杜仲性别相关EST-SSR标记的开发

Development of Gender-Related EST-SSR Markers in Eucommia ulmoides

【目的】杜仲为雌雄异株多年生木本植物,具有重要的药用价值与经济价值,其利用价值因植株性别而异,尤其是杜仲胶含量及其他次生代谢产物在雌雄植株中都存在差别,但杜仲在开花前采用形态学和细胞学方法很难分辨其性别,且需要7年以上才能开花。因此,本研究应用EST-SSR标记开发杜仲性别相关的分子标记,以期能在早期鉴定出雌雄植株,提高杜仲资源利用率。【方法】根据杜仲雌雄植株转录组文库中EST-SSR的两翼序列设计引物,利用来源于7个产地的杜仲雌雄植株各6份DNA样品筛选引物,PCR扩增获得具性别差异的扩增产物,将扩增产物进行克隆及序列分析,应用雌雄植株各12份DNA样品检验性别标记的可靠性。【结果】从140对EST-SSR引物中筛选出1对引物EST-Eu059,在雌、雄植株中产生差异条带,雌、雄植株均有1条约160 bp的序列(分别命名为Eu-f160和Eu-m160),而雄株多1条约120 bp的序列(Eu MSM)。序列分析发现,Eu-f160与Eu-m160均为156 bp,具有重复单元(GT)6,且仅有1个碱基不相同,即雌株重复单元内第3个G碱基变成了A,可能发生了突变;Eu MSM为112 bp,缺失了(GT)6重复单元。经Blastn比对,Eu MSM序列与Gen Bank数据库中的现有序列没有同源性。用杜仲雌、雄植株基因组DNA各12份进行验证表明,在所有雄株中都有1条雄性特异序列。【结论】本研究筛选出了1对可以鉴别杜仲雌雄株性别的引物EST-Eu059,该标记在雄株中具有1条特异序列片段Eu MSM。该标记可在杜仲生长早期快速、可靠地鉴定其性别。

【Objective】Eucommia ulmoides is a dioecious perennial woody plant with high medicinal and economic values. The utilization value was different between male and female of E. ulmoides,especially of the gutta-percha and other secondary metabolites. Also,it was difficult to identify sex by morphological or cytological methods before flowering,and the trees need more than 7 years to bloom. EST-SSR markers were used to find sex-associated ones,and expected to identify sex at early stage for improving the utilization of E. ulmoides resources.【Method】We screened primers by using DNA samples of 6 male and 6 female plants from 7 different regions,and the primers ware designed based on the SSRs locus flanking sequences of dioecious transcriptome library of E. ulmoides. We obtained gender different products by PCR which has been cloned and analyzed,then another 12 male and 12 female plants were used to test reliability of the gender markers.【Result】The result showed that only one pair of primers（ EST-Eu059） out of 140 pairs of EST-SSR primers displayed different bands between male and female,a common band was about 160 bp（ named as Eu-f160 and Eu-m160）for both male and female plants,with an extra band of 120 bp approximately（ named as Eu MSM） in all males. Sequence analysis indicated that Eu-f160 and Eu-m160 were 156 bp,with the repeat unit（ GT）6,and only 1 base was different,that was,the third G base in the female repeat unit became A; and Eu MSM was 112 bp without（ GT）6. By Blastn comparison,Eu MSM was not homologous with the sequences in the Gen Bank database. We confirmed that there was a male-specific band in all male plants. 【Conclusion 】We selected one pair of primers EST-Eu059 that can be used toidentify male and female plants of E. ulmoides,which had a specific band Eu MSM in male plants. This marker can be used to identify sex of E. ulmoides quickly and reliably at an early stage.