Copyright 8 1995 by the Genetics Society of America

New Rust Resistance Specificities Associated With Recombination in the Rpl complex in Maize

Todd E. Richter,* Tony J. Pryor: Jeffrey L. Bemetzed and Scot H. Hulbert" *Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506, 'Division of Plant Industry, CSIRO, Canberra,

Australia ACT 2601, and $Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Manuscript received April 27, 1995

Accepted for publication June 12, 1995

ABSTRACT We address the question of whether genetic reassortment events, including unequal crossing over and

gene conversion, at the Rpl complex are capable of generating novel resistance specificities that were not present in the parents. Some 176 events involving genetic reassortment within the Rpl complex were screened for novel resistance specificities with a set of 11 different rust biotypes. Most (150/176) of the events were susceptible to all tested rust biotypes, providing no evidence for new specificities. Eleven events selected as double-resistant recombinants, when screened with the 11 test biotypes, showed the combined resistance of the two parental types consistent with a simple recombination and pyramiding of the parental resistances. Nine events selected either as having partial resistance or complete susceptibil- ity to a single biotype possessed resistance to a subset of the biotypes that the parents were resistant to, suggesting segregation of resistance genes present in the parental Rpl complex. Four events gave rise to novel specificities being resistant to at least one rust biotype to which both parents were susceptible. All four had flanking marker exchange, demonstrating that crossing over within the Rpl complex is associated with the appearance of new rust resistance specificities.

M AJOR gene resistance to rusts follows the classic gene-for-gene model (FLOR 1956). A resistance gene product from the host plant recognizes (either directly or indirectly) the avirulence gene product from the rust. Thus, mutations to virulence in the rust can occur as a simple loss of function of the avirulence gene. Deletion of DNA at the corresponding avirulence locus has been shown to be associated with mutation to virulence (SWEIGARD et al. 1995; M. A. AYLIFFE, G. J. LAWRENCE, J. G. ELLIS and A. J. PRYOR, personal commu- nication). However, the acquisition of new resistance in the plant host requires the creation of a new specificity. Plant species would benefit from a mechanism capable of generating novel specificities rapidly enough to match the rate of appearance of virulence in fungi, such as the cereal rusts. To date, little is known about how these complex loci evolve new race specificities over time nor is there any experimental evidence that they do.

There is little evidence for conventional point mu- tations leading to the appearance of new genetic speci- ficities in eukaryotes (LANGRIDGE 1991). Loci that do appear capable of generating new specificities have fre- quently been shown to be complex loci that undergo genetic reassortment events, leading to new arrange- ments of preexisting functions. Examples include mat- ing type in basidiomycetes (KUES and CASSELTON 1993) and mammalian immunoglobulins (THOMPSON 1992).

Corresponding author: Scot H. Hulbert, Department of Plant Pathol- ogy, 4024 Throckmorton Plant Sciences Center, Manhattan, Kansas 665065502,

The many plant genes controlling resistance to obli- gate fungal pathogens are commonly clustered in spe- cific regions of the plant genome (SHEPHERD and MAYO 1972; GIESE 1981; CRUTE 1985; HOOKER 1985; FARRARA et al. 1987). A classic example of such a cluster in maize are genes that condition resistance to the common rust fungus, Puccinia sorghi, and map to the distal region of the short arm of chromosome 10 in a complex region that is termed the Rpl complex. There is good evidence that genetic reassortment events occur within the Rpl complex. Recombination experiments have been con- ducted by analyzing the resistances of progeny from crosses of F1 heterozygotes between two Rpl alleles test crossed to a standard recessive q l line. These experi- ments found that two classes of recombinant derivatives could be recovered in test cross populations. These were susceptible derivatives and double resistant deriva- tives, thought to have both parental genes linked in cis ( SAXENA and HOOKER 1968). Subsequent experiments demonstrated that these recombinants were usually as- sociated with crossing over (HULBERT and BENNETZEN 1991). Although most of the Rpl genes recombined as if they were tightly linked or, in some cases, possibly allelic, Rp5 and Rpl-G (now designated RPG') mapped 1-3 cM distally.

Transposon mutagenesis studies of Rpl led to the observation that the frequency of susceptible progeny recovered from test cross progeny of certain Rpl homo- zygotes were significantly higher than expected by an insertion of the transposable element (PRYOR 1987) and that control test crosses also showed high frequencies

Genetics 141: 373-381 (September, 1995)

374 T. E. Richter et al.

of susceptible derivatives (BENNETZEN et al. 1988). Anal- ysis of susceptible derivatives from test cross progeny of Rpl homozygotes constructed with heterozygous flank- ing markers found they were generally associated with crossing over. Both nonparental combinations of flank- ing markers occurred, indicating they arose by unequal crossing over (SUDUPAK et al. 1993; A. J. PRYOR, unpub- lished results). Recombinants from some heterozygotes also showed both nonparental combinations of flanking markers, suggesting that a similar mechanism is respon- sible for the observed instability in both homozygotes and heterozygotes (HULBERT et al. 1993). Susceptible and double-resistant derivatives that were not associated with flanking marker exchange have also been observed in crosses between some Rpl alleles and are thought to represent gene conversion events (Hu and HULBERT 1994). The conversion events were generally less fre- quent than crossover events but occurred at high fre- quencies (7 X in some crosses.

The highly recombinagenic nature of Rpl has led to speculation of the role these events play in creating novel race specificities (PRYOR 1987; BENNETZEN and HULBERT 1992) to provide resistance effective against constantly changing pathogen populations. The pres- ent study was undertaken to determine whether resis- tance genes with novel specificities could be generated at the Rpl locus. We examined 176 genetic stocks de- rived mainly from crossing over in the Rpl complex and possible transposon insertions into Rpl genes. The lines were analyzed with 11 P. sorghi biotypes to determine whether any novel specificities had been generated. Our results confirm the complexity of the Rpl locus and demonstrate that new rust resistance specificities can be recovered from a sample of recombination events at the Rpl complex.

MATERIALS AND METHODS

Origin of maize lines: Some 176 maize lines (Table 1) de- rived from various genetic experiments involving rust resis- tance specified by genes in the Rpl complex were collected for examination of resistance against 11 rust biotypes. The first 130 lines (Table 1A) were selected as susceptible derivatives from Rpl heterozygotes or homozygotes using either a single rust biotype or, in some cases, two rust biotypes sequentially. Many of these derivatives have been described previously (HULBERT and BENNETZEN 1991; HULBERT et al. 1993; SUDU- PAK et al. 1993) with the exception of those from the Rpl-

Rpl-D/Rp5, Rpl-Krl/Rpl-C and Rpl-Krl/Rpl-Jcrosses. An ad- ditional 25 derivatives (Table 1B) came from test crosses of Rpl homozygotes in transposable element backgrounds. Twenty-one of these came from RplD homozygotes in a back- ground with an active Ac element (PRYOR 1993) and four came from an Ql-F homozygote in a Mutator background (BENNETZEN et al. 1988). Three of the four Rpl-F derivatives were originally reported to retain some level of resistance to certain rust biotypes (BENNETZEN et al. 1988), but additional tests indicated the differences observed between the lines were probably due to genetic background effects and/or envi- ronmental differences between the tests (S. H. HULBERT and

D/Rpl-I, Rpl-F/Rpl-A, Rpl-N/Rpl-C, Rpl-I/RpG, Rpl-K/Rpl-C,

J. L. BENNETZEN, unpublished results). Eleven derivatives (Ta- ble lc) were selected from Rpl heterozygotes because of their combined resistance to two biotypes that differentially detect the resistance genes of the parents ( HULBERT et al. 1993). Ten derivatives (Table 1D) were recovered because they showed a reduced level of resistance compared with the parental het- erozygote. Six of these derivatives arose from heterozygotes between a resistance gene (Rpl-K or Rpl-I) and a susceptible rpl allele.

Inoculation and scoring procedures: Seven- to 8-day-old seedlings were inoculated with rust spores and scored for resistance 7- 10 days later. The maize lines were planted side by side to eliminate as much variation as possible. This proce- dure was repeated at least once for each rust biotype. Some infections, particularly the intermediate types, can be difficult to score, and these were repeated several times to confirm the scoring. The reactions of each derivative were scored on a scale of 0 to 4. A rating of 0 indicates a high level of resis- tance with no sporulation. A rating of 1 indicates a high level of resistance with only one or a few pustules per leaf. A rating of 2 indicates a larger number of pustules but with clear hypersensitive reactions, with most of the fungal penetrations resulting in chlorotic or necrotic spots. Pustules are generally smaller than those of compatible interactions and are gener- ally surrounded by chlorosis or necrosis. Ratings of 3 indicate a large number of pustules but less than those rated as 4 or fully susceptible. Ratings of 3 are usually associated with a weak but visible resistance response such as chlorosis around some of the pustules.

Rust biotypes: The 11 biotypes of the common rust fungus, P. sorghz, used were collected from various geographic regions and growing seasons. Each rust biotype was purified by single pustule isolation and tested on all available Rp differential lines to determine their virulence phenotype (HULBERT et al. 1991; this study). The biotypes include eight previously characterized biotypes and three collected during the 1992 summer growing season: OH1 from Ohio was obtained from K. SIMCOX, IAl from Iowa was obtained from P. PETERSON and KS2 was obtained from the summer maize nursery in Manhattan, Kansas. Each biotype had a unique virulence pat- tern. Up to four rust biotypes were in use at a given time. When not in use, rust biotypes were stored, partially desic- cated, at -70". When in use, each rust biotype was propagated in a different greenhouse and, when possible, cultured on maize lines on which the other rust biotypes in use would not grow. Biotypes were checked for contamination periodically by inoculating the series of differential cultivars.

DNA marker analysis: Genomic DNA was isolated and gel blot analysis was performed as previously described (HULBERT and BENNETZEN 1991). The probes npi285, bnl3.04, hu3 and php20075were used to detect restriction fragment length poly- morphism (RFLP) markers flanking the Rpl complex (HONG et al. 1993).

RESULTS

Events that result in a complete loss of resis- tance: The majority (130) of the maize lines analyzed in this study were derived from individuals from test cross families that were susceptible to a single rust bio- type that was avirulent to both parental Rpl genes (Ta- ble 1A). Each of these maize lines was subsequently screened with an additional 11 different rust biotypes to determine whether any of the derivatives that were originally scored as susceptible retained some form of resistance or had altered resistance specificity. Most of

Rpl Specificity Changes

TABU 1

List of the events involving genetic reassortment at the Rpl complex

375

Parental genotype Original biotype Designations

A. Rpl events selected for their susceptibility

Rpl-A/Rpl-A(G.R) Rp 1 -A/ RPG Rpl-A(G.&)/Rpl-I Rpl-B/Rpl-E Rpl-D/Rpl-A(G.K) Rpl-D/Rpl-B Rpl-D/Rpl-F Rpl -D/ Rp5 Rpl-E/Rpl-F Rpl-E/ RPG Rpl -E/ Rp5

Rpl-F/RpG Rpl-F/Rpl-A(G.R) Rpl-F/Rpl-A RpG/Rp5

Rpl-J/Rpl-D Rpl-I/RpG

Rpl-J/RpI-F Rpl-J/Rpl-A(G.R) Rpl-J/Rpl-C Rpl-K/Rpl-C Rpl-L/Rpl-D Rpl-N/Rpl-C RpG/RpG

RPl-J/RPl-J Rpl-Krl/Rpl-C Rpl-Krl/Rpl-J

14 14 14 14 IN2 IN2 14 KSl, IN2 1-4 1-4 IN1

1-4 1-4 IN2 IN3 IN2, IN3 KSl, IN2 KS1, IN2 IN3 KS1, IN2 IN2 IN2 IN2 HI1

KS1 KS1 L41

14, 20 3a, 5a, 6, 8b, 8a, 8c, 8d, 8e, Sf, l l a , 13b 3, 21a 11 4 6 3 3b, 3c, 4b, 4c 2, 6, 11, 56 Id, 3a, 8c la, 6a, 6b, loa, lob, lOc, 10d, 12, 4a, 16, 17, 17b,

2a, 5a, 5b, 5c, 6d, 11, 13b, 16a, 16b, 16c, 16d, 17a 27, 28, 29 9 5b, 6, 10, 18 5b, 6a, 6b, 6d, 7a, 8b, l l a , 7f 18, 42, 43, 50 2, 55, 59 3 4 1 6, 13, 28, 50 1, 6, 8, 13 1, 3a, 3b, 4a, 5, Sa, 9b, loa, lob, 13, 14a, 14b,

14c, 14d, 16a, 16b, 17, 19, 20 12a, 12b, 25 11, 12, 13, 15, 16, 17, 21, 22, 31 7, 13, 94, KrlJ6, KrlJ15, KrlJ92

19, 20, 27a, 27b, 4b

B. Derivatives from Rpl homozygotes in transposable element backgrounds

Rpl-F/Rpl-F Mutator 1-4 Rpl-D/Rpl-D AC R1

A13, D3, D22, A35 1, 2, 3, 4, D5,d 7, 8, 10, 11, 12, 14, 16, 17, 19, 20,

22, 23, 24, 25, 28, 29

C. Derivatives from Rpl heterozygotes with the resistances of both parents

Rpl-J,/Rpl-D Rpl-J/Rpl-F Rpl-I/RpG Rpl-D/Rp5

KSl, IN2 46 Ksl, IN2 11, 58, 69 IN2, IN3 5a, 7c, 7d, 8a, 10b KSl, IN2 3a, 4a

D. Derivatives from Rpl heterozygotes that were selected for a reduced level of resistance to a single rust biotype

Rpl-B/RpI-I IN2 B12 Rpl-D/Rpl-I IN2 DIl, DL28 Rpl-E/Rpl-F 14 EF25 R p l - K / q l IN2 Krl, Krz, Kr3, Kr4, Kr5 R p l - I / q l L41 Ir2

“Biotype originally used to identify mutant or recombinant individuals. The Rpl-A gene originally came from the cultivar “Golden Glow,” whereas the gene designated Rpl-A(GK)

Australian rust biotype. Derivatives with changes in specificity or level of expression are designated in bold type.

came from the cultivar “Golden King.”

376 T. E. Richter et al.

TABLE 2 Resistance reactions of lines carrying Rpl-D and the Rpl-D derivatives from a background with an active

transposable element (Ac) when inoculated with 11 rust biotypes

Rust biotype Rpl-

specificity IN2 KSl 1-4 IN1 IN3 AFl GA2 IAl OH1 KS2 HI1

Ql-D 0 4 0 0 0 4 0 0 0 0 4 Rp 1 -D5 2 4 1 1 0/1 4 2 2 o/ 1 2 4 RPlD 1 to 29 4 4 4 4 4 4 4 4 4 4 4

these derivatives were highly susceptible to all of the rust biotypes (data not shown). Three exceptional de- rivatives came from a cross between Rpl-Jand Rpl-Krl and are described below.

Another 25 Rpl derivatives that arose in transposable element backgrounds were screened with the 11 rust biotypes: 21 were Rpl-D derivatives from lines with ac- tive Ac elements in their background (PRYOR 1993) and four were derivatives from Rpl-F in a Mutator back- ground (Table 1B). All were originally isolated as fully susceptible except one (Rpl-D5, discussed below), which showed partial resistance when compared with the parental Rpl-D allele (PRYOR 1993). The lines de- rived from the 24 fully susceptible derivatives were sus- ceptible to all 11 rust biotypes (Table 2).

Events that result in a combined resistance of the two parental types: Eleven lines (Table 1C) that were originally selected for the combined resistances of the parental types and nonparental combinations of flank- ing markers (Hu and HULBERT 1994) were also tested for resistance to the 11 rust biotypes. The spectrum of resistance exhibited by these lines were those expected for the recombination or pyramiding of closely linked resistance loci. For example, the recombinant RpI-JD46 had the same spectrum of resistance across all the rust biotypes as the parental QI-J/RpI-D heterozygote. It was susceptible only to biotype HI1, the only biotype that is virulent on both Rpl-Jand R p l D (Table 3).

Events that result in altered resistance: In the process of screening progeny from various Rpl heterozygotes, six seedlings were observed that showed a reduced (par- tial) resistance. Analysis of self-fertilized progeny from these seedlings verified that the partial resistance was heritable and segregated as a single gene mapping at rpl. One of these lines, Rpl-Krl, was used as a parent in crosses with Rpl-Jto recover three additional derivatives (Rpl-KrlJ6, Rpl-KrlJl5 and Rpl-KrlJ92) that fall into the same category (Table 4). Unlike the previous six derivatives, these three were originally selected as being completely susceptible to the rust biotype used in the resistance screen. When challenged with the 11 rust biotypes, however, their similarity became apparent: all nine showed resistance to a subset of the biotypes that the parents were resistant to. For example, the Rpl- EF25 derivative showed resistance to a subset of the biotypes the Rpl-E parent was resistant to; it is suscepti-

ble to both rust biotypes (IN3 and GA2) the Rpl-E line is susceptible to, but is resistant only to some of the biotypes the Rpl-E line is resistant to (Table 4).

One additional partially resistant line, Rpl-D5, was selected from an Rpl-D homozygote in a background with an active Ac element. After challenging with the 11 rust biotypes, it appeared different from the above nine partially resistant derivatives because it did not show a change in specificity. The Rpl-D5 line showed resistance reactions to the same rust biotypes as the parental R p l D line, but the resistance was generally less complete (Table 2). Thus, in interactions where the Rpl-D parent was fully resistant (0 reaction), the Rpl-D5 line was partially resistant (0/1 to 2), and in interactions where the e l - D line was fully susceptible, the Rpl-D5 line was also susceptible.

Events that result in novel resistance not present in either parent: Four variants were recovered that showed resistance to at least one rust biotype to which the parental lines were fully susceptible (Table 5). The Rpl-Kr2, 3 and 4 derivatives originated from an RFLP mapping family, derived from the cross R p l - K / q l X ql/$l, which when screened with the rust biotype IN2 segregated 1485 resistant:1211 susceptible:5 partial re- sistance. The other two individuals that showed partial resistance to biotype IN2, Rpl-Krl and Rpl-Kr5, are de- scribed in the previous section. Rpl-Ir2 came from an analogous cross involving the Rpl-I/rpl heterozygote that segregated 1763 resistant: 1631 susceptib1e:l par- tial resistance, when screened with rust biotype IAl. These four, selected as showing partial resistance to one of the rust biotypes (IN2 or IAl), maintain some of the resistances of the parental types but have lost other parental resistances (Table 5). For example, Rpl-Kr2 shows a disease rating 2, or partial resistance, against biotypes IN2 and KS1 but is fully susceptible to the 1- 4 and IN1 biotypes. The parental Rpl-K gene specified complete resistance 0 to these four biotypes. In this regard they were like other variants showing partial re- sistance, but they differ in a most significant way. They show resistance to a biotype to which both parental types were fully susceptible (Figure 1). Whereas the parental types, rpl, Rpl-Kand Rpl-I, were all susceptible to biotype IN3, the four variants were resistant to IN3. The Rpl-Kr4 variant also was resistant to GA2, another biotype to which the Rpl-K parent is fully susceptible.

Rpl Specificity Changes 377

TABLE 3

Reactions of recornbinant Rpl alleles that were selected for their resistance to each of two rust biotypes, one of which was avirulent on each parent

Parents R p l D 0 4 0 0 0 4 0 0 0 0 4 RpI-F 0/1 4 0/1 4 1 4 o/ 1 0/1 0/1 4 4 RPG 4 4 0 1/2 0 4 4 0 0 0 0/1

RP1-J 4 1/2 4 2/3 1/2 3 4 2 2 2/3 4 RP5 4 1/2 2/3 1/2 4 1/2 4 3 1/2 1/2 4

Rpl -I 0 0 0 0 4 0 4 ,3 /4 0 0 0 0

Recombinants" Rpl-JD 46 0 1/2 0 0 0 3 0 0/1 0/1 0 4 R p l - p 11, 58, 69 0/1 1/2 0/1 2/3 0/1 3 o/ 1 0 0 2/3 4 Rpl-IG 5a, 7c, 7d, 8a, 10b 0 0 0 0 1/2 0 4 ,3 /4 0 0 0 0 Rpl-DRp5 3a, 4a 0 1/2 0 0 0 1/2 0 0 0 0 4

Letters in Rpl- allelic designations for recombinants indicate which parental Rpl genes were recombined into the cis arrange- ment. For example, Rpl-JD carries Rpl-Jand Rpl-D.

Association of crossing over with the generation of altered specificities: Of the 127 events that resulted in a complete loss of resistance to all 11 rust biotypes (Ta- ble lA), all but four were associated with an exchange of flanking markers. The exceptions were derivative Rpl- KrlJl3 from a Rpl-Krl/Rpl-J cross, derivatives Ql- KrlC11 and RplKrlCl2from a Rpl-Krl/Rpl-Ccross and a previously described derivative (RPG13 from an RPG homozygote (SUDUPAK et al. 1993). All 11 of the events that were selected for the combined resistances of both parents were crossover events (Table 3). The four deriv- atives with resistance to at least one rust biotype that the parents were susceptible to (Table 5) were also all associated with crossing over (Table 6). The derivative

class with the weakest association with crossing over were those showing resistance to a subset of the parental resistances (Table 4); five of the nine derivatives showed flanking marker exchange (Table 6).

The resistance specificities of individuals from fami- lies that gave several derivatives were correlated with their flanking marker constitution. For example, five individuals with altered resistances were identified from the Rpl-K/qbl X qbl/qbl cross. The two of these that exhibited resistance to a reduced number of rust bio- types when compared with the parents, Rpl-Krl and Rpl-Kr5, were both noncrossover types (Table 6). Of the three derivatives that exhibited resistances not pres- ent in the Rpl-K parent, two (Ql-Kr2 and Rpl-Kr3)

TABLE 4

Reactions of recombinant Rpl alleles with altered specificities that provided resistance to a subset of the rust biotypes that one of their parental genes did

Rpl-specificity IN2 KS1 1 4 IN1 IN3 AFl GA2 IAl OH1 KS2 HI1 ~ ~~ ~

Parents ~~

Rpl-B 1 " 4 3 4 1 3 3 4 4 4 1 Rpl-D 0 4 0 0 0 4 0 0 0 0 4 Rpl-E, -I, K" 0 0 0 0 4 0 4 0 0 0 0 Rpl-F o/ 1 4 o/ 1 4 o/ 1 4 o/ 1 o/ 1 o/ 1 4 4 RP1-J 4 1/2 4 2/3 1/2 3 4 2 2 2 4

Derivatives Rpl -EF25 2 2 1 4 4 2 4 2 3/4 0 Rpl-B12 2 2 1 4 4 2 4 4 4 0 Rpl-DII RPI-D128 2 1 4 4 2 4 3 2 3/4 1

2/3 2

2/3 2

2 2 1 4 4 2 4 3/4 2 4 1

Rpl-Kr5 2 1/2 4 4 2 4 4 3 4 1 Rpl -Krl 2 2 1 4 4 2 4 3 2 4 0 Rpl-KrlJ6 4 3 4 4 1/2 4 4 3/4 4 4 4 Rpl-KrlJ92 3 4 4 4 4 3/4 4 4 4 4 1 Rpl-K~lJ15 3 4 4 4 2 3/4 4 4 4 4 1

a The Rpl-E, Rpl-I and Rpl-K genes have identical reactions to the 11 rust biotypes but were originally distinguished by rust isolates available to HAGAN and HOOKER (1965).

The Rpl-Krl derivative was used as a parent to generate to last three derivatives.

378 T. E. Richter et al.

TABLE 5

Reactions of variant Rpl alleles with altered specificities that provided resistance to rust biotypes that neither parental gene did

R/~l-specificity IN2 KS 1 1 - 4 IN 1 IN3 AFl GA2 IAl OH1 KS2 HI1

Parents R / I I -I, -IT 0 0 0 0 4 0 4 0 0 0 0 r / ~ I 4 4 4 4 4 4 4 4 4 4 4

R/I I - Kr2 2 2 4 4 2 " 3 4 4 2 1 Recombinants

2/3 2 R/1l-Kr3 2 2 4 3 2/3 2 4 4 2/3 1 R / I I-Kr4 3 2 1 1 o/ 1 2 2 1 2 2 3/4 I

Rpl Specificity Changes

TABLE 6 Flanking restriction fragment length polymorphism marker genotypes

of R#d derivatives with altered resistance specificities

RFLP marker

Rpl derivatives Proximal Distal

Rpl-K X rpl Rpl-Krl Rpl-K [285; S; 8Kb] Rpl-K [304; P; 20 Kb] Rpl-Kr2 Rpl-K [285; S; 8 Kb] rpl [304; P; 3 Kb] Rpl -Kr? Rpl-K [285; S; 8 Kb] rpl [304; P; 3 Kb] Rpl -Kr4 rpl [285; S; 6 Kb] Rpl-K [304; P; 20 Kb] Rpl -Kr5 rpl [285; S; 6 Kb] rpl [304; P; 3 Kb]

Rpl-Ir2 rpl [285; S; 9 Kb] Rpl-I [304; H; 15 kb]

Rpl-KrlJ4 Rpl-J [285; S 7, 4 Kb] Rpl-J [3.04, S, 7.5 Kb] Rpl-KrlJ92 Rpl-J [285; S 7, 4 Kb] Rpl-Krl [304; S; 6 Kb] Rpl-KrlJ15 Rpl-Krl [285; S; 8 Kb] Rpl-Krl [304; S; 6 Kb]

Rpl-I x rpl

Rpl-J X Rpl-Krl

Rpl-D X Rpl-I" Rpl-DIl Rpl-D [kt&; H; 2, 3, 15 Kb] Rpl-Z [304; H; 15 Kb] Rpl-DI28 Rpl-D [k&; H; 2, 3, 15 Kb] Rpl-Z [304; H; 15 Kb]

Rpl-B X Rpl-I" Rpl-B12 Rpl-I [285; H; 8 Kb] Rpl-B [304; H; 4 Kb]

Rpl-E X Rpl-F Rpl -EF25 Rpl-F [kst&; H; 3, 5 , 8, 9 Kb] Rpl-E [20075; S; 6 Kb]

"Values are parents with recombinants indented below. Two recombinants were identified in the Rpl-D X Rpl-Z cross from 4202 test cross progeny in which no completely susceptible individuals were found.

Indicates the parental allele that the recombinant carries at the proximal or distal RFLP markers. Informa- tion in brackets refers to the probe used (285 = npi285, 304 = bn13.04 and 20075 = php20075), the enzyme used (H = HindIII, S = ScuI and P = PstI) and the approximate size of the fragment or fragments characteristic of that allele. For some recombinants, such as those from the Rpl-K X rpl cross, both ksu? and npi285 were scored as proximal markers.

Recombinants from the Rpl-B X Rpl-land Rpl-E X Rpl-Fcrosses were previously published (HULBERT and BENNETZEN 1991; HULBERT et u1. 1993). Susceptible recombinants from the Rpl-E/Rpl-F cross (Table 1) had identical flanking marker genotypes to Rpl-EE5.

379

Genetic basis of changes in specificity at Rpl: Analy- sis of recombinants in the Rpl complex with 11 rust bio- types identified 13 variants with a pattern of resistances across the biotypes that were different from either par- ent. Four of these recombinants (Rpl-Kr2, Rpl-Kr3, Rpl- Kr-f and Rpl-Ir2) were resistant to at least one rust bio- type that neither parent showed resistance and repre- sent novel resistance genes (Table 5). Flanking marker analysis indicated that the novel genes arose by cross- over events and could perhaps be representative of an intragenic crossover event.

The nine other Rpl derivatives had altered specifici- ties that provided a pattern of resistance to rust biotypes that appeared to be a subset of the resistances shown by the parental genes (Table 4). Their phenotype is consistent with what would be expected if they arose by a intergenic event separating two parental resistance genes (Figure 2B). Eight of these variants were resistant to a subset of the biotypes that one of their parents was resistant to, whereas Rpl-KrlJ15 appeared to have inherited parts of the resistances present in both par- ents. The occurrence of five of these was associated with an exchange of flanking markers, whereas the other

four were not (Table 6), suggesting they arose by a mechanism other than crossing over, such as gene con- version. Previous analyses of test cross progeny from certain heterozygotes, such as Rpl-J,/Rpl-F and Rpl-J/ Rpl-C, found that roughly 25% of the derivatives were not associated with crossing over. The noncrossover types included susceptible individuals and those with the combined resistance of both parents. It was there- fore concluded that the noncrossover derivatives proba- bly arose from gene conversion type events, because mutation or intrachromosomal crossover events wound be unable to account for resistant types.

Efficient detection of novel specificities: Convincing demonstration of specificity changes that are the result of the creation of novel resistance genes (Figure 2A) and not simply the separation of linked parental genes (Figure 2B) requires testing the derivatives for resis- tance to biotypes that are virulent on both parents. Only two biotypes (IN3 and GA2) were available in the pres- ent study that were virulent on the Rpl-Ior Rpl-Klines, which were used in the crosses from which novel speci- ficities were derived. If the IN3 biotype had not been available, there would be evidence for only one novel

380 T. E. Richter et al.

A Crealh of a new spedficity by intragenic recombhation 1 (;ross-over

R p I - X I R p 1-2

a v - - 2 Gene conversion

R separaabn of two dstinct genes by intergenic recombinatidn

1 cfoss-over

2 Genemversbtl R p l - X R p 1- Y

C. combhation of two dsthct genes by htergeric recombhation

1 cross-over rpl R U

R p l - X e- 2 Geneconversion

FIGURE 2.-Possible mechanisms by which apparent changes in specificity can occur at complex disease resistance loci by crossing over or gene conversion. Boxes represent Rpl resistance genes. Genes designated Rpl provide resistance to one or more biotypes in the current collection. Genes desig- nated rpl represent undetectable or silent alleles. (A) Intra- genic recombination (crossing over or conversion) leading to genes with a new specificities. Novel resistance genes can result from recombination between detectable and silent genes or, presumably, between two detectable genes. (B) In- tergenic recombination can generate nonparental specifici- ties that retain resistance to some rust biotypes if one of the parental specificities is controlled by two detectable resistance genes. (C) Intergenic recombination can generate double- resistant types, with two detectable resistance genes.

resistance gene (Rpl-Kr4). All other novel resistances would have appeared as subsets of one of the parental resistances. This illustrates the importance of a collec- tion of genetically diverse pathogen biotypes when studying specificity changes.

Changes in spectrum of resistances have also been identified when derivatives from flax hybrids that were heterozygous at the L locus were analyzed with a variety of flax rust biotypes (ISLAM et al. 1989; ISLAM and SHEP- HERD 1991a,b). Some of these resistances were modifi- cations of the parental types and probably represent similar events to those reported here for the Rpl com- plex. It was not clear, however, if these altered specifici-

ties represented novel L alleles because they were not demonstrated to be resistant to any rust biotype that was virulent on both parents.

The selection of novel specificities from previously characterized resistance genes, or gene combinations, could have utility in breeding programs where resis- tance genes are not available to control certain patho- gen races. This is illustrated by the Rpl-Kr4 specificity that was resistant to both rust biotypes that were virulent on the parental Rpl-K gene. Generation of new resis- tance genes from lines that are in an agriculturally adapted background has obvious advantages for breed- ing programs over screens of unadapted germplasm. This should allow for more efficient transfer of the gene into agronomically acceptable cultivars by classical breeding techniques and minimize linkage drag of un- desirable alleles.

Whether generating novel resistance alleles for breeding programs or experimental purposes, the effi- ciency with which they can be identified depends both on the genotype of the cross and the pathogen biotype used in the screen. It may be difficult to predict which crosses will generate new resistance genes most fre- quently. Different Rpl homozygotes and heterozygotes have been observed to vary in their tendency to recom- bine. Rpl-Kwas previously considered one of the most stable Rpl alleles, both as a homozygote (BENNETZEN et al. 1988) or in heterozygous combinations (HULBERT and BENNETZEN 1991; HULBERT et al. 1993). It was there- fore surprising that five recombinants with altered re- sistances, representing at least three different specifici- ties, were recovered from only 2701 R p l - K / q l test cross progeny. The results indicate that even the most stable Rpl genes may be found to be very recombinagenic in certain heterozygous combinations. Perhaps the most feasible way to identify novel specificities is to test prog- eny from many crosses, as opposed to very large num- bers from a single cross. Our results indicate that selec- tion of individuals with altered resistance phenotypes when compared with the parents may be more produc- tive than selecting individuals that have completely lost resistance to the biotype being used in the screen.

Changes in resistance gene expression due to trans- poson insertion: Most of the susceptible lines that were derived from Rpl homozygotes in transposable element backgrounds were likely due to recombination events, as opposed to transposon insertion (PRYOR 1993). It was not possible to assay the involvement of crossing over in the generation of these lines because they were derived from Rpl homozygotes in which the flanking markers were homozygous. One of these lines was unique, however, in that it was originally identified by its partial resistance and was subsequently demon- strated to be unstable in a background carrying the transposable element Ac but stable in the absence of Ac (PRYOR 1993). The mutant allele, Rpl-D5, therefore, has the genetic characteristics of a mutation caused by

Rpl Specificity Changes 381

a Ds insertion. The Rpl-D5 line was also found to be unique in the present analysis. It was the only line that gave the same race specificity as the parental line but showed lower levels of resistance to most rust biotypes that the parental line was resistant to. This observation is compatible with the hypothesis that the transposon insertion caused a reduced expression of the Rpl-D gene, which in turn reduced the level of resistance to some biotypes to the extent that limited sporulation is observed. The unique properties of this allele also sup- port the idea that it originated by an event not standard to the Rpl region: transposon insertion (PRYOR 1987, 1993). Alternatively, the unique properties of Rpl-D5 could be due to the partial inactivation of a single Rpl linked locus that is required for the function of several Rpl genes. Such a locus has recently been identified that is linked to the Pto resistance gene and is required both for resistance to Pseudomonas syringae and sensitiv- ity to fenthion (SALMERON et al. 1994).

This work was supported in part by a grant from the U S . Depart- ment of Agriculture (91-37303-5883) and from contribution 95- 460-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan. We are grateful to K. SIMCOX and P. PETERSON for supplying rust biotypes.

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