RESUMO
Background: Strong artificial selection and/or natural bottle necks may limit genetic variation in domesticated species. Lupinus luteus, an orphan temperate crop, has suffered diversity reductions during its bitter/sweet alkaloid domestication history, limiting breeding efforts and making molecular marker development a difficult task. The main goal of this research was to generate new polymorphic insertiondeletion (InDel) markers to aid yellow lupin genetics and breeding. By combining genomic reduction libraries and next generation sequencing, several polymorphic InDel markers were developed for L. luteus L. Results: A total of 118 InDel in silico polymorphic markers were identified. Eighteen InDel primer sets were evaluated in a diverse L. luteus core collection, where amplified between 23 alleles per locus. Observed heterozygosity (HO; 0.0648 to 0.5564) and polymorphic information content (PIC; 0.06 to 0.48) estimations revealed a moderate level of genetic variation across L. luteus accessions. In addition, ten and nine InDel loci amplified successfully Lupinus hispanicus Boiss & Reut, and Lupinus mutabilis Sweet, respectively, two L. luteus close relatives. PCA analysis identified two L. luteus clusters, most likely explained by the domestication species history. Conclusion: The development of InDel markers will facilitate the study of genetic diversity across L. luteus populations, as well as among closely related species.
Assuntos
Variação Genética , Marcadores Genéticos , Lupinus/genética , Mutação INDEL , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
BACKGROUND: Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species. RESULTS: Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession's origin. CONCLUSION: L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discovery, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparisons and putative gene candidates for QTL detection.