利用转录组数据开发意大利蜜蜂的SSR分子标记
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摘要
意大利蜜蜂(简称意蜂)是自然界重要的授粉昆虫,广泛用于世界各国的养蜂生产,具有很高的生态和经济价值。为了开发意蜂的SSR分子标记,利用MISA软件对基于前期获得的意蜂幼虫肠道转录组数据组装得到的12 115条unigenes进行搜索,共预测出分布于2 149条unigenes的6 312个SSR位点,其中主要的重复类型为二核苷酸重复(54.42%)和三核苷酸重复(32.49%),主要基元为AT/AT(31.2%)和AG/CT(18.6%)。进一步利用软件设计出18 444对特异性引物,随机选取24对引物对国内3个不同来源的意蜂幼虫样品进行SSR位点扩增,有22对成功扩增出目的片段。研究开发出的SSR分子标记可用于意蜂的种群遗传和分子进化等研究,结果表明利用转录组数据鉴定非模式生物SSR位点的方法可行且高效。
Apis mellifera ligustica is an important pollinating insect and widely used in beekeeping industry in various countries in the world, thus it has high ecological and economic value. The aim of this study was to develop SSR markers for A. m. ligustica based on our previously obtained transcriptome data. Here, all unigenes were scanned using MISA software and 6 312 SSR loci within 2 149 unigenes were predicted. The largest SSRs were dinucleotide(54.42%) and trinucleotide(32.49%), and for the former, AT/AT(31.2%) and AG/CT(18.6%) were the most abundant. Furthermore, a total of 18 444 primer pairs were designed using softwares, and 24 pairs were randomly selected for amplifying SSR loci in A. m. ligustica from 3 different regions in China, and the result showed 22 target fragments were successfully amplified. The developed SSR markers can be applied to A. m. ligustica studies such as population genetics and molecular evolution. Our data demonstrated that developing SSR markers of non-model organism based on transcriptome data is feasible and efficient.
Apis mellifera ligustica is an important pollinating insect and widely used in beekeeping industry in various countries in the world, thus it has high ecological and economic value. The aim of this study was to develop SSR markers for A. m. ligustica based on our previously obtained transcriptome data. Here, all unigenes were scanned using MISA software and 6 312 SSR loci within 2 149 unigenes were predicted. The largest SSRs were dinucleotide(54.42%) and trinucleotide(32.49%), and for the former, AT/AT(31.2%) and AG/CT(18.6%) were the most abundant. Furthermore, a total of 18 444 primer pairs were designed using softwares, and 24 pairs were randomly selected for amplifying SSR loci in A. m. ligustica from 3 different regions in China, and the result showed 22 target fragments were successfully amplified. The developed SSR markers can be applied to A. m. ligustica studies such as population genetics and molecular evolution. Our data demonstrated that developing SSR markers of non-model organism based on transcriptome data is feasible and efficient.
引文
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[23]XIE W,MENG Q S,WU Q J,et al.Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance[J].PLo S One,2012,7(4):e35181.
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[25]SOLIGNAC M,VAUTRIN D,BAUDRY E,et al.A microsatellite-based linkage map of the honeybee,Apis mellifera[J].Genetics,2004,167(1):253-262.
[26]SOLIGNAC M,MOUGEL F,VAUTRIN D,et al.A thirdgeneration microsatellite-based linkage map of the honey bee,Apis mellifera,and its comparison with the sequence-based physical map[J].Genome Biol,2007,8(4):R66.
[2]ESTOUP A,JARNE P,CORNUET J M.Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis[J].Mol Ecol,2002,11(9):1591-1604.
[3]LU C R,ZOU C S,ZHANG Y P,et al.Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons(Gossypium arboreum L.and Gossypium raimondii Ulbrich)[J].BMC Genomics,2015,6(1):1-12.
[4]HAMILTON M B,PINCUS E L,DI-FIORE A,et al.Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites[J].Biotechniques,1999,27(3):500-507.
[5]ZANE L,BARGELLONI L,PATARNELLO T.Strategies for microsatellite isolation[J].Mol Ecol,2002,11(1):1-16.
[6]O’NEILL E M,SCHWARTZ R,BULLOCK C T,et al.Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander(Ambystoma tigrinum)species complex[J].Mol Ecol,2013,22(1):111-129.
[7]DAVEY J W,HOHENLOHE P A,ETTER P D,et al.Genome-wide genetic marker discovery and genotyping using next-generation sequencing[J].Nat Rev Genet,2013,12(7):499-510.
[8]YU J N,WON C,JUN J,et al.Fast and cost-effective mining of microsatellite markers using NGS technology:an example of a Korean water deer Hydropotes inermis argyropus[J].PLo S One,2011,6(11):e26933.
[9]KISHORE G,GUPTA S,PANDEY A.Assessment of population genetic diversity of Fagopyrum tataricum,using SSR molecular marker[J].Biochem Syst Ecol,2012,43(3):32-41.
[10]张鹏飞,周晓榕,庞保平,等.基于转录组数据高通量发掘沙葱萤叶甲微卫星引物[J].应用昆虫学报,2016,53(5):1058-1064.
[11]武政梅,高朋,温俊宝.沟眶象转录组微卫星特征分析[J].环境昆虫学报,2016,38(5):979-983.
[12]魏明明,陈钰辉,刘富中,等.基于转录组测序的茄子SSR标记开发[J].植物遗传资源学报,2016,17(6):1082-1091.
[13]魏丹丹,石俊霞,张夏瑄,等.基于转录组数据的桔小实蝇微卫星位点信息分析[J].应用生态学报,2014,25(6):1799-1805.
[14]CHEN D F,RUI G,XU X J,et al.Uncovering the immune responses of Apis mellifera ligustica larval gut toAscosphaera apis infection utilizing transcriptome sequencing[J].Gene,2017,621:40-50.
[15]李汶东,熊翠玲,王鸿权,等.基于RNA-seq数据大规模挖掘蜜蜂球囊菌的SSR分子标记[J].福建农林大学学报,2017,46(4):434-438.
[16]朱家颖,吴国星,杨斌.基于转录组数据高通量发掘黄粉甲微卫星引物[J].昆虫学报,2013,56(7):724-728.
[17]罗梅,张鹤,宾淑英,等.基于转录组数据高通量发掘扶桑绵粉蚧微卫星引物[J].昆虫学报,2014,57(4):395-400.
[18]LI L T,ZHU Y B,MA J F,et al.An analysis of the Athetis lepigone transcriptome from four developmental stages[J].PLo S One,2013,8(9):e73911.
[19]袁远,张丽芳,吴国星,等.云南切梢小蠹微卫星的高通量发掘[J].环境昆虫学报,2014,36(2):166-170.
[20]WANG B,EKBLOM R,CASTOE T A,et al.Transcriptome sequencing of black grouse(Tetrao tetrix)for immune gene discovery and microsatellite development[J].Open Biol,2012,2(4):120054.
[21]HUANG Q Y,SUN P D,ZHOU X G,et al.Characterization of head transcriptome and analysis of gene expression involved in caste differentiation and aggression in Odontotermes formosanus(Shiraki)[J].PLo S One,2012,7(11):e50383.
[22]熊翠玲,张璐,付中民,等.基于RNA-seq数据大规模开发中华蜜蜂幼虫的SSR分子标记[J].环境昆虫学报,2017,39(1):68-74.
[23]XIE W,MENG Q S,WU Q J,et al.Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance[J].PLo S One,2012,7(4):e35181.
[24]ESTOUP A,SOLIGNAC M,HARRY M,et al.Characterization of(GT)n and(CT)n microsatellites in two insect species:Apis mellifera and Bombus terrestris[J].Nucleic Acids Res,1993,21960,1427-1431.
[25]SOLIGNAC M,VAUTRIN D,BAUDRY E,et al.A microsatellite-based linkage map of the honeybee,Apis mellifera[J].Genetics,2004,167(1):253-262.
[26]SOLIGNAC M,MOUGEL F,VAUTRIN D,et al.A thirdgeneration microsatellite-based linkage map of the honey bee,Apis mellifera,and its comparison with the sequence-based physical map[J].Genome Biol,2007,8(4):R66.
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