基于荧光标记的紫娟茶树转录组EST-SSR标记开发
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摘要
为开发紫娟茶树转录组EST-SSR标记,基于前期对紫娟茶树芽、第2叶、开面叶、成熟叶转录组高通量测序所得到的242 757条Unigene进行多态性分析与评价,再利用荧光标记PCR技术,规模化开发茶树EST-SSR标记。结果表明:从紫娟茶树转录组中搜索得到46 041条Unigene含有57 976个SSR位点,出现频率为23.88%。EST-SSR类型以一核苷酸、二核苷酸、三核苷酸重复为主,占总SSR的98.54%。设计合成138对荧光SSR引物,采用荧光标记PCR技术对4个差异较大的茶树品种进行引物筛选,其中44对引物得到高质量的EST-SSR标记位点。这些EST-SSR可用于茶树遗传多样性分析、分子育种等。
To develop the transcriptome EST-SSR markers of Zijuan tea tree,the polymorphism of 242 757 unigenes got by high-throughput sequencing based on the earlier-stage study of the bud,the second leaf,the leaves without bud and mature leaves of Zijuan tea tree was analyzed and evaluated,and then fluorescent tags PCR technology was used for scale development of tea tree EST-SSR markers. The results showed that 46 041 unigenes containing 57 976 simple sequence repeats( SSR) loci were obtained by searching from Zijuan tea plant transcriptomes with the frequency of 23. 88%. The main repeat types of EST-SSR were mononucleotide,dinucleotide and trinucleotide accounted for 98. 54% of total SSR. 138 pairs of fluorescent marker primers were designed and synthesized,then four different tea varieties were screened by using fluorescent marker PCR technology,44 pairs of primers got high quality of EST-SSR markers loci. These EST-SSRs can be used for tea tree genetic analysis and molecular breeding.
To develop the transcriptome EST-SSR markers of Zijuan tea tree,the polymorphism of 242 757 unigenes got by high-throughput sequencing based on the earlier-stage study of the bud,the second leaf,the leaves without bud and mature leaves of Zijuan tea tree was analyzed and evaluated,and then fluorescent tags PCR technology was used for scale development of tea tree EST-SSR markers. The results showed that 46 041 unigenes containing 57 976 simple sequence repeats( SSR) loci were obtained by searching from Zijuan tea plant transcriptomes with the frequency of 23. 88%. The main repeat types of EST-SSR were mononucleotide,dinucleotide and trinucleotide accounted for 98. 54% of total SSR. 138 pairs of fluorescent marker primers were designed and synthesized,then four different tea varieties were screened by using fluorescent marker PCR technology,44 pairs of primers got high quality of EST-SSR markers loci. These EST-SSRs can be used for tea tree genetic analysis and molecular breeding.
引文
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[3]敖日格乐,贾晓,葛台明.SSR分子标记的开发策略概述[J].湖北民族学院学报,2009,27(4):462-467.
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[5]方先文,张云辉,肖西林,等.基于重组自交系群体的水稻粒形QTL定位[J].江苏农业学报,2017,33(2):241-247.
[6]张振良,郝德荣,陈国清,等.SSR标记在糯玉米品种鉴定上的应用[J].江苏农业科学,2016,44(6):104-106.
[7]李洋益,雷映霞,高刚,等.应用RAMP标记分析国产姜黄属植物的遗传多样性[J].基因组学与应用生物学,2015,34(8):1778-1783.
[8]张洪源,谈杰,张敏,等.茄子SSR多态性标记筛选及杂交种纯度鉴定[J].南方农业学报,2017,48(7):1148-1154.
[9]曹广英,吴琪,王云云,等.利用SSR标记鉴定花生杂交F1代真假杂种[J].山东农业科学,2016,48(1):7-10,15.
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[12]姚明哲,刘振,陈亮,等.利用EST-SSR分析江北茶区茶树资源的遗传多样性和遗传结构[J].茶叶科学,2009,29(3):243-250.
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[16]罗奕凡,禹双双,陈勤操,等.茶黄素的生理功效研究进展[J].茶叶通讯,2015,42(4):3-8.
[17]李雅,李雅卓,冯慧平,等.茶多酚对冠心病患者血清中s CD146和s CD40水平的影响[J].福建茶叶,2016,38(6):7.
[18]贾琳,张玉梅.儿茶素代谢动力学研究进展[J].食品与药品,2016,18(3):209-218.
[19]邬海桥,丁阳平,周向东.红茶提取物在炎性气道黏液高分泌中的作用[J].上海交通大学学报(医学版),2009,29(2):126-129.
[20]谭婷,刘武嫦,仇云龙,等.茶叶对IBD肠道微生物菌群影响研究进展[J].茶叶通讯,2016,43(3):37-40.
[21]冯强,李毅,夏鹏程,等.茶叶提取物抗肿瘤作用的最新研究进展[J].中国实用医药,2016,11(22):274-275.
[22]乔如颖,李明,郑新强,等.茶叶及其儿茶素对乳腺癌的抑制作用[J].茶叶科学,2016,36(6):557-566.
[23]曲伟,王孟君,于海燕,等.毛尖茶叶多糖及其结合物的抗氧化活性研究[J].天然产物研究与开发,2015,27(2):2010-2013.
[24]李慧,聂枞宁,黄亮,等.分子标记在茶树种质资源遗传多样性分析中的应用研究[J].现代农村科技,2016(2):69-70.
[25]刘本英,李友勇,唐一春,等.云南茶树资源遗传多样性与亲缘关系的ISSR分析[J].作物学报,2010,36(3):391-400.
[26]吴清韩,庄东红,朱慧,等.凤凰单丛茶树资源多样性的ISSR分析[J].热带作物学报,2015,36(3):499-503.
[27]侯渝嘉,常亚丽,何桥,等.应用AFLP分子标记研究茶树品种新材料[J].西南农业学报,2015,28(2):514-518.
[28]陈林波,夏丽飞,周萌,等.基于RNA-Seq技术的“紫娟”茶树转录组分析[J].分子植物育种,2015,13(10):2250-2255.
[29]王希,陈丽,赵春雷.利用MISA工具对不同类型序列进行SSR标记位点挖掘的探讨[J].中国农学通报,2016,32(10):150-156.
[30]王丽鸳,姜燕华,段云裳,等.茶树EST-SSRs分布特征及引物开发[J].植物遗传资源学报,2009,10(4):511-516.
[31]袁阳阳,王青锋,陈进明.基于转录组测序信息的水生植物莕菜SSR标记开发[J].植物科学学报,2013,31(5):485-492.
[32]郁永明,田丹青,潘晓韵,等.基于红掌转录组序列的SSR标记分析与开发[J].分子植物育种,2015,13(6):1349-1354.
[33]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.
[34]张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120.
[35]文亚峰,韩文军,周宏,等.杉木转录组SSR挖掘及EST-SSR标记规模化开发[J].林业科学,2015,51(11):40-49.
[36]程海亮,陆才瑞,邹长松,等.基于10个棉花腺体相关材料转录组的EST-SSR标记开发[J].棉花学报,2015,27(1):1-8.
[37]MA J Q,ZHOU Y H,MA C L,et al.Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant,Camellia sinensis(Theaceae)[J].Am J Bot,2010,97(12):153-156.
[38]段云裳,姜燕华,王丽鸳,等.中国红、绿茶适制品种(系)遗传多样性与亲缘关系的SSR分析[J].中国农业科学,2011,44(1):100-109.
[2]TOTH G,GASPARI Z.Microsatellites in different eukaryotic genomes Surevey and analysis[J].Genome Research,2000,10(7):967-981.
[3]敖日格乐,贾晓,葛台明.SSR分子标记的开发策略概述[J].湖北民族学院学报,2009,27(4):462-467.
[4]POWELL W,MACHRAY G C.PROVAN J.Polymorphism revealed by simple sequence repeats[J].Trends Plant Science,1996,1:215-222.
[5]方先文,张云辉,肖西林,等.基于重组自交系群体的水稻粒形QTL定位[J].江苏农业学报,2017,33(2):241-247.
[6]张振良,郝德荣,陈国清,等.SSR标记在糯玉米品种鉴定上的应用[J].江苏农业科学,2016,44(6):104-106.
[7]李洋益,雷映霞,高刚,等.应用RAMP标记分析国产姜黄属植物的遗传多样性[J].基因组学与应用生物学,2015,34(8):1778-1783.
[8]张洪源,谈杰,张敏,等.茄子SSR多态性标记筛选及杂交种纯度鉴定[J].南方农业学报,2017,48(7):1148-1154.
[9]曹广英,吴琪,王云云,等.利用SSR标记鉴定花生杂交F1代真假杂种[J].山东农业科学,2016,48(1):7-10,15.
[10]冯涛,刘娟,华夏雪.利用SSR、SRAP分子标记鉴定桃早熟芽变[J].江苏农业科学,2017,45(6):42-44.
[11]金基强,崔海瑞,陈文岳,等.茶树EST-SSR的信息分析与标记建立[J].茶叶科学,2006,26(1):17-23.
[12]姚明哲,刘振,陈亮,等.利用EST-SSR分析江北茶区茶树资源的遗传多样性和遗传结构[J].茶叶科学,2009,29(3):243-250.
[13]MA H J,MA H J,Li Q,et al.Effect of theaflavin on intracellular free calcium concentration in rat ventricular myocyte[J].Chin Pharmacol Bull,2009,25(8):1032-1035.
[14]MA H J,MA H J,ZHANG J N,et al.Study of the aflavin on reducing intracellular free calcium concentration in ventricular myocytes in rats with ischemia/reperfusion injury[J].Mod J Integr Tradit Chin West Med,2009,18(25):3024-3029.
[15]MA H J,MA H J,LIU Y,et al.Protective effect of the aflavin against ischemia/reperfusion injury in rat heart[J].Mod J Integr Tradit Chin West Med,2009,18(26):3159-3160.
[16]罗奕凡,禹双双,陈勤操,等.茶黄素的生理功效研究进展[J].茶叶通讯,2015,42(4):3-8.
[17]李雅,李雅卓,冯慧平,等.茶多酚对冠心病患者血清中s CD146和s CD40水平的影响[J].福建茶叶,2016,38(6):7.
[18]贾琳,张玉梅.儿茶素代谢动力学研究进展[J].食品与药品,2016,18(3):209-218.
[19]邬海桥,丁阳平,周向东.红茶提取物在炎性气道黏液高分泌中的作用[J].上海交通大学学报(医学版),2009,29(2):126-129.
[20]谭婷,刘武嫦,仇云龙,等.茶叶对IBD肠道微生物菌群影响研究进展[J].茶叶通讯,2016,43(3):37-40.
[21]冯强,李毅,夏鹏程,等.茶叶提取物抗肿瘤作用的最新研究进展[J].中国实用医药,2016,11(22):274-275.
[22]乔如颖,李明,郑新强,等.茶叶及其儿茶素对乳腺癌的抑制作用[J].茶叶科学,2016,36(6):557-566.
[23]曲伟,王孟君,于海燕,等.毛尖茶叶多糖及其结合物的抗氧化活性研究[J].天然产物研究与开发,2015,27(2):2010-2013.
[24]李慧,聂枞宁,黄亮,等.分子标记在茶树种质资源遗传多样性分析中的应用研究[J].现代农村科技,2016(2):69-70.
[25]刘本英,李友勇,唐一春,等.云南茶树资源遗传多样性与亲缘关系的ISSR分析[J].作物学报,2010,36(3):391-400.
[26]吴清韩,庄东红,朱慧,等.凤凰单丛茶树资源多样性的ISSR分析[J].热带作物学报,2015,36(3):499-503.
[27]侯渝嘉,常亚丽,何桥,等.应用AFLP分子标记研究茶树品种新材料[J].西南农业学报,2015,28(2):514-518.
[28]陈林波,夏丽飞,周萌,等.基于RNA-Seq技术的“紫娟”茶树转录组分析[J].分子植物育种,2015,13(10):2250-2255.
[29]王希,陈丽,赵春雷.利用MISA工具对不同类型序列进行SSR标记位点挖掘的探讨[J].中国农学通报,2016,32(10):150-156.
[30]王丽鸳,姜燕华,段云裳,等.茶树EST-SSRs分布特征及引物开发[J].植物遗传资源学报,2009,10(4):511-516.
[31]袁阳阳,王青锋,陈进明.基于转录组测序信息的水生植物莕菜SSR标记开发[J].植物科学学报,2013,31(5):485-492.
[32]郁永明,田丹青,潘晓韵,等.基于红掌转录组序列的SSR标记分析与开发[J].分子植物育种,2015,13(6):1349-1354.
[33]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.
[34]张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120.
[35]文亚峰,韩文军,周宏,等.杉木转录组SSR挖掘及EST-SSR标记规模化开发[J].林业科学,2015,51(11):40-49.
[36]程海亮,陆才瑞,邹长松,等.基于10个棉花腺体相关材料转录组的EST-SSR标记开发[J].棉花学报,2015,27(1):1-8.
[37]MA J Q,ZHOU Y H,MA C L,et al.Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant,Camellia sinensis(Theaceae)[J].Am J Bot,2010,97(12):153-156.
[38]段云裳,姜燕华,王丽鸳,等.中国红、绿茶适制品种(系)遗传多样性与亲缘关系的SSR分析[J].中国农业科学,2011,44(1):100-109.