云贵川和甘肃地区小麦条锈菌群体的MFLP分析
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摘要
小麦条锈病是由小麦条锈菌Puccinia striiformis f. sp. tritici引起的真菌性病害,是严重危害世界许多国家小麦生产的主要流行病害。目前我国是世界上最大的小麦条锈病流行区。种植抗病品种是防治小麦条锈病最经济有效的措施,但是由于条锈菌新毒性小种的不断产生和发展,抗病品种的抗性丧失现象十分严重。小麦条锈菌属专性寄生菌,无法人工培养,基于鉴别寄主来进行的常规小种鉴定及监测方法繁杂、费工费时,严重制约了小麦条锈菌群体遗传与生物学研究。
微卫星锚锭片段长度多态性(MFLP)是建立在PCR技术基础上、结合了扩增片段长度多态性(AFLP)和微卫星锚锭引物技术(SSR-anchor primer)双重原理的新一代分子标记技术。本研究建立了适用于专性寄生锈菌的MFLP技术,并首次利用该技术对来自云南、贵州、四川和甘肃地区的73个菌系进行了分子多态性分析,初步揭示了以上地区小麦条锈菌群体的MFLP聚类结构及区间关系。主要研究结果如下:
1.通过对DNA提取、限制性内切酶二次酶切、PCR双重扩增等实验因素的选择优化,建立了适用于小麦条锈菌研究的MFLP分子标记分析体系。
2.从456对MFLP引物组合中,筛选出13对带型质量好、分辨率高并具有一定多态性的引物组合,在供试73个小麦条锈菌菌系的基因组中,13对引物共扩增出42条多态性条带。
3.对云南、贵州、四川以及甘肃四个省份的73个小麦条锈菌标样或菌系进行了MFLP聚类分析,结果表明被测菌系的相似系数的基值为0.47,在相似系数0.60处,被测菌系可划分为A,B两组,没发现优势组群。MFLP聚类结果不能明确区分甘肃和云贵川条锈菌群体,可能与区间存在广泛的菌源交流有关。
4.从来自云贵川的27个小麦条锈菌菌系的MFLP聚类分析结果可以看出,其相似系数基值为0.43,在相关系数0.61以上,可划分为2组。在第一组中,除2个四川菌系外,其余均为云贵菌系,第二组中,除3个云贵菌系外,多数为四川菌系,说明云贵川地区的小麦条锈菌群体存在一定的多态性,同时,也可初步推测四川与云南、贵州存在一定程度的菌源交流。
5.甘肃菌系也可划分为两组,分别归属于A、B两大组,每组均包括23个菌系,不同流行区域的亚组群体构成存在差异,可能与近年来水源11类群特别是CYR33在甘肃群体的增长有关。MFLP聚类分析结果不能区分天水甘谷县与武山县的条锈菌系,也不能明确区分洮、岷及陇中流行区与渭河上游自生麦苗越夏区的菌系,说明区间存在较频繁的菌源交流。
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici(Pst), is one of the main important fungal diseases in wheat, which has caused great losses to the wheat production in the world. China is the largest epidemic area for wheat stripe rust. The breeding and applying rust-resistant cultivars has been considered as the most effective and economical strategy for the prevention and control of yellow rust. However, the resistance breakdown of wheat cultivars is very serious due to the occurrence and development of new virulent races. Being an obligate fungus, the race identification and monitoring of Pst were complicated and time-consuming, which have severely restricted the population genetic structure and biology study on Pst.
Microsatellite-anchored fragment length polymorphism(MFLP), is a PCR-based method for DNA fingerprinting, which combines the concept of amplified fragment length polymorphism and microsatellite-anchor primer technique. The MFLP reaction system special for the obligated pathogens, wheat rust fungi, was established successfully in this research. The genetic diversity of 73 isolates of wheat stripe rust collected from Yunnan, Guizhou, Sichuan and Gansu province was analyzed using MFLP method for the first time, and the main results derived from this research were as follows.
1. Using mixed urediniospores of wheat yellow rust, several main factors such as the genomic DNA preparation, enzyme digestion and PCR amplification, which might influence the final MFLP result were studied and optimized in this research. The MFLP reaction system for wheat stipe rust was successfully established.
2. 13 pair primers, which could produce bands with high quality and high polymorphism, were selected out of 456 pair primers. 42 polymorphic bands were detected in the experimental materials with 13 pair MFLP primers.
3. 73 isolates of Pst collected from Yunnan, Guizhou, Sichuan and Gansu province were analyzed with MFLP-clustering method. The basic value of similarity coefficient (SIM)was 0.46. All the isolates could be divided into two groups at SIM 0.60, neither of the two groups was the predominant one. The MFLP-clustering result of Pst collected from the areas above-mentioned was not related with the the geographic areas.Taking the main factors such as climate, geographical location and wheat varieties into account, we can’t draw a clear conclusion whether the stripe rust pathogen is able to exchange between Gansu and Sichuan, especially between Gansu and Yunnan, Guizhou.
4. 27 isolates of Pst collected from Yunnan, Guizhou and Sichuan province were also analyzed, and the basic value of SIM was 0.43. At SIM 0.61, the isolates could be divided into two groups, the first group consisted of 11 isolates from Yunnan and Guizhou, 2 isolates from Sichuan; There were 11 isolates from Sichuan, 3 isolates from Yunnan and Guizhou in the second group. According to the MFLP-clustering result, we can initially speculated that there was a certain level of genetic diversity, and also pathogen exchange exist between Sichuan, Yunnan and Guizhou province.
5. The Gansu Pst population also consisted of two groups, which belonged to Group A and B respectively, each group included 23 isolates. The sub-group constitution was different between Tao-Min-middle Gansu area and the upper reaches of Weihe river basin, which may be ascribed to the increasing of pathotypes of wheat stripe rust virulent to Suwon 11, especially for CYR33. The MFLP-clustering result of isolates from Gangu and Wushan counties of Gansu province showed that the pathogen exchange was undergone frequently within the two areas.
微卫星锚锭片段长度多态性(MFLP)是建立在PCR技术基础上、结合了扩增片段长度多态性(AFLP)和微卫星锚锭引物技术(SSR-anchor primer)双重原理的新一代分子标记技术。本研究建立了适用于专性寄生锈菌的MFLP技术,并首次利用该技术对来自云南、贵州、四川和甘肃地区的73个菌系进行了分子多态性分析,初步揭示了以上地区小麦条锈菌群体的MFLP聚类结构及区间关系。主要研究结果如下:
1.通过对DNA提取、限制性内切酶二次酶切、PCR双重扩增等实验因素的选择优化,建立了适用于小麦条锈菌研究的MFLP分子标记分析体系。
2.从456对MFLP引物组合中,筛选出13对带型质量好、分辨率高并具有一定多态性的引物组合,在供试73个小麦条锈菌菌系的基因组中,13对引物共扩增出42条多态性条带。
3.对云南、贵州、四川以及甘肃四个省份的73个小麦条锈菌标样或菌系进行了MFLP聚类分析,结果表明被测菌系的相似系数的基值为0.47,在相似系数0.60处,被测菌系可划分为A,B两组,没发现优势组群。MFLP聚类结果不能明确区分甘肃和云贵川条锈菌群体,可能与区间存在广泛的菌源交流有关。
4.从来自云贵川的27个小麦条锈菌菌系的MFLP聚类分析结果可以看出,其相似系数基值为0.43,在相关系数0.61以上,可划分为2组。在第一组中,除2个四川菌系外,其余均为云贵菌系,第二组中,除3个云贵菌系外,多数为四川菌系,说明云贵川地区的小麦条锈菌群体存在一定的多态性,同时,也可初步推测四川与云南、贵州存在一定程度的菌源交流。
5.甘肃菌系也可划分为两组,分别归属于A、B两大组,每组均包括23个菌系,不同流行区域的亚组群体构成存在差异,可能与近年来水源11类群特别是CYR33在甘肃群体的增长有关。MFLP聚类分析结果不能区分天水甘谷县与武山县的条锈菌系,也不能明确区分洮、岷及陇中流行区与渭河上游自生麦苗越夏区的菌系,说明区间存在较频繁的菌源交流。
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici(Pst), is one of the main important fungal diseases in wheat, which has caused great losses to the wheat production in the world. China is the largest epidemic area for wheat stripe rust. The breeding and applying rust-resistant cultivars has been considered as the most effective and economical strategy for the prevention and control of yellow rust. However, the resistance breakdown of wheat cultivars is very serious due to the occurrence and development of new virulent races. Being an obligate fungus, the race identification and monitoring of Pst were complicated and time-consuming, which have severely restricted the population genetic structure and biology study on Pst.
Microsatellite-anchored fragment length polymorphism(MFLP), is a PCR-based method for DNA fingerprinting, which combines the concept of amplified fragment length polymorphism and microsatellite-anchor primer technique. The MFLP reaction system special for the obligated pathogens, wheat rust fungi, was established successfully in this research. The genetic diversity of 73 isolates of wheat stripe rust collected from Yunnan, Guizhou, Sichuan and Gansu province was analyzed using MFLP method for the first time, and the main results derived from this research were as follows.
1. Using mixed urediniospores of wheat yellow rust, several main factors such as the genomic DNA preparation, enzyme digestion and PCR amplification, which might influence the final MFLP result were studied and optimized in this research. The MFLP reaction system for wheat stipe rust was successfully established.
2. 13 pair primers, which could produce bands with high quality and high polymorphism, were selected out of 456 pair primers. 42 polymorphic bands were detected in the experimental materials with 13 pair MFLP primers.
3. 73 isolates of Pst collected from Yunnan, Guizhou, Sichuan and Gansu province were analyzed with MFLP-clustering method. The basic value of similarity coefficient (SIM)was 0.46. All the isolates could be divided into two groups at SIM 0.60, neither of the two groups was the predominant one. The MFLP-clustering result of Pst collected from the areas above-mentioned was not related with the the geographic areas.Taking the main factors such as climate, geographical location and wheat varieties into account, we can’t draw a clear conclusion whether the stripe rust pathogen is able to exchange between Gansu and Sichuan, especially between Gansu and Yunnan, Guizhou.
4. 27 isolates of Pst collected from Yunnan, Guizhou and Sichuan province were also analyzed, and the basic value of SIM was 0.43. At SIM 0.61, the isolates could be divided into two groups, the first group consisted of 11 isolates from Yunnan and Guizhou, 2 isolates from Sichuan; There were 11 isolates from Sichuan, 3 isolates from Yunnan and Guizhou in the second group. According to the MFLP-clustering result, we can initially speculated that there was a certain level of genetic diversity, and also pathogen exchange exist between Sichuan, Yunnan and Guizhou province.
5. The Gansu Pst population also consisted of two groups, which belonged to Group A and B respectively, each group included 23 isolates. The sub-group constitution was different between Tao-Min-middle Gansu area and the upper reaches of Weihe river basin, which may be ascribed to the increasing of pathotypes of wheat stripe rust virulent to Suwon 11, especially for CYR33. The MFLP-clustering result of isolates from Gangu and Wushan counties of Gansu province showed that the pathogen exchange was undergone frequently within the two areas.
引文
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