Science, Faculty of
Botany, Department of
DSpace
UBCV
Sidhu, Gurmel Singh
Doctor of Philosophy - PhD
University of British Columbia
The present study was undertaken to learn whether the kind of host-parasite relationships described by H. H. Flor also exist in Hordeum vulgare:Ustilago hordei system. This system provides additional advantages of genetic analysis over most other known parasitic systems, therefore was more suitable for this type of study. Moreover, a complete genetic analysis of a parasitic system involving a smut fungus has never been attempted before, making the system all the more important. To understand gene-for-gene relationships it becomes of fundamental importance to elucidate the genetics of one member of a parasitic system in order to exploit the genetics of the other. This can be efficiently achieved if the genetic purity of interacting members of a parasitic system is ascertained. In cases where sexual reproduction regularly occurs (such as in heterothallic smuts) a greater magnitude of genetic variability is expected as compared to asexually reproducing fungi or fungi with homothallic mating systems. Since one member of the parasitic system with which the present study deals is a heterothallic smut fungus (Ustilago hordei) the study was initiated by establishing which specific virulence genes were borne by the test cultures. The host, barley (Hordeum vulgare), is naturally inbreeding thus creating no serious problem of variability. Two recessive genes for virulence (Uhv₁ and Uhv₂) were discovered in the two test cultures of U. hordei through backcross method. (The back-cross method is more useful than the F₂ ratios obtainable from a tetrad). The culture having virulence gene Uhv₁ was derived from teliospore E through selfing and was effective on both the varieties Hannchen and Vantage. The second culture, possessing virulence gene Uhv₂, however, originated from teliospore D through selfing and was effective on variety Excelsior. Thus the genotype of the two test cultures possessing virulence genes Uhv₁ and Uhv₂ become Uhv₁Uhv₁UhV₂UhV₂ and UhV₁UhV₁Uhv₂Uhv₂ respectively. On the basis of the two virulence genes (Uhv₁ and Uhv₂) the genetics of resistance and of susceptibility of three barley varieties, Excelsior, Hannchen and Vantage was investigated. It was not practical to test single F₂ barley plants to the two test cultures, therefore the study was extended to F₃ progeny. Segregations obtained from F₃ progenies derived from crosses EXH and EXV revealed that resistance and susceptibility to the two test cultures were inherited independently of one another; the resistance was expressed as a dominant characteristic at both loci. The host alleles discovered through the interactions with culture UhV₁UhV₁Uhv₂Uhv₂ were designated as UhR₁ and Uhr₁; those revealed by interactions with culture Uhv₁Uhv₁UhV₂UhV₂ were designated as UhR₂ and Uhr₂ - The genotype UhR₁UhR₁Uhr₂Uhr₂ was thus assigned to varieties Hannchen and Vantage and Uhr₁Uhr₁UhR₂UhR₂ to variety Excelsior. The interrelationships between the two loci for resistance in the host and the two corresponding loci for virulence in the pathogen thus lead to the conclusion that a gene-for-gene relationship also exists in the Hordeum vulgare:Ustilago hordei system.
A GENETIC STUDY OF HOST:PARASITE RELATIONSHIP INVOLVING HORDEUM VULGARE AND USTILAGO HORDEI by GURMEL S. SIDHU B.Sc. Punjab University, India M.Sc. Punjab University, India A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia A p r i l , 1972. In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copy ing o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date i i ct ABSTRACT The present study was undertaken to learn whether the kind of host-parasite r e l a t i o n s h i p s described by H. H. F l o r also e x i s t i n Hordeum vulgare:Ustilago hordei system. This system provides a d d i t i o n a l advantages of genetic analysis over most other known p a r a s i t i c systems, therefore was more suitable f o r t h i s type of study. Moreover, a complete genetic analysis of a p a r a s i t i c system i n v o l v i n g a smut fungus has never been attempted before, making the system a l l the more important. To understand gene-for-gene r e l a t i o n s h i p s i t becomes of funda-mental importance to elucidate the genetics of one member of a p a r a s i t i c system i n order to e x p l o i t the genetics of the other. This can be e f f i -c i e n t l y achieved i f the genetic p u r i t y of i n t e r a c t i n g members of a p a r a s i t i c system i s ascertained. In cases where sexual reproduction r e g u l a r l y occurs (such as i n h e t e r o t h a l l i c smuts) a greater magnitude of genetic v a r i a b i l i t y i s expected as compared to asexually reproducing fungi or fungi with homothallic mating systems. Since one member of the p a r a s i t i c system with which the present study deals i s a h e t e r o t h a l l i c smut fungus (Ustilago hordei) the study was i n i t i a t e d by e s t a b l i s h i n g which s p e c i f i c virulence genes were borne by the test c u ltures. The host, barley (Hordeum vulgare), i s n a t u r a l l y inbreeding thus creating no serious problem of v a r i a b i l i t y . Two recessive genes for virulence (Uhv^ and Uhy^) were discovered i n the two test cultures of U. hordei through backcross method. (The back-cross method i s more us e f u l than the F2 r a t i o s obtainable from a tetrad). The culture having virulence gene Uhv-^ was derived from teliospore E through s e l f i n g and was e f f e c t i v e on both the v a r i e t i e s Hannchen and i i b Vantage. The second culture, possessing virulence gene Uhy^, however, originated from te l i o s p o r e D through s e l f i n g and was e f f e c t i v e on v a r i e t y E x c e l s i o r . Thus the genotype of the two test cultures possessing v i r u -lence genes Uhv^ and Uhv^ become Uhv-^Uhv^ul^UhVo and UhV^UhV-]Uhv„Uhv2 r e s p e c t i v e l y . On the basis of the two virulence genes (Uhv^ and Uhy^) the genetics of resistance and of s u s c e p t i b i l i t y of three barley v a r i e t i e s , E x c e l s i o r , Hannchen and Vantage was investigated. I t was not p r a c t i c a l to test single F 2 barley plants to the two test cultures, therefore the study was extended to F^ progeny. Segregations obtained from F^ progenies derived from crosses EXH and EXV revealed that resistance and s u s c e p t i b i l i t y to the two test cultures were in h e r i t e d independently of one another; the resistance was expressed as a dominant c h a r a c t e r i s t i c at both l o c i . The host a l l e l e s discovered through the i n t e r a c t i o n s with culture UhV^UhV^Uhv^Uhv^ were designated as UhR^ and Uhr^; those revealed by i n t e r a c t i o n s with culture Uhv^Uhv^UhV^UhV^ were designated as UhRp and Uhr2 - The genotype UhR^UhR^Uhr^Uhr^ was thus assigned to v a r i e t i e s Hannchen and Vantage and Uhr^Uhr^UhRpUt^ to v a r i e t y E x c e l s i o r . The i n t e r r e l a t i o n s h i p s between the two l o c i for resistance i n the host and the two corresponding l o c i for virulence i n the pathogen thus lead to the conclusion that a gene-for-gene r e l a t i o n s h i p also exists i n the Hordeum vulgare:Ustilago hordei system. i i i . TABLE OF CONTENTS Page No. INTRODUCTION AND REVIEW OF LITERATURE 1 Genetic basis of virulence 3 Genetic basis of resistance 5 Gene-for-gene r e l a t i o n s h i p s 8 Host-parasite i n t e r r e l a t i o n s h i p s i n smut fungi. . . 13 Genetics of Hordeum vulgare-Ustilago hordei . . . . system 18 Purpose of the present study 20 METHODS AND MATERIALS 22 I The pathogen (Ustilago-hordei) 22 II The host (Hordeum vulgare) 26 RESULTS 28 I The virulence genes 28 II The resistance genes 39 DISCUSSION 44 SUMMARY 61 BIBLIOGRAPHY 62 APPENDIX A 71 APPENDIX B 72 APPENDIX C 75 i v a . LIST OF TABLES Table Page No. I . Resistance genes found i n various graminaceous hosts i n r e l a t i o n to d i f f e r e n t smut species 7 I I . P a r a s i t i c systems i n which gene-for-gene r e l a t i o n s h i p s have been suggested or demonstrated according to F l o r ' s c r i t e r i o n 13 I I I . P a r a s i t i c systems i n which gene-for-gene r e l a t i o n s h i p s have been suggested or demonstrated according to Person's c r i t e r i o n 14 IV. P a r a s i t i c systems i n v o l v i n g smut f u n g i i n which gene-for-gene r e l a t i o n s h i p s have been suggested . . . . 15 V. Disease r e a c t i o n s shown by f i v e b a r l e y c u l t i v a r s f o l l o w i n g i n o c u l a t i o n w i t h c u l t u r e s of U. hordei obtained, through s e l f i n g , from three p a r e n t a l t e l i o s p o r e s 23 VI. Disease r e a c t i o n s of three b a r l e y v a r i e t i e s E x c e l s i o r , Hannchen and Vantage f o l l o w i n g i n o c u l a t i o n w i t h two t e s t c u l t u r e s of U. hordei derived through s e l f i n g , from t e l i o s p o r e s D and E (or F) 27 V I I . I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h 36 i n f e c t i v e l i n e s of U. hordei a l l of which descended from three p a r e n t a l t e l i o s p o r e s 31 V I I I . I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. hordei derived from p a r e n t a l spore E 32 IX. I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. ho r d e i derived from p a r e n t a l spore D 34 X. I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. hordei derived from p a r e n t a l spore D 37 XI. I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. hordei derived from p a r e n t a l spore F 38 ivb. XII. I n f e c t i o n percentages shown on parental v a r i e t i e s Hannche'n,Vantage and E x c e l s i o r following i n o c u l a t i o n with test cultures of U. hordei V|V^V2V2 a n d V 1 V 1 V 2 V 2 4 0 XIII. F 3 barley l i n e s separated into four classes according to t h e i r i n t e r a c t i o n s with two test cultures of U. hordei and the number of l i n e s i n each class . . . . 41 XIV. F 3 barley l i n e s separated into four classes according to t h e i r i n t e r a c t i o n s with two test cultures of U. hordei and the number of l i n e s i n each class . . . . 41 XV. Expectation, based on F l o r ' s hypothesis of gene-for-gene r e l a t i o n s h i p s , of in t e r a c t i o n s i n v o l v i n g parental, F^ and F 2 generations of the E x H and E x V crosses . . 48 XVI. Expectation, based on F l o r ' s hypothesis of gene-for-gene r e l a t i o n s , of inte r a c t i o n s i n v o l v i n g F 3 l i n e s derived from the E x H and E x V crosses 49 V . LIST OF FIGURES Page No. Fig. I. Diagram showing selfing, crossing (F-^ ) and backcrossing (B.C.) scheme of the three parental teliospores D, E and F 25 ACKNOWLEDGEMENTS I would l i k e to express my s i n c e r e thanks to Prof e s s o r Clayton Person f o r h i s i n v a l u a b l e teaching as w e l l as f o r h i s encouragement throughout t h i s t h e s i s p r o j e c t . My thanks are als o due to Margaret Shand f o r her a s s i s t a n c e w i t h l a b o r a t o r y techniques. F i n a l l y I wish to thank Dr. A.J.F. G r i f f i t h s f o r h i s va l u a b l e suggestions during t h e o r e t i c a l d i s -cussions, and other members of the U s t i l a g o l a b o r a t o r y f o r t h e i r h e l p f u l comments. My s p e c i a l thanks go to Dr. R. J . Bandoni f o r a s s i s t i n g me i n w r i t i n g t h i s t h e s i s . INTRODUCTION AND REVIEW OF LITERATURE The terminology used i n the l i t e r a t u r e on parasitism i s not uniform. This i s p a r t i a l l y caused by the v a r i e t y of r e l a t i o n s h i p s between hosts and parasites and by the number of d i f f e r e n t types of organisms involved. Since v a r i a t i o n i n the use of terms occurs, i t would be worthwhile f i r s t to define some of the terms so that consis-tency i n genetic i n t e r p r e t a t i o n s can be maintained throughout t h i s t h e s i s . The term symbiosis r e f e r s to the " l i v i n g together" of two d i f f e r e n t b i o l o g i c a l species. Parasitism r e f e r s to the type of symbiosis i n which one species benefits at the expense of another. Among the many kinds of p a r a s i t i c r e l a t i o n s h i p s are those that lead to disease development. I t i s th i s kind of p a r a s i t i c r e l a t i o n s h i p , which i s the subject of th i s t h e s i s . In such kind of p a r a s i t i c r e l a t i o n s h i p the disease i s often thought of as being expressed by the host; t h i s i s probably because the host i s so often at a clear disadvantage to the par a s i t e . I t i s however, more correct to view disease as a state to which both host and parasite have contributed, since i t i s only through t h e i r i n t e r a c t i o n with one another that the disease can, i n f a c t , develop. The i n t e r a c t i o n between host and parasite can lead to variable degrees of disease development and expression. Where the disease i s f u l l y expressed, the host i s said to be f u l l y susceptible to the para-s i t e and t h i s , at the same time, implies that the para s i t e i s f u l l y v i r u l e n t towards the host. Where, following p a r a s i t i c invasion, the disease develops poorly or not at a l l , the host i s said to be r e s i s t a n t and t h i s , at the same time, implies that the parasite i s avi r u l e n t towards the host. These terms, together with adjectives such as " p a r t i a l " , "intermediate", or "moderate", are used to describe disease reactions. As Fincham and Day (1963) have pointed out, i t i s important to d i s t i n g u i s h between the term "pathogenicity", which ref e r s to the general capacity of the parasite to i n c i t e disease among members of the host species, and "vi r u l e n c e " which ref e r s to the extent of disease development i n any p a r t i c u l a r instance of the disease. From studies over the past 70 years, mainly with fungal diseases of a g r i c u l t u r a l crops, i t i s now recog-nized that these two c h a r a c t e r i s t i c s , pathogenicity and virulence, may both vary from culture to cult u r e . For example, a culture with a wide range of pathogenicity may be f u l l y v i r u l e n t towards only a few v a r i e t i e s (or c u l t i v a r s ) of the host. This behaviour on the part of the parasite, often referred to as " s p e c i f i c i t y " has provided the basis f o r the separa-t i o n of cultures of many p a r a s i t i c species into "physiologic races". Physiologic races are recognized according th the s p e c i f i c i t y ; each race e l i c i t s a s p e c i f i c pattern of disease reactions when i t i s inoculated to a standard group of " d i f f e r e n t i a l " or " t e s t e r " v a r i e t i e s of the host. Physiologic races were f i r s t demonstrated by Barrus (1911) i n the bean anthracnose fungus, Colletotrichum lindemuthianum. He d i s -tinguished two races, a and g, based on t h e i r contrasting pathogenic reactions on two groups of v a r i e t i e s of the common bean, Phaseolus v u l g a r i s . The type of s p e c i f i c i t y , shown by physiologic races of bean anthracnose fungus, has since been described f o r a large number of other pathogenic organisms. 3. Stakman (1914) a p p l i e d the p h y s i o l o g i c race concept exten-s i v e l y to wheat stem r u s t ( P u c c i n i a g. t r i t i c i ) and h i s approach was followed by numerous other workers usi n g other fungal diseases. The concept of p h y s i o l o g i c races has been extended to non-fungal p a r a s i t e s as w e l l . Among the l a t t e r are nematodes (Howard, 1968; Howard and F u l l e r , 1971); b a c t e r i a ( B r i n k e r h o f f , 1970); i n s e c t s (Hatchett and G a l l u n , 1970); and v i r u s e s (Cockerham, 1970). The phenomenon of s p e c i f i c i t y i s found so commonly i n p a r a s i t e s of a g r i c u l t u r a l crops that the view has developed, among many p l a n t p a t h o l o g i s t s , that s p e c i f i c i t y occurs as a n a t u r a l phenomenon i n most, i f not a l l , pathogenic organisms. Genetic basis of v i r u l e n c e With the d i s c o v e r y of sex i n smuts (Kniep, 1919), and i n r u s t s (Cragie, 1927), i t became p o s s i b l e to d e f i n e p a r a s i t i c s p e c i f i c i t y i n terms of the segregating u n i t of genetics - the gene. E a r l y s t u d i e s on the i n h e r i t a n c e of v i r u l e n c e were conducted on U s t i l a g o species p a r a s i t i c on c u l t i v a t e d oats by N i c o l a i s e n , (1934). He i s o l a t e d the products of s i n g l e t e l i o s p o r e s of smuts ( U s t i l a g o avenae) and demon-s t r a t e d , by t e t r a d a n a l y s i s , that the i n h e r i t a n c e of v i r u l e n c e was Mendelian and c o n t r o l l e d by a s i n g l e genetic locus i n some cases. A t e l i o s p o r e may be homozygous or heterozygous at a p a r t i c u l a r locus i n r e l a t i o n to i t s v i r u l e n c e on a p a r t i c u l a r v a r i e t y . N i c o l a i s e n (1934) a l s o mentioned that f a c t o r s f o r p a t h o g e n i c i t y i n the dikaryon derived from two compatible monosporidial c u l t u r e s may be dominant on one v a r i e t y , r e c e s s i v e on a second and intermediate on a t h i r d . The f i r s t convincing genetic explanation for segregations i n v o l v i n g physiologic races was reported by Johnson and Newton (1940); they showed for stem rust of wheat that Mendelian segregations for virulence and avirulence were obtained on i n d i v i d u a l wheat v a r i e t i e s . Their r e s u l t s also showed that genetic studies of virulence and avirulence could only be c a r r i e d out independently of the physiologic race concept. The same was shown to be true i n the case of smuts by Halisky (1956). Through tetrad analysis of three i n d i v i d u a l teliospores of Ustilago avenae, each representing a d i f f e r e n t loose-smut race, Halisky concluded that h e r i t a b l e , contrasting disease reactions on four v a r i e t i e s were under the control of two complementary gene pairs and that on two other v a r i e t i e s such reactions were i n each case c o n t r o l l e d by a s i n g l e genetic locus. Halisky (1956) also concluded that virulence was dominant i n a l l cases. This conclusion was revised i n a l a t e r paper, i n which virulence was stated to be recessive (Holton, 1959; Johnson, 1960). The revised conclusion has since been confirmed by Holton and Halisky (1960) and by Holton (1964). Si m i l a r investigations were undertaken by Thomas and Person (1965) on barley covered smut, U. hordei. With U. hordei, as with other smuts, the percentage of host plants showing the disease i s taken as the measure of virulence of the smut (and, therefore, of resistance of the host). The percentage may f a l l anywhere between zero and a hundred, depending on the genotypes which i n t e r a c t . Thomas and Person (1965) investigated the genetic basis of low virulence, and showed that contrasting disease reactions, avirulence (0%) vs low virulence (5%), were con t r o l l e d by a s i n g l e genetic locus i n U. hordei; the a l l e l e for low ( 5 7 o ) virulence was recessive. Recently Lade ( 1 9 6 9 ) also demon-strated a s i n g l e gene for intermediate ( 5 % to 3 5 % ) virulence i n U. hordei through tetrad analysis and the virulence gene was recessive i n a c t i o n . Inheritance of virulence has been studied i n i n t e r s p e c i f i c crosses of barley smuts by Cherewick ( 1 9 6 7 ) . Twenty seven sets of dikaryons derived from a cross between U. hordei and U. nigra were tested on f i v e barley v a r i e t i e s . He reported f i v e virulence genes located at separate l o c i . Four of them were recessive while the f i f t h one was dominant on t h e i r respective v a r i e t i e s . Virulence genes have been reported i n about a dozen p a r a s i t i c systems which include a varied number of phytopathogenic organisms, mostly fungi, such as rusts, mildews, and smuts. Such genes have recently been reported, for the f i r s t time, i n an insect parasite, the Hessian f l y (Hatchett and Gallun, 1 9 7 0 ) . From the genetic studies of virulence we learn that, i n general, i t i s mostly under simple Mendelian co n t r o l and usually recessive (Person and Sidhu, 1 9 7 0 ) . In contrast with genes f o r resistance, to be described i n the next section, there has been no demonstrated case of multiple a l l e l e s for v i rulence. Genetic basis of resistance As early as 1 8 9 8 Farrer stated that s u s c e p t i b i l i t y to rust i s hereditary i n wheat (Farrer, 1 8 9 8 ) . The rediscovery of Mendelian laws two years l a t e r l a i d the basis f o r studies of the resistance genes i n the host. The f i r s t report came from B i f f e n ( 1 9 0 5 ) , who obtained a 3:1 r a t i o of s u s c e p t i b i l i t y to r e s i s t a n c e among the ?2 progeny of a cross between a r e s i s t a n t and a s u s c e p t i b l e wheat v a r i e t y i n o c u l a t e d a r t i f i c i a l l y w i t h y e l l o w r u s t , P u c c i n i a glumaamm. His r e s u l t s were recei v e d w i t h s c e p t i c i s m by B u t l e r (1905) and Evans (1911), but i n a l a t e r paper B i f f e n (1912) confirmed that the r u s t r e a c t i o n s were i n h e r i t e d according to Mendel's Laws. The d i f f i c u l t y i n accepting the Mendelian basis of disease r e s i s t a n c e perhaps emerged from the use of impure c u l t u r e s of the pathogens. Burkholder (1918) and McRostie (1919, 1921) showed t h i s to be h i g h l y probable i n e a r l y s t u d i e s of disease r e s i s t a n c e . Working w i t h anthracnose of beans caused by C o l l e t o t r i c h u m lindemuthianum, McRostie demonstrated that two patho-genic c u l t u r e s , each capable of g i v i n g a 3:1 r a t i o of r e s i s t a n c e to s u s c e p t i b i l i t y i n a segregating F p o p u l a t i o n , revealed, when inocu-2 l a t e d as a mixture, a 9:7 r a t i o i n the same F po p u l a t i o n . Many 2 stud i e s have s i n c e been c a r r i e d out, i n which r e s i s t a n c e and s u s c e p t i -b i l i t y have been found to segregate according to simple Mendelian p r i n -c i p l e s (See Person and Sidhu, 1970). A summary of the data on r e s i s t a n c e genes, found i n gramina-ceous hosts to operate against d i f f e r e n t smut species i s given i n Table I . This t a b l e shows that i n h e r i t a n c e i s u s u a l l y monogenic, w i t h r e s i s t a n c e to smuts being u s u a l l y dominant. These reports a l s o show, i n review, that wherever a c o l l e c t i o n of t e l i o s p o r e s was used as inoculum f o r d i f f e r e n t i a t i n g r e s i s t a n c e genes, the r e s u l t s were general-l y i n c o n c l u s i v e (Johnston, 1934; W e l l s , 1958). By c o n t r a s t , c u l t u r e s 7. TABLE I Resistance genes found i n various graminaceous hosts i n r e l a t i o n to d i f f e r e n t Smut sp e c i e s . P a r a s i t i c No. of Reports of i n h e r i t a n c e Reports of R-Systems Reports being: genes being: Reviewed Mono- D i - T r i - Poly-genic genic genie genie Dominant Rece; Avena-Ustilago avenae 23 18 8 2 5 17 1 Avena-U.levis 16 10 4 1 7 12 Avena-U.koleri 1 1 1 - - 1 Hordeum-U.nuda 16 16 5 - - 16 Hordeum-U.nigra 3 3 3 Hordeum- U-hordei 3 3 1 - - 3 1 T r i t i c u m -U . t r i t i c i 8 7 3 2 - 7 Triticum-U a g r o p y r i 1 1 1 T r i t i c u m -U r o c y s t i s t r i t i c i 1 1 1 Zea-U.Zeae 1 - 1 Sorghum-Sphacelo-theca s o r g h i 1 - 1 - 1 Sorghum-S.cruenta 1 - 1 - - 1 Eleusine-U.cremeri 1 1 1 76 61 23 6 13 64 2 8. derived from g e n e t i c a l l y s t a b l e and homozygous races ( L a r t e r and Enns, 1962; L a r t e r , 1962; Waud and Metzger, 1970), or from i n d i v i d u a l t e l i o s p o r e s (Metcalf, 1962; M e t c a l f and Helgason, 1962), y i e l d e d c o n c l u s i v e and s t a t i s t i c a l l y acceptable r e s u l t s . Recent st u d i e s have been c a r r i e d out u s i n g , as inoculum, compatible s p o r i d i a l combinations derived from s i n g l e t e l i o s p o r e s (Cherewick and Buchannon, 1969; Cherewick and McKenzie, 1969). The r a t i o s obtained i n these studies were s t a t i s t i c a l l y very c l o s e to the i d e a l r a t i o s . These st u d i e s i l l u s t r a t e the importance of u s i n g g e n e t i c a l l y homogenous c u l t u r e s when attempting to i d e n t i f y r e s i s t a n c e genes i n the host. Gene-for-gene r e l a t i o n s h i p s Although v i r u l e n c e and a v i r u l e n c e on the p a r t of a pathogen and s u s c e p t i b i l i t y and r e s i s t a n c e on the p a r t of a host are both under the c o n t r o l of t h e i r i n d i v i d u a l genetic systems, they are interdependent f o r t h e i r phenotypic expressions. Thus i t becomes evident that the type of r e a c t i o n ( s ) expressed by any combination of host and p a r a s i t e must be the r e s u l t of i n t e r a c t i o n s between two otherwise separate genetic systems. F l o r designed experiments to i n v e s t i g a t e the basis of such i n t e r a c t i o n s u s i n g f l a x (Linum usitatissimum) and f l a x r u s t (Melampsora l i n i ) . In h i s s t u d i e s he obtained a simple 3:1 r a t i o of a v i r u l e n t to v i r u l e n t by i n o c u l a t i n g F2 segregating populations of the f l a x r u s t to appropriate v a r i e t i e s or s e l e c t i o n s of the f l a x host. S i m i l a r l y he obtained a 3:1 r a t i o of r e s i s t a n t to s u s c e p t i b l e p l a n t s when F2 popu-l a t i o n s of f l a x were exposed to appropriate t e s t c u l t u r e s of f l a x r u s t . I t i s important to n o t i c e that the 3:1 r a t i o s were obtained only under appropriate c o n d i t i o n s of t e s t i n g . The reason f o r t h i s was made c l e a r i n genetic experiments i n which F l o r (see F l o r 1956) c a r r i e d out p a r a l l e l s t u d i e s on both members of the h o s t : p a r a s i t e r e l a t i o n -ship . By i n o c u l a t i n g c u l t u r e s 22 and 24 of f l a x r u s t on an F2 segregating p o p u l a t i o n derived from a cross between f l a x v a r i e t i e s Bombay and Ottawa F l o r (1947) obtained a 9:3:3:1 r a t i o , which would be expected i f the two v a r i e t i e s were each homozygous f o r one of the two independently segregating dominant genes f o r r e s i s t a n c e . P r e v i o u s l y F l o r (1946) crossed the two races of M_;_ l i n i (races 22 and 24) which had been used to r e v e a l t h i s segregation i n the host. An F2 segre-ga t i n g r u s t p o p u l a t i o n , derived from the cross of race 22 and 24, was i n o c u l a t e d to v a r i e t i e s Bombay and Ottawa. A r a t i o of 9:3:3:1 was obtained as expected on the basis of two independently segregating v i r u l e n t genes i f each race possessed one of the two homozygous r e c e s s i v e v i r u l e n t genes. From t h i s type of evidence F l o r concluded that disease develops f u l l y only i f the r e s i s t a n c e gene i n the host i s overcome by a s p e c i f i c and r e l a t e d v i r u l e n c e gene- i n the p a r a s i t e . A v i r u l e n c e i s expressed on a r e s i s t a n t host only where there e x i s t s no appropriate and r e l a t e d gene(s) i n the p a r a s i t e to counteract the r e l a t e d r e s i s t a n c e gene(s) i n the host. F l o r (1956), from h i s ex-haustive experimentation demonstrated that r e s i s t a n c e caused by a s i n g l e gene was overcome by a s p e c i f i c and r e l a t e d s i n g l e v i r u l e n c e gene; i f there were two r e s i s t a n c e genes i n the host, the p a r a s i t e a l s o needed to possess two corresponding v i r u l e n c e genes to n u l l i f y the r e s i s t a n c e , and so on. This type of numerical equivalence between the R-genes and V-genes was v e r i f i e d by making a s e r i e s of i n t e r -crosses among the f l a x d i f f e r e n t i a l s ( F l o r , 1941; 1947; 1951; 1954) on one hand, and among the r u s t c u l t u r e s ( F l o r , 1942; 1946; 1955; 1965) on the other. In a l l , 26 p a i r s of r e s i s t a n c e genes were shown to be counteracted by equal and opposite number of v i r u l e n c e genes. This l e d F l o r to formulate h i s now widely accepted hypo-t h e s i s f o r gene-for-gene r e l a t i o n s h i p . Gene-for-gene r e l a t i o n s h i p s have been f u l l y discussed by F l o r (1955, 1956, 1959, 1961, 1971), Person (1959, 1966, 1967), and Day (1960, 1966, 1968). Other reviews have been w r i t t e n by Zadoks (1959), Flangas and Dickson (1961), Ross (1961), Rowell, Loegering and Powers (1963), and Favret (1971). The concept of the gene-for-gene r e l a t i o n s h i p as discussed by Person (1959) i s of s p e c i a l i n t e r e s t here. F l o r ' s c r i t e r i o n of gene-for-gene r e l a t i o n s h i p i s based on the p a r a l l e l genetic st u d i e s of both members of a p a r t i c u l a r system and thus l i m i t s the a p p l i c a t i o n of h i s a n a l y t i c a l approach to systems f o r which the l i f e - c y c l e s of the host as w e l l as of the p a r a s i t e are known. However, there are many p a r a s i t i c systems i n which i t i s not as yet p o s s i b l e to c a r r y out genetic st u d i e s of both i n t e r a c t i n g p a r t n e r s , and where a l t e r n a t i v e a n a l y t i c a l methods are needed. Person (1959), a p p r e c i a t i n g F l o r ' s gene-for-gene hypothesis, took a step forward by t h e o r i z i n g the whole concept. By a n a l y s i n g the published data on the Linum-Melampsora and Solanum-Phytophthora systems, he gave a comprehensive d e s c r i p t i o n of the various features of the gene-for-gene r e l a t i o n s h i p . Person thus concluded that such r e l a t i o n s h i p s should occur as a general r u l e r a t h e r than an exception i n p a r a s i t i c systems, and he devised methods f o r t e s t i n g gene-for-gene r e l a t i o n s h i p s i n systems f o r which genetic data was incomplete or not a v a i l a b l e . Person's concept of i d e a l gene-for-gene r e l a t i o n s h i p pre-supposes two a l t e r n a t i v e c o n d i t i o n s i n expression of r e s i s t a n c e vs. s u s c e p t i b i l i t y i n the host and a v i r u l e n c e vs. v i r u l e n c e i n the p a r a s i t e . I f n i s the number of i n t e r a c t i n g l o c i i n a p a r t i c u l a r system w i t h two a l t e r n a t i v e expressions, and assuming complete dominance, then the t o t a l number of phenotypes e i t h e r i n the host or i n the p a r a s i t e popu-l a t i o n w i l l be 2 n (.cf. Day, 1956). Assuming every host v a r i e t y i s tested against every p a r t i c u l a r race, a p a r t i c u l a r p a t t e r n of i n t e r -a c t i o n s , showing a number of d e f i n i t e c h a r a c t e r i s t i c s , i s p r e d i c t e d . Person concluded that the c h a r a c t e r i s t i c s shown by h i s i d e a l system could be g e n e r a l l y used as c r i t e r i o n s of gene-for-gene r e l a t i o n s h i p s i n other p a r a s i t i c systems. In contrast w i t h F l o r ' s c r i t e r i o n , Person's i s l e s s p r e c i s e . I t has the advantage however, of being a p p l i c a b l e to those p a r a s i t i c systems i n which genetic s t u d i e s have not been, or cannot be, c a r r i e d out. Based on these c r i t e r i a Person, Samborski and Rohringer (1962) formulated a d e f i n i t i o n of the gene-for-gene r e l a t i o n s h i p , which i s reproduced as f o l l o w s : "A gene-for-gene r e l a t i o n s h i p exists when the presence of a gene i n one population i s contingent on the continued presence of a gene i n another population, and where the i n t e r a c t i o n between the two genes leads to a s i n g l e phenotypic expression by which the presence or absence of the relevant gene i n either organism may be recognized". The foregoing discussion brings out two c r i t e r i a f o r the evaluation of p a r a s i t i c systems: (1) F l o r ' s c r i t e r i o n (2) Person's c r i t e r i o n F l o r ' s c r i t e r i o n depends on the demonstration of Mendelian segregations i n both members of the system. Host v a r i e t i e s "A" and "B" are used to generate an F2 population i n which segregation i s revealed through the use of appropriate cultures "x" and "y". Cultures "x" and "y" are then used as parents of a cross i n which genetic segregation i s revealed by v a r i e t i e s "A" and "B". Person's c r i t e r i o n r e l a t e s to the pattern of i n t e r a c t i o n s which i s generated when several host v a r i e t i e s are inoculated with several or many d i f f e r e n t cultures of the pathogen (as i s done i n "race" surveys). Where gene-for-gene r e l a t i o n s h i p s are operating, these patterns w i l l e xhibit a number of c h a r a c t e r i s t i c properties. Person's c r i t e r i o n involves the recognition of these c h a r a c t e r i s t i c properties and t h i s r ecognition can lead to the i d e n t i f i c a t i o n of the "units of i n t e r -a c t i o n " within the system. Person's c r i t e r i o n does not depend on genetic data, and i s g e n e t i c a l l y less e x p l i c i t than F l o r ' s c r i t e r i o n . Its main advantage i s that i t can be used i n systems i n which genetic data are not a v a i l a b l e . And, where genetic studies can be eventually TABLE I I . P a r a s i t i c systems i n which gene-for-gene r e l a t i o n s h i p s have been suggested or demonstrated according to F l o r ' s C r i t e r i o n . System Author(s) Year Linum-Melampsora l i n i F l o r 1942 Triticum-Puccinia g. t r i t i c i Watson 1958 Luig and Watson 1961 Loegering and Powers J r . 1962 Green 1964,1966 Rondon et a l 1966 Williams et a l 1966 Knott and Kao 1968 Kao and Knott 1969 Gough and Williams 1969 Triticum-P. recondita Samborski and Dyck 1968 Dyck and Samborski 1968 Bartos et a l 1969 F r e i t a s 1969 Zea-P. sorghi Flangas and Dickson 1957,1961 Dickson et a l 1959 Hooker and Russell 1962 Triticum-Erysiphe g. t r i t i c i Powers and Sando 1957,1960 Leijerstam 1968 S l e s i n s k i and Ellingboe 1969,1970 Hordeum-E.g.hordei Moseman 1957,1959 Moseman and Schaller 1960,1962 Hiura 1964 Moseman et a l 1965 Hiura and Heta 1969 Avena-Ustilago avenae Holton 1964 Halisky 1965 Malus-Venturia inaequalis Day 1960 Bagga and Boone 1968 Triticum-Mayetiola destructor Hatchett and Gallun 1970 TABLE I I I P a r a s i t i c systems i n which gene-for-gene r e l a t i o n s h i p s have been suggested or demonstrated according to Person's C r i t e r i o n System Author(s) Year T r i t i c u m - P u c c i n i a s t r i i f o r m i s Zadoks 1961 Lewellen et a l 1967 H e l i a n t h u s - P . h e l i a n t h i Sackston 1962 Miah and Sackston 1970 Avena-P.g. avenae Martens et a l 1970 Coffea-Hemileia v a s t a t r i x Noronha-Wagner and Bettencourt 1967 T r i t i c u m - U s t i l a g o t r i t i c i Oort 1963 T r i t i c u m - T i l l e t i a c a r i e s Metzger and Trione 1962 Triticum-T. Controversa Holton et a l 1968 Triticum-T. species S c h a l l e r et a l 1960 Solanum-Phytophthora i n f e s t a n s Toxopeus 1956 Solanum-Synchytrium endobioticum Howard 1968 Solanum-Heterodera r o s t o c h i e n s i s Jones and P a r r o t t 1965 Lycopersicon-Cladosporium fulvum Day 1956 Van Dijkman and Kaars S i j p e s t e l j n 1971 O r y z a - P y r i c u l a r i a oryzae Kiyosawa 1967 Phaseol u s - C o l l e t o t r i c h u m lindemuthianum Albersheim et a l 1969 Malus-Venturia i n a e q u a l i s Boone and K e i t t 1957 Fincham and Day 1963 Gossypium-Xanthomonas malvacearum B r i n k e r h o f f 1970 c a r r i e d out, Person's c r i t e r i o n can be u s e f u l l y employed i n the planning of genetic experiments. Both are u s e f u l and mutually supporting. Evidence of gene-for-gene r e l a t i o n s h i p s , taken from the l i t e r a t u r e and grouped according to whether F l o r ' s or Person' c r i t e r i o n was used, i s presented i n Table I I and I I I . H o s t - p a r a s i t e i n t e r r e l a t i o n s h i p s i n smut fun g i There e x i s t s no convincing evidence f o r gene-for-gene r e l a t i o n s h i p s i n any of the smut f u n g i . A few cases of such r e-l a t i o n s h i p s have been reported (Table IV) and t h e i r b r i e f i n t e r -p r e t a t i o n s f o l l o w : TABLE IV P a r a s i t i c systems i n v o l v i n g smut fun g i i n which gene-for-gene r e l a t i o n s h i p s have been suggested. System Author Year T r i t i c u m - T i l l e t i a species S c h a l l e r et a l 1960 Metzger and Trione 1962 Holton et a l 1968 T r i t i c u m - U s t i l a g o t r i t i c i Oort 1963 Avena-U. avenae Holton 1964 H a l i s k y 1965 16. The T r i t i c u m - T i l l e t l a system: S c h a l l e r et a l (1960) working w i t h one race from each of the bunt organisms, T i l l e t i a c a r r i e s ( T - l ) and T. f o e t i d a (L-9) against two v a r i e t i e s of wheat, M a r t i n and E l g i n , were the f i r s t to i n d i c a t e gene-for-gene r e l a t i o n s h i p s i n smut f u n g i . V a r i e t i e s M a r t i n and E l g i n were r e s i s t a n t and s u s c e p t i b l e r e s p e c t i v e l y to both races. Two r e s i s t a n c e genes (M, M^) were i d e n t i f i e d i n M a r t i n . Genes M and M^ n u l l i f y v i r u l e n c e of races T - l and L-9 r e s p e c t i v e l y . Based on t h i s s p e c i f i c i t y of i n t e r a c t i o n s between the M-genes and respec-t i v e races S c h a l l e r et a l suggested a case of gene-for-gene r e l a t i o n -ship . Metzger and Trione (1962), working w i t h seven R-genes present i n vulgare wheat v a r i e t i e s i n r e l a t i o n to nineteen races of bunt-fungus, T. f o e t i d a , reported a second p o s s i b l e example of gene-for-gene r e l a t i o n s h i p i n v o l v i n g smut f u n g i . The f o l l o w i n g i n t e r a c t i o n s between the s p e c i f i c R-genes and known races were shown to be operative i n the system: M-gene vs. race T-14; M -gene vs. race T - l and 2 Hohenheimer-gene vs. race T-10. S i x t e e n other races showed v i r u l e n c e on v a r i e t i e s w i t h two or more genes f o r r e s i s t a n c e . The t h i r d p o s s i b l e example of gene-for-gene r e l a t i o n s h i p i s based on the observed i n t e r a c t i o n s , i n nature, between R-gene vs. V-gene complexes i n the T r i t i c u m - T i l l e t i a controversa system. In T i l l e t i a more than twenty f i v e v i r u l e n c e patterns (Pathogenic races) are known based on d i f f e r e n t combinations of at l e a s t h a l f a dozen V-genes. About the same number of R-gene complexes are a l s o known i n .the host. Host r e s i s t a n c e to t h i s species i s found to be s p e c i f i c . Consequently, the R-gene complex f u n c t i o n s as a screen against the V-gene complex of the pathogen. During repeated exposures to any given host-r e s i s t a n c e complex, the v i r u l e n c e components of the pathogen pop u l a t i o n come i n t o a c t i o n to n u l l i f y the r e s i s t a n c e by uncovering of one or more r e l e v a n t V-genes p r e v i o u s l y obscured i n the whole complex. Thus, Holton et a l (1968) v i s u a l i z e d a k i n d of h o s t - p a r a s i t e i n t e r p l a y i n nature as e x e m p l i f i e d by T. controversa i n the P a c i f i c North West of the United S t a t e s . The T r i t i c u m - U s t i l a g o t r i t i c i system: An i n s t a n c e of a gene-for-gene r e l a t i o n s h i p i s a l s o i n d i c a t e d f o r the T r i t i c u m - U s t i l a g o t r i t i c i system (Oort, 1963). For the de-l i m i t a t i o n of s p e c i f i c i t y based on a gene-for-gene r e l a t i o n s h i p Oort assumed two sets of r e s i s t a n c e genes and two sets of i n c o m p a t i b i l i t y genes i n the host, corresponding w i t h four sets of v i r u l e n c e f a c t o r s i n the pathogen. I n t e r a c t i o n s based on eight variety-groups of wheat w i t h s i x pathogen races of the loose smut fungus were explained on the b a s i s of gene-for-gene r e l a t i o n s h i p s . The Avena-Ustilago avenae system: Holton (1964) and H a l i s k y (1965) formulated a case of gene-for-gene r e l a t i o n s h i p based on separate and u n r e l a t e d s t u d i e s of r e s i s t a n c e i n the host and v i r u l e n c e i n the pathogen. Monogenic c o n t r o l of r e s i s t a n c e and s u s c e p t i b i l i t y i n c e r t a i n oat v a r i e t i e s (Murphy and Coffman, 1961) and monogenic c o n t r o l of v i r u l e n c e and a v i r u l e n c e of c e r t a i n pathogen races (Holton and H a l i s k y 1960) i s i n d i c a t i v e of gene-18. for-gene r e l a t i o n s h i p s . Because r e s i s t a n c e was found to be u s u a l l y dominant, and v i r u l e n c e u s u a l l y r e c e s s i v e , the case was considered to be a n a t u r a l outcome of h o s t - p a r a s i t e i n t e r r e l a t i o n s h i p s i n t h i s system; but i n the absence of experimental evidence of the type r e q u i r e d by F l o r ' s c r i t e r i o n , such a case i s a matter of record r a t h e r than a proof. In a l l cases of host p a r a s i t e i n t e r a c t i o n s r e f e r r e d to above the data are not s u f f i c i e n t to support any f i r m c o n c l u s i o n . None of the systems attempt to formulate a p u t a t i v e genotype scheme of V-genes i n r e l a t i o n to known R-genes ( c f . Day, 1956; Toxopeus, 1956; Noronha-Wagner and Bettencourt, 196.7) or of R-genes i n r e l a t i o n to known V-genes ( c f . Boone and K e i t t , 1 9 5 7 ; Fincham and Day, 1963). Genetics of Hordeum vulgare - U s t i l a g o hordei System. R-genes:- The f i r s t c o n c l u s i v e evidence of r e s i s t a n c e genes i n b a r l e y came from Shands (1956). Crosses between a r e s i s t a n t v a r i e t y , B r a c h y t i c w i t h s u s c e p t i b l e v a r i e t i e s , Chevron and Colsess were studie d i n F , F and i n backcrosses. Resistance to race 6 of covered 2 3 smut of b a r l e y (U. hordei) was found to be due to one dominant gene i n both crosses. Wells (1958), u s i n g the same race of U. h o r d e i , i n v e s t i g a t e d r e a c t i o n s of F progeny of d i a l l e l e crosses among f i v e r e s i s t a n t 2 v a r i e t i e s (O.A.C. 21, T i t a n , O g a l i t s u , Anoidium and J e t ) and a common s u s c e p t i b l e v a r i e t y , J u n i o r . In every cross h i s conclusions were based on a study of F^ and F^ data. He experienced d i f f i c u l t i e s i n i n t e r -p r e t i n g h i s data due to somewhat low i n f e c t i o n but a f t e r applying a 19. c o r r e c t i o n f a c t o r t o the o b s e r v e d F2 d a t a he c o u l d i d e n t i f y f o u r R-genes; two dominant ( U h l and Uh2) and two r e c e s s i v e (uh3 and uh4) i n a c t i o n . C o t t i n g h a m (1958), u s i n g r a c e 6, d e m o n s t r a t e d two f a c t o r p a i r s f o r r e s i s t a n c e i n a c r o s s between v a r i e t i e s P a n n i e r and Odessa. Other s t u d i e s on i n h e r i t a n c e of r e s i s t a n c e i n b a r l e y c u l t i v a r s a g a i n s t more t h a n one b a r l e y smut have been r e p o r t e d by M e t c a l f ( 1 9 6 2 ) , and by C h e r e w i c k and Buchannon (1969). M e t c a l f s t u d i e d t h e F^ , F2 and F^ g e n e r a t i o n s o f c r o s s e s between a r e s i s t a n t v a r i e t y , J e t , and s u s c e p -t i b l e v a r i e t i e s , Vantage and P l u s h . The p r o b a b l e e x i s t e n c e o f a s i n g l e dominant gene o p e r a t i n g a g a i n s t U. h o r d e i was r e p o r t e d . L i k e w i s e , C h e r e w i c k and Buchannon (1969) r e p o r t e d a s i n g l e dominant r e s i s t a n t gene, e f f e c t i v e a g a i n s t r a c e 4 of U. h o r d e i . T h i s gene was p r e s e n t i n two v a r i e t i e s , P a n n i e r and E x c e l s i o r , and c o n f e r r e d r e s i s t a n c e t o two s p e c i e s o f b a r l e y smut, U. h o r d e i and U. n i g r a . Such s t u d i e s have an i m p o r t a n t b e a r i n g on t h e b r e e d i n g f o r r e s i s t a n c e a g a i n s t more t h a n one d i s e a s e . V-genes: T h e r e seem: t o be o n l y two r e p o r t s c o n c e r n i n g t h e i n h e r i t a n c e o f v i r u l e n c e genes i n U s t i l a g o h o r d e i . The f i r s t r e p o r t , by Thomas and P e r s o n (1965), d e m o n s t r a t e d one gene f o r low v i r u l e n c e and t h e secon d , by Lade (1969), i d e n t i f i e d a n o t h e r gene f o r i n t e r m e d i a t e v i r u l e n c e . Thomas and P e r s o n o b t a i n e d t h e i r r e s u l t s from F2 s e g r e g a t i o n s o f t h e V-gene on two c u l t i v a r s , Gateway and O l l i w h i l e Lade s t u d i e d F3 p r o g e n i e s i n a d d i t i o n t o F2 s e g r e g a t i o n s on t h e v a r i e t y P a n n i e r . V i r u l e n c e was r e c e s s i v e i n b o t h t h e c a s e s . 20. Purpose of the Present Study The Hordeum vulgare - U s t i l a g o hordei system provides an e x c e l l e n t opportunity f o r studying the genetic basis of host-p a r a s i t e i n t e r a c t i o n s . The host, b a r l e y (Hordeum vulgare L.) i s g e n e t i c a l l y w e l l known. Since i t i s d i p l o i d and s e l f f e r t i l i z i n g , i t i s p o s s i b l e to develop l i n e s which are homozygous, or n e a r l y so, and thus to o b t a i n u n i f o r m i t y i n disease r e a c t i o n s . Crossing can be achieved e a s i l y , w i t h p r a c t i c e . The p a r a s i t e , covered smut ( U s t i l a g o hordei (Pers.) Lagerh.), i s a l s o s u i t e d to genetic s t u d i e s f o r a number of reasons: ( i ) on germination a t e l i o s p o r e immediately undergoes meiosis and forms an ordered, l i n e a r t e t r a d of h a p l o i d s p o r i d i a ; ( i i ) s p o r i d i a can be i s o l a t e d through micromanipulation and each gives r i s e through y e a s t - l i k e budding to a compact colony; s p o r i d i a l c o l o n i e s can be r e p l i c a - p l a t e d ; ( i i i ) the s p o r i d i a l c u l t u r e s can be grown on completely defined medium, e i t h e r on p l a t e s or i n shake c u l t u r e s ; ( i v ) i t i s r e l a t i v e l y easy to make c o n t r o l l e d matings of compatible s p o r i d i a l c u l t u r e s (the mating system i s b i p o l a r , and compatible matings lead to the production of d i k a r y o t i c i n f e c t i o u s hyphae, or " s u c h f c l d e n " , which i n i t i a t e the p a r a s i t i c stage of the l i f e c y c l e ) ; (v) i n f e c t i o n normally occurs i n the e a r l y s e e d l i n g stage; o p p o r t u n i t i e s f o r contamination are t h e r e f o r e r e s t r i c t e d to the time of germination and e a r l y s e e d l i n g development; ( v i ) the disease culminates i n the formation of t e l i o s p o r e s which i n U. hordei and other covered smuts, are contained w i t h i n host t i s s u e , thereby reducing the p o s s i b i l i t y of contamination through wind d i s p e r s a l . Having these s e v e r a l advantages i n mind, i t was the purpose of t h i s study t o : ( i ) e s t a b l i s h the genetic basis f o r the c o n t r a s t i n g patterns of pathogenic s p e c i f i c i t y shown by two c u l t u r e s of U. h o r d e i ; ( i i ) to i n v e s t i g a t e the genetic basis of the d i f f e r e n t p atterns of r e s i s t a n c e and s u s c e p t i b i l i t y shown by the host i n r e l a t i o n to these two c u l t u r e s ; and ( i i i ) to examine the r e s u l t s of these i n v e s t i g a t i o n s i n order to determine whether gene-for-gene r e l a t i o n s h i p s of the k i n d described by F l o r and other i n v e s t i g a t o r s ( c f . Table II) are a l s o operating i n Hordeum vulgare - U s t i l a g o hordei system. METHODS AND MATERIALS I . The Pathogen ( U s t i l a g o hordei (Pers.) Lagerh.) A set of four s p o r i d i a produced by a s i n g l e t e l i o s p o r e of U s t i l a g o hordei i s numbered from one to four, number one being borne at the apex of the promycelium. Since the mating system i n t h i s organism i s b i p o l a r (+ and - ) , two out of four s p o r i d i a are plus and two minus. A compatible combination o r i g i n a t e s where plus and minus come together and an i n f e c t i v e dikaryon i s formed; the dikaryon so formed i s an o b l i g a t e p a r a s i t e on b a r l e y . Four i n f e c t i v e d i k a r y o t i c l i n e s are produced when the s p o r i d i a (gamete) of a s i n g l e t e l i o s p o r e are brought together i n a l l p o s s i b l e combinations ( " s e l f i n g " ) and eight such l i n e s are p o s s i b l e when the gametic products of two i n d i v i d u a l t e l i o s p o r e s are combined i n a l l combinations ("crossing"). The disease culminates upon formation of monkaryotic d i p l o i d t e l i o -spores i n the b a r l e y s p i k e s . Three sets of l i n e a r t e t r a d s derived from three t e l i o s p o r e s , D, E, and F, formed the basis of the present study. Twelve s p o r i d i a l l i n e s thus derived were c u l t u r e d on complete medium (Appendix A) as modified by Hood (1966) from Vogel (1956). Stock c u l t u r e s on agar medium as w e l l as s i l i c a - g e l c u l t u r e s were prepared f o r short term and long term uses r e s p e c t i v e l y . Both types of c u l t u r e s were stored i n a r e f r i g e r a t o r at 2-5° C. The mating types of a l l c u l t u r e s were determined by t e s t i n g them against the two standard mating types, I + 4 and E^; matings which r e s u l t e d i n the formation of "suchfHden" were judged to be compatible. (Bauch, 1923). 23. In a l l , 36 d i k a r y o t i c l i n e s were obtained by " s e l f i n g " and " c r o s s i n g " the products of the t e l i o s p o r e s i n a l l compatible combina-t i o n s . To t e s t f o r p a t h o g e n i c i t y , ( i e . f o r v i r u l e n t or a v i r u l e n t r e a c t i o n s ) , each d i k a r y o t i c l i n e was i n o c u l a t e d to f i v e b a r l e y c u l t i v a r s : CI-1248 E x c e l s i o r , CI-541 Hannchen, CI-923 L i o n , CI-936 T r e b i and CI-7324 Vantage. These are f u r t h e r l i s t e d i n Table V, together w i t h t h e i r disease r e a c t i o n s to the cultureswhich were derive d , by s e l f i n g , from the three p a r e n t a l t e l i o s p o r e s . TABLE V Disease r e a c t i o n s shown by f i v e b a r l e y c u l t i v a r s f o l l o w i n g i n o c u l a t i o n w i t h c u l t u r e s of U. hordei obtained, through s e l f i n g , from three p a r e n t a l t e l i o s p o r e s . S e l f e d Cultures of: Disease r e a c t i o n s * of: Teliospo r e s E x c e l s i o r Hannchen Vantage T r e b i L i o n D S R R S S E R S S S S F R S S S S * R = r e s i s t a n t ; S = s u s c e p t i b l e S p o r i d i a l c u l t u r e s (Haploid) were grown i n l i q u i d medium (Appendix A) at 22° C i n shake c u l t u r e s f o r 24-30 hours. Appropriate d i k a r y o t i c l i n e s ( d i p l o i d ) , synthesized by mixing equal amounts of two mating types, were f u r t h e r grown i n shakers f o r about 12 hours. Barley seeds 24. to be i n o c u l a t e d were surface s t e r i l i z e d w i t h formaldehyde, washed, d r i e d and placed i n 3 dram v i a l s , 100 seeds to a v i a l . For i n o c u l a -t i o n , 8 ml of inoculum mixture, c o n t a i n i n g 4 ml of each of the two mating types was poured over the seeds. A negative pressure of 20 inches was a p p l i e d f o r 25-30 minutes i n a vacuum chamber to ensure i n t i m a t e contact of s p o r i d i a w i t h the p e r i c a r p l a y e r of the seeds. Excess inoculum was poured from the v i a l s and the seeds were put i n t o small c o i n envelopes keeping the tops open to a i r dry the seeds f o r about 36-40 hours. The seeds thus i n o c u l a t e d were planted i n s o i l , e i t h e r i n the greenhouse or the f i e l d . The percentages of i n f e c t i o n were based on the number of p l a n t s i n f e c t e d and a p l a n t was considered i n f e c t e d i f one or more spikes were smutted. These percentages were based on 100 seeds sown under f i e l d c o n d i t i o n s or 50 seeds sown under greenhouse c o n d i t i o n s . During the summer of 1969, the f i r s t experiment was designed to der i v e maximum i n f o r m a t i o n concerning the nature of v i r u l e n c e of a l l the s p o r i d i a l l i n e s obtained from the three p a r e n t a l t e l i o s p o r e s . Two experiments were run co n c u r r e n t l y to v e r i f y the r e s u l t s . On the basis of i n f o r m a t i o n obtained, a plan of f u t u r e work f o r i n v e s t i g a t i n g the genetics of v i r u l e n c e was formulated; t h i s i s shown d i a g r a m a t i c a l l y i n Figure 1. This diagram attempts to show that the three p a r e n t a l t e l i o s p o r e s were crossed i n a l l p o s s i b l e ways. The three crosses each gave r i s e to eight F-L dikaryons. S i n g l e t e l i o s p o r e s , taken from s e l e c t e d F^ dikaryons, were then backcrossed to the p a r e n t a l t e l i o s p o r e s ; each of these backcrosses a l s o gave r i s e to eight d i k a r y o t i c l i n e s . A 4:4 r a t i o , 25. F i g . 1. Diagram showing s e l f i n g , c r o s s i n g (F^) and backcrossing (B.C.) scheme of the three p a r e n t a l t e l i o s p o r e s , D,E and F. (For e x p l a n a t i o n see page 24). of v i r u l e n t to a v i r u l e n t , o c c u r r i n g w i t h i n a group of e i g h t -progeny dikaryons, would i n d i c a t e that one of the t e l i o s p o r e s p a r e n t a l to the group was heterozygous f o r p a t h o g e n i c i t y . With t h i s p lan of experimentation i t was there f o r e p o s s i b l e to t e s t each of the three o r i g i n a l p a r e n t a l spores f o r h e t e r o z y g o s i t y , as w e l l as to t e s t f o r genetic d i f f e r e n c e s between the p a r e n t a l spores. I I . The Host (Hordeum vulgare L.) D i a l l e l e crosses among f i v e b a r l e y v a r i e t i e s , CI-1248 E x c e l s i o r (E), CI-541 Hannchen (H), CI-7324 Vantage (V), CI-936 Tr e b i (T), and CI-923 L i o n ( L ) , were made. The study however was confined to three .crosses (ExH, ExV and HxV) i n v o l v i n g three v a r i e t i e s which showed c o n t r a s t i n g r e a c t i o n s to two s e l e c t e d t e s t c u l t u r e s of U s t i l a g o h o r d e i . One of the t e s t c u l t u r e s had been derived through s e l f i n g of t e l i o s p o r e D, the other through s e l f i n g of t e l i o s p o r e E; v a r i e t y E x c e l s i o r was s u s c e p t i b l e to the f i r s t , and r e s i s t a n t to the second of these, whereas v a r i e t i e s Hannchen and Vantage j o i n t l y gave the opposite and c o n t r a s t i n g r e a c t i o n s to the same two t e s t c u l t u r e s (Table V I ) . 27. TABLE VI. Disease r e a c t i o n s of three b a r l e y v a r i e t i e s Vantage f o l l o w i n g i n o c u l a t i o n w i t h two t e s t derived through s e l f i n g , from t e l i o s p o r e s D E x c e l s i o r , Hannchen and c u l t u r e s of U. hordei and E (or F ) . Test c u l t u r e s derived from spore: Disease r e a c t i o n s of: E x c e l s i o r Hannchen and Vantage D S R E (or F) R S I t was not p r a c t i c a l to t e s t a s i n g l e F j p l a n t against both the t e s t c u l t u r e s , t h e r e f o r e , i t was necessary to grow F2 progeny f o r the e l u c i d a t i o n of genotypes of the i n d i v i d u a l F2 p l a n t s . Therefore, the crosses were advanced to F2 seed l e v e l , harvested from s i n g l e F2 p l a n t s . A t o t a l of 150 F2 p l a n t s from each of the three crosses was randomly s e l e c t e d and each pla n t was d i v i d e d i n t o two l o t s of 100 seeds each. One l o t was i n o c u l a t e d w i t h a c u l t u r e derived from Spore D and the other l o t w i t h a second c u l t u r e derived from Spore E. The F-j progenies were planted i n rows i n C a l i f o r n i a during 1970. The r e s u l t s were repeated at the U n i v e r s i t y of B r i t i s h Columbia during the summer of 1971, t h i s time u s i n g 75 F2 p l a n t s from each of the three crosses. F^ l i n e s which showed no disease f o l l o w i n g i n o c u l a t i o n were c l a s s i f i e d as r e s i s t a n t to the c u l t u r e i n question; those w i t h one or more diseased p l a n t s were c l a s s i f i e d as s u s c e p t i b l e . The i n f e c t i o n percentages were c a l c u l a t e d on the basis of number of p l a n t s i n f e c t e d vs. t o t a l number of pla n t s i n a row. 28. RESULTS I The virulence-genes The three p a r e n t a l t e l i o s p o r e s were designated D, E, and F. The r e s u l t s obtained when the products of these three spores were brought together through s e l f i n g , i n a l l compatible combinations and i n o c u l a t e d to the f i v e b a r l e y v a r i e t i e s are presented i n Table V I I . They show that the four l i n e s derived from spore D were v i r u l e n t on v a r i e t y E x c e l s i o r but a v i r u l e n t on Hannchen and Vantage; by con t r a s t the l i n e s d e rived by s e l f i n g from spores E and F were v i r u l e n t on Hannchen and Vantage but a v i r u l e n t on E x c e l s i o r . A l l derived c u l t u r e s from three t e l i o s p o r e s were v i r u l e n t on v a r i e t i e s T r e b i and L i o n . In no case i s there evidence of segregation among the four l i n e s derived by s e l f i n g from a s i n g l e p a r e n t a l spore. The r e s u l t s obtained when the h a p l o i d products of the three p a r e n t a l spores were crossed i n a l l compatible combinations are a l s o presented i n Table V I I . From the data of the D x E and D x F crosses i t i s evident that p a t h o g e n i c i t y on v a r i e t i e s E x c e l s i o r , Hannchen and Vantage i s r e c e s s i v e . The d i k a r y o t i c F^ l i n e s produced through s e l f i n g and c r o s s i n g of the p a r e n t a l t e l i o s p o r e s were numbered from 1 to 36 i n c l u s i v e , as shown i n Table V I I . S i n g l e t e l i o s p o r e s from c e r t a i n of these l i n e s were t e s t e d by backcrossing them to one or more of the p a r e n t a l spores. The r e s u l t s of these t e s t crosses are presented i n Table V I I I , f r o m which i t i s evident that t e l i o s p o r e s taken from l i n e s 13 and 14 (three from 29. each l i n e ) were heterozygous at a s i n g l e genetic locus determining v i r u l e n c e on v a r i e t i e s Hannchen and Vantage. The f a c t that the segregations i n a l l cases a f f e c t e d the two v a r i e t i e s j o i n t l y leads to the c o n c l u s i o n that v i r u l e n c e on e i t h e r , or on both Hannchen and Vantage, i s c o n t r o l l e d by a s i n g l e genetic locus t e n t a t i v e l y designated as the V^ lo c u s . The data obtained i n the t e s t crosses described above provided no new genetic i n f o r m a t i o n about v i r u l e n c e on v a r i e t i e s E x c e l s i o r , T r e b i , and L i o n . V i r u l e n c e on these v a r i e t i e s was examined f u r t h e r i n another s e r i e s of backcrosses. S i n g l e t e l i o s p o r e s i s o l a t e d from s u i t -able F-^ crosses were backcrossed to an appropriate p a r e n t a l t e l i o s p o r e . The data obtained from these t e s t crosses i s presented i n Table IX. The r e s u l t s show that i n d i v i d u a l t e l i o s p o r e s taken from l i n e 21 were heterozygous, at a s i n g l e genetic locus r e s p o n s i b l e f o r v i r u l e n c e on v a r i e t y E x c e l s i o r . Likewise, f i v e more sets of tetrads i s o l a t e d from t e l i o s p o r e s taken from l i n e s 13, 14, 22, 23 were a l s o t e s t e d on a l l f i v e v a r i e t i e s . In these crosses i t was p o s s i b l e to t e s t only four of the eight p o s s i b l e t e s t cross combinations; however, si n c e a l l four products of the F^ t e l i o s p o r e s are used i n each backcross, the data do provide dependable genetic i n f o r m a t i o n . As w e l l , f o r some of the crosses i t was a l s o not p o s s i b l e to t e s t f o r v i r u l e n c e on v a r i e t i e s Hannchen and Vantage. The data of these t e s t c r o s s e s are presented i n Table X. The 1:1 segregations show that v i r u l e n c e on v a r i e t y E x c e l s i o r i s a l s o c o n t r o l l e d at a s i n g l e genetic locus l a b e l l e d , f o r the time being, as the V. l o c u s . 30. A f i n a l s e r i e s of backcrosses was designed p r i m a r i l y to determine whether any segregation had gone undetected i n the E x F c r o s s . The r e s u l t s of these t e s t c rosses, presented i n Table XI show no good evidence of segregation on any of the f i v e host v a r i e t i e s which indicates,that t e l i o s p o r e s E and F are i d e n t i c a l i n a c t i o n . V i r u l e n c e genes v^ and V 2 were r e s p e c t i v e l y revealed i n the c u l t u r e s derived from t e l i o s p o r e s E and D. Both the v i r u l e n c e genes were independent and r e c e s s i v e i n a c t i o n . The genotypes of c u l t u r e s derived from spore E and D, thus become v^v^V^V^and ^ 1 ^ 1 V 2 V 2 r e s P e c t i v e l y » a n d genotypes appropriate to the i n d i v i d u a l segregants are l i s t e d on Table V I I I , IX and X. 31. TABLE VII I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h 36 i n f e c t i v e l i n e s of U. hordei a l l of which descended from three p a r e n t a l t e l i o s p o r e s P a r e n t a l Spori- D i k a r y o t i c P a t h o g e n i c i t y recorded on v a r i e t i e s : spore(s) d i a l Line No. E x c e l s i o r Hannchen Vantage T r e b i L i o n Mating D D-2 X D-l 1 49 0 0 17 19 D-2 X D-3 2 70 0 0 18 14 D-4 X D-l 3 65 0 0 17 21 D-4 X D-3 4 55 0 0 23 17 E E-2 X E - l 5 0 34 42 28 18 E-2 X E-3 6 0 43 81 22 22 E-4 X E - l 7 0 42 53 23 19 E-4 X E-3 8 0 43 54 17 19 F F-2 X F - l 9 0 47 66 20 22 F-2 X F-4 10 0 43 66 42 13 F-3 X F - l 11 0 35 60 17 13 F-3 X F-4 12 0 52 58 30 20 D x E D-2 X E - l 13 0 0 0 17 57 D-2 X E-3 14 0 0 0 25 28 D-4 X E - l 15 0 0 0 27 37 D-4 X E-3 16 0 0 0 24 39 D-l X E-2 17 0 0 0 34 39 D-3 X E-2 18 0 0 0 34 51 D-l X E-4 19 0 0 0 45 43 D-3 X E-4 20 0 0 0 41 30 D x F D-2 X F - l 21 0 0 0 40 44 D-2 X F-4 22 0 0 0 58 39 D-4 X F - l 23 0 0 0 60 53 D-4 X F-4 24 0 0 0 43 40 D-l X F-2 25 0 0 0 53 45 D-3 X F-2 26 0 0 0 50 46 D-l X F-3 27 0 0 0 53 35 D-3 X F-3 28 0 0 0 45 47 E x F E-2 X F - l 29 0 49 81.. 49 18 E-2 X F-4 30 0 63 83 35 36 E-4 X F - l 31 0 54 65 54 32 E-4 X F-4 32 0 49 67 45 19 E - l X F-2 33 0 57 83 38 27 E-3 X F-2 34 0 61 75 56 40 E - l X F-3 35 0 49 53 25 17 E-3 X F-3 36 0 43 40 28 29 * The data shown are averages of two separate t e s t s . TABLE V I I I I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. hordei derived from p a r e n t a l spore E. S p o r i d i a l P a t h o g e n i c i t y recorded on v a r i e t i e s ; Genotype of Mating E x c e l s i o r Hannchen Vantage T r e b i L i o n Pathogen 13A-1 x E - l 13A-2 x E - l 13A-3 x E-2 13A-4 x E-2 13A-1 x E-3 13A-2 x E-3 13A-3 x E-4 13A-4 x E-4 0 0 0 0 0 0 0 0 0 0 29 60 0 0 20 29 0 0 32 80 0 0 63 56 18 20 24 33 22 38 22 10 37 22 13 17 29 34 10 12 V l V v 1 1 V v 1 1 V V 1 1 V v 1 1 V i v l v l V, V, 13B-1 x E - l 0 13B-4 x E - l 0 13B-2 x E-2 0 13B-3 x E-2 0 13B-1 x. E-3 0 13B-4 x E-3 0 13B-2 x E-4 0 13B-3 x E-4 0 0 0 25 35 0 0 49 23 0 0 28 37 0 0 46 30 19 17 19 8 14 21 23 9 25 25 31 23 30 20 25 17 V l V i v i v i V i V i V V 1 1 v l v l 13C-1 x E - l 0 13C-3 x E - l 0 13C-2 x E-2 0 13C-4 x E-2 0 13C-1 x E-3 0 13C-3 x E-3 0 13C-2 x E -4 0 13C-4 x E -4 0 86 69 0 0 50 67 0 0 57 45 0 0 73 45 0 0 69 43 82 60 67 10 33 36 64 25 14 50 25 14 18 25 V l v l v l V l V v 1 1 v l v l V i V i V i TABLE V I I I (continued) 14A-1 x E - l 0 0 0 10 21 v i v 1 14A-4 x E - l 0 0 0 12 17 V 1v ] L 14A-2 x E-2 0 17 50 15 29 14A-3 x E-2 0 15 61 23 22 14A-2 x E-4 0 37 79 30 14 14A-3 x E-4 0 14 66 18 18 14B-2 x E - l 0 32 58 17 15 14B-3 x E-2 0 54 68 30 25 14B-2 x E-3 0 27 58 37 45 14B-3 x E-4 0 38 80 22 25 14C-1 x E - l 0 36 40 6 13 14C-3 x E-2 0 29 50 11 41 14C-1 x E-3 0 31 40 4 19 14C-3 : x E-4 0 11 17 3 7 v l v l V l 14A-1 x E-3 0 0 0 14 22 V 1 v 1 14A-4 x E-3 0 0 0 6 26 V ^ V-i v l v lV i 14Brl x E - l 0 0 0 14 23 V ^ V l V l 14B-4 x E-2 0 0 0 33 41 V 1 v 1 14B-1 x E-3 0 0 0 19 30 V v 1 1 V l v l v l 14B-4 x E-4 0 0 0 15 32 V ^ v v 1 1 14C-4 x E - l 0 0 0 3 25 V v 1 1 14C-2 x E-2 0 0 0 19 16 V ^ v l v l v l v l 14C-4 x E-3 0 0 0 0 19 V ^ 14C-2 x E-4 0 0 0 5 13 V ^ V 1 V 1 * f o r crosses i n v o l v i n g spores 13A and 13B the data are based on two t e s t s ; f o r the cross i n v o l v i n g spore 13C the data are based on a s i n g l e t e s t . 34. TABLE IX I n f e c t i o n percentages obtained on f i v e barley v a r i e t i e s following i n o c u l a t i o n with backcross l i n e s of U. hordei derived from parental spore D. S p o r i d i a l Pathogenicity recorded on v a r i e t i e s Genotype of Mating Ex c e l s i o r Hannchen Vantage Trebi Lion Pathogen 21A-1 X D-2 0 0 0 11 18 V 2 v 2 21A-1 X D-4 0 0 0 12 21 V 2 V 2 21 A-2 X D-l 23 0 0 16 21 v 2 v 2 21A-2 X D-3 24 0 0 17 20 V 2 V 2 21A-3 X D-2 0 0 0 11 19 V.v, 21 A-3 X D-4 0 0 0 9 15 V 2 V 2 21 A-4 X D-l 11 0 0 13 19 V 2 V 2 21 A-4 X D-3 18 0 0 12 20 V 2 V 2 21B-1 X D-2 0 0 0 23 19 V 2 v 2 21B-1 X D-4 0 0 0 18 20 V 2 V 2 21B-2 X D-2 48 0 0 11 9 v 2 v 2 21B-2 X D-4 46 0 0 16 15 V 2 V 2 21B-3 X D-l 14 0 0 10 13 V 2 V 2 21B-3 X d-3 88 0 0 21 18 v 2 v 2 21B-4 X D-l 0 0 0 17 21 V 2 V 2 21B-4 X D-3 0 0 0 18 22 V 2 v 2 21C-1 X D-2 11 0 0 11 9 v 2 v 2 21C-1 X D-4 6 0 0 13 14 v 2 V 2 21 C-2 X D-2 0 0 0 18 20 V 2 V 2 21 C-2 X D-4 0 0 0 11 21 V 2 v 2 21C-3 X D-l 19 0 0 11 20 V 2 V 2 21C-3 X D-3 46 0 0 18 12 v 2 v 2 21 C-4 X D-l 0 0 0 9 10 V 2 v 2 21 C-4 X D-3 0 0 0 . 18 21 V 2 V 2 TABLE IX (Continued) 21D-1 X D-2 27 0 0 18 18 V 2 V : 21D-1 X D-4 20 0 0 21 21 v 2v. 21D-2 X D-l 10 0 0 22 22 v 2v. 21D-2 X D-3 18 0 0 24 24 v 2v. 21D-3 X D-l 0 0 0 11 32 V : 21D-3 X D-3 0 0 0 13 18 V : 21D-4 X D-2 0 0 0 15 19 V : 21D-4 X D-4 0 0 0 16 25 V : 21E-1 X D-2 22 0 0 21 18 V 2 V : 21E-1 X D-4 17 0 0 11 12 v 2v. 21E-2 X D-2 75 0 0 12 13 V 2 V : 21E-2 X D-4 50 0 o • 14 14 v 2v. 21 E-3 X D-l 0 0 0 19 18 V : 21E-3 X D-3 0 0 0 10 21 V : 21E-4 X D-l 0 0 0 13 18 V : 21E-4 X D-3 0 0 0 11 12 V : 21F-1 X D-2 0 0 0 11 13 V : 21F-1 X D-4 0 0 0 14 18 V : 21F-2 X D-l 0 0 0 18 15 V . 21F-2 X D-3 0 0 0 11 18 V 21F-3 X D-l 17 0 0 21 20 v 2 v 21F-3 X D-3 30 0 0 20 21 V 2 V 21F-4 X D-2 31 0 0 19 21 v 2 v 2 IF-4 X D-4 32 0 0 10 24 v 2 v TABLE IX (Continued) 21G-1 X D-2 0 21G-1 X D-4 0 21G-2 X D-2 22 21G-2 X D-4 25 21G-3 X D-l 24 21G-3 X D-3 25 21G-4 X D-l 0 21G-4 X D-3 0 21H-1 X D-2 18 21H-1 X D-4 20 21H-2 X D-l 18 21H-2 X D-3 26 21H-3 X D-l 0 21H-3 X D-3 0 21H-4 X D-2 0 21H-4 X D-4 0 211-1 X D-2 37 211-1 X D-4 45 211-2 X D-2 0 211-2 X D-4 0 211-3 X D-l 20 211-3 X D-3 37 211-4 X D-l 0 211-4 X D-3 0 21J-1 X D-2 31 21J-1 X D-4 24 21J-2 X D-l 0 21J-2 X D-3 0 21 J-3 X D-l 50 21J-3 X D-3 28 21J-4 X D-2 0 21J-4 X D-4 0 0 0 18 19 V 2 V 2 0 0 10 12 V 2 V 2 0 0 19 10 V 2 V 2 0 0 21 14 V 2 V 2 0 0 11 15 V 2 V 2 0 0 14 20 V 2 V 2 0 0 15 15 V 2 V 2 0 0 18 18 V2 V2 0 0 18 13 V 2 V 2 0 0 12 18 V 2 V 2 0 0 14 20 v 2 v 2 0 0 16 10 V 2 V 2 0 0 11 14 V 2 V 2 0 0 2 18 V 2 V 2 0 0 14 19 V 2 V 2 0 0 12 20 V v 2 2 0 0 14 18 V 2 V 2 0 0 18 20 V 2 V 2 0 0 12 25 V 2 V 2 0 0 12 30 V 2 V 2 0 0 14 21 V 2 V 2 0 0 13 25 V 2 V 2 0 0 19 20 V 2 V 2 0 0 21 20 V v 2 2 0 0 11 18 V 2 V 2 0 0 12 21 V 2 V 2 0 0 18 13 V 2 V 2 0 0 19 15 V 2 V 2 0 0 12 18 V 2 V 2 0 0 14 21 v 2 v 2 0 0 21 24 V 2 V 2 0 0 22 31 V2 V2 * The data shown are based on a si n g l e t e s t . TABLE X 37. I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s : of U .hordei derived from p a r e n t a l spore D S p o r i d i a l P a t h o g e n i c i t y ' recorded on v a r i e t i e s : Genotype of Mating E x c e l s i o r Hannchen Vantage T r e b i L i o n Pathogen 13-1 x D-l 0 0 0 -** - 2 2 13-2 x D-2 0 0 0 - - V2V2 13-3 x D-4 50 0 0 - - v 2 v 2 13-4 x D-3 20 0 0 - - V 2 V 2 14-1 x D-3 0 0 0 - - V v n 2 2 14-2 x D-4 0 0 0 - - V 2 V 2 14-3 x D-2 28 0 0 - - v 2 v 2 14-4 x D-l 25 0 0 - - V 2 V 2 22A-1 x D-l 0 0 0 9 17 V 2 v 2 22A-2 x D-3 48 0 0 10 21 V V 2 2 22A-3 x D-2 55 0 0 15 23 V 2 V 2 22A-4 x D-4 0 0 0 21 14 V 2 V 2 22B-1 x D-l 0 0 0 15 10 V 2 v 2 22B-2 x D-2 41 0 0 29 16 V 2 V 2 22B-3 x D-4 58 0 0 21 24 V 2 V 2 22B-4 x D-3 0 0 0 11 18 V 2 V 2 23-1 x D-l 61 0 0 14 21 V V 2 2 23-2 x D-2 71 0 0 18 24 V 2 V 2 23-3 x D-4 0 0 0 11 13 V 2 v 2 23-4 x D-3 0 0 0 17 30 V 2 v 2 * The data shown are averages of two separate t e s t s , except where no number i s given. Not t e s t e d . 38. TABLE XI I n f e c t i o n percentages obtained on f i v e b a r l e y v a r i e t i e s f o l l o w i n g i n o c u l a t i o n w i t h backcross l i n e s of U. hordei derived from p a r e n t a l spore F. S p o r i d i a l P a t h o g e n i c i t y recorded on v a r i e t i e s : Mating E x c e l s i o r Hannchen Vantage Tr e b i L i o n 33A-2X F - l 0 59 60 11 16 33A-3X F - l 0 44 45 7 3 33A-1X F-2 0 64 59 25 19 33A-4X F-2 0 38 61 9 10 33B-2 x F - l 0 35 60 5 14 33B-4 x F - l 0 35 67 15 20 33B-1 x F-2 0 64 55 9 6 33B-3 x F-2 0 63 45 19 11 33C-1 x F - l 0 36 46 2 10 33C-2 x F - l 0 28 58 5 18 33C-3 x F-2 0 48 61 12 10 33C-4 x F-2 0 60 81 30 18 33D-2 x F - l 0 65 44 8 15 33D-4 x F - l 0 50 45 5 13 33D-1 x F-2 0 70 38 15 30 33D-3 x F-2 0 59 60 17 28 34A-2 x F - l 0 61 61 6 13 34A-2 x F - l 0 50 65 9 14 34A-1 x F-2 0 75 68 6 18 34A-4 x F-2 0 63 71 21 18 39. Table XT (continued) 34B-1 X F - l 0 42 50 18 20 34B-2 X F - l 0 44 56 19 8 34B-3 X F-2 0 65 60 17 10 34B -4 X F-2 0 69 43 22 13 34C-1 X F - l 0 30 55 14 10 34C-3 X F - l 0 43 48 8 6 34C-2 X F-2 0 45 42 14 21 34C-4 X F-2 0 49 72 21 16 34D-1 X F - l 0 53 60 19 13 34D-4 X F - l 0 40 54 24 14 34D-2 X F-2 0 63 51 17 20 34D-3 X F-2 0 50 45 15 14 *The data shown are averages of two separate t e s t s . I I The Resistance genes Resistance genes were revealed through the i n t e r a c t i o n of a l r e a d y known v i r u l e n c e genes designated as v^ and v^ . Recessive a l l e l e v^ determines v i r u l e n c e on v a r i e t i e s Hannchen (H) and Vantage (V), whereas r e c e s s i v e a l l e l e v^ determines v i r u l e n c e on the v a r i e t y E x c e l s i o r : dominant a l l e l e s V and V determine a v i r u l e n c e on v a r i e t i e s 1 2 Hannchen and Vantage, and on E x c e l s i o r , r e s p e c t i v e l y . The disease i n t e r a c t i o n s between the two t e s t c u l t u r e s and the three b a r l e y v a r i e t i e s are summarized i n Table X I I . 40. TABLE X I I I n f e c t i o n percentages shown on p a r e n t a l v a r i e t i e s Hannchen, Vantage, and E x c e l s i o r f o l l o w i n g i n o c u l a t i o n w i t h t e s t c u l t u r e s of U. hordei v l V 1 ^ 2 ^ 2 a n c l V - ^ V ^ V 2 V 2 Disease r e a c t i o n s of: Test Cultures Year and Place of t e s t i n g Hannchen Vantage E x c e l s i o r V 1 V 1 V 2 V 2 1970 ( C a l i f . ) ' 1971 (UBC)** 44 47 Average 46 38 48 43 0 0 0 V 1 V 1 v 2 v 2 1970 ( C a l i f . ) ' 1971 (UBC)** 0 0 Average 0 0 0 40 73 57 * = Tested w i t h F^ l i n e s at C a l i f o r n i a ** = Tasted w i t h F 3 l i n e s at UBC The data obtained, f o l l o w i n g i n o c u l a t i o n of F^ b a r l e y l i n e s w i t h both t e s t c u l t u r e s of U. h o r d e i , are presented i n Appendices B and C. The r e s u l t s show that f o r two of the three crosses E x V and E x H, the F^ l i n e s were separable i n t o four c l a s s e s ( S - S , S - R , R - S , and R -R), depending on t h e i r s u s c e p t i b i l i t y to both, or to one, or the other, or to n e i t h e r of the two t e s t c u l t u r e s . For convenience of i n t e r p r e t a t i o n of r e s u l t s , the number of F^ l i n e s f a l l i n g i n t o each of these four cl a s s e s as shown i n Appendices B and C are summarized i n Tables X I I I and XIV, r e s p e c t i v e l y . From the c o n s o l i d a t e d data i t i s evident that the number of F^ l i n e s f a l l i n g i n t o these four c l a s s e s gave acceptable f i t s to a 9:3:3:1 r a t i o t y p i c a l of two independently segregating dominant f a c t o r s . 41. TABLE X I I I F^ b a r l e y l i n e s separated i n t o four c l a s s e s tions- w i t h two t e s t c u l t u r e s of U. hordei each c l a s s according to t h e i r i n t e r a c -and the number of l i n e s i n Reaction of Fg l i n e s to t e s t c u l t u r e : Designation of cros s , and numbers of Fg l i n e s i n each c l a s s v l v 1 ^ 2 ^ 2 V-^V^V2V2 Class E x H E x V H x V S S S - S 70 72 0 S R S - R 31 28 150 R S R - S 32 30 0 R R R - R 15 13 0 Tot a l s 148* 143** 150 * x 2 = 6.71 P f o r 9:3:3:1 r a t i o = 0. 10 and 0.05 ** x 2 = 3.15 P f o r 9:3:3:1 r a t i o = 0. 50 and TABLE 0.30 XIV Fg b a r l e y l i n e s separated i n t o a c t i o n s w i t h two t e s t c u l t u r e s i n four c l a s s e s of U. hordei each c l a s s according to t h e i r i n t e r -and the number of l i n e s R eaction of Fg l i n e s to t e s t c u l t u r e : Designation of cr o s s , and numbers of F^ l i n e s i n each c l a s s v l v l v 2 ^ 2 V^V^V2V2 Class E x H E x V H x V S S S - S 35 34 0 S R S - R 15 12 75 R S R - S 17 21 0 R R R - R 8 8 0 Tot a l s 75* 75** 75 * x 2 = 4.24 P f o r 9:3:3:1 r a t i o ** x 2 = 7.66 = 0 . 3 0 - 0 1.20 P f o r 9:3:3:1 r a t i o = 0. 10 - 0. 05 42. The r e s p e c t i v e p-values f o r both the crosses, E x H and E x V, as revealed by the two separate t e s t s , are presented i n Tables X I I I and XIV; they show that the data give s a t i s f a c t o r y s t a t i s t i c a l f i t s to a 9:3:3:1 r a t i o . ( I t w i l l be noted that the p-value i s i n the 5-10 percent range f o r one of the crosses i n each of these two t a b l e s . This i s a s s o c i a t e d , i n both cases, w i t h an excess of l i n e s showing r e s i s t a n t r e a c t i o n s , and i s most probably due to the f a c t that any s u s c e p t i b l e l i n e which has "escaped" the disease has a u t o m a t i c a l l y been included w i t h the r e s i s t a n t c l a s s ) . Segregations a t one of these l o c i was r e -vealed through the i n t e r a c t i o n w i t h c u l t u r e v^ v^^2^2' a n (^ a t t* l e other locus through the i n t e r a c t i o n w i t h c u l t u r e V^V^v 2v 2. The data of the H x V cross gave no evidence of segregation. The F^ l i n e s were s u s c e p t i b l e and r e s i s t a n t to c u l t u r e s v-LV-^V2V2and ^ T l V 2 V 2 ' r e sP e c ti- v ely» a s w e r e the two parents Hannchen and Vantage. The lack of segregation i n d i c a t e s that both of these v a r i e t i e s share the same r e s i s t a n t gene or very c l o s e l y l i n k e d r e s i s t a n c e genes. From the key i n t e r a c t i o n s of Table VI i t i s obvious that one of the r e s i s t a n c e f a c t o r s which segregated i n the E x H and E x V crosses was c o n t r i b u t e d by v a r i e t y E x c e l s i o r and that the second segre-g a t i n g f a c t o r i s he l d i n common by v a r i e t i e s Hannchen and Vantage. The E x c e l s i o r f a c t o r was revealed i n i n t e r a c t i o n w i t h c u l t u r e v^ v^^2^2 wh i l e Hannchen-Vantage f a c t o r was expressed i n i n t e r a c t i o n w i t h V^V-^v2v, Both the f a c t o r s were dominant. The r e s i s t a n c e a l l e l e which was con-t r i b u t e d to both crosses by E x c e l s i o r , and whose expression i s conditioned by v i r u l e n c e a l l e l e s V„ and v 9 of U. h o r d e i ; i s t h e r e f o r e designated R^. For s i m i l a r reasons the second r e s i s t a n c e a l l e l e which i s shared by the v a r i e t i e s Hannchen and Vantage, and which segregated i n the presence of V^ (but not i n the presence of v-^v i s designated R^. The genotypes of Hannchen and Vantage, and of E x c e l s i o r thus become R ^ R - j ^ ^ a n& r l r l R 2 R 2 r e s P e c t i v e l y • 44 DISCUSSION Two r e c e s s i v e genes f o r v i r u l e n c e , v^ and v^, were demon-s t r a t e d i n t h i s study. For reasons o u t l i n e d by Sidhu and Person (1971), these two genes have been redesignated Uhv^ and Uhv 2. Cultures of U s t i l a g o h o r d e i which were homozygous f o r the r e c e s s i v e gene Uhv^ were v i r u l e n t on two v a r i e t i e s , Hannchen and Vantage, w h i l e those which were homozygous f o r the r e c e s s i v e gene Uhv 2 were v i r u l e n t on the v a r i e t y E x c e l s i o r . The genotypic designations of the two c u l t u r e s which were chosen to serve as t e s t c u l t u r e s f o r i n v e s t i g a t i n g the genetics of host r e s i s t a n c e thus become Uhv-^ mw 1UjhV 2UhV 2 and UhV^UhV^Uhv^t^. Each of the two c u l t u r e s e x h i b i t e d a high l e v e l of v i r u l e n c e on the v a r i e t y (or v a r i e t i e s ) to which i t was s p e c i f i c a l l y v i r u l e n t as shown i n Table X I I ; the a v i r u l e n t combinations gave no disease at a l l . This provided a favourable opportunity f o r studying the genetics of r e s i s t a n c e and s u s c e p t i b i l i t y i n the host. The existence of v i r u l e n c e genes (Uhv-^ and Uhv 2) was esta-b l i s h e d from backcross data ( c f . Tables V I I I , IX and X). The back-cross method has c e r t a i n advantages over F 2 r a t i o s obtainable from a t e t r a d . V i r u l e n c e r a t i o s i n the dikaryophase are dependent on the arrangement of v i r u l e n c e and mating type f a c t o r s brought about by f i r s t and second d i v i s i o n s of meiosis. Based on these m e i o t i c events, F 2 progeny derived by s e l f i n g from s i n g l e F^ t e l i o s p o r e s may e x h i b i t e i t h e r of two types of r a t i o , i . e . 4A:0V or 3A:IV (where "A" and "V" stands f o r a v i r u l e n c e and v i r u l e n c e r e s p e c t i v e l y ) . Since the 4:0 45 r a t i o i s also obtainable from a homozygous spore, i t provides no useful genetic information. The 3:1 r a t i o , which does provide useful genetic information, requires at least one crossover event, leading to the formation of a "tetratype" tetrad. The crossover event needed to produce a tetratype segregation and a 3:1 r a t i o on s e l f i n g can take place between the gene for virulence and i t s centromere, or between the mating locus and i t s centromere; i f these two l o c i happen to be linked, the crossover must then take place i n the i n t e r -vening region. Consequently, the frequency of occurrence of " u s e f u l " ( i . e . tetratype) tetrads w i l l be e n t i r e l y dependent on the linkage r e l a t i o n s h i p s of the gene(s) f o r virulence, the mating type gene, and t h e i r respective centromeres. The backcross method bypasses these genetic l i m i t a t i o n s and ensures the type of information sought for the virulence genes. Having i d e n t i f i e d the two virulence genes i n the pathogen U. hordei, the stage was set for i n v e s t i g a t i n g the genetics of r e s i s t -ance and s u s c e p t i b i l i t y i n the host, Hordeum vulgare. The i d e a l s i t u a t i o n would have been ei t h e r to test i n d i v i d u a l genotype of back-cross progeny or F 2 progeny derived from three d i a l l e l e crosses (EXH, EXV, and HXV), against both virulence genes. Since i t was not p r a c t i c a l (though practicable) either to r a i s e enough backcross seed or to test each genotype of the F 2 progeny against the two v i r u l e n t genes, the study was extended to F 3 progeny. Half the Fg progeny of s i n g l e F 2 plants were inoculated with culture V ^ V ^ V 2 V 2 a n <* t' i e other h a l f with V V v v . The hypothesis under test was: 1 1 2 2 46. i ) that the expression of r e s i s t a n c e would be determined by s i n g l e dominant genes i n the host; and i i ) that the i n t e r a c t i o n s would be based on gene-for-gene s p e c i f i c a t i o n s . The r e s u l t s ( c f . Tables X I I I and XIV) of t h i s study were i n agreement w i t h t h i s hypothesis. One of the r e s i s t a n c e f a c t o r s , possessed by v a r i e t y E x c e l s i o r , was expressed i n i n t e r a c t i o n w i t h c u l t u r e v^v^V 2V2 and t h i s second f a c t o r , held i n common by v a r i e t i e s Hannchen and Vantage, was expressed i n the i n t e r a c t i o n w i t h c u l t u r e V^V^v^v^. The hypothesis being t e s t e d would p r e d i c t a 1:3:3:9 r a t i o f o r the four r e a c t i o n c l a s s e s (S-S, S-R, R-S, and R-R) i n the F2 generation. However, w i t h t e s t i n g delayed to the Fg generation, the expectation i s that Fg l i n e s which have descended from r e s i s t a n t but heterozygous F 2 p l a n t s w i l l c o n t a i n some ( i . e . 25 per cent) sus-c e p t i b l e segregants. These Fg l i n e s w i l l then be c l a s s i f i e d as s u s c e p t i b l e and, i n consequence, the p r e d i c t e d Fg r a t i o f o r the four r e a c t i o n c l a s s e s l i s t e d above w i l l become 9:3:3:1. In f a c t , t h i s was the r a t i o a c t u a l l y obtained i n Fg progeny and l e d to c o n c l u s i o n that the r e s i s t a n c e was expressed as a dominant c h a r a c t e r i s t i c at both segregating l o c i . (Moreover, i f the r e s i s t a n c e genes had been r e c e s s i v e , the r a t i o f o r r e a c t i o n c l a s s e s (S-S, S-R, R-S, and R-R) would have been 9:3:3:1 i n the F2 progeny and 1:3:3:9 i n the Fg progeny; or i n the event of r e s i s t a n c e being p o l y g e n i c , there would have been a complete l a c k of any k i n d of r a t i o ) . 47. I t was of i n t e r e s t to determine whether the i n t e r a c t i o n i n t h i s system, between a l l e l e s f o r r e s i s t a n c e and s u s c e p t i b i l i t y i n the host and f o r v i r u l e n c e and a v i r u l e n c e i n the p a r a s i t e , are i n accord w i t h F l o r ' s hypothesis of gene-for-gene r e l a t i o n s ( F l o r , 1942, 1955). The r e s u l t s of t h i s study have shown that the r e s i s t a n c e of E x c e l s i o r , when i t i s expressed, i s due t o the presence of a dominant r e s i s t a n c e a l l e l e . However, e a r l i e r study had shown that the expression of E x c e l -s i o r r e s i s t a n c e can a l s o be conditioned by a l l e l e s V-> and v 2 of U. hor d e i ; w i t h V 2 present, E x c e l s i o r r e s i s t a n c e was expressed, but wi t h V 2 absent ( i . e . U. hordei was v 2 v 2 ) , i t was not expressed. I t i s e v i -dent that a gene-for-gene r e l a t i o n s h i p does e x i s t . The r e s i s t a n c e a l l e l e which was co n t r i b u t e d to both crosses by E x c e l s i o r , and whose expression i s conditioned by a l l e l e s V 2 and v 2 of U. ho r d e i , i s there-f o r e designated UhR 2. For s i m i l a r reasons the second r e s i s t a n c e a l l e l e , which i s common to v a r i e t i e s Hannchen and Vantage, and which segregated i n the presence of V^ but not i n the presence of v ^ v ^ i s designated UhR^. The genotypes of Hannchen and Vantage and of E x c e l s i o r , thus become UhR-jUhRjUhr 2Uhr 2 and Uhr^Uhr^UhR 2UhR 2 r e s p e c t i v e l y . The disease i n t e r a c t i o n s between the two t e s t c u l t u r e s and these p a r e n t a l host genotypes, as w e l l as those p r e d i c t e d on the ba s i s of F l o r ' s hypothesis f o r the F^ and F 2 p o p u l a t i o n s , are summarized i n Table XV. Although i t was not p o s s i b l e to i d e n t i f y host phenotypes i n the F2 generation, the i n t e r a c t i o n s expected of the F 2 d i d serve as a basis f o r p r e d i c t i n g the disease r e a c t i o n s expected of the F^ l i n e s . This p r e d i c t i o n , which i s summarized i n Table XVI, d i d accommodate the experimental TABLE XV Expect a t i o n , based on F l o r ' s hypothesis of gene-for-gene r e l a t i o n s h i p s , of i n t e r a c t i o n s i n v o l v i n g p a r e n t a l , F^ and F generations of the E x H and E x V crosses. I n t e r a c t i o n w i t h Host: * Genotype: Hannchen) Vantage ) R R r r -1-1-2-2 R s E x c e l s i o r S R F l R R 9/16 £1-1*2" R R 3/16 - l " - 2 - 2 R S F 2 3/16 £lZlR 2" S R 1/16 LILIL2L2 S S * Designations of host a l l e l e s UhR^, Uhr^, UhRo and Uhr^ abbreviated to R^,r^, R2 and r2> r e s p e c t i v e l y . ** R = r e s i s t a n t ; S = s u s c e p t i b l e . observations ( c f . Table X I I I and XIV). The co n c l u s i o n was the r e f o r e drawn that the genetic i n t e r a c t i o n s observed i n t h i s study are i n complete accord w i t h F l o r ' s hypothesis of gene-for-gene r e l a t i o n s . 49. TABLE XVI Exp e c t a t i o n , based on F l o r ' s hypothesis of gene-for-gene r e l a t i o n s , of i n t e r a c t i o n s i n v o l v i n g F l i n e s d e r i v e d from the E x H and E x V crosses. Genetic composition of I n t e r a c t i o n of F3 genotypes w i t h C l a s s i f i c a t i o n of F3 l i n e s F 2 p o p u l a t i o n F 3 l i n e s Class f 1/16 R 1R 1R 2R 2 R l R l R 2 l 2 R R R-R 1/16 2/16 RiRiR_2£2 3/4 R1I1R2— 1/4 R ^ r ^ R R R S R-S 3/16 1/16 R]_R-jr 2r2 R 1 I 1Z 2^2 R S R-S 2/16 R j r ^ R ^ 3/4 R]_-R2R2 1/4 r 1 r 1 R 2 R 2 R R S R S-R 3/16 1/16 r l X l R 2 R 2 -1-1-2-2 S R S-R 4/16 R 1 r 1 R 2 r 2 9/16 R — R — 3/16 Rx-v2r2 -V16 r l £ l R 2 -1/16 I_\I.\lL2—2 R R R S S R S S S-S -* •0 9/16 2/16 R ^ r ^ 3/4 R - r 2 r 2 1/4 r 1 x 1 r 2 r 2 R S S S S-S 2/16 r^r^R 2_r 2 3/4 r_iL-\^2~ 1/4 1 1 X ^ 2 S R S S S-S 1/16 1 ^ X ^ r ^ S S S-S * Designations of host a l l e l e s UhR-^ , Uhr^, UhR 2 and Uhr_2 are abbreviated to R^,r^,R 2 and r_ 2 r e s p e c t i v e l y . ** R = r e s i s t a n t ; S = s u s c e p t i b l e . I t i s an i n t e r e s t i n g feature of gene-for-gene int e r a c t i o n s that the expression of resistance a l l e l e s ( R - a l l e l e s ) and of virulence a l l e l e s ( v - a l l e l e s ) i s , i n both cases, conditioned by the genotype of the other i n t e r a c t i n g organism. For expression of an R - a l l e l e i t i s required that the rela t e d v - a l l e l e be absent. And, although the expression of a v - a l l e l e i s not conditioned by a l l e l e s at the r e l a t e d resistance locus i n the host, a v - a l l e l e can be expressed only i f other R - a l l e l e s at host l o c i unrelated to the v--a l l e l e i n question, are a l l confronted with t h e i r s p e c i f i c a l l y -r e l a t e d v - a l l e l e s . Resistance can be conferred on the host by any R - a l l e l e that i s not matched by a rela t e d v - a l l e l e , whereas sus-c e p t i b i l i t y (=virulence) requires that each and every host R - a l l e l e be confronted by matching v - a l l e l e s i n the p a r a s i t e . At the present moment gene-for-gene r e l a t i o n s h i p s have been either demonstrated or suggested to operate, i n about twenty para-s i t i c systems. These systems show c e r t a i n features i n common: i ) resistance of the host i s u s u a l l y i n h e r i t e d as a dominant c h a r a c t e r i s t i c , whereas virulence of the pathogen i s us u a l l y i n h e r i t e d as a recessive c h a r a c t e r i s t i c ; i i ) although there are several published reports of a l l e l i s m and/or linkage of resistance genes i n the host, t h i s has not been reported f o r virulence genes i n the pathogen. An exhaustive survey of the l i t e r a t u r e on resistance and virulence genes, i n systems where gene-for-gene r e l a t i o n s h i p s have not been 51. shown, has also revealed that these features are i n fact held i n common regardless of the species i n t e r a c t i n g i n the p a r a s i t i c system (Person and Sidhu, 1971). The genetic r e s u l t s of this study are i n agreement with point ( i ) above. I t w i l l therefore be of i n t e r e s t i n future work to look for a l l e l i s m , and for linkage, of resistance genes i n barley to covered smut. I t i s not known whether the features ( l i s t e d above), which on the basis of present knowledge appear to occur commonly i n p a r a s i t i c systems, w i l l i n f a c t turn out to be of general occurrence i n systems of parasitism. The reports which have been published are a l l concerned with diseases of a g r i c u l t u r a l crops. Very l i t t l e i s known of p a r a s i t i c systems which have been under natural, rather than a r t i f i c i a l s e l e c t i o n and i t i s therefore possible that these features may not be discovered i n natural systems of parasitism. The reasons why they should be found i n a g r i c u l t u r a l systems are not immediately apparent. A g r i c u l t u r a l experience has shown that newly-introduced crop v a r i e t i e s frequently lose t h e i r resistance a f t e r a few years of c u l t i v a t i o n , and that loss of resistance i n many cases i s known to be due to genetic changes which have taken place within the p a r a s i t i c populations. Where the resistance i s governed by a s i n g l e gene i n the host, and where th i s resistance i s l a t e r overcome by virulence a l l e l e s at a s i n g l e genetic locus i n the parasite, the basis for gene-for-gene r e l a t i o n s h i p i s established. But t h i s kind of event does not provide a ready explanation for the facts that resistance genes are u s u a l l y dominant whereas genes for virulence are usually recessive, or for the f a c t that a l l e l i s m and 52. close linkage have been reported f o r resistance genes, but not for virulence genes. In his studies of resistance i n f l a x to f l a x rust, F l o r (1971) found 26 d i f f e r e n t genes for resistance. These occurred at only f i v e genetic l o c i . At one of these l o c i , eleven d i f f e r e n t a l l e l e s were demonstrated. Genetic polymorphism of t h i s kind, i n v o l v i n g a l l e l e s for r esistance to disease, has also been demonstrated i n barley (for resistance to barley mildew) by Moseman, 1966; i n r i c e (for resistance to r i c e b l ast) by Kiyosawa, 1967; i n Malus (for resistance to apple scab) by Williams and Shay, 1966; and i n other s i m i l a r cases. Examples of a l l e l i s m i n c e r t a i n other a g r i c u l t u r a l hosts has r a r e l y been shown. This i s , perhaps, due to the lack of co-ordination among the i n v e s t i g a t o r s . I t w i l l not be s u r p r i s i n g i f the a l l e l i c series i n such hosts w i l l also be discovered when resistance genes from d i f f e r e n t sources are studied i n r e l a t i o n to each other. Papers have been published by Mode (1958) and by Person (1966) which deal with the possible r o l e of genetic polymorphism ( i . e . of a l l e l i s m of genes for resistance) i n plant p a r a s i t i c systems. These authors both examine the question of how hosts and t h e i r parasites have managed to co-exist over long periods of time; both conclude that genetic polymorphism can lead to the maintenance of a dynamic equilibrium, i n which the host and parasite are kept i n balance so that t h e i r long-term co-evolution i s p o s s i b l e . However, i t i s assumed by both these authors that the host populations are geneti-c a l l y heterogeneous, and that t h i s heterogeneity i s maintained 53. through outcrossing. A serious argument against t h i s assumption i s the f a c t that outcrossing i s not an important feature i n any of the systems i n which polymorphism has been thus f a r demonstrated. Mode (1958) concludes that the genetic polymorphism which now exis t s ( i n f l a x ) i s " r e l i c t " polymorphism, dating to an e a r l i e r time when host populations were outbreeders. Person (1966) on the other hand considers that polymorphism can be maintained i n inbreeding host populations such as f l a x or barley, so long as a small but r e l a t i v e l y constant l e v e l of outcrossing i s maintained. The model systems described by Mode (1958) and by Person (1966) both involve the maintenance, within a s i n g l e interbreeding host population, of a number of d i f f e r e n t a l l e l e s at a sing l e genetic locus for host r e s i s t a n c e . No system of th i s kind has yet been found, either i n natural or i n a g r i c u l t u r a l systems of parasitism. Where a l l e l i s m has been reported, the d i f f e r e n t a l l e l e s for resistance have been discovered i n d i f f e r e n t c u l t i v a r s of the host, whose immediate ancestry has also been d i f f e r e n t ( i . e . the a l l e l e s have not been found together i n a s i n g l e host population). From the evidence on hand, i t appears to be too early to assign a d e f i n i t i v e r o l e to genetic polymorphism, or to come to any fir m conclusions concerning the importance of genetic polymorphism, i n the co-evolution of hosts and t h e i r p a r a s i t e s . At the same time i t i s c l e a r that studies should be undertaken to investigate further the possible r o l e of genetic polymorphism i n maintaining host and parasite i n equilibrium. Such studies could lead to a much better understanding of how to 54. b r i n g t h e d i s e a s e s of c u l t i v a t e d c r o p s under b e t t e r l o n g - t e r m c o n t r o l . D e m o n s t r a t i o n of g e n e - f o r - g e n e r e l a t i o n s h i p s i s a m a t t e r o f c o n v e n i e n c e r a t h e r t h a n an e v a l u a t i o n o f t r u e s i t u a t i o n s o p e r a t i n g i n n a t u r a l systems o f p a r a s i t i s m . S e l e c t i o n o f r a c e s on a s e t o f d i f f e r -e n t i a l s i s q u a l i t a t i v e r a t h e r t h a n q u a n t i t a t i v e . I n o t h e r words t h e r a c e s a r e o n l y d e f i n e d a r b i t r a r i l y and a r e made t o d i f f e r i n t h e i r l e v e l s o f p a t h o g e n i c i t y by as l i t t l e d i f f e r e n c e s as can come w i t h i n t h e c o m p r e h e n s i o n o f man. But t h e f a c t r e m a i n s t h a t i s o l a t e s show c o n t i n u o u s v a r i a t i o n f r o m one end o f t h e s c a l e t o t h e o t h e r . The g e n e - f o r - g e n e i n t e r a c t i o n s on t h e o t h e r hand, a r e d e f i n e d on t h e b a s i s o f m e a s u r a b l e d i f f e r e n c e s i n terms of v e r t i c a l r e s i s t a n c e ( = v i r u l e n c e ) c o n f e r r e d by m a j o r genes. But i t would be r a t h e r o v e r s i m p l i f i c a t i o n t o v i e w t h a t i n t e r a c t i o n s between t h e members o f a p a r a s i t i c s y s t e m a r e always m e a s u r a b l e . Genes w h i c h a r e known t o t a k e p a r t i n g e n e - f o r - g e n e r e l a t i o n -s h i p s a r e a l s o genes w h i c h have l a r g e e f f e c t s . The r e s i s t a n c e genes a r e " major" genes ( u s i n g Van der P l a n k ' s t e r m i n o l o g y ) w h i c h c o n f e r a h i g h l e v e l o f r e s i s t a n c e , t h e r e l a t e d v-genes, i n whose p r e s e n c e t h i s r e s i s t a n c e i s n o t e x p r e s s e d , must a l s o be c o n s i d e r e d as h a v i n g l a r g e e f f e c t s . The q u e s t i o n can be a s k e d whether t h e g e n e - f o r - g e n e r e l a t i o n s h i p depends on t h e f a c t t h a t t h e genes c o n c e r n e d have l a r g e e f f e c t s , and whether t h e g e n e - f o r - g e n e r e l a t i o n s h i p would a r i s e i f t h e genes c o n c e r n e d d i d n o t have l a r g e e f f e c t s . A t t h e moment t h e s e q u e s t i o n s cannot be answered. I t w i l l t h e r e f o r e be i n t e r e s t i n g t o 55. look for gene-for-gene r e l a t i o n s h i p s under conditions i n which the genes concerned control small, rather than large, differences i n disease r e a c t i o n . Van der Plank (1968), i n his book "Disease Resistance i n Plants", has recently introduced a number of t h e o r e t i c a l concepts concerning the nature of disease resistance, and of i n t e r a c t i o n s between hosts and t h e i r p a r a s i t e s . Van der Plank distinguishes two kinds of host resistance: ( i ) v e r t i c a l resistance - t h i s i s associated with genes, referred to as "major genes", which give Mendelian segregations; i t i s resistance of t h i s kind which permits the recognition of physiologic races and which can give r i s e to gene-for-gene r e l a t i o n s h i p s . ( i i ) h o r i z o n t a l resistance - t h i s i s associated with "minor" genes or polygenes which act a d d i t i v e l y together to control a type of resistance which i s non-specific, (which does not permit recognition of physiologic races, and does not give r i s e to gene-for-gene r e l a t i o n s h i p s ) , and which, according to Van der Plank, w i l l be stable resistance, ( i . e . not having the tendency to i n c i t e , i n the pathogenic population, changes whose e f f e c t w i l l be to lessen the l e v e l of resistance conferred). The pathogenic counterpart of h o r i z o n t a l resistance, termed "aggressiveness" by Van der Plank, i s also associated with polygenes, and i s also n o n - s p e c i f i c . One of Van der Plank's most important predictions i s that the use, by plant breeders, of horizontal rather 56. than v e r t i c a l resistance would provide long-term solutions to the problems of disease control i n a g r i c u l t u r a l crops. Van der Plank's i n t e r e s t i n g theories are based l a r g e l y on h i s t o r i c a l and on e p i d e m i o l o g i c a l considerations. The v a l i d i t y of his assertions regarding the genetic nature of h o r i z o n t a l resistance, and of p a r a s i t i c aggressiveness, has yet to be demonstrated. The search for gene-for-gene r e l a t i o n s h i p s , under conditions i n which the genes concerned have small e f f e c t s , could y i e l d important i n f o r -mation r e l a t i n g to Van der Plank's theories. From the p h y s i o l o g i c a l and biochemical points of view the argument developed above has a useful extension. Both types of resistance, v e r t i c a l or h o r i z o n t a l , w i l l depend on the type of defence mechanism b u i l t into the host and the type of attack mechan-ism the pathogen w i l l incorporate to n u l l i f y the resistance. The phenomenon of gene-for-gene r e l a t i o n s h i p i s only relevant to major R-genes i n the host and physiologic races defined by them (Day, 1968). If the defence mechanism, as Van der Plank v i s u a l i z e s , incorporated i n the host requires the pathogen to increase aggressiveness a l l around i n order to match i t , the resistance w i l l be h o r i z o n t a l even i f i t i s only monogenic. Thus the p h y s i o l o g i c a l basis for the v e r t i c a l and h o r i z o n t a l resistance ( i f these are two kinds of r e s i s t -ance which e x i s t , separately, as d i s c r e t e e n t i t i e s ) should be quite d i f f e r e n t . However Zadocks (1972)argues persuasively that h o r i z o n t a l and v e r t i c a l are the two extremes of a continuous range of resistance. Apart from a l l these complexities of problem the nature of resistance i t s e l f i s an important and i n t r i g u i n g question. What t r i g g e r s the h o s t - r e s i s t a n c e mechanism under one set of c o n d i t i o n s but not others? Although gene-for-gene r e l a t i o n s h i p s have been rel e g a t e d o c c a s i o n a l l y to s u b s i d i a r y importance, the f a c t remains that any l o g i c a l clue about the mechanism of r e s i s t a n c e must emerge from systems that are g e n e t i c a l l y simple, and p r e f e r a b l y which e x h i b i t gene-for-gene r e l a t i o n s h i p s . The gene-for-gene hypothesis aids i n understanding the i n t r i -cate r e l a t i o n s h i p which e x i s t s between the host and the p a r a s i t e ; both from e v o l u t i o n a r y and p r a c t i c a l p o i n t s of view. To l e a r n how para-s i t i c systems operate i n nature would undoubtedly r e s u l t i n a b e t t e r understanding of the man-guided e v o l u t i o n of a g r i c u l t u r a l p a r a s i t e s (Johnson, 1961 and Yarwood, 1970) as w e l l as of r e s i s t a n c e mechanisms which o f t e n breaks down over short periods of time. The experimental approach of Pimentel (1961) i s of s p e c i a l i n t e r e s t , where he developed a theory of genetic feed-back by which a s t a b l e e q u i l i b r i u m among para-s i t i c populations i s achieved ( c f . Mode, 1958). In Pimentel's (1965) view"a g e n e t i c a l l y d i v e r s e host p o p u l a t i o n would lead to a g e n e t i c a l l y d i v e r s e pathogen p o p u l a t i o n ? " D i v e r s i t y i n the host p o p u l a t i o n would fo r c e a corresponding d i v e r s i t y i n the pathogen p o p u l a t i o n . Such a s i t u a t i o n , as f u r t h e r viewed by Pimentel, would e v e n t u a l l y lead to a s t a b l e e q u i l i b r i u m i n the p a r a s i t i c system. The genetic feed-back hypothesis of Pimentfel r e c e i v e s i t s strongest support from the concept of gene-for-gene r e l a t i o n s h i p s . Mode (1958) and Person (1966, 1968) as p r e v i o u s l y discussed have ijhown, t h e o r e t i c a l l y , the relevance of a 58. study of gene-for-gene r e l a t i o n s h i p s to the understanding of n a t u r a l l y o c c u r r i n g p a r a s i t i c systems, but experimental data i n support of t h e i r r e s p e c t i v e hypotheses i s d i f f i c u l t to o b t a i n ( F l o r , 1971). Pimentel (1968) has c a r r i e d out some i n t e r e s t i n g and f r u i t f u l s t u d i e s i n t h i s d i r e c t i o n , and u s e f u l s t u d i e s could p o s s i b l y be done a l s o i n p a r a s i t i c systems i n v o l v i n g smut species. Some of the smuts possess most of the a t t r i b u t e s s u i t a b l e f o r p o p u l a t i o n s t u d i e s (Person, 1968). B a r l e y covered smut ( U s t i l a g o h o r d e i ) , w i t h which the present t h e s i s d e a l s , o f f e r s good oppor-t u n i t i e s f o r d i s c o v e r i n g f a c t o r s which may act to maintain balance among p a r a s i t i c systems. From a p r a c t i c a l p o i n t of view the study of gene-for-gene r e l a t i o n s h i p s can be u t i l i z e d to evaluate: i ) the sources of pathogenic v a r i a b i l i t y i n p a r a s i t i c systems, i i ) the m u t a b i l i t y of r e s i s t a n c e and v i r u l e n c e genes, i i i ) the problem of why host r e s i s t a n c e i s expressed under one set of c o n d i t i o n s and not under others. These and other aspects of h o s t - p a r a s i t e r e l a t i o n s h i p s have been discussed by Day (1960, 1966, 1968) and more r e c e n t l y , by F l o r (1971). T h i s t h e s i s describee the f i r s t case of a gene-for-gene r e l a t i o n s h i p ever demonstrated i n a p a r a s i t i c system i n v o l v i n g a smut fungus. Gene-for-gene r e l a t i o n s h i p s have been suggested f o r other smut species ( c f . Table IV) but i n a l l these cases the 59. suggestions are based on evidence which i s i n s u f f i c i e n t and i n -c o n c l u s i v e . There are very few p a r a s i t i c systems i n which i t i s p o s s i b l e to ca r r y out p r e c i s e and w e l l c o n t r o l l e d experimentation w i t h both the host and the p a r a s i t e . Among these few, the Hordeum-U s t i l a g o system, i n v e s t i g a t e d i n t h i s t h e s i s i s one of the most fa v o r a b l e . Because of the advantages o f f e r e d by t h i s system, i t i s now p o s s i b l e to f o l l o w s e v e r a l new avenues i n f u t u r e research. Some suggestions are as f o l l o w s : i ) I d e n t i f i c a t i o n of two v i r u l e n c e genes i n U. h o r d e i , now makes i t p o s s i b l e to determine whether or not they w i l l i n t e r a c t when brought together i n a s i n g l e dikaryon ( i . e . to l e a r n whether they act independently, or a d d i t i v e l y , or, p o s s i b l y , a n t a g o n i s t i c a l l y ) ; i i ) Three d i f f e r e n t l e v e l s of v i r u l e n c e have been a r b i -t r a r i l y defined i n U. hordei by Tapke (1937). The two genes reported here represent higher l e v e l s of v i r u l e n c e . P r e v i o u s l y s i n g l e genes f o r low as w e l l as fo r intermediate l e v e l s of v i r u l e n c e have been reported, i n U. h o r d e i , by Thomas and Person (1965) and by Lade (1969), r e s p e c t i v e l y . I t w i l l be of i n t e r e s t , now, to study the i n t e r a c t i o n s between genes c o n t r o l l i n g d i f f e r e n t l e v e l s of v i r u l e n c e on the same host v a r i e t y when these are put together i n the same dikaryon. This would a l s o open the p o s s i b i l i t y of l o o k i n g f o r a l l e l i s m of v i r u l e n c e genes. Furthermore, such s i t u a t i o n s may be 60. amenable to biochemical a n a l y s i s . A s i n g l e v i r u l e n c e locus g i v i n g d i f f e r e n t l e v e l s of v i r u l e n c e on d i f f e r e n t hosts, or a s i n g l e r e s i s t a n c e gene e x h i b i t i n g d i f f e r e n t l e v e l s of r e s i s t a n c e against d i f f e r e n t c u l t u r e of the p a r a s i t e would suggest biochemical d i v e r i s i t e s at d i f f e r e n t l e v e l s of i n t e r a c t i o n s i n a p a r a s i t i c system, i i i ) Through i n c o r p o r a t i o n of g e n e t i c a l l y - c o n t r o l l e d biochemical d e f i c i e n c i e s i n t o c u l t u r e s having known v i r u l e n c e genes, i t i s now p o s s i b l e to study the r e l a t i o n s h i p s , ( i f any e x i s t ) , between the n u t r i t i o n a l needs of the pathogen and i t s v i r u -lence towards s p e c i f i c v a r i e t i e s or genotypes of the host, i v ) I t can now be asce r t a i n e d whether the v a r i a b i l i t y range of v i r u l e n c e shown by the two v i r u l e n c e genes i s due i n part to the segregation of polygenes i n a s s o c i a t i o n w i t h the major gene, whether t h i s v a r i a b i l i t y i s due to environmental cause. The l e v e l of s o p h i s t i c a t i o n of genetics a n a l y s i s , such as t e t r a d a n a l y s i s a v a i l a b l e i n smut spe c i e s , f a c i l i t a t e such an approach, v) I t i s now p o s s i b l e to look f o r gene-for-gene r e l a t i o n s h i p s i n v o l v i n g d i f f e r e n t l e v e l s , i n c l u d i n g very low l e v e l s , of p a t h o g e n i c i t y to see i f there i s any l e v e l at which the gene-for-gene r e l a t i o n s h i p ceases to e x i s t . Such in f o r m a t i o n could c o n t r i b u t e to a b e t t e r understanding of the e v o l u t i o n a r y problems as s o c i a t e d w i t h p a r a s i t i s m . 61. SUMMARY The genetics of v i r u l e n c e of three t e l i o s p o r e s of U s t i l a g o h o r d e i towards f i v e b a r l e y v a r i e t i e s ( E x c e l s i o r , Vantage, Hannchen, T r e b i and Lion) was i n v e s t i g a t e d . Two v i r u l e n c e genes, Uhv^ and Uhv 2 were i d e n t i f i e d ; Uhv^ was e f f e c t i v e on both Hannchen and Vantage and Uhv 2 on E x c e l s i o r . Both the V-genes were r e c e s s i v e . V a r i e t i e s T r e b i and L i o n were s u s c e p t i b l e to both the V-genes. On the basis of the known V-genes (Uhv^ and Uhv^ the genetics of r e s i s t a n c e of three b a r l e y v a r i e t i e s , E x c e l s i o r , Hannchen and Vantage was i n v e s t i g a t e d . Two r e s i s t a n c e genes, UhR-^ and UhR 2 were discovered. UhR^ was shared by v a r i e t i e s Hannchen and Vantage and was found through the i n t e r a c t i o n s of the V-gene Uhv^ while UhR 2, present i n v a r i e t y E x c e l s i o r , was discovered i n response to the V-gene Uhv 2. 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D i s t i l l e d water 1 1. Tryptophane 50 mg. Casein hydrolysate 5 gm. Yeast e x t r a c t (Difco) 5 gm. Sucrose 20 gm. or Dextrose 10 gm. Vitamin S o l u t i o n 10 ml. Note: 1. Vitamin s o l u t i o n to be added a f t e r a u t o c l a v i n g 2. To make complete p l a t e s add 20 gm. Bacto-agar before a u t o c l a v i n g . (B) VOGEL'S SOLUTION (concentrate) Na 3 c i t r a t e . 2H2O 123 gm. KH2PO4 anhydrous 250 gm. NH4NO3 anhydrous 100 gm. MgSO, . 7H 20 10 gm. C a C l 2 • 2H 20 5 gm. Trace element s o l u t i o n 5 ml. D i s t i l l e d water 750 ml. Chloroform 2 ml. Note: 1. Add chemicals s u c c e s s i v e l y w i t h s t i r r i n g . 2. Store at room temperature. 3. D i l u t e 50-fold w i t h d i s t i l l e d water before use (C) VITAMIN SOLUTION Thiamin 100 mg. R i b o f l a v i n . 50 mg. P y r i d o x i n 50 mg. Calcium pantothenate 200 mg. Benzoic a c i d 50 mg. N i c o t i n i c a c i d 200 mg. Choline c h l o r i d e 200 mg. I n o s i t o l 400 mg. F o l i c a c i d 50 mg. D i s t i l l e d water to a t o t a l of 1 1. Note: 1. Store at 4°C. 2. Use 10 ml. of v i t a m i n s o l u t i o n per l i t r e of s t e r i l e medium. APPENDIX B 72. F^ l i n e s derived from the crosses E x c e l s i o r x Hannchen and E x c e l s i o r x Vantage showing r e s p e c t i v e numbers of p l a n t s t e s t e d (NPT) and i n f e c t i o n percentages (%ages) against the t e s t c u l t u r e s v^v-^V2V2and V^V-^v2v2 F^ l i n e Reactions of F^ l i n e s derived Reactions of Fg l i n e s derived Number from the cross EXV to t e s t from the cross EXH to t e s t c u l t u r e s : c u l t u r e s : v 1 v 1 V 2 V 2 V 1 V 1 V 2 V 2 v 1 v 1 V 2 V 2 V i V i v 2 v 2 NPT %ages NPT %ages C l a s s * * NPT %ages NPT %ages Class 1 48 0 54 33 R-S 52 12 48 16 S-S 2 48 0 40 13 R-S 63 0 54 41 R-S 3 64 0 51 0 R-R 51 0 36 8 R-S 4 45 33 48 15 S-S 55 0 42 14 R-S 5 19 5 61 25 S-S 42 7 42 38 S-S 6 36 11 45 13 S-S 30 23 50 28 S-S 7 53 0 49 39 R-S 61 0 51 4 R-S 8 42 50 58 45 S-S 24 4 61 0 S-R 9 46 13 50 8 S-S 42 10 55 0 S-R 10 51 0 34 6 R-S 66 0 48 0 R-R 11 45 9 54 15 S-S 45 33 46 9 S-S 12 46 15 45 29 S-S 48 8 63 0 S-R 13 39 44 55 38 S-S 50 24 58 12 S-S 14 32 9 46 28 S-S 32 6 49 0 S-R 15 43 12 58 0 S-R 29 7 51 0 S-R 16 34 9 50 10 S-S 48 13 60 0 S-R 17 48 25 55 9 S-S 58 12 29 28 S-S 18 61 0 60 0 R-R 58 0 52 40 R-S 19 45 7 61 0 S-R 62 6 35 34 S-S 20 62 0 48 8 R-S 49 0 61 0 R-R 21 48 15 61 33 S-S miss i n g 41 7 -22 63 0 55 0 R-R 63 0 23 4 R-S 23 48 15 63 0 S-R 56 0 51 0 R-R 24 53 0 50 0 R-R 61 0 55 36 R-S 25 59 0 68 47 R-S 48 10 48 13 S-S 26 48 13 48 16 S-S 25 4 51 10 S-S 27 52 12 48 0 S-R 42 10 58 0 S-R 28 76 0 59 0 R-R 58 0 49 0 R-R 29 50 24 46 35 S-S 59 0 62 5 R-S 30 51 0 40 12 R-S 57 16 52 19 S-S 31 42 33 63 0 S-R 48 25 48 15 S-S 32 48 38 60 0 S-R 61 0 41 8 R-S 33 50 32 42 27 S-S 35 51 65 0 S-R 34 63 0 70 0 R-R 50 10 60 0 S-R 35 13 8 38 5 S-S 42 31 38 16 S-S 36 48 0 52 27 R-S 58 38 62 48 S-S 37 21 14 50 8 S-S 48 8 41 12 S-S 38 42 17 66 33 S-S 58 16 48 38 S-S 39 48 13 48 19 S-S 58 31 38 5 S-S 40 50 34 55 27 S-S 48 0 61 0 R-R 41 40 8 59 0 S-R 49 0 58 0 R-R 42 31 3 66 0 S-R 51 0 48 0 R-R 43 41 0 58 21 R-S 33 33 52 15 S-S 44 48 0 49 12 R-S 52 0 49 0 R-R 45 38 11 40 38 S-S 42 29 66 0 S-R 46 35 9 49 0 S-R 48 13 49 0 S-R 47 40 0 58 0 R-R 49 0 58 0 R-R 48 30 40 68 25 S-S 64 0 33 9 R-S 49 60 0 17 6 R-S 61 0 71 49 R-S 50 40 18 51 20 S-S 60 13 60 33 S-S 51 63 0 61 0 R-R 56 0 38 16 R-S 52 38 47 60 40 S-S 58 9 64 39 S-S 53 42 19 55 22 S-S 28 7 56 25 S-S 54 42 14 54 33 S-S 38 32 61 0 S-R 55 45 7 21 5 S-S 63 0 41 2 R-S 56 53 0 48 27 R-S 39 46 48 6 S-S 57 60 0 35 11 R-S 60 8 58 0 S-R 58 48 15 45 24 S-S 62 0 53 0 R-R 59 51 25 38 16 S-S 59 0 52 12 R-S 60 52 8 48 15 S-S 38 8 58 10 S-S 61 50 8 49 0 S-R 64 0 54 26 R-S 62 42 33 55 13 S-S 58 14 50 20 S-S 63 55 13 58 16 S-S 55 9 55 22 S-S 64 63 0 68 0 R-R 61 0 61 5 R-S 65 52 12 60 8 S-S m i s s i n g 52 25 -66 38 13 60 25 S-S 52 10 58 14 S-S 67 48 8 61 0 S-R 55 11 48 0 S-R 68 52 13 58 17 S-S 51 0 40 8 R-S 69 48 0 48 29 R-S 48 13 42 21 S-S 70 45 16 60 0 S-R 38 8 50 6 S-S 71 42 33 54 31 S-S 61 11 58 14 S-S 72 56 0 40 10 R-S 48 0 60 0 R-R 73 48 13 45 13 S-S 49 0 50 36 R-S 74 43 35 50 24 S-S 38 5 63 33 S-S 75 40 3 28 7 S-S 26 4 63 0 S-R 76 35 34 56 0 S-R 38 3 54 0 S-R 77 38 39 70 0 S-R 42 33 50 0 S-R 78 40 40 62 0 S-R 58 7 60 8 S-S 79 32 3 33 3 S-S 61 0 48 10 R-S 80 16 13 48 25 S-S 64 0 66 0 R-R 81 60 0 62 0 R-R 38 53 57 16 S-S 82 60 13 63 0 S-R 60 12 60 13 S-S 83 53 0 35 6 R-S 56 0 31 3 R-S 84 58 10 39 23 S-S 53 0 38 37 R-S 85 56 11 m i s s i n g - 45 13 50 40 S-S 86 62 0 " - 48 8 42 10 S-S 87 52 9 " - 28 4 52 15 S-S 88 63 0 - 39 8 48 38 S-S 89 31 10 - 55 13 56 14 S-S 90 42 10 " - 56 0 60 0 R-R 91 51 0 36 33 R-S 50 10 51 14 S-S 92 48 0 45 24 R-S 44 25 60 8 S-S 93 38 8 50 6 S-S 63 0 31 3 R-S 94 39 18 45 20 S-S 38 24 54 0 S-R 95 40 30 44 34 S-S 69 0 38 5 R-S 96 51 0 33 6 R-S 28 7 42 5 S-S 97 16 13 18 6 S-S 70 0 52 0 R-R 98 45 44 65 0 S-R 51 0 58 41 R-S 99 36 11 57 11 S-S 48 8 33 9 S-S 100 42 38 51 22 S-S 45 9 28 7 S-S 101 40 28 65 0 S-R 41 5 61 0 S-R 102 40 5 42 7 S-S 30 3 39 10 S-S 103 48 10 49 20 S-S 61 0 43 49 R-S 104 52 8 46 0 S-R 58 0 38 8 R-S 105 52 23 48 38 S-S 45 11 42 19 S-S 106 50 0 55 18 R-S 48 38 55 36 S-S 107 48 0 65 0 R-R 48 23 48 17 S-S 108 40 3 38 5 S-S 46 35 56 13 S-S 109 48 4 41 0 S-R 50 12 58 9 S-S 110 41 0 34 12 R-S 52 15 60 13 S-S 111 52 0 48 8 R-S 61 11 61 0 S-R 112 54 0 56 14 R-S 56 7 61 11 S-S 113 38 32 49 0 S-R 28 4 39 31 S-S 114 48 13 50 0 S-R 46 46 51 47 S-S 115 50 36 45 27 S-S 53 0 48 8 R-S 116 50 22 48 0 S-R 52 27 48 38 S-S 117 61 0 35 6 R-S 33 9 38 16 S-S 118 48 13 38 16 S-S 54 0 51 37 R-S 119 66 0 46 0 R-R 48 29 49 33 S-S 120 missing 30 3 - 68 0 39 3 R-S 121 38 16 40 30 S-S 48 0 68 0 R-R 122 11 9 25 8 S-S 58 7 25 8 S-S 123 48 23 32 6 S-S 50 36 58 0 S-R 124 50 38 62 37 S-S 60 13 63 14 S-S 125 48 0 28 14 R-S 58 29 61 34 S-S 126 54 17 49 18 S-S 46 46 63 0 S-R 127 45 40 52 19 S-S 40 13 48 15 S-S 128 48 33 55 0 S-R 38 16 59 0 S-R 129 38 29 52 13 S-S 52 42 58 45 S-S 130 35 34 45 11 S-S 55 9 50 40 S-S 131 40 13 46 0 S-R 58 9 61 0 S-R 132 49 14 56 16 S-S 61 0 40 5 R-S 133 38 42 56 32 S-S 55 9 66 0 S-R 134 35 34 50 20 S-S 52 15 68 0 S-R 135 46 0 31 6 R-S 48 25 36 8 S-S 136 32 6 28 21 S-S 50 36 45 11 S-S 137 52 0 48 27 R-S 45 7 38 32 S-S 138 38 39 36 27 S-S 38 7 42 26 S-S 139 41 27 35 9 S-S 45 24 60 0 S-R 140 40 40 42 17 S-S 40 38 35 6 S-S 141 66 0 30 7 T-S 48 25 58 0 S-R 142 52 19 45 24 S-S 28 18 36 11 S-S 143 48 25 42 10 S-S 36 22 48 0 S-R 144 63 0 61 28 R-S 48 15 53 0 S-R 145 55 11 46 0 S-R 64 0 32 3 R-S 146 43 5 48 0 S-R 56 14 54 0 S-R 147 58 0 38 16 R-S 50 28 32 9 S-S 148 40 5 32 6 S-S 23 4 48 15 S-S 149 29 3 61 0 S-R 57 12 60 13 S-S 150 65 0 64 0 R-R 60 0 42 2 R-S * Tested at C a l i f o r n i a (1970) ** R - R e s i s t a n t ; S = S u s c e p t i b l e APPENDIX C 75. l i n e s derived from the crosses E x c e l s i o r x Hannchen and E x c e l s i o r x Vantage showing r e s p e c t i v e numbers of p l a n t s tested(NPT) and i n f e c t i o n percentages (70ages) against the t e s t c u l t u r e s v^v-^V2V2 and V-^V^V2V2 Fg l i n e Reactions of F 3 l i n e s derived Reaction of F^ l i n e s derived Number from the cross E x V to t e s t from the cross E x H to t e s t c u l t u r e s : c u l t u r e s V 1 V 1 V 2 V 2 V 1 V 1 V 2 V 2 v - ^ v j ^ ^ V 1 V 1 V 2 V 2 1 NPT Xages NPT %ages C l a s s * * NPT Xages NPT 7»ages Clas 1 32 13 29 38 S-S 22 9 31 13 S-S 2 38 0 40 0 R-R 32 6 38 0 S-R 3 30 7 37 11 S-S 36 0 27 22 R-S 4 41 0 22 5 R-S 48 4 25 4 S-S 5 16 38 38 56 S-S 49 4 36 36 S-S 6 45 0 22 55 R-S 40 0 36 6 R-S 7 15 33 50 0 S-R 39 0 39 36 R-S 8 48 0 39 33 R-S 29 3 42 0 S-R 9 35 9 40 5 S-S 42 5 46 7 S-S 10 38 0 42 0 R-R 38 0 37 59 R-S 11 22 32 36 12 S-S 32 41 36 69 S-S 12 30 13 50 24 S-S 42 0 15 13 R-S 13 42 0 31 32 R-S 42 2 45 0 S-R 14 25 16 22 9 S-S 30 7 30 0 S-R 15 4 25 56 30 S-S 35 14 31 3 S-S 16' 28 4 34 12 S-S 32 6 45 2 S-S 17 21 5 21 68 S-S 34 9 39 0 S-R 18 21 19 50 0 S-R 49 0 19 42 R-S 19 20 5 23 4 S-S 37 0 31 0 R-R 20 39 0 24 21 R-S 30 3 37 3 S-S 21 17 6 33 15 S-S 38 3 32 6 S-S 22 25 4 48 0 S-R 46 0 28 25 R-S 23 20 10 52 0 S-R 27 33 30 7 S-S 24 38 0 48 0 R-R 38 3 34 12 S-S 25 27 4 43 5 S-S 41 0 22 9 R-S 26 24 8 40 0 S-R 38 0 39 0 R-R 27 28 4 35 49 S-S 38 11 21 24 S-S 28 38 0 28 7 T-S 39 0 31 13 R-S 29 22 9 19 11 S-S 40 48 25 24 S-S 30 44 0 44 14 T-S 44 25 48 0 S-R 31 40 0 6 33 R-S 42 31 32 3 S-S 32 45 0 26 19 R-S 34 3 38 0 S-R 33 25 8 24 63 S-S 48 8 35 0 S-R 34 24 8 29 10 S-S 46 0 24 25 R-S 35 28 4 45 33 S-S 18 33 18 6 S-S 36 33 3 34 53 S-S 45 0 25 40 R-S 37 36 31 32 72 S-S 41 12 22 5 S-S 38 30 3 40 7 S-S 42 10 15 53 S-S 39 46 8 17 29 S-S 43 0 42 0 R-R 40 41 0 38 0 R-R 34 15 36 0 S-R •sV* 41 30 3 32 6 S-S 39 0 44 0 R-R 42 32 6 11 9 S-S 38 8 24 17 S-S 43 36 3 18 17 S-S 41 0 46 0 R-R 44 31 32 41 0 S-R 32 3 48 19 S-S 45 38 0 42 0 R-R 45 2 20 5 S-S 46 35 0 30 33 R-S 37 16 42 10 S-S 47 28 0 27 7 R-S 15 13 30 13 S-S 48 37 0 18 67 R-S 46 0 22 9 R-S 49 36 17 16 25 S-S 25 4 33 0 S-R 50 41 0 40 0 R-R 19 5 52 12 S-S 51 45 2 42 0 S-R 36 8 40 0 S-R 52 32 31 15 20 S-S 25 4 41 15 S-S 53 41 0 26 54 R-S 26 4 31 13 S-S 54 27 0 27 7 R-S 42 0 42 0 R-R 55 25 4 36 28 S-S 24 9 31 13 S-S 56 42 0 19 16 R-S 35 0 27 4 R-S 57 55 20 28 18 S-S 22 5 34 6 S-S 58 41 0 39 8 R-S 36 0 41 17 R-S 59 45 0 41 0 R-R 26 4 57 47 S-S 60 39 0 48 0 R-R 32 0 49 29 R-S 61 32 3 50 0 S-R 34 3 58 0 S-R 62 28 4 14 14 S-S 30 3 50 2 S-S 63 41 5 12 17 S-S 30 3 30 3 S-S 64 40 0 25 20 R-S 24 25 35 0 S-R 65 33 0 27 4 R-S 34 0 42 5 R-S 66 29 0 21 48 R-S 28 32 41 2 S-S 67 19 21 40 0 S-R 22 14 46 ' 2 S-S 68 18 0 19 11 R-S 28 0 35 0 R-R 69 38 3 43 26 S-S 21 38 45 20 S-S 70 43 0 15 7 R-S 41 0 46 0 R-R 71 38 29 28 0 S-R 23 4 39 0 S-R 72 21 29 12 42 S-S 22 36 32 9 S-S 73 20 30 38 0 S-R 18 27 44 2 S-S 74 36 3 41 0 S-R 45 0 49 4 R-S 75 30 3 22 5 S-S 24 8 35 0 S-R * Tested at U n i v e r s i t y of B r i t i s h Columbia (1971) '* R = R e s i s t a n t ; S = S u s c e p t i b l e PUBLICATIONS S i d h u , Gurmel . 19 66. A n i m a l B i o l o g y . Lahore P u b l i s h i n g House, Ludhiana I n d i a . 351 pp . (Pb. E d i . ) S i d h u , G . , F . Wellenmann and V . C . B r i n k , 1968. C a r y o p s i s c o l o r s as markers f o r s p e c i a l i t y wheats . WIS S i d h u , G. and C l a y t o n P e r s o n . 1971'. G e n e t i c s of h o s t - p a r a s i t e i n t e r -r e l a t i o n s h i p s . Symp. " M u t a t i o n b r e e d i n g f o r r e s i s t a n c e " , IAEA, V i e n n a . 31-38 . S i d h u , Gurmel and C l a y t o n P e r s o n . 1971. G e n e t i c c o n t r o l of v i r u l e n c e i n U s t i l a g o h o r d e i . I I . S e g r e g a t i o n s f o r h i g h e r l e v e l s of v i r u l e n c e . Can. J . Genet . C y t o l . 13 : 173. S i d h u , Gurmel and C l a y t o n P e r s o n . 1972. G e n e t i c c o n t r o l of v i r u l e n c e i n U s t i l a g o h o r d e i . I I I . I d e n t i f i c a t i o n of gene f o r h o s t r e s i s t a n c e and demonst ra t ion o f . g e n e - f o r - g e n e r e -l a t i o n s . Can. J . Genet . C y t o l (In p r e s s ) . S i d h u , Gurmel and (Y .A . Emara and C. P e r s o n ) . P o l y g e n i c m o d i f i c a t i o n s of v i r u l e n c e i n U s t i l a g o h o r d e i . Submit ted to N a t u r e . S i d h u , Gurmel and C l a y t o n P e r s o n . 1972. G e n e t i c c o n t r o l of v i r u l e n c e i n U s t i l a g o h o r d e i . . IV . Monogenic c o n t r o l of two l e v e l s of v i r u l e n c e . 17th Ann. M e e t i n g , G e n e t i c s S o c . Canada. ( A b s t r a c t ) .
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A genetic study of host : parasite relationship involving Hordeum vulgare and Ustilago hordei
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