Chandra Amaresh
Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi - 284 003, India.
J Environ Biol. 2011 May;32(3):347-54.
In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60 degreesC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. Atotal of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNAfrom 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55 degreesC and 32.3% at 60 degreesC annealing temperature. 24 alleles of the total 50 (48%) at 55 degreesC and 27 of 51 (53%) at 60 degreesC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where SequentialAgglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.
一般来说，热带饲用豆科植物缺乏微卫星或简单序列重复（SSR）标记。从表达序列标签（EST）数据库开发基因SSR标记是为这类作物物种的基因组分析生成标准DNA标记的一种替代且有效的方法。在本文中，从蒺藜苜蓿的187763个EST中鉴定出总共816个EST - SSR，其中包含单核苷酸（33.5%）、二核苷酸（14.7%）、三核苷酸（39.3%）、四核苷酸（2.7%）、五核苷酸（0.7%）和六核苷酸（0.4%）的完美重复序列。此外，还观察到70个（8.5%）复合型SSR。测试了七对三核苷酸重复的引物对在包含11个属的19份饲用豆科植物材料中的交叉转移性。在两个不同的退火温度（55和60℃）下，除AJ410087外的所有引物对都能与许多饲用豆科植物材料发生反应。用六个蒺藜苜蓿EST - SSR引物对检测来自代表11个属的19份材料的DNA，共检测到51个等位基因，等位基因数量范围为2至13个。在55℃退火温度下这些EST - SSR的交叉转移性为40.6%，在60℃退火温度下为32.3%。在55℃时，50个等位基因中的24个（48%）以及在60℃时51个等位基因中的27个（53%）在这些材料中具有多态性。这些鉴定出的27个多态性扩增产物可作为DNA标记。本研究证明从蒺藜苜蓿EST开发的SSR标记是有价值的遗传标记，并且还提出了在具有饲用重要性的豆科不同属物种之间转移这些标记的可能性。从一组多花合欢材料获得的结果可以明显看出，基于Dice相似性的顺序凝聚层次和嵌套（SAHN）聚类分析以及算术平均的非加权配对组方法（UPGMA）揭示了材料之间存在显著的变异性（24%至74%）。高自展值（>30）支持了材料聚类分析生成的节点。
