山地虎耳草转录组SSR信息分析

doi:10.7525/j.issn.1673-5102.2018.06.018

摘要/Abstract

摘要： 利用MISA（MicroSatellite）软件对山地虎耳草转录组拼接序列进行微卫星位点信息分析，为后期SSR标记的开发和物种遗传多样性检测提供候选序列。结果发现，在拼接得到的63 763条Unigene序列中含有4 622个SSR，发生频率为7.25%，有110种重复基元，平均每10.00 kB出现一个SSR位点。山地虎耳草转录组序列的SSR主要集中在三核苷酸重复（55.50%），其次为二核苷酸重复（30.23%）。二核苷酸重复和三核苷酸重复中的优势重复基元分别为AG/TC和AAG/TTC。二核苷酸重复基元的重复次数类型最多，跨度最大，具有更高的多态性，三核苷酸次之，而四、五、六核苷酸重复类型很少。山地虎耳草转录组SSR以5~9次重复为主，且SSR数量随着重复次数的增加逐渐减少，基序长度主要集中于12~30 bp，多态性均在中等以上。

关键词: 山地虎耳草, 转录组, SSR信息分析

Abstract: MISA (MicroSatellite) software was used to analyze the microsatellites information from transcriptome sequences of Saxifraga sinomontana J.T.Pan & Gornall, intending to provide candidate sequences for the development of SSR markers and detection of genetic diversity. In total, 4 622 SSRs were detected in 63 763 unigenes, which was represented by 110 repeat motifs. The frequency of SSRs was detected to be 7.25%, with an average occurrence of per 10.00 kB in length. The tri-nucleotide was the most abundant repeat motif (55.50%), followed by di-nucleotide (30.23%). The dominant repeat motifs in di-nucleotide and tri-nucleotide were AG/TC and AAG/TTC, respectively. In SSRs, the di-nucleotide possessed the most abundant repeat types, the longest span, as well as higher polymorphism, followed by tri-nucleotide. However, for the tetra-nucleotide, penta-nucleotide and hexa-nucleotide, there were few repeat types. For S.sinomontana, the repeat numbers of SSRs were mainly from five to nine, of which the number gradually decreased along with the repeat numbers increased. Besides, the motif length mostly distributed in 12 to 30 bp, and the polymorphism of SSRs was above medium level.

Key words: Saxifraga sinomontana, transcriptome, SSR analysis

中图分类号:

Q949.751.2

李彦, 更吉卓玛, 贾留坤, 王智华, 陈世龙, 高庆波. 山地虎耳草转录组SSR信息分析[J]. 植物研究, 2018, 38(6): 939-947.

LI Yan, GENGJI Zhuo-Ma, JIA Liu-Kun, WANG Zhi-Hua, CHEN Shi-Long, GAO Qing-Bo. Analysis of SSR Information in Transcriptome Sequences of Saxifraga sinomontana[J]. Bulletin of Botanical Research, 2018, 38(6): 939-947.

参考文献

1. Myers N,Mittermeier R A,Mittermeier C G,et al. Biodiversity hotspots for conservation priorities[J]. Nature,2000,403(6772):853-858.
2. Hewitt G M. Post-glacial re-colonization of European biota[J]. Biological Journal of the Linnean Society,1999,68(1-2):87-112.
3. Hewitt G M. Speciation,hybrid zones and phylogeography-or seeing genes in space and time[J]. Molecular Ecology,2001,10(3):537-549.
4. Wu Z Y,Raven P H,Hong D Y. Flora of China[M]. Beijing:Science Press & Missouri;Missouri Botanical Garden Press,2001:208-344.
5. Gao Q B,Li Y H,Gornall R J,et al. Phylogeny and speciation in Saxifraga sect. Ciliatae (Saxifragaceae):evidence from psb A-trn H, trn L-F and ITS sequences[J]. Taxon,2015,64(4):703-713.
6. Ebersbach J,Muellner-Riehl A N,Michalak I,et al. In and out of the Qinghai-Tibet Plateau:divergence time estimation and historical biogeography of the large arctic-alpine genus Saxifraga L. [J]. Journal of Biogeography,2017,44(4):900-910.
7. Abbott R J,Comes H P. Evolution in the Arctic:a phylogeographic analysis of the circumarctic plant, Saxifraga oppositifolia (Purple saxifrage)[J]. New Phytologist Trust,2004,161(1):211-224.
8. Dechaine E G,Anderson S A,Mcnew J M,et al. On the evolutionary and biogeographic history of Saxifraga sect. Trachyphyllum (Gaud.) Koch(Saxifragaceae Juss.)[J]. PLoS One,2013,8(7):e69814.
9. Ebersbach J,Schnitzler J,Favre A,et al. Evolutionary radiations in the species-rich mountain genus Saxifraga L. [J]. BMC Evolutionary Biology,2017,17:119.
10. Gao Q B,Li Y,Gengji Z M,et al. Population genetic differentiation and taxonomy of three closely related species of Saxifraga (Saxifragaceae) from southern Tibet and the Hengduan Mountains[J]. Frontiers in Plant Science,2017,8:1325.
11. 更吉卓玛,李彦,贾留坤,等. 唐古特虎耳草谱系地理学研究[J]. 西北植物学报,2018,38(2):370-380.Gengji Z M,Li Y,Jia L K,et al. Phylogeography of Saxifraga tangutica Engl. (Saxifragaceae)[J]. Acta Botanica Boreali-Occidentalia Sinica,2018,38(2):370-380.
12. Litt M,Luty J A. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene[J]. American Journal of Human Genetics,1989,44(3):397-401.
13. Tautz D,Trick M,Dover G A. Cryptic simplicity in DNA is a major source of genetic variation[J]. Nature,1986,322(6080):652-656.
14. 陈相艳,李伟,戴海英,等. 大豆EST资源的SSR信息分析[J]. 大豆科学,2009,28(3):394-399.Chen X Y,Li W,Dai H Y,et al. Analysis of SSR information in EST resource of soybean(Glycine max)[J]. Soybean Science,2009,28(3):394-399.
15. Weber J L. Informativeness of human(dC-dA) n·(dG-dT)n polymorphisms[J]. Genomics,1990,7(4):524-530.
16. Wang Z,Weber J L,Zhong G,et al. Survey of plant short tandem DNA repeats[J]. Theoretical and Applied Genetics,1994,88(1):1-6.
17. Maroof M A S,Biyashev R M,Yang G P,et al. Extraordinarily polymorphic microsatellite DNA in barley:species diversity,chromosomal locations,and population dynamics[J]. Proceedings of the National Academy of Sciences of the United States of America,1994,91(12):5466-5470.
18. 雷淑芸,高庆波,付鹏程,等. 基于Solexa高通量测序的唐古特红景天(Rhodiola algida)微卫星信息分析[J]. 植物研究,2014,34(6):829-834.Lei S Y,Gao Q B,Fu P C,et al. Analysis on Microsatellites in Rhodiola algida based on Solexa Sequencing[J]. Bulletin of Botanical Research,2014,34(6):829-834.
19. 田尊哲,高庆波,陈世龙,等. 基于高通量测序分析青藏高原特有植物蓝玉簪龙胆(Gentiana veitchiorum)的SSR和SNP特征[J]. 植物研究,2016,36(5):747-752.Tian Z Z,Gao Q B,Chen S L,et al. Characteristics of SSR and SNP in Gentiana veitchiorum in QingHai-Tibetan Plateau,by high-throughput sequencing[J]. Bulletin of Botanical Research,2016,36(5):747-752.
20. 王久利,雷淑芸,陈世龙,等. 基于磁珠富集法开发并筛选西川红景天的微卫星标记[J]. 广西植物,2017,37(3):342-347.Wang J L,Lei S Y,Chen S L,et al. Development and screening of SSR markers in Rhodiola alsia with magnetic beads method[J]. Guihaia,2017,37(3):342-347.
21. Grabherr M G,Haas B J,Yassour M,et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology,2011,29(7):644-652.
22. 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.
23. Erlich Y,Mitra P P,Delabastide M,et al. Alta-Cyclic:a self-optimizing base caller for next-generation sequencing[J]. Nature Methods,2008,5(8):679-682.
24. Jiang L C,Schlesinger F,Davis C A,et al. Synthetic spike-in standards for RNA-seq experiments[J]. Genome Research,2011,21(9):1543-1551.
25. 张水仙. 基于蒙药冷蒿基因组低通量测序的SSR遗传多样性分析[D]. 北京:中央民族大学,2013.Zhang S X. SSR genetic diversity analysis of Mongolia medicinal plant Artemisia frigida based on low-throughput genome sequencing[D]. Beijing:Minzu University of China,2013.
26. 刘越,范增华,孙洪波,等. 裂叶牵牛获得表达序列标签资源的简单序列重复信息分析[J]. 中国药学杂志,2011,46(23):1790-1794.Liu Y,Fan Z H,Sun H B,et al. Analysis of SSR information in EST resource of Pharbitis nil[J]. Chinese Pharmaceutical Journal,2011,46(23):1790-1794.
27. 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.
28. Dreisigacker S,Zhang P,Warburton M L,et al. SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different megaenvironments[J]. Crop Science,2004,44(2):381-388.
29. 岳春江,陈川川,郭凤仙,等. 蒙药冷蒿转录组SSR信息分析[J]. 中国农业科技导报,2016,18(6):31-43.Yue C J,Chen C C,Guo F X,et al. Data mining of simple sequence repeats in transcriptome sequences of Mongolia medicinal plant Artemisia frigida Willd[J]. Journal of Agricultural Science and Technology,2016,18(6):31-43.
30. 冯法节,李明杰,古力,等. 地黄EST-SSR标记的开发及扩增体系的建立[J]. 广东农业科学,2015,42(10):120-126.Feng F J,Li M J,Gu L,et al. Development of EST-SSR marker and amplification system of Rehmannia glutinosa[J]. Guangdong Agricultural Sciences,2015,42(10):120-126.
31. 黄海燕,杜红岩,乌云塔娜,等. 基于杜仲转录组序列的SSR分子标记的开发[J]. 林业科学,2013,49(5):176-181.Huang H Y,Du H Y,Wuyun T N,et al. Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides[J]. Scientia Silvae Sinicae,2013,49(5):176-181.
32. 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 L,Cheng Y F,Gan X Y,et al. Mining EST-SSRs based on potato transcriptome and analyzing their polymorphism[J]. Molecular Plant Breeding,2015,13(7):1535-1544.
33. 张华丽,王涛,宋利娜,等. 菊花EST-SSR标记开发及其对万寿菊的可转移性研究[J]. 核农学报,2016,30(1):42-49.Zhang H L,Wang T,Song L N,et al. Study on EST-derived SSR marker development of Chrysanthemum morifolium and its transferability to Tagetes erecta L. [J]. Journal of Nuclear Agricultural Sciences,2016,30(1):42-49.
34. 蒋超,袁媛,刘贵明,等. 基于EST-SSR的金银花分子鉴别方法研究[J]. 药学学报,2012,47(6):803-810.Jiang C,Yuan Y,Liu G M,et al. EST-SSR identification of Lonicera japonica Thunb[J]. Acta Pharmaceutica Sinica,2012,47(6):803-810.
35. 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 X Q,Lu M,An H M. Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii[J]. Acta Horticulturae Sinica,2015,42(2):341-349.
36. 刘越,岳春江,王翊,等. 藏茵陈川西獐牙菜转录组SSR信息分析[J]. 中国中药杂志,2015,40(11):2068-2076.Liu Y,Yue C J,Wang Y,et al. Data mining of simple sequence repeats in transcriptome sequences of Tibetan medicinal plant Zangyinchen Swertia mussotii[J]. China Journal of Chinese Materia Medica,2015,40(11):2068-2076.
37. 陈茵,李翠婷,姜倪皓,等. 灯盏花转录组中SSR位点信息分析及其多态性研究[J]. 中国中药杂志,2014,39(7):1220-1224.Chen Y,Li C T,Jiang N H,et al. SSR information in Erigeron breviscapus transcriptome and polymorphism analysis[J]. China Journal of Chinese Materia Medica,2014,39(7):1220-1224.
38. Wei W L,Qi X Q,Wang L H,et al. Characterization of the sesame(Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers[J]. BMC Genomics,2011,12:451.
39. 王森,张震,姜倪皓,等. 半夏转录组中的SSR位点信息分析[J]. 中药材,2014,37(9):1566-1569.Wang S,Zhang Z,Jiang N H,et al. SSR Information in Transcriptome of Pinellia ternata[J]. Journal of Chinese Medicinal Materials,2014,37(9):1566-1569.
40. 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.
41. Metzgar D,Bytof J,Wills C. Selection against frameshift mutations limits microsatellite expansion in coding DNA[J]. Genome Research,2000,10(1):72-80.
42. Blanca J,Cañizares J,Roig C,et al. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae)[J]. BMC Genomics,2011,12:104.
43. Garg R,Patel R K,Tyagi A K,et al. De novo assembly of chickpea transcriptome using short reads for gene discovery and marker identification[J]. DNA Research,2011,18(1):53-63.
44. 范三红,郭蔼光,单丽伟,等. 拟南芥基因密码子偏爱性分析[J]. 生物化学与生物物理进展,2003,30(2):221-225.Fan S H,Guo A G,Shan L W,et al. Analysis of genetic code preference in Arabidopsis thaliana[J]. Progress in Biochemistry and Biophysics,2003,30(2):221-225.
45. 贾新平,叶晓青,梁丽建,等. 基于高通量测序的海滨雀稗转录组学研究[J]. 草业学报,2014,23(6):242-252.Jia X P,Ye X Q,Liang L J,et al. Transcriptome characteristics of Paspalum vaginatum analyzed with Illumina sequencing technology[J]. Acta Prataculturae Sinica,2014,23(6):242-252.
46. Morgante M,Hanafey M,Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nature Genetics,2002,30(2):194-200.
47. Gao L F,Tang J F,Li H W,et al. Analysis of microsatellites in major crops assessed by computational and experimental approaches[J]. Molecular Breeding,2003,12(3):245-261.
48. Cardle L,Ramsay L,Milbourne D,et al. Computational and experimental characterization of physically clustered simple sequence repeats in plants[J]. Genetics,2000,156(2):847-854.

[1]	倪馨宇, 贺俊英, 燕孟娇, 杜超. RNA-Seq技术在珍稀濒危植物研究中的应用进展[J]. 植物研究, 2023, 43(4): 481-492.
[2]	郑占敏, 商玉冰, 周广波, 肖迪, 刘轶, 由香玲. PsnHB13与PsnHB15在小黑杨中的遗传转化与功能分析[J]. 植物研究, 2023, 43(3): 340-350.
[3]	黄东梅, 陈颖, 白露, 倪迪安, 徐奕扬, 张志国, 秦巧平. 萱草叶片响应低温胁迫的转录组分析[J]. 植物研究, 2022, 42(3): 424-436.
[4]	余静雅, 夏铭泽, 徐浩, 张发起. 青藏高原地区3种蒿属植物转录组比较分析[J]. 植物研究, 2022, 42(2): 200-210.
[5]	赵雪艳, 李思锋, 柏国清, 李为民. 百合属5种植物的比较转录组分析[J]. 植物研究, 2021, 41(4): 614-625.
[6]	滕云, 邱宗波, 王稳利, 张雪如. 泡桐维管形成层及木质部区的转录组分析[J]. 植物研究, 2021, 41(3): 354-361.
[7]	周晨晨, 李义良, 王哲, 赵奋成, 钟岁英, 林昌明, 谭志强, 李福明, 吴惠姗, 郭文冰, 廖仿炎, 王建革. 基于RNA-Seq技术的湿加松特定时期产脂差异基因筛选及表达分析[J]. 植物研究, 2021, 41(3): 419-428.
[8]	鲁琳, 杨尚谕, 刘维东, 鲁黎明. 基于转录组测序的花烟草低温胁迫响应转录因子挖掘[J]. 植物研究, 2021, 41(1): 119-129.
[9]	赵历强, 单春苗, 张声祥, 施圆圆, 马克龙, 吴家文. 基于转录组测序的细风轮花青素合成途径及关键酶基因分析[J]. 植物研究, 2020, 40(6): 886-896.
[10]	刘丹, 曾钦朦, 刘斌, 李煜, 陈世品. 闽楠转录组分析及基因功能注释[J]. 植物研究, 2020, 40(4): 613-622.
[11]	张雨, 夏铭泽, 张发起. 药用资源植物山莨菪的转录组信息分析[J]. 植物研究, 2020, 40(3): 458-467.
[12]	朱利利, 杜庆鑫, 何凤, 庆军, 杜红岩. 杜仲雄花芽2个发育时期转录组分析[J]. 植物研究, 2020, 40(2): 284-292.
[13]	叶兴状, 刘丹, 罗佳佳, 范辉华, 张国防, 刘宝, 陈世品. 濒危珍稀植物半枫荷的转录组分析[J]. 植物研究, 2019, 39(2): 276-286.
[14]	刘雄芳, 李太强, 张序, 李正红, 万友名, 安静, 刘秀贤, 马宏. 重要食药同源植物余甘子转录组微卫星特征分析[J]. 植物研究, 2019, 39(2): 294-302.
[15]	王大玮, 周凡, 沈兵琪, 王连春. 基于高通量测序的怒江红山茶微卫星位点特征分析[J]. 植物研究, 2019, 39(1): 148-155.