This is to certify that the
dissertation entitled
GENETIC ANALYSIS OF THE Ml-a LOCUS

IN BARLEY CONDITIONING REACTION
TO ERYSIPHE GRAMINIS F. SP. HORDEI

 

presented by

Roger Philip Wise

has been accepted towards fulfillment
of the requirements for

Ph.D. Genetics
degree in

 

 

EM»; (u. Qua/W
Ma jorfirofessor/
Dennis W. Fulbright

Date //- 7’ 83

 

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GENETIC ANALYSIS OF THE Ml—a LOCUS
IN BARLEY CONDITIONING REACTION

TO ERYSIPHE GRAMINIS F. SP. HORDEI

 

BY

Roger Philip Wise

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Interdisciplinary Genetics Program

1983

ABSTRACT

GENETIC ANALYSIS OF THE Ml—a LOCUS
IN BARLEY CONDITIONING REACTION
TO ERYSIPHE GRAMINIS F. SP. HORDEI

 

BY

Roger Philip Wise

The Ml-a locus on chromosome 5 in barley (Hordeum

 

vulgare L.) confers specific resistance to powdery mildew

caused by Erysiphe graminis f. sp. hordei. There are a

 

large number of naturally-occurring alleles or closely-
linked genes at this locus. An investigation was undertaken
to determine the effect of several different host genes on
pathogen development during primary infection. The
infection kinetics were determined for race CR3 of Erysiphe
graminis f. sp. hordei on six sets of isolines of barley.

Ml—a, Ml-a6 or Ml-a13 lines gave a type 0 final infection

 

whereas Ml-a7(Mu), M1-a7(LG) or Ml-alo lines gave a type

 

 

1-2 or type 2 final infection. The percent elongating
secondary hyphae (ESH) ranged from 8 to 21 at 28 hours after
inoculation. A significant difference in the percent ESH

was found between three groups of lines; (Ml-a, M1-a6,

 

Ml-a13), Ml-a7(Mu), and [Ml-310, Ml—a7(LG)]. A

 

 

 

significant difference was also found in the rate of

Roger Philip Wise

development of ESH between Ml-a7(LG) and the other five

 

lines examined, suggesting a difference in specificity. It
is proposed that the 53:3 gene in Long Glumes conferring
resistance to race CR3 of E. graminis f. sp. hordei be
designated Ml-alS.

The genetic fine structure of this locus was also
investigated. Crosses were made among six isolines of

barley having different specificities at the Ml-a locus.

 

Rare susceptible recombinants were recovered by F3

family analysis from the Ml-alO x Ml-a, Ml—a x Ml-a7(LG),

 

 

and Ml-a6 x Ml-a13 crosses. Exchange of the flanking

markers Hor-l and Her-2 accompanied exchange within the Ml-a

 

region in these crosses. In the M1-a6 x Ml-a13 cross, a
number of susceptible recombinants did not fit the expected
F3 family segregation ratios. These recombinants often
reverted to resistance in subsequent generations. In a
number of cases, reversion was incomplete. No recombinants
were recovered in the reciprocal cross, Ml-a13 x Ml-a6.

The possibility of an autonomous regulatory element, and a

possible linear order of six "alleles" at the Ml-a locus is

 

discussed.

DEDICATED
to
my family
and in loving memory of

my grandparents, Claude and Shirley Wise

ii

ACKNOWLEDGEMENTS

I wish to express my gratitude for the guidance and
support of Dr. Albert H. Ellingboe and Dr. Dennis W.
Fulbright, my major professors.

I am grateful for the hours of discussion and the
contributions made by each of the members of my guidance
committee: Dr. Jon F. Fobes, Dr. Peter S. Carlson, Dr. Hsing
Jien Kung, Dr. Leonard G. Robbins, and Dr. Chris R.
Somerville.

I would like to give special thanks for the
technical assistance of Joseph Clayton and Al Ravenscroft.
Without them, this work could not have been accomplished.

Very special thanks goes to Molly Stack, for her
love and support during both good and difficult times.

This research was supported in part by National
Science Foundation Grant PCM77-053403, BRSG Grant #2-807
RRO7049-15 awarded by the Biomedical Research Support Grant
Program, Division of Research Resources, National Institutes
of Health, and by a grant from the International Plant

Research Institute.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES ........................................ v
LIST OF FIGURES . ....... ............................... vii
INTRODUCTION .......................................... 1
LITERATURE REVIEW .. ...... ............................. 4
Multiple allelic systems ..... ................. 10
Summary ....................................... 14
Literature Cited .............................. 16
CHAPTER
I. Infection kinetics of Erysiphe graminis

f. sp. hordei on barley with different
alleles at the Ml-a IOCUS 00.000000000000000... 18

 

Abstract ............. ...... ................ 18
Introduction ............................... 19
Materials and Methods ...................... 21
Results .................................... 25
Discussion ................................. 30
Literature Cited ........................... 34

II. Investigations on recombination at the Ml-a
locus in barley conditioning reaction to
Erysiphe graminis f. sp. hordei. Fine
structure analysis and ev1dence for
controlling-element alleles ................... 35

 

Abstract ................................... 35
Introduction ............................... 36
Materials and Methods ...................... 39
Results .................................... 50
Discussion ................................. 74
Summary .................................... 83
Literature Cited ........................... 86

iv

LIST OF

LIST OF

TABLES .....

FIGURES ...

INTRODUCTION ......

LITERATURE REVIEW ..

CHAPTER

I.

II.

TABLE OF CONTENTS

Multiple allelic systems

summary ......OOOOOOOOIOOOOO
Literature Cited

Infection kinetics of Erysiphe graminis
f. sp. hordei on barley with different
alleles at the Ml-a lOCUS oooooooooooooooooo

Abstract

 

Introduction
Materials and Methods

Results

Discussion
Literature

Cited

Investigations on recombination at the Ml-a
locus in barley conditioning reaction to
Erysiphe graminis f. sp. hordei.

 

Fine

structure analysis and ev1dence for

controlling-element alleles

Abstract

Introduction
Materials and Methods

Results

Discussion

Summary

Literature Cited

iv

Page

vii

10
l4
16

18

18
19
21
25
3O
34

35

35
36
39
50
74
83
86

LIST OF TABLES

Page

The reaction of 3 host lines with 4
strains of a pathogen........................... 5

The basic pattern of inheritance observed
for interactions between host and parasite...... 7

The basic pattern of interactions between
host and paraSite...0....OOOOOOOOOOOOOOOOOOOOOOO 8

The interaction between the Sr6 gene in

Triticum aestivum and the COFFEsponding

P6 gene in Puccinia graminis f. sp. tritici

at two temperatures............................. 9

 

 

Six pairs of near-isogenic barley lines
and their reaction to culture CR3 of
Erysiphe graminis f. sp. hordei................. 22

 

Percent elongating secondary hyphae (ESH)

of Erysiphe graminis f. sp. hordei race CR3

on six resistant isolines of barley cultivar
Manchuria....................................... 24

 

Six near-isogenic barley lines and cultivar
Manchuria and their reaction to culture CR3
of Erysiphe graminis f. sp. hordei.............. 40

 

Reciprocal parental crosses and the

segregation pattern of F families 7 days

after inoculation with race CR3 of Erysiphe

graminis f. sp. hordei.......................... 51

 

 

Results of powdery mildew tests of F
segregating progeny from reciprocal grosses
between Ml-BG and M1"al3ooocoo-00.000.000.000... 56

Table

10.

11.

12.

13.

140

Page

Reciprocal parental crosses of Ml—a13 and

four other isolines and the pattern of

segregation 7 days after inoculation with

race CR3 of Erysiphe graminis f. sp. hordei.... 58

 

Frequency of exchange between Hor—l and Her-2

in reciprocal crosses between barley isolines

M1-a6 and Ml-a13 and a cross between Ml-a6

and Ml-a....................................... 63

 

 

Results of inoculation of progeny from
OR:IS and 3R:1S F families with race
CR3 of Erysiphe graminis f. sp. hordei......... 67

 

Results of powdery mildew tests with progeny
of crosses between M1-a6/Ml-a13

recombinants and Manchuria and between
Ml-a6/M1-a13 recombinants and the

 

 

susceptible fill-86 iSOIineoooooooooooooococoon.o 69

Results of powdery mildew tests on progeny
of intercrosses among different
Ml-aé/Ml-a13 recombinant—8000000000oooooooooooooo 7O

 

vi

Figure

1.

10.

LIST OF FIGURES

Page

Comparison of cis and trans heterozygotes for
codominance for specific disease resistance ..... 12

Formation of elongating secondary hyphae
(ESH) on susceptible isolines of barley by
E. graminis f. sp. hordei race CR3............... 26

 

Formation of elongating secondary hyphae

(ESH) by E. graminis f. sp. hordei race

CR3 on near-isogenic barley lines that possess
different specificities at the Ml-a locus........ 27

 

Formation of elongating secondary hyphae
(ESH) on isogenic barley lines M1-a7(Mu) and
Ml—a7(LG)OOO....0.....0O..........OOOOOCOCOOOOOOO 29

 

 

Selection scheme for recovery of recombinants
at the Ml-a lOCUSooooooooooo0.0000000000000000... 41

Intercrossing the two kinds of recombinants to
recover the original parental specificities...... 44

Hordein banding patterns of the six Ml—a lines
and cu1tivar ManChuriaOIOOOO......OOOOOOOOOOOOIOO 47

SDS—PAGE of hordein polypeptides of progeny of
recombinants within the Ml-a region.............. 52

SDS-PAGE of hordein polypeptides of single
seeds of F progeny from a cross between
barley isoIines Ml-a6 and Ml—a13................. 61

Phenotype of revertant barley seedlings 8-9 days
after inoculation with race CR3 of Erysiphe
graminis f. sp. hordei........................... 68

 

 

vii

Figure Page

11. Flow chart of crosses between M1-a6 and Ml-a13
and the results of seedling tests with Erysiphe
graminis f. sp. hordei for six generations....... 72

12. Model of Ml—a region on chromosome 5............. 84

viii

INTRODUCTION

During the course of research and breeding for

resistance to pathogens in barley, Hordeum vulgare L., a

 

considerable number of genes have been identified which
confer resistance to the powdery mildew fungus, Erysiphe

graminis f. sp. hordei. The application of Flor's (1956)

 

gene—for-gene hypothesis has facilitated the identification
of specific resistance genes in the host and their
corresponding pathogenicity genes in the pathogen. The
genes in the barley host have been given the designation M1.
A number of M1 variants in barley appear to be alleles or
tightly-linked genes at the M123 locus on chromosome five
(Moseman and Schaller, 1960). Fourteen different naturally-
occurring variants have been identified using many strains

of Erysiphe graminis f. sp. hordei (Giese et a1., 1981).

 

 

Jorgensen and Moseman (1972) estimated that there are about
twenty different resistance sites in the M123 region and
suggested that the region consists of a cluster of closely-
linked loci each with two or more alleles.

If the M113 locus has many different variants, each
specific in its function, what is the nature of their
specificity? Do differences in gene structure lead to
differences in phenotype? It is well known that identical

1

2
phenotypes may be derived from very different biochemical
pathways, or conversely small changes in gene structure can
lead to large phenotypic differences.
The purpose of this study was to investigate the
nature of specificity conditioned by different variants at

the Ml-a locus. There were three basic phases in this

 

investigation. The first examined the effect of the
different host genes on pathogen development during primary
infection. By following the pathogen through its distinct
morphological changes during the ontogeny of infection, it
is possible to monitor differences in expression of the host
genes in response to a challenge by the parasite (Ellingboe,
1972). Chapter I is devoted to these experiments.
Intragenic recombination is a valuable tool with
which to explore the nature of the cistron. When applied to
a host-pathogen system, the analysis of specificity among
naturally-occurring variants may be approached. In the
second phase, an attempt was made to genetically recombine

the different variants at the Ml-a locus. The purposes of

 

this were twofold: 1) to investigate the effect of
recombination on the specificity of the host-pathogen
interaction and 2) to establish a fine structure map of the
M123 locus. In the third phase the action of the M123
derivatives was investigated by genetic analysis of the
recombinants recovered in the second phase. Chapter II is

devoted to the experiments in phases two and three.

3
Chapter I has been accepted for publication (Wise
and Ellingboe, 1983). Chapter II is being prepared for

publication.

LI TERATURE REVIEW

In 1942 Flor published an account of segregation in
the progenies of crosses between different races of the rust

fungus Melampsora lini (Ehrenb.) Lev, a pathogen on flax

 

(Linum usitatissimum L.) (Flor, 1942). Flor studied

 

virulence on two host varieties. He found that virulence on
each host variety was determined by a recessive allele at a
single locus, and that the virulence determinants for each
host variety were independent. Flor (1947) continued this
analysis and described the inheritance of resistance to rust
races 22 and 24 in the two flax varieties Ottawa 770B and
Bombay. He also analyzed the corresponding inheritance of
virulence in these two races on the two host varieties
(Flor, 1946). The basic, fundamental pattern that emerged
was a one for one relationship between genes in host and
pathogen. This led to the proposal of the gene-for-gene
hypothesis. This hypothesis states that for each gene that
conditions reaction in the host, there is a complementary
and specific gene that conditions pathogenicity in the
parasite (Flor, 1956).

Most studies of host-parasite interactions begin
with a demonstration of variability in host and parasite.
A set of host lines is usually inoculated with a number of

4

5
strains of a pathogen. A simplified set of data is
presented in Table 1. A (-) signifies that the host is
resistant and/or the pathogen is avirulent. Collectively,
this is referred to as an incompatible interaction. A (+)
signifies that the host is susceptible and the pathogen is
virulent. This is referred to as a compatible interaction.
We see from Table 1 that no host line is resistant to all
strains of a pathogen and that no pathogen strain is
virulent on all host lines. This is not surprising. It
simply shows that there is genetic variability both in the
host and in the pathogen. The main point of Table l is to
demonstrate that the interactions between host and parasite
are determined by the action of both host and parasite
genotypes. How do the action of these genotypes specify the

types of interactions observed?

Table 1. The reaction of 3 host lines with 4 strains of a
pathogen.

 

Host Lines

 

Parasite Strains A B C

 

bump-a
+

+I+l
I

 

6

The most common pattern of inheritance for plants
and plant pathogenic fungi is shown in Table 2 (Ellingboe,
1976). Resistance and avirulence are usually dominant.
However, whether a trait is dominant or recessive is of
little consequence in determining which combinations are
specific. Note that in Table 2 only when the host plant
contains gene 51 and the pathogen possesses gene P1 is
incompatibility specified. A host with gene RI cannot
express resistance unless the complementary PI gene is
present in the pathogen. A pathogen with gene 21 is only
avirulent on hosts with gene 51. If a host had only gene
52 it would still give a compatible relationship with a
pathogen with gene 31. Only the interaction of RI and P1
result in incompatibility. Thus, it is important to
consider both host and pathogen genotypes when analyzing the
types of interactions between them. Simplified, the basic
pattern of interactions between host and parasite is shown
in Table 3. The interpretation of this pattern is that
specific recognition between host and parasite is for

incompatibility.

 

+ + Hmam

 

+ I Hmam
+ : Hmam mm
+ : Hmam Hm
+ + + + + + HmHm m ucmumm
+ I I I + I Hmam H #thmm
Huflu Hufim Hmfim Hufim Heap Hmam
mm Hm m ucmumm H ucmuma

ommuocow ouflwmumm

 

omxuocoo amom

 

.muflmmumm new umoc cmmeoQ mcofluomuoucw
uOm ©m>uwmno oucmpfluwncfl m0 cumuumm Damon 0:? .N canoe

Table 3. The basic pattern of interactions between host and
parasite.

 

Host Genotype

 

 

Parasite Genotype Rx rx
Px - +
px + +

 

Two other lines of evidence suggest that specific
recognition is for incompatibility. The first is
temperature sensitivity of the interactions between host and
parasite. Temperature-sensitive mutations are considered to
be missense mutations Which affect the stability of the
tertiary structure of the gene product and thus its
specificity. The protein is stable at the permissive
temperature. However, at the nonpermissive temperature the
tertiary structure is destabilized affecting the biological
activity of the protein. The interaction of the BER gene in
wheat for stem rust resistance and the corresponding 26 gene

in Puccinia graminis f. sp. tritici is temperature sensitive

 

(Loegering, 1966). This is illustrated in Table 4. The
interpretation of these data is that the permissive
temperature is 20°C, which allows for specific reCOgnition

between genes in host and parasite. An incompatible

Table 4. The interaction between the Sr6 gene in Triticum
aestivum and the corresponding 26 gene in Puccinia graminis
f. sp. tritici at two temperatures.

 

 

 

 

 

 

 

Host
Parasite 20°C 25°C
Sr6 sr6 Sr6 sr6
P6 - + + +
p6 + + + +
relationship is the result. The nonpermissive temperature

is 25°C which does not allow for a specific interaction
between host and parasite and therefore gives a compatible
relationship. Note that the interactions between S36 and 26
are unchanged by the temperature shift. The interactions
between SE6 and 26 or between 556 and 26 are interpreted as
being nonspecific.

The second line of evidence is provided by mutations
to increased virulence. In temperature-sensitive
interactions, the interpretation is loss of specificity at
the nonpermissive temperature. If this interpretation is
correct, any mutation of a 2 gene which leads to loss of

recognition of its corresponding 3 gene should result in

10
compatibility between host and parasite. Gabriel et al.
(1981) were able to recover a number of mutations to

increased virulence in avirulent strains of Erysiphe

 

graminis f. sp. tritici using nitrosoguanidine mutagenesis.

 

The pathogen was fully compatible with the once resistant
host lines possessing the corresponding 5 genes. The
relative ease in which these mutations were obtained was
interpreted as the result of alteration of specificity
leading to compatibility.

Multiple allelic systems. It has frequently been

 

reported that genes conferring resistance to an obligate
parasite are grouped together along a small segment of a
host chromosome. In flax, dominant genes conferring
resistance to flax rust have been assigned to five loci
labeled 5, L, M, E, and E with 1, 13, 7, 3, and 4

"alleles" respectively (Mayo and Shepherd, 1980). The M
group appears to be a series of closely-linked genes whereas
the L group consists of many codominant alleles (Shepherd

and Mayo, 1972). There are also five loci in maize (Zea

mays L.) conferring resistance to maize rust (Puccinia

 

sorghi schw.) with as many as 14 "alleles" at the Rpl locus

(Saxena and Hooker, 1968). In barley (Hordeum vulgare L.)

 

there are seven loci conferring resistance to powdery mildew

(Erysiple graminis f. sp. hordei) with the Ml-a region

 

possessing as many as 30 different alleles or closely-linked

genes (Giese, 1981).

11

Studies on the fine structure of resistance genes
has been restricted to relatively few systems despite the
available variability in both host and parasite. Flor
(1965) was the first to recover recombinants between
different sites within the "allelic series" of resistance
genes in flax. Subsequently, an analysis was made of the
"allelic" series at the RBI locus in maize. Saxena and
Hooker (1968) tested seven large testcross families, ranging
from 4170 to 19641 individuals. In all cases recombinants
were recovered. From their results a tentative linear map
was proposed, in which Epic lies between Rpla and 521k.
However, since flanking markers were not used and the 95%
confidence limits showed almost complete overlap the order
may not be completely accurate.

The question arises whether multiple alleles for
disease resistance represent one complex gene with multiple
sites or a series of closely-linked cistrons. Since the
sites are differentiated by many physiologic races of the
pathogen, one may question whether they represent entirely
different functions. Flor's analysis of rust resistance
genes in flax was extended when a modified cis-trans test
for codominant genes was introduced by Shepherd and Mayo
(1972). The test is illustrated in Figure 1. In this
model, it would be expected that if two "alleles" are in
different cistrons, the phenotypes of the cis and trans

heterozygotes would be the same. If the alleles are in the

12

 

 

 

 

 

 

 

 

 

 

 

 

Closely—linked genes Allelic genes

R1 _ _ + Rla +
Trans _ _

+ R2 + Rlb
Phenotype R1 R2 Rla Rlb

R1 _ _ R2 Rla Rlb
Cis _ _

+ + + +
Phenotype R1 R2 R1? or +
Figure 1. Comparison of cis and trans heterozygotes for

codominance for specific disease resistance.
After Shepherd and Mayo, 1972.

13

same cistron, the cis heterozygote may not have the same
phenotype as the trans heterozygote. The test to illustrate
this hypothesis is as follows: If recombination occurs
between two closely-linked genes, when testcrossed, only one
class of recombinant, namely ii' would give a compatible
relationship with a pathogen that possessed the
complementary E genes for El and 52. If specific
recognition is for incompatibility, then intracistronic
recombination between two host alleles should produce two
classes of recombinants (RlaRlb and ii) that are not
recognized by the corresponding parasite genes £13 and

212. When testcrossed a compatible relationship would
result (Ellingboe, 1976).

Reciprocal recombinants were recovered from
interallelic crosses at the M locus in flax. However in
similar crosses at the L locus, only recombinants giving a
compatible relationship with the appropriate strains of the
pathogen were recovered (Shepherd and Mayo, 1972). Upon
selfing six of the recombinants at the L locus, one of the
parental specificities was recovered. From these data it
was concluded that the M locus was a series of tightly—
linked genes whereas the L locus contained a number of
alleles within one cistron. A review of the data from
crosses at the 521 locus (Saxena and Hooker, 1968) reveals

that only one of the two classes of recombinants gave a

l4
compatible relationship with the appropriate rust strains.
Therefore, the Rpl locus is now considered to consist of a
number of separate but closely-linked cistrons.

Summary. The underlying justification for
performing genetic analyses is to make predictions about the
nature of genes and their products. At this point we may
speculate about the structure of resistance genes and the
function of their primary products. It has been suggested
that allelic and closely-linked duplicate genes may have the
same origin, evolving from a single host gene by tandem
duplication and unequal crossing over (Shepherd and Mayo,
1972). It follows that the primary products of these genes
would represent variants of a common product. These
products may be expected to behave similarly in their
function. Working with the powdery mildews of wheat and
barley Ellingboe (1972) and co-workers have defined the
unique stages of infection at which resistance is expressed.

The functions of host "alleles" Pm3a and Pm3b in Wheat were

 

very similar in their restriction of pathogen development.
If evolution for close linkage had occurred the genes
involved would most likely have very different primary
products. Different products might be expected to have
different functions. The P223 gene displays a very
different functional response to infection than that of nga

and Pm3b. Could Pm3c be a separate gene with a different

 

primary product? Each of the other unlinked Pm genes has

15
unique behavior in their restriction of pathogen
development. By the use of genetic and physiological tests
we should be able to make predictions about the structure
and function of these genes. These types of predictions are
necessary to direct our efforts if we are to uncover the

underlying mechanism of host—parasite interaction.

LITERATURE CITED

LITERATURE CITED

Ellingboe, A.H. 1972. Genetics and physiology of primary
infection by Erysiphe graminis. Phytopathology
62:401-406.

 

Ellingboe, A.H. 1976. Genetics of host-parasite
interactions. In Physiological Plant Pathology, Vol. 4.
R. Heitefus and—P.H. Williams, eds. Springer-Verlag,
N.Y. 890 pp.

Flor, H.H. 1942. Inheritance of pathogenicity in
Melampsora lini. Phytopathology 32:653-669.

 

Flor, H.H. 1946. Genetics of pathogenicity in Melampsora
lini. J. Agr. Res. 73:335-357.

 

Flor, H.H. 1947. Inheritance of reaction to rust in flax.

Flor, H.H. 1956. The complementary genic systems in flax
and flax rust. Adv. Genet. 8:29-54.

Flor, H.H. 1965. Tests for allelism of rust-resistance
genes in flax. Crop Sci. 5:415-418.

Gabriel, D.W., N. Lisker, and A.H. Ellingboe. 1982. The
induction and analysis of two classes of mutations in an
obligate parasite. Phytopathology 72:1026-1028.

Giese, H. 1981. Powdery mildew resistance genes in the
Ml-a and Ml-k regions on barley chromosome 5. Hereditas

Giese, H., J.H. Jorgensen, H.P. Jensen, and J. Jensen.
1981. Linkage relationships of ten powdery mildew
resistance genes on barley chromosome 5. Hereditas
95:43-50.

Jorgensen, J.H. and J.G. Moseman. 1972. Recombination at
the Ml-a locus in barley conditioning resistance to
Erys1phe graminis f. sp. hordei. Can. J. Genet.
Cytol. 14:43-48.

 

16

17

Loegering, W.Q. 1966. The relationship between host and
pathogen in stem rust of wheat. Proc. 2nd Int. Wheat
Genetics Symp. (Lund, 1963). Hereditas, Suppl.
2:167-177.

Moseman, J.G. and G. W. Schaller. 1960. Genetics of the
allelic series at the Ml-a locus in barley and cultures
of Erysiphe graminis f. sp. hordei that differentiate
these alleles. Crop Sci. 50:736-741.

 

Mayo, G.M.E. and K.W. Shepherd. 1980. Studies of genes
controlling specific host-parasite interactions in flax
and its rust. 1. Fine structure analysis of the M group
in the host. Heredity 44:211-227. —

Saxena, K.M. and A.L. Hooker. 1968. On the structure of a
gene for disease resistance in maize. Proc. Nat. Acad.
Sci. USA 61:1300-1305.

Shepherd, K.W. and G.M.E. Mayo. 1972. Genes conferring
specific plant disease resistance. Science
175:375-380.

Wise, R.P. and A.H. Ellingboe. 1983. Infection kinetics of
Erysiphe graminis f. sp. hordei on barley with different

alleles at the Ml-a locus. Phytopathology 73:1220-1222.

 

CHAPTER I

Infection kinetics of Erysiphe graminis f. sp. hordei

 

on barley with different alleles at the Ml-a locus.

CHAPTER I

Infection kinetics of Erysiphe graminis f. sp. hordei
on barley with different alleles at the Ml-a locus.

 

 

Abstract
The infection kinetics were determined for race CR3

of Erysiphe graminis f. sp. hordei on six sets of isolines

 

of barley. Lines with genes Ml-a, Ml-a6 or Ml-a13 gave a

final infection type 0 whereas lines with genes M1-a7(Mu),

 

Ml-a7(LG) or Ml-alO gave a final infection type of 1-2 or 2.

 

The percent elongating secondary hyphae (ESH) ranged from 8
to 21 at 28 hours after inoculation. A significant
difference in the percent ESH was found between three groups

of genes; (Ml-a, Ml-a6, Ml-a13), Ml-a7(Mu), and

 

 

[Ml-alO, M1-a7(LG)]. A significant difference was

 

also found in the rate of development of ESH between

Ml—a7(LG) and the other five genes examined, suggesting a

 

difference in specificity. It is proposed that the gene in
Long Glumes at the Ml-a locus conferring resistance to race

CR3 of E. graminis f. sp. hordei be designated Ml-aIS.

18

19

Introduction

 

Genes in plants for resistance to pathogens are
commonly identified by the reactions produced when the
plants are inoculated with a set of races of the pathogen.
Final infection type is the usual criterion for evaluation
of interactions. It has also been possible to determine the
effects of host and parasite genes during the first few

hours of interaction of Erysiphe graminis f. sp. hordei with

 

barley, Hordeum vulgare (Masri and Ellingboe, 1966b; Yang et

 

al., 1972). It has been posssible to study the effects of
the host and parasite genes on early interactions because
procedures have been developed to get high infection
efficiency and synchronized development of the pathogen
during the early stages of pathogenesis (Masri and
Ellingboe, 1966a). The infection kinetics of E. graminis
represent a very sensitive assay of the effects of host and
parasite genes on the development of the parasite and the
response of the host.

A number of the M1 genes in barley for resistance to

E. graminis f. sp. hordei appear to be alleles at the Ml-a

 

locus (Giese, 1981). If a gene for resistance affects a
distinct stage in the ontogeny of the interactions between
host and parasite, one may expect an allele of that gene to
affect the same stage of the interactions. Different

alleles would be expected to produce the same gene product

20

although their products would have different specificities.
The different specificities may account for the intensity of
the interaction although the time of expression should be
the same (Ellingboe, 1972). If the different alleles at the
M123 locus are separate but closely—linked cistrons, the
different genes might be expected to affect unique stages in
the ontogeny of interactions.

The objective of the research reported herein was to
determine the effect of six M123 "alleles" on the process of

primary infection and final infection type.

21

Materials and Methods

 

The six isogenic paired lines and cultivar Manchuria
(Table 5) were obtained from J.G. Moseman (1972). The
paired lines were developed by crossing each of six
resistant lines with Manchuria, backcrossing the resistant
progeny to Manchuria for three generations, selfing the
heterozygotes for 13 to 15 generations, and selecting
homozygous susceptible and homozygous resistant lines for
increase and use. The cultivar Manchuria was used as the
susceptible control. The designations for each line is
given in Table 5.

Culture CR3 of Erysiphe graminis DC. Merat f. sp.

 

hordei em. Marchal is avirulent on each of six host lines
with dominant M123 genes. CR3, therefore, is considered to
have the corresponding 2 gene for each of the six Ml genes.
Culture CR3 was propagated as described previously (Masri
and Ellingboe, 1966b).

Individual seedlings 5 to 6 days old were inoculated
by the rolling method (Nair and Ellingboe, 1962). A
reasonably uniform distribution of approximately 100 to 200
single conidia per cm length of leaf was obtained.
Following inoculation, the plants were kept under
environmental conditions that favor high infection
efficiencies (70% on Manchuria) and give reasonably

synchronous development of the parasite (Yang et al., 1972).

22

.<.Q.w.D

.cofluosoouscH Hmmuwu u .H.Un

.Amhoav :mEowoz Eoum ooummom manned

 

 

 

 

 

 

 

 

e mm cmmm mauszocmz
e mmmumm omHoH Ame .cmz Amflmv «e\mmmsm
o mmmumm mmflofl Ame .cmz Amaze .e\mmmsm
v Aoqvkmnas amass Amv .cmz Amamv .e\mmssao mcoq
mud “caveats: mmHmH Lav .cmz Amamv .¢\mmssuo mcoq
v Aszvsmnas mefioa Amy .cmz Amamv .¢\cmuasz
mus Aszvsmuaz nvumH Ame .cmz amass .e\:muasz
a mmmnmm omHmH “my .cmz Amflmv .e\acmuso
m mmmnmm_ oeamfi Amy .cmz Amamv .e\acmuso
v mmnmm «mama Amy .cmz “many .¢\uwm:mum
o mmumm HmHmH Ame .cmz Amamv .¢\ummcmum
a wide mmHoH “we .cmz Aeamv .v\:maummaa
0 min: hmHmH Amy .amz Aeflmv .e\cmflumma<
mmo
mmO 0v coHuomou .oc .H.U mocHH vasomOmH
zuw3 mama mcflcofluwocoo D . .
GOflUOOMCH OGOO

 

M

 

.woouon .mm .m mficfiEmum osmflmmum mo mmu muouaoo

on cofiuommu uflwflp one mocfla hmaumn vasomOmfllumo: mo muwmm xflm .m canoe

23
At a given hour after inoculation, a 1 cm section of a leaf
was examined under a light microscope (250x). Parasite
units with secondary hyphae longer than 10 um were
classified as elongating secondary hyphae (ESH). The
percentage of ESH was recorded. The percent ESH was used as
a measure of infection efficiency since it has been shown
that the formation of ESH is dependent on the establishment
of haustoria in the host epidermal cells (Masri and
Ellingboe 1966a). Each determination was replicated at
least four times with at least 80-100 spores observed in
each determination.

Data for 24, 26, and 28 hours after inoculation were
analyzed in a three way mixed model analysis of variance
(fixed lines and times, random replicates) for lines with
dominant Ml:a genes. Time effects were analyzed by
comparing slopes of regression lines of percent ESH on time.

Data were compared using Tukey's HSD test (Table 6).

24

.Amo.xmv oumN EOMM ucwuommao zagcmowmacmwm no: mm3 onIHz ocm
.AszVBMIHz .MIHZ .mMIHS .MHMIHZ zufl3 mmcwa you :oflmmmumoh

onu mo omon one .Aaoo.vmv macaw mo ummu m am an ooGAEuwuoo

mm newsmasoocw “dump choc: mm on om Eoum w>Hm umfiuo on» scum vacuommwo
Saucmoflmwcmflm mm3 Aqubmlaz Lufl3 nosed How GOAmwmnmmu may no oQOHm 059m

.Ho.
o
.mo. u m .¢N.N u mocmuomwflo unmowwflcmflm ESEHCHZU

 

m .mh.m u monouowmwo unmowmacmflm Esfiflcflz

.ummu Qmm m.>mxse 0p mcflouooom Hofiuo sumo Eoum

vomMoMMHo hapGMmeacmfim no: mum mafia mEMm ofiu hp omuomccoo mcfisaoon

.coflumasoocfl Hound
mason mm ocm .mm .¢N How mcoflumoflammu NH Eoum mmm mo ommucwoumm :mmZM

 

 

 

 

GHQ.

08 .

 

 

AN

H.6H s.mH «.mH s.m m.s m.> mmmm w

 

 

 

mAUAVBMIHZ OHMIHE ADZVFMIHZ MIHZ mmIHZ MHMIHZ OCHHOmfi

woahwm

 

.wahococwz
Hm>flpaoo hoaumn mo mmcflHOmfi acmumflmou Raw :0 mmu momu awoke: .mm .m

 

 

mflcflfimnm mnmwwNwm mo Rummy mafia»; humocooom mcflummCOHm unmoumm .m canoe

25

Results

Barley lines with Ml-a, Ml—a6 or Ml-a13 all gave

 

an infection type 0 reaction following inoculation with CR3

(Table 5). The three lines with Ml-alO, Ml-a7(Mu) or

 

Ml-a7(LG) each gave an infection type 2 or 1-2. The

 

infection type 0 signifies no macroscopically visible
pathogen development on the leaf surface. The infection
type 1-2 signifies a very small amount of macroscopically
visible mycelial development. Infection type 2 signifies a
moderate amount of macroscopically visible mycelial
development with slight sporulation. Infection type 1—2 and
2 are both accompanied by visible host cell necrosis at the
infection site, ie. a hypersensitive reaction. All of the
host lines with the recessive ml genes gave an infection
type 4, and were indistinguishable. Infection type 4 is
characterized by abundant mycelial development and strong
sporulation by the pathogen.

The kinetics of the formation of elongating
secondary hyphae (ESH) on each of the seven susceptible
lines with the recessive ml genes are given in Figure 2.
There were no significant differences among the seven host
lines. The kinetics of the formation of ESH on each of the
six resistant host lines with the dominant Ml genes are
given in Figure 3. All six Ml genes significantly reduced

the percent ESH with respect to the susceptible member of

26

 

o mvmo
A mI-a7(Mu) ._
o ml-a7(LG) .
o Manchuria

jj

70

%ESH

 

 

 

% ESH

 

 

 

 

J\§L l l l l
o 22 24 26 28
HOURS AFTER INOCULATION

Figure 2. Formation of elongating secondary hyphae (ESH) on
susceptible isolines of barley by E. graminis f. sp.
hordei race CR3.

27

 

 

 

 

 

 

 

 

l
r c>Mkm0
20.. ‘ MI'OT(MU) _
‘ M|:07(LG)
l5- _
IO— _
57 _
I:
:3 JVL l l 1 l
32 " (IMFO
_ "ML06 _
20 O Ml—al3
l5- _
IO— _
5- _
My; 1 I I 1
20 22 24 26 28

HOURS AFTER INOCULATION

Figure 3. Formation of elongating secondary hyphae (ESH) by

graminis f. sp. hordei race CR3 on near- isogenic barley
lines that possess different specificities at the Ml-a
locus. The standard error for 24. 26, and 28 hours after
inoculation is .91. The slope of Ml-a7(LG) was
significantly different from the other five from 24 to 28
hours after inoculation as determined by an f test of slope
(P<.001).

 

28
each isoline pair (Figures 2 and 3). The three host lines

with genes Ml-a, M1—a6 or Ml-a13 gave lower percentages of

 

ESH than the host lines with genes Ml-alO, Ml-a7(Mu) or

 

 

Ml—a7(LG) (Table 6). The first three had infection types 0,

 

the latter had infection types 2 or 1—2.
There was a significant difference in the slope of

the regression for host lines with Ml-a7(LG) and Ml-a7(Mu)

 

 

during the first 28 hours after inoculation (Figure 3). A
maximum of 13 percent ESH was obtained on the host line with

Ml-a7(Mu). This was significantly different from a

 

maximum of 21 percent ESH which was obtained on the host

line with Ml-a7(LG) (Table 6). In an additional experiment

 

the percent ESH was recorded from 24 to 36 hours after
inoculation (Figure 4). An analysis of variance for 28, 32,
and 36 hours after inoculation using a Student's t test

showed a significant (P<.001) difference between M1-a7(Mu)

 

and M1-a7(LG) in their percentages of ESH with race CR3 of

 

E. graminis f. sp. hordei.

On all six Mg_alleles there was collapse of some
parasite units at approximately 24-26 hours after
inoculation. The percent that collapse seems to be related

to the particular M1 allele.

29

 

   
 

 

 

 

 

 

l
70— —
60*— A Ml-07(Mu) -
o Ml-a7(LG)
o Manchuria
1.50- —
U)
u:
40— —
32
30— _
20_ ./////,a—7 —#£—: ., _
10— f fl —
A 1 1 I 1
24 28 32 36

HOURS AFTER INOCULATION

Figure 4. Formation of elongating secondary hyphae (ESH) on
isogenic barley lines M1-a7(Mu) and Ml—a7(LG). The

standard error for 28, 32, and 36 hours after inoculation is
.78.

 

 

30

Discussion

 

Approximately 30 different genes conferring
resistance to E. graminis f. sp. hordei have been mapped
to the M123 locus on chromosome 5 in barley (Giese, 1981).
Thus far, 14 different alleles (or closely—linked genes)
have been designated M123 through Ml-a14 based on their

differential reactions to many strains of E. graminis f.

 

sp. hordei. If the six alleles used in this study were

true alleles, we would expect that they all would affect the
same stage in the ontogeny of the interaction (Ellingboe,
1972). The different levels of infection efficiency may be
attributed to modification of the gene product specified by
the different alleles. The results of the infection
kinetics are consistent with this hypothesis, though they do
not exclude the possibility that the six lines may each have

a gene in a unique cistron. For example, lines with M1-a6

 

and Ml-al3 display identical phenotypes during primary
infection by E. graminis f. sp. hordei (Figure 3). However,
recent data on recombination in this region suggests that
H1239 and Ml-a13 are in separate cistrons (R. Wise,
unpublished). The fact that there are three different final
infection types suggests that there may be three cistrons,
though previous results have suggested that a single host
gene can condition different phenotypes with different

strains of a pathogen (Martin and Ellingboe, 1976).

31
All six Ml alleles affect the formation of ESH.
Collapse of some parasite units occurs at approximately the
same time. Based on the pattern of development of ESH,
there appears to be four groups of genes in terms of how
they affect primary infection (Table 6). A significant
difference in the percent ESH was found between the three

groups of genes; (Ml-a, Ml-a6, Ml-al3), Ml—a7(Mu),

 

 

and [Ml-alo, Ml-a7(LG)]. In addition a significant

 

difference was found in the rate of development of ESH

between Ml-a7(LG) and the other five. The different levels

 

of compatibility may be attributed to the interaction of
each specific host/parasite gene pair since each of the host
lines with recessive ml genes had kinetics indistinguishable
from Manchuria. It was interesting to find a significant

difference between Ml-a7(Mu) and Ml—a7(LG) in their degree

 

 

of compatibility with E. graminis f. sp. hordei.

Moseman and Jorgensen (1973) suggested that these two genes
may be identical based on their final infection type using
differential races of E. graminis f. sp. hordei. In
addition, tests with about 65 different isolates of E.
graminis f. sp. hordei from Europe, NOrth America, Japan,
and Israel have shown that alleles in 'Long Glumes' and
'Multan' are susceptible only to cultures virulent on Mlzal
(J.H. Jorgensen, personal communication). Although final
infection type is satisfactory for differentiating gross

qualitative differences it is not very useful, however, for

32
determining when incompatibility is expressed during the
early stages of disease development.

Measurement of the primary infection kinetics of E.
graminis is a quantitative method of determining the degree
of compatibility in the interaction between host and
parasite. Using this method, we have demonstrated a
repeatable difference in compatibility in the isolines

Ml-a7(Mu) and Ml—a7(LG) with race CR3 of E. graminis f.

 

 

sp. hordei. The specificity of the interaction between

host and parasite is presumably determined by the product of
the host gene and the product of the parasite gene and hence
their nucleotide sequences. A different specificity most
likely reflects a different nucleotide sequence. Recent
experiments at Riso National Laboratory have tentatively
revealed an additional resistance gene in the Multan isoline
with a complementary avirulence gene in CR3 (J.H. Jorgensen,
personal communication). If the primary resistance gene in

the isolines Ml-a7(Mu) and Ml-a7(LG) is M1-a7, then the

 

 

lower infection efficiency observed on Ml—a7(Mu) as compared

 

to Ml-a7(LG) may be due to a synergistic effect of the gene

 

at the Ml-a locus and a second gene in Ml—a7(Mu) due to

 

their interaction with possibly two complementary avirulence
genes in CR3. Alternatively, the difference observed

between Ml-a7(Mu) and Ml-a7(LG) may represent two different

 

 

alleles at the Ml-a gene complex. To our knowledge, these

two genes have not been differentiated by recombination or

33
by different races of E. graminis f. sp. hordei (Giese,
1981; Moseman and Jorgensen, 1973). Based on the evidence
presented here, we feel that a distinction is warranted and

suggest the gene from cultivar Long Glumes at the Ml-a locus

 

tentatively be designated Ml-alS.

34

Literature Cited

 

Ellingboe, A.H. 1972. Genetics and physiology of primary
infection by Erysiphe graminis. Phytopathology
62:401—406.

 

Giese, H. 1981. Powdery mildew resistance genes in the
Ml-a and Ml-k regions on barley chromosome 5.
Hereditas 95:51-62.

Martin, T.J., and A.H. Ellingboe. 1976. Differences
between compatible parasite/host genotypes involving
the Pm-4 locus of wheat and the corresponding genes in
Erys1phe graminis f. sp. tritici. Phytopathology
66:1435-1438.

 

Masri, S.S., and A.H. Ellingboe. 1966a. Germination of
conidia and formation of appressoria and secondary
hyphae in Erysiphe graminis f. sp. tritici.
Phytopathology 56:304-308.

 

Masri, S.S., and A.H. Ellingboe. 1966b. Primary infection
of Wheat and barley by Erysiphe graminis.
Phytopathology 56:389-395.

 

Moseman, J.G. 1972. Isogenic barley lines for reaction to
Erysiphe graminis f. sp. hordei. Crop Science
12:681-682.

 

Moseman J.G., and J.H. Jorgensen. 1973. Differentiation of
resistance genes at the Ml-a locus in six pairs of
isogenic barley lines. Euphytica 22:189—196.

Nair, K.R.S., and A.H. Ellingboe. 1962. A method of
controlled inoculations with conidiospores of Erysiphe
graminis var. tritici. Phytopathology 52:714.

 

Yang, S.L., J.G. Moseman, and A.H. Ellingboe. 1972. The
formation of elongating secondary hyphae of Erysiphe

graminis and the segregation of El genes in barley.
Phytopathology 82:1219-1223.

 

 

CHAPTER II

Investigations on recombination at the Ml-a locus in barley
conditioning reaction to Erysiphe graminis f. sp. hordei.

Fine structure analysis and evidence for controlling—element

alleles.

CHAPTER II

Investigations on recombination at the Ml-a locus in barley
conditioning reaction to Erysiphe gramin1s f. sp. hordei.
Fine structure analysis and eVidence for controlling-element
alleles.

 

Abstract
There are a large number of naturally-occurring

variants at the Ml-a locus in barley conferring resistance

 

to Erysiphe graminis f. sp. hordei. The Ml-a locus is

 

also bracketed by Hor-l and Hor-2, genes which encode
storage proteins in the barley endosperm. These properties
make the Elli locus amenable to fine structure analysis.
Rare susceptible recombinants were recovered by F

3
family analysis from the Ml—alO x Ml-a, Ml-a x Ml-a7(LG),

 

 

and M1—a6 x Ml-a13 crosses. A number of susceptible
recombinants were recovered from the Ml-a6 x Ml-a13 cross

that did not fit the expected F family segregation

3
ratios. These recombinants often reverted to resistance in
subsequent generations. No recombinants were recovered in
the reciprocal cross, Ml-a13 x Ml—a6. The possibility of

an autonomous regulatory element, and a possible linear

order of six "alleles" at the Ml-a locus is discussed.

35

36

Introduction

 

Although genes conferring specific disease
resistance are often used in basic and applied breeding
experiments, there have been few analyses of their fine
structure. One of the interesting features of genes
conferring resistance to an obligate parasite is the way
they are arranged in groups along a particular host
chromosome (Flor, 1956; Saxena and Hooker, 1968: Moseman,
1971; Shepherd and Mayo, 1972). One of the primary
questions about many of these loci is whether they are
single genes with many alleles or clusters of closely-linked
cistrons.

Gene-for-gene specificity, in addition to naturally-
occurring genetic variability, is useful in the study of the
fine structure of these loci. When applied to groups of
codominant genes, the modified cis-trans test described by
Shepherd and Mayo (1972) is particularly useful in
differentiating between functional alleles and closely-
linked cistrons. The results of their investigations of the

L and E loci in flax (Linum usitatissimum L.) controlling

 

 

specific interactions with flax rust [Melampsora lini

 

(Ehrenb.) Lev.] demonstrated important functional
differences among genes of the E group as distinct from
genes of the E group. The interpretation of those data and

that of later experiments (Mayo and Shepherd, 1980) was that

37
the E_group is a series of separate but closely-linked genes
whereas variants in the E group are functionally allelic.
A large number of variants have been identified in

barley (Hordeum vulgare L.) conferring resistance to

 

 

powdery mildew (Erysiphe g;aminis DC. Merat f. sp. hordei
em. Marchal). These are distributed among seven groups;

Ml-at, Ml—a, Ml-k, Ml-nn, and Ml-p on chromosome 5,

 

 

and Elzg and ELSE on chromosome 4 (Jorgensen and Jensen,
1976; Jensen, 1980). There is a large cluster of allelic or
closely-linked variants Within the £123 region, 14 of which
have been differentiated by their reactions to many strains
of E. graminis f. sp. hordei (For review see Giese, 1981).
Jorgensen and Moseman (1972) were able to detect rare

recombination between Ml—a and Ml-a3 and Giese et al. (1981)

 

recovered a possible recombinant between Ml-alz and Ml-al3.

Since then, we have extended the analysis of the
Elli locus using the concept of the modified cis—trans test.
The El:3 locus was chosen for fine structure analysis of a
gene conferring specific resistance for a number of reasons.
One of these is the large number of naturally-occurring
variants in the region, six of which have been made near
isogenic to the cultivar Manchuria, minimizing differences
in genetic background (Moseman, 1972). Manchuria has no

known Ml-a resistance. The six isogenic lines are all

 

resistant to a single race (CR3) of E. graminis f. sp.

hordei enabling large numbers of progeny to be screened with

38
one pathogen, thus excluding the possibility of
contamination with other cultures. The El:g locus is also
bracketed by the hordein genes which encode endosperm
storage proteins (Jensen et al., 1980). The hordein
proteins can be assayed by polyacrylamide gel
electrophoresis and therefore can be used as flanking
markers. The objectives of the research reported here were
examination of the fine structure of the Elli locus and
exploration of the nature of the specificity conferred by

the naturally-occurring variants in this region.

39

Materials and Methods

 

Table 7 lists the six near-isogenic barley lines and
cultivar Manchuria followed by their reaction seven days

after inoculation with Erysiphe graminis f. sp. hordei race

 

CR3. Infection types 0, 1-2, or 2 signify resistant
reactions. Infection type 4 signifies a fully susceptible
reaction. All barley lines were obtained from J.G. Moseman,
Small Grains Collection, U.S.D.A., Beltsville, MD.

Culture CR3 of E. graminis f. sp. hordei is
avirulent on each of the six host Elzg-dominant lines.
CR3, therefore is presumed to have the corresponding
avirulence (23) gene for each of the six Elli
specificities. Culture CR3 was propagated as described
previously (Masri and Ellingboe, 1966). Purity of the
mildew culture was monitored by periodic inoculation of sets
of differential host lines and checking of infection types.

Experimental design. The selection scheme for

 

recovering recombinants in the Elli region is presented in
Figure 5. Vertical lines represent the chromosomal segment
encompassing the Elli region. Horizontal slash marks
represent putative mutant sites conferring specific
resistance to race CR3 of E. graminis f. sp. hordei.

These sites could be allelic or in closely-linked cistrons.
Once a large number of parental crosses are made, natural

self pollination may be exploited to produce large numbers

4O

.¢.Q.m.D .uonfisc cofluosoonucH Hmouou I .H.Un
.vcoEQon>mo BooHflE unmocsnw u o .ucmEQOHo>oo
3ooHflE cw COHposomu pcmuwmwcmfim n m .COHuomwu UprHUwc u m .mGAXUmHm
OMuOungo n H .ucmfimon>wo 3moHME manm>uomno o: u O «max» GOAUUoMGHm

 

 

o HE ommm mausnocmz
o mdm1az mmamfi Ame .cmz Amflmv .v\momsm
N1H AoAVsM1Hz mmflmfi Amy .cmz Amamv .v\mmssao macs
~1H Aszvsm1az seHoH Amy .amz Amuse .e\cmuasz
m on1Hz aefiofl Ame .emz Amfimv .¢\flcmusa
o onus: amass Ame .cmz Amflmv .¢\ummcmum
o M1Hz smHmH Ame .cmz Avamv .¢\:maummam
ammo mmu o» 20wuomou 9.0: .H.O mmcwa vasomOmH
nufl3 waxy mcflcoHufiocoo
C0flfiOOHCH OCOO

 

 

.flwouon .mm .m mfl:HEmum mzmwmhum mo mmo onsuaso Op GOAuumon
uflonp ocm ownsfiocwz um>Huaso can mmcfla hoaumn oflcomOmfllumwc Raw .5 wanna

41

 

 

 

 

. V 1111311 111111
111111 IIUII

. : I
1?: 3R 3B

Figure 5. Selection scheme for recovery of recombinants at
the Ml-a locus. Vertical lines represent the chromosomal
segment encompassing the Ml-a region. Horizontal slash
marks represent sites conferring specific resistance to race
CR3 of Erysiphe graminis f. sp. hordei. P, F1 and F2 plants
are all resistant to CR3. All the F2 plants are selfed'
however all nonrecombinant F families are nonsegregating
for resistance to CR3. For gimplicity, only the recombinant
F families are diagrammed. The expected phenotypic ratio

1 illustrated below F family types 1-4.

R = resistant, S = susceptible.

 

42
of segregating F2 progeny. Rare recombination may occur
in the F1 generation between different sites conferring
specific resistance during gamete formation. During
fertilization, recombinant gametes will most likely fuse

with wild type gametes to form four types of recombinant"
seed. Types 1 and 2 will possess both sites for specific
resistance whereas types 3 and 4 will possess neither.
Since the recombinant chromosome is paired with a
nonrecombinant chromosome carrying a gene for resistance,
all of the F2 seedlings will be resistant to race CR3 of
E. graminis f. sp. hordei. However, when these F

2

plants are progeny tested, segregation will yield offspring
homozygous for the recombinant chromosome. Since CR3
possesses avirulence specificities for both resistances used
in the parental cross, recombinants with a double resistant
phenotype will be indistinguishable from the parentals.
Recombinants with a susceptible phenotype will, however, be
readily distinguishable.

Two recombinant types will be produced: those
bearing both resistance sites (left-most elements in F3
families 1 and 2) and those bearing neither site (left-most
elements in F3 families 3 and 4). The latter will
necessarily be susceptible in phenotype, but the phenotype
of the former, possessing both resistance sites, will depend

both on whether the resistance determinants are in distinct

cistrons or are alleles and, in the case of allelic

43
determinants on the unknown molecular recognition mechanism.
The easiest outcome to interpret would be that in which the
two-site recombinant is recovered as susceptible. In that
case, the two variants are necessarily allelic because the
cis—combination disrupts resistance rather than conferring
both. Should this class prove to be resistant, which in
these crosses would be indicated by failure to recover them,
it would be impossible to distinguish between the two
models; variants in two loci would yield resistance in cis,
but so could variants at two sites within one gene product
if multiple sites within that product can be recognized by
the pathogen.

Recovery of susceptible cis-recombinants can be
conferred as shown in Figure 6 where intercrossing of the
recombinants followed by a second round of recombination
will yield both parental types.

Breeding program. Parental crosses were made

 

among the six host lines with dominant resistance

specificities at the Ml-a locus. Individual parental

 

crosses resulted in approximately 18 hybrid seed per head.
The parental crosses were numbered by head and all
subsequent progeny resulting from a particular parental
cross were kept separate throughout the analysis. To be
sure parental plants were homozygous for their respective
genes for resistance, approximately 20 progeny seed from

each parental plant used in a cross were tested for

44

 

F“ i|-—>I><l

 

v is

EIIII lHIIl 11H

Figure 6. Intercrossing the two kinds of recombinants to
recover the original parental specificities.

45

segregation by inoculation with CR3. The resulting F1

generation was selfed in the greenhouse during the winter to

obtain a maximum F2. To prevent contamination by

outcrossing, no susceptible barley was grown in the same

greenhouse or in the near vicinity. The F2 seeds were

space planted in the field and a single head was harvested

off each F2 plant to represent the F3 families.

This procedure was repeated twice (from P to F3) in
successive years.

Screening procedure. F3 families were
screened for susceptible segregants by planting the heads in

 

flats and inoculating the seedlings with culture CR3 of
E. graminis f. sp. hordei when they were approximately
three inches high. Forty-seven (47) families as well as a
susceptible control (Manchuria) were planted per flat.
Flats of inoculated seedlings were placed in a Sherer-
Gillett Model CEL 512-37 or Model CEL 25-7 controlled
environment chamber with 660 ft-c of light (600 ft-c from
White VHO flourescent tubes and 60 ft-c from 25 watt
incandescent bulbs) with a 15 hour photoperiod. The
temperature was kept at 16-18°C and the relative

humidity was 65 i 5%. These conditions are optimal for
development of the pathogen. Six to seven days after
inoculation the flats were screened for susceptible

segregants. Segregating F3 families were repotted and

grown to maturity. Five F4 seedlings from the mature

46
recombinant plants were retested with CR3. Five seeds from
each putative recombinant were assayed electrophoretically
for their hordein banding patterns by running endosperm
protein extracts on 12% SDS-polyacrylamide slab gels
(Doll and Andersen, 1981). This served two purposes:
1) If recombination occurred within the Elli region, it
would often be accompanied by exchange of outside markers
depending on whether exchange was reciprocal or a
nonreciprocal, gene conversion, event.
2) The hordein patterns served as a "fingerprint" of each
parental line to further rule out any contamination
(Figure 7).

Recombination estimates. Recombination

 

frequencies were estimated by the maximum likelihood

 

formula:
p = n2 + 2n3
n1 + n2 + n3
where p = recombination frequency,

nl = P3 families segregating 1 resistant:0 susceptible,

n2=F3

n =F

families segregating 3 resistant:l susceptible, and
3 families segregating 0 resistant:1 susceptible.

In the event no recombinant plants were obtained, the upper
95% confidence limits were estimated by the method of Giese
et a1. (1981). The limits will only be correct if the genes

involved segregate in a 3:1 or 1:2:1 ratio When tested with

47

1 2 3 4 5 6 7 8

Hordein-l

Hordein-2

 

Figure 7. Hordein banding patterns of the six Ml-a lines
and cultivar Manchuria. 1, Ml-a: 2. Ml-a6; 3, fll-a7(Mu);

 

 

48
the appropriate mildew strains. The formula used to obtain
these limits is as follows:
p 1 2/3(l-(1-1.5(l-exp(l/N 1no.os)))°'5)
where p = upper limit of recombination and N = the number of
nonrecombinant families observed.

Studies on the action of recombinants. Progeny

 

seed from the recombinant plants were grown up and used to
make intercrosses among the different recombinants in most
pairwise combinations. In the event that only one
recombinant was recovered it was crossed to Manchuria.
Manchuria represented the type of recombinant with neither
resistance site. Selected recombinants from the Ml-a6 x
Ml-a13 hybrid were also crossed to each of the parental
resistant lines and to Manchuria. Approximately 10 hybrid
seed per cross were planted in the field and allowed to
self-pollinate. The progeny were planted in flats and
tested with CR3 as described previously.

Preparation of hordein for electrophoresis. The

 

method of Doll and Andersen (1981) was used with minor
modification. The distal third of a single seed was
excised, crushed with pliers, and placed in a 15 ml
nitrocellulose centrifuge tube with a small magnetic spin
bar. The embryo portions were saved in a microtiter plate
corresponding to the order the sample was to be loaded on
the gel. The endosperm was then stirred for 1 hour at room

temperature in 0.8 ml 50% (v/v) isopropanol containing 41mM

49
Tris, 40mM boric acid, and SmM dithiothreitol (DTT), pH 8.6.
The sample was centrifuged at 12,000 x g for 10 minutes.
The protein in the supernatant was alkylated by adding 10 ul
of 8M iodoacetamide and heating in a 50°C water bath for
25 minutes. The hordein polypeptides were precipitated by
the addition of 1 ml of glass distilled water and
refrigerated overnight. The precipitate was centrifuged at
12.000 x g for 10 minutes and the pellet was resuspended in
0.6 m1 electrophoresis buffer containing 41mM Tris, 40mM
boric acid, 10% sucrose and 0.01% bromphenol blue (pH 8.6).
Ten microliters were layered on the gel for electrophoresis.

Electrophoresis was carried out in 174 x 183 x 1.5
mm gels using a discontinuous buffer system described by
Chua and Bennoun (1975). The stacking and separation gels
respectively contained 5% and 12% monomer.consisting of
acrylamide and N,N'-methylene-bis-acrylamide in a 15:1 and
40:1 ratio respectively. Electrophoresis was performed at a
constant current of lOmA until the sample had passed through
the stacking gel and then the current was raised to l6mA
until the tracking dye had moved 12 cm into the separation
gel.

Gels were fixed for at least 2 hours in an aqueous
solution containing 50% methanol and 10% acetic acid, washed
in water for 30 minutes, and then stained overnight in 200
m1 of a mixture of 15% trichloroacetic acid and 300 ul

methanol containing 2% Coomasie brilliant blue R-250.

50

Results

F3 families for each type of cross were derived from

approximately sixty F plants. Five to fifteen F1 plants

1
were derived from each set of parents. The F3 family data
from each parental cross type were homogeneous and the results
are pooled in Table 8.

Families segregating 3 resistantzl susceptible seven

days after inoculation with race CR3 of Erysiphe graminis f.

 

sp. hordei were observed in the Ml-alO x Ml-a, Ml-a x

 

Ml-a7(LG), and Ml—a6 x Ml-al3 crosses. Exchange of flanking

 

markers accompanied exchange within the Ml-a region in these
crosses. The flanking marker constitution for progeny of

the Ml-alo/Ml-a recombinant was Hor-1(a10) and Hor-2(a).

 

In progeny of the M1—a/M1-a7(LG) recombinant the flanking

 

marker constitution was Hor-1[a7(LG)] and Hor-2(a) and for

progeny of the Ml-a6/Ml-al3 recombinants it was Hor-1(a13)

 

and Hor—2(a6) (Figure 8). Three or four progeny from each
of the putative recombinants were retested with CR3. All

the progeny tested from the Ml-alO/Ml-a and Ml-a6/Ml-a13

 

 

recombinants were susceptible. Surprisingly, all the

progeny tested from the Ml-a7(LG)/Ml-a recombinant

 

displayed type 2 reactions which were accompanied by

exchange between Hor-l and Hor-2. Progeny from one of the

 

plants giving a type 2 reaction also gave mostly type 2

 

(3:0 mmouo mHmer x 001.2 spam H38 :0 oommn magnum c0305}
. 89$de gm: 3 8.538 coaumfifioommmu

.038 in m 8h $063 38366 bandaged
.m 9.3 5 mmcfioomm H33 mm mo 1.50 manflummoma N no H has .muqmcafimucoo mafimmgn

$5qu mama x vacuum mam—ammo

Esau m

 

 

 

51

 

sSm. 28 o 0 came 6TH: x STE m S moan STE x 6T2
S38 S38 83: S80 Sin 8::
EzKTE “SETS:

80. $9 pH 88 x SEATS: 3m x AEKTE

80. 8mm 93 6TH: x EerE Sam BEATS: x 6T2

80. $8 68 6T2 x STE SS STE x 6T2

So. SE S SS To. an STE mmoe STE x TE

$8. «82 8mm TE x ASKTE S $va ASKTE x TE

80. 3mm am :8 TE x 32:sz mSN AEKTE x TE

mg. 68 8%.. TE x oTE am 88 6TH: on T2
GOflqumn—FOOQQ MO
SEE 883mg

3&2 wmm 323% Sam... mos: Sam mos:
ocoflmfinficoomw HBO? cofipmmoumvom mmOQO aflcohmm coflummoumom mmmOnU Hmucmumm

 

 

 

48.8: .mm .m madam mfiflmbm mo 96 8B 53 833585
Houmm goo b 3:326.“ mm mo Ewuumm cowummmumwm o5 cam mommouo Amanda Hwoohfiomm .m wanna.

52

_ cwfiufi3 mucmcflnfioumu mo mammoum mo mmcflummmmaom Camcuo: mo madmlmam

.msooH Nunom mnp >9 cwcoucm mmcflummmhaom mum mccmn mcflgmumflE
umpmmm $39 .msUOH Hauom m£# >9 cmooocm mmwfiumwmmaom mum momma mcaumumflfi

um3OHm $59 .mamuaz x mmlaz mm3 mmouo Hapcmumm Hmcwmwuo mSB .coflmmu mud: m:#
.m musmflm

 

53

.m wusmflm

.
.

. .

«Eu—.3532... 2.2135;
mm aka cum caravan u 3:
ml; 9a on 2.30:3 to:

I...» ...-3‘“! I...»_ I u S...

p ..O...

 

52

. cwfipfl3 mgcmcwneoomu mo mammoum mo mmcflummmhaom Camouon mo mo<mlmom

.msuoH mluom mnu an cwcoocm mmofiummmmaom mum mwcmn mafiumumfle
“mammw m .msoOH alhom 03w >9 Umooucm mwcwummmhaom mum momma mcaumumas

H030Hm mSB .mHMIHz x owlaz mm? mmOno kucmnmm Hmcflmwuo mnh .cofimmu MIHZ m:#
.m musmfim

 

53

.m musmflm

A
_

2:33:33: 22:23
W m...lu . elm 023285 N 3:
a; . . a; . on «niacom to:

 

_. ho...—

54
reactions, however two seedlings displayed a type 3 reaction
with severe necrosis.
Possible segregating F families were also

3
observed in the Ml-a7(Mu) x Ml-a, Ml-a7(LG) x M1-a7(Mu),

 

 

 

and Ml—a x M1-a6 crosses. For both of the Ml-a7(Mu)/Ml-a

 

apparent recombinants, only 1 out of 25 seedlings in the
family was susceptible (Table 8). Progeny from one of these
susceptible plants displayed a hordein banding pattern
unlike either of the parents and unlike the pattern expected
if there were exchange between EEEZL and E2313. It is
suspected that this was a stray control seed. The other

displayed a pattern characteristic of Ml—a7(Mu). Since

 

Ml-a7(Mu) has the same hordein banding as Manchuria, this

 

also could be the result of a stray control seed. In the

apparent recombinant families from the Ml-a7(LG) x Ml-a7(Mu)

 

 

and Ml-a x Ml-a6 crosses, 1 seedling out of 25 was

 

 

susceptible. These susceptible seedlings did not reach
maturity because of mildew infection so the hordein banding
patterns were not tested. In an attempt to verify if the
susceptible segregants were true recombinants in the above
crosses, 16 resistant seedlings from the segregating families
were grown to maturity. Approximately one-half of these
plants should be heterozygous for the recombinant chromosome.
Thus, progeny from these plants should include susceptible
seedlings. Twenty to thirty progeny from each plant were

powdery mildew tested, but no segregation was Observed.

55

F3 families displaying a 0 resistant:l susceptible
segregation were observed in the Ellié x Ml-a13 cross.
Progeny seeds from these plants had hordein banding patterns
identical to the 3R:18 families recovered from the same
cross (Figure 8). The recovery of 0R:lS families was
completely unexpected given the frequency of 3R:lS families
observed. If resistance is dominant it is reasonable to

assume that an entirely susceptible F family would have

3

been derived from a homozygous F plant. Such F

2 2

plants would be the product of recombinant gametes from both
pollen and ova. To test this possibility, additional F2
seed from the same parental crosses were planted out in
flats and inoculated with CR3. The results are presented in
Table 9. Twenty-two susceptible plants were observed from

crosses in which Ml-a6 was the female parent. No

 

susceptible plants were observed from crosses in which
Ml-al3 was the female parent. The hordein banding patterns
of progeny from the susceptible plants were identical to

those observed in the progeny of the F segregants thus

3
demonstrating true recombination and not contamination. It
is not known Why these data are heterogeneous.

Occasionally the hordein banding patterns in progeny

from the Ml-a6/Ml-a13 susceptible segregants were

 

characteristic of the pattern displayed by M1-a13. This
is the pattern expected if there was an additional crossover

in the region between the first exchange and the Hor-2 gene.

.ycmumm mama x ucmuwm mHmEmmm

 

56

 

 

 

o m¢mH . Havoe
o Nmm 5mm
0 pom «NH
0 on Nod mMIHz x manna:
mm mama Sauce
0 0mm Hmm
mm» v 50H moH
mm» ma Nam mma
O was omH manna: x mwlaz
wmcmnoxm manfiumoomsm acmumflwmm .oz mmOuU wmwouu
umxumz
mcwxcmHm

 

.MHMIHZ UCM WMIHS CG03#GQ mwmmOHU HMUOHQHUwH

mm mo muwmu 3m©HflE mump3om mo muasmmm .m manna

EOHM hammoum mafiummmummm

57
This pattern occurred in one of the 3R:lS families and in
one of the 0R:lS families. In the 0R:lS family it was
evident that the Ml-al3 pattern came from only one of the
parents since the recombinant pattern and the Ml-a13 pattern
segregated in progeny of the recombinants. The Ml-a13
pattern was also present in progeny of two of the F2
plants susceptible to CR3. It was also evident in the
progeny from the susceptible F2 plants that the Ml-a13
pattern came from only one of the parents.

Since Ml-a13 recombined with Ellié at an easily
observable frequency, it was also crossed with the other
five isolines. The F1 was selfed to obtain a maximum
F2 and the F2 seeds were space planted in the field.
Single heads were harvested from each F2 plant and the
F3 families were screened as described previously. The

results are presented in Table 10. Only one 3R:lS

segregating family was observed in the cross Ml-a7(LG) x

 

Ml-a13. In croSSes with Ml-a13 and Ml-alO, 10 families
segregated 3R:lS, however the seedlings that were scored as
susceptible had a type 3 reaction with severe necrosis but
the mildew did not continue to spread to the third and
fourth leaves. Thus, these were not true susceptible
plants.

Further tests on the Ml-alO/Ml-a recombinant.

 

Susceptible recombinants could possess both allelic sites

for specific resistance or neither site. If the lone

58

. mflmouowc 896m £33 20308.“ m waxy m mums 353 $8.59

.ucmumm mama x Summon mam—5mm

 

moo. moo. SS S mom STE x ASKTE Sm ASKTE x STE
coo. ommm SS STE x EEKTE 03 AEKTE x STE
Go. Be. emvm pm omS STE x STE pm ms: STE x STE
«8. 2.8 Sm STE x TE 8S TE x STE

 

GOHHEE mo
”3E3 mocwowmcoo
3%: mmo 8.25.8 Summ mos: Summ mos:
Godumcfigbommw H506 soHpmmmumwm mmouu aficmhmm cofiammmnmwm mmeuU Hmucmnmm

 

 

 

Among .mw .m mwcfizwmm SEES mo 95 womu 5H3 8335005 umufim gap 5
coflpwmmumvmm mo Emuumm mg was $5303 .850 .50.“ EB mHTHz mo mommohu amusmua Hmoonmflomm .oH manna.

59

Ml-alO/Ml-a recombinant possessed both allelic sites, it

 

should be possible to reconstruct the parental specificity
by crossing it to a similar host line without any sites for
specifc resistance. Recombination between the two sites
might be expected to restore both of the parental
specificities.

Progeny from the Ml-alO/Ml-a recombinant were

 

crossed with Manchuria. The hybrid seed were planted out
and allowed to self-pollinate. The resulting progeny were
tested with CR3 as described in the Materials and Methods.
Nine resistant seedlings were recovered out of 3922 that
were screened. These seedlings gave a type 0-1 reaction
with no necrosis. This is similar to the reaction given by

the Ml—a allele.

 

Expression of Ml-a6/Ml-a13 recombinants. The

 

occurrence of an excess of 0R:lS families in the Ml-a6 x
Ml-al3 cross as well as the lack of any segregating families
in the reciprocal cross, was significantly different (p<.05)
than expected if normal recombination were followed by

normal meiotic segregation. The difference in the frequency
of segregating families among reciprocal crosses with Ml-a6
and Ml-al3 could be due to a maternally acting recombination
suppressor/enhancer. To test this possibility, it was
necessary to examine the recombination of genes on chromosome
five in which the phenotypes of all parental and recombinant

types could be distinguished. This was possible utilizing

60
the hordein genes as all phenotypes are distinguishable by
their characteristic banding patterns. It is possible to
distinguish homozygous and heterozygous parental types as
well as homozygous and heterozygous recombinant types.
Heterozygous recombinant seed originate from one recombinant
gamete and one of either parental gametes. Homozygous
recombinant types originate from recombinant gametes from
both pollen and ova.

The distal third of individual F2 seeds from
reciprocal crosses between Ellié and Ml-a13 were extracted
using the standard procedure (Doll and Andersen, 1981).
The embryo portion was saved and the extracts were
electrophoresed on 12% SDS-polyacrylamide slab gels. The
gels were scored for exchange between Hor-l and £2523
(Figure 9). After the gels were scored, the recombinants
were recovered and grown up from the embryo portion.

Subsequent mildew tests can be carried out on progeny from a

parent in which the constitution of Hor-l and Hor-2 is

 

 

known. The data are presented in Table 11. There was no
significant difference (p>.05) in the frequency of exchange
between £932; and EEEZZ between reciprocal crosses of ELISE
and Ml-al3 lines. There was, however, a significant
difference in the frequency of exchange of EEEZL and ESEZZ
between crosses with Ellié and Ml-a13 and crosses with El:3§
and Elli' The percentage recombination between E2521 and

Hor-2 in crosses between the Ml-a6 and Ml-a13 isolines

61

.mDUOH mluom mcu >3 cocoons mopflummmxaom mum

mocmn mcflumumflfi uwummm 0:9 .msuoH Hluom mg» >3 Umooocm mmbflummmxaom mum mocmfl
mcflpmumHE um3on mfie .MHMIHZ cam omlaz mwcHHOmfl mwaumn cmw3umn meuU m Eouw
mammoum mm m0 mommm mamcww wo mwoflumwmxaom Campuon mo mo<mlmom .o musmfim

62

35:3 «Scan
- ano: 33:... an. .. 9.30:
or: on a; mm
a; on on 9a
.m m. .1. E. m. .m
a; a; on own a; on
on on arm
on on . mp:
’ i
.i I...) g.»

.m musmflm

«930:3 a to:

onzocom w .5:

m

 

 

 

 

63

 

 

.mHCMCHDEOUmucoc u a
cam .mucmcHQEoowu msom%N0EO£ u NC .mucmcHQEOUwu msomxuouwumn n H: mums?
Hm: + m: + Hcvm
Nam + H: mHsfiuOM mfiu an owumEHumm :oHpmcHQEOUmmn
.ucmumm mHmE N ucmumm mHmEmmm
o¢H.h wIHz\wMIHz
oHo.mH mHmnHz\mmnHz
DcoHumcHnfioomu w
00H me 0 mm Hb MIHE x @mIHz
mow 00H H mm Hmuoe

HHH mm H 5H Nmm
0H m o m mmH

¢m mm o wH moH mMIHz x mHmnHz

Nam mam N. ha HmuOB

m¢ mm H mH mmm
mp Ho H mH NhH
mmH mNH ¢ om mmH

om «o H mH mw mMHMIHZ x wmle

Hmuoe mucmcHnEoumucoz muCMCHQEOUmm mucmcHQEoowm .oz mmouu mmouo

msom>NOEom msomxuoumumm

 

 

.mqu can wMIHz :wm3umn mmouo m cam mHMIHz cam olez mmcHHOmH hmHumn :mm3umn
mmmmouo Hmooumwomu CH Nlhom can thom :wm3umn wmcmnuxm mo hocmswwum .HH anmB

64
was 12.016. The percentage recombination between EEEZl and
EEEZZ in the cross between the ELISE and Elli isolines was
7.140.
To examine the effect recombination in the area

between the hordein genes might have had on the Ml-a

 

specificities, embryo portions of 32 of the heterozygous
recombinants and 6 of the homozygous recombinants from
reciprocal crosses between Ml-a6 and Ml—al3 lines were grown
to maturity. Progeny from these plants were tested with
CR3. Three of the six homozygous recombinant plants were
susceptible. Progeny from the resistant homozygous
recombinant plants were resistant. Progeny from the 32
heterozygous recombinant plants were all resistant. Thus,
recombination in the area between the hordein genes had no
significant effect.

Recombination within the Elli region might affect
the dominance relationship of these variants. To examine

this possibility, progeny from all the Ml-a6/Ml-a13

 

recombinants were crossed to the Ml-a6 and Ml-al3 parental
lines. Three seedlings from each cross were tested with
CR3. All seedlings in this test were resistant. Hence, the
dominance relationship was not affected.

The recombinants might complement one another just
as different point mutations complement either intergenically
or intragenically. To test this possibility, all the

Ml-a6/Ml-a13 recombinants were intercrossed to each other in

 

65
most pairwise combinations. Three seedlings from each cross
were inoculated with CR3. All seedlings in this test were
susceptible. Hence, no complementation was observed.

Instability of alleles. It is conceivable that

 

the reason for the 0R:lS families in the Eilflé x Ml-al3
cross is due to a suppressor of Ml-a13 resistance, since the
hordein banding pattern characteristic of lines with Ml-al3
was observed in some of the recombinants. The suppressor
would normally not be active, but would be active in the
Ellié cytotype. It would also have to be cis-acting.
Trans-acting suppression would have likely been detected in
the dominance'test described earlier. If all these
postulates were true, the hordein banding pattern
characteristic of Ml-a13 lines should segregate in the
progeny of 0R:lS F3 families and in progeny of F2
susceptible seedlings. Ml-a13 resistance might also be
expected to segregate in these progeny. Selfed progeny of
crosses between these recombinants and Manchuria might be
expected to produce rare resistant plants due to the
recombination of the suppressor into the Manchuria
chromosome leaving the Ml-a13 resistance exposed. To
explore this possibility, l4 progeny seed each from two of
the 0R:lS families and from two of the F2 susceptible
plants were assayed for their hordein banding patterns. All
58 lanes were of the recombinant type. No segregation was

observed in these tests or in any subsequent tests of this

66
type. However, some resistant seedlings were Observed when
the mildew inoculation was carried out on sister seedlings
from these same recombinants. Further testing showed that
resistance was recovered in some of the progeny from nearly
all the recombinants, regardless of whether they originated
from 0R:lS or 3R:lS families. This was surprising
considering that the progeny from the 3R:lS families
originated from a selfed homozygous susceptible plant with
the recombinant hordein pattern. Mildew tests of the

progeny from F susceptible recombinants were repeated

3
three times. Intact heads were planted in flats and
inoculated heavily with CR3 on day 0 and day l. The results
from one of the tests is presented in Table 12. These are
representative of the results seen each time. The results
were not always distinct. In some of the cases an
intermediate infection was observed. This was not necrotic
like a type 2 infection but the overall development of the
mildew was severely limited as compared to the control
(Figure 10). Some plants that appeared immune at 7 to 8
days developed various degrees of infection by 14 days. By

the tenth day, control plants were dead. Similar results

were obtained in progeny of the crosses of Ml-aG/Ml-a13

 

recombinants with Manchuria (Table 13). However, resistant
progeny among intercrosses of the recombinants were rare
(Table 14). This high frequency of reversion might be

expected if a controlling element had inserted into a site

67

Table 12. Results of inoculation of progeny from 0R:lS and
3R:lS F families with race CR3 of Erysiphe graminis f.

sp. hor ei. Each line represents a single head or a
portion of a single head.

 

 

 

Recombinant family Resistant Intermediate Susceptible

0R:lS

193-1 5 12
193-1 17 2
193-1 18 1
193-4 7 2 5
193-4 6 2
193-4 2 4 7
193-4 9 0
193-4 16 2 4
193-4 5 13
193-4 ' 20 9
193-4 8 10
168-4 18 0
168-4 10 2 5
168-4 6 2
168-4 8 2
168-4 5 4
168-4 1 5
3R:1S

193-2 20 0
193-2 16 4 2
193-2 20 8
168-1 14 0
168-1 14 1
168-1 15 0
168-3 8 1
168-5 10 0
168-5 25 1
193-3 0 20

 

68

 

m/HVCHUAM

Figure 10. Phenotype of revertant barley seedlings 8-9 days
after inoculation with race CR3 of Erysiphe graminis f. sp.
hordei.
'TT_PFSgeny of a cross between the Ml-a6/Ml-a13 recombinants
193-1 and 168-4. All seedlings from one group are from a
single head. Reaction type: a, susceptible;

b, revertant-intermediate type; c, revertant—resistant type.
2. Manchuria control.

 

 

69

 

 

m .mmHHHsmm
m mH.mm scum wmumchHHo 6cm .sume .mnmoH .HummH .msmmHu

.mmHHHEmH mm mH.mc
scum cmpmchHuo a. .HnmmH .onmoH .vummH .mummH .muHummHn
.ucmumm mHmE x Hcmumm mHMEwhm

mum 5mm mHm mmuHE x HummH mHs

mmm H muHummH x mmuHe NH.
oNoH NH v omuHs x mummH mom
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71
at or near a resistance locus to cause a susceptible
phenotype and was excising in later generations. A diagram
of the kinds of results observed in each generation is

presented in Figure 11.

 

72

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73

 

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74

Discussion

 

This analysis of a group of genes conferring
specific resistance at the E1:3 locus was based a priori on
two assumptions:

1. Each parental host variety possesses a different
single dominant variant conferring specific
resistance.

2. Susceptible segregants are the result of
reciprocal recombination during gametogenesis.

These assumptions will be examined in more detail as we
proceed through an interpretation of the data.

Although the genes used in this analysis behave as
units of segregation (Moseman and Jorgensen, 1973), it has
recently been suggested that some of the six host lines may
have more than one gene for resistance (J.H. Jorgensen,
personal communication). The question arises whether the
use of a variety which possesses more than one E123 gene
invalidates the analysis. This would be true only if the
powdery mildew culture used (CR3) possessed an avirulence
gene complementary to the additional resistance gene.
Without complementary avirulence, the resistance reaction
cannot be expressed. If the above were true and the two
genes in one variety were positioned on either side of a
gene in the other variety, products of a single crossover

event between two of the genes in repulsion would not be

75
detected. This is because the additional gene would still
confer resistance to the "double recessive" progeny
resulting from recombination. The varieties Multan and

Rupee which possess the genes M1-a7(Mu) and M1-al3,

 

respectively, have been reported to have additional
resistance genes with complementary avirulence genes in CR3
(J.H. Jorgensen, personal communication). Interpretations
of their effect on the data will be discussed.

The absence of verifiable recombinants in many of
the crosses was not expected. This may reflect the

closeness of the alleles at the Ml-a locus or it may

 

represent an inability of the different alleles to recombine
at all. Studies on the genetic fine structure of the Waxy
locus in maize by pollen analysis resulted in a marked
non-additivity of recombination frequencies (Nelson, 1968).
Nelson proposed that this may have been due to small
differences in gene structure. The Ex progenitors in the
different inbred lines were all able to support amylose
synthesis. Different Ex alleles could conceivably contain a
small duplication or deletion and still remain functional.
Apparently attempts to place all Hi mutants in similar
genetic background by backcrossing to M14 would not correct
these differences. A similar phenomenon occurred in studies
on intragenic recombination at the maize édhl locus which
codes for the enzyme alcohol dehydrogenase. Freeling (1976)

found that the ability of different Adhl‘ mutants to

 

76
recombine depended on their origin. Different Adhl—
alleles recombined only if derived from the same parental

Adhl+ allele. Mutants derived from different Adhl+

 

 

alleles did not show recombination by pollen analysis.

Structural differences in the different Ml-a "alleles" may

 

be an explanation for their lack of recombination. However,
the number of progeny screened may have been insufficient to
detect recombination. Based on the upper 95% confidence
limits of recombination the M123 alleles in Table 8 would
then be no more than .056 map units apart.

Crosses with the Multan isoline may be interpreted

as the Ml-a7(Mu) allele being tightly linked with Ml-a,

 

Ml-a6, and Ml-a7(LG). Alternatively, an additional gene

 

may be masking the effect of recombination between the two
primary genes involved in the cross. If this were true, the
upper 95% confidence limit may not reflect an accurate
estimate. Recombinants were recovered in some of the
crosses involving the Rupee isoline. These results are
interpreted with caution (see below), however the additional
genes may be distal to the Ml-al3 gene if Ml-a6 and

Ml-a7(LG) are used as a point of reference.

 

Restoration of specificity in the Ml-alO/Ml-a

 

recombinant. One susceptible recombinant was observed in

 

the Ml-alo x Ml-a cross. This recombinant could possess
both allelic sites for specific resistance or neither site

(double recessive). However, resistant seedlings (9 out of

77
3922) were Observed in the progeny of crosses between the

Ml-alO/Ml-a recombinant and the Manchuria isoline

 

possessing recessive ml-alo. These may be due to a
recombination event restoring M113 type specificity (Figure
6). If this were true, then the original recombinant most
likely contained both dominant sites for specific
resistance, M1:3_and Ml-alo. The restoration of

resistance is interpreted as evidence that these two sites

are allelic. If intracistronic recombination between the

 

two allelic sites had led to an altered specificity, then in ‘
fact the original selection in this particular cross was for

both kinds of recombinants, not just the double recessive

(Figure 5). This would alter the maximum likelihood formula

by doubling the denominator resulting in a linkage value of

.0065. Alternatively, the resistant seedlings observed may

represent the excision of an insertion element (see

interpretation for Ml-a6/Ml-al3 recombinants).

 

Insertion of the element would have lead to dysfunction of
the M123 allele resulting in a compatible interaction with
the pathogen. The excision event in later generations might
restore function leading to an incompatible interaction.

On the occurrence of mutable alleles. One

 

segregating family was observed in the Ml-a x M1-a7(LG)

 

cross. Type 4 infections were observed in the susceptible
segregants in this cross. Exchange of outside markers was

observed in progeny of these plants. However, type 2

78
infections resulted from inoculation of these progeny.

In an analogous fashion, progeny of M1-a6/Ml-a13

 

susceptible segregants also showed exchange of outside
markers. These progeny also reverted in significant
frequencies. Reversion occurred at a much higher rate than
expected if standard recombination or mutation were
occurring. Furthermore, susceptible recombinants were only

observed in F families where the Ml-a6 line was the

3
original female parent. How can we explain these results?
A maternally acting recombination suppressor/enhancer is
unlikely since the hordein alleles recombined in
approximately equal frequencies in reciprocal crosses. A
cytoplasmic suppressor of the resistance phenotype is
plausible, however it should have been expressed in the
backcrosses to the parental lines possessing M1-a6 and
Ml-a13.

If an unlinked nuclear suppressor were present,

seedlings resulting from a testcross of a Ml—a6/Ml-a13

 

recombinant to Manchuria would be expected to segregate
1R:1S. All seedlings in this test, however, were
susceptible. A closely-linked resistance suppressor would
not be expected to segregate in progeny of the recombinants.
Resistant progeny were recovered at a significant frequency,

however (Table 12).

79

There are a significant number of anomalies

associated with the Ml-a6/Ml-a13 recombinants that

 

cannot be explained by Mendelian genetics:

1.

Susceptible segregants were only observed in

F3 families where Ml-a6 was the original

female parent.

 

If each recombinant is the result of an
independent event, then based on the 12 3R:1S
families we would expect less than one 0R:lS
family. Nine 0R:lS segregating families were
observed (Table 8). This difference cannot be
ascribed to chance.

Resistant revertants were recovered in high
frequencies showing variagation in infection type
among progeny from the same homozygous
susceptible "recombinants". These revertants had
no change in their flanking marker constitution.
Furthermore, resistant revertants appeared in the
F5 and F6 generations and not all of these

revertants are stable.

The reciprocal cross difference and mitotic recombination

(see below) phenomena display parallels to what has been

termed "hybrid dysgenesis" in Drosophila which has been

associated with controlling elements (Kidwell et al., 1977).

First described by McClintock (1951) "controlling elements"

are transposable elements which make themselves

80

visible by their adventitious control of standard genes (see
reviews by Fincham and Sastry, 1974; Nevers and Saedler,
1977; Burr and Burr, 1981). Insertion of an element into or
near a gene may produce a mutant phenotype, while accurate
excision may lead to restored expression. The initial
susceptible segregants may be due to insertion of an element
during development with later excision yielding reversion to
resistance. This also may explain the lack of other
verifiable recombinants in crosses of other alleles with
M1236 (Table 8) and with Ml-a13 (Table 10).

The initial recombination in the M123 region in
isolines possessing M1236 and M1-a13 may be associated with
the transposition event. Laughnan (1952) found that mutable

Ad

alleles coding for anthocyanin synthesis in maize

were closely associated with recombination in that region.
In numerous instances of newly-induced mutable alleles, the
locus is first seen as autonomously mutable. This means
that the regulatory element is at the affected locus and in
a cis position (Friedemann and Peterson, 1982). It is
possible that the initial recombination event positioned a

regulatory element in cis with a resistance allele. Some of

the M1-a6/Ml-a13 revertant plants were transplanted and

 

tested with race A27 of E. graminis f. sp. hordei. Race
A27 is avirulent on Ml-a13 and virulent on M1-a6. A27
produces a type 2-3n reaction on Ml-al4 which is also

present in the Ml-a6 isoline (Giese, 1981). However, since

 

81

a type 2-3n reaction was not observed in inoculations of

progeny of the M1-a6/Ml-a13 recombinants with A27, it is

 

interpreted that M1-al4 is not present in these plants.

These M1-a6/Ml-a13 revertants also had a type 0 reaction

 

with A27. This is interpreted as meaning that at least
Ml-a13 resistance was restored by the reversion event. It is

possible that Ml-a6 was also restored in the Ml-a6/Ml-a13

 

revertants, however other mildew cultures would be necessary
to confirm this. Alternatively, Ml-a6 specificity might
never have been present in these recombinants. Suppression

of Ml-al3 resistance in the Ml-a6/Ml-a13 recombinants

 

supports the cis-acting regulatory element model.
Alternatively, insertion of the element may have
caused the recombination. Additional evidence, however,
supports the converse interpretation. Susceptible
segregants were not observed in two of the six independent
crosses of the M1-a6 x M1-a13 type, but, analysis of the

hordein alleles using F segregating progeny showed that

2
recombination was occurring at the same rate in mutable and
non-mutable crosses.

On the nature of the 0R:lS F3 families.
Controlling elements can alter the nature of coding sequence
expression in a variety of ways (see Shapiro and Cordell,
1982). One of these is by creating somatic instability

(McClintock, 1951). Somatic recombination in heterozygous

cells in the apical meristem would clearly alter the

82
developmental possibilities of the progeny clones. If these
clones were in a portion of the meristem which gave rise to
germinal tissue, half of the gametes that descended from
that event would be "recombinant". Depending on how early
in development the recombination event occurred, it may give
rise to any number of “recombinant" gametes, greatly
increasing the chances of two "recombinant" gametes fusing
with each other during fertilization. Since both pollen and
ova are present in every floret, this might give rise to
what appeared to be 0R:lS F3 families.

What is the basis for the high frequency of
resistant revertants? The simplest explanation for
reversion is excision of the insertion element to restore
normal function Of the gene. Integration into specific Ml:g
alleles could either lead to complete loss of the gene
product or to the loss of its distal end, resulting in a
modified product. Accurate excision would lead to normal
function. Inaccurate excision may lead to partial or
modified function and could explain the observation of a

number of intermediate infection types seen in later

generations.

83

Summary

This study began with the question of whether the
variants of the Ml:3 region were alleles or closely-linked
cistrons. The data have not answered that question. The
data have provided a model in which the likelihood of a
transposable element is presented.

Based on the recombination estimates, flanking
marker constitution, and interpretation of reversion events,
a model of the M122 locus is presented in Figure 12. We
interpret the recombination events in the Ml-a6 x Ml-a13
crosses to be between the M1-al3 allele and an autonomous
regulatory element. Ml-al3 specificity was positively
identified by raCe A27 of E. graminis f. sp. hordei in the

revertant progeny from M1-a6/Ml-al3 recombinants. If

 

somatic recombination did occur in Ml-a6 x Ml-al3 crosses,

then it is likely that the 0R:lS F families originated

3
from progeny clones of the original recombination events.
If this is the case, the recombination estimate would be
lower.

The flanking marker constitution in progeny of the

Ml-alO/Ml-a recombinant was Hor-1(a10) and Hor-2(a).

 

For this reason Ml-alO is positioned between H2521 and Mlla.
In addition, it was possible to recover M123 type
specificity in the Ml-alo x M123 cross. Ml-alo type
specificity may also have been recovered but it may have

been masked by the more resistant Ml-a type.

84

 

 

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85

In progeny of the M1-a/M1-a7(LG) recombinant the

 

flanking marker constitution was §g£:l[a7(LG)] and §2£:2(a).
‘Ml:aZ(LG) type specificity was recovered in these prOgeny.
The reasons for this are unclear. The other variants are
positioned based on their recombination estimates. These
results show the complex nature of the M123 region.
Controlling elements have been shown to be associated with
other complex loci (Laughnan, 1952: Nelson, 1968: Freeling,
1976), although to our knowledge this is the only example in

a locus conditioning resistance to an obligate parasite.

86

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Doll, H. and B. Andersen. 1981. Preparation of barley
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