CN116555280A - 西瓜隐性抗白粉病基因Clpm-2WF及其应用 - Google Patents
西瓜隐性抗白粉病基因Clpm-2WF及其应用 Download PDFInfo
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Abstract
本发明公开了一种隐性遗传的西瓜抗白粉病基因Clpm‑2WF的发掘及在抗病西瓜新品种培育上的应用。本发明利用抗白粉病西瓜自交系R23(P1)和感白粉病自交系R12(P2)构建了6世代遗传群体(P1、P2、F1、BC1P1、BC1P2、F2),通过接种白粉病生理小种2WF并进行病害鉴定确定了白粉病抗性为隐性遗传。利用极端抗感F2单株重测序数据,通过GWAS分析获得抗病基因初定位区间后,再利用F2:3家系进行精细定位。进一步根据抗感亲本接菌前后转录组数据分析,筛选抗病基因。综合精细定位结果和转录组分析结果,鉴定到一个隐性遗传的西瓜抗白粉病基因Clpm‑2WF,并根据抗感材料差异位点开发了分子标记引物应用于抗白粉病材料辅助选择,为西瓜选育抗白粉病品种提供基因材料和方法。
Description
技术领域
本发明涉及分子生物学领域,特别涉及一种用于检测西瓜抗白粉病基因的引物及其应用。
背景技术
白粉病是一种严重危害西瓜生产的真菌病害,整个生育期均可发病,症状表现为在叶片、茎蔓、叶柄和果实上形成白色粉状霉层并不断扩散,导致叶片枯死,产量下降,造成经济损失。施用农药防治是应对白粉病的主要方法,但过量施用农药会导致环境污染,并且使白粉病病原菌产生抗药性。此外,白粉病菌生理小种众多,给防治带来严峻挑战。综上原因,发掘抗病基因,培育抗病品种是控制西瓜白粉病最经济环保的方法。
目前已报道的抗白粉病生理小种1W基因有3个,Pm1.1是一个不完全显性基因,连锁标记OP-483与其抗性位点相距3.6cM1;pmr2.1位于西瓜2号染色体上,对表型变异的贡献率为80.0%(LOD=30.76),该QTL的两侧有两个CAPS标记wsb2-24(4.00cM)和wsb2-39(13.97cM)2;ClaPMR2是通过分析西瓜抗感材料接种生理小种1W后的转录组差异表达基因而获得的一个抗病蛋白,该蛋白与拟南芥中调控白粉病抗性的蛋白RPW8同源,是NBS-LRR类型的抗病蛋白3。西瓜种质资源PI 270545中存在两个抗白粉病2WU.S.生理小种的基因:隐性高抗基因pmr-1和显性中抗基因Pmr-24。上述发掘的抗病基因只针对白粉病生理小种1W和2W-U.S.,而2WF生理小种在我国分布更广5-6,目前还没有抗生理小种2WF抗病基因的报道。
参考文献:
1.Kim,K.H.et al.Inheritance of resistance to powdery mildew in thewatermelon and development of a molecular marker for selecting resistantplants.Horticulture,Environment and Biotechnology 54,134-140(2013).
2.Kim,K.H.et al.Major quantitative trait loci and putative candidategenes for powdery mildew resistance and fruit-related traits revealed by anintraspecific genetic map for watermelon(Citrullus lanatus var.Lanatus).PloSOne 10,e0145665(2015).
3.Mandal,M.K.,Suren,H.,Kousik,C.Elucidation of resistance signalingand identification of powdery mildew resistant mapping loci(ClaPMR2)duringwatermelon-Podosphaera xanthii interaction using RNA-Seq and whole-genomeresequencing approach.Scientific Reports 10,14038(2020).
4.Tetteh,A.Y.,Wehner,T.C.,Davis,A.R.Inheritance of resistance topowdery mildew race 2in Citrullus lanatus var.lanatus.HortScience 48,1227-1230(2013).
5.王娟,宫国义,郭绍贵等.北京地区瓜类蔬菜白粉病菌生理小种分化的初步鉴定.中国蔬菜2006,7-9(2006).
6.王建设,陈杭.甜瓜抗白粉病鉴定.华北农学报.2000,15(1):125-128.
7.刘欣,程瑞,徐兵划,白甜,许文钊,张朝阳,罗德旭,赵建锋,张兴平,孙玉东.基于KASP技术的SNP标记用于西瓜品种指纹图谱构建和种子纯度检测[J].江苏农业报,2022,38(05):1348-1356.
发明内容
本发明的目的在于提供一种西瓜隐性抗白粉病基因Clpm-2WF及其检测引物和应用。
本发明利用西瓜抗病自交系R23(P1)为抗原,与感病亲本R12(P2)构建了F1、BC1P1、BC1P2和F2群体(474个单株)。根据F1、BC1P1和BC1P2对白粉病生理小种2WF的抗病反应,确定来自R23的抗白粉病基因是一个隐性抗病基因。利用474个F2单株对白粉病生理小种2WF的抗感表型数据和重测序数据,通过GWAS分析,结合抗感亲本不同侵染时间的转录组数据分析结果,发明人定位到了该隐性抗白粉病基因,命名为Clpm-2WF。
为实现上述目的,本发明采取的技术方案为:
一种西瓜隐性抗白粉病基因Clpm-2WF,该基因Clpm-2WF的核苷酸序列如SEQ IDNO:1所示。
用于检测上述西瓜隐性抗白粉病基因Clpm-2WF的引物对,该引物对上游引物的核苷酸序列如SEQ ID NO:3所示,下游引物的核苷酸序列如SEQ ID NO:4所示。
一种用于检测西瓜隐性抗白粉病基因Clpm-2WF的试剂盒,包含上述的引物对和DdeI限制性内切酶。
进一步优选的:还包含PCR缓冲液、dNTP、MgCl2、TaqDNA聚合酶和ddH2O。
上述的西瓜隐性抗白粉病基因Clpm-2WF,上述的引物对,或上述的试剂盒在检测抗白粉病西瓜或辅助选育抗白粉病西瓜品种中的应用。
一种检测抗白粉病西瓜的方法,以西瓜基因组DNA作为模板,采用上述的引物对或上述的试剂盒进行PCR扩增,使用DdeI限制性内切酶对扩增产物进行酶切,并对酶切产物进行检测,若只出现一条168bp的条带,则待测西瓜对白粉病具有抗性。
优选的,所述PCR扩增反应条件如下:94℃预变性2分钟后;94℃变性30秒,62℃退火30秒,72℃延伸30秒,共35个循环;最后72℃彻底延伸2分钟。
与现有技术相比,本发明具有如下有益效果:
一、本发明发现了西瓜对白粉病2WF生理小种的抗病性由一个隐性基因控制,通过200个极端抗感表型F2单株的重测序数据进行GWAS分析、遗传图谱构建以及抗感亲本不同侵染时间的转录组数据分析结果,定位到了该基因(ID号:ClG42_02g016130,参考基因组:http://watermelondb.cn/#/map),命名为Clpm-2WF。通过检测该基因序列,能有效检测出西瓜材料是否抗白粉病生理小种2WF,即能够在苗期或种子萌发阶段,不接种白粉病的情况下,快速高通量的筛选出抗白粉病生理小种2WF的西瓜材料,提高西瓜抗白粉病育种效率,缩短育种年限。利用抗病西瓜品种极大地避免了过量施用农药而导致的环境污染,有效预防白粉病病原菌产生抗药性,降低生产成本,增加种植收益;
二、本发明成功定位了西瓜隐性抗白粉病生理小种2WF基因Clpm-2WF,该基因的编码区第2308位碱基在抗病材料中为G,感病材料中为A(见SEQ ID NO:1~2),该位点的改变引起氨基酸的改变,从而导致西瓜对白粉病抗性的差异;
三、本发明中,基于抗病和感病材料Clpm-2WF基因差异位点进一步开发了一对dCAPS分子标记引物,其核苷酸序列如实施例1中所示;该分子标记在分离群体中的准确率达到100%,检测方便、快速,扩增稳定,在不用接种白粉病的情况下筛选出含有抗病基因的西瓜材料,能有效提高育种效率,降低育种成本。
附图说明
图1利用GWAS分析初步定位抗白粉病基因位于2号染色体;
图2精细定位抗白粉病基因于2号染色体物理位置29581906至29591789区间内;
图3抗、感材料Clpm-2WF基因编码区序列差异;
图4使用dCAPS-9839F/R标记检测抗西瓜白粉病病基因。
注:本专利中的R23为参考文献7中的实验材料Wm13(材料名称:2012WMH0980),R12为参考文献7中的实验材料Wm33(材料名称:G38)。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。
实施例1
西瓜白粉病抗性遗传规律及抗病基因的定位和克隆:
1)白粉病抗性遗传规律和抗病基因的定位:
以西瓜抗病自交系R23(P1)与感病自交系R12(P2)构建F1、BC1P1、BC1P2及包含474个单株的F2群体。在F2群体幼苗期提取单株叶片DNA保存用于后期测序。待幼苗长至2-3片真叶时通过喷雾法对F1、、BC1P1、BC1P2及包含474个单株的F2群体接种白粉病菌2WF(悬浮菌液浓度为1×106个/ml),置于温度为25℃,相对湿度为80%的环境中,发病后调查病情,利用扫描仪对发病叶片进行扫描,并用图像分析软件评估V2.0(ASSESS2.0)对叶片病斑面积进行统计和分析,根据叶片感染白粉面积同时与抗感亲本发病面积对比,进而确定植株抗病性。病情调查2-3次,间隔3-5天左右,并对F2群体中出现极端表型的抗、感单株(各100株)和双亲进行重测序。试验结果表明:接菌白粉病后F1表型为全部感病,BC1P1和BC1P2抗、感分离比均为1:1,因此确定R23抗病性由隐性基因控制。通过对F2群体鉴定结果结合200个极端抗、感单株重测序数据,通过GWAS分析将抗病基因初步定位于2号染色体末端1M区间内(如图1)。利用在该区间发生重组的F2:3家系继续接种鉴定,根据表型结果,进一步精细定位抗病基因,确定该基因位于2号染色体物理位置29581906至29591789区间内(如图2)。同时对抗、感亲本进行接种前后转录组测序,分析定位区间内差异表达基因,综合精细定位结果及转录组数据筛选,发明人在2号染色体上鉴定到一个西瓜抗白粉病基因,基因ID号:ClG42_02g016130(参考基因组网站:http://watermelondb.cn/#/map),命名为Clpm-2WF。
2)Clpm-2WF克隆:
采用Trizol法提取抗病自交系R23和感病自交系R12的叶片总RNA,利用TAKARA反转录试剂盒(货号:RR037A)按照说明书操作步骤获得cDNA。通过PCR扩增Clpm-2WF基因从翻译起始位点ATG到终止位点TAA的CDS序列。扩增引物序列如下:
Clpm-2WF-F:5’-ATGTTGATTGGAAGCATTG-3’
Clpm-2WF-R:5’-TTAAATGGAAATGCTATTAGGA-3’
PCR扩增使用的是诺唯赞的Phanta Max Master Mix,具体反应体系如下:25μL的2×Phanta Max Master Mix,各2μL的Clpm-2WF-F/R(100μmol/L),20μL的ddH2O,最后加入稀释2倍后cDNA 1μL,混匀进行PCR,全程冰上操作。
PCR反应程序如下:
将PCR产物送至青岛擎科生物技术有限公司进行Sanger测序,获得抗、感自交系中Clpm-2WF基因的序列如下:
抗病自交系Clpm-2WF基因CDS序列如SEQ ID NO:1所示。
感病自交系Clpm-2WF基因CDS序列如SEQ ID NO:2所示。
分析二者序列差异,发现在Clpm-2WF编码区第2308位(2号染色体第29586231位),抗病自交系R23为碱基G,感病自交系R12为碱基A(如图3),其结果与重测序结果一致,该差异位点与白粉病抗性连锁。
实施例2检测西瓜抗白粉病基因Clpm-2WF的分子标记
在本发明中,针对获得的抗、感材料Clpm-2WF基因编码区第2308位差异位点,利用dCAPS Finder 2.0网站(http://helix.wustl.edu/dcaps/)设计合适的引物,该引物存在一个错配碱基,从而引入酶切位点DdeI,开发出1对分子标记:
dCAPS-9839F(SEQ ID NO:3):
5’-GACAAAATCTCAATAACTGACACCCCCC-3’
dCAPS-9839R(SEQ ID NO:4):
5’-TTACGGTGGCTTTGCTCCCAATAGA-3’
PCR扩增产物为168bp。用DdeI酶切PCR产物,若被测样品含有抗病基因Clpm-2WF,则PCR产物不能被DdeI酶切,只有一条带,即168bp;否则PCR产物可被DdeI酶切成两条带:140bp和28bp或三条带:168bp、140bp和28bp。上述产物通过聚丙烯酰胺凝胶电泳(PAGE)或4%琼脂糖凝胶电泳检测的方法即可获得。
实施例3分子标记的应用
为了验证上述标记的有效性,利用36份育种材料进行Clpm-2WF等位基因的检测。
(1)采用CTAB法提取西瓜基因组DNA。
(2)PCR扩增。PCR扩增使用的是睿博兴科的Taq Mix,具体反应体系如下:5μL的2×Taq Plus Master Mix,各0.4μL的dCAPS-9839F/R(100μmol/L),3.2μL的ddH2O,最后加入模板DNA 1μL,混匀进行PCR,全程冰上操作。
PCR反应程序如下:
(3)酶切。以上述PCR扩增产物为底物,用DdeI进行酶切,酶切体系如下:
将上述酶切体系置于PCR仪或恒温水浴锅中37℃温浴3-4小时,随后65℃温浴20分钟终止酶切反应。
(4)酶切产物进行聚丙烯酰胺凝胶电泳。具体步骤如下:
a)将长短两块玻璃板用蒸馏水洗净、晾干后,用无水乙醇再擦拭,晾干。将两块玻璃板叠合紧密后在左右两边夹上夹子固定玻璃板。
b)制备50mL凝胶液:ddH2O 30mL+30%丙烯酰胺13.3mL+5×TBE 10mL+10%过硫酸铵400μL+TEMED 33μL,充分混匀后,立即灌入两玻璃板的中间缝隙内。灌胶时应小心缓慢,避免产生气泡。灌胶完成后插入梳子,室温下凝胶0.5-1小时。
c)将胶板固定于电泳槽上,加入1×TBE缓冲液,小心拔掉梳子。在酶切产物中加入2μL的6×DNA Loading Buffer充分混匀,取2-3μL加入到点样孔中,220V电泳跑50分钟。
d)电泳结束后,将胶块从玻璃板中取出,浸泡在核酸染料(每100mL电泳缓冲液中加入30μL StarStain Red Plus,制成3×的工作浓度染色液)中染色15min。
e)将染色处理后的凝胶置于紫外光下分析、拍照。
(5)根据预期试验结果,dCAPS-9839F/R在抗病材料(R)中仅有一条168bp的条带,而在感病材料(S)中,则存在三条带:168bp,140bp,28bp(条带不可见)。根据胶图显示,可以发现所测样品中抗病材料有13份,感病材料有23份,基因型与表型一致(如图4)。因此,该dCAPS标记可以作为Clpm-2WF等位基因检测的理想标记,有助于在西瓜苗期筛选抗白粉病生理小种2WF的材料或品种,在西瓜抗白粉病育种中具有重要的应用价值。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
SEQ ID NO:1
ATGTTGATTGGAAGCATTGTGGGGGCTATCTCTGGGTTGAAGCATGGAGGGAAGAAAATCCATGGAACTGTGGTTTTGATGAGAGACAATGTTTTGGACTTCAATGATTTTGGTTCCACTGTTCTGGATAATCTTCATGAGCTTTTAGGGGGTGGTGTTTCTCTTCGACTTGTTAGCGCTCATCATGGAGACCCTTCTAAAGATTTTGAAGGGAAAGTGGGAGAGGCAGCATACTTGGAGAATTGGGTTGGGAATACAATAATCCCAATTTTTGCTGGAGAAACAGCCTTCAGTATTACATTTGATTGGGATGAAGAAATTGGAGTTCCAGGTGCTTTCTTCATTAGAAATGAACATTTTAGTGAATTCTTTCTCAAATCTCTCACTCTTGAGGATGTCCCTGGCCATGGTAGACTTCATTTTGATTGCAATTCTTGGGTTTATCCTGCTGATAAATACAAAAATGATCGTATATTTTTTGCCAATCAGGCATACCTTCCTAATGAAACACCGGAGCCACTTCGCAAATATAGGGCTGATGAACTATTGAATCTTAGAGGAAATGGAAAAGGAGAGAGAAAGGAATGGGATAGAATTTATGATTATGATGTATACAATGATATTGGTGACCCAGATAGTAATTTGGACCTTGGTCGTCCTATACTTGGAGGTTCATCCAAATATCCGTACCCTCGTAGAGGAAGAACGGGAAGACCACCTTCCAAGAAAGATCCCAAAAGTGAAAGTAGATTACCAGGTTCAAGTAAACCAACTGATATTTACGTTCCAAGAGATGAAAGATTTGGTCACTTGAAGATGTCAGACTTTCTTGCTTATGGCCTAAAATCAATTTCAAGATCAATCAAACCAAAATTGGATGATCTATTTGATAGCACCCCAGGGGAATTTGATGACTTTAATGATGTCTTTGACCTCTTTGAAAAAGGTTTGCCTGTTCCAAGAACTTTGCTTGAGAGTATCACTGACAACATTCCAGCTCCATTGCTTAAAGAAATCTTTAGAACTGATGGTGAAAGATTTCTTAGATTCCCAACTCCTCAGCTAATCCATGAGGATAAGTTTGCATGGAGCACTGATGAAGAATTTGCAAGAGAAATGTTGGCCGGAGTTAACCCCGTAGTTATTGCTCGTCTTCAAGAATTTCCTCCTACAAGCAAACTGGATCCTAACATTTATGGTGATCAAACAAGCAAAATAACTGAAGAACACATAAAAGATGGCTTAGATGGGCTAACTGTGGATGAGGCAGTTGAGAAGAACAAGCTATACATATTGAATCACCATGATTCATTGATTCCATATCTTAGAAGAATAAATACAACTCCCACAAAGACTTATGCTACAAGAACAGTTTTGTTCTTGAAAAATGATGGGACTTTAAAGCCTTTGGCAATTGAATTAAGCTTACCACATCCTCAAGGGGACAAATTTGGGGTCATTAGTAGAGTAATTTTGCCTGCTAAGACAGGAGTTGATGGCACAATTTGGCAGCTAGCTAAAGCTTATGTTACTGTTAATGATACTGGCTATCATCAACTTATTAGCCATTGGTTGAATACTCATGCAACAATCGAACCATTTGTGATAGCAACCAACAGACAATTAAGTGTTCTTCATCCAATTCACAAGTTGCTTGTTCCTCATTTTCGAGACACAATGAACATTAATGCTTTAGCTAGACAATCTCTTATTAATGCTGATGGAATTATTGAAACAACTCATTATCCAGCTAAATACTCCATGGAGCTGTCTTCTTTTGTTTACAAAACTTGGGTCTTCCCTCAACAAGCTCTCCCTGCTGATCTAATCAAAAGAGGAGTTGCAACAGAGGATTCAAGCTCTCCCCATGGACTTCAACTACTAATAGAAGATTATCCATATGCTGTTGATGGTCTTGAGATTTGGTCAGCAATCAAAACATGGGTACAAGATTATTGCTCTTTCTACTACAAAGATGATCAAACACTACATAATGATACAGAGCTCCAATCATGGTGGAAAGAGCTTCGCGAGAAAGGCCATGCAGATAAGAAAGATGAACCATGGTGGCCAAAAATGCAATCCGTTCAAGACCTAATACAAAGTTGTACAATCATTATATGGATCTCTTCAGCTCTTCATGCTGCAGTTAACTTTGGACAATATCCTTACGGTGGCTTTGCTCCCAATAGACCATCCACTAGTCGTCGGTTCCTACCAGAAAACGGTACTCCCGACTACAAAGAGCTCGAGACGAATCCCGAAAAGGCGTTCTTGAGAACAATCACTTCACAATTACAAGCTCTTGTGGGGGTGTCAGTTATTGAGATTTTGTCAAGGCATTCTTCAGATGAAGTGTATTTAGGGCAAAGAAGCGACCCTGAATGGACTTTGGATAAGGAAGCTTTGGAAGCATTTGAGAAGTTTGGGAAAAAGTTGGCTGAAATTGAAGGGAAGATTGCTATGAGAAATAAAGATCCTCAATTGAAAAATAGAGTAGGACCTGTGGATATGCCATATACTTTGTTGTTTCCTACTAGTTCAGAAGGTCTCACAGGTAGAGGAATTCCTAATAGCATTTCCATTTAA
SEQ ID NO:2
ATGTTGATTGGAAGCATTGTGGGGGCTATCTCTGGGTTGAAGCATGGAGGGAAGAAAATCCATGGAACTGTGGTTTTGATGAGAGACAATGTTTTGGACTTCAATGATTTTGGTTCCACTGTTCTGGATAATCTTCATGAGCTTTTAGGGGGTGGTGTTTCTCTTCGACTTGTTAGCGCTCATCATGGAGACCCTTCTAAAGATTTTGAAGGGAAAGTGGGAGAGGCAGCATACTTGGAGAATTGGGTTGGGAATACAATAATCCCAATTTTTGCTGGAGAAACAGCCTTCAGTATTACATTTGATTGGGATGAAGAAATTGGAGTTCCAGGTGCTTTCTTCATTAGAAATGAACATTTTAGTGAATTCTTTCTCAAATCTCTCACTCTTGAGGATGTCCCTGGCCATGGTAGACTTCATTTTGATTGCAATTCTTGGGTTTATCCTGCTGATAAATACAAAAATGATCGTATATTTTTTGCCAATCAGGCATACCTTCCTAATGAAACACCGGAGCCACTTCGCAAATATAGGGCTGATGAACTATTGAATCTTAGAGGAAATGGAAAAGGAGAGAGAAAGGAATGGGATAGAATTTATGATTATGATGTATACAATGATATTGGTGACCCAGATAGTAATTTGGACCTTGGTCGTCCTATACTTGGAGGTTCATCCAAATATCCGTACCCTCGTAGAGGAAGAACGGGAAGACCACCTTCCAAGAAAGATCCCAAAAGTGAAAGTAGATTACCAGGTTCAAGTAAACCAACTGATATTTACGTTCCAAGAGATGAAAGATTTGGTCACTTGAAGATGTCAGACTTTCTTGCTTATGGCCTAAAATCAATTTCAAGATCAATCAAACCAAAATTGGATGATCTATTTGATAGCACCCCAGGGGAATTTGATGACTTTAATGATGTCTTTGACCTCTTTGAAAAAGGTTTGCCTGTTCCAAGAACTTTGCTTGAGAGTATCACTGACAACATTCCAGCTCCATTGCTTAAAGAAATCTTTAGAACTGATGGTGAAAGATTTCTTAGATTCCCAACTCCTCAGCTAATCCATGAGGATAAGTTTGCATGGAGCACTGATGAAGAATTTGCAAGAGAAATGTTGGCCGGAGTTAACCCCGTAGTTATTGCTCGTCTTCAAGAATTTCCTCCTACAAGCAAACTGGATCCTAACATTTATGGTGATCAAACAAGCAAAATAACTGAAGAACACATAAAAGATGGCTTAGATGGGCTAACTGTGGATGAGGCAGTTGAGAAGAACAAGCTATACATATTGAATCACCATGATTCATTGATTCCATATCTTAGAAGAATAAATACAACTCCCACAAAGACTTATGCTACAAGAACAGTTTTGTTCTTGAAAAATGATGGGACTTTAAAGCCTTTGGCAATTGAATTAAGCTTACCACATCCTCAAGGGGACAAATTTGGGGTCATTAGTAGAGTAATTTTGCCTGCTAAGACAGGAGTTGATGGCACAATTTGGCAGCTAGCTAAAGCTTATGTTACTGTTAATGATACTGGCTATCATCAACTTATTAGCCATTGGTTGAATACTCATGCAACAATCGAACCATTTGTGATAGCAACCAACAGACAATTAAGTGTTCTTCATCCAATTCACAAGTTGCTTGTTCCTCATTTTCGAGACACAATGAACATTAATGCTTTAGCTAGACAATCTCTTATTAATGCTGATGGAATTATTGAAACAACTCATTATCCAGCTAAATACTCCATGGAGCTGTCTTCTTTTGTTTACAAAACTTGGGTCTTCCCTCAACAAGCTCTCCCTGCTGATCTAATCAAAAGAGGAGTTGCAACAGAGGATTCAAGCTCTCCCCATGGACTTCAACTACTAATAGAAGATTATCCATATGCTGTTGATGGTCTTGAGATTTGGTCAGCAATCAAAACATGGGTACAAGATTATTGCTCTTTCTACTACAAAGATGATCAAACACTACATAATGATACAGAGCTCCAATCATGGTGGAAAGAGCTTCGCGAGAAAGGCCATGCAGATAAGAAAGATGAACCATGGTGGCCAAAAATGCAATCCGTTCAAGACCTAATACAAAGTTGTACAATCATTATATGGATCTCTTCAGCTCTTCATGCTGCAGTTAACTTTGGACAATATCCTTACGGTGGCTTTGCTCCCAATAGACCATCCACTAGTCGTCGGTTCCTACCAGAAAACGGTACTCCCGACTACAAAGAGCTCGAGACGAATCCCGAAAAGGCGTTCTTGAGAACAATCACTTCACAATTACAAGCTCTTATGGGGGTGTCAGTTATTGAGATTTTGTCAAGGCATTCTTCAGATGAAGTGTATTTAGGGCAAAGAAGCGACCCTGAATGGACTTTGGATAAGGAAGCTTTGGAAGCATTTGAGAAGTTTGGGAAAAAGTTGGCTGAAATTGAAGGGAAGATTGCTATGAGAAATAAAGATCCTCAATTGAAAAATAGAGTAGGACCTGTGGATATGCCATATACTTTGTTGTTTCCTACTAGTTCAGAAGGTCTCACAGGTAGAGGAATTCC。
Claims (7)
1. 一种西瓜抗白粉病基因Clpm-2WF,该基因Clpm-2WF的核苷酸序列如SEQ ID NO:1所示。
2. 用于检测权利要求1所述西瓜抗白粉病基因Clpm-2WF的引物对,其特征在于,该引物对上游引物的核苷酸序列如SEQ ID NO:3所示,下游引物的核苷酸序列如SEQ ID NO:4所示。
3.一种用于检测西瓜抗白粉病基因Clpm-2WF的试剂盒,其特征在于:包含权利要求2所述的引物对和DdeI限制性内切酶。
4.根据权利要求3所述的试剂盒,其特征在于:还包含PCR缓冲液、dNTP、MgCl2、TaqDNA聚合酶和ddH2O。
5.权利要求1中所述的西瓜抗白粉病基因Clpm-2WF,权利要求2中所述的引物对,或权利要求3中所述的试剂盒在检测抗白粉病西瓜或辅助选育抗白粉病西瓜品种中的应用。
6.一种检测抗白粉病西瓜的方法,其特征在于:以西瓜基因组DNA作为模板,采用权利要求2所述的引物对或权利要求3所述的试剂盒进行PCR扩增,使用DdeI限制性内切酶对扩增产物进行酶切,并对酶切产物进行检测,若只出现一条168 bp的条带,则待测西瓜对白粉病具有抗性。
7.根据权利要求6所述的方法,其特征在于:所述PCR扩增反应条件如下:94℃预变性2分钟后;94℃变性30秒,62℃退火30秒,72℃延伸30秒,共35个循环;最后72℃彻底延伸2分钟。
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