Sequence‐based SNP genotyping in durum wheat

Marker development for marker‐assisted selection in plant breeding is increasingly based on next‐generation sequencing (NGS). However, marker development in crops with highly repetitive, complex genomes is still challenging. Here we applied sequence‐based genotyping (SBG), which couples AFLP^®‐based complexity reduction to NGS, for de novo single nucleotide polymorphisms (SNP) marker discovery in and genotyping of a biparental durum wheat population. We identified 9983 putative SNPs in 6372 contigs between the two parents and used these SNPs for genotyping 91 recombinant inbred lines (RILs). Excluding redundant information from multiple SNPs per contig, 2606 (41%) markers were used for integration in a pre‐existing framework map, resulting in the integration of 2365 markers over 2607 cM. Of the 2606 markers available for mapping, 91% were integrated in the pre‐existing map, containing 708 SSRs, DArT markers, and SNPs from CRoPS technology, with a map‐size increase of 492 cM (23%). These results demonstrate the high quality of the discovered SNP markers. With this methodology, it was possible to saturate the map at a final marker density of 0.8 cM/marker. Looking at the binned marker distribution (Figure 2), 63 of the 268 10‐cM bins contained only SBG markers, showing that these markers are filling in gaps in the framework map. As to the markers that could not be used for mapping, the main reason was the low sequencing coverage used for genotyping. We conclude that SBG is a valuable tool for efficient, high‐throughput and high‐quality marker discovery and genotyping for complex genomes such as that of durum wheat.     

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     

Distribution of DArT, AFLP, and SSR markers in a genetic linkage map of a doubled-haploid hexaploid wheat population.

A genetic linkage mapping study was conducted in 93 doubled-haploid lines derived from a cross between Triticum aestivum L. em. Thell 'Arina' and a Norwegian spring wheat breeding line, NK93604, using diversity arrays technology (DArT), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. The objective of this study was to understand the distribution, redundancy, and segregation distortion of DArT markers in comparison with AFLP and SSR markers. The map contains a total of 624 markers with 189 DArTs, 165 AFLPs and 270 SSRs, and spans 2595.5 cM. All 3 marker types showed significant (p < 0.01) segregation distortion, but it was higher for AFLPs (24.2%) and SSRs (22.6%) than for DArTs (13.8%). The overall segregation distortion was 20.4%. DArTs showed the highest frequency of clustering (27.0%) at < 0.5 cM intervals between consecutive markers, which is 3 and 15 times higher than SSRs (8.9%) and AFLPs (1.8%), respectively. This high proportion of clustering of DArT markers may be indicative of gene-rich regions and (or) the result of inclusion of redundant clones in the genomic representations, which was supported by the presence of very high correlation coefficients (r > 0.98) and multicollinearity among the clustered markers. The present study is the first to compare the utility of DArT with AFLP and SSR markers, and the present map has been successfully used to identify novel QTLs for resistance to Fusarium head blight and powdery mildew and for anther extrusion, leaf segment incubation, and latency.     

Development of a molecular linkage map of pearl millet integrating DArT and SSR markers

Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F₇ Recombinant Inbred Lines (RILs) from cross H 77/833-2 × PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available.     

Genetic Diversity and Population Structure of Tetraploid Wheats (Triticum turgidum L.) Estimated by SSR,DArT and Pedigree Data

Levels of genetic diversity and population genetic structure of a collection of 230 accessions of seven tetraploid Triticum turgidum L. subspecies were investigated using six morphological, nine seed storage protein loci, 26 SSRs and 970 DArT markers. The genetic diversity of the morphological traits and seed storage proteins was always lower in the durum wheat compared to the wild and domesticated emmer. Using Bayesian clustering (K = 2), both of the sets of molecular markers distinguished the durum wheat cultivars from the other tetraploid subspecies, and two distinct subgroups were detected within the durum wheat subspecies, which is in agreement with their origin and year of release. The genetic diversity of morphological traits and seed storage proteins was always lower in the improved durum cultivars registered after 1990, than in the intermediate and older ones. This marked effect on diversity was not observed for molecular markers, where there was only a weak reduction. At K >2, the SSR markers showed a greater degree of resolution than for DArT, with their identification of a greater number of groups within each subspecies. Analysis of DArT marker differentiation between the wheat subspecies indicated outlier loci that are potentially linked to genes controlling some important agronomic traits. Among the 211 loci identified under selection, 109 markers were recently mapped, and some of these markers were clustered into specific regions on chromosome arms 2BL, 3BS and 4AL, where several genes/quantitative trait loci (QTLs) are involved in the domestication of tetraploid wheats, such as the tenacious glumes (Tg) and brittle rachis (Br) characteristics. On the basis of these results, it can be assumed that the population structure of the tetraploid wheat collection partially reflects the evolutionary history of Triticum turgidum L. subspecies and the genetic potential of landraces and wild accessions for the detection of unexplored alleles.     

Comparison of genetic and cytogenetic maps of hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR and DArT markers

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20–34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and assessment of DArT markers in triticale

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.     

Resistance to <Emphasis Type="Italic">Soil-borne cereal mosaic virus</Emphasis> in durum wheat is controlled by a major QTL on chromosome arm 2BS and minor loci

Soil-borne cereal mosaic (SBCM) is a viral disease, which seriously affects hexaploid as well as tetraploid wheat crops in Europe. In durum wheat (Triticum durum Desf.), the elite germplasm is characterized by a wide range of responses to SBCMV, from susceptibility to almost complete resistance. In this study, the genetic analysis of SBCMV resistance was carried out using a population of 181 durum wheat recombinant inbred lines (RILs) obtained from Meridiano (resistant) × Claudio (moderately susceptible), which were profiled with SSR and DArT markers. The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection during 2007 and 2008. A wide range of disease reactions (as estimated by symptom severity and DAS-ELISA) was observed. A large portion of the variability for SBCMV response was explained by a major QTL (QSbm.ubo-2BS) located in the distal telomeric region of chromosome 2BS near the marker triplet Xbarc35–Xwmc661–Xgwm210, with R ² values ranging from 51.6 to 91.6%. The favorable allele was contributed by Meridiano. Several QTLs with minor effects on SBCMV response were also detected. Consistently with the observed transgressive segregation, the resistance alleles at minor QTLs were contributed by both parents. The presence and effects of QSbm.ubo-2BS were validated through association mapping in a panel of 111 elite durum wheat accessions.     

Development, identification, and validation of markers for marker-assisted selection against the Stagonospora nodorum toxin sensitivity genes Tsn1 and Snn2 in wheat

The wheat-Stagonospora nodorum pathosystem involves a number of pathogen-produced host-selective toxins that interact with host genes in an inverse gene-for-gene manner to cause disease. The wheat intervarietal recombinant inbred population derived from BR34 and Grandin (BG population) segregates for the toxin sensitivity genes Tsn1, Snn2, and Snn3, which confer sensitivity to the toxins ToxA, SnTox2, and SnTox3, respectively. Here, we report the addition of 141 molecular markers to the BG population linkage maps, the identification and/or development of markers tightly linked to Tsn1 and Snn2, and the validation of the markers using a set of diverse wheat accessions. The BG population maps now contain 787 markers, and new simple sequence repeat (SSR) markers closely linked to Snn2 on chromosome arm 2DS were identified. In an effort to target more markers to the Snn2 locus, STS markers were developed from 2DS bin-mapped ESTs resulting in the development and mapping of 36 markers mostly to the short arms of group 2 chromosomes. Together, SSR and EST-STS markers delineated Snn2 to a 4.0 cM interval. SSRs developed in related work for Tsn1 were mapped in the BG population and delineated the gene to a 1.0 cM interval. Evaluation of the markers for Tsn1 and Snn2 in a diverse set of wheat genotypes validated their utility for marker-assisted selection, which is particularly efficient for removing toxin sensitivity alleles from elite germplasm and varieties. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

A genetic map of 1,000 SSR and DArT markers in a wide barley cross

A high-density genetic map was developed from an F1-derived doubled haploid population generated from a cross between cultivated barley (Hordeum vulgare) and the subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using 536 SSR (558 loci) and 442 DArT markers. Of the SSRs, 149 markers (153 loci) were derived from barley ESTs, and 7 from wheat ESTs. A high level of polymorphism (∼70%) was observed, which facilitated the mapping of 197 SSRs for which genetic assignments had not been previously reported. Comparison with a published composite map showed a high level of co-linearity and telomeric coverage on all seven chromosomes. This map provides access to previously unmapped SSRs, improved genome coverage due to the integration of DArT and EST-SSRs and overcomes locus order issues of composite maps constructed from the alignment of several genetic maps. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Population structure and long-range linkage disequilibrium in a durum wheat elite collection

A collection of 134 durum wheat accessions, mainly including cultivars (cvs.) representative of the major gene pools, was assembled and characterized with 70 SSRs for genetic diversity and level of long-range linkage disequilibrium (LD). Results of both a distance-based and a model-based (Bayesian) cluster analysis evidenced the presence of a structured diversity. In the model-based analysis, six to eight main distinct subpopulations were identified based on the molecular data. Only a relatively small portion (20%) of the molecular variation was accounted for by the geographical origin of the accessions. Major differences were detected between the North American and the Mediterranean cvs., while a considerable overlap characterized the cvs. from CIMMYT-ICARDA and Italy. The North American cvs. showed the highest within group mean genetic similarity (GS_m = 0.68). French cvs. revealed sizeable similarities with both the North American as well as the Italian and CIMMYT-ICARDA pools. Considering the germplasm as a whole, high levels of LD were found both at locus pairs with an intrachromosomal distance <50 cM as well as at those with distances more than 50 cM and independent (86, 52 and 54% of SSR pairs at p < 0.01, respectively). After re-evaluating LD within each of the three main subgroups identified through the analysis of the germplasm structure, the LD level remained high for tightly to moderately linked locus pairs (<20 cM apart), but was greatly reduced in the loosely linked (more than 50 cM apart) and independent locus pairs. The implications of these findings as to the possibility of using association mapping for gene/QTL discovery in durum wheat are discussed.     

Association mapping of leaf rust response in durum wheat

Resistance to leaf rust (Puccinia triticina Eriks.) is a main objective for durum wheat (Triticum durum Desf.) breeding. Association mapping on germplasm collections is now being used as an additional approach for the discovery and validation of major genes/QTLs. In this study, a collection of 164 elite durum wheat accessions suitable for association mapping has been tested for leaf rust response at the seedling stage and under field conditions (adult plant stage). Seedling tests were carried out with 25 selected isolates from durum wheat, bread wheat and triticale, while field experiments were carried out in artificially inoculated plots in Italy and in Mexico. The collection has been profiled with 225 simple sequence repeat (SSR) loci of known map position and a PCR assay targeting Ppd-A1. Associations showing highly consistent experiment-wise significances across leaf rust isolates and field trials were mainly detected for the 7BL distal chromosome (chr.) region (harbouring Lr14 from cultivar Llareta INIA and QLr.ubo-7B.2 from cultivar Creso) and for two chr. regions located in chrs. 2A and 2B. Additionally, isolate-specific associations and/or associations with smaller effects in the field trials were identified in most of the chromosomes. The chr. 7BL distal region was investigated in detail through haplotyping with 15 SSR markers, revealing that the Creso and Llareta INIA alleles are identical by descent at 6 adjacent SSR loci in the most distal 7BL region spanning 8 cM. Association mapping allowed us to further refine the map location of the Lr14/QLr.ubo-7B.2 resistance gene to the most distal region of the linkage group, tagged by Xcfa2257.2, Xgwm344.2 and Xwmc10. The resistant haplotype is present in a number of accessions (ca. 15% of the accessions included in the collection) from the Italian, CIMMYT and ICARDA breeding programmes. Therefore, this chr. 7BL region can be considered as the most important source of resistance to leaf rust currently exploited by durum breeders in the Mediterranean areas. Furthermore, the field trials at the adult plant stage allowed us to identify marker associations (e.g. chrs. 2BL and 3BS, proximal regions; chr. 7BS, distal region) which suggest the presence of minor QTLs for slow-rusting resistance.     

Development and genetic mapping of SSR markers in foxtail millet [Setaria italica (L.) P. Beauv.]

SSR markers are desirable markers in analysis of genetic diversity, quantitative trait loci mapping and gene locating. In this study, SSR markers were developed from two genomic libraries enriched for (GA)n and (CA)n of foxtail millet [Setaria italica (L.) P. Beauv.], a crop of historical importance in China. A total of 100 SSR markers among the 193 primer pairs detected polymorphism between two mapping parents of an F₂ population, i.e. “B100” of cultivated S. italica and “A10” of wild S. viridis. Excluding 14 markers with unclear amplifications, and five markers unlinked with any linkage group, a foxtail millet SSR linkage map was constructed by integrating 81 new developed SSR markers with 20 RFLP anchored markers. The 81 SSRs covered nine chromosomes of foxtail millet. The length of the map was 1,654 cM, with an average interval distance between markers of 16.4 cM. The 81 SSR markers were not evenly distributed throughout the nine chromosomes, with Ch.8 harbouring the least (3 markers) and Ch.9 harbouring the most (18 markers). To verify the usefulness of the SSR markers developed, 37 SSR markers were randomly chosen to analyze genetic diversity of 40 foxtail millet accessions. Totally 228 alleles were detected, with an average 6.16 alleles per locus. Polymorphism information content (PIC) value for each locus ranged from 0.413 to 0.847, with an average of 0.697. A positive correlation between PIC and number of alleles and between PIC and number of repeat unit were found [0.802 and 0.429, respectively (P < 0.01)]. UPGMA analysis revealed that the 40 foxtail millet cultivars could be grouped into five clusters in which the landraces’ grouping was largely consistent with ecotypes while the breeding varieties from different provinces in China tended to be grouped together. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Association mapping of agronomic traits on chromosome 2A of wheat

Association mapping is a method to test the association between molecular markers and quantitative trait loci (QTL) based on linkage disequilibrium (LD). In this study, the collection of 108 wheat germplasm accessions form China were evaluated for their plant heights, spike length, spikelets per spike, grains per spike, thousand kernel weight and spikelets density in 3 years at three locations. And they were genotyped with 85 SSR markers and 40 EST-SSR markers. The population structure was inferred on the basis of unlinked 48 SSR markers and 40 EST-SSR markers. The extent of LD on chromosome 2A was 2.3 cM. Association of 37 SSR loci on chromosomes 2A with six agronomic traits was analysed with a mixed linear model. A total of 14 SSR loci were significantly associated with agronomic traits. Some of the associated markers were located in the QTL region detected in previous linkage mapping analysis. Our results demonstrated that association mapping can enhance QTL information and achieves higher resolution with short LD extent.     

Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

Genetic and physical mapping of new EST-derived SSRs on the A and B genome chromosomes of wheat

The availability of genetic maps and phenotypic data of segregating populations allows to localize and map agronomically important genes, and to identify closely associated molecular markers to be used in marker-assisted selection and positional cloning. The objective of the present work was to develop a durum wheat intervarietal genetic and physical map based on genomic microsatellite or genomic simple sequence repeats (gSSR) markers and expressed sequence tag (EST)-derived microsatellite (EST-SSR) markers. A set of 122 new EST-SSR loci amplified by 100 primer pairs was genetically mapped on the wheat A and B genome chromosomes. The whole map also comprises 149 gSSR markers amplified by 120 primer pairs used as anchor chromosome loci, two morphological markers (Black colour, Bla1, and spike glaucousness, Ws) and two seed storage protein loci (Gli-A2 and Gli-B2). The majority of SSR markers tested (182) was chromosome-specific. Out of 275 loci 241 loci assembled in 25 linkage groups assigned to the chromosomes of the A and B genome and 34 remained unlinked. A higher percentage of markers (54.4%), localized on the B genome chromosomes, in comparison to 45.6% distributed on the A genome. The whole map covered 1,605 cM. The B genome accounted for 852.2 cM of genetic distance; the A genome basic map spanned 753.1 cM with a minimum length of 46.6 cM for chromosome 5A and a maximum of 156.2 cM for chromosome 3A and an average value of 114.5 cM. The primer sets that amplified two or more loci mapped to homoeologous as well as to non-homoeologous sites. Out of 241 genetically mapped loci 213 (88.4%) were physically mapped by using the nulli-tetrasomic, ditelosomic and a stock of 58 deletion lines dividing the A and B genome chromosomes in 94 bins. No discrepancies concerning marker order were observed but the cytogenetic maps revealed in some cases small genetic distance covered large physical regions. Putative function for mapped SSRs were assigned by searching against GenBank nonredundant database using TBLASTX algorithms.     

Molecular Characterization and Comparative Analysis of Six Durum Wheat Accessions Including Graziella Ra

Genetic identity and relatedness of the durum wheat Graziella Ra, four Italian commercial durum cultivars (Cappelli, Grazia, Flaminio and Svevo) and Kamut were evaluated using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs) and α-gliadin gene sequence analysis. Our primary objective was to study molecular genetic diversity in such a set of wheats including three modern (Grazia, Flaminio and Svevo) and three older (Cappelli, Kamut and Graziella Ra) durum accessions. Specifically, we aimed at determining the relationship between the historic accession Graziella Ra and Kamut, which is considered an ancient relative of the durum subspecies. Obtained results revealed that (1) both AFLP and SSR molecular markers detected highly congruent patterns of genetic diversity among the accessions showing nearly similar efficiency; (2) for AFLPs, percentage of polymorphic loci within accession ranged from 6.57% to 19.71% (mean, 12.77%) and for SSRs, from 0% to 57.14% (mean, 28.57%); (3) principal component analysis of genetic distance among accessions showed the first two axes accounting for 58.03% (for AFLPs) and 61.60% (for SSRs) of the total variability; (4) for AFLPs, molecular variance was partitioned into 80% (variance among accessions) and 20% (within accession) and for SSRs, into 73% (variance among accessions) and 27% (within accession); (5) cluster analysis of AFLPs and SSRs datasets displayed Graziella Ra and Kamut constantly grouped into the same cluster; and (6) molecular comparison of α-gliadin gene sequences showed Graziella Ra and Kamut in separate clusters. All these findings support the hypothesis that Graziella Ra and Kamut, although very similar, at least in the little part of the genome investigated by molecular markers employed in this study, might be considered as distinct accessions.