利用SNP标记构建茶树品种资源分子身份证

doi:10.3864/j.issn.0578-1752.2021.08.014

中国农业科学 ›› 2021, Vol. 54 ›› Issue (8): 1751-1760.doi: 10.3864/j.issn.0578-1752.2021.08.014

利用SNP标记构建茶树品种资源分子身份证

樊晓静¹(),于文涛²(),蔡春平²,林浥¹,王泽涵¹,房婉萍³,张见明⁴,叶乃兴¹()

¹福建农林大学园艺学院/茶学福建省高校重点实验室,福州 350002
²福州海关技术中心/福建省检验检疫技术研究重点实验室,福州 350001
³南京农业大学园艺学院,南京 210095
⁴武夷学院科研处,福建武夷山 354300

收稿日期:2020-08-28 接受日期:2020-09-27 出版日期:2021-04-16 发布日期:2021-04-25
通讯作者: 于文涛,叶乃兴
作者简介:樊晓静,E-mail： 1187248076@qq.com。
基金资助:
福建省“2011协同创新中心”中国乌龙茶产业协同创新中心专项(闽教科〔2015〕75号);海关总署科技项目(2020HK187);福建农林大学茶产业链科技创新与服务体系建设项目(2020-01);福建张天福茶叶发展基金会科技创新基金(FJZTF01)

Construction of Molecular ID for Tea Cultivars by Using of Single- nucleotide Polymorphism (SNP) Markers

FAN XiaoJing¹(),YU WenTao²(),CAI ChunPing²,LIN Yi¹,WANG ZeHan¹,FANG WanPing³,ZHANG JianMing⁴,YE NaiXing¹()

¹College of Horticulture, Fujian Agriculture and Forestry University/Key Laboratory of Tea Science in Universities of Fujian Province, Fuzhou 350002
²Technology Centre of Fuzhou Customs District/Fujian Key Laboratory for Technology Research of Inspection and Quarantine, Fuzhou 350001
³College of Horticulture, Nanjing Agricultural University, Nanjing 210095
⁴Wuyi University, Wuyishan 354300, Fujian

Received:2020-08-28 Accepted:2020-09-27 Online:2021-04-16 Published:2021-04-25
Contact: WenTao YU,NaiXing YE

摘要/Abstract

摘要：

【目的】建立茶树品种的SNP分子标记数据库,结合茶树品种基本信息,将SNP位点组成的DNA指纹图谱构建28位数字组成的茶树品种资源分子身份证,便于茶树品种资源的保护与精准管理,避免“同名异物、同物异名”的现象。【方法】通过挖掘茶树的表达序列标签,获得大量的高质量表达序列标签,将其进行装配后,开发候选位点,将候选位点与茶树全基因组进行BLAST,得到其在全基因组染色体上的位置与具体关联基因。以铁观音、福鼎大白茶、龙井43、云抗10号等103份国内外不同类型的茶树品种资源为供试材料,提取基因组DNA,利用预扩增技术和微流体芯片法对供试茶树品种资源进行SNP基因分型,获得SNP位点数据及候选SNP位点的信息指数、观测杂合度、期望杂合度等信息,将多态性从高到低进行排序,进行SNP位点组合筛选,得到最优SNP位点组合后,结合茶树品种基本信息构建茶树品种资源分子身份证。【结果】从茶树的表达序列标签数据库中挖掘出1 786个候选SNP位点。根据序列保守性,筛选出96个SNP标记位点,与最新茶树基因组比对发现候选位点较均匀地分布于茶树全基因组的15条染色体上;对茶树品种资源的候选SNP位点的多态性信息进行分析,剔除10个不具多态性的位点,剩余86个位点的信息指数平均值为0.517,观测杂合度平均值为0.370,期望杂合度平均值为0.346,固定指数平均值为-0.036,次等位基因频率平均值为0.269。从86个SNP位点中筛选出24个多态性高的SNP位点,组成DNA指纹图谱,可区分出全部参试茶树品种资源。对24个SNP位点组成的DNA指纹图谱并结合茶树品种资源基本信息进行数字编码,最终形成由28位数字组成的茶树品种资源分子身份证。【结论】依据SNP标记的多态性信息,筛选SNP位点,精准区分全部供试茶树品种,并将24个SNP位点所构建的茶树品种资源DNA指纹图谱及品种资源的基本属性信息编码成特定的数字串,使每份茶树品种资源具有唯一的分子身份证,并生成相应的条形码和二维码,可快速被扫码设备识别。

关键词: 茶树, 品种资源, SNP, 分子身份证, DNA指纹图谱

Abstract:

【Objective】In order to facilitate the protection and precise management of tea cultivars and avoid the phenomenon of homonyms and synonyms, single-nucleotide polymorphism (SNP) molecular marker database of tea cultivars was established, and the 28 digit molecular identities of tea cultivars were constructed by DNA fingerprinting of SNP loci and the basic information of tea cultivars. 【Method】By mining the expressed sequence tags (EST) of tea plants, a large number of high-quality ESTs were obtained. Then, the ESTs were assembled to develop candidate SNP loci. And, high-quality SNPs for tea plants were screened. Furthermore, the candidate SNP loci were compared with the whole genome of tea plant to confirm their positions on the chromosomes and specific genes. The genomic DNA was extracted from fresh leaves of 103 tea cultivars. Subsequently, the genotyping of accessions was carried out on microfluidic chips. Information index, observed heterozygosity and expected heterozygosity of the candidate SNPs were obtained. The SNP loci were further screened by their polymorphism, obtaining the optimal combination of SNP loci. The molecular identities of tea cultivars were finally constructed by combining the basic information of tea cultivars. 【Result】A total of 1 786 candidate SNP loci were selected from the EST database of Camellia sinensis. According to the sequence conservation, 96 SNP loci were selected. Compared with the latest tea plant genome, the candidate loci were evenly distributed on 15 chromosomes of the whole tea plant genome. The polymorphism information of candidate SNP loci of tea cultivars were analyzed, and 10 non-polymorphic loci were eliminated. The average values of information index, observed heterozygosity, expected heterozygosity, fixed index and minor allele frequency of the remaining 86 loci were 0.517, 0.370, 0.346, -0.036, 0.269, respectively. 24 SNPs, with high polymorphism, were screened out from 86 SNPs to distinguish all tea cultivars. Based on the fingerprint of 24 SNP markers and the basic information of tea cultivars, the tea molecular ID, which composed of 28 digits, was formed finally. 【Conclusion】According to the polymorphism information of SNP markers, the candidate SNP loci were screened. And all tea cultivars were accurately distinguished. Furthermore, The DNA fingerprints of 103 tea cultivars were constructed by the 24 SNP markers and the converted serial codes from information of the tea cultivars, each germplasm thus has a unique molecular identity code, and the bar codes and quick response (QR) codes are generated as the molecular ID card, which can be quickly identified by the code scanning equipment.

Key words: Camellia sinensis, cultivar, SNP, molecular ID, DNA fingerprint

樊晓静, 于文涛, 蔡春平, 林浥, 王泽涵, 房婉萍, 张见明, 叶乃兴. 利用SNP标记构建茶树品种资源分子身份证[J]. 中国农业科学, 2021, 54(8): 1751-1760.

FAN XiaoJing, YU WenTao, CAI ChunPing, LIN Yi, WANG ZeHan, FANG WanPing, ZHANG JianMing, YE NaiXing. Construction of Molecular ID for Tea Cultivars by Using of Single- nucleotide Polymorphism (SNP) Markers[J]. Scientia Agricultura Sinica, 2021, 54(8): 1751-1760.

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https://www.chinaagrisci.com/CN/Y2021/V54/I8/1751

图/表 4

表1

103份茶树品种资源的分子身份证"

品种资源 Cultivars	分子身份证 Molecular ID	品种资源 Cultivars	分子身份证 Molecular ID
蕉城苦茶1号 Jiaocheng Kucha 1	1351232113232131123233232332	紫牡丹 Zimudan	1353112323322131312131212111
蕉城苦茶4号 Jiaocheng Kucha 4	1351232123232331223233232332	凤圆春 Fengyuanchun	1353111111111121311211112211
蕉城苦茶5号 Jiaocheng Kucha 5	1351132121112131123213212131	大叶乌龙 Dayewulong	1353112112313211113231111111
寿宁地洋1号 Shouning Diyang 1	1351321113222111121122111223	蜀永1号 Shuyong 1	1503323111113131121212111223
寿宁地洋2号 Shouning Diyang 1	1351311131323131112113111211	蜀永2号 Shuyong 2	1503321313212133222232312222
寿宁芎坑1号 Shouning Xiongkeng 1	1351121132321111233112213221	蜀永3号 Shuyong 2	1503113223211111211211122312
寿宁芎坑2号 Shouning Xiongkeng 2	1351113211321131113113213212	蜀永808 Shuyong 808	1503123223212131221131112322
凤凰苦茶 Fenghuangkucha	1441213233232111323233211111	蜀永703 Shuyong 703	1503123213221121132231121221
佛手 Foshou	1353211232221111311213311211	名山白毫131 Mingshanbaihao 131	1513111231313121112112313212
白鸡冠 Baijiguan	1352113111121111211121213113	安徽7号 Anhui 7	1343311131232121211111313113
慢奇兰 Manqilan	1352122113122111323311311121	蒙山9号 Mengshan 9	1343111131311111112122113212
金面奇兰 Jinmianqilan	1352111131122131331111312111	名山早311 Mingshanzao 311	1513131132311121332112313232
寿宁桃眉 Shouning Taomei	1352131121122111332131111232	川茶2号 Chuancha 2	1513123223121111111211312322
寿宁黄叶茶 Shouning Huangyecha	1352113331331111113211312213	川茶3号 Chuancha 2	1513121233221131112221311221
吴山清明茶 Wushanqingmingcha	1352321132333131321212311221	紫嫣 Ziyan	1513112211311131311211113212
水古茶 Shuigucha	1332323211323111312332112222	云抗10号 Yunkang 10	1533113123211131123211112332
夜来香单丛 Yelaixiang Dancong	1442232122121111311233113131	云茶1号 Yuncha 1	1533311332323121312331313211
芝兰香单丛 Zhilanxiang Dancong	1442132223232121223233211331	长叶白毫 Changyebaihao	1533223123232111223233212322
八仙香单丛 Baxianxiang Dancong	1442233123132121123233232131	紫娟 Zijuan	1533111111211231121211112132
老仙翁单丛 Langxianweng Dancong	1442232223212121111231232331	鄂茶11号 Echa 11	1423123131311121132113321123
红帝单丛 Hongdi Dancong	1442231123232221113232212331	千年雪 Qiannianxue	1333311212121131312311333113
城门单丛 Chengmen Dancong	1442113123211111312211112111	平阳特早 Pingyangtezao	1333123233121121132132112123
探春香单丛 Tanchunxiang Dancong	1442233123232121113231213331	中茶102 Zhongcha 102	1333121111321111332311113221
贡香单丛 Gongxiang Dancong	1442211211232111323212212111	碧云 Biyun	1333131231332131311322311233
棕榈香单丛 Zonglvxiang Dancong	1442111121211211121211111111	白叶1号 Baiye 1	1333113131113131112131111211
鸭屎香单丛 Yashaxiang Dancong	1442131133132311113232211331	龙井43 Longjing 43	1333113313213111331132312113
姜母香单丛 Jiangmuxiang Dancong	1442233331232321121233232331	中茶108 Zhongcha 108	1333311331313111112222311211
青心大冇 Qingxindamao	1712112121132111312213112311	嘉茗1号 Jiaming 1	1333322233111111332112113223
软枝乌龙 Ruanzhiwulong	1712112321312131312233112112	龙井长叶 Longjingchangye	1333313131123121211111313212
四季春 Sijichun	1712122231322111312223111223	黄金芽 Huangjinya	1333123111322111131212311122
福鼎大白茶 Fuding Dabaicha	1353131132311121332112313231	安徽3号 Anhui 3	1343111231313121112112313211
政和大白茶 Zhenghe Dabaicha	1353123111121111121212111221	凫早2号 Fuzao 2	1343122332123121331231313121
霞浦春波绿 Xiapu Chunbolv	1353321131321211122123311121	舒茶早 Shuchazao	1343121231311111322212313123
早逢春 Zaofengchun	1353111221212111223212211333	农抗早 Nongkangzao	1343313211121121132112311211
霞浦元宵茶 Xiapu Yuanxiaolv	1353313233121131211311311211	白毫早 Baihaozao	1433332232121111111312113232
福云6号 Fuyun 6	1353111111111111111231111111	槠叶齐 Chuyeqi	1433112123223111221222122112
福云7号 Fuyun 7	1353213211132111111111111131	桃源大叶 Taoyuandaye	1433121133223111111211311121
福云10号 Fuyun 10	1353113311131111111211311131	涟源奇曲 Lianyuanqiqu	1432121123211131111112112121
福云20号 Fuyun 20	1353133131231111332213131131	湘波绿 Xiangbolv	1433213111132211111111113112
福云595 Fuyun 595	1353211211112211111111113111	尖波黄 Jianbohuang	1433132111111111111212111232
大红袍 Dahongpao	1353322212313131322213112223	保靖黄金茶1号 Baojing Huangjincha 1	1433323212322131211211212221
铁观音 Tieguanyin	1353112131312111332211311112	乌叶单丛 Wuye Dancong	1443132121232121113233212331
黄棪 Huangdan	1353212221113111313231213111	金萱 Jinxuan	1713122222121111322212211122
肉桂 Rougui	1353211133312321333111212211	翠玉 Cuiyu	1713321321231111331213312321
本山 Benshan	1353212331312111331111311112	福云8号 Fuyun 8	1354213311111111111231111132
梅占 Meizhan	1353122122112111311112312121	福云591 Fuyun 591	1354111331311211311212111111
毛蟹 Maoxie	1353132331311121331321311131	寿宁凤阳种 Shouning Fengyangzhong	1354112332321121112112311111
白芽奇兰 Baiyaqilan	1353122321322331311231112221	金茗早 Jinmingzao	1354112222111121113232213111
九龙大白茶 Jiulongdabaicha	1353213311111111111231111131	长乐种 Changlezhong	1354213211132111111311111131
九龙袍 Jiulongpao	1353322212313131322213112123	玉绿 Yulv	1005112231111131231112111211
福建水仙 Fujian Shuixian	1353311131311311211111311211	格鲁吉亚1号 Gelujiya 1	1005221323212111212112312121
八仙茶 Baxiancha	1353111123232121222232213211

表1

图1

图2

图3

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利用SNP标记构建茶树品种资源分子身份证

Construction of Molecular ID for Tea Cultivars by Using of Single- nucleotide Polymorphism (SNP) Markers

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