Agriaquaculture Nutritional Genomic Center, CGNA, Genomics and Bioinformatics Unit, Km 10 Camino Cajón-Vilcún, INIA, Temuco, Chile.
BMC Genomics. 2012 Aug 24;13:425. doi: 10.1186/1471-2164-13-425.
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.
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.
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.
黄羽扇豆（ Lupinus luteus L. ）是一种小豆类作物，其种子蛋白质含量高。尽管在几个温带国家种植，但由于其孤儿状态，限制了基因组工具的生成，以帮助提高产量和营养品质的育种工作。在这项研究中，我们报告了 454 表达序列标签（ EST ）文库的构建，进行了黄羽扇豆与模式豆科植物的比较研究，开发了一套综合的 EST-简单重复序列（ SSR ）标记，并验证了它们在多样性研究和向相关物种转移的有效性。
两轮 454 焦磷酸测序分别为 L1 （幼叶、芽和花）和 L2 （未成熟种子） EST 文库产生了 205 Mb 和 530 Mb 的序列数据。一个组合的组装（ L1L2 ）产生了 71655 个 contigs ，平均 contig 长度为 632 个核苷酸。 L1L2 contigs 被聚类为 55309 个 isotigs 。 38200 个 isotigs 翻译成蛋白质，其中 8741 个是全长的。约 57%的黄羽扇豆序列与至少一个 Medicago 、 Lotus 、 Arabidopsis 或 Glycine 的序列具有显著相似性，40.17%与所有这些物种都有阳性匹配。黄羽扇豆 isotigs 也被筛选出 SSR 序列。共有 2572 个 isotigs 含有至少一个 EST-SSR ，每个 SSR 间隔 17.75 kbp 。对 EST-SSR 候选标记的实证评估产生了 222 个多态性 EST-SSR 。 254 （ 65.7%）和 113 （ 30%）个 SSR 引物对能够扩增 L. hispanicus 和 L. mutabilis DNA 的片段，分别。从 64 个黄羽扇豆品系中，使用 50 个多态性 EST-SSR 对一个样本进行了基因型分析。邻接法距离分析检测到黄羽扇豆品系之间存在几个聚类，强烈表明存在种群分支。然而，没有明确的聚类模式遵循品系的起源。
L. luteus 深度转录组测序将有助于进一步开发基因组工具和羽扇豆种质资源。 cDNA 文库的大规模测序将继续为基因发现、多态性（ SNPs 、 EST-SSR 、 INDELs 等）鉴定、基因组比较的锚定序列和 QTL 检测的候选基因提供原材料。
