摘要
转录组SSR引物的开发为接骨木遗传多样性分析、遗传图谱构建和功能基因的挖掘提供丰富的遗传标记。以一株成年西洋接骨木的果实为实验材料,提取RNA后,采用Illumina HiSeq 2000高通量测序获得西洋接骨木转录组数据,共得到西洋接骨木转录组的257975条Unigene,检测到64148个SSR位点,分布于51336条Unigene,SSR位点出现频率为24.87%(SSR位点数与总的Unigene数的比值),其中,单核苷酸重复是主要类型,占总SSR的51.13%。其次是二、三核苷酸重复,分别占总SSR的38.63%和9.11%。A/T和AG/CT是单核酸和二核苷酸的优势重复基元。通过试验优化获得最优的两步荧光引物PCR扩增体系。加M13接头PCR扩增体系:10μL PCR体系中含DNA原液1μL,2×Mix 5μL,10μM引物各0.1μL,灭菌超纯水3.8μL;荧光引物PCR扩增体系:20μL PCR体系中含第一步扩增产物DNA原液2μL,2×Mix 10μL,10μM M13接头及反向引物各0.15μL,灭菌超纯水7.7μL。利用已筛选的100对荧光SSR引物对8个不同种接骨木个体进行SSR扩增,80对可扩增出条带,有效扩增率为80%,25对可成功扩增出多态性,多态性比率为31.25%,从25对具有多态性引物中选择14对多态性较好的引物对不同种接骨木进行遗传多样性分析,14对多态性引物共检测出67个等位基因(Na),每位点3~7个,平均等位基因4.786个;每位点有效等位基因数(Ne)1.684~6.095个,平均有效等位基因数3.413个,观测杂合度(Ho)及期望杂合度(He)分别在0~0.875和0.406~0.836之间,平均值分别为0.313、0.664;Shannon多样性指数(I)在0.736~1.862之间,平均1.310。不同种接骨木具有较高的遗传多样性,25对具有多态性荧光SSR标记开发可为接骨木分子标记辅助育种、杂种优势预测等提供理论基础。
The development of transcriptome SSR primers provides abundant genetic markers for analysis of genetic diversity,genetic map construction,and functional gene mining of Sambucus williamsii.Taking an adult fruit of Sambucus nigra as experimental material,After extraction of RNA,the Sambucus nigra transcriptome were obtained by Illumina HiSeq 2000 high-throughput sequencing.257,975 Unigenes and 64148 SSR loci were detected and distributed in 51336 Unigenes.The frequency of SSR loci was 24.87%,Among them,mononucleotide repeats are the main type,accounting for 51.13%of the total SSR.Followed by two or three nucleotide repeats,accounting for 38.63%and 9.11%of the total SSR.A/T and AG/CT are the dominant repetitive motifs of single and dinucleotides.The optimal two-step fluorescent PCR amplification system was obtained through experiment optimization.Plus M13 adapter PCR amplification system:10μL reaction volumes containing 1μL template DNA,5μL 2×Taq Master Mix,0.1μL of each primer which concentration is 10μM and 3.8μL sterile ultrapure water;fluorescent PCR amplification system:20μL PCR system containing 2μL DNA from the first step amplification product,10μL 2×Taq Master Mix,0.15μL of M13 adapter and reverse primer which concentration is 10μM,7.7μL sterilized ultrapure water.Using the screened 100 pairs of fluorescent SSR primers to perform SSR amplification on 8 different species of Sambucus williamsii,80 pairs of primers can amplify the band,the effective amplification rate is 80%,25 pairs can successfully amplify the polymorphism,and the polymorphism ratio is 31.25%.A total of 14 pairs of polymorphic primers were used to analyze the genetic diversity of different species of Sambucus williamsii.A total of 67 alleles were detected,average number of alleles and effective number of alleles were 4.786 and 3.413 respectively.The mean observed heterozygosity was 0.313,the mean expected heterozygosity was 0.664 and Shannon information index was 1.310 on average.Different species of Sambucus williamsii have higher genetic diversity,and 25 pairs of polymorphic fluorescent SSR markers provide the theoretical basis for molecular marker-assisted breeding and heterosis prediction in Sambucus williamsii.
作者
姚俊修
李善文
任飞
李庆华
刘学良
吴德军
燕丽萍
YAO Junxiu;LI Shanwen;REN Fei;LI Qinghua;LIU Xueliang;WU Dejun;YAN Liping(Shandong Academy of Forestry,Jinan 250014,Shandong,China;Shandong Province Key Laboratory of Forest Tree Genetic Improvement,Jinan 250014,Shandong,China;Shandong Agricultural University,Taian 271018,Shandong,China)
出处
《中南林业科技大学学报》
CAS
CSCD
北大核心
2019年第12期123-129,147,共8页
Journal of Central South University of Forestry & Technology
基金
山东省农业重大关键技术“耐盐碱林木及经济作物品种选育及示范推广”(2015ZDJS03003)
山东省农业良种工程“高抗逆药用接骨木新品种选育与示范”(2016LZGC017)
山东省林业科技创新项目“接骨木、元宝枫种质资源创新与高值化利用”(LYCX05-2018-24)
关键词
西洋接骨木
转录组
SSR
荧光标记
Sambucus nigra L.
transcriptome
SSR
fluorescent marker
