果树学报2017，Vol.34Issue(10)：1258-1265,8.DOI:10.13925/j.cnki.gsxb.20170034
榧树转录组SSR信息分析及其分子标记开发
Analysis of SSR information in transcriptome and development of molecular markers in Torreya grandis
摘要
Abstract
[Objective]The genus Torreya is an important member of Taxaceae with only 9 species distributed in China,the United States of American,Japan and Korea.T.grandis Fort.ex Lindl.has been famous of its nut seeds with high oil for economic usage and as food for thousands years in China.Breeding of T.grandis is currently limited on selection of the wild resources derived from spontaneous mutation and occational seedling.The genetic characteristics of the wild resources are great concern for the efficiency of improvement of T.grandis.To get insight of hereditary information for commercial utilization and cultivar discrimination of genetic resource of this species,microsatellite loci were exploited based on compared transcriptomic profiles de novo assembled for RNA-seq data of T.grandis.[Methods] Seeds of T.grandis‘Xifei’ and T.grandis ‘Dielsii’ were sampled from a orchard (Fuyang,Zhejiang province,China).Based on the biochemistry analysis of fatty acids biosynthesis,seeds at initiation stage (449 days after pollination,DAP),fastigium stage (469 DAP) and steady stage (512 DAP) were separately mixed as sample pools for RNA extraction;followed by comparative transcriptomic analysis through RNA-Seq.De novo transcriptome assembly was achieved using Trinity.Microsatellite mining was performed using the software MIcroSAtellite (MISA).Primers (20 base pairs) were designed using Primer 3 were used.49 pairs of primers were randomly selected and were used for morphological amplification with product sizes ranging from 100 to 300 bp.Ten individuals of each species,including T.nucifera,T.fargesii,T.jackii,T.grandis and T.grandis var.jiulongshanensis,were selected for primary assess on the microsatellite polymorphism.Genomic DNA was extracted from young leaves by the cetyltrimethylammonium bromide (CTAB) modified method.[Results] All high quality reads were assembled into 142 213 unigene.A total of 5 458 SSRs containing inserts with various microsatellite motifs (di-to penta nucleotides) were identified from 22 976 unigenes (＞ 1 kb).The distribution density was 105.4 SSRs per Mb.Mono nucleotide was the most abundant type of repeat motif with a frequency of 64.95％,followed by the tri-nucleotide (18.56％),di (14.77％),tetra (0.97％),hexa (0.42％) and pentanucleotides (0.33％).The AT/AT was the most frequent repeat motif (3.85％),followed by TA/TA (3.57％) and AG/CT (2.29％);Among the tri-nucleotide repeats,the dominant repeat motif in EST-SSRs was GAA/TTC (2.36％),followed by CTT/AAG (1.32％) and AAT/ATT (1.26％).The frequency of tetra-and hexanucleotide were much lower than that of di-and tri-nucleotide.Numbers of each repetitive unit ranged from 5 to 21.Units repeated less than or equal to 10 times and greater than 10 times were predominantly at mono nucleotide type with 1 990 (36.46％) and 1 555 (28.49％),and followed by 5 (12.90％),6 (11.95％),7 (5.42％) and 8 (2.53％) times among di-to hexanucleotide type.Repeat number was mainly 6,7 and 8 in Di nucleotide unit as well as 5,6 and 7 in tri-nucleotide unit.A total of 4 633 prime pairs were designed,among them 49 primer pairs were selected randomly to test in Torreya.From these,16 polymorphic SSRs were characterized.The number of alleles of each locus ranged from 2 to 6,which was averagely 3 per locus.The average value of polymorphism information content (PIC),observed heterozygosity (Ho) and expected heterozygosity (He) were 0.464 4,0.283 7 and 0.500 6 respectively.[Conclusion] The transcriptome sequencing data of Torreya can provide abundant SSR locus,which can be used into develop SSR primer rapidly.The primer developed can provide marker selection for Torreya genetic diversity,the construction of fingerprint pattern of variety and molecular mark assisted breeding.关键词
榧树/转录组/SSRKey words
Torreya grandis/RNA-Seq/SSR分类
农业科技引用本文复制引用
张敏,周彩红,陈焘,戴文圣,张迟..榧树转录组SSR信息分析及其分子标记开发[J].果树学报,2017,34(10):1258-1265,8.基金项目
林业公益性行业科研专项(201504708) （201504708）
浙江省团队科技特派员服务计划项目(浙科发农[2013]215-122) （浙科发农[2013]215-122）
浙江省科技计划项目(2012C22095) （2012C22095）
浙江省农业新品种选育重大科技专项(2016C02052-12) （2016C02052-12）
