Development of chickpea EST-SSR markers and analysis of allelic variation across related species
Development of chickpea EST-SSR markers and analysis of allelic variation across related species
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DOI：
10.1007/s00122-008-0923-z
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发表时间：
2009-02-01
影响因子：
5.4
通讯作者：
Bhatia, Sabhyata
中科院分区：
文献类型：
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作者：
Choudhary, Shalu;Sethy, Niroj Kumar;Bhatia, Sabhyata
尽管鹰嘴豆是第三重要的豆科粮食作物，但基因组资源有限，尤其是基于表达序列标签（EST）的标记。在本研究中，我们从未成熟种子中产生了822个鹰嘴豆EST，并利用鹰嘴豆数据库中的1309个EST，总共使用2131个EST序列来开发功能性EST - SSR标记。鉴定出246个简单序列重复（SSR）基序，据此设计了183对引物，其中60对被验证为功能性标记。对30份鹰嘴豆种质进行的遗传多样性分析显示，有10个标记具有多态性，共产生29个等位基因，观察到的杂合度平均值为0.16，从而表现出较低的种内多态性水平。然而，这些标记在6个一年生鹰嘴豆属物种间的跨物种转移性较高，范围为68.3% - 96.6%，在7个豆科属间为29.4% - 61.7%。对来自不同物种的大小变异扩增子的序列分析表明，大小多态性是由多种事件导致的，例如微卫星重复序列以及侧翼区域的拷贝数变异、点突变和插入/缺失。有趣的是，在具有系统发育信息的鹰嘴豆属物种中观察到广泛存在的杂交群特异性序列变异。邻接树状图清楚地将鹰嘴豆栽培品种与野生鹰嘴豆区分开来，并验证了犹太鹰嘴豆（C. judaicum）与羽裂叶鹰嘴豆（C. pinnatifidum）的亲缘关系。因此，本研究首次深入了解了鹰嘴豆转录区域中SSR的分布，并展示了基因SSR的开发和利用。除了证明它们适用于遗传多样性分析外，它们的高转移性也证明了它们在比较基因组研究以及追踪野生种的基因渗入和进化方面的潜力，野生种构成了鹰嘴豆宝贵的二级基因库。
Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.