Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

An RFLP map of diploid Hordeum bulbosum L. and comparison with maps of barley (H. vulgare L.) and wheat (Triticum aestivum L.)

This paper describes the first extensive genetic map of Hordeum bulbosum, the closest wild relative of cultivated barley. H. bulbosum is valuable for haploid production in barley breeding, and because of desirable agronomic characteristics, it also has potential for trait introgression into barley. A H. bulbosum map will assist introgression and provide a basis for the identification of QTLs for crossability with barley and other potentially useful genes. The present study used a population of 111 individuals from a PB1×PB11 cross to develop a genetic linkage map of diploid H. bulbosum (2n=2x=14) based on barley, wheat and other ”anchor” cereal RFLP markers previously mapped in other species. Because of the cross-pollinating and highly polymorphic nature of H. bulbosum, up to four alleles showed segregation at any one locus, and five different segregation types were found. This enabled maps to be developed for the PB1 and PB11 parents, as well as a combined map. In total, 136 RFLP loci were mapped with a marker coverage of 621 cM. The markers were generally colinear with barley but H. bulbosum had less recombination in the centromeric regions and similar or more in the distal regions. Cytological studies on pollen mother cells at metaphase-I showed marked distal localization of chiasmata and a frequency consistent with the genetic map length. This study showed that H. bulbosum was highly polymorphic, making it suitable for trait analysis and supplementing maps of barley. Received: 20 November 2000 / Accepted: 5 January 2001     

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.     

Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense

Background

Hordeum chilense, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (Hordeum vulgare) represents a promising source to increase the number of genetic markers available for the H. chilense genome.

Results

All of the 82 barley EST-derived SSR primer pairs tested for transferability to H. chilense amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among H. chilense lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing H. chilense phylogenetic relationships. Triticum aestivum-H. chilense addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.

Conclusion

From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of H. chilense, related wild barleys like H. murinum, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.     

Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markers

A selection of 147 wheat D-genome and 130 barley genomic simple sequence repeat (gSSR) markers were screened for their utility in Hordeum chilense, as an alien donor genome for cereal breeding. Fifty-eight wheat D-genome and 71 barley PCR primer pairs consistently amplified products from H. chilense. Nineteen wheat D-genome and 20 barley gSSR markers were polymorphic and allowed wide genome coverage of the H. chilense genome. Twenty-three of the wheat D-genome and 11 barley PCR primer pairs were suitable for studying the introgressions of H. chilense into wheat, amplifying H. chilense products of distinct size. In 88% of the markers tested, H. chilense products were maintained in the expected homeologous linkage group, as revealed by the analysis of wheat/H. chilense addition lines. Twenty-nine microsatellite markers (eight gSSRs and 21 expressed sequence tags-SSRs) uniformly distributed across the genome were tested for their utility in genetic diversity analysis within the species. Three genetic clusters are reported, in accordance with previous morphological and amplified fragment length polymorphism data. These results show that it is possible to discriminate the three previously established germplasm groups with microsatellite markers. The reported markers represent a valuable resource for the genetic characterisation of H. chilense, for the analysis of its genetic variability, and as a tool for wheat introgression. This is the first intraspecific study in a collection of H. chilense germplasm using microsatellite markers.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Fine-mapping of the BaMMV, BaYMV-1 and BaYMV-2 resistance of barley (Hordeum vulgare) accession PI1963

Barley yellow mosaic disease caused by the bymoviruses barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) is one of the economically most important diseases of winter barley in Europe. In European barley breeding programmes, resistance is currently due to only two genes—rym4, which is effective against viruses BaMMV and BaYMV-1, and rym5, which is effective against BaYMV-2. Diversification of resistance is therefore an important task. Because the accession PI1963 confers immunity against all European strains of barley yellow mosaic disease and is not allelic to rym5, we have attempted to develop closely linked markers in order to facilitate the efficient introgression of this resistance into adapted germplasm. By means of restriction fragment length polymorphism analysis, we located a gene locus for resistance to BaMMV, BaYMV-1 and BaYMV-2 of PI1963 on chromosome 4HL using a mapping population (W757) comprising 57 doubled haploid (DH) lines. Subsequent tests for allelism indicated that the BaMMV resistance gene in PI1963 is allelic to rym11. Two DH populations, IPK1 and IPK2, comprising 191 and 161 DH lines, respectively, were derived from the initial mapping population W757 and used for further analysis. As random amplified polymorphic DNA development did not facilitate the identification of more closely linked markers, simple sequence repeat (SSR) analyses were conducted. For population IPK1, the closest SSRs detected were Bmac181 and Bmag353, which flank the gene at 2.1 cM and 2.7 cM, respectively. For the IPK2 population, the SSR markers HVM3 and Bmag353 are located proximally at 2.5 cM and distally at 8.2 cM, respectively. In order to develop markers more tightly linked to rym11, a targeted amplified fragment length polymorphism (AFLP) marker identification approach was adopted using bulks comprising lines carrying recombination events proximal and distal to the target interval. Using this approach we identified six AFLP markers closely linked to rym11, with the two markers, E56M32 and E49M33, co-segregating with rym11 in both populations. The SSRs and AFLPs identified in this study represent useful tools for marker-assisted selection.     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work     

Genetic structure and ecogeographical adaptation in wild barley (<Emphasis Type="Italic">Hordeum chilense</Emphasis>Roemer et Schultes) as revealed by microsatellite markers

Background  

Multi-allelic microsatellite markers have become the markers of choice for the determination of genetic structure in plants. Synteny across cereals has allowed the cross-species and cross-genera transferability of SSR markers, which constitute a valuable and cost-effective tool for the genetic analysis and marker-assisted introgression of wild related species. Hordeum chilense is one of the wild relatives with a high potential for cereal breeding, due to its high crossability (both interspecies and intergenera) and polymorphism for adaptation traits. In order to analyze the genetic structure and ecogeographical adaptation of this wild species, it is necessary to increase the number of polymorphic markers currently available for the species. In this work, the possibility of using syntenic wheat SSRs as a new source of markers for this purpose has been explored.     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Molecular mapping of the rf1 gene for pollen fertility restoration in sorghum (Sorghum bicolor L.)

We report the molecular mapping of a gene for pollen fertility in A1 (milo) type cytoplasm of sorghum using AFLP and SSR marker analysis. DNA from an F₂ population comprised of 84 individuals was screened with AFLP genetic markers to detect polymorphic DNAs linked to fertility restoration. Fifteen AFLP markers were linked to fertility restoration from the initial screening with 49 unique AFLP primer combinations (+3/+3 selective bases). As many of these AFLP markers had been previously mapped to a high-density genetic map of sorghum, the target gene (rf1) could be mapped to linkage group H. Confirmation of the map location of rf1 was obtained by demonstrating that additional linkage group-H markers (SSR, STS, AFLP) were linked to fertility restoration. The closest marker, AFLP Xtxa2582, mapped within 2.4 cM of the target loci while two SSRs, Xtxp18 and Xtxp250, flanked the rf1 locus at 12 cM and 10.8 cM, respectively. The availability of molecular markers will facilitate the selection of pollen fertility restoration in sorghum inbred-line development and provide the foundation for map-based gene isolation. Received: 22 August 2000 / Accepted: 18 October 2000     

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.     

First RFLP linkage map of red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) based on cDNA probes and its transferability to other red clover germplasm

We constructed a genetic linkage map of red clover (Trifolium pratense L., 2n=2x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7 cM and the average distance between two markers was 3.4 cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.Communicated by H.C. Becker     

Hordeum chilense repetitive sequences. Genome characterization using biotinylated probes

Summary A library of random DNA fragment clones of wild barley Hordeum chilense was screened for clones of repeated nucleotide sequences. Five clones were isolated that gave a stronger hybridization signal in colony and dot blot hybridization with total H. chilense DNA in comparison to Triticum aestivum DNA. Clones labelled with biotinylated nucleotides were used as probes to investigate the repeated sequences organization in the H. chilense genome. Tandemly arranged and interspersed sequences have been found, together with homology differences with related sequences present in T. Aestivum, which could allow the differentiation of H. chilense DNA when it is present in wheat. We show that biotin can replace the use of ³²P in preparing repeated sequence probes for Southern and DNA dot blot analyses.     

Variation in the high-molecular-weight glutenin subunits coded at the Glu-Hch1 locus in Hordeum chilense

Hordeum chilense Roem. et Schult. is a native South American diploid wild barley included in the section Anisolepis Nevski. H. chilense occurs exclusively in Chile and Argentina and has been used in the synthesis of a new amphiploid named tritordeum (×Tritordeum Ascherson et Graebner). The HMW glutenin subunits of H. chilense have a great influence on gluten strength of tritordeum. The variability of these proteins has been analysed electrophoretically, and up to ten allelic variants have been detected in a world collection of this species. This genetic variability has been included in 121 lines of tritordeum and could be used for widening the genetic basis of tritordeum and wheat. Received: 22 March 2000 / Accepted: 14 April 2000     

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi]

To make progress in genome analysis of azuki bean (Vigna angularis) a genetic linkage map was constructed from a backcross population of (V. nepalensis x V. angularis) x V.angularis consisting of 187 individuals. A total of 486 markers—205 simple sequence repeats (SSRs), 187 amplified fragment length polymorphisms (AFLPs) and 94 restriction fragment length polymorphisms (RFLPs) —were mapped onto 11 linkage groups corresponding to the haploid chromosome number of azuki bean. This map spans a total length of 832.1 cM with an average marker distance of 1.85 cM and is the most saturated map for a Vigna species to date. In addition, RFLP markers from other legumes facilitated finding several orthologous linkage groups based on previously published RFLP linkage maps. Most SSR primers that have been developed from SSR-enriched libraries detected a single locus. The SSR loci identified are distributed throughout the azuki bean genome. This moderately dense linkage map equipped with many SSR markers will be useful for mapping a range of useful traits such as those related to domestication and stress resistance. The mapping population will be used to develop advanced backcross lines for high resolution QTL mapping of these traits. O.K. Han, A. Kaga, T. Isemura have contributed equally to this paper.     

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.     

A molecular,isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.This research was also supported by other members of the NABGMP: K. Kasha, Department of Crop Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1; W. Kim, Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9; A. Laroche, Agriculture Canada Research Station, P.O. Box 3000 Main, Lethbridge, Alberta, Canada,TU 4B1; S. Molnar, Plant Research Centre Agriculture Canada, Central Experimental farm, Ottawa, Ontario, Canada K1A 0C6; G. Scoles, Department of Crop Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWOThis research is part of the North American Barley Genome Mapping Project, R. A. Nilan and K. Kasha, Coordinator and Associate Coordinator, respectively Permanent address: Department of Plant Genetics, NI Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow