荔枝EST资源的SSR信息分析及EST-SSR标记开发

 【目的】明确荔枝EST序列中SSR的总体特点，开发荔枝EST-SSR引物，为利用EST-SSR分子标记进行荔枝种质资源遗传多样性、连锁图谱构建及亲缘关系研究奠定基础。【方法】应用SSRIT软件，按照设定标准从自行构建的一个荔枝果皮发育关键期的cDNA文库中的1 331条Unigene（惟一序列）中搜索SSR位点。利用软件Primer primer5.0设计EST-SSR引物。选用16份表型差异较大的荔枝种质资源检测引物的有效性及多态性，利用聚丙烯酰胺凝胶(PAGE)进行片段分离。【结果】荔枝的1 331条EST序列中共搜索出220个SSR位点，分布于189条EST中，出现频率是16.53%。这些EST-SSR的平均长度为18.43 bp，平均分布频率1/4.42 kb。在1—6 bp的重复基元中，二核苷酸和三核苷酸重复类型占主导地位，共占总SSR的69.09%，二者出现的频率分别为37.27%和31.82%。共观察到72种重复基元，出现频率最高的是GA/TC（16.82%）；其次是AG/CT、A/T、AT/TA及GAA/TTC，频率分别为14.55%、11.82%、5.00%和3.64%。不同核苷酸数目的重复基元的重复次数差异很大。设计、合成150对EST-SSR引物，122对（81.33%）引物在荔枝中获得有效扩增，100对引物具有多态性。【结论】荔枝EST资源中含有高频率的SSR位点，且EST-SSR 标记开发效率较高。本研究为利用EST-SSR标记开展荔枝遗传研究提供了100 对EST-SSR引物，并为进一步开发荔枝EST-SSR标记提供了含SSR位点的候选序列。

Characteristics of SSRs Derived from ESTs and Development of EST-SSR Markers in Litchi (Litchi chinensis Sonn.)

 【Objective】 The objective of this study was to characterize litchi SSRs derived from ESTs and to develop EST-SSR markers for possible use of the markers in the studies of litchi genetic diversities, linkage mapping and phylogenetic relationships.【Method】 SSRIT (simple sequence repeat identification tool) was used to search SSRs in 1 331 unigenes obtained from a cDNA library of pericarp cell enlargement period of litchi. EST-SSR primers were subsequently designed using Primer5.0. The validity and revealing power of the primers were tested by PCR amplification of 16 litchi germplasm accessions showing apparent morphological variations and by PAGE analysis of the PCR products. 【Result】 A search of 1 331 Unigene sequences of litchi identified a total of 220 SSRs that occurred in 189 Unigenes. The average SSR density was one SSR per 4.42 kb of EST sequences, and the average SSR length was 18.43 bp. The dinucleotide and trinucleotide repeats, which were 37.27% and 31.82%, respectively, were the most abundant SSRs detected and accounted for 69.09% of the total. Among the 72 observed repeat motifs, GA/CT was the most abundant (16.82%), followed by AG/TC(14.55%), A/T(11.82%)，AT/TA(5.00%) and GAA/CTT (3.64%). The number of repeats in a SSR was quite different with different repeat motifs. Among the 150 EST-SSR primer pairs designed, 122 (81.33%) yielded ideal PCR products, and 100 of them showed polymorphic bands. 【Conclusion】 There were abundant SSR loci in ESTs of litchi, and the efficiency of developing SSR markers from litchi ESTs was high. The study has generated 100 EST-SSR markers which are potentially useful in the analysis of genetic variations of litchi. More SSR markers could be developed from EST-SSRs identified in this study.