT AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTACCAGCCCGGGCCGTCGACCACGCGTGCCCT AT >2AnnieBGilroyGenomeWalkingHooded1L/2L AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACAC TAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACT TGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACC CCCACCCATTCAAATATTGCTTTCTAAACCGAAAGATTTTTTTAGACAAAGAAGAGG ACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGA TTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAA GTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAAC GAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCC ATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGA AGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAA AGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCA TTAATCCATCATCTTCCTTCAATTCGACAATGCGTATAATATCTCTCCACCACAACCA GACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCTTCAAATGTATAGG GCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTAG ATTTTGCATGATA  64  >3AnnieBGilroyGenomeWalkingHooded1L/2L TTAATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTT CTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTAC ACCAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCA CTTGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCA CCCCCACCCATTCAAATATTGCTTTCCAAACCAAAAGATTTTTTTAGACAAAGAAGA GGACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAG GATTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTT AAGTCTTTAATCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACA ACGAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATT CCATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAA GAAGAAGCAGCATACATATATCAAAATCTTGTACGCAAAGGTGATCATTCAAACCA AAAGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTT CATTAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAAC CAGACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATA GGGCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATT AGATTTTGCATGATA >4AnnieBGilroyGenomeWalkingHooded1L/2L AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAGCCAATTCTAAAAAATGAAAATAATTTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACAC CAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACT TGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACC CCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGG ACATGTTTCAAATATTTTATATGATGAAAACGAAAAACACATACCAGCTCATGAGGA TTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAA GTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAAC GAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCC ATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGA AGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAA AGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCA TTAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATTTCTCCACCACAACCA GACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAGG GCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATTTAGTATAATATTATTAG ATTTTGCATGAT  65  TATCATGCAAAATCTAATAATATTATACTATATGAATCTTACACTATATATCTCTTTT ATTTTGGGGGGCCCTATACATTTGAGGCTCTGTGGTGTTGCACTTGTTGCATAGACAT ATGATCGAGTCTGGTTGTGGTGGAGAGATATTATACGCATTGTTGAATTGAAGGAAG ATGATGGATTAATGAAGCAATTAGTGTTCAAGCTAGGTGACAGCGCAACAGTTTCAT TTTGGCATGCCTTTTGGTTTGAATGATCACTTTTGCATACAAGATTTTGATATATGTA TGCTGCTTCTTCTTATAAATACAAAACTGTTAGTAACTCGGGCATGTGGGAGGATAA AAATTGGCAATGGAATTTGGGAACATTGAATACACGGTTTGTAAATGATCAATTAGA GGAATTGTATTCGTTGTTGGCACAGGTGGAACCATTCACTGAAAGGGATGACACTTT TTGGTTAAAGACTTAAATTGGATAAACTTCCGACTATATTGAATTTGGAAGTCAGAG GAGTAATATTTAATCCTCATGAGCTGGTATGTGTTTTTTGTTTTCATCATATAAAATA TTTGAAACATGTCCTCTTCTTTGTCTAAAAAAATCTTTTGGTTTAGAAAGCAATATTT GAATGGGTGGGGGTGGAGTGAGGGGCGACGAGTGGTTGTTGTCAATATATGCTTTA ATGGTTTGTGCAAGTGGTGGGAAGTTCATCAAAGAAGACGGCATGTTTGATTTGGTT TTGTACATGTTGGTGTAGTTGGGTCATATGAATCAAAAAAAATTTAATGACAATTTC GGATGTGAGGAGGAGATTGTATACAAAATTATTTTCATTTTTTAGAATTGGTTAGCC ATTAATTTGTATCCTAGAATTAGGTGTAATTTATACGAATGATTTAAACAGCCTTGA GAAGTGTAAATCTTGCTGTGCCGGTCTTTTTTTGCGGGTTTCTTAGTAAGGAAACAA GAAATTCCATAGTGAGAACCACCACCAAGAGTTAAAGGATCTTGTTTCGGCATCGG AACCGCAATGTTATTA >6AnnieBGilroyGenomeWalkingHooded1L/2L ATAACATTGCGGTTCCGATGCCGAAACGAGATCCTTTAACTCTTGGTGGTGGTCCTC ACTATGGAATTTCTTGTTTCCTTACTAAGGAACCCGCAAAAAAAGACCGGCACAGCA AGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGG ATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTC CTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACCACACC AACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTT GCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCC CCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGA CATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGAT TAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAAG TCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAACG AATACAGTTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCCA TTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGAA GAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAAA GGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCAT TAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAACCAG ACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAGGG CCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTAGA TTTTGCATGATA  66  >7AnnieBGilroyGenomeWalkingHooded1L/2L AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATCTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACAC CAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACT CGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACC CCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGG ACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGA TTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAA GTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAAC GAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCC ATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGA AGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAA AGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCA TTAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAACCA GACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAGG GCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTAG ATTTTGCATGAT >8AnnieBGilroyGenomeWalkingHooded1L/2L TAATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTC TCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAG CAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTA GGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATC TCCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACA CCAACATGTACAAAACCAAATCAAACATGCCGTCCTCTTTGATGAACTTCCCACCAC TTGCACAAACTATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCAC CCCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAG GACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGG ATTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTA AGTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAA CGAGTACGATTCCTCTAATTGATCATTTGCAAACCGTGTATTCAATGTTCCCAAATTC CATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAG AAGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAA AAGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTC ATTAATCCACCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAACC AGACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAG GGCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTA GATTTTGCATGATA  67  >9AnnieBGilroyGenomeWalkingHooded1L/2L TTAATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTT CTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAATAAATGAAGATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTAC ACCAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCA CTTGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCA CCCCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGA GGACATGTTCCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGA GGATTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATT TAAGTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAAC AACGAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAA TTCCATTGCCAATTTTTATCCTCCCACGTGCCTGAGTTGCTAACAGTTTTGTATTTAT AAGAAGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAAC CAAAAGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGC TTCATTAATCCATCATCTTCCTTCAATTCAACGATGCGTATAATATCTCTCCACCACA ACCAGACTCGATCATATGTCTATGCAACAAGTGCAACATCACGGAGCCTCAAATGTA TAGGGCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTA TTAGATTTTGCATGATA >10AnnieBGilroyGenomeWalkingHooded1L/2L AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACAC CAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACT TGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCGCC CCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGG ACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGA TTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAA GTCTTTAACCAAAAAGTGTCACCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAAC GAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCC ATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGA AGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAA AGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCA TTAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAACCA GACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAGG GCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTAG ATTTTGCATGAT  68  >11AnnieBGilroyGenomeWalkingHooded1L/2L AATAACATTGCGGTTCCGATGCCGAAACAAGATCCTTTAACTCTTGGTGGTGGTTCT CACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATCTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACAC CAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACT CGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACC CCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGG ACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGA TTAAATATTACTCCTCTGACTTCCAAATTCAATATAGTCGGAAGTTTATCCAATTTAA GTCTTTAACCAAAAAGTGTCATCCCTTTCAGTGAATGGTTCCACCTGTGCCAACAAC GAATACAATTCCTCTAATTGATCATTTACAAACCGTGTATTCAATGTTCCCAAATTCC ATTGCCAATTTTTATCCTCCCACATGCCCGAGTTGCTAACAGTTTTGTATTTATAAGA AGAAGCAGCATACATATATCAAAATCTTGTATGCAAAAGTGATCATTCAAACCAAA AGGCATGCCAAAATGAAACTGTTGCGCTGTCACCTAGCTTGAACACTAATTGCTTCA TTAATCCATCATCTTCCTTCAATTCAACAATGCGTATAATATCTCTCCACCACAACCA GACTCGATCATATGTCTATGCAACAAGTGCAACATCACAGAGCCTCAAATGTATAGG GCCCCCCAAAATAAAAGAGATATATAGTGTAAGATTCATATAGTATAATATTATTAG ATTTTGCATGAT The F2 Segregation Ratio  Sum: Expected Ratio Percentage Expected:  Cupani 62 9 0.5625 66.375  Hooded Dark Painted Annie B Cupani Lady Gilroy 31 21 4 3 3 1 0.1875 0.1875 0.0625 22.125 22.125 7.375  0.288371 3.560028 0.057203 1.544492  Degrees of Freedom α=0.05  5.450094 3 7.81  **Since the observed χ2 = 5.45 is less than 7.81, P>0.05, we fail to reject the null hypothesis. Data is consistent with a 9:3:3:1 segregation ratio.  69  F2 PCR Amplification Results Row  Plant  Cross  Phenotype  1 1 1 1  1 2 3 4  AC14 AC14 AC14 AC4  1  5  AC14  1 1 1  6 7 8  AC14 AC4 AC14  1  9  AC14  1  10  AC14  1  11  AC4  1 1  12 13  AC14 AC14  1 1 1 1  14 15 16 17  AC14 AC4 AC14 AC14  1  18  AC4  1 1  19 20  AC14 AC14  1  21  AC14  1  22  AC4  Cupani Cupani Cupani Dark Cupani Hooded Painted Lady Cupani Cupani Hooded Painted Lady Annie B Gilroy Dark Cupani Dark Cupani Cupani Burgundy Hooded Cupani Cupani Cupani Dark Cupani Hooded Painted Lady Cupani Dark Cupani Dark Cupani Cupani  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp)  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp)  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp)  -  (Not used)  (Not used)  + + -  +  +  -  +  +  +  +  +  +  + + + + Dark Flower  + + + + + + + Variation in flower shape  -  + + + +  70  Row  Plant  Cross  Phenotype  1  23  AC14  1  24  AC14  1  25  AC4  1  26  AC14  1  27  AC14  1  28  AC4  1  29  AC14  1  30  AC4  2 2 2 2 2  1 2 3 4 5  AC14 AC14 AC4 AC4 AC14  2  6  AC14  Annie B Gilroy Hooded Painted Lady Dark Cupani Hooded Painted Lady Violet Hooded Dark Cupani Dark Cupani Hooded Painted Lady Cupani Cupani Cupani Cupani Annie B Gilroy Cupani  2 2  7 8  AC4 AC14  2 2  9 10  AC4 AC4  2 2  11 12  AC14 AC14  2  13  AC14  Cupani Dark Cupani Cupani N/A Cupani Hooded Painted Lady Red Flake  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp) -  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp) +  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp) +  -  +  +  -  +  +  -  +  +  -  +  +  + + + + -  +  +  +  +  +  + + Variation in flower shape  Mildly hooded Mild PCR amplification  + + +  Plant did not flower Mildly hooded  + + + -  +  71  Row  Plant  Cross  Phenotype  2  14  AC14  2  15  AC14  2 2  16 17  AC4 AC14  2 2 2 2  18 19 20 21  AC4 AC14 AC14 AC4  2 2 2  22 23 24  AC4 AC4 AC4  2 2 2  25 26 27  AC4 AC14 AC14  2  28  AC14  Hooded Purple flake Hooded Painted Lady Cupani Dark Cupani Cupani Cupani Cupani Dark Cupani Cupani Cupani Dark Cupani Cupani Cupani Dark Cupani Cupani  2 2 3  29 30 1  AC4 AC14 AC14  3  2  AC14  3 3 3 3 3 3  3 4 5 6 7 8  AC14 AC4 AC4 AC4 AC14 AC4  Cupani Cupani Burgundy Hooded Coral Cerise Bicolour Cupani Cupani Cupani Cupani Cupani Cupani  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp) -  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp) +  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp) +  -  +  +  +  +  + + + + + + + + + + + + Plant has a virus. Flowers have spots  +  + + +  + + + + + +  72  Row  Plant  Cross  Phenotype  3 3  9 10  AC14 AC4  3  11  AC14  3  12  AC4  3 3 3 3  13 14 15 16  AC4 AC4 AC4 AC14  Cupani Dark Cupani Dark Cupani Dark Cupani Cupani Cupani Cupani Dark Cupani  3  17  AC4  Hooded Painted Lady  3  18  AC14  3 3 3  19 20 21  AC4 AC4 AC4  3 3 3 3 3  22 23 24 25 26  AC4 AC4 AC14 AC14 AC14  Dark Cupani Cupani Cupani Dark Cupani Cupani Cupani Cupani Cupani Hooded Painted Lady  3  27  AC14  3  28  AC14  3  29  AC4  Hooded Pink Flake Dark Cupani Cupani  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp)  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp)  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp)  +  +  + + + + -  +  +  -  +  +  + + + + + + + +  Variation in flower shape, notched standard petal  -  + + + +  Notched standard petal. Plant has a virus. Flowers have spots  + +  73  Row  Plant  Cross  Phenotype  3  30  AC14  4  1  AC14  Dark Cupani Cupani  4  2  AC4  Cupani  4  3  AC4  Cupani  4  4  AC14  Cupani  4  5  AC14  Cupani  4  6  AC14  Cupani  4  7  AC14  4  8  AC14  Dark Cupani Cupani  4  9  AC4  Cupani  4  10  AC4  Cupani  4  11  AC14  4  12  N/A  Coral Cerise Bicolour Shell Pink  4  13  AC14  Cupani  4  14  AC14  Cupani  4  15  AC4  4  16  AC4  Dark Cupani Cupani  4  17  AC14  4  18  AC14  Dark Cupani Dark Cupani  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp)  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp)  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp)  +  +  + Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality  +  Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality  -  + + + + + + + + + +  + + + + + +  74  Row  Plant  Cross  Phenotype  Notes  PCR Amplification (Primers: CYC2GENE4/9, fragment ~125bp)  4  19  AC14  Gel is poor quality  4  20  AC14  4  21  AC14  Hooded Painted Lady Hooded Painted Lady Cupani  4  22  AC14  4  23  AC14  4  24  AC14  4  25  AC14  4  26  AC4  Hooded Painted Lady Annie B Gilroy Hooded Painted Lady Dark Cupani Cupani  4  27  N/A  N/A  4  28  AC14  4  29  AC4  Dark Cupani Cupani  4  30  AC14  Hooded Painted Lady  -  PCR Amplification (Primers: LoHoodedGen e4L/4R, fragment ~205bp) +  PCR Amplification (Primers: LoHoodedGen e3L/2R, fragment ~480bp) +  Gel is poor quality  Smear  +  +  Gel is poor quality Gel is poor quality  + -  (No amplification)  (No Amplification)  Gel is poor quality Gel is poor quality  -  (No Amplification) +  (No Amplification) +  Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality Gel is poor quality  +  +  +  -  +  + + -  75  F2 gels  76  F2 hooded Sequence Primers: LoHoodedGene4L/4R  >A1July30_Row2-5_Annie_B_Gilroy TCACTATGGAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTT >A2July30_Row2-12_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTTCATTAAATTTT >A3July30_Row2-14_Hooded_Purple_Flake TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >A4July30_Row2-15_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >A5July30_Row3-1_Burgundy_Hooded TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >A6July30_Row3-17_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >A7July30_Row3-26_Hooded_Painted_Lady TTCACTATGGAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAG CAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTA GGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATC TCCTCCTCACATCCGAAATGTATTAAATTTT 77  >A8July30_Row3-27_Hooded_Pink_Flake TTCACTATGGAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAG CAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTA GGATACAAATTAATAGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTTCATTAAATTTT ****Row1-5 is hooded, but sequencing was not attempted**** >B1July30_Row1-8_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAAGAAAATAATTTTTATACAATCT CCTCCTCACATCCGAAATTTATTAAA >B2July30_Row1-9_Annie_B_Gilroy TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >B3July30_Row1-13_Burgundy_Hooded TCACTATGGAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGC AAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAG GATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCT CCTCCTCACATCCGAAATTGTCATTAAATTTT >B4July30_Row1-18_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >B5July30_Row1-23_Annie_B_Gilroy TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAAGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATC TCCTCCTCACATCCGAAATTTCATTAAATTTT >B6July30_Row1-24_Hooded_Painted_Lady TCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAG CAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTA GGATACAAATTAAGGCTAACCAATTCTAAAAAAGAAAATAATTTTGTATACAATCTC CTCCTCACATCCGAAAT  78  >B7July30_Row1-26_Hooded_Painted_Lady TCACTATGGAAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >B8July30_Row1-27_Violet_Hooded CTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAGATTTACACTTC TCAAGGCTGTTTAAATCATTCGTWYAWWYKWMWMMWWWWYMYMKRATTCTAG GATACAAATTAATAGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATC TCCTCCTCACATCCGAAA >B9July30_Row1-30_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATAGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACA ATCTCCTCCTCACATCCGAAAT >B10July30_Row4-12_Shell_Pink TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT >B11July30_Row4-19_Hooded_Painted_Lady TTCACTATGGAAATTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAAGAAAATAATTTTGTATACAATC TCCTCCTCACATCCGAAA >B12July30_Row4-20_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT ****Row4-22 and Row 4-23 are hooded, but no sequence was acquired**** >B15July30_Row4-24_Hooded_Painted_Lady CTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAA GATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGA TACAAATTAATGGCTAACCAATTCTAAAAAATGAAATAATTTTGTATACAATCTCCT CCTCACATCCGAAATTGTCATTAAATTTT  79  >B16July30_Row4-30_Hooded_Painted_Lady TTCACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACA GCAAGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCT AGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAAT CTCCTCCTCACATCCGAAATTGTCATTAAATTTT Primers: LoHoodedGene3L/2R  ****Row1-5 is hooded, but sequencing was not attempted**** >C1July30_Row1-8_Hooded_Painted_Lady ATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAG ATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGAT ACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCT CCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAA CATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGC ACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCC ACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACA TGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTA AATATTACTCCTCTGACTTCCAA >C2July30_Row1-9_Annie_B_Gilroy GTGTTCTCACTATGGAATTTTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGG CACAGCAAGATTTACACTTTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAA TTCTAGGATACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATA CAATCTCCTCCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAA CTACACCAACATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCC ACCACTTGCACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCAC TCCACCCCCACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAG AAGAGGACATGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTC ATGAGGATTAAATATTACTCCTCTGACTTCCAAATT >C3July30_Row1-13_Burgundy_Hooded TATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAG ATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGAT ACAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCT CCTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAA CATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGC ACAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCC ACCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACA TGTTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCAGAGGATTAA ATATTACTCCTCTGACTTCCAAAT  80  >C4July30_Row1-18_Hooded_Painted_Lady GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCAAA >C5July30_Row1-23_Annie_B_Gilroy GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCAAA >C6July30_Row1-24_Hooded_Painted_Lady GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCAA >C7July30_Row1-26_Hooded_Painted_Lady TGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGAT TTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATAC AAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCC TCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACA TGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCAC AAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCAC CCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATG TTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAA TATTACTCCTCTGACTTCCAAA  81  >C8July30_Row1-27_Violet_Hooded GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCAGAGGATTAAAT ATTACTCCTCTGACTTCCA >C9July30_Row1-30_Hooded_Painted_Lady GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCAA >C10July30_Row2-5_Annie_B_Gilroy AATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTA CACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAA ATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTC ACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACATG TACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACAA ACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACCC ATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTT TCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCC >C11July30_Row2-12_Hooded_Painted_Lady GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCAGAGGATTAAAT ATTACTCCTCTGACTTCCA  82  >C12July30_Row2-14_Hooded_Purple_Flake TACACTTCTCAAGGCTGTTTAAATCATTGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTG >C13July30_Row2-15_Hooded_Painted_Lady TTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTACAC TTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAAATTA ATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTCACAT CCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACATGTACA AAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACAAACCA TTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACCCATTC AAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTTTCAA ATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAATATTAC TCCTCTGACT >C14July30_Row3-1_Burgundy_Hooded AATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTA CACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAA ATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTC ACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACATG TACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACAA ACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACCC ATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTT TCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCA >C15July30_Row3-17_Hooded_Painted_Lady ACTATGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCA AGATTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGG ATACAAATTAATGGCTAACCAATTTAAAAAAGAAAAAATTTTGTATACAATTCCTCC TCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACA TGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCAC AAACCATTAAAGCATATATTGACAACAACCACTGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGAC  83  >C16July30_Row3-26_Hooded_Painted_Lady ATGGAATTTCTTGTTTCCTTATAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGA TTTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATA CAAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTC CTCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAAC ATGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCA CAAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCA CCCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACAT GTTTCAATATTTTATATGATGAAAACAAAAAACACATA >C17July30_Row3-27_Hooded_Pink_Flake TGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGAT TTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATAC AAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCC TCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACA TGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCAC AAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCAC CCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATG TTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAA TATTACTCCTCTGACTTCCA >C18July30_Row4-12_Shell_Pink AATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTA CACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAA ATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTC ACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACATG TACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACAA ACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACCC ATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTT TCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCA >C19July30_Row4-19_Hooded_Painted_Lady TGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGAT TTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATAC AAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCC TCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACA TGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCAC AAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCAC CCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATG TTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAA TATTACTCCTCTGACTTCCAAAT  84  >C20July30_Row4-20_Hooded_Painted_Lady GAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTT ACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACA AATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCT CACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACAT GTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACA AACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACC CATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGT TTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAAT ATTACTCCTCTGACTTCCAAA ****Row4-22 and Row 4-23 are hooded, but no sequence was acquired**** >C23July30_Row4-24_Hooded_Painted_Lady CTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGATTTACACTT CTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATACAAATTAA TGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCCTCACATC CGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACATGTACAA AACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCACAAACCAT TAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCACCCATTCA AATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATGTTTCAAA TATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAATATTACT CCTCTGACTTCCAA >C24July30_Row4-30_Hooded_Painted_Lady TGGAATTTCTTGTTTCCTTACTAAGAAACCCGCAAAAAAAGACCGGCACAGCAAGAT TTACACTTCTCAAGGCTGTTTAAATCATTCGTATAAATTACACCTAATTCTAGGATAC AAATTAATGGCTAACCAATTCTAAAAAATGAAAATAATTTTGTATACAATCTCCTCC TCACATCCGAAATTGTCATTAAATTTTTTTTGATTCATATGACCCAACTACACCAACA TGTACAAAACCAAATCAAACATGCCGTCTTCTTTGATGAACTTCCCACCACTTGCAC AAACCATTAAAGCATATATTGACAACAACCACTCGTCGCCCCTCACTCCACCCCCAC CCATTCAAATATTGCTTTCTAAACCAAAAGATTTTTTTAGACAAAGAAGAGGACATG TTTCAAATATTTTATATGATGAAAACAAAAAACACATACCAGCTCATGAGGATTAAA TATTACTCCTCTGACTTCCAAA  85  Localized Growth Patterns in the Standard Petal of Lathyrus odoratus  86  87  88  89  90  91  92  93  94  95  Petal Micromorphology in Lathyrus odoratus – scanning electron micrographs  Miss Wilmott standard petal, abaxial surface  Miss Wilmott wing petal, abaxial surface 96  Miss Wilmott keel petal, abaxial surface  Lady Grisel Hamilton standard petal, adaxial surface  97  Lady Grisel Hamilton wing petal, adaxial surface  Lady Grisel Hamilton keel petal, adaxial surface  98  Lady’s Bonnet standard petal, adaxial surface  Lady’s Bonnet wing petal, adaxial surface  99  Lady’s Bonnet keel petal, adaxial surface  Scarlet NAB standard petal, adaxial surface  100  Scarlet NAB wing petal, adaxial surface  Scarlet NAB keel petal, adaxial surface  101  Painted Lady standard petal, adaxial surface  Painted Lady wing petal, adaxial surface  102  Painted Lady keel petal, adaxial surface  103  

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