Sethy Niroj Kumar, Shokeen Bhumika, Edwards Keith J, Bhatia Sabhyata
National Centre for Plant Genome Research, Jawaharlal Nehru University Campus, Post Box No. 10531, New Delhi, 110067, India.
Theor Appl Genet. 2006 May;112(8):1416-28. doi: 10.1007/s00122-006-0243-0. Epub 2006 Mar 14.
Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT)n and (CT/GA)n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08-0.91) and 0.74 (0.23-0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.
鹰嘴豆（Cicer arietinum L.）中多态性分子标记的匮乏一直是改良这种重要豆类的主要限制因素。因此，为了从鹰嘴豆中开发序列标签微卫星位点（STMS）标记，构建了来自鹰嘴豆品种Pusa362核基因组的微卫星富集文库，用于鉴定（CA/GT）n和（CT/GA）n微卫星基序。共鉴定出92个新的微卫星，其中开发了74对功能性STMS引物对。使用9个鹰嘴豆品种和1个网脉鹰嘴豆种质对这些标记进行了验证。在开发的STMS标记中，25个多态性标记用于分析36个地理来源不同的鹰嘴豆种质的种内遗传多样性。25对引物扩增出单一位点，产生的等位基因最少为2个，最多为11个。共检测到159个等位基因，平均每个位点6.4个等位基因。观察到的杂合度值和预期杂合度值平均分别为0.32（0.08 - 0.91）和0.74（0.23 - 0.89）。基于UPGMA的聚类图能够区分除两个来自阿富汗的种质外的所有种质，这表明微卫星能够成功检测鹰嘴豆的种内遗传多样性。此外，对两个微卫星位点大小变异等位基因的克隆和测序表明，不同等位基因中AG重复序列数量的变化是多态性的主要来源。发现在完美重复序列的长片段内部和紧邻上游均发生了点突变，从而导致完美基序转变为不完美或复合基序。此类事件可能是为了限制微卫星的扩展，同时也导致了新微卫星的产生。本研究中开发的微卫星标记将有助于遗传多样性分析、连锁图谱构建以及描绘种内微卫星进化。
