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^,11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

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

FAN XiaoJing^,1, YU WenTao^,2, CAI ChunPing², LIN Yi¹, WANG ZeHan¹, FANG WanPing³, ZHANG JianMing⁴, YE NaiXing^,11College 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

通讯作者: * 叶乃兴,E-mail： ynxtea@126.com; 于文涛,E-mail： wtyu@foxmail.com

责任编辑: 李莉
收稿日期:2020-08-28接受日期:2020-09-27网络出版日期:2021-04-16

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

Corresponding authors: *$con.vcortext
Received:2020-08-28Accepted:2020-09-27Online:2021-04-16
作者简介 About authors
樊晓静,E-mail： 1187248076@qq.com。










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.
Keywords：Camellia sinensis;cultivar;SNP;molecular ID;DNA fingerprint


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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2021.08.014
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 Acricultura Sinica, 2021, 54(8): 1751-1760 doi:10.3864/j.issn.0578-1752.2021.08.014


开放科学（资源服务）标识码（OSID）：

0 引言

【研究意义】茶树（Camellia sinensis (L.) O. Kuntze）为多年生异花授粉植物,具有高度异质性和杂合性^[1]。中国西南地区是茶树的起源地^[2],茶树从起源地向中国其他地区和国外的自然传播和人为传播过程中,积累了自然演化和人工选择的变异,从而在各茶区形成了丰富的茶树品种资源^[3]。然而在茶树品种资源的大量引种及频繁的品种资源交换过程中,造成同名异物、同物异名的现象^[4,5],不仅给茶树品种资源保护利用带来诸多困难,也损害了消费者的权益。因而科学准确地区分和鉴定茶树品种资源具有重要意义。【前人研究进展】传统使用形态标记、细胞标记等方法来区分、鉴定植物品种均有一定局限性。如形态学标记容易受环境和植物发育阶段等影响;细胞学标记容易受制片技术的影响,且对于染色体小数量多的物种,其染色体核型不易区分清楚^[6]。随着生物信息技术的进步,以生物的遗传物质核酸的多态性为基础的分子标记得到了快速发展,DNA分析技术开始大量应用于植物学研究。RAPD、SSR等分子标记技术在研究茶树遗传多样性、指纹图谱及分子身份证构建等方面已有较多运用^{[7,8,9,10,11,12,13]}。如安徽茶树群体中的SSR遗传多样性分析^[8]、茶树SSR指纹图谱的构建^[9,10]、野生茶树的RAPD分子标记鉴定^[11]和名山茶树基因身份证的构建^[13]。LANDER^[14]在1996年提出SNP的遗传标记技术,作为继SSR为代表的第二代分子标记技术之后发展起来的第三代分子标记技术,是指个体间基因组DNA序列同一位置单个核苷酸变异所引起的多态性。SNP标记具有高密度性,且广泛存在于基因组中,如人类基因组中平均每1 000 bp就会出现一个SNP,玉米中平均每57 bp出现一个SNP^[15]。SNP作为第三代分子标记,其优点在于快速、自动化、高通量^[16],其多态性几乎在所有被研究物种中都被用作有效的遗传标记,包括动物^[17]和植物^[18]。【本研究切入点】目前,关于茶树SNP分子标记的研究,主要集中在品种资源鉴定、遗传多样性、遗传关系的分析及指纹图谱的构建^[19,20],对茶树品种资源的分子身份证进行构建鲜见报道。【拟解决的关键问题】本研究通过收集国内外103份茶树种质,利用SNP标记并结合茶树品种资源基本信息构建茶树品种资源分子身份证,以期为茶树品种资源保护鉴定提供一种新思路。

1 材料与方法

1.1 供试材料

供试茶树品种资源来源于武夷学院茶树种质资源圃（福建省武夷山市）和福建省农业科学院茶叶研究所茶树种质资源圃（福建省福安市）,其中,中国茶树品种资源有101份,来自华南、西南、江南、江北四大茶产区的10个茶叶主产区省份;国外茶树品种2份,包括日本的玉绿和格鲁吉亚的格鲁吉亚1号。茶树品种资源详细信息见电子附表1。

Table 1
表1
表1103份茶树品种资源的分子身份证
Table 1Molecular ID of 103 tea plant cultivars

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

新窗口打开|下载CSV

1.2 DNA的提取

采用新型植物基因组DNA提取试剂盒（TIANGEN,DP320,北京）提取基因组DNA,利用超微量紫外分光光度计（Implen,S60716,德国）测定DNA浓度和纯度。提取的基因组DNA于-80℃保存备用。

1.3 SNP基因分型

中国乌龙茶产业协同创新中心课题组前期从国家生物信息中心（national center of biotechnology information,NCBI）的数据库（http://www.ncbi.nlm.nih.gov/）中下载了茶树的表达序列标签（express sequence tags,EST）,获得大量可用于数据挖掘的高质量EST。经过软件CAP3程序将序列装配,再利用Quality SNP在含有6条以上同源序列重叠群中开发候选SNP位点,得到了1 786个候选EST-SNP位点。为确保获得高质量的SNP用于后期验证,之后进入人工选择,要求筛选出的SNP位点前后有60 bp碱基完全保守,最终筛选出用于茶树种质资源的96个SNP标记位点^[20,21]。目前,已有多个茶树全基因组数据公布^{[22,23,24,25]},将96个候选位点与最新发表的茶树全基因组^[25]进行局部序列比对搜索^[26],得到候选SNP位点序列在全基因组染色体与具体基因上的位置。利用Fluidigm 96.96 Dynamic Array? IFC芯片（Integrated Fluidic Circuit; Fluidigm? Corp,USA）进行基因分型,该芯片可同时检测96个样品和96个SNP位点,且上样后可自动进行PCR反应,芯片读取图如电子附图1。96.96 Dynamic Array? IFC芯片使用方法需根据样品实际情况进行改进;而后使用EP1?成像仪（Fluidigm? Corp, USA）获得96.96 IFC荧光图像。

图1

新窗口打开|下载原图ZIP|生成PPT
图124个茶树SNP位点多态性读取图

蓝色：杂合子（XY）;红色：纯合子（XX）;绿色：纯合子（YY）
Fig. 1Polymorphism plots of 24 SNP loci

Blue: Heterozygote (XY); Red: Homozygote (XX); Green: Homozygote (YY)

1.4 数据处理

利用Fluidigm SNP Genotyping Analysis软件（https://www.fluidigm.com/software）进行数据导出和分析,并确定每个SNP位点处的样品基因型是纯合子或杂合子。使用GenAlEx6.503软件分析等位基因频率（allele frequency）、信息指数（information index,I）、观察杂合度（observed heterozygosity,Ho）、预期杂合度（expected heterozygosity,He）、固定指数（fixation index,F）和次等位基因频率（minor allele frequency,MAF）。将茶树品种资源基本信息与其DNA指纹图谱信息结合,利用在线条码生成器（http://barcode.cnaidc.com）和二维码在线生成软件（http://qr-batch.com/）以数字条码的形式构建茶树品种资源分子身份证。

2 结果

2.1 茶树SNP标记多态性描述统计

采用的96个SNP标记位点较均匀地分布于茶树全基因组15条染色体上（电子附表2）。对103份茶树品种资源进行分析,剔除10个不具多态性的引物后,剩余86个SNP位点多态性的相关信息列于电子附表3。根据统计,这些SNP标记多态性信息指数（I）为0.071—0.693,平均值为0.517。观测杂合度（Ho）范围为0.027—0.982,平均值为0.370。期望杂合度（He）的范围在0.026—0.500,平均值为0.346。固定指数（F）为-0.964—0.462,平均值为-0.036。次等位基因频率（MAF）范围在0.013—0.500,平均值为0.269。

2.2 茶树品种资源最佳SNP位点的筛选及指纹图谱的构建

信息指数常用来评价群落的遗传多样性,如果每一个体都属于不同的种,多样性指数就最大;如果每一个体都属于同一种,则其多样性指数就最小,即多样性指数越高,其区分品种能力却强。因此,基于103份茶树品种资源材料,对SNP数据进行统计分析,从86个SNP位点中优化筛选了24个多态性较高的SNP位点（电子附表4,图1）,可将103份茶树品种资源完全区分开。DNA指纹图谱（图2）显示每个SNP位点处的样品是杂合子（XY）或纯合子（XX,YY）。

图2

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图2103份茶树品种资源SNP指纹图谱

蓝色：杂合子（XY）;红色：纯合子（XX）;绿色：纯合子（YY）
Fig. 2SNP fingerprints of 103 tea germplasms

Blue: Heterozygote (XY); Red: Homozygote (XX); Green: Homozygote (YY)

2.3 茶树DNA指纹图谱编码

对筛选获得的最佳SNP数据进行数字编码,作为构建分子身份证基本信息。在24个SNP标记,共有24种基因型,3种等位基因（XY、XX和YY）,因此,每个位点分别用1—3代表等位基因多态性。以福鼎大白茶为例,在CS3处的基因型为TC,对应XY,编码为1,以此类推,将基因型全部转换成24数字编码为131132311121332112313231。

2.4 茶树品种资源信息编码

茶树的基本信息由4位数字组成,其中,茶树品种资源类型分类参考陈亮等^[27,28]方法。第1位数字代表茶组植物具体物种,茶树（Camellia sinensis (L.) O.Kuntze）为1,大厂茶（C. tachangensis F. C. Zhang）为2,厚轴茶（C. crassicolumna Chang）为3,大理茶（C. taliensis (W. W. Smith) Melchior）为4,秃房茶（C. gymnogyna Chang）为5;第2—3位数字代表行政区划代码,如福建为35,浙江为33,云南为53,国外为00;第4位数字代表茶树品种资源类型,野生茶树为1,地方品种和地方种质为2,优良品种（含育成品种）为3,新选品系、株系为4,国外品种为5。以福鼎大白茶举例,4位数字商品码为1353,其中“1”表示是分类上茶组植物种类,“35”代表该茶树种质原产地是福建,“3”表示为优良品种。

2.5 茶树品种资源分子身份证的构建

基本商品信息和DNA指纹图谱共同组成由28位数字编码的茶树品种资源分子身份证（表1）。以国家级品种福鼎大白茶举例,其品种资源基本信息为：属茶组植物中的茶树（C. sinensis）,原产于福建,品种资源类型为优良品种,转换成数字码为1353;其24个SNP分子标记的基因型分别为TC、TT、AT、CT、GG、GG、CC、TC、TG、TC、TT、AG、GG、TT、CC、AG、CT、TT、CC、AT、GG、GG、CC、TC,转换成24位数字码为131132311121332112313231。则福鼎大白茶的分子身份证为1353131132311121332112313231（图3-A）,将其转化为条形码和二维码如图3-B所示。

图3

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图3福鼎大白茶分子身份证

Fig. 3Molecular ID of Camellia sinensis Fuding Dabaicha

3 讨论

3.1 茶树品种资源SNP标记的基因分型

本研究103份茶树品种资源样品来源于中国的四大茶产区,以及日本和格鲁吉亚,来源范围广,茶树品种资源遗传多样性丰富,其中也有遗传背景相近的茶树品种亲子代,如福鼎大白茶和云南大叶种的自然杂交后代福云6号、福云7号和福云595,及以四川中叶种为母本、云南大叶种为父本的蜀永2号、蜀永3号和蜀永703。本研究中所选用的96个SNP标记位点中,多态性标记为86个,占比为89.6%。SNP标记一般只有2种碱基组成,因此,被认为具有二态性;由于具有等位基因性的特点,其等位基因在任何种群中都可被估算出来。除此之外,SNP标记在不同条件下具有较高的重复性和准确性^[29,30],这是指纹图谱和分子身份证建立的重要前提。本研究通过SNP技术对这些茶树品种资源进行基因分型及统计分析,使每份品种资源具有唯一的SNP基因型,验证了SNP标记技术的准确性。与SSR相比,SNP分析不基于DNA大小片段的分离,因此,可通过高通量的形式自动化检测。目前常用的高通量、自动化程度较高的检测分析SNP的方法之一是DNA芯片法^[31]。本研究所采用的高通量微流体芯片法具有高通量、试剂和样品用量少、IFC技术自动操作、试验重复性好的优点^[32]。

3.2 茶树最佳引物的筛选确认

筛选较少的引物,可以缩短茶树品种资源鉴定的时间,降低应用成本。最佳引物的选择是构建指纹图谱和分子身份证的重要步骤。PAN^[33]利用21对引物对1 025份甘蔗种质构建了分子身份证。李春花等^[34]采用15对引物构建了48份苦荞资源的分子身份证。冉昆等^[35]利用10对引物构建了45份山东地方种质梨的分子身份证。高源等^[36]利用TP-M13-SSR分子标记技术,通过筛选引物,对苹果分子身份证进行了构建。在本研究的96个SNP标记中,去除10个在试验样品中表现出多态性差的标记位点,再依据引物的信息指数（I）,通过从高到低排序,逐步增加引物组合数量,使其能够区分更多的品种资源,以获得最佳的引物。经过分析,本研究最终筛选了24个SNP标记位点能够将103份茶树品种资源完全区分,24个SNP标记位点位于茶树全基因组的12条染色体上,染色体覆盖度为80%,覆盖度较高,有利于茶树品种资源的有效鉴别。

3.3 茶树品种资源DNA分子身份证的价值与应用

作物品种资源的分子身份证与DNA指纹图谱的功能相同,都是为了区分不同生物个体。相对于指纹图谱,分子身份证是将作物品种资源的基本信息用特定的数字编码为数字串,并且辅以条形码、二维码的形式展现,更加简明直观地区分品种资源之间的差异。分子身份证因其自身所具有特性,可利用机器进行扫描,达到方便快捷地识别大量品种资源,提高品种资源鉴定和评价的效率。另外,分子身份证的唯一性,也可有效甄别市场上同名异物、同物异名现象,有利于品种识别与保护。许多植物已利用分子标记技术构建分子身份证,如水稻^[37]、百合^[38]、桃^[39]、枸杞^[40]等。本研究首次利用SNP标记技术将DNA指纹图谱与品种资源基本信息相结合,可为每份茶树品种资源构建独有的分子身份证。该身份证信息包括了茶树品种资源的分子指纹码和属性码,可快速了解其分子信息、来源等基本信息。本研究结果对于茶树品种资源区分和精准鉴定、分子数据数字化建立具有重要的意义和实际应用价值,为茶树品种资源DNA分子身份证的构建提供了思路。

4 结论

筛选出24个最佳SNP位点组合,可精准区分全部103份供试茶树野生种、地方品种和地方种质、优良品种、新选品系和株系以及国外品种。将24个SNP位点组成茶树品种资源DNA指纹图谱编码,与茶树品种资源的基本属性信息编码组成28位数字的茶树品种资源DNA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。

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