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为研究山桐子遗传多样性,对山桐子转录组数据进行分析,开发SSR分子标记技术.将山桐子高通量转录组测序获得的84 213条Unigene进行简单重复序列(SSR)位点挖掘和分析,并结合引物验证,初步证明其可行性.通过软件分析,共获得含SSR位点的序列数23 077,出现频率为27.40%,涉及序列数量为29 953条,发生频率为35%.SSR序列中包括1 620种重复基元类型,其中单核苷酸、二核苷酸和三核苷酸为优势重复类型,SSR位点数分别为9 782(32.67%)、6 921(23.11%)和5 420(18.10%).单核苷酸中A/T类型比例最高,为9 630(32.15%),二核苷酸和三核苷酸中以类型AG/CT(4 679;15.62%)和AAG/CCT(1 220;1.53%)为主.SSR位点重复次数差别较大,主要是3次(4 748;15.70%),其次为6次(3 707;12.25%)和5次(3 475;11.60%).运用多态性分析的方式初步验证SSR位点在不同地区山桐子标记中的可行性.同时,利用Primer 5.0进行引物设计,随机筛选100对引物进行验证,31对引物可以扩增出条带,19对引物扩增出预期大小的条带,8对引物能特异性区分宜宾、资阳、宁强、成都4个不同地区的山桐子.本研究通过分析山桐子高通量转录组序列的SSR信息,筛选出8对引物验证不同地区山桐子的遗传多样性,将有助于山桐子基因挖掘、分子标记育种和资源保护等后续工作的开展.
In order to study the genetic diversity of Jatropha curcas, the SSR molecular marker technology was developed for the analysis of the Shanhaiguanzi transcriptome data, and 84 213 Unigene sequences obtained from the high-throughput transcriptome of the Jatropha curcas were excavated and analyzed by Simple Repeat (SSR) , And combined with primer verification, initially proved its feasibility.A total of 23 077 SSR loci were obtained by software analysis, the frequency of occurrence was 27.40%, involving 29 953 sequences and the frequency of occurrence was 35% Among them, there are 1 620 types of repeat motifs, of which mononucleotide, dinucleotide and trinucleotide are dominant repeat types, and the number of SSR loci is 9 782 (32.67%), 6 921 (23.11%) and 5 420 (18.10%). The highest percentage of A / T types was found in single nucleotide, which was 9 630 (32.15%). Among the dinucleotide and trinucleotide types, AG / CT / CCT (1 220; 1.53%). There were significant differences in the number of repetitions of SSR loci, mainly three times (4 748; 15.70%) followed by 6 times (3 707; 12.25%) and 5 times ; 11.60%). The polymorphism analysis method was used to verify the feasibility of SSR loci in different regions of Jatropha curcas. At the same time, Primer 5.0 primer design, random screening of 100 pairs of primers for testing , 31 pairs of primers could amplify the bands and 19 pairs of primers amplified the bands of expected sizes and 8 pairs of primers could specifically distinguish C. yacon, Ziyang, Ningqiang and Chengdu jacaranda in four different regions.Through analysis The results showed that the genetic diversity of Jatropha curcas in different areas was screened by 8 pairs of primers, which would be helpful for subsequent researches on gene excavated, molecular marker breeding and resource conservation of Jatropha curcas.