CN109929942B - 与葡萄果皮颜色相关的snp分子标记及应用 - Google Patents
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Abstract
本发明提供一种与葡萄果皮颜色相关的SNP分子标记，所述SNP分子标记位于葡萄VvmybA3基因上，SNP位点对应于VvmybA3基因cDNA序列第2688位碱基，该处碱基为A的葡萄果皮为无色，该处碱基为C的葡萄果皮为有色。通过基因分型检测，若该SNP位点的基因型为C/C或C/A，葡萄果皮表现为有色，若该SNP位点的基因型为A/A，葡萄果皮表现为无色，以此标记开展葡萄果皮早期辅助选择，可以大大提高育种进程。
Description
技术领域
本发明涉及分子生物学及作物育种技术领域，具体地说，涉及一种与葡萄果皮颜色相关的SNP分子标记及应用。
背景技术
葡萄是我国主要水果之一，我国鲜食葡萄产量多年稳居世界首位，栽培面积位居世界第二，对葡萄而言，果皮颜色是吸引消费者得一种重要果实性状。
目前，育种人员在葡萄育种工作中，主要采用的是传统的杂交育种手段，杂交种子培育成苗，经过数年童期后，再对杂种单株的生长结果习性和果实性状进行评价，以此来选择和淘汰杂种单株，该方法育种周期长，耗时耗力，耗土地资源，育种效率低，随着分子生物学技术的迅速发展，以及葡萄基因组测序的完成，分子标记辅助育种技术，可以对杂种单株进行早期选择，提前淘汰非目标性状单株。
葡萄果皮颜色主要受到花青苷(花青素)含量的影响，花青苷的合成是由多种合成酶基因调节，都属于多基因家族MYB，研究表明MYB结构和功能变异与果皮颜色有着密切关系，MYB基因上插入的一个逆转座子导致了葡萄果皮无色，果实呈现出绿色，而没有该转座子的葡萄果皮有色，表现出从粉色、紫色到黑色的连续变化，但是有色与无色类似质量性状，开发有色/无色分子标记辅助选择，可以提高育种效率，节省育种成本。
发明内容
本发明的目的是提供一种与葡萄果皮颜色相关的SNP分子标记及应用。
为了实现本发明目的，本发明提供一种与葡萄果皮颜色相关的SNP分子标记，所述SNP分子标记位于葡萄VvmybA3基因上，SNP位点位于VvmybA3基因cDNA序列第2688位碱基，该处碱基为A或C。
其中，葡萄VvmybA3基因cDNA序列如SEQ ID NO:1所示(GenBank：AB442011.1)。SEQID NO:1所示序列的第2688位碱基为n，n为a或c。
本发明所述SNP分子标记的AA基因型对应的葡萄果皮为无色，所述SNP分子标记的CC或AC基因型对应的葡萄果皮为有色。
本发明还提供基于KASP技术开发的用于扩增权利要求1或2所述分子标记的引物，包括正向引物F1、正向引物F2和反向引物R，引物序列分别如下(SEQ ID NO:2-4)：
正向引物F1：5′-CTCCTTAAGAAGATATATACTCCATCATA-3′
正向引物F2：5′-TCCTTAAGAAGATATATACTCCATCATC-3′
反向引物R：5′-GCCGCATCAAAGGCTAGAATTATGC-3′
本发明还提供含有所述KASP引物的检测试剂或试剂盒。
本发明还提供所述SNP分子标记、所述KASP引物或所述检测试剂或试剂盒在葡萄果皮早期筛选中的应用。包括以下步骤：
1)提取待测葡萄样品的DNA；
2)KASP反应：以步骤1)提取的DNA为模板，向其中加入特异的KASP Primer mix和通用的KASP Mastermix，进行PCR扩增；
3)分析PCR扩增产物。
优选地，KASP反应在384微孔板中进行(货号04729749001，Roche)，PCR反应体系为：总体系3μL，DNA模板1μL，浓度为4ng/ul，KASP Primer mix 1μL，2×KASP Mastermix 1μL；其中所述KASP Primer mix中正向引物F1、F2和反向引物R的浓度均为10μM，KASPMastermix(购自LGC公司,www.lgcgroup.com，货号KBS-1016-002)。
PCR反应条件如下：
优选地，步骤3)对PCR扩增产物进行荧光检测分析：AA基因型对应的葡萄果皮为无色，CC或CA基因型对应的葡萄果皮为有色。
本发明还提供所述SNP分子标记、所述KASP引物或所述检测试剂或试剂盒在葡萄分子标记辅助育种中的应用。
本发明还提供所述SNP分子标记、所述KASP引物或所述检测试剂或试剂盒在葡萄果皮颜色基因分型中的应用。
本发明首次公开了一种用于筛选葡萄果皮无色/有色的SNP分子标记，该SNP位点位于葡萄VvmybA3基因中，通过基因分型检测，若该位点核苷酸为C/C或C/A，葡萄果皮则表现为有色，若该位点核苷酸为A/A，葡萄果皮表现为无色，以此标记开展葡萄果皮早期辅助选择，可以大大提高育种进程。
附图说明
图1为本发明实施例2中基于KASP技术的葡萄VvmybA3基因分型结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1与葡萄果皮颜色相关的SNP分子标记的开发
通过对北京市林业果树科学研究院葡萄资源圃内的23个绿色葡萄品种和25个有色葡萄品种进行重测序数据分析，结果发现在VvmybA3基因cDNA序列(AB442011.1)第2688位碱基为A/C突变的SNP位点。命名为VVMYBA32688A/C。设计可用于KASP技术的特异引物：
正向引物F1：5′-CTCCTTAAGAAGATATATACTCCATCATA-3′
正向引物F2：5′-TCCTTAAGAAGATATATACTCCATCATC-3′
反向引物R：5′-GCCGCATCAAAGGCTAGAATTATGC-3′
上述引物由LGC公司合成。
绿色葡萄品种为：莎丽、新玫瑰、康能玫瑰、葡萄园皇后、戈尔比、维多利亚、亚历山大、瑞都香玉、牛奶、凤凰51号、香妃、艾莫无核、黎明无核、喜乐无核、国立一号、王妃、意大利、白罗莎、京香玉、奥古斯特、贵妃玫瑰、沙芭珍珠、无核白鸡心。
有色葡萄品种为：京秀、瑞都脆霞、艳红、早玫瑰香、玫瑰香、美人指、红双味、奇妙无核、爱神玫瑰、金星无核、圣诞玫瑰、塔米娜、里扎马特、瑞都早红、瑞都红玉、红鸠、绯红、凤凰51号、奥迪亚无核、摩尔多瓦、火焰无核、红宝石无核、克瑞森无核、红地球、瑞比尔。
实施例2基于KASP技术开发的用于扩增SNP分子标记的引物及应用
用该SNP标记验证了北京市农林科学院林业果树研究所葡萄资源圃保存的119个葡萄品种和251个杂种后代植株，
1、DNA提取
选取待测品种的幼嫩叶片，约0.1g，使用新型植物基因组DNA提取试剂盒(aidlab，货号DN15),用检测DNA质量，A260/280介于1.7-1.9之间，将DNA稀释成10ng/μL备用。
2、基于KASP技术的PCR反应
按照LGC公司提供的标准实验流程，整个实验步骤按照
genetyping assay,manual part#15004070进行。KASP反应在384微孔板中进行，反应体系为3μL。
PCR反应条件如下：
3、SNP分子标记与葡萄果皮颜色性状的关联度分析
用该SNP标记验证了北京市农林科学院林业果树研究所葡萄资源圃保存的119个葡萄品种和杂交后代251个植株，基因分型和表型统计结果见表1，由表1可以看出，该分子标记用于鉴定有色/绿色植株葡萄的准确率为100％。
表1
基于KASP技术的葡萄VvmybA3基因分型结果如图1所示，图中左上角的分型结果为CC，对应的材料果皮颜色为有色，图中部分型结果为CA，对应的材料果皮颜色为有色，图中右下角的分型结果为AA，对应的材料果皮颜色为绿色，可见，利用该SNP标记可用于区分葡萄果皮颜色的有无。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 北京市林业果树科学研究院
<120> 与葡萄果皮颜色相关的SNP分子标记及应用
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gaaagataaa gcccaacaca attctgaaaa ccatctgata aagcctaagc cttgcaacat 4560
ctttctaggg ttccaattca aacatcaagc tgtggatgct tatgagcaca aaggcagtca 4620
ttgtagtgag ctgcacacca ccatggtcca cacaacacag gatattttat ggtgggaaag 4680
cctattcgct gcacatcaca ttgaagatca ggagcttatt gccagcctct tggctgcaga 4740
acatgcatca gctgtagata ccaatggagg tgggaattat gattttcctt ttgaaatggg 4800
cctcattgat gaagtcatga gataaatgag aagttcattc gtagttaata tttgattaat 4860
gtacagcatt tttattctga ttatataata aaggcatcaa aatagcaaaa atgcatggga 4920
catcaatgcc gtccaatctg ccatattcag attatgtagc aagataaaat ggggatggga 4980
aaatccgaga taaaatttaa ccatgaggca tacactcgta ggcattgagt ttatcatttg 5040
tatcaagtct tttgagatta ccaatagata ttgaattggg aaacaaaata cattttctta 5100
ggcttaatga ataaaggata tagcaattca ccacaggtaa ttaaatacaa atattcaaat 5160
tttttttaca tttaaatatt aagtcaaatt aatttttttt ccttaatctg aatttttttt 5220
ttatcttatt tgctcaataa tagcatgttt tataatcata atttccaatc aataattttt 5280
tttaaataaa tctattatat tttatgtatt tataatcatt taaatattta tgtttacaca 5340
taaatttgat aatttaaaat aaattttata gtaataaaaa aaatgttata gattttatat 5400
ttcatgttca tattttaatt tctgaaatat tatatttttc ttttttgctt ttttttgttt 5460
tttttctctg ctttttcagt atttgccgcc gtcgctgccg ccgcccaccc cacccccggc 5520
attcgttttt ggggacacct ccccccccgg cattcgtttt ccgggatggt tgcaacacaa 5580
aaaaattgct tctcccgttt ttttctcccg tgttttctgg atctgccgcc gccgtccccc 5640
ccccccccgg cattcgtttt ctgccgccgc cccgccccat cctcgtatgc tttccgagaa 5700
aattcttgca aacaccccaa ggaaacacaa aaaaattgtt gtttttttgc tcctcatgtt 5760
ttttggatct tggatctgcc gtgcgccccc aagagaaaaa taaaatttct ggtagggaga 5820
cccttacacc ccgagagaaa gaaaaatttt ttccgatagg gaggctttga gggtaatttt 5880
gaaaattaaa atataacaag taaatagtgc atcaatcctt taatattttt cttattatca 5940
cacaaaatct atatttcaat tatatggatt ttcaaaattt tatcgtatag gagcctataa 6000
cacaacattt ccctataagt aatatttggt tatataacac ctcttccctt ccaatgaggc 6060
aatttgcccc aaacattgta ctctccaatg ttcgcaaatt aatttcactc ttcccttcca 6120
atgaggcaat ttgccccaaa cattgtcatc tccaatgttc gcaaattaat ttcactaaaa 6180
cattgtcaat gtcacctctt cccttccaat gaggcaattt gccccaaaca ttgtcatctc 6240
caatgttcga caatcatgga aaattcagtg atgaaaatgt cgtctggtcc agtagccagc 6300
caaaacaaga ggtgctcatt gcaggagttt tgggccaaaa tgtcccatat ctacatcatt 6360
tataaaattt atgttggtct cttgagtgcc gcatcagagc atctcaaaaa tatatatttt 6420
ttaaacaata tagttaagat ctcattcaaa ttataattga gcatttattt ttgaattaaa 6480
cgtatattta ttaattaaga cttcggtttt tgaattgaag tcaaaattat caatggatgc 6540
ttcgttttaa aattaaagct ataattgtca attaagattc cattttttaa ttgaagtttc 6600
aatctggttc ttcattgaaa cattagaagt ctcataaaaa attttcttgg tacacttatg 6660
gctatatttg gttcccggaa aatttgaggg aaaatgcgag ggaaagaaaa tcaaaaggaa 6720
acatagaagg aaagaaaaag tgaaggaaaa taaaaaaaat agattaaaag ttgataaatt 6780
atttttttgt tgcttcaaca tcattttatt tattttacat catcaatata aaaattaaat 6840
aatttaaaaa tcaataagtt tttaattagt tttaattata ttttattttc tttgatattt 6900
tttcataaga cacacaacaa tgagaaaata attttcttta caattttttt ctttcctttg 6960
tcatttctga gacacaacaa tagccatatg ttttttgttt tttcggaaat ttaagaattt 7020
gcttccaatg gttttttaga caacattttt attatttaga ataaaaaaca caaaaaatat 7080
gtttgcaaca cataaatttt tttttttttt tatgtttcta agaataaaaa ataaagtttt 7140
ttttagataa catatgattt aaaaataaaa cattggcatt aaaaagttat ttttaaaaaa 7200
tatttaaaaa tattaaaata tgttaaaaat attttatgtt ttaaacacaa cttttatttt 7260
ataaacaatt ataaaataat tttcaaaaca tatttttcag acatatttta aaaaatagta 7320
ggtctacacc tccattcttt tttggcttca ttttggtata aatatcctca cttttttctt 7380
ataaaaataa tcatttttct taaaattatg tataaatcat tgagatgcaa agtgcaattt 7440
atataaaaaa aatattcatt ttcaagtaaa aagaaagaag cgataaattc cacaaaatca 7500
aaggagtatt gaatctcttt tacataatta atatttttca attcccaatt aaagcaataa 7560
aaaatgaagg acatttggtt ttgtaattta cattaacaca ataatcacaa ccgattgaat 7620
agcttagatt tagaaaatga tatatttgga atacaacatt tttttaatta agaaaaaaag 7680
aaagataaaa atgatattat aaaagtttca ttaaagccca taatgttccc caataagtct 7740
aaaacagtct tcttcaagaa gatatcttat tgctgcctct atatgcaaca gtctttttct 7800
cttttttttt ccttctttaa tgtcaaaagg tctttggaga aaatgatgca caggcctgca 7860
gaattaagaa attattggtg tgcaaaatca tacatttctt ctatttttat taataataca 7920
ttgaaacatg atatttgtta ttttattaat tttttaaaaa tataataatt taatatctca 7980
caattcatta ttttatcaaa aatttcaaag gttcaggtga aatttttata cttttatcaa 8040
tttccatcat ttttcaaatt ttaaacaaat tttaatttat atgcaaattt ctcatgatat 8100
ttccataaaa ctcgaaaatc agtgagggtg caaaagtcaa tttttttaca attgggtata 8160
tgtatatatt tgagggaaaa taattgtcag tgagaagcaa ccagtcccta tcacaagatg 8220
gatggttgag aatgacaccg gtcattgaat tgcaaataga aaaagttcta aaatatattt 8280
ttatcgcagt tcatggtcag aacaaaagtc ctccgcgagc cagaggcata tcctaatgat 8340
tgtcactttc ttccacgcaa attcccatta attagctgct gcccatgcat agcggctgta 8400
atataatggt agaggcacca tggagctccc cttttcagtg acaatgggtt agtcgcaaaa 8460
agaaaatgtt aaagttgaaa gaggagcggt ggccctcaaa ggttcccatc cattggttgc 8520
tttttgtcaa ggaacaagtg gtatcagaat ccaaatcttc tcagtaatgt cccattcatc 8580
ctcacaatgt ccatatgaat tcctctggca attaaaacat ggtagccagt ggttgtcttc 8640
aggatccaca cagtttatca attttgcaca caaaatagag attgttcata aaggatcata 8700
atcagcaatt aattcctaaa ttttgcgctg tcaatttata gtaagttgat caataatggg 8760
taaatatctc ttatgcacaa ccacctttgt ccatgatctc cagcgcattc ggaagccagg 8820
taatgccaca taagaacagt gtcgaatcaa ccaattaggg gtctggtgtc cgagtcatga 8880
gatagacaag gttcgaggtt gttatatatc aatcaataat tagagaagga gccggtctct 8940
tgtgttgagt tgcatcgatg gagagcttag gagttagaaa gggtgcatgg caccaagaag 9000
aggatgttct cctgaggaaa tgcattgaga aatatggaga aggaaagtgg catctggttc 9060
ccctccgagc aggtacaatg aaagagaaag ggatcagtat tagtttgtgt ttttttcatt 9120
ctgttttgct taaagagttt cgttttcttg agtttgcagg gttgaataga tgccgaaaaa 9180
gctgcaggtt gagatggctc aattatttga agccggatat caagagagga gagtttgcat 9240
tagcagaggt tgatctcatg attaggcttc caaatttgtt ggggacaagg caagtctata 9300
atacatcaag tcatagcttg ataatgatat tatattagtt ctgaagctgt tcagacatta 9360
caaaagagct gttcagttga tcatttgtct gatgttgtgc gtgtatagat ggtccttgat 9420
tgcgggtagg cttccaggga ggcatgctaa tgatgtcaag acatattggc atggtccaca 9480
cttgaaaaag aaggttcagt tccaggaaga agggagagat aacacccaaa ccaattctaa 9540
acacaaagct ataaagtctc gcaagcctct ggcaagaaga acatgcacaa cagcaaaagg 9600
gcaccatgga tggtatggta gacaaaatcc agggaggtga gggtgatttt ccatttgatg 9660
tgggcttctg ggattttccc acacaagaag taaatccatt gatctgattt agcacatttc 9720
aatcttaatg tttaagttca aaagttgttc ttgcattcca catctcatct ctaatcattt 9780
taagttccaa agttgttaat cttagaaata aaacaaaaat tcaataaata tatcaatcag 9840
tagaatggtg gatatggagg taacaagtgg atgtagcaaa cagttcaaaa tgattcatgt 9900
cgcaatccaa catcaagcat tgaatgaatg gttaagatta atgacaccaa tcatttgaag 9960
tgcaaaaaga agcaatttta aaatattgtt acaaatattt gaatccccta cataaaatct 10020
atgcaatgaa aaaattattt ctatcggttt ccacacttta ataatcctct aatggttaat 10080
gaatattggc caatctctct ttctagcttg ttagcgaata tacacattct ccattttgag 10140
ggccctattt ggtaattatt ttttaaaata gttatgaaaa ataattttta gaaataattt 10200
tataatattt tatagacaaa aattctcttt aagacactga attgtcttta cactattttt 10260
aatgttttta aaaataattt ttatagttat taatttattt ttaatcattt ttcaaacata 10320
ccataaaaaa attatgtgaa acaaattgaa aatcattaaa caatgttttt tgaaaatcaa 10380
tgttttttgt ttttaagaat aaaaaataga aacaaatttt ataattataa aaaaaaaatg 10440
ttctagagaa caaaaaattg ttctagaaaa taattgtttt ctaatcatgt cttttttttt 10500
tttggagaaa atacagtaag catcaaaaat aaaatctcac ttggtccaaa agagcaataa 10560
gtctccccat ttatataaaa tttataaatt gatagttggg ttgttgtatc gtcatatgaa 10620
aaattggtat cagttccaag gtggcaccaa tatttaatct tttgggttta catgggcaca 10680
cctcaatttt acatattgag cagttcccat aatgccatcc catcaattta cctcaacaaa 10740
ggtattgtgc tcagtttttt gggtggaaat ttcaagtatt aattaacact ctcaaggaga 10800
gttttaggtt caatgaatgt ttatatgtga tttcaaaatt ctaacaatag ttcaatagaa 10860
ttcttaaata tgggtttcca cattttattg atccttaaaa aaaaattggg cacacaaatt 10920
aattttatta cattaaaatt atctttattt tcaatcaaaa catttttaat atgtccaatt 10980
gtcaaatata tatataattt agtagcatag tgtggcaccc gccattcgct tgatgcgttc 11040
ccattcgaaa gtgaacatcg ctttttattt tatttgatga aaatttgatt tttagaaaaa 11100
gatttggagt cgcccattat ttttgtttta ttattttaag ggaaaacaca aataagaaag 11160
aaacacctaa gtgtgcatcc tgaaggaaaa acagggcatg tgaaaacata agtttgggtc 11220
cgggggttag gttcattatt gggaaggtca gatggtgagc cgtacacacc ctctaagccc 11280
gtatcagcca ggcctgtcat aacagaattg aggaattgtg gcaattaatt aattaattat 11340
ggatcacaag aaaaataatc aatatcaaag ttatggtgaa agttaatatg atgaaatcta 11400
attcaaaaag tcaataaaat aaataataag agaattatgc aaaatgattg attgaattaa 11460
ataaataaaa gatattaaaa aaaggtttta aagaaatttc aaaggatttt atttaaaaat 11520
gattttgaat taatgatttc aatttattta cttcaaaaaa aagaatcaat ttattttctc 11580
aatttcagtt atattcaatt ttcaaaaaag ttatttcaat ttattgtatc aaaagaattt 11640
attcaaaaat ttcaatttgg gccaaaaaat tatttttcat tgcttttcat agaaaataat 11700
gaatttttca aattttattt caaaaagtat ttagattcaa tttcattttt ttttaaaaaa 11760
aagtgatttt tcaaaattat ttaaaatgca atacatttat ttgcaaaata aattttacaa 11820
aaaaaaaggt taaatgaaat cttctatttc taaataatca ctttaatcca attttaatta 11880
aggaaaaaga attcattaaa aaagaaatat atatatctat agttagcaaa ttttattaaa 11940
acaaattctt taaattctat aaaaaaaaca aattctttgg gatttttctg aatctctata 12000
aataaataaa aacatttctt ttataaaaaa aaatattgta aaattattat tttattaaaa 12060
cctatttatc gaattcttcc tctcatttaa caaaaatttc tagtttgctc gatattcaat 12120
tctgtcacaa ctcaataaat atatcaaaat gaatatttca ataacactat aaaattaaaa 12180
taagataaaa gtaggcaata aacaatgtaa aataacatta caataagttc ccagtgttat 12240
caattcgtca tcagccacaa agattcaaga atgggtccat gcaaaacaaa gaatagccaa 12300
gatgtaaata tccagagatg tataaaatct caacaaaata ttcaacacca aattccaatt 12360
tcaaattagt cccataaatt tcgtcaatta aaatgagcca aaattcatat agtttttaca 12420
ctagacaatc caaattaata cacaaaaata caacacaaat cacacaacat taaaatggga 12480
taaattaaaa taaattctca tagcactaaa tttaatattt cataatctaa gcctagttta 12540
atatcacaca taattgtccc atgtcccaat taacaaattc aaattggcca acaccatatc 12600
aaatattcaa ataatccaca aaatctccac ccaattatgg gctccaaaat tcgtatcaat 12660
tacaaagccc caataaaaaa aaaatcaata gtcaagagaa ataatatttt aagcccaatc 12720
caatgagccc aacaaaataa cacattacag atagcactaa gtccaaataa caaatatgca 12780
attgatgtaa tccaaatatc ctcaaattaa tccacaaata caatatcccc caaacaccaa 12840
attacaaaat ttcaaattaa ttccatacaa ataaattaaa ttgaattcag tattaatcta 12900
ccacaaaaaa attacaccca aatttcatat taattcacaa atccaaattt ccaaatcaat 12960
aatccaaatt tccaaacaaa caattcacat taaaataaaa taaaatacaa caaatacaaa 13020
taataatcat acaaataata atgcaaataa taataataat aataataata ataataataa 13080
taatgggaag ggggttgccg catggcgtca tgcccacgtc gtggagcgag agaaaaaatg 13140
agggaaggag aagaggaaat gagaggctcg caccactaaa aaaaatggaa aagaaaagaa 13200
aagaagaaaa aaacacaaaa tccaaaggga gaaatgagaa gagagaaaga gggaagagat 13260
gagagcgtgg gtgtgtacac ggccaggaag ggaagtggag tgtggggaga gagaaggaaa 13320
aaaatggaga gaaaaaaaat aaaataaaat ccatggggcc atgtgtgtct ttgggtggag 13380
aaaaaagagt agaagagtga gggaggggga aggagtggag cgggcagcaa agaaagggtg 13440
gaaaagagga aaaaatggag ggaaaatctt gagaattcga gggaaaagct ggagggcaaa 13500
atgggaagaa caataaaaat aggaggaaca ggaaatgagg gggggccggc tgtgtgggaa 13560
gaatgggaag agagagaaga aaatggtggg gtagttgggg aataggaaag agagaggaga 13620
gaggagagag gagagagaaa agaataaaaa aataaaaata aaatagatat aataataata 13680
ataatagtaa taataataat aaaataatag taataataat aataaaataa aagtattagt 13740
aataataata ataaaataaa agtattagta ataaaataaa ataaaatgat atgcaaaata 13800
ataaaagcta ttaaatggtg agtgagccaa aaaatatatg taattggaat ttaaacatgg 13860
gctcaaaatt aatgtaaaga taaaattagg caaaattttg gggtctcaaa atatgcccct 13920
cttcggtaga gttccagagt aaagtgagcg tatcacagaa caaacagaag tgacatttgc 13980
cgaagacaaa gaaagattgc caaatttgtc ctagtatgca atgggctctc tcgaatgata 14040
aggctgggga aaaatgcatc tgacagccaa aaaggaggaa agatggtgca tttaggtgcc 14100
tgaatgagta tgccagatag ctttgacagc caaacataaa ggaagaaaat gcaattgaat 14160
gccaagctgg caaatggctc tgaatgccaa aatgaagcaa cgcatctgaa tgcctaaaca 14220
tgataaaatg aaagcatctg acagcctaaa attggaaaag atggctttga cagcttagga 14280
aagaaaattg tggctctgaa tgccaaagga agatc 14315
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctccttaaga agatatatac tccatcata 29
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tccttaagaa gatatatact ccatcatc 28
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gccgcatcaa aggctagaat tatgc 25
Claims (7)
1.基于KASP技术开发的用于扩增与葡萄果皮颜色相关的SNP 分子标记的引物，其特征在于，包括正向引物F1、正向引物F2和反向引物R，引物序列分别如SEQ ID NO:2-4所示；
其中，与葡萄果皮颜色相关的SNP分子标记位于葡萄VvmybA3基因上，VvmybA3基因参见GenBank：AB442011.1。
2.含有权利要求1所述引物的检测试剂或试剂盒。
3.权利要求1所述引物或权利要求2所述检测试剂或试剂盒在葡萄果皮有色或无色早期筛选中的应用。
4.根据权利要求3所述的应用，其特征在于，包括以下步骤：
1)提取待测葡萄样品的DNA；
2)KASP反应：以步骤1)提取的DNA为模板，向其中加入特异的KASP Primer mix和通用的KASP Mastermix，进行PCR扩增；
3)分析PCR扩增产物；
步骤3)对PCR扩增产物进行荧光检测分析：AA基因型对应的葡萄果皮为无色，CC或AC基因型对应的葡萄果皮为有色。
5.根据权利要求4所述的应用，其特征在于，步骤2)中KASP反应在384微孔板中进行，PCR反应体系为：总体系3μL，DNA模板1μL，KASP Primer mix 1μL，1×KASP Mastermix 1μL；其中所述KASP Primer mix中正向引物F1、F2和反向引物R的浓度均为10μM；
PCR反应条件如下：
6.权利要求1所述引物或权利要求2所述检测试剂或试剂盒在葡萄果皮有色或无色分子标记辅助育种中的应用。
7.权利要求1所述引物或权利要求2所述检测试剂或试剂盒在葡萄果皮有色或无色基因分型中的应用。
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Citations (2)
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|---|---|---|---|---|
| EP1272621A1 (en) * | 2000-03-31 | 2003-01-08 | INTERNATIONAL FLOWER DEVELOPMENTS Pty. Ltd. | Plant anthocyanidin rutinoside aromatic acyl transferases |
| CN103276057A (zh) * | 2013-04-03 | 2013-09-04 | 南京农业大学 | 一种基于lamp技术快速检测灰葡萄孢的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1272621A1 (en) * | 2000-03-31 | 2003-01-08 | INTERNATIONAL FLOWER DEVELOPMENTS Pty. Ltd. | Plant anthocyanidin rutinoside aromatic acyl transferases |
| CN103276057A (zh) * | 2013-04-03 | 2013-09-04 | 南京农业大学 | 一种基于lamp技术快速检测灰葡萄孢的方法 |
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| retrontransposon-induced mutations in grape skin color;Kobayashi S等;《science》;20041231;第304卷(第5673期);第982页 * |
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