Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.)

Nineteen novel full-ORF α-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical α-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced α-gliadins demonstrated that 14 of these contained 1–5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 α-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all α-gliadins had high content of β-strands and most of the α-helixes and β-strands were present in two unique domains. Phylogenetic analysis demonstrated that α-gliadin genes had a high homology with γ-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five α-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the α-gliadin genes can express in a high level under the control of T₇ promoter.     

Molecular characterisation of the low-molecular weight glutenin subunit genes of tall wheatgrass and functional properties of one clone Ee34

Wild tall wheatgrass (Lophopyrum elongatum L., 2x = 14) is an important resource for improving bread wheat (Titicum aestivum L.), including HMW-GS and LMW-GS relevant to end-use quality of the wheat flour. A set of 14 distinct sequences were amplified from the genomic DNA of the tall wheatgrass, using degenerate primers targeted at Glu-3, the locus containing the genes encoding the low-molecular weight glutenin subunits (LMW-GS). Three sequences contained an internal stop codon and were classified as pseudogenes. The other 11 all consisted of a single intron-less intact open-reading frame. An alignment of deduced protein sequences showed that the primary structure of all 11 sequences was similar to that of wheat and other wheat-related grass Glu-3 genes. All 11 sequences carried the 14 amino acid residue N-terminal motif MESNIIISFLK/RPWL, and were classified as LMW-m genes, based on the identity of the first amino acid of the mature protein. All but one of the sequences contained seven cysteine residues (the exception had 6). Their repetitive domain differs significantly from that present in Glu-3 genes isolated from the close relative intermediate wheatgrass (Thinopyrum Intermedium, 6x). A phylogenetic analysis showed that the tall wheatgrass sequences were closely related to those of the intermediate wheatgrass, but only distantly so to those from decaploid tall wheatgrass. One of the 11 LMW-GS peptides with a free-cysteine residue was heterologously expressed in E. coli and purified in sufficient scale to perform a flour supplementation test. This showed that the dough strength of bread wheat flour was significantly increased by the presence of the tall wheatgrass LMW-GS.     

Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli–tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.     

PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of novel low-molecular-weight glutenin genes inAegilops longissima

Extensive genetic variations of low-molecular-weight glutenin subunits (LMW-GS) and their coding genes were found in the wild diploid A- and D-genome donors of common wheat. In this study, we reported the isolation and characterization of 8 novel LMW-GS genes fromAe.longissima Schweinf. & Muschl., a species of the sectionSitopsis of the genusAegilops, which is closely related to the B genome of common wheat. Based on the N-terminal domain sequences, the 8 genes were divided into 3 groups. A consensus alignment of the extremely conserved domains with known gene groups and the subsequent cluster analysis showed that 2 out of the 3 groups of LMW-GS genes were closely related to those from the B genome, and the remaining was related to those from A and D genomes of wheat andAe. tauschii. Using 3 sets of gene-group-specific primers, PCRs in diploid, tetraploid and hexaploid wheats andAe. tauschii failed to obtain the expected products, indicating that the 3 groups of LMW-GS genes obtained in this study were new members of LMW-GS multi-gene families. These results suggested that theSitopsis species of the genusAegilops with novel gene variations could be used as valuable gene resources of LMW-GS. The 3 sets of group-specific primers could be utilized as molecular markers to investigate the introgression of novel alien LMW-GS genes fromAe. longissima into wheat.     

Molecular markers for tracking variation in lipoxygenase activity in wheat breeding

Lipoxygenase (LOX) activity is an important factor determining the color of flour and end-use products of wheat. In the present study, quantitative trait loci (QTL) for LOX activity in common wheat were mapped using 71 doubled haploid (DH) lines derived from a Zhongyou 9507 × CA9632 cross, and SSR markers. Two QTL, QLpx.caas.1AL and QLpx.caas-4B, were identified on chromosomes 1AL and 4B, closely associated with LOX activity. The SSR loci Xwmc312 and Xgwm251 proved to be diagnostic and explained 13.4–25.2% of the phenotypic variance for the 1AL locus and 14.3–27.0% for the 4B locus across four environments. The SSR markers Xgwm251 and Xwmc312 were validated across 198 Chinese wheat cultivars and advanced lines and showed highly significant (P < 0.01) association with LOX activity. We further established a multiplexed PCR with SSR marker combination Xwmc312/Xgwm251 to test these wheat cultivars and advanced lines. The results suggested that the marker combination Xwmc312/Xgwm251 is efficient and reliable for evaluating LOX activity and can be used in marker-assisted selection (MAS) for targeting flour color attributes to noodle and other wheat-based products.     

Length variation of i-type low-molecular-weight glutenin subunit genes in diploid wheats

Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei’s genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei’s genetic variation index of 0.806 and 0.783, respectively. Less variation was detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variation found in this study could be used as valuable source for the enrichment of genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted. This article was submitted by the authors in English.     

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.     

A Novel STS Marker for Polyphenol Oxidase Activity in Bread Wheat

The enzyme activity of polyphenol oxidase (PPO) in grain has been related to undersirable brown discoloration of bread wheat (Triticum aestivum L.) based end-products, particularly for Asian noodles. Breeding wheat cultivars with low PPO activity is the best approach to reduce the undesirable darkening. Molecular markers could greatly improve selection efficiency in breeding programs. Based on the sequences of PPO genes (GenBank Accession Numbers AY596268, AY596269 and AY596270) conditioning PPO activity during kernel development, 28 pairs of primers were designed using the software ‘DNAMAN’. One of the markers from AY596268, designated as PPO18, can amplify a 685-bp and an 876-bp fragment in the cultivars with high and low PPO activity, respectively. The difference of 191-bp size was detected in the intron region of the PPO gene. The STS marker PPO18 was mapped to chromosome 2AL using a DH population derived from a cross Zhongyou 9507× CA9632, a set of nulli-tetrasomic lines and ditelosomic line 2AS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the STS marker PPO18 and is closely linked to Xgwm312 and Xgwm294 on chromosome 2AL, explaining 28–43% of phenotypic variance for PPO activity across three environments. A total of 233 Chinese wheat cultivars and advanced lines were used to validate the correlation between the polymorphic fragments of PPO18 and grain PPO activity. The results showed that PPO18 is a co-dominant, efficient and reliable molecular marker for PPO activity and can be used in wheat breeding programs targeted for noodle quality improvement.     

Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.     

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat were used for callus induction on media containing four different doses (0, 1, 2 and 4 mg L^?1) of 2,4-D and dicamba supplemented with five different boron concentrations (0, 6.2, 12.4, 24.8, and 37.2 mg L^?1). The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L^?1) different BAP doses with five boron concentrations for further regeneration. The maximum callus weight in einkorn wheat was in culture media with 1 mg L^?1 dicamba and 6.2 mg L^?1 (3.71?±?0.13 g). Bread wheat had the maximum callus weight on culture media with 4 mg L^?1 dicamba and 12.4 mg L^?1 (3.46?±?0.40 g). The highest plantlet numbers were in only 2 mg L^?1 BAP (2.92?±?0.88) for einkorn wheat and 0.5 mg L^?1 BAP supplemented with 6.2 mg L^?1 boron (3.71?±?1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses expected to be useful for future wheat breeding studies.

     

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5Mg of Aegilops geniculata

Next‐generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole‐genome shotgun sequencing is cost‐prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow‐sorted chromosome 5M^g from a wheat/Aegilops geniculata disomic substitution line [DS5M^g (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired‐end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M^g, in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single‐gene FISH indicated no major chromosomal rearrangements between chromosomes 5M^g and 5D. Comparing chromosome 5M^g with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M^g‐specific SNPs and cytogenetic probe‐based resources were developed and validated. Deletion bin‐mapped and ordered 5M^g SNP markers will be useful to track 5M‐specific introgressions and translocations. This study provides a detailed sequence‐based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

Cereal asparagine synthetase genes

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (nonprotein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high‐temperature cooking and processing. Here we review publicly available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dIstachyon) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species.     

The in silico identification and characterization of a bread wheat/Triticum militinae introgression line

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross‐bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.     

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1^t and 1Dx2^t) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.     

Microsatellite-based molecular diversity of bread wheat germplasm and association mapping of wheat resistance to the Russian wheat aphid

Genetic diversity of a set of 71 wheat accessions, including 53 biotype 2 Russian wheat aphid (RWA2)-resistant landraces and 18 RWA2 susceptible accessions, was assessed by examining molecular variation at multiple microsatellite (SSR) loci. Fifty-one wheat SSR primer pairs were used, 81 SSR loci were determined, and 545 SSR alleles were detected. These SSR loci covered all the three genomes, 21 chromosomes, and at least 41 of the 42 chromosome arms. Diversity values averaged over SSR loci were high with mean number of SSR alleles/locus = 6.7, mean Shannon’s index (H) = 1.291, and mean Nei’s gene diversity (He) = 0.609. The three wheat genomes ranked as A > D > B and the homoeologous groups ranked as 7 > 3  > 1 > 2 > 6 > 5 > 4 based on the number of alleles per locus. Xgwm136 on chromosome arm 1AS is the most polymorphic SSR locus with the largest number of observed and effective alleles and the highest H and He. Among all 2485 pairs of wheat accessions, genetic distance (GD) ranged from 0.054 to 1.933 and averaged 0.9832. A dendrogram based on GD matrix showed that all the wheat accessions could be grouped into distinct clusters. Most of the susceptible cultivars (13/18) were clustered into groups that contains all or mostly susceptible accessions. Most of the U.S. cultivars belong to a group that is distinguishable from all the different RWA2 resistant groups. Diversity analysis was also conducted separately for subgroups containing 53 RWA2-resistant accessions and 18 RWA2-susceptible accessions. Association mapping revealed 28 SSR loci significantly associated with leaf chlorosis, and 8 with leaf rolling. New chromosome regions associated with RWA2 resistance were detected, and indicated existence of new RWA resistance genes located on chromosomes of all other homoeologous groups in addition to the groups 1 and 7 in bread wheat. This information is helpful for development of mapping populations for RWA2 resistance genes from different phylogenetic groups, and for wise utilization of the RWA-resistant germplasm in wheat breeding programs.