黑果枸杞转录组SSR信息分析及分子标记开发

SSR information in transcriptome and development of molecular markers in Lycium ruthenicum

分析黑果枸杞Lycium ruthenicum转录组中简单重复序列(SSR)位点信息,开发SSR分子标记。采用MISA软件在黑果枸杞转录组中共检测到73 896个SSR位点,分布于56 170条单基因簇(unigenes)中,出现频率为26.36%,平均分布距离为3.55 kb。优势重复基序为单核苷酸、二核苷酸和三核苷酸,分别占总SSR位点的74.33%, 13.30%和11.81%;共发现84种重复基序,重复次数为5～33次,其中出现频率最高的基序为A/T, AG/CT, AT/AT, C/G和AAC/GTT。针对SSR位点设计了12 674对引物,随机挑选128对引物进行多态性验证,其中74对(57.8%)得到清晰的扩增产物;用其中的28对高多态性引物对24份枸杞种质进行扩增,共检测到等位基因256个,平均为9.1个。平均主效等位基因频率、观察杂合度、期望杂合度和多态信息量分别为0.432, 0.439, 0.712和0.678。以上结果表明:基于黑果枸杞转录组测序得到的Unigene信息可作为开发SSR标记的有效来源,获得的大批量SSR标记可为枸杞遗传多样性分析和遗传图谱构建提供可靠的标记选择。

The objective was to analyze Simple Sequence Repeat(SSR)loci information and to develop SSR molecular markers in Lycium ruthenicum(wolfberry)for further analysis with genetic diversity.A total of 73 896 SSRs were identified in L.ruthenicum transcriptome by MISA software,and primer pairs were designed from the SSR loci by BatchPrimer 3 software.Results showed that distribution was in 56 170 unigenes with a frequency of 26.36%and an average density of one SSR per 3.55 kb.Among these SSR loci,the most abundant motifs were mononucleotide(74.33%),dinucleotide(13.30%),and trinucleotide(11.81%)repeat types.Among 73 896 SSR loci,84 repeat motifs with iteration numbers from 5 to 33 were discovered.The most abundant repeat motifs were A/T,AG/CT,AT/AT,C/G,and AAC/GTT.A total of 12 674 primer pairs were designed from the SSR loci by BatchPrimer 3 software,and 128 primers pairs of randomly selected SSR were validated,of which 74(57.8%)pairs of primers were able to produce Polymerase Chain Reaction(PCR)products.Among the 74 SSR loci,28 high polymorphic SSR loci were used to amplify 24 accessions of wolfberry germplasm.A total of 256 alleles were detected for an average of 9.1.The average major allele frequency(0.432),observed heterozygosity(0.439),expected heterozygosity(0.712),and polymorphism information content(PIC)(0.678)were noted.Thus,the development of SSR markers based on the transcriptome of L.ruthenicum could provide reliable marker selection for genetic diversity analysis and genetic map construction of wolfberry germplasm.[Ch,1 fig.4 tab.31 ref.].