重要食药同源植物余甘子转录组微卫星特征分析
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摘要
为全面了解余甘子转录组SSR位点的分布特征和变异规律,本研究利用Illumina Hiseq 4000平台对余甘子叶片转录组进行测序,通过MISA软件对获得的Unigenes进行SSR位点搜索和统计分析。结果发现9 538条包含SSR位点的Unigenes,共检测到9 991个SSR位点,平均每5. 49 k B出现1个SSR。单碱基和二碱基为余甘子转录组SSR主要重复类型,分别占SSR总数的42. 3%和30. 79%。位于基因编码区的SSR位点共有1 731个,出现频率为0. 039 SSRs/k B,优势重复类型为三碱基重复。余甘子转录组SSR中共有169种重复基元,其中所占比例最高的是A/T(42. 10%),其次是AG/CT(22. 91%)和AAG/CTT(5. 02%)。SSR各基元的重复次数波动于4～75次,且多数集中于4～20次。重复片段长度≥20 bp的SSR占21. 20%,且SSR发生频率与片段长度呈显著负相关(P <0. 01),相关系数为-0. 561。本研究获得的余甘子转录组SSR位点出现频率较高、分布密度较大、低级重复基元较多,重复次数较高、长片段较多,大多数SSR位点的多态性潜能较高,用于余甘子遗传多样性分析的潜力较大,为下一步余甘子转录组SSR标记的大规模开发和群体遗传学研究提供了重要的数据信息,进而为余甘子野生资源的保护和合理开发利用提供了参考依据。
In order to comprehensively understand the characteristics of distribution and variation of the SSR loci in the transcriptome of Phyllanthus emblica L.,transcriptome sequencing was conducted on young leaves of P. emblica by using Illumina Hiseq 4000. The SSR loci were studied using MISA software from the obtained unigenes. A total of 9 991 SSRs distributed on 9 538 unigenes were detected,with an average density of one SSR per 5. 49 kB. Mononucleotide and dinucleotide repeats were the main SSR types,accounting for 42. 3%and 30. 79% of all SSRs,respectively. There were 1 731 SSR loci distributed across the protein-coding regions,with an overall density of 0. 039 SSRs/k B. The dominant repeat type was trinucleotide repeat type. Among all the 169 repeat motifs,A/T(42. 10%) was the richest motif,followed in order by AG/CT(22. 91%) and AAG/CTT(5. 02%). The number of repeats ranged from 4 to 75,most of them were concentrated on four to20. The SSRs with repeat fragment length ≥20 bp accounted for 21. 20%. There was significant negative correlation(P < 0. 01) between the frequency of SSRs and the repeat fragment length,with correlation coefficient of-0. 561. The SSR loci in the transcriptome of P. emblica showed high frequency and density of distribution,rich low-level repeat motifs,large repeat number and long fragments,most of which exhibited high potential of polymorphisms in our study. These SSR loci would lay an important foundation for further developing a large number of EST-SSRs and studying on population genetics of P. emblica,and they would also provide a reference for conservation and rational exploitation of genetic resources of the species.
In order to comprehensively understand the characteristics of distribution and variation of the SSR loci in the transcriptome of Phyllanthus emblica L.,transcriptome sequencing was conducted on young leaves of P. emblica by using Illumina Hiseq 4000. The SSR loci were studied using MISA software from the obtained unigenes. A total of 9 991 SSRs distributed on 9 538 unigenes were detected,with an average density of one SSR per 5. 49 kB. Mononucleotide and dinucleotide repeats were the main SSR types,accounting for 42. 3%and 30. 79% of all SSRs,respectively. There were 1 731 SSR loci distributed across the protein-coding regions,with an overall density of 0. 039 SSRs/k B. The dominant repeat type was trinucleotide repeat type. Among all the 169 repeat motifs,A/T(42. 10%) was the richest motif,followed in order by AG/CT(22. 91%) and AAG/CTT(5. 02%). The number of repeats ranged from 4 to 75,most of them were concentrated on four to20. The SSRs with repeat fragment length ≥20 bp accounted for 21. 20%. There was significant negative correlation(P < 0. 01) between the frequency of SSRs and the repeat fragment length,with correlation coefficient of-0. 561. The SSR loci in the transcriptome of P. emblica showed high frequency and density of distribution,rich low-level repeat motifs,large repeat number and long fragments,most of which exhibited high potential of polymorphisms in our study. These SSR loci would lay an important foundation for further developing a large number of EST-SSRs and studying on population genetics of P. emblica,and they would also provide a reference for conservation and rational exploitation of genetic resources of the species.
引文
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4.Variya B C,Bakrania A K,Patel S S.Emblica officinalis(Amla):a review for its phytochemistry,ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms[J].Pharmacological Research,2016,111:180-200.
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9.Kumar A,Singh K.Isolation of high quality RNA from Phyllanthus emblica and its evaluation by downstream applications[J].Molecular Biotechnology,2012,52(3):269-275.
10.陈林,李龙娜,杨国栋,等.特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析[J].分子植物育种,2016,14(4):959-965.Chen L,Li L N,Yang G D,et al.Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus[J].Molecular Plant Breeding,2016,14(4):959-965.
11.刘玉林,李伟,董树斌,等.文冠果转录组SSR特征分析及EST-SSR标记开发[J].西北农林科技大学学报:自然科学版,2017,45(3):89-95.Liu Y L,Li W,Dong S B,et al.Characterization of SSRs and development of EST-SSR markers in transcriptome of Xanthoceras sorbifolia Bunge[J].Journal of Northwest A&F University:Natural Science Edition,2017,45(3):89-95.
12.杨洁.特有濒危植物青檀微卫星(EST-SSR)分子标记开发及其小尺度空间遗传结构研究[D].南京:南京大学,2016.Yang J.Development of polymorphic microsatellite loci and study on fine-scale spatial genetic structure of Pteroceltis tatarinowii,an endangered plant endemic to China[D].Nanjing:Nanjing University,2016.
13.Cavagnaro P F,Senalik D A,Yang L M,et al.Genomewide characterization of simple sequence repeats in cucumber(Cucumis sativus L.)[J].BMC Genomics,2010,11:569.
14.Bai T D,Xu L A,Xu M,et al.Characterization of masson pine(Pinus massoniana Lamb.)microsatellite DNA by454 genome shotgun sequencing[J].Tree Genetics&Genomes,2014,10(2):429-437.
15.Harr B,Schl9tterer C.Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide under representation[J].Genetics,2000,155(3):1213-1220.
16.李太强,刘雄芳,万友名,等.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,2017,30(4):533-541.Li T Q,Liu X F,Wan Y M,et al.Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to Southeastern Yunnan,China[J].Forest Research,2017,30(4):533-541.
17.刘菁菁,戴晓港,王洁,等.杨树微卫星序列对基因表达频率的影响及表达序列中微卫星特征的分析[J].南京林业大学学报:自然科学版,2011,35(1):11-14.Liu J J,Dai X G,Wang J,et al.Effect of microsatellites on gene expression level and characteristics of expressed SSRs in poplars[J].Journal of Nanjing Forestry University:Natural Science Edition,2011,35(1):11-14.
18.王丽鸳,韦康,张成才,等.茶树花转录组微卫星分布特征[J].作物学报,2014,40(1):80-85.Wang L Y,Wei K,Zhang C C,et al.Characterization of micorsatellites in tea(Camellia sinensis)floral transcriptome[J].Acta Agronomica Sinica,2014,40(1):80-85.
19.Reddy P S,Housman D E.The complex pathology of trinucleotide repeats[J].Current Opinion in Cell Biology,1997,9(3):364-372.
20.童治军,肖炳光.3种烟草基因组SSR位点信息分析和标记开发[J].西北植物学报,2014,34(8):1549-1558.Tong Z J,Xiao B G.Survey of SSR loci information in three tobacco genomes and development of SSR markers[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(8):1549-1558.
21.马秋月,廖卓毅,张得芳,等.碧桃花瓣转录组微卫星特征分析[J].南京林业大学学报:自然科学版,2015,39(3):34-38.Ma Q Y,Liao Z Y,Zhang D F,et al.Deep sequencedbased transcriptome analysis of microsatellites in peach(Prunus persica cv.duplex)flowers[J].Journal of Nanjing Forestry University:Natural Sciences Edition,2015,39(3):34-38.
22.魏琦琦,林青,贾宝光,等.枣转录组序列的微卫星特征分析[J].中南林业科技大学学报,2015,35(6):93-97.Wei Q Q,Lin Q,Jia B G,et al.Microsatellites characteristics of transcriptomic sequences from Ziziphus jujuba cv.‘Zhongqiusucui’[J].Journal of Central South University of Forestry&Technology,2015,35(6):93-97.
23.Blanca J,Ca1izares J,Roig C,et al.Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo(Cucurbitaceae)[J].BMC Genomics,2011,12:104.
24.李炎林,杨星星,张家银,等.南方红豆杉转录组SSR挖掘及分子标记的研究[J].园艺学报,2014,41(4):735-745.Li Y L,Yang X X,Zhang J Y,et al.Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei[J].Acta Horticulturae Sinica,2014,41(4):735-745.
25.许玉兰,蔡年辉,康向阳,等.EST-SSR标记的开发及其在木本植物中的分布特点[J].中国农学通报,2012,28(4):1-7.Xu Y L,Cai N H,Kang X Y,et al.Development and characterization of EST-SSR markers in woody plants[J].Chinese Agricultural Science Bulletin,2012,28(4):1-7.
26.张得芳,李淑娴,夏涛.蔷薇科6个属植物EST-SSR特征分析[J].植物研究,2014,34(6):810-815.Zhang D F,Li S X,Xia T.Characterization of EST-SSR among six genera of Rosaceae[J].Bulletin of Botanical Research,2014,34(6):810-815.
27.Morgante M,Hanafey M,Powell W.Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J].Nature Genetics,2002,30(2):194-200.
28.Li S X,Yin T M.Map and analysis of microsatellites in the genome of Populus:the first sequenced perennial plant[J].Science in China Series C:Life Sciences,2007,50(5):690-699.
29.Schorderet D F,Gartler S M.Analysis of Cp G suppression in methylated and nonmethylated species[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(3):957-961.
30.Sharopova N.Plant simple sequence repeats:distribution,variation,and effects on gene expression[J].Genome,2008,51(2):79-90.
31.Samadi S,Artiguebielle E,Estoup A,et al.Density and variability of dinucleotide microsatellites in the parthenogenetic polyploid snail Melanoides tuberculata[J].Molecular E-cology,1998,7(9):1233-1236.
32.Thao D V,Yamashita M,Watanabe A,et al.Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon[J].Conservation Genetics Resources,2013,5(2):405-407.
33.Weber J L.Informativeness of human(d C-d A)n·(d G-d T)npolymorphisms[J].Genomics,1990,7(4):524-530.
34.Liu X F,Ma H,Li T Q,et al.Development of novel EST-SSR markers for Phyllanthus emblica(Phyllanthaceae)and cross-amplification in two related species[J].Applications in Plant Sciences,2018,6(7):e01169.
35.Temnykh S,Declerck G,Lukashova A,et al.Computational and experimental analysis of microsatellites in rice(Oryza sativa L.):frequency,length variation,transposon associations,and genetic marker potential[J].Genome Research,2001,11(8):1441-1452.
2.李巧明,赵建立.云南干热河谷地区余甘子居群的遗传多样性研究[J].生物多样性,2007,15(1):84-91.Li Q M,Zhao J L.Genetic diversity of Phyllanthus emblica populations in dry-hot valleys in Yunnan[J].Biodiversity Science,2007,15(1):84-91.
3.杨顺楷,杨亚力,杨维力.余甘子资源植物的研究与开发进展[J].应用与环境生物学报,2008,14(6):846-854.Yang S K,Yang Y L,Yang W L.Advances in research and development of Phyllanthus emblica L.[J].Chinese Journal of Applied&Environmental Biology,2008,14(6):846-854.
4.Variya B C,Bakrania A K,Patel S S.Emblica officinalis(Amla):a review for its phytochemistry,ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms[J].Pharmacological Research,2016,111:180-200.
5.Chaphalkar R,Apte K G,Talekar Y,et al.Antioxidants of Phyllanthus emblica L.bark extract provide hepatoprotection against ethanol-induced hepatic damage:a comparison with Silymarin[J].Oxidative Medicine and Cellular Longevity,2017,2017:3876040.
6.Pandey M,Changtragoon S.Isolation and characterization of microsatellites in a medicinal plant,Phyllanthus emblica(Euphorbiaceae)[J].American Journal of Botany,2012,99(12):e468-e469.
7.Solbrig O T.From genes to ecosystems:a research agenda for biodiversity[R].Paris:IUBS,1991:124.
8.Mawalagedera S M U P,Janaththani P,Dunuwille S W MB,et al.DNA marker analysis reveals genomic diversity and putative QTL associated with drupe traits in Phyllanthus emblica L.[J].Ceylon Journal of Science,2014,43(2):31-46.
9.Kumar A,Singh K.Isolation of high quality RNA from Phyllanthus emblica and its evaluation by downstream applications[J].Molecular Biotechnology,2012,52(3):269-275.
10.陈林,李龙娜,杨国栋,等.特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析[J].分子植物育种,2016,14(4):959-965.Chen L,Li L N,Yang G D,et al.Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus[J].Molecular Plant Breeding,2016,14(4):959-965.
11.刘玉林,李伟,董树斌,等.文冠果转录组SSR特征分析及EST-SSR标记开发[J].西北农林科技大学学报:自然科学版,2017,45(3):89-95.Liu Y L,Li W,Dong S B,et al.Characterization of SSRs and development of EST-SSR markers in transcriptome of Xanthoceras sorbifolia Bunge[J].Journal of Northwest A&F University:Natural Science Edition,2017,45(3):89-95.
12.杨洁.特有濒危植物青檀微卫星(EST-SSR)分子标记开发及其小尺度空间遗传结构研究[D].南京:南京大学,2016.Yang J.Development of polymorphic microsatellite loci and study on fine-scale spatial genetic structure of Pteroceltis tatarinowii,an endangered plant endemic to China[D].Nanjing:Nanjing University,2016.
13.Cavagnaro P F,Senalik D A,Yang L M,et al.Genomewide characterization of simple sequence repeats in cucumber(Cucumis sativus L.)[J].BMC Genomics,2010,11:569.
14.Bai T D,Xu L A,Xu M,et al.Characterization of masson pine(Pinus massoniana Lamb.)microsatellite DNA by454 genome shotgun sequencing[J].Tree Genetics&Genomes,2014,10(2):429-437.
15.Harr B,Schl9tterer C.Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide under representation[J].Genetics,2000,155(3):1213-1220.
16.李太强,刘雄芳,万友名,等.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,2017,30(4):533-541.Li T Q,Liu X F,Wan Y M,et al.Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to Southeastern Yunnan,China[J].Forest Research,2017,30(4):533-541.
17.刘菁菁,戴晓港,王洁,等.杨树微卫星序列对基因表达频率的影响及表达序列中微卫星特征的分析[J].南京林业大学学报:自然科学版,2011,35(1):11-14.Liu J J,Dai X G,Wang J,et al.Effect of microsatellites on gene expression level and characteristics of expressed SSRs in poplars[J].Journal of Nanjing Forestry University:Natural Science Edition,2011,35(1):11-14.
18.王丽鸳,韦康,张成才,等.茶树花转录组微卫星分布特征[J].作物学报,2014,40(1):80-85.Wang L Y,Wei K,Zhang C C,et al.Characterization of micorsatellites in tea(Camellia sinensis)floral transcriptome[J].Acta Agronomica Sinica,2014,40(1):80-85.
19.Reddy P S,Housman D E.The complex pathology of trinucleotide repeats[J].Current Opinion in Cell Biology,1997,9(3):364-372.
20.童治军,肖炳光.3种烟草基因组SSR位点信息分析和标记开发[J].西北植物学报,2014,34(8):1549-1558.Tong Z J,Xiao B G.Survey of SSR loci information in three tobacco genomes and development of SSR markers[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(8):1549-1558.
21.马秋月,廖卓毅,张得芳,等.碧桃花瓣转录组微卫星特征分析[J].南京林业大学学报:自然科学版,2015,39(3):34-38.Ma Q Y,Liao Z Y,Zhang D F,et al.Deep sequencedbased transcriptome analysis of microsatellites in peach(Prunus persica cv.duplex)flowers[J].Journal of Nanjing Forestry University:Natural Sciences Edition,2015,39(3):34-38.
22.魏琦琦,林青,贾宝光,等.枣转录组序列的微卫星特征分析[J].中南林业科技大学学报,2015,35(6):93-97.Wei Q Q,Lin Q,Jia B G,et al.Microsatellites characteristics of transcriptomic sequences from Ziziphus jujuba cv.‘Zhongqiusucui’[J].Journal of Central South University of Forestry&Technology,2015,35(6):93-97.
23.Blanca J,Ca1izares J,Roig C,et al.Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo(Cucurbitaceae)[J].BMC Genomics,2011,12:104.
24.李炎林,杨星星,张家银,等.南方红豆杉转录组SSR挖掘及分子标记的研究[J].园艺学报,2014,41(4):735-745.Li Y L,Yang X X,Zhang J Y,et al.Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei[J].Acta Horticulturae Sinica,2014,41(4):735-745.
25.许玉兰,蔡年辉,康向阳,等.EST-SSR标记的开发及其在木本植物中的分布特点[J].中国农学通报,2012,28(4):1-7.Xu Y L,Cai N H,Kang X Y,et al.Development and characterization of EST-SSR markers in woody plants[J].Chinese Agricultural Science Bulletin,2012,28(4):1-7.
26.张得芳,李淑娴,夏涛.蔷薇科6个属植物EST-SSR特征分析[J].植物研究,2014,34(6):810-815.Zhang D F,Li S X,Xia T.Characterization of EST-SSR among six genera of Rosaceae[J].Bulletin of Botanical Research,2014,34(6):810-815.
27.Morgante M,Hanafey M,Powell W.Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J].Nature Genetics,2002,30(2):194-200.
28.Li S X,Yin T M.Map and analysis of microsatellites in the genome of Populus:the first sequenced perennial plant[J].Science in China Series C:Life Sciences,2007,50(5):690-699.
29.Schorderet D F,Gartler S M.Analysis of Cp G suppression in methylated and nonmethylated species[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(3):957-961.
30.Sharopova N.Plant simple sequence repeats:distribution,variation,and effects on gene expression[J].Genome,2008,51(2):79-90.
31.Samadi S,Artiguebielle E,Estoup A,et al.Density and variability of dinucleotide microsatellites in the parthenogenetic polyploid snail Melanoides tuberculata[J].Molecular E-cology,1998,7(9):1233-1236.
32.Thao D V,Yamashita M,Watanabe A,et al.Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon[J].Conservation Genetics Resources,2013,5(2):405-407.
33.Weber J L.Informativeness of human(d C-d A)n·(d G-d T)npolymorphisms[J].Genomics,1990,7(4):524-530.
34.Liu X F,Ma H,Li T Q,et al.Development of novel EST-SSR markers for Phyllanthus emblica(Phyllanthaceae)and cross-amplification in two related species[J].Applications in Plant Sciences,2018,6(7):e01169.
35.Temnykh S,Declerck G,Lukashova A,et al.Computational and experimental analysis of microsatellites in rice(Oryza sativa L.):frequency,length variation,transposon associations,and genetic marker potential[J].Genome Research,2001,11(8):1441-1452.