红松转录组SSR分析及EST-SSR标记开发

doi:10.11707/j.1001-7488.20150815

摘要/Abstract

摘要：

[目的] 红松已开发的分子标记缺乏共显性的遗传标记,尚不能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,本研究利用高通量测序技术开发红松EST-SSR标记,掌握其在转录组序列中的分布类型及特征,为红松的SSR多样性分析及变异分析提供基础。[方法] 利用SSR检索程序从红松转录组41 476条Unigenes中筛选得到1 757个SSR位点,并对SSR位点的数量、分布特征进行统计分析。设计合成101对SSR引物,采用琼脂糖电泳初步检查和聚丙烯酰胺凝胶电泳分离检测方法确定引物的多态性,并收集扩增产物,送测序验证,最终开发出16对SSR引物。合成6对荧光引物,采用荧光标记技术对来自黑龙江省鹤岗、林口、铁力、苇河4个种子园的53份自由授粉子代进行遗传多样性分析。[结果] 基于转录组序列开发出的EST-SSR的分布频率(SSR的个数与总Unigene的数量比)为4.24%。单、二、三核苷酸重复单元分别占总SSR的46.90%,17.12%和34.66%; SSR重复单元的重复次数分布在5~24次之间。参试的101对引物中有21对引物扩增可检测出多态性位点,占引物总数的20.8%; 重测序验证,有16对引物能够扩增出目标序列。6对荧光引物共检测出18个等位基因,多态性信息量(PIC)为0.036 3~0.667 4,平均为0.325。[结论] 红松属于基因组序列比较庞大的裸子植物,本研究可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。所研究红松种子园子代属于中等多态性水平。本研究印证了利用红松转录组数据开发SSR标记的可行性,同时采用荧光标记技术检测红松自由授粉子代材料,为红松种质资源的多样性水平分析及变异水平的研究提供基础。

关键词: 红松, 转录组, EST-SSR, 遗传多样性

Abstract:

[Objective] In Korean pine(Pinus koraiensis) breeding programs, lack of co-dominant genetic markers constrained the development of molecular marker assisted breeding. At present, development of SSR markers based on transcriptome data is still an economic and efficient development strategy of DNA molecular markers. In this study, we used high-throughput sequencing technology to develop EST-SSR markers for Korean pine. Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed, in order to provide a basis for analysis of SSR diversity and mutation of Korean pine. [Method] A total of 1 757 SSR sites were identified from 41 476 unigenes in Korean pine transcriptome by using SSR searching program. Statistical analyses were conducted for number, distribution and characteristics of the SSR loci. And 101 pairs of SSR primers were designed and synthesized. Agarose electrophoresis was used for initial check and polyacrylamide gel electrophoresis for separation and detection of the polymorphisms of primers. Then amplification products were collected and sequenced for validation. Finally, 16 pairs of SSR primers and 6 pairs of fluorescence primers were identified. To study the genetic variation, 53 samples of open-pollinated progeny were collected from four seed orchards respectively in Hegang, Linkou, Tieli and Weihe. [Result] The distribution frequency of EST-SSRs (ratio of the number of SSRs to the total number of unigenes) was 4.24%, based on the transcriptome sequences. Mononucleotide dinucleotide and trinucleotide repeats were 46.90%, 17.12% and 34.66% of total SSR, respectively. The SSR repeat number of SSR repeat units was between 5 and 24. Twenty-one pairs of primers showed polymorphism among 101 pairs of primer, which accounting for 20.8% of the total number of primer pairs. By sequencing validation, 16 pairs of primers amplified the target sequence. Eighteen alleles were tested from 6 pairs of fluorescence primers. Polymorphic information content (PIC) was 0.036 3-0.667 4 and the mean was 0.325 0. [Conclusion] Korean pine has a relatively large genome in gymnosperms. The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats. The progeny of the Korean pine seed orchard revealed a medium level of polymorphism. This study demonstrated that using Korean pine transcriptome data to develop SSR markers was feasible. The technique of using fluorescent markers to analyze the progeny materials provided a basis for studies of genetic diversity and variation of the Korean pine germplasm resources.

Key words: Pinus koraiensis, transcriptome, EST-SSR, genetic diversity

中图分类号:

S718.46

张振, 张含国, 莫迟, 张磊. 红松转录组SSR分析及EST-SSR标记开发[J]. 林业科学, 2015, 51(8): 114-120.

Zhang Zhen, Zhang Hanguo, Mo Chi, Zhang Lei. Transcriptome Sequencing Analysis and Development of EST-SSR Markers for Pinus koraiensis[J]. Scientia Silvae Sinicae, 2015, 51(8): 114-120.

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