黑果枸杞转录组SSR信息分析及分子标记开发
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摘要
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 of73 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.
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 of73 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.
引文
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[10]REBAA F,ABID G,AOUIDA M,et al.Genetic variability in Tunisian populations of faba bean(Vicia faba L.var major)assessed by morphological and SSR markers[J].Physiol Mol Biol Plant,2017,23(2):397-409.
[11]LI Zhigang,CHEN Feng,HUANG Chunhua,et al.Genome-wide mapping and characterization of microsatellites in the swamp eel genome[J].Sci Rep,2017,7(1):3157.
[12]AMBREEN H,KUMAR S,VARIATH M T,et al.Development of genomic microsatellite markers in Carthamus tinctorius L.(safflower)using next generation sequencing and assessment of their cross-species transferability and utility for diversity analysis[J].PLoS One,2015,10(8):e0135443.
[13]鄢秀芹,鲁敏,安华明.刺梨转录组SSR信息分析及其分子标记开发[J].园艺学报,2015,42(2):341-349.YAN Xiuqin,LU Min,AN Huaming.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii[J].Acta Hortic Sin,2015,42(2):341-349.
[14]宗宇,王月,朱友银,等.基于中国樱桃转录组的SSR分子标记开发与鉴定[J].园艺学报,2016,43(8):1566-1576.ZONG Yu,WANG Yue,ZHU Youyin,et al.Development and validation of SSR markers based on transcriptomic data of Chinese cherry(Prunus pseudocerasus)[J].Acta Hortic Sin,2016,43(8):1566-1576.
[15]巩檑,程永芳,甘晓燕,等.马铃薯转录组EST-SSR挖掘及其多样性分析[J].分子植物育种,2015,13(7):1535-1544.GONG Lei,CHENG Yongfang,GAN Xiaoyan,et al.Mining EST-SSR based on potato transcriptome and analyzing their polymorphism[J].Mol Plant Breed,2015,13(7):1535-1544.
[16]CHEN Jinhuan,ZHANG Dongzhi,ZHANG Chong,et al.Physiological characterization,transcriptomic profiling,and microsatellite marker mining of Lycium ruthenicum[J].J Zhejiang Univ-Sci B Biom Biotechnol,2017,18(11):1002-1021.
[17]GRABHERR M,HAAS B J,YASSOUR M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nat Biotechnol,2011,29(7):644-652.
[18]樊文强,盖红梅,孙鑫,等.SSR数据格式转换软件DataFormater[J].分子植物育种,2016,14(1):265-270.FAN Wenqiang,GAI Hongmei,SUN Xin,et al.DataFormater,a software for SSR data formatting to development population genetic analysis[J].Mol Plant Breed,2016,14(1):265-270.
[19]LIU Kejun,MUSE S V.PowerMarker:an integrated analysis environment for genetic marker analysis[J].Bioinformatics,2005,21(9):2128-2129.
[20]BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].Am J Human Genet,1980,32(3):314-331.
[21]尹跃,安巍,赵建华,等.枸杞种质资源遗传多样性的i PBS分析[J].福建农林大学学报(自然科学版),2017,46(6):612-617.YIN Yue,AN Wei,ZHAO Jianhua,et al.Genetic diversity analysis of wolfberry germplasm using iPBS makers[J].JFujian Agric For Univ Nat Sci Ed,2017,46(6):612-617.
[22]黄耀辉,张超,周莉花,等.基于转录组序列的夏蜡梅SSR位点特征与引物开发[J].浙江农林大学学报,2017,34(4):589-596.HUANG Yaohui,ZHANG Chao,ZHOU Lihua,et al.Development and primer screening of SSR markers based on transcriptome sequences in Sinocalycanthus chinensis[J].J Zhejiang A&F Univ,2017,34(4):589-596.
[23]梅利那,范付华,崔博文,等.基于马尾松转录组的SSR分子标记开发及种质鉴定[J].农业生物技术学报,2017,25(6):991-1002.MEI Lina,FAN Fuhua,CUI Bowen,et al.Development of SSR molecular markers based on transcriptome sequences and germplasm identification in Masson pine(Pinus massoniana)[J].J Agric Biotechnol,2017,25(6):991-1002.
[24]郑如玉,刘坤,杨杞,等.中间锦鸡儿干旱转录组SSR标记的开发及遗传多样性研究[J].分子植物育种,2017,15(6):2236-2243.ZHENG Ruyu,LIU Kun,YANG Qi,et al.SSR marker development and the genetic diversity of Caragana intermedia in based on transcriptome under drought stress[J].Mol Plant Breed,2017,15(6):2236-2243.
[25]张庆田,李晓艳,杨义明,等.蓝靛果忍冬转录组SSR信息分析及其分子标记开发[J].园艺学报,2016,43(3):557-563.ZHANG Qingtian,LI Xiaoyan,YANG Yiming,et al.Analysis on SSR information in transcriptome and development of molecular markers in Lonicera caerulea[J].Acta Hortic Sin,2016,43(3):557-563.
[26]李娜,姚民,梅兰菊,等.基于山桐子转录组序列的SSR分子标记开发[J].应用与环境生物学报,2017,23(5):952-958.LI Na,YAO Min,MEI Lanju,et al.Development of SSR molecular markers based on transcriptome se quencing of Idesia polycarpa Maxim[J].Chin J Appl Environ Biol,2017,23(5):952-958.
[27]李海波,王珊,丁红梅,等.普通油茶转录组EST-SSR分子标记开发[J].植物生理学报,2017,53(7):1267-1278.LI Haibo,WANG Shan,DING Hongmei,et al.Development of EST-SSR molecular markers based on transcriptome sequencing of Camellia oleifera[J].Plant Physiol J,2017,53(7):1267-1278.
[28]方智振,叶新福,周丹蓉,等.‘芙蓉李’转录组SSR信息分析与分子标记开发[J].果树学报,2016,33(4):416-424.FANG Zhizhen,YE Xinfu,ZHOU Danrong,et al.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.[J].J Fruit Sci,2016,33(4):416-424.
[29]ZHONG Ruichun,ZHOU Meili,ZHAO Chuanzhi,et al.SSR marker development from peanut gynophore transcriptome sequencing[J].Plant Breed,2016,135(1):111-117.
[30]LIU Touming,ZHU Siyuan,FU Lili,et al.Development and characterization of 1,827 expressed sequence tag-derived simple sequence repeat markers for ramie(Boehmeria nivea L.Gaud)[J].PLoS One,2013,8(4):e60346.
[31]CLOUTIER S,NIU Zhixia,DATLA R,et al.Development and analysis of EST-SSRs for flax(Linum usitatissimum L.)[J].Theor Appl Genet,2009,119(1):53-63.
[2]WANG Hanqing,LI Jiaoning,TAO Weiwei,et al.Lycium ruthenicum studies:molecular biology,phytochemistry and pharmacology[J].Food Chem,2018,240:759-666.
[3]WU Tao,L譈Haiyang,WANG Fengzhong,et al.Characterization of polyphenols from Lycium ruthenicum fruit by U-PLC-Q-TOF/MSEand their antioxidant activity in Caco-2 Cells[J].J Agric Food Chem,2016,64(11):2280-2288.
[4]王锦楠,陈进福,陈武生,等.柴达木地区野生黑果枸杞种群遗传多样性的AFLP分析[J].植物生态学报,2015,39(10):1003-1011.WANG Jinnan,CHEN Jinfu,CHEN Wusheng,et al.Population genetic diversity of wild Lycium ruthenicum in Qaidam inferred from AFLP markers[J].Chin J Plant Ecol,2015,39(10):1003-1011.
[5]阿力同·其米克,王青锋,杨春锋,等.新疆产药用植物黑果枸杞遗传多样性的ISSR分析[J].植物科学学报,2013,31(5):517-524.ALITONG Qimike,WANG Qingfeng,YANG Chunfeng,et al.ISSR analysis on genetic diversity of medically important Lycium ruthenicum Murr.in Xinjiang[J].Plant Sci J,2013,31(5):517-524.
[6]LIU Zenggen,SHU Qingyan,WANG Lei,et al.Genetic diversity of the endangered and medically important Lycium ruthenicum Murr.revealed by sequence-related amplified polymorphism(SRAP)markers[J].Biochem Syst Ecol2012,45:86-97.
[7]GUO Qi,GUO Lili,ZHANG Lin,et al.Construction of a genetic linkage map in tree peony(Paeonia Sect.Moutan)using simple sequence repeat(SSR)markers[J].Sci Hortic,2017,219:294-301.
[8]WANG Long,LI Xiugen,WANG Lei,et al.Construction of a high-density genetic linkage map in pear(Pyrus communis×Pyrus pyrifolia Nakai)using SSRs and SNPs developed by SLAF-seq[J].Sci Hortic,2017,218:198-204.
[9]SUNGIRAI M,BARON S,van der MERWE N A,et al.Population structure and genetic diversity of Rhipicephalus microplus in Zimbabwe[J].Acta Trop,2018,180:42-46.
[10]REBAA F,ABID G,AOUIDA M,et al.Genetic variability in Tunisian populations of faba bean(Vicia faba L.var major)assessed by morphological and SSR markers[J].Physiol Mol Biol Plant,2017,23(2):397-409.
[11]LI Zhigang,CHEN Feng,HUANG Chunhua,et al.Genome-wide mapping and characterization of microsatellites in the swamp eel genome[J].Sci Rep,2017,7(1):3157.
[12]AMBREEN H,KUMAR S,VARIATH M T,et al.Development of genomic microsatellite markers in Carthamus tinctorius L.(safflower)using next generation sequencing and assessment of their cross-species transferability and utility for diversity analysis[J].PLoS One,2015,10(8):e0135443.
[13]鄢秀芹,鲁敏,安华明.刺梨转录组SSR信息分析及其分子标记开发[J].园艺学报,2015,42(2):341-349.YAN Xiuqin,LU Min,AN Huaming.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii[J].Acta Hortic Sin,2015,42(2):341-349.
[14]宗宇,王月,朱友银,等.基于中国樱桃转录组的SSR分子标记开发与鉴定[J].园艺学报,2016,43(8):1566-1576.ZONG Yu,WANG Yue,ZHU Youyin,et al.Development and validation of SSR markers based on transcriptomic data of Chinese cherry(Prunus pseudocerasus)[J].Acta Hortic Sin,2016,43(8):1566-1576.
[15]巩檑,程永芳,甘晓燕,等.马铃薯转录组EST-SSR挖掘及其多样性分析[J].分子植物育种,2015,13(7):1535-1544.GONG Lei,CHENG Yongfang,GAN Xiaoyan,et al.Mining EST-SSR based on potato transcriptome and analyzing their polymorphism[J].Mol Plant Breed,2015,13(7):1535-1544.
[16]CHEN Jinhuan,ZHANG Dongzhi,ZHANG Chong,et al.Physiological characterization,transcriptomic profiling,and microsatellite marker mining of Lycium ruthenicum[J].J Zhejiang Univ-Sci B Biom Biotechnol,2017,18(11):1002-1021.
[17]GRABHERR M,HAAS B J,YASSOUR M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nat Biotechnol,2011,29(7):644-652.
[18]樊文强,盖红梅,孙鑫,等.SSR数据格式转换软件DataFormater[J].分子植物育种,2016,14(1):265-270.FAN Wenqiang,GAI Hongmei,SUN Xin,et al.DataFormater,a software for SSR data formatting to development population genetic analysis[J].Mol Plant Breed,2016,14(1):265-270.
[19]LIU Kejun,MUSE S V.PowerMarker:an integrated analysis environment for genetic marker analysis[J].Bioinformatics,2005,21(9):2128-2129.
[20]BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].Am J Human Genet,1980,32(3):314-331.
[21]尹跃,安巍,赵建华,等.枸杞种质资源遗传多样性的i PBS分析[J].福建农林大学学报(自然科学版),2017,46(6):612-617.YIN Yue,AN Wei,ZHAO Jianhua,et al.Genetic diversity analysis of wolfberry germplasm using iPBS makers[J].JFujian Agric For Univ Nat Sci Ed,2017,46(6):612-617.
[22]黄耀辉,张超,周莉花,等.基于转录组序列的夏蜡梅SSR位点特征与引物开发[J].浙江农林大学学报,2017,34(4):589-596.HUANG Yaohui,ZHANG Chao,ZHOU Lihua,et al.Development and primer screening of SSR markers based on transcriptome sequences in Sinocalycanthus chinensis[J].J Zhejiang A&F Univ,2017,34(4):589-596.
[23]梅利那,范付华,崔博文,等.基于马尾松转录组的SSR分子标记开发及种质鉴定[J].农业生物技术学报,2017,25(6):991-1002.MEI Lina,FAN Fuhua,CUI Bowen,et al.Development of SSR molecular markers based on transcriptome sequences and germplasm identification in Masson pine(Pinus massoniana)[J].J Agric Biotechnol,2017,25(6):991-1002.
[24]郑如玉,刘坤,杨杞,等.中间锦鸡儿干旱转录组SSR标记的开发及遗传多样性研究[J].分子植物育种,2017,15(6):2236-2243.ZHENG Ruyu,LIU Kun,YANG Qi,et al.SSR marker development and the genetic diversity of Caragana intermedia in based on transcriptome under drought stress[J].Mol Plant Breed,2017,15(6):2236-2243.
[25]张庆田,李晓艳,杨义明,等.蓝靛果忍冬转录组SSR信息分析及其分子标记开发[J].园艺学报,2016,43(3):557-563.ZHANG Qingtian,LI Xiaoyan,YANG Yiming,et al.Analysis on SSR information in transcriptome and development of molecular markers in Lonicera caerulea[J].Acta Hortic Sin,2016,43(3):557-563.
[26]李娜,姚民,梅兰菊,等.基于山桐子转录组序列的SSR分子标记开发[J].应用与环境生物学报,2017,23(5):952-958.LI Na,YAO Min,MEI Lanju,et al.Development of SSR molecular markers based on transcriptome se quencing of Idesia polycarpa Maxim[J].Chin J Appl Environ Biol,2017,23(5):952-958.
[27]李海波,王珊,丁红梅,等.普通油茶转录组EST-SSR分子标记开发[J].植物生理学报,2017,53(7):1267-1278.LI Haibo,WANG Shan,DING Hongmei,et al.Development of EST-SSR molecular markers based on transcriptome sequencing of Camellia oleifera[J].Plant Physiol J,2017,53(7):1267-1278.
[28]方智振,叶新福,周丹蓉,等.‘芙蓉李’转录组SSR信息分析与分子标记开发[J].果树学报,2016,33(4):416-424.FANG Zhizhen,YE Xinfu,ZHOU Danrong,et al.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.[J].J Fruit Sci,2016,33(4):416-424.
[29]ZHONG Ruichun,ZHOU Meili,ZHAO Chuanzhi,et al.SSR marker development from peanut gynophore transcriptome sequencing[J].Plant Breed,2016,135(1):111-117.
[30]LIU Touming,ZHU Siyuan,FU Lili,et al.Development and characterization of 1,827 expressed sequence tag-derived simple sequence repeat markers for ramie(Boehmeria nivea L.Gaud)[J].PLoS One,2013,8(4):e60346.
[31]CLOUTIER S,NIU Zhixia,DATLA R,et al.Development and analysis of EST-SSRs for flax(Linum usitatissimum L.)[J].Theor Appl Genet,2009,119(1):53-63.