油菜EST-SSR标记的建立

在油菜17987条非冗余EST中,共发掘出了2083个EST-SSR,分布于2443条EST中,发生频率是13.58%,平均分布距离为4.34kb。在油菜EST-SSR中,二、三核苷酸重复是主要的重复类型,二者出现的频率相近,占总SSR的89.05%。AG/CT和AAG/CTT是二、三核苷酸中的优势重复类型,分别占二、三核苷酸重复类型的84.31%和37.71%。进一步设计了23对SSR引物,通过梯度PCR试验确定了各引物的适宜退火温度,并利用非变性聚丙烯酰胺凝胶银染对这些引物在10个油菜品种中的扩增情况和多态性进行了检测。有21对引物显示扩增,引物可用率为91.30%;有12对引物显现出多态性,占可扩增引物的57.14%。本研究结果证明根据油菜EST建立SSR标记是有效、可行的。

DATA MINING FOR SSRs IN ESTs AND DEVELOPMENT OF EST-SSR MARKER IN OILSEED RAPE

Totally 2803 SSRs distributed in 2443 ESTs were mined out and accounted for 13.58% of 17987 non-redundant ESTs from oilseeed rape,with the average distance of distribution about 4.26 kb. Dinucleotide and trinucleotide repeats are the dominant type, with similar frequency and accounting for 89.05% together in all SSRs. AG/CT and AAG/CTT are the most frequent motifs, accounting for 84.31% and 37.71% in dinucleotide and trinucleotide repeats respectively. Further, 23 primer pairs for EST-SSRs were designed and the suitable annealing temperature for each primer pair was determined by gradient PCR. The amplification and polymorphism displayed by these primers in 10 varieties of oilseed rape were detected by using silver staining of nondenaturing polyacrylamide gel. 21 primer pairs showed the amplification,accounting for 91.30% of total primers, and 12 primer sets showed polymorphisms, accounting for 57.14% of primers available. These results indicate that it is an effective and feasible approach to develop SSR markers based on ESTs in oilseed rape.