椭圆叶花锚简化基因组的SSR信息分析及SSR引物开发
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摘要
基于RAD-seq简化基因组测序技术获得椭圆叶花锚(Halenia ellipitica D.Don)简化基因组水平上的序列信息并开发SSR分子标记。利用SR search软件检测而得到双端各有至少100 bp的五种类型的SSR(二、三、四、五、六核苷酸)位点共6 201个,并成功设计其中3 865个SSR引物。在能成功设计引物的SSR位点中,三核苷酸SSR位点最多;重复序列长度包括17种(12~36 bp);重复序列的基序共达316种,其中五核苷酸基序种类最多(91种)。从中挑选65对可对应五种SSR类型的引物,经梯度PCR检验后,利用椭圆叶花锚的4个居群32个个对可扩增的引物进行PCR和聚丙烯酰氨凝胶电泳检测,其中14对引物能在绝大多数个体中扩增,且13对具多态性。13个多态性位点的等位基因数量均值为5.462,多态性较高且不连锁(P<0.05);其中10个位点在多数居群中偏离哈迪温伯格平衡(P<0.01)且存在较高的纯合子数量(观测杂合度Ho均值0.226);近交系数在-0.443~1,均值为0.656;基因流N_m为0.474。
The restriction-site associated DNA sequencing( RAD-seq) technology was used to analyze simple sequence repeats( SSR) information of Halenia ellipitica D. Don; SSR markers for this species were developed based on RAD-seq data. We identified 6 201 SSR loci with at least 100 bp at two ends,including five types( dinucleotide,tri-nucleotide,tetra-nucleotide,penta-nucleotide,and hexa-nucleotide). Primers of 3 865 loci were designed successfully. Among which,amount of tri-nucleotide SSR loci is the most. Repeat motif type number is 316,and type number of penta-nucleotide motif is the most( 91). Afterward,65 designed primers were randomly selected to represent the five SSR types,and tested using annealing temperature gradient PCR.Subsequently,32 individuals from four natural populations of H. elliptica were employed to estimate the SSR primers that can be amplified. One monomorphic and 13 polymorphic SSR loci were developed according to theresult of PCR and Polyacrylamide gel electrophoresis. The number of alleles of the 13 polymorphic SSR loci is high( mean 5. 462),and no linkage relation was found( P < 0. 01); ten loci deviate from HWE( P < 0. 01) and have many homozygotes( observed heterozygosity mean of 0. 226); the range of inbreeding coefficient is in-0. 443-1( mean of 0. 656); the gene flow N_m is 0. 474.
The restriction-site associated DNA sequencing( RAD-seq) technology was used to analyze simple sequence repeats( SSR) information of Halenia ellipitica D. Don; SSR markers for this species were developed based on RAD-seq data. We identified 6 201 SSR loci with at least 100 bp at two ends,including five types( dinucleotide,tri-nucleotide,tetra-nucleotide,penta-nucleotide,and hexa-nucleotide). Primers of 3 865 loci were designed successfully. Among which,amount of tri-nucleotide SSR loci is the most. Repeat motif type number is 316,and type number of penta-nucleotide motif is the most( 91). Afterward,65 designed primers were randomly selected to represent the five SSR types,and tested using annealing temperature gradient PCR.Subsequently,32 individuals from four natural populations of H. elliptica were employed to estimate the SSR primers that can be amplified. One monomorphic and 13 polymorphic SSR loci were developed according to theresult of PCR and Polyacrylamide gel electrophoresis. The number of alleles of the 13 polymorphic SSR loci is high( mean 5. 462),and no linkage relation was found( P < 0. 01); ten loci deviate from HWE( P < 0. 01) and have many homozygotes( observed heterozygosity mean of 0. 226); the range of inbreeding coefficient is in-0. 443-1( mean of 0. 656); the gene flow N_m is 0. 474.
引文
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2.中国科学院西北高原生物研究所.藏药志[M].西宁:青海人民出版社,1991:113-114.Northwest Plateau Institute of Biology,Chinese Academy of Sciences.Handbook of Tibetan medicine[M].Xining:Qinghai People’s Publishing Press,1991:113-114.
3.Huang B,Ban X Q,He J S,et al.Hepatoprotective and antioxidant effects of the methanolic extract from Halenia elliptica[J].Journal of Ethnopharmacology,2010,131(2):276-281.
4.古锐,王昌华,刘翔,等.藏药椭圆叶花锚的资源学研究[J].时珍国医国药,2011,22(3):720-722.Gu R,Wang C H,Liu X,et al.Resources research of Tibetan medicinal Halenia elliptica[J].Lishizhen Medicine and Materia Medica Research,2011,22(3):720-722.
5.张婉,崔继哲,于拴仓,等.白菜品种的SSR指纹图谱数据库的构建[J].分子植物育种,2013,11(6):843-857.Zhang W,Cui J Z,Yu S C,et al.Construction of SSR fingerprint database of Chinese cabbage varieties(Brassica campestris L.ssp.Pekinensis)[J].Molecular Plant Breeding,2013,11(6):843-857.
6.Zhang Z F,Bian Q L,Luo P,et al.Ethnopharmacological,chemical,and pharmacological aspects of Halenia elliptica:a comprehensive review[J].Pharmacognosy Reviews,2015,9(18):114-119.
7.Liu Y L,Wang P,Chen T,et al.One-Step isolation and purification of four Xanthone Glycosides from Tibetan medicinal plant Halenia elliptica by high-speed counter-current chromatography[J].Separation Science and Technology,2014,49(7):1119-1124.
8.Sun Y W,Liu G M,Huang H,et al.Chromone derivatives from Halenia elliptica and their anti-HBV activities[J].Phytochemistry,2012,75:169-176.
9.Huang B,Ke H B,He J S,et al.Extracts of Halenia elliptica exhibit antioxidant properties in vitro and in vivo[J].Food and Chemical Toxicology,2011,49(1):185-190.
10.Zhang Z R,Yang J,Sun Y,et al.A set of novel microsatellite markers developed for the traditional tibetan medicinal plant Halenia elliptica(Gentianaceae)[J].American Journal of Botany,2011,98(7):173-175.
11.Davey J W,Blaxter M L.RADSeq:next-generation population genetics[J].Briefings in Functional Genomic,2010,9(5-6):416-423.
12.Miller M R,Dunham J P,Amores A,et al.Rapid and costeffective polymorphism identification and genotyping using restriction site associated DNA(RAD)markers[J].Genome Research,2007,17(2):240-248.
13.王洋坤,胡艳,张天真.RAD-seq技术在基因组研究中的现状及展望[J].遗传,2014,36(1):41-49.Wang Y K,Hu Y,Zhang T Z.Current status and perspective of RAD-seq in genomic research[J].Beijing:Hereditas,2014,36(1):41-49.
14.王久利,朱明星,徐明行,等.基于RAD-seq技术的异型花SSR信息分析[J].植物研究,2017,37(3):447-452,460.Wang J L,Zhu M X,Xu M H,et al.Analysis on SSR in Sinoswertia tetraptera base on RAD-seq[J].Bulletin of Botanical Research,2017,37(3):447-452,460.
15.Hosseini P,Tremblay A,Matthews B F,et al.An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets[J].BMC Research Notes,2010,3:183.
16.Li W Z,Godzik A.Cd-hit:a fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,2006,22(13):1658-1659.
17.Zerbino D R,Birney E.Velvet:algorithms for de novo short read assembly using de Bruijn graphs[J].Genome Research,2008,18(5):821-829.
18.Rozen S,Skaletsky H.Primer3 on the WWW for general users and for biologist programmers[J].Bioinformatics Methods and Protocols,2000,132:365-386.
19.Li H,Durbin R.Fast and accurate long-read alignment with burrows-wheeler transform[J].Bioinformatics,2010,26(5):589-595.
20.Li H,Handsaker B,Wysoker A,et al.The sequence alignment/map format and SAMtools[J].Bioinformatics,2009,25(16):2078-2079.
21.Ohta T.Linkage disequilibrium with the island model[J].Genetics,1982,101(1):139-155.