杜仲转录组SSR发掘及标记开发

SSR Mining and Marker Development in Eucommia ulmoides Oliver Transcriptome

为了加强对杜仲资源的有效保护和可持续利用,了解杜仲的遗传背景,我们基于杜仲雌雄株转录组数据库开发SSR标记,能够应用于杜仲的功能基因发掘、分子标记辅助育种、遗传多样性分析、遗传图谱构建等研究。基于杜仲雌雄株转录组数据库Unigenes序列,利用MISA软件进行SSRs序列查找及其特征分析;根据微卫星的两翼序列设计引物,以1份随机挑选的杜仲基因组DNA为PCR扩增模板,采用L9(34)正交试验设计优化SSR-PCR反应体系,检验引物扩增效果和各引物扩增条件,进一步以10份不同来源的杜仲DNA为模板检测扩增引物的有效性。发掘了74 230个SSR位点,分布在61 629条Unigenes中,占总Unigenes 33.99%,其中,12~24 bp长度的重复基序最多,基序重复次数以11~15次居多;重复基序种类主要以一、二、三重复基元类型为主,占98.6%,其中出现最多的3种重复类型分别为:A/T(73.16%)、AG/CT(10.91%)、AAG/CTT(1.14%)。采用L9(34)正交试验获得的最优SSR-PCR体系为:20μL体系中含2×Premix Taq酶(0.05μmol·min-1·μL-1)10μL、DNA模板(25 ng/μL)1μL,引物(10μmol/L)0.5μL,以dd H2O补足。从210对引物中筛选获得140对引物,成功用于杜仲基因组PCR的有效扩增,占66.66%,进一步以9个地区的10份杜仲资源,检验上述引物多态性,最终获得15个稳定、可重复的多态性SSR位点,上述位点共检测到57个等位基因,平均每个位点包含3.8个等位基因,杂合度(Ho)为0~1.0,期望杂合度(He)为0.28~0.78,多态信息量(PIC)为0.26~0.70,平均值为0.44。经测序验证,该15个SSR位点都含有预期的重复基序序列。杜仲雌雄株转录组序列中含有高频率的SSR位点,以期开发获得的多态性SSR标记能够应用于杜仲不同种群间的遗传结构和遗传多样性分析,为杜仲的合理保护提供科学依据。

In order to enhance the effective conservation and sustainable utilization for Eucommia ulmoides Oliver germplasm resources, we need to reseach genetic background. Therefore, we based on the female and male transcriptome datum of E. ulmoides to develop SSR molecular markers, which could be used to research functional gene mining, molecular marker-assisted breeding, genetic diversity analysis, genetic map construction, etc. SSRs were mined and its characterization were analyzed by MISA software based on the unigenes of the female and male E. ulmoides transcriptome datum; microsatellite primers were designed based on the flanking sequences, one DNA sample of E. ulmoides was used to optimize the SSR-PCR reaction system by the orthogonal test of L9(34), and detect the amplification effect and optimize annealing temperature of 210 primers that were randomly selected, and then 10 DNA samples from 9 different regions were used to detect the efficiency of SSR primers. We obtained74 230 SSRs that distributed in 61 629 unigenes, with the frequency was 33.99%. Among of these SSRs, the most repeat motifs with length of 12~24 bp were discowered, and the most repeat numbers of the motif were 11 to 15.Mononucleotides, dinucleotides and trinucleotides appeared to be the most abundant repeated motifs, accounted for 98.6% in total SSRs, in which A/T(73.16%)、AG/CT(10.91%)、AAG/CTT(1.14%) were the most abundant respectively. According to the orthogonal test of L9(34), the optimized amplifications were performed in 20 μL volume containing 10 μL of 2 × Premix Taq DNA polymerase(0.05 μmol·min-1·μL-1), 1 μL of DNA(25 ng/μL), 0.5 μL(10 μmol/L) of each primers, 8 μL dd H2 O. One hundred and forty pairs of primers in 210 primers(66.66%) were successful amplificated, and then we used 10 DNA samples from 9 different regions to detected the efficiency of SSR primers, obtained 15 stable, repeatable polymorphic SSR loci and 57 alleles were observed with 3.8 alleles in one locus on average; observed heterozygosity(Ho), expected heterozygosity(He) and the polymorphism information content valued(PIC) range from 0 to 1.00, 0.28 to 0.78 and 0.26 to 0.70, respectively, and the mean value of PIC was 0.44. All of the 15 SSRs loci had expected repeat motif sequences by sequencing indicated. There was high frequency SSR loci in the transcriptome data of E. ulmoides, based on the datum to developed SSR markers had relatively high polymorphism, and those markers could be utilized to analyze genetic structure of different populations and genetic diversity in E. ulmoides, and providing a reasonable scientific basis to protect resources of E. ulmoides.