USDA-Agricultural Research Service, Plant Science Research Unit, St, Paul, MN 55108, USA.
BMC Genomics. 2011 Apr 19;12:199. doi: 10.1186/1471-2164-12-199.
Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.
Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.
Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.
紫花苜蓿（Medicago sativa (L.) sativa），一种广泛种植的多年生饲料，具有作为纤维素乙醇原料的发展潜力。然而，非模式物种苜蓿的基因组学仍处于起步阶段。最近出现的 RNA-Seq 是一种大规模平行测序方法，用于转录组分析，为鉴定苜蓿基因和多态性以及进行深入的转录谱分析提供了机会。
与 773 基因型的茎细胞壁相比，708 基因型的茎细胞壁的纤维素浓度更高，木质素浓度更低。使用 Illumina GA-II 平台，从 708 和 773 的伸长茎（ES）和伸长后茎（PES）节段的 cDNA 文库中生成了总共 198,861,304 个表达序列标签（EST，长度为 76bp）。此外，使用 GS FLX Titanium 平台从 773 的 ES 和 PES 节段中生成了 341,984 个 EST。使用 GenBank 中可用的 Sanger EST、GS FLX Titanium EST 序列和从头组装的 Illumina 序列组装了第一个紫花苜蓿（Medicago sativa）基因索引（MSGI 1.0）。MSGI 1.0 包含 124,025 个独特序列，包括 22,729 个暂定共识序列（TC）、22,315 个单序列和 78,981 个拟单序列。我们在 MSGI 1.0 中的序列中总共鉴定了 1,294 个简单序列重复（SSR）。此外，在这两个基因型之间总共预测了 10,826 个单核苷酸多态性（SNP）。在随机选择用于实验验证的 55 个 SNP 中，有 47 个（85%）在两个基因型之间表现出多态性。我们还在每个基因型内鉴定了许多等位基因变异。数字基因表达分析确定了许多可能在茎发育中起作用的候选基因，以及可能导致两个基因型茎细胞壁组成差异的候选基因。
我们的结果表明，RNA-Seq 可成功用于鉴定非模式、异花授粉、自四倍体物种苜蓿中的基因、多态性检测和转录谱分析。本研究组装的苜蓿基因索引以及鉴定的 SNP、SSR 和候选基因可用于改良紫花苜蓿作为饲料作物和纤维素饲料。
