CN105400866B - 一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用 - Google Patents

一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用 Download PDF

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CN105400866B
CN105400866B CN201510560617.3A CN201510560617A CN105400866B CN 105400866 B CN105400866 B CN 105400866B CN 201510560617 A CN201510560617 A CN 201510560617A CN 105400866 B CN105400866 B CN 105400866B
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寇姝燕
袁平荣
邹茜
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Institute Of Food Crops Yunnan Academy Of Agricultural Sciences
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Abstract

本发明公开了一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用，利用PCR分子标记技术从3份Pi40粳稻材料中扩增，筛选获得特异性片段，然后经回收、测序、引物设计，最终转化为一个pi40特异性SCAR标记；该标记在单基因系、及186份水稻育成品种中进一步检测，发现其对Pi40基因具有很高的特异性；在分子标记辅助育种中，利用该标记通过PCR可快速准确筛选出后代群体的抗性单株。

Description

一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用
技术领域
本发明属于基因工程技术领域，涉及一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用。
背景技术
稻瘟病(rice blast)由子囊菌(Magnaporthe grisea)的无性世代(Pyriculariagrisea)引起，是水稻生产中危害最严重的世界性的水稻病害。我国每年因稻瘟病而损失的稻谷约10亿千克，稻瘟病已成为制约我国粮食安全及水稻稳产高产的瓶颈。有效防控稻瘟病对保障我国粮食安全意义重大。我国对水稻稻瘟病抗性育种也十分重视，在水稻品种区试中，高感的水稻品种不能通过区试，具有一票否决的作用。水稻育种实践证明：选育利用抗病新品种(组合)是防止该病最行之有效的措施，同时，也是减少农业污染、降低农业生产投入、增加农民收入的可持续发展的最为经济有效的方法；而水稻抗稻瘟病育种的核心是稻瘟病抗性基因，尤其是广谱持久抗稻瘟基因，的发掘与利用。
水稻上，对多个稻瘟菌生理小种表现抗性的基因，称为广谱抗瘟基因。一般认为，广谱抗瘟基因与水稻的水平抗性有关，由多基因控制。但是有报道表明，在水稻生产中某些携带一个主效抗瘟基因的栽培品种对病原菌有较广的抗谱，同时在长时间栽种过程中抗性也不会丧失，具有较好的广谱持久抗性。水稻广谱持久抗瘟基因Pi40来源于澳洲野生稻，Pi40位于水稻第6号染色体着丝粒附近的抗瘟基因簇内，位于标记S2539和RM3330之间，遗传距离分别为3.8cM和2.4cM，对来自韩国和菲律宾毒性最强的稻瘟病菌株表现较强的抗性。通过电子着陆(e-Landing)技术分析，在70Kb的染色体区域内，DNA标记9871.T7E2b与Pi40(t)紧密连锁(Khush and Jena,2007)。
SCAR(sequence characterized amplified regions特定序列扩增)标记通常是从RAPD、SRAP、SSR标记转化而来。SCAR标记是将特异标记片断从凝胶上回收并进行克隆和测序，根据其碱基序列设计一对特异引物(18-24碱基)；也可对RAPD标记末端进行测序，在原RAPD所用10碱基引物的末端增加14个左右的碱基，成为与原RAPD片断末端互补的特异引物。SCAR标记一般表现为扩增片断的有无，是一种显性标记。抗稻瘟病基因Pi40的特异SCAR分子标记的开发为pi40分子辅助选择育种提供技术基础。
发明内容
为了解决现有技术中存在的技术问题，本发明提供了一种广谱稻瘟病抗病基因Pi40的SCAR标记及其应用。利用PCR分子标记技术从3份Pi40粳稻材料中扩增，筛选获得特异性片段，然后经回收、测序、引物设计，最终转化为一个pi40特异性SCAR标记；该标记在单基因系、及186份水稻育成品种(系)中进一步检测，发现其对Pi40基因具有很高的特异性；在分子标记辅助育种中，利用该标记通过PCR可快速准确筛选出后代群体的抗性单株。
其技术方案如下：
一种广谱稻瘟病抗病基因Pi40的SCAR标记，引物的序列为：5’-tgcacctgaagaagatctact-3’、5’-cccccaggtcgtgataccttc-3’。
本发明所述广谱稻瘟病抗病基因Pi40的SCAR标记在分子标记鉴定过程中的应用。
与现有技术相比，本发明利用PCR分子标记技术从3份Pi40粳稻材料中扩增，筛选获得特异性片段，然后经回收、测序、引物设计，最终转化为一个pi40特异性SCAR标记；该标记在单基因系、及186份水稻育成品种(系)中进一步检测，发现其对Pi40基因具有很高的特异性；在分子标记辅助育种中，利用该标记通过PCR可快速准确筛选出后代群体的抗性单株。
附图说明
图1是3F引物对pi单基因系的扩增结果；
图2是Pi40特异SCAR标记对部分群体材料的扩增结果。
具体实施方式
下面结合具体实施例进一步说明本发明的技术方案。
利用pi40特异性SCAR标记进行分子标记鉴定的具体方法如下：
1、以中国普感稻瘟病水稻品种—丽江新团黑谷、携带Pi40的水稻品系、Pii、pi9、pita、Piz等单基因系为对照，分析SCAR标记的特异性；
(1)材料：丽江新团黑谷、3份Pi40高代粳稻回交系(4145、4150和4163)和pita、piz、pikm、pii和pib等单基因系材料；
(2)DNA提取：上述材料取幼苗叶片，液氮研磨，利用2×CTAB法提取每个样品的基因组总DNA；
(3)开发的Pi40特异SCARf引物的序列为：5’-tgcacctgaagaagatctact-3’、5’-cccccaggtcgtgataccttc-3’；
(4)PCR反应程序为：PCR反应体系共15μl，其中10×PCRbuffer 1.5ul，dNTP1.125ul，Taq 0.15ul，Forward Reverse Primer各0.1ul,模板DNA 1ul，加ddH2O补足15ul。PCR循环程序：94℃预变性5min，94℃变性30s，60℃退火30s，72℃延伸1min，25个循环。末次循环结束后，将反应管置于72℃温育10min，以确保充分延伸；
(5)上述PCR反应产物放置于1.5％琼脂糖凝胶上电泳，花青素染色，0.5×TBE电泳缓冲液，电压6V/cm，电泳时间40min，电泳完成后，在凝胶呈像系统拍照并分析结果；
(6)Pi40特异性SCAR引物仅在3份Pi40高代粳稻回交系中均能够扩增出一条约550bp的特征带型，而在轰早生(云南地方老品种，携带pii)、合系24号(云南粳稻课题组育成品种，携带pii和pikm)、IR24(国际水稻所育成品种，携带pib)、地谷B和K22(外省引进品种，携带pi9)、三磅七十箩(云南地方老品种，抗病，基因未知)以及其它单基因系材料中均没有扩增出特异带型，如图1所示：
2、以186份水稻品种为模板，分析Pi40特异SCAR标记的应用前景；
(1)材料：水稻材料186份，具体如表1所示：
(2)基因组总DNA提取、PCR反应和电泳检测方法同1所述相同；
(3)结果：如表1，在186份云南育成水稻品种中的9份(楚粳22号、楚粳27号、岫粳11号、岫粳14号、岫糯3号、临籼21号、临籼26号、红优8号)中扩增出了特异性的目标带型，而在其它176份材料中均没有扩增产物出现，3F对云南育成水稻品种的检测准确性达到94.6％，表明3F可以作为今后在云南水稻育种生产中的pi40抗性育种的MAS标记加以应用。
表1：186份云南水稻材料对Pi40特异SCAR引物的扩增反应一览表
Figure BDA0000796810190000031
Figure BDA0000796810190000041
Figure BDA0000796810190000051
3、在抗稻瘟病遗传改良群体中利用Pi40特异SCAR标记快速筛选抗性单株。
(1)材料：合系30号/4163杂交F2群体，214个单株；
(2)分单株提取DNA，提取方法、PCR反应和电泳检测方法同1所述相同；
(3)结果：在214份单株中，应用开发的Pi40特异SCAR标记从中检测出带有Pi40基因的单株120份，如图2所示。
以上所述，仅为本发明最佳实施方式，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Figure BDA0000796810190000061
Figure IDA0000796810290000011

Claims (1)

1.一种广谱稻瘟病抗病基因Pi40的SCAR标记在水稻辅助育种中的应用，其特征在于，所述广谱稻瘟病抗病基因Pi40的SCAR标记检测引物的序列为：5’-tgcacctgaagaagatctact-3’、5’-cccccaggtcgtgataccttc-3’。
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