EST-SSR标记在水曲柳雌雄鉴定中的应用

Application of EST-SSR Markers in the Sex Identification of Fraxinus mandshurica Rupr.

选用黑龙江省青山林木种子园成年水曲柳200棵树为实验材料,在来自NCBI中的5 423条白蜡属EST序列和该实验室测得的水曲柳转录组的179 502条序列中查找SSR位点并设计引物,通过PCR扩增筛选有效扩增和有差异片段的引物,用于水曲柳雌雄的鉴别。结果显示:(1)白蜡属EST和水曲柳转录组中的5 423条和179 502条序列中,分别查找到9 488个和3 557个SSR位点,SSR出现的频率分别是174.96%和1.98%,SSR的类型都是以二核苷酸为主。(2)232对引物中有208对引物获得有效扩增,74对引物在雌雄DNA中有差异,27对扩增片段符合预定大小片段。(3)经过3轮筛选,选出19号和56号引物用于200棵水曲柳中,雌雄差异率分别是64.5%和89.5%。研究表明,利用EST-SSR分子标记对水曲柳性别进行鉴定是一种有效可行的方法。

200adults of Fraxinusmandshurica were selected from Qingshan seed orchard,Heilongjiang Province to screen primers which can result in different PCR products between male and female ash.PCR primers were designed based on the SSR sites of 5 423 ESTs of Fraxinus from NCBI and 179 502 sequences of F.mandshuricatranscriptome from our lab.The results showed that：（1）9 488 and 3 557 SSR sites（174.96% and 1.98%in relative frequency,repectively）were found in ESTs from NCBI and transcriptome from our lab,respectively,with a dominancy of dinucleotide repeat in SSR type;（2）208of 232 pairs of primer got PCR products,74 of which got different products between male and female and 27 of which got products with expected size;（3）After 3rounds of PCRs,primer 19 and 56got different products between two sexes in 64%and 89.5%trees,respectively.These results indicated that EST-SSR is a feasible method to identity the sex of F.mandshurica.