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A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

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Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F(1)(NA(6) x AU(6)) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F(1)(NA(6) x AU(6)) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.

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The combination of homologous, homoeologous and paralogous classes of sequence variation presents major challenges for SNP discovery in outbreeding allopolyploid species. Previous in vitro gene-associated SNP discovery studies in the allotetraploid forage legume white clover (Trifolium repens L.) were vulnerable to such effects, leading to prohibitive levels of attrition during SNP validation. Identification of T. occidentale and T. pallescens as the putative diploid progenitors of white clover has permitted discrimination of the different sequence variant categories. Amplicons from selected abiotic stress tolerance-related genes were obtained using mapping family parents and individuals from each diploid species. Following cloning, progenitor comparison allowed tentative assignment of individual haplotypes to one or other sub-genome, as well as to gene copies within sub-genomes. A high degree of coincidence and identity between SNPs and HSVs was observed. Close similarity was observed between the genome of T. occidentale and one white clover sub-genome, but the affinity between T. pallescens and the other sub-genome was weaker, suggesting that a currently uncharacterised taxon may be the true second progenitor. Selected validated SNPs were attributed to individual sub-genomes by assignment to and naming of homoeologous linkage groups, providing the basis for improved genetic trait-dissection studies. The approach described in this study is broadly applicable to a range of allopolyploid taxa of equivocal ancestry.

SummaryThe application of genomics in crops has the ability to significantly improve genetic gain for agriculture. Many marker‐dense tools have been developed, but few have seen broad adoption in plant genomics due to issues of significant variations of genome size, levels of ploidy, single nucleotide polymorphism (SNP) frequency and reproductive habit. When combined with limited breeding activities, small research communities and scant sequence resources, the suitability of popular systems is often suboptimal and routinely fails to effectively balance cost‐effectiveness and sample throughput. Genotyping‐by‐sequencing (GBS) encompasses a range of protocols including resequencing of the transcriptome. This study describes a skim GBS‐transcriptomics (GBS‐t) approach developed to be broadly applicable, cost‐effective and high‐throughput while still assaying a significant number of SNP loci. A range of crop species with differing levels of ploidy and degree of inbreeding/outbreeding were chosen, including perennial ryegrass, a diploid outbreeding forage grass; phalaris, a putative segmental allotetraploid outbreeding forage grass; lentil, a diploid inbreeding grain legume; and canola, an allotetraploid partially outbreeding oilseed. GBS‐t was validated as a simple and largely automated, cost‐effective method which generates sufficient SNPs (from 89 738 to 231 977) with acceptable levels of missing data and even genome coverage from c. 3 million sequence reads per sample. GBS‐t is therefore a broadly applicable system suitable for many crops, offering advantages over other systems. The correct choice of subsequent sequence analysis software is important, and the bioinformatics process should be iterative and tailored to the specific challenges posed by ploidy variation and extent of heterozygosity.

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