Kalyana Babu B, Pandey Dinesh, Agrawal P K, Sood Salej, Kumar Anil
Department of Molecular Biology and Genetic Engineering, College of Basic Sciences & Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, 263 145, India,
Mol Biol Rep. 2014 May;41(5):3081-90. doi: 10.1007/s11033-014-3168-8. Epub 2014 Jan 30.
In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS-LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS-LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.
近年来，DNA序列可用性的增加使得开发和探索源自表达序列标签（EST）的简单序列重复（SSR）标记成为可能。在本研究中，总共使用了1956个龙爪稷的EST来查找微卫星类型、分布、频率，并从龙爪稷的EST中开发了总共545对引物。32个EST序列具有两个以上的微卫星，1357个序列没有任何SSR重复。最常见的重复类型是三聚体基序，然而第二位是二聚体基序，其次是四聚体、六聚体和五聚体重复基序。最常见的二聚体重复基序是GA，对于三聚体SSR，是CGG。龙爪稷和水稻NBS-LRR区域的EST序列显示出更高的共线性，并且在水稻染色体图谱上的位置几乎相同。在选定的抗感龙爪稷基因型中，15个基于EST的SSR引物中有8个具有多态性。引物FMBLEST5能够将它们区分为抗感基因型。使用ABI 3130XL基因分析仪对抗感基因型特有的等位基因进行测序，发现与水稻和龙爪稷的NBS-LRR区域相似，并且包含属于NBS-LRR区域的植物R基因的特征性激酶-2和激酶3a基序。电子分析和比较分析表明，负责稻瘟病抗性的基因可以通过分子育种方法进行鉴定、定位并进一步导入，以增强龙爪稷的稻瘟病抗性。
