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| Development and Polymorphism Analysis of EST-SSR Markers Based on Transcriptome of Non-heading Chinese Cabbage (Brassica rapa ssp. chinensis) |
| YANG Dan-Qing1, HE Xiao-Li1, DU Zhi-Jie1, WANG Shu-Bin1, SUN Li-Wei1, LIN Yi-Zhang1, SHAO Gui-Rong3, ZHONG Feng-Lin1,2,* |
1 College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
2 The Vegetable Institute of Fujian Agricultural and Forestry University, Fuzhou 350002, China;
3 Fujian Jinpin Agricultural Technology Co., Ltd., Fuzhou 350002, China |
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Abstract The EST-SSR markers obtained from transcriptome sequencing information have the advantages of good stability, high polymorphism, co-dominant inheritance, and simple operation, and are widely used in genetic diversity analysis of plant germplasm resources. In this study, high-throughput sequencing of the transcriptome of non-heading Chinese cabbage (Brassica rapa ssp. chinensis) was used to seek SSR loci and develop EST-SSR markers, and preliminary primer verification and polymorphism analysis were performed. A total of 17 009 SSR loci were found in the 48 817 unigene sequences of the transcriptional set of non-heading Chinese cabbage, with an occurrence frequency of 18.44% and a SSR distribution frequency of 34.84%. The SSR sites of non-heading Chinese cabbage contained 212 repeat elements whose SSR types were mainly concentrated on single, binary and trinucleotide repeats. Among them, single nucleotide repeats accounted for the most, accounting for 39.70% of the total, and the dominant repeating unit was T/A (26.83%). The EST-SSR primers designed by Primer Premier 3.0 soft were used to randomly selected for 40 pairs in batch design primers for synthesis and preliminary verification. Twenty-five pairs of primers could amplify the expected size bands in 92 non-heading Chinese cabbage germplasm resources. The average number of alleles was 3.72, and the effective number of alleles (Ne), Shannon's index (I), polymorphism information content (PIC) and gene diversity (GD) were 2.710 8, 0.999 1, 0.488 4 and 0.545 2, respectively. The obtained EST-SSR primers could provide effective molecular markers for genetic structure analysis, germplasm utilization and genetic map construction of non-heading Chinese cabbage and other Brassica plants.
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Received: 01 August 2019
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Corresponding Authors:
*zhong591@fafu.edu.cn
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[1] 曹学伟. 2016. 不结球白菜分蘖性状的发生机理及其候选基因挖掘[D]. 博士学位论文,南京农业大学, 导师: 李英. pp. 7-16.
(Cao X W.2016. The formation mechanism and candidate gene identification of tillering in non-heading chinese cabbage [D]. Thesis for Ph.D.,Nanjing Agricultural University, Superviser: Li Y. pp. 7-16.)
[2] 郭晶心, 曹鸣庆. 2001. 芸薹属植物起源、演化及分类的分子标记研究进展[J]. 生物技术通报,(01): 26-30.
(Guo J X, Cao M Q. 2001. Progress of molecular maker researches on the taxonomy, origin and evolution of Brassica[J].Biotechnology Bulletin, (01): 26-30.)
[3] 甘霖, 吴景, 杨玉双, 等. 2019. 橡胶草转录组SSR信息分析与标记开发[J]. 热带农业科学, 39(01): 42-46.
(Gan L, W J, Yang Y S, et al.2019. Transcriptome sequences SSR information analysis of russian dandelion[J]. Journal of Tropical Agriculture, 39(01): 42-46.)
[4] 何宇宁. 2017. 铜胁迫下海州香薷酸性转化酶基因启动子的甲基化研究[D]. 武汉大学, 导师: 熊治廷. pp. 13-17.
(He Y N.2017. The methylation patterns of acid invertase gene promoters from Cu-tolerant and non-tolerant populations of Elsholtzia haichowensis under copper stress[D]. Thesis for Ph.D., Wuhan University, Superviser: Xiong Z T.pp.13-17.)
[5] 李桂花, 陈汉才, 张艳, 等. 2017. 小白菜种质遗传多样性与亲缘关系的SRAP和SSR分析[J]. 广东农业科学, 44(05): 37-45.
(Li G H, Chen H C, Zhang Y, et al.2017. SSR fingerprinting and genetic distinctness of pak-choi (Brassica rapa L.ssp.chinensis Makino)[J]. Guangdong Agricultural Sciences, 44(05): 37-45.)
[6] 李鸥, 赵莹莹, 郭娜, 等. 2009. 草鱼种群SSR分析中样本量及标记数量对遗传多度的影响[J]. 动物学研究, 30(02): 121-130.
(Li O, Zhao Y Y, Guo N, et al.2009. Effects of sample size and loci number on genetic diversity in wild population of grass carp revealed by SSR[J]. Zoology Research, 30(02): 121-130.)
[7] 刘冬梅, 娄喜艳, 吴狄, 等. 2019. 基于棉花转录组测序的SSR分子标记的开发[J]. 江苏农业科学, 47(07): 32~35.
(Liu D M, Yan X Y, Wu D, et al.2019. Excavation of SSR molecular markers based on transcriptome sequencing of cotton[J]. Jiangsu Agricultural Sciences, 47(07): 32-35.)
[8] 刘峰, 王运生, 田雪亮, 等. 2012. 辣椒转录组SSR挖掘及其多态性分析[J]. 园艺学报, 39(01): 168~174.
(Liu F, Wang Y S, Tian X L, et al.2012. SSR mining in pepper (Capsicum annuum L.) transcriptome and the polymorphism analysis[J]. Journal of Horticulture, 39(01): 168-174.)
[9] 陆云峰, 杨安娜, 张俊红, 等. 2018. 紫楠转录组EST-SSR标记开发及通用性分析[J]. 农业生物技术学报, v.26(6): 108-118.
(Lu Y F, Yang A N, Zhang J H, et al.Development and transferability evaluation of EST-SSR markers based on transcriptome data of Phoebe sheareri[J]. Journal of Agricultural Biotechnology, v.26(6): 108-118.)
[10] 罗丹, 吴委林, 周波, 等. 2012. 芜菁SSR引物的开发及多态性分析[J]. 生物技术通讯, 23(03): 402-406.
(Luo D, Wu W L, Zhou B, et al.2012. Development of simple sequence repeats in Brassica rapa and poly morphism analysis[J]. Biotechnology Communication, 23(03): 402-406.)
[11] 潜宗伟, 陈海丽, 崔彦玲. 2016. 菠菜转录组SSR位点分析及其分子标记的开发[J]. 农业生物技术学报, 24(11): 1688-1697.
(Qian Z W, Chen H L, Cui Y L.2016. Analysis of the SSR loci and development of molecular markers in Spinacia oleracea transcriptome[J]. Journal of Agricultural Biotechnology, 24(11): 1688-1697.)
[12] 时小东, 朱学慧, 盛玉珍, 等. 2016. 基于转录组序列的楠木SSR分子标记开发[J]. 林业科学, 52(11): 71-78.
(Shi X D, Zhu X H, Sheng Y Z, et al.2016. Development of SSR markers based on transcriptome sequence of Phoebe zhennan[J]. Forestry Science, 52(11): 71-78.)
[13] 王果, 胡正, 张保缺, 等. 2008. 山西省野生大豆资源遗传多样性分析[J]. 中国农业科学, (07): 2182-2190.
(Wang G, Hu Z, Zhang B W, et al.2008. Genetic diversity analysis of shanxi's wild soybean (Glycine soja)[J]. Chinese Journal of Agricultural Sciences, (07): 2182-2190.)
[14] 王红梅, 张正英, 陈玉梁. 2003. SSR标记技术及其在植物遗传学中的应用[J]. 西北师范大学学报(自然科学版), (01): 113-116.
(Wang H M, Zhang Z Y, Chen Y L.2003. SSR markers and their application in plant genetics'[J]. Journal of Northwest Normal University(Natural Science), (01): 113-116.)
[15] 王笑一, 于拴仓, 张凤兰, 等. 2008. 小白菜品种的SSR指纹图谱及遗传特异性分析[J]. 华北农学报, (05): 97-103.
(Wang X Y, Yu Z C, Zhang F L, et al.2008. SSR fingerprinting and genetic distinctness of pak-choi (Brassica rapa L.ssp.chinensis Makino)[J]. 华北农业学报, (05): 97-103.)
[16] 王新华, 陈火英. 2010. 不结球白菜SRAP标记体系的建立与作图亲本筛选[J]. 上海交通大学学报(农业科学版), 28(05): 397-401.
(Wang X H, Chen H Y.2010. Developing a technical protocol for pak-choi SRAP assays and screening for highly polymorphic parents for mapping[J]. Journal of Shanghai Jiaotong University, Agricultural Sciences, 28(05): 397-401.)
[17] 韦德群, 周涛, 江维克, 等. 2018. 太子参转录组中SSR位点信息分析及其多态性分析[J]. 分子植物育种, 16(23): 7754-7759.
(Wei D Q, Zhou T, Jiang W L, et al.2018. Analysis on SSR loci information in transcriptome of Pseudostellaria heterophylla and its polymorphi[J]. Molecular Plant Breeding, 16(23): 7754-7759.)
[18] 薛思鸣, 成丹, 解洪杰, 等. 2016. 利用拟南芥种群间转录组和表皮毛密度差异信息筛选表皮毛相关功能基因[J]. 农业生物技术学报, 24(08): 1138-1146.
(Xue S M, Cheng D, Xie H J, et al.2016. Screening trichome-related functional genes using difference of transcriptome and trichome density among Arabidopsis thaliana populations[J]. Journal of Agricultural Biotechnology, 24(08): 1138-1146.)
[19] 张海焕. 2013. 基于转录组序列的芸薹属SSR标记的开发[D]. 硕士学位论文.江西农业大学,导师:贺浩华. pp.15-18.
(Zhang H H.2013. Development of SSR markers based on transcriptome sequences in Brassica species[D]. Thesis for Ph.D., Jiangxi Agricultural University, Superviser: He H H.pp.15-18.)
[20] 张婉, 李丽.2013. 白菜SSR分子标记的研究进展[J]. 科技创新与应用, (08): 9-10.
(Zhang W, Li L. 2013. Research progress of SSR molecular markers in chinese cabbage[J]. Science and Technology Innovation and Application, (08): 9-10.)
[21] 朱海生, 黄丽芳, 王彬, 等. 2018. 美洲南瓜转录组SSR信息分析及其分子标记开发[J]. 中国细胞生物学学报, 40(01): 99-107.
(Zhu H S, Huang L F, Wang B, et al.2018. Analysis on SSR information in transcriptome and development of molecular markers in summer squash[J]. Chinese Journal of Cell Biology, 40(01): 99-107.)
[22] 周杰. 2009.白菜遗传多样性研究[D].硕士学位论文, 华中农业大学,导师:刘克德. pp.4-8.
(Zhou J.2009. Genetic diversity study of Brassica comepestris[D].Thesis for Ph.D., Huazhong Agricultural University, Superviser: Liu K D.pp.4-8.)
[23] An Z W, Zhao Y H, Cheng H, et al.2009. Development and application of EST-SSR markers in Hevea brasiliensis Muell. Arg[J]. Hereditas, 31(3): 311-319.
[24] Chen J, Li R, Xia Y, et al.2017. Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies[J]. PLOS ONE, 12(9): e184736.
[25] Liu C, Dou Y, Guan X, et al.2017. De novo transcriptomic analysis and development of EST-SSRs for Sorbus pohuashanensis (Hance) Hedl[J]. PLOS ONE, 12(6): e179219.
[26] Jones C J, Edwards K J, Castaglione S, et al.1997. Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories[J]. Molecular Breeding, 3(5): 381-390.
[27] Nagy S, Poczai P, Cernák I, et al.2012. PICcalc: An online program to calculate polymorphic information content for molecular genetic studies[J]. Biochemical Genetics, 50(9-10): 670-672.
[28] Pathaichindachote W, Panyawut N, Sikaewtung K, et al.2019. Genetic diversity and allelic frequency of selected thai and exotic rice germplasm using SSR markers[J]. Rice Science, 26(6): 393-403.
[29] Varshney R K, Andreas G, Sorrells M E.2005. Genic microsatellite markers in plants: Features and applications[J]. Trends in Biotechnology, 23(1): 48-55.
[30] Wang S, Wang X, He Q, et al.2012. Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish[J]. Plant Cell Reports, 31(8): 1437-1447.
[31] Zerbo G C, Konrad H, Ouedraogo M, et al.2017. Fourteen simple-sequence repeats newly developed for population genetic studies in Prosopis africana (Fabaceae-Mimosoideae)[J]. BMC Research Notes, 10(1): 437.
[32] Zietkiewicz E, Rafalski A, Labuda D.1994. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J]. Genomics, 20(2): 176-183. |
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