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马尾松(Pinus massoniana)在已开发的分子标记中存在缺乏共显性的遗传标记的不足,无法满足分子标记辅助育种的需要,利用转录组数据开发SSR标记目前仍然是较为经济高效的DNA分子标记开发策略,为了全面解析马尾松种质的遗传信息,开发更多适用于马尾松的新型分子标记。本研究利用高通量测序技术开发马尾松EST-SSR标记,掌握其在转录组序列中的分布类型及特征,建立马尾松SSRPCR技术体系,并以27份马尾松无性系为材料进行验证。结果显示,在获得的70 896条基因簇(Unigene)中共检测出3 329个SSR位点,分布在3 074条Unigene上,发生频率为4.69%,平均距离为20.94 kb。马尾松转录组中优势重复基序为单核苷酸、三核苷酸和二核苷酸,分别占总SSR位点的40.16%、32.83%和20.94%。A/T、AT/AT和AAG/CTT分别是单核苷酸、二核苷酸和三核苷酸的优势重复基元,分别占总SSR重复类型的97.68%、71.45%和21.70%。随机挑选合成的200对引物中,引物扩增率为78.5%。用检测获得的24对多态性较好的引物对27份马尾松进行种质鉴定,总共扩增出137个SSR标记,多态信息含量(polymorphism information content,PIC)为0.703,平均观测等位基因数(average observed number of alleles,Na)和有效等位基因数(effective number of alleles,Ne)的平均值分别为6和3.9,香农信息指数(Shannon’s information index,I)和Nei’s基因多样性指数(Nei’s gene diversity index,H)在不同SSR位点之间分别为1.338和0.66。利用引物Pms164和Pms184可将27份无性系完全区分开:采用SSR标记人工绘制植物种质鉴别图(manual cultivar identification diagram,MCID)。供试种质的遗传相似系数为0.32~0.92,以相似系数为基础,进行了非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)聚类分析,以0.63为阈值可将供试种质分为3类。本研究基于马尾松转录组测序序列设计了一批具有高多态性潜力的SSR引物,该引物将27个马尾松无性系基本按材性或种质来源地进行了分类,利用较少的引物就获得了丰富的遗传信息。即基于转录组信息设计的SSR标记是可行的。本研究结果能够为开展马尾松种质资源构建遗传图谱、绘制指纹图谱和标定目标基因等研究提供理论依据。
The lack of co-dominant genetic markers in the developed molecular markers of Pinus massoniana can not meet the needs of molecular marker-assisted breeding. The development of SSR markers using transcriptome data is still a more cost-effective DNA molecular marker Development strategy, in order to comprehensively resolve the genetic information of Pinus massoniana germplasm development, develop more new molecular markers suitable for masson pine. In this study, EST-SSR markers of Pinus massoniana were developed using high-throughput sequencing technology to understand the distribution types and characteristics of EST-SSR markers in the transcriptome. The SSRPCR system was established and 27 Pinus massoniana clones were used for validation. The results showed that 3,329 SSR loci were detected in 70,896 gene clusters (Unigene) distributed in 3 074 Unigene, with a frequency of 4.69% and an average distance of 20.94 kb. The dominant repeat motifs in Pinus massoniana transcriptome were single nucleotide, trinucleotide and dinucleotide, accounting for 40.16%, 32.83% and 20.94% of the total SSR loci, respectively. A / T, AT / AT and AAG / CTT were the dominant repeat units of mononucleotide, dinucleotide and trinucleotide respectively accounting for 97.68%, 71.45% and 21.70% of the total SSR repeat types, respectively. Of 200 randomly selected synthetic primers, the primer amplification rate was 78.5%. Twenty-seven SSR markers were amplified using 24 pairs of primers with good polymorphism. The polymorphism information content (PIC) was 0.703 and the average observed allele Average observed number of alleles (Na) and effective number of alleles (Ne) were 6 and 3.9, respectively. Shannon’s information index (I) and Nei’s gene diversity index (Nei’s gene diversity index, H) were 1.338 and 0.66 between different SSR loci, respectively. 27 clones were completely distinguished by using primers Pms164 and Pms184: The manual cultivation identification (MCID) was drawn by using SSR markers. The genetic similarity coefficient of tested germplasm was 0.32 ~ 0.92. On the basis of similarity coefficient, the unweighted pair-group method with arithmetic means (UPGMA) Germplasm is divided into 3 categories. In this study, a series of SSR primers with high polymorphism potential were designed based on sequencing of P. massoniana transcriptome. The primer was used to classify 27 Pinus massoniana clones based on their woody or germplasm origin. By using fewer primers Get a wealth of genetic information. That is based on transcriptome information designed SSR markers is feasible. The results of this study can provide a theoretical basis for the research on genetic map, fingerprinting and target gene identification of germplasm resources of Pinus massoniana.