蛭类转录组中EST-SSR分析及抗凝血相关分子标记的挖掘

Analysis on EST-SSR in transcriptome of medicinal leeches and mining of anticoagulant related functional molecular markers

目的对蛭类转录组进行SSR分析,是开发功能分子标记、开展对药用蛭类资源的开发利用及进行遗传背景监测的必要手段。方法利用与医学密切相关的医蛭科棒纹牛蛭Poecilobdella javanica和日本医蛭Hirudo nipponia、黄蛭科宽体金线蛭Whitmania pigra和山蛭科洞穴山蛭Heamadipsa cavatuses sp.nov.共14个样本,通过RNA测序及转录组分析,筛选吸血差异unigene,并比较4个种的SSR标记特征和分布,用于挖掘抗凝血相关功能EST-SSR。结果通过SSR密度分析发现,SSR在棒纹牛蛭、日本医蛭、宽体金线蛭和洞穴山蛭转录组中的间隔长度为4 723、6 026、8 059、8 144 bp;SSR组成分析发现三碱基重复的基序占蛭类SSR类型组成中绝大多数;另外,洞穴山蛭的SSR组成与其他3种存在明显区别。比较4种吸血蛭吸血前后转录组中表达丰度显著差异的unigene群,发现4种蛭中包含SSR且抗凝血相关的重叠unigene类群在转录组中呈较高的片段多态性。结论 unigene片段长度、SSR的密度、组成和分布特点能反映物种之间的分类关系;4种蛭重叠的抗凝血相关分子功能对应的EST-SSR,可作为抗凝血相关的功能分子标记侯选EST-SSR。

Objective To expand the exploitation and genetic diversity conservation of medicinal leech resources, the effective functional molecular markers for identification are needed to be developed by transcriptome-SSR analysis. Methods Totally 14 samples including Poecilobdella javanica and Hirudo nipponia of Hirudinidae, Whitmania pigra Whitman of Haemopidae and Heamadipsa cavatuses sp. nov. of Haemadipsidae were used in transcriptomic analysis by RNA-seqencing, the simple sequence repeats （SSR） distribution characteristics of four species were compared, and transcriptomic comparation ofhematophagia samples and hungry samples were carried out to investigate the anticoagulation involved unigene. Finally, the data in two groups were combined to mining functional EST-SSR involved in anticoagulation. Results The SSR density analysis showed the gap lengths of SSR in transcriptome of Poecilobdella javanica, Hirudo nipponia, Whitmania pigra Whitman, and Heamadipsa cavatuses were 4 723, 6 026, 8 059, and 8 144 bp. In the four species, trinucleotide repeats occupied overwhelming majority in composition of EST-SSR; On the whole, distribution of SSR types call distinct from each other, and Heamadipsa cavatuses was particularly different from others. Moreover, we found that the anticoagulation involved unigene group containing SSR had high fragment-polymophism. Conclusion For the four leech species, the characteristics of density, composition, and distribution of EST-SSR can reflect its classification relationship. Meanwhile, the EST-SSRs in unigenes which involved in anticoagulation and overlaped in four leech species are ideal fragment group for screening anticoagulation functional marker.