坛紫菜EST-SSR筛选及其在遗传多样性分析中的实用性

Development and Practicability of EST-SSR Markers in Porphyra haitanensis

微卫星标记作为1种重要的分子标记,在分子标记辅助育种及遗传图谱构建中都起到重要的作用。EST文库搜索法是获得微卫星标记的途径之一,本研究对坛紫菜EST数据库中6035条序列进行搜索,发现340条EST包含SSR序列,其中三碱基重复最多,占总数45.95%;其次是两碱基重复,占总数的45.38%;再次是六碱基和五碱基,分别占总数的5.78%和2.02%;四碱基最少,仅占总数的0.87%。在两碱基中AG,GA,TC,CT类型数量最多,占所有两碱基SSR的82.8%;在三碱基中CCG,CGC,GCC,GGC,GCG,CGG类型数量最多,占所有三碱基SSR的52.83%;其它重复次数中,各种类型所占比例相差不大。进一步比较坛紫菜与条斑紫菜EST-SSR序列,发现2种紫菜SSR类型及比例存在明显差异。根据EST-SSR序列设计合成了10对引物,其中4个位点在8个不同来源坛紫菜叶状体间存在多态性,说明从坛紫菜EST中筛选的SSR标记可以用于进行坛紫菜遗传多样性分析。

Simple sequence repeats （SSRs） or microsatellites are important molecular markers, which have been applied to marker-assisted selection and construction of genetic mapping. Microsatellite markers can be developed from expressed sequence tag（EST） in public databases directly. In this paper we analyzed 6 035 EST sequences of the Porphyra haitanensis dbEST from NCBI database, in which 340 EST were found to contain SSRs. The length of repeat motifs ranged from 2 bp to 6 bp, among which trinucleotide and dinucleotide repeat motifs were most common with abundance of 45.95 % and 45.38 %, respectively; followed by hexanucleotide and pentanucleotide（respectively 5.81% and 2.03 % ） and tetranucleotide（0.87 % ）. AG, GA, TC, CT units were found to be the most abundant in dinucleotide（82.8% ）, so did CCG, CGC, GCC, GGC, GCG, CGG units in trinucleotide（52.83 % ）, however there were no significant differences among other types. Furthermore, EST-SSRs from P. yezoensis were compared to that of P. haitanensis and the difference existed not only in SSR types but also in quantity of repeat motifs. Ten EST-SSR primers were designed to analyze the genetic polymorphisms of eight different sources thallus and four loci showed polymorphisms, which indicated that SSRs from P. haitanensis dbEST are feasible in polymorphism analysis.