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为获得有效的分子标记,并应用于柱花草的遗传学研究中。本研究基于柱花草转录组测序获得的36 558条EST序列,利用MISA和PRIMER 3.0软件进行EST-SSR标记开发,对所开发标记进行特征分析,最终获得柱花草新EST-SSR标记2 008对,然后随机合成523对SSR标记并对其有效性及应用效果进行评价。其中主要重复类型是二核苷酸、三核苷酸,分别占30.50%和50.33%,在二、三核苷酸中的优势重复类型分别为AG/CT,AAG/CTT。在随机合成的523对引物中,472对引物都有扩增产物,扩增效率达到90.25%,其中有多态性的引物179对,占可扩增引物的37.92%,具有较高的多态性。利用新EST-SSR标记分析表明55份F_1单株均为真杂种,并能对F_2群体单株进行基因型鉴定。本研究开发的新EST-SSR标记将为柱花草遗传图谱的构建、基因克隆、指纹图谱构建奠定基础。
In order to obtain effective molecular markers, and applied to the genetics of Stylosanthes. In this study, 36 558 EST sequences were obtained from the transcriptome of Stylosanthes. EST-SSR markers were developed using MISA and PRIMER 3.0 software. The developed markers were characterized and finally 2 008 EST-SSR markers were obtained. Then 523 pairs of SSR markers were randomly synthesized and their effectiveness and application effect were evaluated. The main repeat types were dinucleotide and trinucleotide, accounting for 30.50% and 50.33%, respectively. The dominant repeat types in the two and three nucleotides were AG / CT and AAG / CTT respectively. Of the 523 randomly synthesized primers, 472 pairs of primers all had amplification products with an amplification efficiency of 90.25%, of which 179 pairs were polymorphic, accounting for 37.92% of the amplifiable primers with high polymorphism Sex. The analysis of EST-SSR markers showed that 55 F1 plants were all true hybrids and could be used to genotype F 2 individuals. The new EST-SSR marker developed in this study will lay the foundation for the construction of Stylosanthes guianensis genetic map, gene cloning and fingerprinting.