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Published online 1 September 2007
Published in Crop Sci 47:1995-2003 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

A Cryptic Wheat–Aegilops triuncialis Translocation with Leaf Rust Resistance Gene Lr58

Vasu Kuraparthya, Shilpa Sooda,e, Parveen Chhunejab,e, Harcharan S. Dhaliwalc, Satinder Kaura, Robert L. Bowdend and Bikram S. Gilla,*

a Wheat Genetic and Genomic Resources Center, Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506
b Dep. of Plant Breeding, Genetics & Biotechnology, Punjab Agricultural Univ., Ludhiana, 141004, Punjab, India
c Dep. of Biotechnology, Indian Institute of Technology, Roorkee, 247667, Uttaranchal, India
d Plant Science and Entomology Research Unit, USDA-ARS, Kansas State Univ., Manhattan, KS 66506
e contributed equally to this work

* Corresponding author (bsgill{at}ksu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genes transferred to crop plants from wild species are often associated with deleterious traits. Using molecular markers, we detected a cryptic introgression with a leaf rust resistance gene transferred from Aegilops triuncialis L. into common wheat (Triticum aestivum L.). One agronomically desirable rust-resistant introgression line was selected and advanced to BC3F11 from a cross of hexaploid wheat and A. triuncialis. In situ hybridization using A. triuncialis genomic DNA as a probe failed to detect the alien introgression. The translocation line was resistant to the most prevalent races of leaf rust in India and Kansas. Genetic mapping in a segregating F2:3 population showed that the rust resistance was monogenically inherited. Homeologous group 2 restriction fragment length polymorphism markers XksuF11, XksuH16, and Xbg123 showed diagnostically polymorphic alleles between the resistant and susceptible bulks. The alien transfer originated from homeologous chromosome recombination. The A. triuncialis-specific alleles of XksuH16, XksuF11, Xbg123, and one simple sequence repeat marker Xcfd50 cosegregated with the rust resistance, suggesting that the wheat–A. triuncialis translocation occurred in the distal region of chromosome arm 2BL. This translocation was designated T2BS·2BL-2tL(0.95). The unique source and map location of the introgression on chromosome 2B indicated that the leaf rust resistance gene is new and was designated Lr58.

Abbreviations: BSA, bulk segregant analysis • CS, Chinese Spring • GISH, genomic in situ hybridization • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat

A Cryptic Wheat–Aegilops triuncialis Translocation with Leaf Rust Resistance Gene Lr58

Vasu Kuraparthya, Shilpa Sooda,e, Parveen Chhunejab,e, Harcharan S. Dhaliwalc, Satinder Kaura, Robert L. Bowdend and Bikram S. Gilla,*

a Wheat Genetic and Genomic Resources Center, Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506
b Dep. of Plant Breeding, Genetics & Biotechnology, Punjab Agricultural Univ., Ludhiana, 141004, Punjab, India
c Dep. of Biotechnology, Indian Institute of Technology, Roorkee, 247667, Uttaranchal, India
d Plant Science and Entomology Research Unit, USDA-ARS, Kansas State Univ., Manhattan, KS 66506
e contributed equally to this work

* Corresponding author (bsgill{at}ksu.edu).

Genes transferred to crop plants from wild species are often associated with deleterious traits. Using molecular markers, we detected a cryptic introgression with a leaf rust resistance gene transferred from Aegilops triuncialis L. into common wheat (Triticum aestivum L.). One agronomically desirable rust-resistant introgression line was selected and advanced to BC3F11 from a cross of hexaploid wheat and A. triuncialis. In situ hybridization using A. triuncialis genomic DNA as a probe failed to detect the alien introgression. The translocation line was resistant to the most prevalent races of leaf rust in India and Kansas. Genetic mapping in a segregating F2:3 population showed that the rust resistance was monogenically inherited. Homeologous group 2 restriction fragment length polymorphism markers XksuF11, XksuH16, and Xbg123 showed diagnostically polymorphic alleles between the resistant and susceptible bulks. The alien transfer originated from homeologous chromosome recombination. The A. triuncialis-specific alleles of XksuH16, XksuF11, Xbg123, and one simple sequence repeat marker Xcfd50 cosegregated with the rust resistance, suggesting that the wheat–A. triuncialis translocation occurred in the distal region of chromosome arm 2BL. This translocation was designated T2BS·2BL-2tL(0.95). The unique source and map location of the introgression on chromosome 2B indicated that the leaf rust resistance gene is new and was designated Lr58.

Abbreviations: BSA, bulk segregant analysis • CS, Chinese Spring • GISH, genomic in situ hybridization • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LEAF RUST or brown rust (caused by Puccinia triticina Eriks.) is one of the most common diseases affecting wheat production worldwide. Development and deployment of resistant cultivars has been the most successful, environmentally sound, and economically viable approach to combat leaf rust. Incorporating host genetic resistance to this pathogen into adapted elite germplasm lines is therefore a major objective of most wheat breeding programs. Numerous resistance genes have been identified and introgressed into released cultivars (McIntosh et al., 1995, 2005), yet the continuous emergence of new races of the pathogen has been a substantial challenge to breeders attempting to produce cultivars with durable resistance. Thus, it is necessary to continue to identify further sources of resistance and incorporate them into elite breeding lines. Wheat has a narrow genetic base, and its wild relatives can be used as a source of new genes for disease resistance (Dvorak, 1977; Sharma and Gill, 1983; Gale and Miller, 1987; Jiang et al., 1994; Friebe et al., 1996).

Several strategies have been used for transferring alien segments that are smaller than complete chromosome arms into wheat from nonprogenitor wild species. Sears (1956) used radiation treatment to transfer a leaf rust resistance gene (Lr9) from Aegilops umbellulata Zhuk. to wheat. Recently, Masoudi-Nejiad et al. (2002) exploited the action of gametocidal genes to transfer alien chromosome segments to wheat. Both ionizing radiation treatment and gametocidal genes induce random chromosome breakage and fusion of the broken segments, resulting in translocation chromosomes. The majority of translocations were between nonhomeologous chromosomes, which leads to duplication or deficiencies and thus were noncompensating and agronomically undesirable. Alien genes from nonprogenitor species have been transferred to wheat through induced homeologous chromosome pairing between wheat and alien chromosomes (see the reviews by Friebe et al., 1996; Jiang et al., 1994) by making the Ph1 gene ineffective. This was done either by using mutant or null alleles of the Ph1 gene (Sears, 1972, 1981) or by using PhI, an epistatic inhibitor of the Ph1 gene from Aegilops speltoides Tausch (Riley et al., 1968a, 1968b). These transfers were genetically compensating because they involved homeologous recombination. Their agronomic desirability, however, depended on the size of the alien segments transferred, which determined the degree of linkage drag (Jiang et al., 1994; Friebe et al., 1996). Small interstitial secondary recombinants could be isolated by further chromosome engineering using the primary recombinants (Sears, 1972, 1981; Lukaszewski, 2000, 2006). The identification and characterization of a cytologically undetectable primary recombinant, with a cryptic wheat–Aegilops geniculata Roth introgression, suggested that it is feasible to transfer small alien segments without linkage drag (Kuraparthy et al., 2007a).

Aegilops triuncialis L. (2n = 4x = 28, UtUtCtCt), a nonprogenitor tetraploid species, was found to be an excellent source of resistance to various pests and diseases (Dhaliwal et al., 1991; El Bouhssini et al., 1998; Romero et al., 1998; Harjit-Singh and Dhaliwal, 2000; Martin-Sanchez et al., 2003). Previously, rust resistance of A. triuncialis was transferred to wheat using the induced homeologous pairing effect of the PhI gene (Aghaee-Sarbarzeh et al., 2002). Genomic in situ hybridization (GISH) and simple sequence repeat (SSR) marker analysis identified only one leaf rust resistant wheat–A. triuncialis recombinant, consisting of most of the complete 5Ut chromosome with a small terminal segment derived from 5AS (Aghaee-Sarbarzeh et al., 2002).

Rust resistance of A. triuncialis also was transferred to wheat without inducing homeologous pairing between chromosomes of wheat and A. triuncialis (Harjit-Singh et al., 2000; Aghaee-Sarbarzeh et al., 2001). In one leaf rust resistant line, an introgressed A. triuncialis segment was identified on chromosome arm 4BS (Aghaee-Sarbarzeh et al., 2001).

We selected one leaf rust resistant introgression line derived from the wheat–A. triuncialis cross of Harjit-Singh et al. (2000) for further backcrossing, molecular characterization, and mapping of the alien introgression. We identified and mapped the cryptic wheat–A. triuncialis rust-resistant translocation using cytogenetic and molecular mapping in a segregating population.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
The introgression line was developed by crossing the susceptible hexaploid wheat cultivar WL711 with rust-resistant A. triuncialis (TA10438, PAU no. 3549) and backcrossing the resultant F1 with WL711 (Harjit-Singh et al., 2000; Aghaee-Sarbarzeh et al., 2001). Leaf rust resistant BC1F1 plants were selected, backcrossed further to WL711 and selfed to develop BC3F11 lines. In the BC2F1 and BC3F1 generations, leaf rust resistant plants with the full complement of wheat chromosomes were selected for further selfing. In the backcross and segregating generations, selection for rust resistance was made by screening the seedling progenies using the Indian races 77–5 (avirulent on plants with Lr9, Lr19, Lr24, and Lr25, and virulent on Lr1, Lr3, Lr10, Lr13, Lr15, Lr20, Lr23, Lr26, Lr30, Lr33, Lr36, Lr48 and Lr49) and 104–2 (avirulent for Lr9, Lr15, Lr19, Lr24, and Lr25, and virulent for Lr1, Lr3, Lr10, Lr13, Lr14, Lr16, Lr17, Lr18, Lr20, Lr23, and Lr26). The same plants were screened as adults under artificial rust epiphytotic conditions at Punjab Agricultural University, Ludhiana, India. The BC3F11 resistant introgression line with normal plant growth and development was selected and further screened for resistance to five U.S. leaf rust races (for virulence/avirulence formulae, see Long et al., 2000) at Kansas State University, Manhattan (Table 1 ).


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  		Table 1. Seedling{dagger} and adult plant{ddagger} reactions of TA5605 and parents to five races of leaf rust.

 
One leaf rust resistant wheat–A. triuncialis introgression line (TA5605), along with the original A. triuncialis accession (TA10438), ‘Chinese Spring’ (CS), and the parental cultivars WL711 and Jagger were used for cytogenetic and molecular genetic analysis.

The hard red winter wheat cultivar Jagger (seedling susceptible to U.S. leaf rust races PRTUS25 and MCDL) was crossed as a female with the introgression line (TA5605). A total of 118 F2 plants were used for genetic analysis and molecular mapping of leaf rust resistance. From each F2 plant, 18 to 20 F3 seedlings were screened for leaf rust reaction at the seedling stage. All the plants were grown in square pots filled with Scotts Metro Mix 200 (Scotts Miracle-Gro Co., Marysville, OH).

Screening the Plants for Leaf Rust Reaction
The seedling and adult reactions of the parental lines inoculated with the five leaf rust races are shown in Table 1. Rust inoculations, incubation of the infected plants, and rust scoring followed Browder (1971). All F2 plants, their parents, and line TA5605 were inoculated with race PRTUS25 at the two-leaf seedling stage to screen for segregation of rust reaction. For progeny testing, 18 to 20 F3 seedlings from each F2 plant were grown and screened with the same race.

Molecular Characterization and Mapping
Genomic in situ hybridization was used to determine the size of the alien introgression in line TA5605, as described in Zhang et al. (2001), using A. triuncialis genomic DNA as a probe and CS genomic DNA as a blocker.

Based on previous reports on the association of chromosomes 5A (Aghaee-Sarbarzeh et al., 2002) and 4B (Aghaee-Sarbarzeh et al., 2001) with A. triuncialis-derived rust resistance, we initially selected nine and 14 SSRs mapping on chromosome 5A and 4B of wheat, respectively, for characterizing the introgression line. Bulk segregant analysis (BSA) with distally mapped restriction fragment length polymorphism (RFLP) markers that detect orthologous alleles among the three genomes was then used to diagnostically identify markers and chromosomes associated with the rust resistance. Three DNA bulks each for resistant and susceptible phenotypes were made by pooling the DNA of 10 homozygous resistant and 10 susceptible F2 plants. These DNA bulks along with DNA from the susceptible cultivars WL711 and Jagger and TA5605 were digested with six restriction enzymes (BamHI, DraI, EcoRI, EcoRV, HindIII, and XbaI). The DNA isolation, Southern blotting, and hybridizations were as reported in Kuraparthy et al. (2007b). In the first attempt, a total of 17 RFLP markers mapping distally on homoeolgous groups 1 and 2 (Appels, 1997; Sharp, 1996; GrainGenes maps for wheat available at wheat.pw.usda.gov/GG2/maps.shtml#wheat [verified 6 July 2007]), were used for BSA. To physically characterize the wheat–alien translocation in line TA5605 and determine the translocation breakpoint with respect to the fraction length of CS deletion bins, 18 RFLP markers mapping distally in homeologous group 2 chromosomes of wheat (Delaney et al., 1995; Nelson et al., 1995; Dubcovsky et al., 1996; Sharp, 1996; Erayman et al., 2004) were used. A total of 34 SSRs physically or genetically mapped on the long arms of homeologous group 2 chromosomes (Roder et al., 1998; Sourdille et al., 2004; Somers et al., 2004) were used for molecular mapping of the rust-resistant introgression to a specific chromosome in TA5605. The RFLP and SSR markers diagnostically identifying the A. triuncialis segment in TA5605 were mapped in the F2 population to genetically map the leaf rust resistance.

Linkage Analysis
The computer program MAPMAKER (Lander et al., 1987) Version 2.0 for Macintosh was used to calculate linkage using the Kosambi mapping function (Kosambi, 1944) with a logarithm of odds threshold of 3.00.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rust Reaction of the Introgression Line
At the seedling stage, the introgression line showed a clear, hypersensitive resistant reaction (Fig. 1a ) to leaf rust races PRTUS6, PRTUS25, and MCDL, and a susceptible reaction to races PNMQ and PRTUS35 (Table 1). The recipient wheat cultivar WL711 was highly susceptible at the seedling stage (Table 1, Fig. 1a). Line TA5605 was resistant (Fig. 1b) to races PRTUS6, PRTUS25, MCDL, and PNMQ at the adult plant stage but was susceptible to race PRTUS35 (Table 1). Cultivar WL711 (having Lr13) was highly susceptible to all races of leaf rust except PNMQ (avirulent on Lr13) at the adult plant stage (Table 1).


Figure 1
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  		Figure 1. Rust resistance in introgression line TA5605 with T2BS.2BL-2tL(0.95): (a) leaf rust (race PRTUS25) reaction of the parents and TA5605 at the seedling stage; (b) leaf rust (race MCDL) reaction of the parents and TA5605 line at the adult stage.

 
Segregation for leaf rust reaction was analyzed by screening the F2 and F3 populations at the seedling stage using race PRTUS25. The F2 plants and progenies showed clearly different infection types for resistance (; to ;1C) and susceptibility (3+ to 4) (data not shown).

Genetic Analysis of Rust Resistance
The F2 population of 118 plants developed from Jagger x TA5605 segregated 86 resistant and 32 susceptible plants, a good fit for dominant monogenic (3:1) segregation. Progeny of these F2 plants when tested with race PRTUS25 gave 34 homozygous resistant, 52 heterogygous resistant, and 32 homozygous susceptible lines. This further indicated that leaf rust resistance in the introgression line was monogenically inherited.

Molecular Characterization of the Alien Introgression
No signal could be detected in the introgression line TA5605 when A. triuncialis DNA was used as a probe in the in situ hybridization experiments (data not shown). This indicated that the introgressed A. triuncialis chromatin in the leaf rust resistant translocation TA5605 was very small and cytologically undetectable.

None of the tested 23 SSRs of chromosomes 5A and 4B identified the A. triuncialis-specific introgression in the translocation line TA5605 (data not shown). This suggested that either the leaf rust resistant introgression in TA5605 is different from previous reports of Aghaee-Sarbarzeh et al. (2001, 2002) or the marker density might not be enough to detect the leaf rust resistant introgression of A. triuncialis in TA5605. Bulked segregant analysis of the homozygous resistant and susceptible F2 bulks was used for chromosome mapping and tagging of the leaf rust resistant introgression using distally mapped RFLP markers. Of the initial 17 RFLP markers for homeologous groups 1 and 2 used in the BSA, 13 were polymorphic between Jagger and TA5605 with one or more restriction enzymes. None of the seven polymorphic RFLP probes of homeologous group 1 chromosomes identified the diagnostic polymorphism between resistant and susceptible bulks. Probe KSUF11, mapped on homeologous group 2 chromosomes of wheat, identified diagnostically polymorphic alleles between the resistant and susceptible bulks. The polymorphic fragments detected by probe KSUF11 in the resistant bulks were specific to A. triuncialis with all the six enzymes used in the BSA (Table 2 ). This indicated the presence of A. triuncialis chromatin and its association with rust resistance in the translocation line TA5605.


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  		Table 2. Diagnostic restriction fragment length polymorphism marker patterns in the resistant and susceptible bulks and introgression line TA5605.

 
We further selected 18 additional RFLP markers that were genetically or physically mapped on the homeologous chromosome arm 2L for characterizing the translocation in TA5605. The diagnostic marker patterns of some of the informative probes are given in Table 2. From this set of markers, two RFLP probes KSUH16 and BG123 further showed A. triuncialis-specific diagnostic polymorphism between resistant and susceptible bulks with all six enzymes used (Table 2). Probe KSUH16 detected the replacement of one of the wheat group 2L chromosome alleles by the A. triuncialis homeologous chromosome (2t) in line TA5605 (Fig. 2 ). Furthermore, A. triuncialis-specific alleles of RFLP probes KSUH16, BG123, and KSUF11 cosegregated with leaf rust resistance in the F2 mapping population. This unequivocally indicated that the rust resistance of the translocation line TA5605 was derived from homeologous group 2 chromosomes of A. triuncialis, and the introgression occurred onto the homeologous chromosome arm 2L of wheat through homeologous recombination. The diagnostically polymorphic alleles between resistant and susceptible bulks generated by RFLP markers XksuD23, Xbcd410, and Xpsr609 were not specific to A. triuncialis (Table 2), suggesting that these markers were linked with the rust resistance and physically mapped proximal to the breakpoint of the wheat–A. triuncialis translocation.


Figure 2
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  		Figure 2. Molecular mapping of rust-resistant introgression T2BS·2BL-2tL(0.95) using bulked segregant analysis. Southern hybridization pattern of EcoRV-digested genomic DNA of parents and bulks from homozygous resistant and susceptible F2 plants using probe KSUH16. The restriction fragment length polymorphism fragments diagnostically polymorphic between resistant and susceptible bulks are indicated by arrows. The WL711 allele of XksuH16 replaced by an Aegilops triuncialis-specific allele is indicated with an asterisk.

 
The wheat–alien translocation in line TA5605 was physically characterized and the translocation breakpoint was determined with respect to the fraction length of the CS deletion bins based on the presence or absence of diagnostic polymorphisms between chromosomes 2tL of A. triuncialis and group 2 chromosomes of wheat using physically mapped RFLP markers. None of the markers, physically mapped in the deletion bins 2L-0.69–0.70, 2L-0.70–0.76, and 2L-0.76–0.85, diagnostically identified the A. triuncialis segments (Table 2, Fig. 3 ) in line TA5605. Only three (XksuH16, XksuF11, and Xbg123) out of 10 informative RFLP markers mapped in the deletion bin 2L-0.89–1.00 diagnostically identified the A. triuncialis-specific chromatin in translocation line TA5605. This suggested that the breakpoint of the translocation in line TA5605 was located in the deletion bin 2L-0.89–1.00 of the consensus physical map and that the size of the introgressed segment was <10% of the long arm of wheat chromosome 2L (Fig. 3). Because the deletion bin 2L-0.89–1.00 of the consensus map was the same as the deletion bin 2BL6-0.89–1.00 of 2B (Delaney et al., 1995), the introgressed segment is actually <10% of the long arm of wheat chromosome 2BL.


Figure 3
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  		Figure 3. Physical map of chromosome 2B of wheat and inferred genomic in situ hybridization, restriction fragment length polymorphism, and simple sequence repeat marker-based physical map of recombinant wheat–Aegilops triuncialis chromosomes 2B and 2t in WL711 background. In the inferred physical map of the introgression line T2BS·2BL-2tL(0.95), A. triuncialis 2t chromatin is indicated in gray. The solid black bands represent the C-banding pattern of chromosome 2B. The 2L consensus physical map was based on Delaney et al. (1995), Nelson et al. (1995), Dubcovsky et al. (1996), Sharp (1996), Sourdille et al. (2004), and Erayman et al. (2004). Markers used in the present study are shown in bold.

 
To identify and establish the specific wheat chromosome involved in the wheat–A. triuncialis translocation in TA5605, physically and genetically mapped homeologous group 2, chromosome-specific SSR markers were used. Only Xcfd50 of 34 SSR markers surveyed diagnostically identified the A. triuncialis-specific segment in TA5605. Marker Xcfd50 amplified a single, high-molecular-weight band specific to A. triuncialis in TA5605, and a low-molecular-weight band in Jagger. Molecular mapping in the F2 population showed that the A. triuncialis-specific allele of Xcfd50 cosegregated with the leaf rust resistance gene. Previously, Xcfd50 was mapped physically and genetically to the distal region of chromosome arms 2BL and physically in 2DL of wheat (Sourdille et al., 2004). To allocate the Xcfd50 allele associated with rust resistance to a specific homeologous group 2 chromosome, SSR markers were further used for molecular mapping. Of the 34 SSRs surveyed for polymorphism between TA5605 and Jagger, three were codominant, six were dominant, and 24 were not polymorphic. Four dominant (Xgwm365, Xgwm265, Xgwm501, and Xcfd267) and the three codominant SSR markers (Xgwm311, Xgwm294, and Xbarc76) and four RFLP markers (XksuD23, XksuH16, XksuF11, and Xbg123) were then mapped in the F2 population to identify linkage of the rust resistance gene with chromosome-specific SSRs. None of the SSRs specific to chromosome 2A (Xgwm365, Xgwm265, Xgwm294, or Xbarc76) or 2D (Xgwm311) showed linkage with the leaf rust resistance gene or with the diagnostic markers XksuF11, XksuH16, or Xbg123, thus suggesting that the diagnostically polymorphic allele of Xcfd50 was associated with chromosome 2B. Hence, the rust resistance gene from A. triuncialis in TA5605 was in chromosome arm 2BL. The identity of the A. triuncialis chromosome arm (2UtL or 2CtL) involved in the translocation T2BS·2BL-2tL(0.95) is unknown.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified and mapped a small alien translocation with a leaf rust resistance gene transferred from A. triuncialis to wheat without disruption of the normal bivalent pairing control. Because the Ph1 gene suppresses homeologous pairing between wheat and alien chromosomes, such a transfer of alien chromatin was unexpected. There are two possible mechanisms for the origin of wheat–alien translocations. One is through spontaneous breakage and reunion of wheat and alien chromosomes during introgressive hybridization. The second is through homeologous pairing and recombination between homeologous chromosomes. The molecular marker data showed that wheat homoeoloci were substituted by alien homoeoloci in a precise recombination-like manner (Fig. 2).

Spontaneous transfers due to a low level of pairing were frequently observed in hybrids of hexaploid wheat and A. triuncialis (Romero et al., 1998; Harjit-Singh et al., 1993), and of hexaploid or tetraploid wheat and A. peregrina (Hack.) Maire & Weiller (Yu et al., 1990; Spetsov et al., 1997). Although wide variation existed among various homeologous chromosomes and genotypes, meiotic pairing frequencies, as high as 80 to 85%, were observed in wheat–A. geniculata hybrids (Cifuentes et al., 2006). This low level of pairing in wheat–alien hybrids was used to transfer Hessian fly resistance (Martin-Sanchez et al., 2003) and cereal cyst nematode resistance (Romero et al., 1998) genes from A. triuncialis, and powdery mildew resistance (Spetsov et al., 1997) and root-knot nematode resistance (Yu et al., 1990) genes from A. peregrina to wheat. Such low levels of chromosome pairing in wheat wide crosses could be due to partial homology between wheat and alien chromosomes, or to ineffectiveness of the Ph1 gene in preventing homeologous chromosome pairing in the distal high recombination gene-rich regions of wheat. Romero et al. (1998) and Martin-Sanchez et al. (2003) speculated that transfers derived from wheat–A. triuncialis hybrids could be due to the ability of the C genome to suppress the Ph1 diploidization mechanism (Kimber and Feldman, 1987). Such an effect might not be the case in this study, however, because a low level of chromosome pairing was observed in the wheat–A. triuncialis hybrid originally used to transfer the rust resistance (Harjit-Singh et al., 2000). Previously, Jena et al. (1992) reported the spontaneous introgression of chromosomal segments conferring resistance to brown planthopper from Oryza officinalis Wall. ex G. Watt chromosomes to those of rice (O. sativa L.).

We identified one rust-resistant alien translocation line (TA5605) with a cytologically undetectable alien segment from A. triuncialis. Based on GISH and molecular mapping, we previously described a method for estimating the size of alien introgressions (Kuraparthy et al., 2007a). The translocation in the present study was described as T2BS·2BL-2tL(0.95). Because the specific homeologous group 2 chromosome of A. triuncialis involved in the translocation was unknown, we identified the donor chromosome as 2tL, where superscript t refers to an A. triuncialis chromosome. We termed this small introgression, undetected by cytological analysis, as a "cryptic alien introgression" (Kuraparthy et al., 2007a). The "cryptic" nature of the A. triuncialis introgression in T2BS·2BL-2tL(0.95) was supported by molecular mapping, where only three of 10 otherwise informative RFLPs in deletion bin 2BL6-0.89–1.00 diagnostically identified the A. triuncialis chromatin (Table 2, Fig. 3). An unconventional recombination mechanism was speculated for such introgressions in rice (Jena et al., 1992). It is not known if a cryptic alien introgression can occur in the absence of a chiasmatic meiotic association in wheat, but the precise exchange indicates a recombination-like event.

Previously, XksuH16 was placed in bin 2L-0.85–0.89 and XksuF41 was mapped in the distal deletion bin 2L-0.89–1.00 in a consensus physical map of homeologous group 2 chromosomes (Delaney et al., 1995). This order is highly unlikely because most of the genetic maps indicated that XksuF41 was proximal to XksuH16 (Nelson et al., 1995; Gill et al., 1991; Gale and Miller, 1987; Sharp, 1996) and XksuH16 incorrectly was placed in the deletion bin apparently due to the lack of polymorphism between homoeoalleles in the physical mapping experiments of Delaney et al. (1995). The absence of diagnostically polymorphic alleles between the resistant and susceptible bulks for XksuF41 and the identification of A. triuncialis-specific alleles by XksuH16, and by the most terminally mapped marker Xbg123 of Dubcovsky et al. (1996), suggest that the wheat–A. triuncialis introgression in TA5605 is a terminal transfer. Our results also suggest that XksuF41 should be proximal to XksuH16 and that XksuH16 is in deletion bin 2L-0.89–1.00.

Identification of cryptic alien introgressions with disease resistance from A. triuncialis in the present study and from A. geniculata reported previously (Kuraparthy et al., 2007a) suggest that it is feasible to transfer disease resistance genes with minimal linkage drag from wild species by selecting rust-resistant backcross derivatives with no obvious effect on plant growth, and by characterizing lines using GISH and terminally mapped molecular markers from genetic and physical maps. Using this strategy, we showed conclusively that cryptic wheat–alien introgressions with rust resistance can be produced in wheat.

Previously, for the detection and characterization of critical recombinants in targeted chromosome engineering, diagnostic cytological or molecular markers specific to the chromosome or chromosome arm targeted for alien gene transfer were used (Lukaszewski and Xu, 1995; Lukaszewski, 2000; Iqbal et al., 2000; Qi et al., 2007). Characterization and mapping of cytologically undetectable cryptic alien introgressions from wheat–alien direct crosses, however, require rapid and efficient strategies such as BSA (Michelmore et al., 1991). Using BSA, we not only quickly detected the wheat chromosome involved in TA5605, but also identified the A. triuncialis-specific segment associated with leaf rust resistance gene Lr58 in T2BS·2BL-2tL(0.95).

Cryptic wheat–alien introgressions, especially terminal segments, are the most desirable and feasible translocations for transferring disease resistance genes in wheat, because disease resistance genes are mostly located in the terminal recombination-rich regions of grass chromosomes (Leister et al., 1998). The physical localization of expressed resistance gene analogs on wheat chromosomes showed that about 75% of the R genes mapped in the distal 20% of the chromosomes; most of the wheat R genes were present in the telomeric or subtelomeric regions (Dilbirligi et al., 2004). This also was supported by physical mapping of linked markers and expressed sequence tags (ESTs) in a core set of CS deletion lines (Qi et al., 2004). In wheat, recombination also is unevenly distributed; 90% of the recombination occurs in the distal regions toward the telomeric ends of the chromosomes (Gill et al., 1993; Lukaszewski and Curtis, 1993; Lukaszewski, 1995). Furthermore, homeologous recombination appears to be highly localized and occurs distal to homologous recombination (Luo et al., 2000; Lukaszewski et al., 2003, 2005). Wheat–alien transfers were mostly derived from single crossover events. Only two breakpoints were detected by a single RFLP marker in a sample of eight wheat–rye (Secale cereale L.) recombinants for the 1RL arm of rye probed with 36 RFLP markers (Rogowsky et al., 1993). All recombination events were restricted to the distal 18% of the arm in wheat–wheatgrass [Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey] recombinants with wheat streak mosaic virus resistance (Qi et al., 2007). Kuraparthy et al. (2007a) provided further evidence for such transfers where the smallest wheat–A. geniculata cryptic terminal introgression with Lr57 and Yr40 was found to be <3.5% of the chromosome arm 5DS. Identification of such terminal single-breakpoint transfers needs molecular or cytological markers mapped at or near the telomeric ends of the wheat chromosomes. Physically and genetically mapped RFLPs and SSRs (wheat.pw.usda.gov/GG2/maps.shtml#wheat [verified 10 July 2007]) and bin-mapped EST markers (wheat.pw.usda.gov/NSF/project/mapping_data.html [verified 10 July 2007]) could be an ideal resource for such markers. The rice genomic sequence also may be useful to develop markers if the wheat–rice synteny is conserved in such regions.

Although the resistance gene Lr58 transferred from A. triuncialis to wheat in this study has not been deployed in any cultivar, virulence to this gene exists in races PNMQ and PRTUS35 (Table 1) of North America. These two races are virulent on Lr58 at both the seedling and the adult plant stages (Table 1). Such virulence to genes transferred to wheat from A. tauschii (Lr39 and Lr41) and Triticum monococcum L. (an unnamed gene in KS92WGRC23) has been detected before deployment of these genes in agriculture (Hussien et al., 1997; Raupp et al., 2001). In each of these cases, virulence was found in Puccinia triticina race PNMQ. Interestingly, the race PNMQ is also virulent to the genes Lr9 and Lr24 that were transferred to wheat from Aegilops umbellulata and Thinopyrum ponticum. The presence of virulence to new genes derived from wheat relatives before development of resistant cultivars will limit the usefulness of these genes unless they are deployed in combination with other effective genes for resistance to leaf rust. Identification of markers (SSR marker Xcfd50 and RFLP markers XksuH16, Xbg123, and XksuF11) linked to Lr58 provide a tool to incorporate this gene into pyramids that include other effective resistance genes.

Homeologous group 2 chromosomes of wheat contain at least 19 cataloged genes for leaf rust resistance. Except for Lr11 (2A) and Lr35 (2B), whose arm location is unknown, most of the resistance genes were mapped on the short arms of homeologous group 2 chromosomes (see www.ars.usda.gov/Main/docs.htm?docid=10342 [verified 10 July 2007]). Only three leaf rust resistance genes have been mapped to homeologous chromosome arm 2L of wheat, and all three were derived from wild related species. Resistance gene Lr38 mapped on chromosome 2AL was a noncompensating translocation from Agropyron intermedium (Host) P. Beauv. (Friebe et al., 1993). Resistance gene Lr54 mapped on 2DL was derived from a whole-arm translocation from Aegilops kotschyi Boiss. (Marais et al., 2005). Gene Lr50 mapped on 2BL was introgressed from Triticum timopheevii (Zhuk.) Zhuk. ssp. armeniacum (Jakubz.) Slageren (Brown-Guedira et al., 2003). Furthermore, chromosome arm 2L contains at least three stripe rust resistance genes (Yr5, Yr7, and Yr37) and five stem rust resistance genes (Sr9 allelic series, Sr16, Sr20, Sr21, and Sr28) (see http://www.ars.usda.gov/Main/docs.htm?docid=10342). In this study, mapping of the leaf rust resistance gene Lr58 in the distal region of chromosome arm 2BL suggests either the presence of conserved orthologous R loci in 2L or that the genomic region of 2L is rich in resistance genes. Precise genetic mapping using linked RFLP markers that produce orthologous alleles in the chromosome arms 2L is necessary to characterize such regions.


   ACKNOWLEDGMENTS
 
We thank Robert McIntosh for reviewing the manuscript and providing the new gene designation for the rust-resistant gene reported in this study. We extend a special note of thanks to Duane Wilson and Jon Raupp for their excellent technical assistance. We thank Bernd Friebe for the helpful discussions and Peng Zhang for her guidance in the GISH technique. Research supported by Kansas Wheat Commission and USDA special grant to WGGRC. This paper is Contribution no. 07-152-J from the Kansas Agricultural Experimentation Station, Kansas State University, Manhattan.


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Received for publication January 22, 2007.


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V. Kuraparthy, S. Sood, D. R. See, and B. S. Gill
Development of a PCR Assay and Marker-Assisted Transfer of Leaf Rust and Stripe Rust Resistance Genes Lr57 and Yr40 into Hard Red Winter Wheats
Crop Sci., January 28, 2009; 49(1): 120 - 126.
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