Identification and Mapping of Adult Plant Resistance Loci to Leaf Rust and Stripe Rust in Common Wheat Cultivar Kundan.

Leaf rust (LR) and stripe rust (YR) are important diseases of wheat worldwide. We used 148 recombinant inbred lines (RIL) from the cross of Avocet × Kundan for determining and mapping the genetic basis of adult plant resistance (APR) loci. The population was phenotyped LR and YR for three seasons in field trials conducted in Mexico and genotyped with the diversity arrays technology sequencing (DArT-Seq) and simple sequence repeat markers. The final genetic map was constructed using 2,937 polymorphic markers with an average distance of 1.29 centimorgans between markers. Inclusive composite interval mapping identified two co-located APR quantitative trait loci (QTL) for LR and YR, two LR QTL, and three YR QTL. The co-located resistance QTL on chromosome 1BL corresponded to the pleiotropic APR gene Lr46/Yr29. QLr.cim-2BL, QYr.cim-2AL, and QYr.cim-5AS could be identified as new resistance loci in this population. Lr46/Yr29 contributed 49.5 to 65.1 and 49.2 to 66.1% of LR and YR variations, respectively. The additive interaction between detected QTL showed that LR severities for RIL combining four QTL ranged between 5.3 and 25.8%, whereas the lowest YR severities were for RIL carrying QTL on chromosomes 1BL + 2AL + 6AL. The high-density DArT-Seq markers across chromosomes can be used in fine mapping of the targeted loci and development SNP markers.

Yr60, a Gene Conferring Moderate Resistance to Stripe Rust in Wheat.

Stripe rust, caused by Puccinia striiformis f. sp. tritici W., is a devastating disease of wheat worldwide. A new stripe rust resistance gene with moderate seedling and adult plant resistance was mapped using an F5 recombinant inbred line (RIL) population developed from the cross of the resistant parent 'Almop' with the susceptible parent 'Avocet'. The parents and RILs were phenotyped for seedling stripe rust response variation in a greenhouse and in field trials at Toluca, Mexico for 2 years. Almop showed moderate levels of resistance at both seedling and adult plant stages compared with the highly susceptible response of Avocet. The distribution of homozygous resistant, homozygous susceptible, and segregating RILs conformed to segregation at a single locus. Seedlings and adult plant responses were correlated, indicating that the same gene conferred resistance at both stages. A bulk segregant analysis approach with widely distributed simple sequence repeat (SSR) markers mapped the resistance gene to the distal region of the long arm of chromosome 4A. The SSR marker wmc776 cosegregated with this gene, whereas markers wmc219 and wmc313 were tightly linked and both located at 0.6 centimorgans. The resistance locus was designated Yr60.

A multiple resistance locus on chromosome arm 3BS in wheat confers resistance to stem rust (Sr2), leaf rust (Lr27) and powdery mildew.

Sr2 is the only known durable, race non-specific adult plant stem rust resistance gene in wheat. The Sr2 gene was shown to be tightly linked to the leaf rust resistance gene Lr27 and to powdery mildew resistance. An analysis of recombinants and mutants suggests that a single gene on chromosome arm 3BS may be responsible for resistance to these three fungal pathogens. The resistance functions of the Sr2 locus are compared and contrasted with those of the adult plant resistance gene Lr34.

An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat.

The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis Pers. f. sp. tritici) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurately predicts Sr2 in diverse wheat germplasm. Using DNA sequence derived from the vicinity of the Sr2 locus, we developed a cleaved amplified polymorphic sequence (CAPS) marker that is associated with the presence or absence of the gene in 115 of 122 (95%) diverse wheat lines. The marker genotype predicted the absence of the gene in 100% of lines which were considered to lack Sr2. Discrepancies were observed in lines that were predicted to carry Sr2 but failed to show the CAPS marker. Given the high level of accuracy observed, the marker provides breeders with a selection tool for one of the most important disease resistance genes of wheat.

Fine scale genetic and physical mapping using interstitial deletion mutants of Lr34 /Yr18: a disease resistance locus effective against multiple pathogens in wheat.

The Lr34/Yr18 locus has contributed to durable, non-race specific resistance against leaf rust (Puccinia triticina) and stripe rust (P. striiformis f. sp. tritici) in wheat (Triticum aestivum). Lr34/Yr18 also cosegregates with resistance to powdery mildew (Pm38) and a leaf tip necrosis phenotype (Ltn1). Using a high resolution mapping family from a cross between near-isogenic lines in the "Thatcher" background we demonstrated that Lr34/Yr18 also cosegregated with stem rust resistance in the field. Lr34/Yr18 probably interacts with unlinked genes to provide enhanced stem rust resistance in "Thatcher". In view of the relatively low levels of DNA polymorphism reported in the Lr34/Yr18 region, gamma irradiation of the single chromosome substitution line, Lalbahadur(Parula7D) that carries Lr34/Yr18 was used to generate several mutant lines. Characterisation of the mutants revealed a range of highly informative genotypes, which included variable size deletions and an overlapping set of interstitial deletions. The mutants enabled a large number of wheat EST derived markers to be mapped and define a relatively small physical region on chromosome 7DS that carried Lr34/Yr18. Fine scale genetic mapping confirmed the physical mapping and identified a genetic interval of less than 0.5 cM, which contained Lr34/Yr18. Both rice and Brachypodium genome sequences provided useful information for fine mapping of ESTs in wheat. Gene order was more conserved between wheat and Brachypodium than with rice but these smaller grass genomes did not reveal sequence information that could be used to identify a candidate gene for rust resistance in wheat. We predict that Lr34/Yr18 is located within a large insertion in wheat not found at syntenic positions in Brachypodium and rice.

Molecular genetic characterization of the Lr34/Yr18 slow rusting resistance gene region in wheat.

Wheat expressed sequence tags (wESTs) were identified in a genomic interval predicted to span the Lr34/Yr18 slow rusting region on chromosome 7DS and that corresponded to genes located in the syntenic region of rice chromosome 6 (between 2.02 and 2.38 Mb). A subset of the wESTs was also used to identify corresponding bacterial artificial chromosome (BAC) clones from the diploid D genome of wheat (Aegilops tauschii). Conservation and deviation of micro-colinearity within blocks of genes were found in the D genome BACs relative to the orthologous sequences in rice. Extensive RFLP analysis using the wEST derived clones as probes on a panel of wheat genetic stocks with or without Lr34/Yr18 revealed monomorphic patterns as the norm in this region of the wheat genome. A similar pattern was observed with single nucleotide polymorphism analysis on a subset of the wEST derived clones and subclones from corresponding D genome BACs. One exception was a wEST derived clone that produced a consistent RFLP pattern that distinguished the Lr34/Yr18 genetic stocks and well-established cultivars known either to possess or lack Lr34/Yr18. Conversion of the RFLP to a codominant sequence tagged site (csLV34) revealed a bi-allelic locus, where a variant size of 79 bp insertion in an intron sequence was associated with lines or cultivars that lacked Lr34/Yr18. This association with Lr34/Yr18 was validated in wheat cultivars from diverse backgrounds. Genetic linkage between csLV34 and Lr34/Yr18 was estimated at 0.4 cM.

Resistance gene analogues of wheat: molecular genetic analysis of ESTs.

Using two divergent nucleotide binding site (NBS) regions from wheat sequences of the NBS-LRR (leucine rich repeat) class, we retrieved 211 wheat and barley NBS-containing resistance gene analogue (RGA) expressed sequence tags (ESTs). These ESTs were grouped into 129 gene sequence groups that contained ESTs that were at least 70% identical at the DNA level over at least 200 bp. Probes were obtained for 89 of these RGA families and chromosome locations were determined for 72 of these probes using nullitetrasomic Chinese Spring wheat lines. RFLP analysis of 49 of these RGA probes revealed 65 mappable polymorphic bands in the doubled haploid Cranbrook x Halberd wheat population (C x H). These bands mapped to 49 loci in C x H. RGA loci were detected on all 21 chromosomes using the nullitetrasomic lines and on 18 chromosomes (linkage groups) in the C x H map. This identified a set of potential markers that could be developed further for use in mapping and ultimately cloning NBS-LRR-type disease resistance genes in wheat.

Fine genetic mapping fails to dissociate durable stem rust resistance gene Sr2 from pseudo-black chaff in common wheat (Triticum aestivum L.).

The broad-spectrum stem rust resistance gene Sr2 has provided protection in wheat against Puccinia graminis Pers. f. sp. tritici for over 80 years. The Sr2 gene and an associated dark pigmentation trait, pseudo-black chaff (PBC), have previously been localized to the short arm of chromosome 3B. In a first step towards the positional-based cloning of Sr2, we constructed a high-resolution map of this region. The wheat EST (wEST) deletion bin mapping project provided tightly linked cDNA markers. The rice genome sequence was used to infer the putative gene order for orthologous wheat genes and provide additional markers once the syntenic interval in rice was identified. We used this approach to map six wESTs that were collinear with the physical order of the corresponding genes on rice chromosome 1 suggesting there are no major re-arrangements between wheat and rice in this region. We were unable to separate by recombination the tightly linked morphological trait, PBC from the stem rust resistance gene suggesting that either a single gene or two tightly linked genes control both traits.

Leaf tip necrosis, molecular markers and beta1-proteasome subunits associated with the slow rusting resistance genes Lr46/Yr29.

Resistance based on slow-rusting genes has proven to be a useful strategy to develop wheat cultivars with durable resistance to rust diseases in wheat. However this type of resistance is often difficult to incorporate into a single genetic background due to the polygenic and additive nature of the genes involved. Therefore, markers, both molecular and phenotypic, are useful tools to facilitate the use of this type of resistance in wheat breeding programs. We have used field assays to score for both leaf and yellow rust in an Avocet-YrA x Attila population that segregates for several slow-rusting leaf and yellow rust resistance genes. This population was analyzed with the AFLP technique and the slow-rusting resistance locus Lr46/Yr29 was identified. A common set of AFLP and SSR markers linked to the Lr46/Yr29 locus was identified and validated in other recombinant inbred families developed from single chromosome recombinant populations that segregated for Lr46. These populations segregated for leaf tip necrosis (LTN) in the field, a trait that had previously been associated with Lr34/Yr18. We show that LTN is also pleiotropic or closely linked to the Lr46/Yr29 locus and suggest that a new Ltn gene designation should be given to this locus, in addition to the one that already exists for Lr34/Yr18. Coincidentally, members of a small gene family encoding beta-1 proteasome subunits located on group 1L and 7S chromosomes implicated in plant defense were linked to the Lr34/Yr18 and Lr46/Yr29 loci.

Powdery mildew resistance and Lr34/Yr18 genes for durable resistance to leaf and stripe rust cosegregate at a locus on the short arm of chromosome 7D of wheat.

The incorporation of effective and durable disease resistance is an important breeding objective for wheat improvement. The leaf rust resistance gene Lr34 and stripe rust resistance gene Yr18 are effective at the adult plant stage and have provided moderate levels of durable resistance to leaf rust caused by Puccinia triticina Eriks. and to stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. These genes have not been separated by recombination and map to chromosome 7DS in wheat. In a population of 110 F(7) lines derived from a Thatcher x Thatcher isogenic line with Lr34/Yr18, field resistance to leaf rust conferred by Lr34 and to stripe rust resistance conferred by Yr18 cosegregated with adult plant resistance to powdery mildew caused by Blumeria graminis (DC) EO Speer f. sp. tritici. Lr34 and Yr18 were previously shown to be associated with enhanced stem rust resistance and tolerance to barley yellow dwarf virus infection. This chromosomal region in wheat has now been linked with resistance to five different pathogens. The Lr34/Yr18 phenotypes and associated powdery mildew resistance were mapped to a single locus flanked by microsatellite loci Xgwm1220 and Xgwm295 on chromosome 7DS.

The wheat D-genome HMW-glutenin locus: BAC sequencing, gene distribution, and retrotransposon clusters.

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5' end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5' to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters.

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii.

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2(t) and T2, and was considered as part of the same allele at the Glu-D (t) 1locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2(t) and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an omega-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of omega-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F(2)grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii ( Glu-D (t) 1 and Gli-D (t) 1) segregated as alleles of the Glu-D1 and Gli-D1loci of bread wheat. A new locus encoding the omega-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively.

Physical arrangement of retrotransposon-related repeats in centromeric regions of wheat.

Cereal centromeres commonly contain many repetitive sequences that are derived from Ty3/gypsy retrotransposon. FISH analysis using a large DNA insert library of wheat identified a 67-kb clone (R11H) that showed strong hybridization signals on the centromeres. The R11H clone contains Ty3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified. Subsequently, we isolated additional 23 large-insert clones which also contained the integrase and CCS1 sequences. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca. 55-kb interval on average. This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.

Highly recombinogenic regions at seed storage protein loci on chromosome 1DS of Aegilops tauschii, the D-genome donor of wheat.

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii, the diploid D-genome donor species of hexaploid wheat. Ae. tauschii was used to overcome some of the limitations commonly associated with molecular studies of wheat such as low levels of DNA polymorphism. Detection of multiple loci by most RFLP probes suggests that gene duplication events have occurred throughout this chromosomal region. Large DNA fragments isolated from a BAC library of Ae. tauschii were used to determine the relationship between physical and genetic distance at seed storage protein loci located at the distal end of chromosome 1DS. Highly recombinogenic regions were identified where the ratio of physical to genetic distance was estimated to be <20 kb/cM. These results are discussed in relation to the genome-wide estimate of the relationship between physical and genetic distance.

Resistance gene analogs within an introgressed chromosomal segment derived from Triticum ventricosum that confers resistance to nematode and rust pathogens in wheat.

A resistance (R) gene-rich 2S chromosomal segment from Triticum ventricosum contains a cereal cyst nematode (CCN; Heterodera avenae) R gene locus CreX and a closely linked group of genes (Sr38, Yr17, and Lr37) that confer resistance to stem rust (Puccinia graminis f. sp. tritici), stripe rust (P. striiformis f. sp. tritici), and leaf rust (P. recondita f. sp. tritici) when introgressed into wheat. The 2S chromosomal segment from T. ventricosum is further delineated in translocations onto chromosome 2A of bread wheat, where the rust genes are retained but not the CreX gene. Using these critical genetic stocks, we have isolated family members of R gene analogs that are associated with either the 2S segment from T. ventricosum carrying the CreX locus or the rust genes. Derivatives of the Cre3 candidate R gene sequence and a rice (Oryza sativa) R gene analog that mapped to the 2S homologous chromosome groups in wheat were used to isolate related gene sequences from T. ventricosum that contain a nucleotide binding site-leucine rich repeat domain. The potential of these gene sequences as entry points for isolating candidate genes or gene family members of the CreX or rust genes and their further applications to plant breeding are discussed.

Map-based cloning of a gene sequence encoding a nucleotide-binding domain and a leucine-rich region at the Cre3 nematode resistance locus of wheat.

The Cre3 gene confers a high level of resistance to the root endoparasitic nematode Heterodera avenae in wheat. A DNA marker cosegregating with H. avenae resistance was used as an entry point for map-based cloning of a disease resistance gene family at the Cre3 locus. Two related gene sequences have been analysed at the Cre3 locus. One, identified as a cDNA clone, encodes a polypeptide with a nucleotide binding site (NBS) and a leucine-rich region; this member of the disease resistance gene family is expressed in roots. A second Cre3 gene sequence, cloned as genomic DNA, appears to be a pseudogene, with a frame shift caused by a deletion event. These two genes, related to members of the cytoplasmic NBS-leucine rich repeat class of plant disease resistance genes were physically mapped to the distal 0.06 fragment of the long arm of wheat chromosome 2D and cosegregated with nematode resistance.

Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances.

Zhong 5 is a partial amphiploid (2n = 56) between Triticum aestivum (2n = 42) and Thinopyrum intermedium (2n = 42) carrying all the chromosomes of wheat and seven pairs of chromosomes from Th. intermedium. Following further backcrossing to wheat, six independent stable 2n = 44 lines were obtained representing 4 disomic chromosome addition lines. One chromosome confers barley yellow dwarf virus (BYDV) resistance, whereas two other chromosomes carry leaf and stem rust resistance; one of the latter also confers stripe rust resistance. Using RFLP and isozyme markers we have shown that the extra chromosome in the Zhong 5-derived BYDV resistant disomic addition lines (Z1, Z2, or Z6) belongs to the homoeologous group 2. It therefore carries a different locus to the BYDV resistant group 7 addition, L1, described previously. The leaf, stem, and stripe rust resistant line (Z4) carries an added group 7 chromosome. The line Z3 has neither BYDV nor rust resistance, is not a group 2 or group 7 addition, and is probably a group 1 addition. The line Z5 is leaf and stem rust resistant, is not stripe rust resistant, and its homoeology remains unknown.

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.

Identification of RFLPs flanking a scald resistance gene on barley chromosome 6.

We report the map location of the scald resistance gene, Rrs13, from wild barley (Hordeum vulgare ssp. spontaneum) with respect to RFLP loci on barley chromosome 6 (= 6H). The identification of two RFLP loci (Cxp3 and ABG458) flanking the Rrs13 locus will assist selection for the resistant allele in barley breeding programs.

Molecular-genetic maps for group 1 chromosomes of Triticeae species and their relation to chromosomes in rice and oat.

Group 1 chromosomes of the Triticeae tribe have been studied extensively because many important genes have been assigned to them. In this paper, chromosome 1 linkage maps of Triticum aestivum, T. tauschii, and T. monococcum are compared with existing barley and rye maps to develop a consensus map for Triticeae species and thus facilitate the mapping of agronomic genes in this tribe. The consensus map that was developed consists of 14 agronomically important genes, 17 DNA markers that were derived from known-function clones, and 76 DNA markers derived from anonymous clones. There are 12 inconsistencies in the order of markers among seven wheat, four barley, and two rye maps. A comparison of the Triticeae group 1 chromosome consensus map with linkage maps of homoeologous chromosomes in rice indicates that the linkage maps for the long arm and the proximal portion of the short arm of group 1 chromosomes are conserved among these species. Similarly, gene order is conserved between Triticeae chromosome 1 and its homoeologous chromosome in oat. The location of the centromere in rice and oat chromosomes is estimated from its position in homoeologous group 1 chromosomes of Triticeae.