铁皮石斛EST-SSR分子标记的开发

Development of EST-SSR Molecular Markers in Dendrobium officinale

为了开发铁皮石斛EST-SSR分子标记,利用SSRIT软件对铁皮石斛近缘物种-金钗石斛的15 383条EST序列进行SSR位点查找,利用Primer 5和Oligo 7软件进行引物设计和评价,之后经PCR扩增和琼脂糖凝胶电泳对引物进行初步筛选,并通过聚丙烯酰氨凝胶电泳对所筛选的引物进行有效性检测。结果表明,SSRIT软件共查找到370条SSR位点序列,共有379个SSR位点,候选SSR位点出现的频率为2.46%。二核苷酸、三核苷酸和六核苷酸重复是最主要的重复类型,分别占41.42%、26.91%和27.44%,其中二核苷酸重复中AG/CT(20.84%)和GA/TC(18.73%)最丰富;三核苷酸重复中TCT/AGA(4.22%)和GA/TC(3.96%)最丰富;而六核苷酸重复基元较多。EST-SSR平均长度为24 bp,平均每8.5条EST序列包含1个SSR位点。设计的106对EST-SSR引物中有50对能扩增出理想的PCR产物,引物有效扩增率为47.17%,其中有43对能够扩增出多态性条带,占可扩增引物的86%。本研究开发的43对引物为铁皮石斛遗传多样性分析、种质鉴定、遗传图谱构建和功能基因研究等提供了重要的参考价值。

To develop EST-SSR molecular markers for Dendrobium officinale, 15 383 ESTs from Dendrobium nobile （Related Species of Dendrobium officinale） in NCBI were screened using SSRIT software. The primers were designed and evaluated by using Primer 5 and Oligo 7 software, followed by agarose gel electrophoresis for preliminary screening after PCR amplification, and reusing polyacrylamide gel electrophoresis to detect the effectiveness of the selected primers. The results showed that 379 SSRs were mined from 370 ESTs with a frequency of 2.46%. Dinucleotide, Trinucleotide and Hexanucleotide nucleotide repeats EST-SSRs were dominant, which account for 41.42%, 26.91% and 27.44%, respectively. Among them, AG/CT （20.84%） and GA/TC （18.73%） were highly abundant in Dinucleotide nucleotide repeats, TCT/AGA（4.22%）and GA/TC（3.96%）were highly abundant in Trinucleotide nucleotide repeats, and Hexanucleotide repeat motifs were higher. The average length of EST-SSR was 24bp, and every 8.5 EST sequences contained a SSR site. Among 106 designed primer pairs, 56 primer pairs showed ideal amplifications and the effective amplification rate was 47.17%. Moreover, 43 primer pairs displayed polymorphic, which accounts for 86% of the effective amplification. Taken together, 43 primer pairs developed in this study could be used in genetic diversity analysis, germplasm identification, genetic map construction and functional gene study of Dendrobium officinale to provide an important reference value.