Evaluation of Polymorphism at Microsatellite Loci of Spring Durum Wheat (Triticum durum Desf.) Varieties and the Use of SSR-Based Analysis in Phylogenetic Studies

Polymorphism at 28 SSR loci was analyzed and described in 45 cultivars of spring durum wheat created in the former USSR and Russia during the last 80 years. Each cultivar was shown to have a unique allele combination. This allows SSR markers to be used to identify durum wheat varieties. Meanwhile, these markers can hardly be used to detect phylogenetic relationships among varieties and to specify their pedigrees, because genetic distances calculated on the basis of these markers do not correlate with the distance calculated by coefficient of parentage.     

Intravarietal heterogeneity of durum wheat is an important component of species biodiversity

Sixty-four durum wheat varieties of the domestic breeding (USSR and Russia) were studied for heterogeneity using various genetic markers: storage proteins (gliadins), RAPD, and microsatellite (SSR) markers. About a third of the studied varieties (24) were shown to be heterogeneous at the protein markers. These varieties contained from two to six biotypes. Using the molecular markers, the biotypes were found to differ not only in the gliadin-coding genes as determined with the protein markers, but also in other chromosome regions. Moreover, using SSR markers, some additional subbiotypes were detected within the biotypes defined with the gliadin markers. Thus, the intravarietal durum wheat heterogeneity is an important component of general species biodiversity.     

Intravarietal heterogeneity of durum wheat is an important component of the species biodiversity

Sixty-four durum wheat varieties of the domestic breeding (USSR and Russia) were studied for herogeneity using various genetic markers: storage proteins (gliadins), RAPD, and microsatellite (SSR) markers. About a third of the studied varieties (24) were shown to be heterogeneous at the protein markers. These varieties contained from two to six biotypes. Using the molecular markers, the biotypes were found to differ not only in the gliadin-coding genes as determined with the protein markers, but also in other chromosome regions. Moreover, using SSR markers, some additional subbiotypes were detected within the biotypes defined with the gliadin markers. Thus, the intravarietal durum wheat heterogeneity is an important component of general biodiversity of the species.     

An integrated DArT-SSR linkage map of durum wheat

Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.     

QTL mapping of resistance to race Ug99 of Puccinia graminis f. sp. tritici in durum wheat (Triticum durum Desf.)

Stem rust caused by Puccinia graminis f. sp. tritici was historically one of the most destructive diseases of wheat worldwide. The evolution and rapid migration of race TTKSK (Ug99) and derivatives, first detected in Uganda in 1999, are of international concern due to the virulence of these races to widely used stem rust resistance genes. In attempts to identify quantitative trait loci (QTL) linked with resistance to stem rust race Ug99, 95 recombinant inbred lines that were developed from a cross between two durum wheat varieties, Kristal and Sebatel, were evaluated for reaction to stem rust. Seven field trials at two locations were carried out in main and off seasons. In addition to the natural infection, the nursery was also artificially inoculated with urediniospores of stem rust race Ug99 and a mixture of locally collected stem rust urediniospores. A genetic map was constructed based on 207 simple sequence repeat (SSR) and two sequence tagged site loci. Using composite interval mapping, nine QTL for resistance to stem rust were identified on chromosomes 1AL, 2AS, 3BS, 4BL, 5BL, 6AL 7A, 7AL and 7BL. These results suggest that durum wheat resistance to stem rust is oligogenic and that there is potential to identify previously uncharacterized resistance genes with minor effects. The SSR markers that are closely linked to the QTL can be used for marker-assisted selection for stem rust resistance in durum wheat.     

Identification of Canadian durum wheat varieties using a single PCR

Accurate and reliable means of variety identification are necessary to assess purity of seed supplies, to support claims relating to plant breeders rights and, in Canada, to provide quality assurances in the grain handling system. A single, multiplexed set of seven simple-sequence-repeat (SSR) markers was found to uniquely identify all 18 durum wheat varieties that have been developed in Canada and are currently, or were formerly, registered for commercial production. Significant features of this multiplexed set include an allele that is specific, within Canadian durum varieties, to those having high gluten strength, and redundancy that was included in an effort to increase the capacity to accommodate future varieties. In combination with a reasonably rapid individual-kernel DNA extraction protocol and automated allele calling, this marker system offers a higher resolution alternative to complement established protein-based variety identification methods.Communicated by P. Langridge     

EST-SSR DNA polymorphism in durum wheat (Triticum durum L.) collections

SSRs derived from EST were molecular markers belonging to the transcribed region of the genome. Therefore, any polymorphism detected using EST-SSRs might reflect the better relationship among species or varieties. Using wheat EST-SSR markers, 60 durum wheat (Triticum durum L.) accessions from seven countries were investigated. Twenty-five primer pairs could amplify successfully in the 60 durum wheat accessions, of which tri-nucleotide repeats were the dominant type, and revealed 26 loci on all seven wheat homologous chromosome groups. A total of 87 eSSR alleles were detected, and the number of alleles detected by a single pair of primers ranged from 1 to 11, with an average of 3.3 alleles per locus. Higher numbers of alleles and PIC were identified on the B genome than those on the A genome.     

Nucleotide-binding site (NBS) profiling of genetic diversity in durum wheat.

Molecular markers are effective tools to investigate genetic diversity for resistance to pathogens. NBS (nucleotide-binding site) profiling is a PCR (polymerase chain reaction)-based approach to studying genetic variability that specifically targets chromosome regions containing R-genes and R-gene analogues. We used NBS profiling to measure genetic diversity among 58 accessions of durum wheat. Mean polymorphism rates detected using MseI and AluI as restriction enzymes were 34% and 22%, respectively. Mean number of polymorphisms per enzyme-primer combination was equal to 23.8 +/- 5.9, ranging from 13 to 31 polymorphic bands. In total, 96 markers over 190 indicated a good capacity to discriminate between accessions (the polymorphic index content ranging from 0.30 to 0.50). The results obtained with NBS profiling were compared with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data of the same set of accessions. The genetic distances computed with 190 NBS profiling markers were in close agreement with those obtained with AFLP and SSR markers (r = 0.73 and 0.76, respectively). Our results indicate that NBS profiling provides an effective means to investigate genetic diversity in durum wheat.     

Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)

Gliadins are seed storage proteins which are characterized by high intervarietal polymorphism and can be used as genetic markers. As a result of our work, a considerably extended catalogue of allelic variants of gliadin component blocks was compiled for durum wheat; 74 allelic variants for four gliadin-coding loci were identified for the first time. The extended catalogue includes a total of 131 allelic variants: 16 for locus Gli-A1(d), 19 for locus Gli-B1(d), 41 for locus Gli-A2(d), and 55 for locus Gli-B2(d). The electrophoretic pattern of the standard cultivar and a diagram are provided for every block identified. The number of alleles per family is quite small for loci Gli-A1(d) and Gli-B1(d) of durum wheat, as contrasted to loci Gli-A2(d) and Gli-B2(d) that are characterized by large families including many alleles. The presence of large block families determines a higher diversity of durum wheat for loci Gli-A2(d) and Gli-B2(d) as compared to Gli-A1(d) and Gli-B1(d). The catalogue of allelic variants of gliadin component blocks can be used by seed farmers to identify durum wheat cultivars and evaluate their purity; by breeders, to obtain homogenous cultivars and control the initial stages of selection; by gene bank experts, to preserve native varieties and the original biotypic composition of cultivars.     

Genetic mapping of the gene affecting polyphenol oxidase activity in tetraploid durum wheat

The quality of durum wheat (Triticum turgidum ssp. durum) is influenced by polyphenol oxidase(PPO) activity and its corresponding substrates. A saturated molecular-marker linkage map was constructed previously by using a set of recombinant inbred (RI) lines, derived from a cross between durum wheat cultivars Jennah Khetifa and Cham 1. Quantitative trait loci (QTL) for PPO activity in seeds were mapped in this population. PPO activity in seeds of the parents and 110 RI lines was measured spectrophotometrically. The PPO activity of Cham 1 was significantly lower than that of Jennah Khetifa. QTL analysis of these data indicated that most of PPO activity was associated with major loci on the long arm of chromosome 2A. The trait was found to be strongly associated with the SSR marker Xgwm312@2A. With this knowledge, marker-assisted selection can be used to select genotypes with lower PPO activity in durum wheat populations.     

Assessment of genetic diversity among Syrian durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

Relationships among durum wheat accessions. II. A comparison of molecular and pedigree information.

The study of direct ancestry relationships provides information with which to determine essential derivation. SSR profiles were used to determine the pattern of relatedness among 134 durum wheat accessions, representing the most important modern durum wheat gene pools. Simple sequence repeat (SSR)- and amplified fragment length polymorphism (AFLP)-based genetic similarities among cultivars with accurate pedigrees were compared with pedigree-based coefficients of parentage. Sizeable departures of molecular similarities from the expected ones were observed, indicating the unreliability of inferring the pattern of genetic relatedness from the coefficient of parentage. Case studies consisting of parent-progeny cultivar trios and pairs, identified on the basis of their registered pedigree, were studied to evaluate the probability of ancestry of each progeny cultivar, compared with all the remaining accessions. Rare alleles and haplotype sharing were also explored. When the results did not agree with the registered parentages, SSR markers provided information with which to identify the most probable parents (or the corresponding "breeding lineages") in the collection.     

Global diversity of durum wheat Triticum durum Desf. for alleles of gliadin-coding loci

Genetic diversity for the alleles of gliadin-coding loci was studied with 465 durum wheat accessions from 42 countries. A total of 108 alleles were identified for four loci; 60 alleles were described for the first time. Broad diversity of rare gliadin-coding alleles was observed. The highest genetic diversity was characteristic of durum wheat accessions from the Middle East, Trans-Caucasia, the Pyrenean Peninsula, and the Balkans. Two genetically isolated ancient branches of durum wheat were isolated. A “southern” branch included mostly accessions from the Mediterranean region, the Middle East, and Trans-Caucasia. A “northern” branch included Russian and Ukrainian durum wheat accessions and varieties obtained on their basis. An additional group included durum wheat accessions that had been obtained in several past decades on the basis of the material of international breeding centers (CIMMYT and ICARDA) and had low genetic diversity.     

Identification of a threshold level to assess essential derivation in durum wheat

Essentially derived varieties (EDV) are considered to be those that are obtained from an original variety by means of methods that do not substantially modify its genetic structure, and whose characteristics are therefore derived from the original. The identification of EDV requires the definition of a threshold value of genetic similarity between the new and the original variety that if exceeded would suggest derivation. Although protocols for estimating genetic similarities based on molecular markers have been proposed for EDV identification in some species, no information was available for durum wheat (Triticum durum). A set of 60 genotypes (F8 or F9 lines and their parents) representing different levels of relatedness were profiled using 14 (later reduced to 13 by excluding the most deviant one) amplified fragment length polymorphism (AFLP) primer combinations and 109 simple sequence repeat (SSR) loci evenly distributed in the genome. For both marker types, an EDV threshold was calculated according to the “tail principle” on the distributions of Jaccard similarities among the subset of 39 independent genotypes. For all pairs of closely related lines branched in advanced generations (F7–F8), or for all but one pairs of lines deriving from the same F4 family, similarities exceeded the thresholds for both marker types, indicating a very good agreement in showing cases of suspected derivation. Compared to SSR markers, AFLP markers appear more suitable for assessing essential derivation because of their superior cost-efficiency. Based on 13 AFLP primer combinations, a threshold of 0.96 Jaccard similarity is proposed, below which a variety should be considered to be independently derived.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

A consensus framework map of durum wheat (Triticum durum Desf.) suitable for linkage disequilibrium analysis and genome-wide association mapping

Background

Durum wheat (Triticum durum Desf.) is a tetraploid cereal grown in the medium to low-precipitation areas of the Mediterranean Basin, North America and South-West Asia. Genomics applications in durum wheat have the potential to boost exploitation of genetic resources and to advance understanding of the genetics of important complex traits (e.g. resilience to environmental and biotic stresses). A dense and accurate consensus map specific for T. durum will greatly facilitate genetic mapping, functional genomics and marker-assisted improvement.

Results

High quality genotypic data from six core recombinant inbred line populations were used to obtain a consensus framework map of 598 simple sequence repeats (SSR) and Diversity Array Technology^® (DArT) anchor markers (common across populations). Interpolation of unique markers from 14 maps allowed us to position a total of 2,575 markers in a consensus map of 2,463 cM. The T. durum A and B genomes were covered in their near totality based on the reference SSR hexaploid wheat map. The consensus locus order compared to those of the single component maps showed good correspondence, (average Spearman’s rank correlation rho ρ value of 0.96). Differences in marker order and local recombination rate were observed between the durum and hexaploid wheat consensus maps. The consensus map was used to carry out a whole-genome search for genetic differentiation signatures and association to heading date in a panel of 183 accessions adapted to the Mediterranean areas. Linkage disequilibrium was found to decay below the r² threshold = 0.3 within 2.20 cM, on average. Strong molecular differentiations among sub-populations were mapped to 87 chromosome regions. A genome-wide association scan for heading date from 27 field trials in the Mediterranean Basin and in Mexico yielded 50 chromosome regions with evidences of association in multiple environments.

Conclusions

The consensus map presented here was used as a reference for genetic diversity and mapping analyses in T. durum, providing nearly complete genome coverage and even marker density. Markers previously mapped in hexaploid wheat constitute a strong link between the two species. The consensus map provides the basis for high-density single nucleotide polymorphic (SNP) marker implementation in durum wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-873) contains supplementary material, which is available to authorized users.     

Molecular markers as a complementary tool in risk assessments: quantifying interspecific gene flow from triticale to spring wheat and durum wheat

Triticale is being considered as a bioindustrial crop in Canada using genetic modification. Because related spring wheat (Triticum aestivum) and durum wheat (T. durum) may exhibit synchronous flowering and grow in proximity, determination of interspecific gene flow when triticale is the pollen donor is necessary to evaluate potential risk. Pollen-mediated gene flow risk assessments generally rely on phenotypic markers to detect hybridization but DNA markers could be powerful and less ambiguous in quantifying rare interspecific gene flow. Six cultivars representing four species [spring wheat, durum wheat, triticale and rye (Secale cereale)] were screened with 235 spring wheat and 27 rye SSR markers to evaluate transferability and polymorphism. Fifty-five polymorphic markers were used in conjunction with morphological characterization to quantify interspecific gene flow from a blue aleurone (BA) triticale line to two spring wheat cultivars (AC Barrie and AC Crystal) and one durum wheat cultivar (AC Avonlea). Approximately 1.9 Million seeds from small plot experiments were visually screened in comparison with known hybrid seed. In total 2031 putative hybrids were identified and 448 germinated. Morphological analysis of putative hybrid plants identified five hybrids while molecular analysis identified 11 hybrids and two were common to both. Combined, 14 hybrids were confirmed: 10 spring wheat × triticale (0.0008 % of harvested seed): seven AC Barrie × BA triticale (0.001 %) and three AC Crystal × BA triticale (0.0005 %); and four durum wheat × triticale (0.0006 %). The occurrence of rare hybrids does not present a substantial risk to the development of GM triticale.     

Mapping gibberellin-sensitive dwarfing locus <Emphasis Type="Italic">Rht18</Emphasis> in durum wheat and development of SSR and SNP markers for selection in breeding

Gibberellin-sensitive dwarfing gene Rht18 was mapped in two durum wheat recombinant inbred lines (RIL) populations developed from crosses, Bijaga Yellow/Icaro and HI 8498/Icaro. Rht18 was mapped within genetic interval of 1.8 cM on chromosome 6A. Simple sequence repeat (SSR) markers S470865SSR4, barc37 and TdGA2ox-A9 specific marker showed co-segregation with Rht18 in Bijaga Yellow/Icaro population consisting 256 RILs. Effect of Rht18 on plant height was validated in HI 8498/Icaro RIL population which segregated for Rht18 and Rht-B1b. Rht-B1b from HI 8498 showed pleiotropic effect on plant height and coleoptile length, on the other hand, Rht18 did not show effect on coleoptile length. The SSR and SNP markers linked to Rht18 were also validated by assessing their allelic frequency in 89 diverse durum and bread wheat accessions. It was observed that 204 bp allele of S470865SSR4 could differentiate Icaro from rest of the wheat accessions except HI 8498, suggesting its utility for selection of Rht18 in wheat improvement programs. Rht18 associated alleles of TdGA2ox-A9, IAW4371 and IAW7940 were absent in most of the tall Indian local durum wheat and bread wheat, hence could be used to transfer Rht18 to bread wheat and local durum wheat. SSR marker barc3 showed high recombination frequency with Rht18, though it showed allele unique to Icaro. Since semidwarf wheat with GA-sensitive dwarfing genes are useful in dry environments owing to their longer coleoptile, better emergence and seedling vigor, Rht18 may provide a useful alternative to widely used GA-insensitive dwarfing genes under dry environments.