Development and mapping of EST-SSR markers in wheat | 小麦EST-SSR标记的开发和遗传作图

Chino. 【目的】利用小麦EST序列数据库开发EST-SSR标记。【方法】对GenBank/dbEST注册的普通小麦EST序列（2006.4.18―2007.2.4）进行SSR查找,采用Primer5.0软件设计EST-SSR引物,选用3个小麦品种进行有效性检测,利用RIL群体和Mapmaker/Exp3.0软件进行遗传作图。【结果】在265 362条普通小麦EST序列中,共发现6 314个SSR,占整个EST数据库的2.38%。其中二核苷酸、三核苷酸重复序列最多,分别为2 237（35.43%）和2 084（33.01%）个。二核苷酸重复中,以GA/CT和AG/TC出现频率最高、分别占SSR总数的17.85%和10.37%,其次是CA/GT（4.07%）和AC/TG（2.53%）;三核苷酸重复中,CAA/GTT（3.93%）、CGG/GCC（3.83%）、CGC/GCG（3.36%）、GGC/CCG（3.14%）、CTT/GAA（2.53%）、TGC/ACG（2.27%）以较高的频率出现。根据筛选得到的微卫星序列共设计了596个EST-SSR引物对,选择其中95分以上的194个合成。PCR检测表明,165个引物对（85%）可以扩增出稳定清晰的带型;在RIL群体中检测到21个EST-SSR引物26个位点有多态性,将其中的23个位点整合到已有的小麦遗传图谱上。【结论】开发了165个小麦EST-SSR新标记,EST序列是小麦SSR标记的重要来源。

Inglés. 【Objective】 The objective of this study was to develop new simple sequence repeat (SSR) markers from EST (Expressed sequence tag) sequences (EST-SSR markers) for wheat. 【Method】 The wheat EST sequences from GenBank/dbEST (from April 18, 2006 to February 4, 2007) were used to search for SSRs, EST-SSR primer pairs were designed using Primer5.0 software, the validity of primer pairs were detected using three wheat varieties, and the marker loci were mapped using RIL population and Mapmaker/Exp3.0 software.【Result】From the 265 362 wheat EST sequences released in GenBank/dbEST, 6 314 SSRs were found which amounts to 2.38% of the total number of ESTs. Among those EST-SSRs, the most motifs were dinucleotides and trinucleotides, which were 2 237 (35.43%) and 2 084 (33.01%), respectively. For dinucleotides, GA/CT (17.85%) and AG/TC (10.37%) were the most frequent repeat, followed by CA/GT (4.07%) and AC/TG (2.53%); and for trinucleotides, CAA/GTT (3.93%), CGG/GCC (3.83%), CGC/GCG (3.36%), GGC/CCG (3.14%), CTT/GAA (2.53%) and TGC/ACG (2.27%) appeared high frequency. Based on these EST-SSR sequences, 596 EST-SSR primer pairs were designed, 194 of which were synthesized, and 165（85%）led to PCR amplification products. Using a RIL population, the polymorphism of 21 EST-SSR primer pairs with 26 loci were detected, and 23 of those loci were integrated on the wheat genetic map. 【Conclusion】 A total of 165 new wheat EST-SSR markers have been developed. EST sequences are important sources for developing SSR markers.