橡胶树EST-SSR标记的开发与应用

通过对橡胶树10 778条EST序列进行拼接, 共得到Unigene 3 090条, 从中发掘出分布于353条Unigene的430个EST-SSR位点, SSR发生频率为11.42%, 平均分布距离为3.93 kb。在橡胶树EST-SSR中, 二核苷酸和三核苷酸重复是主要的重复类型, 分别占63.49%、32.09%。TC/AG、CT/GA和CTT/GAA、AAG/TTC、AGA/TCT是二、三核苷酸的优势重复类型。利用PRIMER5.0设计引物148对, 随机挑选21对引物进行合成, 并用其中15对扩增较好的引物, 通过变性聚丙烯酰胺凝胶结合银染的方法对44个橡胶树无性系进行了遗传多样性检测, 构建了聚类分析树状图。研究结果表明, 从橡胶树EST中开发SSR标记是有效、可行的, 文章所开发的EST-SSR引物可用于橡胶树遗传分析研究。

Development and application of EST-SSR markers in Hevea brasiliensis Muell. Arg.

Three thousand and ninety Unigenes were obtained from 10 778 Hevea brasiliensis ESTs. Four hundred and thirty SSRs were distributed in 353 Unigenes, which accounts for 11.42% of the total number of Unigenes. The frequency of SSRs was 1/3.93 kb. Dinucleotide and trinucleotide repeats were the dominant types among the obtained unigenes, ac-counting for 63.49% and 32.09%, respectively. TC/AG, CT/GA and CTT/GAA, AAG/TTC, and AGA/TCT were the most abundant motifs for dinucleotide and trinucleotide motifs. One hundred and forty-eight primer pairs were designed by PRIMER5.0 and 21 primer pairs were synthesized. Among them, 15 primer pairs can produce clear and stable bands, and the PCR products were screened in denaturing polyacrylamide gel following silver staining. Genetic diversity of 44 rubber clones were investigated with these primer pairs, and a dendrogram of 44 rubber clones was built. The results indicated that it is an effective and feasible way to develop EST-SSR markers from H. brasiliensis EST sequences, and the primers de-signed in this study can be used in genetic study of rubber tree.