山融3号小麦BC_2代群体耐盐主效QTL相关分子标记的筛选定位
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摘要
由于自然环境的不断恶化,耕地土壤盐渍化已成为制约世界农业生产的主要的环境因子。小麦是世界上重要的粮食作物之一,对盐渍环境十分敏感,盐胁迫往往造成其产量和品质的大幅下降。如何运用生物技术研究小麦的耐盐机理、定位克隆耐盐基因、培育耐盐新品种、提高小麦产量是育种学家和生物技术工作者共同面临的重大课题。本课题组首创的小麦(Triticum aestivum L.2n=42)不对称体细胞杂交技术,将耐盐禾草(长穗偃麦草Agropyron elongatum 2n=70)的染色体小片段或／和DNA整合入普通小麦济南177(JN177)基因组,选育出耐盐渐渗小麦新品种山融3号(SR3)。前期对该品种的初步研究表明,其耐盐性由5A染色体上的主效基因控制。
本研究以济南177作为轮回亲本,对山融三号与济南177的回交一代(BC1)进行回交,以所获BC2代作为研究材料。利用5A染色体上存在的新的分子标记,对山融三号小麦的耐盐相关QTL进一步深入分析,初步确定了耐盐主效基因的性质和位置。实验中,利用SSR／EST-SSR等分子标记技术,设计211对标记引物(小麦5A染色体上59对SSR引物、26对EST-SSR引物和65对STS引物；小麦其余染色体上30对标记引物以及31对与小麦5A具有高同源性的水稻第9染色体STS引物)对BC2群体的双亲进行PCR扩增,其中46对引物的PCR扩增呈微卫星DNA多态性,多态性指数为25.4%。用这46对引物继续对以BC2群体构建的耐盐池和盐敏感池进行PCR扩增,筛选出5对位于5A染色体上的微卫星DNA扩增呈多态性的SSR标记引物(Xwmc713-5A, Xgwm304, Xgwm666,(?)Xcfa2141, XBarc144),在BC2群体中个体扩增后显示与耐盐基因连锁。利用MapMaker 3.0及MapDraw V2.1软件推算出耐盐主效基因位于5AL染色体上,与最近的XBarc144标记的相对距离为12.7cM,可利用这5个分子标记在缺失图Chinese_Spring_Deletion_5A以及连锁图Wheat Ta-SSR-2004-5A上的整合信息,将主效QTL(Gene)进一步定位在5A染色体长臂距着丝点5-6cM左右。本工作利用不同的群体,进一步验证了山融三号耐盐主效基因的位置；缩小了分子标记与耐盐主效基因的距离。本文为进一步寻找与耐盐主效基因更紧密连锁的分子标记,克隆耐盐主效基因,研究其耐盐机理奠定了基础。
Current status of agriculture land becomes worse and worse due to man-made and natural changes. Moreover, solid degradation, especially aridity and edaphic salinization, has been a very serious problem in the world for years. Common wheat (Triticum asetivum L.) is one of the most important cereal crops in the world. It is very sensitive to salt-stress which can lead to low productivity of wheat. Therefore, it is very necessary to work on the botanic salt-tolerant mechanisms, to map salt-tolerant genes and to breed new salt-tolerant species by using biotechnology. Years ago, a novel salt-tolerant wheat variety Shanrong No.3 (SR3) generated by using asymmetric somatic hybridization between common wheat Jinan 177 and Agropyron elongatum in our lab, which posses strong tolerance to salt stress. Specifically, one piece of salt-tolerance chromatin from Argopyron elogatum was intrgressed into the Jinan 177 genome, which is salt-tolerent, drought-resistant and high productive. The earlier experiments revealed that the salt-tolerance of SR3 is highly related with the major quantitative trait locus (QTL) located near the centromere of wheat on the long arm of chromosome 5A.
In this study, an advanced BC2 population consisted of 171 individuals was constructed from the backcross between SR3 and Jinan 177 after large scale screening under salt stress. For salt-tolerance QTL analysis, microsatellite (SSR), expressed sequence tag-simple sequence repeats (EST-SSR) and BSA (bulked segregant analysis) techniques in combination with the SSR map of wheat were used.221 5A polymorphic markers including 59 SSR primers,26 EST-SSR primers,65 STS primers on wheat chromosome 5A and 31 STS primers on rice chromosome No.9, along with 30 non-5A markers were used to construct a linkage map. Among all of the 211 markers,46 show polymorphism in SR3 and Jinan 177, with a polymorphic index 25.4%. Then PCR amplification has been carried out among the salt tolerance pond and sensitive pond with those 46 marker pairs. A novel molecular marker XBarcl44 was identified. Combined with the SSR high density genetic linkage map (Wheat Ta-SSR-2004-5A) and deletion map (Chinese_Spring_Deletion_5A) of wheat, the major gene for salt tolerance has been located on the 5A long arm of wheat chromosome, the distance to the XBarc144 marker is 12.7 cM, and to the centromere is 5-6cM. Our findings not only narrow the further search scope, laying foundation for mapping salt-tolerance QTL, but also shed light on identification of plant mechanisms for salt tolerance and breeding of new cultivars.
本研究以济南177作为轮回亲本,对山融三号与济南177的回交一代(BC1)进行回交,以所获BC2代作为研究材料。利用5A染色体上存在的新的分子标记,对山融三号小麦的耐盐相关QTL进一步深入分析,初步确定了耐盐主效基因的性质和位置。实验中,利用SSR／EST-SSR等分子标记技术,设计211对标记引物(小麦5A染色体上59对SSR引物、26对EST-SSR引物和65对STS引物；小麦其余染色体上30对标记引物以及31对与小麦5A具有高同源性的水稻第9染色体STS引物)对BC2群体的双亲进行PCR扩增,其中46对引物的PCR扩增呈微卫星DNA多态性,多态性指数为25.4%。用这46对引物继续对以BC2群体构建的耐盐池和盐敏感池进行PCR扩增,筛选出5对位于5A染色体上的微卫星DNA扩增呈多态性的SSR标记引物(Xwmc713-5A, Xgwm304, Xgwm666,(?)Xcfa2141, XBarc144),在BC2群体中个体扩增后显示与耐盐基因连锁。利用MapMaker 3.0及MapDraw V2.1软件推算出耐盐主效基因位于5AL染色体上,与最近的XBarc144标记的相对距离为12.7cM,可利用这5个分子标记在缺失图Chinese_Spring_Deletion_5A以及连锁图Wheat Ta-SSR-2004-5A上的整合信息,将主效QTL(Gene)进一步定位在5A染色体长臂距着丝点5-6cM左右。本工作利用不同的群体,进一步验证了山融三号耐盐主效基因的位置；缩小了分子标记与耐盐主效基因的距离。本文为进一步寻找与耐盐主效基因更紧密连锁的分子标记,克隆耐盐主效基因,研究其耐盐机理奠定了基础。
Current status of agriculture land becomes worse and worse due to man-made and natural changes. Moreover, solid degradation, especially aridity and edaphic salinization, has been a very serious problem in the world for years. Common wheat (Triticum asetivum L.) is one of the most important cereal crops in the world. It is very sensitive to salt-stress which can lead to low productivity of wheat. Therefore, it is very necessary to work on the botanic salt-tolerant mechanisms, to map salt-tolerant genes and to breed new salt-tolerant species by using biotechnology. Years ago, a novel salt-tolerant wheat variety Shanrong No.3 (SR3) generated by using asymmetric somatic hybridization between common wheat Jinan 177 and Agropyron elongatum in our lab, which posses strong tolerance to salt stress. Specifically, one piece of salt-tolerance chromatin from Argopyron elogatum was intrgressed into the Jinan 177 genome, which is salt-tolerent, drought-resistant and high productive. The earlier experiments revealed that the salt-tolerance of SR3 is highly related with the major quantitative trait locus (QTL) located near the centromere of wheat on the long arm of chromosome 5A.
In this study, an advanced BC2 population consisted of 171 individuals was constructed from the backcross between SR3 and Jinan 177 after large scale screening under salt stress. For salt-tolerance QTL analysis, microsatellite (SSR), expressed sequence tag-simple sequence repeats (EST-SSR) and BSA (bulked segregant analysis) techniques in combination with the SSR map of wheat were used.221 5A polymorphic markers including 59 SSR primers,26 EST-SSR primers,65 STS primers on wheat chromosome 5A and 31 STS primers on rice chromosome No.9, along with 30 non-5A markers were used to construct a linkage map. Among all of the 211 markers,46 show polymorphism in SR3 and Jinan 177, with a polymorphic index 25.4%. Then PCR amplification has been carried out among the salt tolerance pond and sensitive pond with those 46 marker pairs. A novel molecular marker XBarcl44 was identified. Combined with the SSR high density genetic linkage map (Wheat Ta-SSR-2004-5A) and deletion map (Chinese_Spring_Deletion_5A) of wheat, the major gene for salt tolerance has been located on the 5A long arm of wheat chromosome, the distance to the XBarc144 marker is 12.7 cM, and to the centromere is 5-6cM. Our findings not only narrow the further search scope, laying foundation for mapping salt-tolerance QTL, but also shed light on identification of plant mechanisms for salt tolerance and breeding of new cultivars.
引文
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