苹果斑点落叶病抗性基因RAPD标记筛选研究
摘要
苹果早期落叶病是我国苹果产区普遍发生的一种重要病害,利用抗性资源,开展其抗性育种研究具有重要理论价值和实践意义。本研究以苹果“秦冠”和“富士”及其杂交群体为材料,通过对亲本及杂交F1代单株抗病性的田间鉴定与室内鉴定,分析了秦冠、富士苹果对早期落叶病的抗性遗传规律。在此基础上,开展了苹果早期落叶病抗性基因RAPD标记筛选,获得了与苹果早期落叶病抗性基因连锁的分子标记,为苹果抗病育种早期性状选择及抗病基因的挖掘利用提供技术依据。
本研究获得的主要结果如下:
(1)经田间鉴定,秦冠苹果对于早期落叶病的抗性明显高于富士。
(2)在斑点落叶病抗性遗传方面,“秦冠×富士”杂交F1代1039株群体出现抗病、感病两种表现型,其中抗病和感病单株数分别为544和495株,经卡方检验,分离比例符合1:1的比例(χ2 = 2.645,χ20.01 = 3.84);“富士×秦冠”杂交F1代489株群体出现抗病、感病两种表现型,其中抗病和感病单株数分别为235和254株,经卡方检验,分离比例基本符合1:1的理论比例(χ2 = 0.7434,χ20.01 = 3.84)。2个组合杂交后代病情指数呈连续分布,带有数量性状的特征。因此,苹果斑点落叶病抗性遗传抗性遗传可能是由一对主效基因和若干微效基因共同作用的结果。
(3)以秦冠为母本的组合后代斑点落叶病和褐斑病超高亲株数都多于反交组合,表明秦冠对早期落叶病抗性较富士具有遗传优势。
(4)应用325个RAPD引物对秦冠、富士进行了筛选,其中92个引物在双亲间能扩展出差异条带,在这些引物中有18个引物在两个亲本和杂交后代的抗、感基因池(每池7个单株)中扩增出了稳定、可重复的多态性条带。其中S428-854片段在秦冠和抗病基因池中出现,而在富士和感病基因池中不出现。利用引物S428在71个F1代中进行验证,有56个单株获得的结果与此一致,占78.9%,与田间病害鉴定结果也一致。因此,说明S428-854片段为与苹果抗斑点落叶病基因连锁的RAPD标记。
(5)首次获得了一个与苹果抗斑点落叶病基因连锁的S428-854标记。对S428-854特异条带进行了测序,其准确长度为854bp。应用NCBI和DNASTAR软件对该片段进行了分析,结果表明没有与此片段同源性高于30%的序列,不含开放阅读框架,不具有基因结构,因此该片段为苹果一个新的DNA序列,GenBank登录号为EU710766。
The early defoliation disease of apple is one of the most serious diseases in the apple production area in China. It is important to process the resistance breeding program using the resistance germplasm. A F1 population from the cross of Fuji (a susceptible parent to Alternaria leaf spot) and‘Qinguan’(a resistant parent) is used in this study. Under the natural conditions, the resistance level in the parents and their F1 individuals were identified to analyze the inheritance of resistance to Alternaria leaf spot in apples. Based on the field data, RAPD molecular markers linked to Alternaria leaf spot resistant gene in apples were detected.
These are the main results.
1. Resistance to Alternaria leaf spot in‘Qinguan’was much higher than that in‘Fuji’from the field identification data.
2. In the cross of‘Qinguan×Fuji’, 544 plants proved to be resistant and 495 plants proved to be susceptible; and in the“Fuji×Qinguan”cross, the number of the resistant and susceptible plants was 235 and 254. The ratio of resistant vines to susceptible vines in both crosses have been tested in accordance withχ20.01 distribution of 1:1 segregation (χ2 Qinguan×Fuji = 2.645;χ2 Fuji×Qinguan = 0.743;χ20.01=3.84). The SI values of F1 progenies in both crosses revealed genetic features of quantitative traits demonstrating successive distribution. This indicates that Alternaria leaf spot resistance in these two crosses may be controlled by a major gene in combination with some minor genes.
3. Distribution features of the SI values of the progenies in two crosses also revealed that‘Qinguan’may have stronger inheritance than‘Fuji’for Alternaria leaf spot resistance.
4. In this study, 325 RAPD primers were used to screen the polymorphism between‘Qinguan’and‘Fuji’92 primers can produce polymorphic PCR bands between two parents. Among these 92 primers, 18 primers can produce polymorphic PCR bands between the resistant gene pool and the susceptible gene pool (each gene pool contained 10 F1 individuals). A fragment, S428-854, presented in the‘Qinguan’and the resistance DNA pool, absented in the‘Fuji’and susceptible DNA pool. The distribution of S428 in the F1 individuals showed that the molecular data of the 56 individuals (78.9%) were consistent with the field identification data. 5. The S428-854 RAPD marker was sequenced. Its actual length is 854bp. Results of blasting this sequence with the NCBI database indicated that their homology is no more than 30%. Structure analysis showed that this sequence has no opening read frame, meaning that it does not have a complete structure. Therefore, it could be a new sequence found in apples. Its accession in GeneBank is EU710766.
本研究获得的主要结果如下:
(1)经田间鉴定,秦冠苹果对于早期落叶病的抗性明显高于富士。
(2)在斑点落叶病抗性遗传方面,“秦冠×富士”杂交F1代1039株群体出现抗病、感病两种表现型,其中抗病和感病单株数分别为544和495株,经卡方检验,分离比例符合1:1的比例(χ2 = 2.645,χ20.01 = 3.84);“富士×秦冠”杂交F1代489株群体出现抗病、感病两种表现型,其中抗病和感病单株数分别为235和254株,经卡方检验,分离比例基本符合1:1的理论比例(χ2 = 0.7434,χ20.01 = 3.84)。2个组合杂交后代病情指数呈连续分布,带有数量性状的特征。因此,苹果斑点落叶病抗性遗传抗性遗传可能是由一对主效基因和若干微效基因共同作用的结果。
(3)以秦冠为母本的组合后代斑点落叶病和褐斑病超高亲株数都多于反交组合,表明秦冠对早期落叶病抗性较富士具有遗传优势。
(4)应用325个RAPD引物对秦冠、富士进行了筛选,其中92个引物在双亲间能扩展出差异条带,在这些引物中有18个引物在两个亲本和杂交后代的抗、感基因池(每池7个单株)中扩增出了稳定、可重复的多态性条带。其中S428-854片段在秦冠和抗病基因池中出现,而在富士和感病基因池中不出现。利用引物S428在71个F1代中进行验证,有56个单株获得的结果与此一致,占78.9%,与田间病害鉴定结果也一致。因此,说明S428-854片段为与苹果抗斑点落叶病基因连锁的RAPD标记。
(5)首次获得了一个与苹果抗斑点落叶病基因连锁的S428-854标记。对S428-854特异条带进行了测序,其准确长度为854bp。应用NCBI和DNASTAR软件对该片段进行了分析,结果表明没有与此片段同源性高于30%的序列,不含开放阅读框架,不具有基因结构,因此该片段为苹果一个新的DNA序列,GenBank登录号为EU710766。
The early defoliation disease of apple is one of the most serious diseases in the apple production area in China. It is important to process the resistance breeding program using the resistance germplasm. A F1 population from the cross of Fuji (a susceptible parent to Alternaria leaf spot) and‘Qinguan’(a resistant parent) is used in this study. Under the natural conditions, the resistance level in the parents and their F1 individuals were identified to analyze the inheritance of resistance to Alternaria leaf spot in apples. Based on the field data, RAPD molecular markers linked to Alternaria leaf spot resistant gene in apples were detected.
These are the main results.
1. Resistance to Alternaria leaf spot in‘Qinguan’was much higher than that in‘Fuji’from the field identification data.
2. In the cross of‘Qinguan×Fuji’, 544 plants proved to be resistant and 495 plants proved to be susceptible; and in the“Fuji×Qinguan”cross, the number of the resistant and susceptible plants was 235 and 254. The ratio of resistant vines to susceptible vines in both crosses have been tested in accordance withχ20.01 distribution of 1:1 segregation (χ2 Qinguan×Fuji = 2.645;χ2 Fuji×Qinguan = 0.743;χ20.01=3.84). The SI values of F1 progenies in both crosses revealed genetic features of quantitative traits demonstrating successive distribution. This indicates that Alternaria leaf spot resistance in these two crosses may be controlled by a major gene in combination with some minor genes.
3. Distribution features of the SI values of the progenies in two crosses also revealed that‘Qinguan’may have stronger inheritance than‘Fuji’for Alternaria leaf spot resistance.
4. In this study, 325 RAPD primers were used to screen the polymorphism between‘Qinguan’and‘Fuji’92 primers can produce polymorphic PCR bands between two parents. Among these 92 primers, 18 primers can produce polymorphic PCR bands between the resistant gene pool and the susceptible gene pool (each gene pool contained 10 F1 individuals). A fragment, S428-854, presented in the‘Qinguan’and the resistance DNA pool, absented in the‘Fuji’and susceptible DNA pool. The distribution of S428 in the F1 individuals showed that the molecular data of the 56 individuals (78.9%) were consistent with the field identification data. 5. The S428-854 RAPD marker was sequenced. Its actual length is 854bp. Results of blasting this sequence with the NCBI database indicated that their homology is no more than 30%. Structure analysis showed that this sequence has no opening read frame, meaning that it does not have a complete structure. Therefore, it could be a new sequence found in apples. Its accession in GeneBank is EU710766.
引文
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