基于沙棘转录组序列开发EST-SSR分子标记

Development of EST-SSR Markers Based on Seabuckthorn Transcriptomic Sequences

[目的]本研究通过对蒙古沙棘"向阳"品种的转录组序列进行SSR引物开发,为沙棘亲本分析、遗传多样性和遗传育种等研究提供支持。[方法]利用已测得的转录组序列进行分析和筛选,整理具有SSR位点的序列,进行引物设计,随机挑选179条引物进行验证。[结果]得到具有SSR位点的EST序列17 383条,其中,单核苷酸、二核苷酸和三核苷酸重复基元分别占62.77%、21.82%和13.77%;二核苷酸重复基元类型以AT和AG为主,三核苷酸重复基元类型以AAG、ATC和ATA为主。9 291条序列设计出扩增EST-SSR位点的引物,随机合成的179对引物中,142对扩增成功,选取其中92个位点的扩增产物上机检测,40个SSR位点(43.48%)呈现多态。对扩增稳定且峰图清晰的17个多态位点的进一步分析,得到蒙古沙棘天然群体在各位点的观测杂合度(HO)为0.083 0.875,期望杂合度(HE)为0.180 0.750。[结论]本研究开发出的EST-SSR标记,为后期进行沙棘属植物遗传多样性分析、遗传图谱构建及分子育种等研究提供了重要支持。

[Objective]The transcriptomes of Hippophae rhamnoides subsp. mongolica cv‘Xiangyang＇were analyzed to design primers and develop EST-SSR（ Expressed Sequence Tags-Simple Sequence Repeat） markers.[Method]The primers were designedand the SSR was developed based on the Expressed Sequence. 179 pairs of primers were validated randomly. [Result]17 383 SSR-ESTs（ SSR-containing EST） were identified. Of the total EST-SSRs,the mononucleotiderepeat was the most dominant type,accounting for 62. 77%,followed by dinucleotiderepeats and trinucleotiderepeats,accounting for 21. 82% and 13. 77%,respectively. AT and AG were the most abundant dinucleotide motifs; AAG,ATC and ATA were repeated dominant motifs in trinucleotide. Based on these SSR-ESTs,9 291 pairs of EST-SSR primers were designed; 179 pairs of primers were randomly selected and compounded for PCR amplification,among which 142 loci were amplified successfully. Amplificationproductions of 92 loci were genotyped using the DNA analyzer,of which 40 loci（ 43. 48%） were identified as polymorphism. At last,17 loci that amplified highly successfully and genotyped easily were recommend and analyzed for natural H. rham-noides subsp. mongolica individuals,with observedheterozygosity（ HO） ranged from 0. 083 to 0. 875 and expected heterozygosity（ HE） ranged from 0. 180 to 0. 750. [Conclusion]These EST-SSR markers would be valuable for further research on genetic diversity analysis,linkage mapping construction and molecular breeding of the genus Hippophae.