CN110106275B - 一种茶叶紫芽紧密连锁的InDel分子标记及其应用 - Google Patents
一种茶叶紫芽紧密连锁的InDel分子标记及其应用 Download PDFInfo
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Abstract
一种茶叶紫芽紧密连锁的InDel分子标记及其应用，属于分子生物学技术领域。本发明公开了一段与紫芽性状紧密相关的插入片段，并提供了鉴定茶树紫芽性状的功能标记，解决了目前紫芽基因精确定位和遗传机理研究在国内外几乎空白这一现状，将本发明中的InDel标记应用于分子标记辅助选择，能快速筛选出紫娟后代中具有紫芽性状的茶树材料，提高紫芽茶树品种的选育效率，为茶树新品种的选育奠定基础。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种茶叶紫芽紧密连锁的InDel分子标记及其应用。
背景技术
富含花青素的紫娟茶是我国培育的重要紫芽茶树品种。与普通绿芽茶叶相比，紫芽茶有助于人类抗氧化消除自由基，预防心脑血管疾病及癌症，降低神经退行性疾病的患病风险等。因此研究和开发利用紫芽茶受到人们的密切关注。
近年来，茶叶科学家对于紫芽茶的成色机理开展了大量研究。目前基于遗传群体的QTL定位已发现CsMYB75位置附近的序列差异可以解释28.1-40.8%的紫芽性状遗传变异。但是有关该位点具体的序列变异是什么目前尚不明确，也未开展相关分子标记的研究。因此，确定影响茶叶紫芽的关键序列变异并开发对应的分子标记，可以为紫芽茶的分子标记辅助选择育种奠定应用基础。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种茶叶紫芽紧密连锁的InDel分子标记及其应用的技术方案。
所述的茶叶紫芽紧密连锁的InDel分子标记作为紫芽茶筛选标记的应用，所述InDel分子标记为具有长度为182bp的1处特异插入/缺失片段，其序列如SEQ ID NO.1所示。
所述的可识别茶叶紫芽紧密连锁的InDel分子标记的引物组在紫芽茶筛选中的应用，所述茶叶紫芽紧密连锁的InDel分子标记序列如SEQ ID NO.1所示。
所述的应用，其特征在于所述引物组包括紫芽F1和紫芽R1，所述紫芽F1的序列如SEQ ID NO.3所示，所述紫芽R1的序列如SEQ ID NO.4所示。
所述的一种茶树紫芽性状筛选的方法，其特征在于检测茶树第8连锁群上茶叶紫芽紧密连锁的InDel分子标记的插入/缺失情况，根据茶叶紫芽紧密连锁的InDel分子标记的插入/缺失情况确定茶树紫芽性状，所述茶叶紫芽紧密连锁的InDel分子标记序列如SEQID NO.1所示。
所述的方法，其特征在于采用可识别茶叶紫芽紧密连锁的InDel分子标记的引物组对茶树基因组DNA进行PCR扩增，所述引物组包括紫芽F1和紫芽R1，所述紫芽F1的序列如SEQ ID NO.3所示，所述紫芽R1的序列如SEQ ID NO.4所示。
所述的方法，其特征在于若扩增得到1kb左右的扩增产物，则该植株具有能诱发紫芽的关键序列，如果没有扩增产物，则该植株不具备诱发紫芽的关键序列。
所述的一种培育紫芽茶树的方法，其特征在于包括在茶树第8连锁群上引入茶叶紫芽紧密连锁的InDel分子标记插入/缺失，所述茶叶紫芽紧密连锁的InDel分子标记序列如SEQ ID NO.1所示。
本发明将控制茶叶紫芽的1个关键基因CsMYB75定位于茶树的第8连锁群上的较小关联区域内，并提供了与紫芽关键基因CsMYB75紧密连锁的分子标记InDel紫芽，可直接用于紫芽关键基因CsMYB75分子标记辅助育种体系的建立。本发明的分子标记及分子标记扩增引物可以简便、快速、高通量地应用于紫芽茶选育实践中，同时为紫芽关键基因CsMYB75的功能研究奠定了良好的基础。
序列表
<110> 中国农业科学院茶叶研究所
<120> 一种茶叶紫芽紧密连锁的InDel分子标记及其应用
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caacaatgat tcatcccact aaaaatatca ccactaatag cactaatgca ttaaaaaagt 60
aaaaaaaaaa aaaaaaaata caactccaag accaaaacat cacatcatta acggtgggac 120
ccactgccac ctaattagtg ggagcactaa ttagtgctcc caagcatttt tctttttttt 180
tt 182
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gtgaatctgt cctccctgtg tgtccttgtg gatgttaagc gatgtaaaac ttggacttgg 60
ctctagcact aacactacaa acttttcttt ggctctttgt tttctcggcg cgacttttaa 120
tttaagcaga atttgtttta agagatcaga gtaagttctt aaattatagt atcttcgttc 180
gaaacggcta acaggatatt ctaataatct ttacaagggg cgttatcttg aatttgtgaa 240
agaataggga tgattttcct gtaatcaaaa aaaaaaaaaa aactaacact acaaactttt 300
ggtttaaatt ttcaggatta actttttaaa aatttaaaaa tttttatttt tcatattttt 360
ttatttttat tggatttttt taatatttta aataattttt tcttaatatt tagatgcatc 420
taatataaat aaactaaaaa tgataatatt tgtgaaaaaa atctttaaaa aatcactaaa 480
tatcaaaatt ataaaaaaat atacgagaat tcaatatatt ttttatattc ttatattttt 540
attgattttt taaaatattt ttttattaat attttacttt ttaaatatat ttaagtaagg 600
tgtatctaaa aaatttaaaa aattacttaa aattttaaaa atattcacta aaaatataaa 660
aaaattaatt ttcaaaaaat taaatctaaa aaattaaaat caaatacact gattattatt 720
tatcttaaat ttatttagaa ttattatttt atagtttaga tttattttca caccgcataa 780
atttaatcac aaacatgcaa tgtcattcga aataaatttt gaataggtac gtaaatttat 840
tcttttctta aaattcctgc aaataaggtg gaaaaaaaaa atattattat ttaatatctt 900
gtccgcatct aataatcttt ccatataagg gagagttttt ttttttttca acaatgattc 960
atcccactaa aaatatcacc actaatagca ctaatgcatt aaaaaagtaa aaaaaaaaaa 1020
aaaaaataca actccaagac caaaacatc 1049
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gtgaatctgt cctccctgtg t 21
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gatgttttgg tcttggagtt g 21
附图说明
图1紫娟茶与龙井43紫芽关键位点核苷酸序列比对；
图2 紫娟茶子代6个芽叶分化单株及紫娟一芽一叶表型；
图3紫娟茶子代6个芽叶分化单株及紫娟InDel分子标记PCR结果；
图4紫娟与龙井43InDel分子标记PCR结果。
实施方式
在已有参考文献(Wei K, Wang L, Zhang Y, Ruan L, Li H, Wu L, Xu L,Zhang C, Zhou X, Cheng H, Edwards R. A coupled role for CsMYB75 and CsGSTF1in anthocyanin hyperaccumulation in purple tea. The Plant Journal,2019,97:825-840)中把控制茶叶紫芽的关键转录因子命名为CsMYB75基因，本研究根据亲本材料表型特征及遗传规律沿用该基因命名。
下面结合实验对本发明作进一步的说明，但并不局限于此。
以下实施例中所采用的分子生物学实验技术包括DNA提取、PCR扩增、PAGE凝胶电泳等实验，如无特殊说明，通常按照常规方法操作，具体可参见《分子克隆实验指南》(第三版) (Sambrook J ,Russell DW，Janssen K ,Argentine J .黄培堂等译，2002，北京：科学出版社)，或按照制造厂商所建议的条件。
实施例1：茶叶紫芽紧密连锁的InDel分子标记的开发
1、供试材料
植物材料：以中国农业科学院茶叶研究所种植的茶树品种紫娟、龙井43及紫娟子代芽叶分化的6个单株（4个春季绿色芽叶，2个春季紫色芽叶）为试验材料。
2、基因组DNA的提取：
取1g新鲜嫩梢，加液氮研磨至粉末状。利用植物基因组DNA提取试剂盒进行基因组DNA的提取。提取完毕后用1.1%的琼脂糖凝胶电泳检测提取的完整性，之后利用Nanodrop2000微量核酸定量仪测定DNA的浓度和纯度，经鉴定合格的DNA，将样品DNA浓度统一稀释至100ng/µl，-20℃冰箱中保存。
3、茶树CsMYB75启动子的克隆：
基于前期研究我们已经明确了CsMYB75基因与控制茶树紫芽的关键QTL位点重合，是控制紫芽的关键基因。由于其在紫芽中的表达量远高于绿芽，推测其启动子序列差异可能对表达量有重要影响。因此根据启动子序列，设计引物。正向引物：AGCAGATGGAAAGATAGAGTG，反向引物：GCACCTTTTCTCACTCCTAAT，通过PCR扩增样品中CsMYB75约2kb的启动子序列。扩增结束后将扩增产物克隆到pMD18-T载体中进行转化、培养和阳性筛选，并对阳性克隆进行测序。通过基因克隆，从龙井43和紫娟的基因组DNA中获得了CsMYB75启动子序列，分别记为LJ43-MYB75-pro和ZJ-MYB75-pro，它们的相似性为83.77 %，图1显示了它们序列的比对结果，可以看出它们除有个别碱基的替换、缺失和小片段的插入缺失外，在3’端（接近起始密码子端）ZJ存在一处182bp大片段的插入缺失。MethPrimer预测结果显示都没有CpG岛的存在。利用在线软件PLACE和PlantCARE对LJ43-MYB75-pro和ZJ-MYB75-pro所具有的顺式作用元件进行预测，结果表明它们除具有TAAT-box和CAAT-box等核心启动子元件，还具有响应光（BOXCPSAS1、CACGTGMOTIF、GT1CORE、LTRECOREATCOR15、TCCC-motif等）、低温（CBFHV、LTREATLTI78等）、热（CCAATBOX1、CPBCSPOR等）、干旱（ACGTATERD1等）、激素（ABRELATERD1、ARR1AT、AUXREPSIAA4、EBOXBNNAPA、TCA-element等）和损伤（WUN-motif）等功能元件。另外，LJ43-MYB75-pro和ZJ-MYB75-pro都具有多个MYB的识别和结合位点（MYBCORE、MYBCOREATCYCB1、MYB26PS、MYBPLANT、MYBST1、MYBPZM、MYB1AT、MYB1LEPR、MYB2CONSENSUSAT等），除这些元件外还具有一些与组织特异性、胚乳发育、碳代谢等等相关的位点。通过序列分析，发现ZJ中CsMYB75启动子182bp的插入区域包含多种顺式作用元件，如光、热、激素等响应元件，还有核心元件（CAATBOX1）和一些其它功能的元件，另外还含有3个MYB转录因子识别和结合序列位点（MYBCORE、MYBCOREATCYCB1、MYB2CONSENSUSAT）。
4、分子标记InDel紫芽开发
基于紫娟、龙井43的CsMYB75基因启动子测序结果，找到了1个InDel标记，其在紫娟中比龙井43中多了1个182个碱基的插入，该InDel标记序列如SEQ ID NO.1所示。根据上述InDel标记设计了一组特异性引物对(紫芽F1：5 '-GTGAATCTGTCCTCCCTGTGT-3 '，如SEQID NO.3所示；紫芽R1：5 '-GATGTTTTGGTCTTGGAGTTG-3 '，如SEQ ID NO.4所示)，该引物对可在紫娟中扩增出长度为1049bp的片段，该片段序列如SEQ ID NO.2所示；而在龙井43中无法扩增出该片段。基于紫娟子代自身的芽叶颜色分离情况（如图2），比较这些种质材料芽叶与PCR扩展结果（如图3）发现，具有该1049bp扩展片段的都为紫芽品种，而没有该片段的都为绿芽品种，说明该InDel标记与茶树紫芽性状紧密连锁，可应用于紫芽茶选育实践。
实施例2：茶叶紫芽紧密连锁的InDel分子标记的应用
采摘10g紫娟与龙井43品种的鲜叶，加液氮研磨至粉末状。利用植物基因组DNA提取试剂盒进行基因组DNA的提取。提取完毕后用1.1%的琼脂糖凝胶电泳检测提取的完整性，之后利用Nanodrop 2000微量核酸定量仪测定DNA的浓度和纯度，经鉴定合格的DNA，将样品DNA浓度统一稀释至100ng/µl，-20℃冰箱中保存。根据上述InDel标记的特异性引物对(紫芽F1：5 '-GTGAATCTGTCCTCCCTGTGT-3 '；紫芽R1：5 '-GATGTTTTGGTCTTGGAGTTG-3 ')，该引物对可在紫娟中扩增出长度为1049bp的片段，而在龙井43中无法扩增出该片段，如图4所示。
Claims (3)
1. 检测茶叶紫芽紧密连锁的InDel分子标记的试剂在紫芽茶筛选中的应用，所述InDel分子标记为182bp的1处特异插入/缺失片段，其序列如SEQ ID NO.1所示，若存在所述182bp片段，则植株具备诱发紫芽的关键序列，若不存在所述182bp片段，则植株不具备诱发紫芽的关键序列。
2. 特异性检测茶叶紫芽紧密连锁的InDel分子标记的引物组在紫芽茶筛选中的应用，所述InDel分子标记为182bp的1处特异插入/缺失片段，其序列如SEQ ID NO.1所示，采用识别茶叶紫芽紧密连锁的InDel分子标记的引物组对茶树基因组DNA进行PCR扩增，所述引物组包括紫芽F1和紫芽R1，所述紫芽F1的序列如SEQ ID NO.3所示，所述紫芽R1的序列如SEQID NO.4所示；
若扩增得到1049 bp的扩增产物，则该植株具有能诱发紫芽的关键序列，如果没有扩增产物，则该植株不具备诱发紫芽的关键序列。
3. 一种茶树紫芽性状筛选的方法，其特征在于检测茶树第8连锁群上茶叶紫芽紧密连锁的InDel分子标记的插入/缺失情况，根据茶叶紫芽紧密连锁的InDel分子标记的插入/缺失情况确定茶树紫芽性状，所述茶叶紫芽紧密连锁的InDel分子标记序列如SEQ ID NO.1所示；
采用识别茶叶紫芽紧密连锁的InDel分子标记的引物组对茶树基因组DNA进行PCR扩增，所述引物组包括紫芽F1和紫芽R1，所述紫芽F1的序列如SEQ ID NO.3所示，所述紫芽R1的序列如SEQ ID NO.4所示；
若扩增得到1049 bp的扩增产物，则该植株具有能诱发紫芽的关键序列，如果没有扩增产物，则该植株不具备诱发紫芽的关键序列。
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