基于石蒜属转录组序列的SSR分子标记开发应用

为开发石蒜属简单重复序列(SSR)分子标记,并研究SSR引物在石蒜属内的通用性,本研究对石蒜属石蒜、忽地笑、中国石蒜、长筒石蒜、换锦花、香石蒜6个种质转录组测序,检测SSR位点并设计引物,通过PCR扩增和毛细管电泳判断引物的有效性和多态性,绘制石蒜属17个种质资源的指纹图谱并对杂交后代的真实性进行早期检测。结果表明,共获得404 481条Unigenes,利用数据库进行同源比对和功能注释,并对Unigene进行SSR位点挖掘和分析,共检测到59 612个SSR位点。其中,单核苷酸重复>二核苷酸重复>三核苷酸重复,分别占SSR总数的62.88%、20.06%和14.66%,四核苷酸及以上重复单元相对较少。选取并合成8对荧光引物进行PCR扩增,通过毛细管电泳检测发现,8对荧光引物共检测到60个多态位点,多态位点数平均为7.50,多态性信息含量(PIC)值变化范围为0.148 0~0.940 8,平均值为0.593 0。利用引物扩增带型组合法构建了石蒜属17个种资源的指纹图谱,其中引物QZ209可区分所有供试材料,并可用于杂交后代鉴定。本研究开发的SSR标记具有丰富的多态性,在石蒜属植物的资源多样性分析、杂交种鉴定及遗传图谱的构建应用中具有重要意义。

Development and Application of SSR Molecular Markers Based on Transcriptome Sequencing of Lycoris spp.

In order to develop SSR molecular markers and to study the universality of SSR primers in Lycoris spp., in this study, six species of Lycoris spp. including Lycoris radiata, Lycoris aurea, Lycoris chinensis, Lycoris longituba, Lycoris sprengeri, and Lycoris incarnata were RNA sequenced. SSR loci were detected and primers were designed. The validity and polymorphism of primers were detected by PCR amplification and capillary electrophoresis. The fingerprints of 17 species of Lycoris were drawn, and early detection of offspring authenticity in hybrids was carried out. The results showed that, a total of 404 481 Unigenes were obtained after de novo assembly. Five public databases were used for homology comparison and functional annotation. A total of 59 612 SSR loci were detected by simple sequence repeat analysis. Among them, the number of single nucleotide repeats>dinucleotide repeats>trinucleotide repeats, which accounted for 62.88%, 20.06%, and 14.66% of the total SSRs, respectively. There were relatively few repeats of four bases or more. Eight pairs of fluorescent primers were selected and synthesized for PCR amplification. In total, 60 polymorphic alleles were detected using eight SSR fluorescent labeling markers, with an average of 7.50 alleles per locus. The polymorphism information content ranged from 0.148 0~0.940 8, with an average of 0.593 0. The fingerprints of 17 species ofLycoris spp. were constructed according to the combinations of amplified bands. Primer QZ209 could distinguish all tested materials, and which could be used for the identification of hybrid offspring. In conclusion, the SSR markers developed in this study are rich in polymorphisms, which are of great significance in genetic diversity analysis, hybrid identification, and construction of a genetic map of Lycoris spp.