结球甘蓝杂种后代的分子标记辅助选择
摘要
结球甘蓝(Brassica oleracea var.capitata),原产于地中海沿岸,16世纪传入我国,因其适应性广,抗逆性强,在世界各地均有栽培,也是我国普遍种植的一种重要蔬菜。根据我国现阶段蔬菜育种目标,培育抗病、优质、高产的新品种是首要的使命。因此,培育聚合多个优良基因的甘蓝新品种,集优质、抗病为一体是甘蓝育种的当务之急。传统的表型鉴定方法难以准确快速的鉴定出2个以上优良基因的聚合个体,应用分子标记技术可以解决这一问题,快速准确地鉴定出含有多个优良基因的聚合体。本研究采用分子标记辅助选择手段,在甘蓝的传统的杂交和回交后代选择聚合有抗病基因和耐抽薹基因的单株,以期为甘蓝分子聚合育种奠定理论基础和获得优异的甘蓝种质资源。
本试验主要包括两个部分：(1)利用结球甘蓝自交系A21(高抗黑腐病和TuMV)和P02(冬性强的迟抽薹材料)组配试验群体和性状选择、评价群体；在BC1F1代(F1与P02回交得到),结合田间抽薹性状调查,检测与结球甘蓝迟抽薹基因连锁的RAPD标记与目的基因的连锁关系,将RAPD标记转化成SCAR标记,建立SCAR标记体系。(2)通过迟抽薹基因、抗黑腐病基因和抗TuMV基因的分子标记分析,结合分子标记辅助选择,在BC1F2和F3代中筛含有多个优良基因的聚合体。
通过研究得出以下结论：
(1)以耐抽薹材料P02和易抽薹材料春化处理后的时间为标准,确定BC1F1群体在春化处理后,迟抽薹株数和早抽薹株数的田间鉴定结果与RAPD标记N1的鉴定结果相一致,证实了N1标记与迟抽薹基因连锁的准确性。
(2)对RAPD标记特异片段进行回收、克隆和测序,依据测序结果设计SCAR引物,对SCAR-PCR反应条件进行优化,建立了稳定的SCAR标记体系。用SCAR引物对BC1F1代迟抽薹单株和早抽薹单株进行分析,结果与RAPD标记的验证结果完全符合,且稳定性大大提高,表明RAPD标记已经被成功转化成为SCAR标记,可以应用于迟抽薹性状的分子标记辅助选择。
(3)在聚合杂交的基础上,采用早代进行抗病性鉴定的方法和分子标记辅助选择的策略,进行迟抽薹基因、抗黑腐病基因和抗TuMV基因的聚合体选择。利用已验证的SCAR和SSR标记,对回交组合BC1F2和杂交组合F3进行分子标记辅助选择,选出38个具有迟抽薹基因、抗黑腐病基因和抗TuMV基因的单株,132个具有迟抽薹基因和抗黑腐病基因的单株,60个含有迟抽薹基因和抗TuMV基因的单株。这些聚合体后代的获得,为进一步选育优质、抗病的甘蓝新品种提供了优良的基础材料。
(4)借助甘蓝迟抽薹基因和抗病基因的标记分析,初步建立了甘蓝迟抽薹、抗黑腐病、抗TuMV的分子辅助选择体系,在育种过程中可以早期进行这些性状的分子标记辅助选择,能够在很大程度上提高杂种后代的准确性和选择效率,从而缩短育种年限,加快甘蓝品种的繁育速度。
Cabbage (Brassica oleracea var.capitata), which native to the Mediterranean region with the characters of great resistance and adaptability, has been cultivated all around the world and in China as an importan vegetable crop after being introduced in the 16th century. Accroding to current vegetable breeding goals in China, it is a mission to cultivate disease resistance, high quality and high productivity new lines to vegetable breeders. Therefore, nurturing a number of pyramiding varieties of cabbage which combining high quality and disease resistance is the most pressing matter of the moment. Identification of the phenotype of traditional methods is difficult to accuretely select two or more excellent genes in one individual. Application of molecular markers can solve this problem, then rapid and accurate selection of individuals containing several targent genes can be achieved. Marker assisted selection was used in this research for screening individuals possessing disease resistance genes and later bolting gene in traditional cross and backcross progenies, which was aimed to provide a basis for gene pyramiding breeding and to obtain excellent cabbage germplasm.
This experiment was mainly composed of two parts:
(1) A trail group and two selecting populations were constructed with materials A21 (with high resistance to TuMV and black rot) and P02 (with later bolting gene). BC1F1 population derived from F1 and P02 backcross was used to verify the linkage relation between RAPD marker N1 and later bolting gene with reference to bolting trait in the field. And then converted RAPD marker into SCAR marker.
(2) Markers linked to later bolting gene, black rot resistance gene and TuMV resistance gene were analyzed and used to selecte cultivars possessing multiple gene of interes, which provided a basis for marker assisted breeding in headed cabbage.
The main results are summarized as follows:
(1) The linkage relation between N1 marker and later bolting gene was verified accroding to the result that the identification of phenotype in the field is identicial to those of N1 marker.
(2) RAPD specific fragment was recovered, cloned and sequenced, and a pair of SCAR markers was designed based on the obtained sequence. An stable SCAR marker system was established after the optimization of SCAR-PCR reaction. SCAR amplification result were fully identicial with that by RAPD marker in 166 BC1F1 individuals, which demonstrated the accuracy of SCAR marker.
(3) Based on the hybridzation, it has become available to choose target genes plants using MAS combined with inoculation and odservation in early generation. Using the SCAR markers linked to later bolting gene and TuMV resistance gene previously recovered and the SSR marker linked to black rot resistance gene identified,38 pyramiding individuals containing later bolting gene, black rot resistance gene and TuMV resistance gene,132 pyramiding individuals containing later bolting gene and black rot resiatance gene, and 60 pyramiding individuals containing later bolting gene and TuMV resistance gene were selected from the progenies of BC1F2 and F3. And the pyramiding plants can be used as excellent germplasm in cabbage quality and disease resistance breeding.
(4) The system of later bolting, black rot resistance and TuMV resistance marker assisted breeding has been established preliminarily. The result showed that MAS pyramiding breeding can not only increase the accuricy and efficiency of progeny selection but also save time required during breeding cycles to a large extent.
本试验主要包括两个部分：(1)利用结球甘蓝自交系A21(高抗黑腐病和TuMV)和P02(冬性强的迟抽薹材料)组配试验群体和性状选择、评价群体；在BC1F1代(F1与P02回交得到),结合田间抽薹性状调查,检测与结球甘蓝迟抽薹基因连锁的RAPD标记与目的基因的连锁关系,将RAPD标记转化成SCAR标记,建立SCAR标记体系。(2)通过迟抽薹基因、抗黑腐病基因和抗TuMV基因的分子标记分析,结合分子标记辅助选择,在BC1F2和F3代中筛含有多个优良基因的聚合体。
通过研究得出以下结论：
(1)以耐抽薹材料P02和易抽薹材料春化处理后的时间为标准,确定BC1F1群体在春化处理后,迟抽薹株数和早抽薹株数的田间鉴定结果与RAPD标记N1的鉴定结果相一致,证实了N1标记与迟抽薹基因连锁的准确性。
(2)对RAPD标记特异片段进行回收、克隆和测序,依据测序结果设计SCAR引物,对SCAR-PCR反应条件进行优化,建立了稳定的SCAR标记体系。用SCAR引物对BC1F1代迟抽薹单株和早抽薹单株进行分析,结果与RAPD标记的验证结果完全符合,且稳定性大大提高,表明RAPD标记已经被成功转化成为SCAR标记,可以应用于迟抽薹性状的分子标记辅助选择。
(3)在聚合杂交的基础上,采用早代进行抗病性鉴定的方法和分子标记辅助选择的策略,进行迟抽薹基因、抗黑腐病基因和抗TuMV基因的聚合体选择。利用已验证的SCAR和SSR标记,对回交组合BC1F2和杂交组合F3进行分子标记辅助选择,选出38个具有迟抽薹基因、抗黑腐病基因和抗TuMV基因的单株,132个具有迟抽薹基因和抗黑腐病基因的单株,60个含有迟抽薹基因和抗TuMV基因的单株。这些聚合体后代的获得,为进一步选育优质、抗病的甘蓝新品种提供了优良的基础材料。
(4)借助甘蓝迟抽薹基因和抗病基因的标记分析,初步建立了甘蓝迟抽薹、抗黑腐病、抗TuMV的分子辅助选择体系,在育种过程中可以早期进行这些性状的分子标记辅助选择,能够在很大程度上提高杂种后代的准确性和选择效率,从而缩短育种年限,加快甘蓝品种的繁育速度。
Cabbage (Brassica oleracea var.capitata), which native to the Mediterranean region with the characters of great resistance and adaptability, has been cultivated all around the world and in China as an importan vegetable crop after being introduced in the 16th century. Accroding to current vegetable breeding goals in China, it is a mission to cultivate disease resistance, high quality and high productivity new lines to vegetable breeders. Therefore, nurturing a number of pyramiding varieties of cabbage which combining high quality and disease resistance is the most pressing matter of the moment. Identification of the phenotype of traditional methods is difficult to accuretely select two or more excellent genes in one individual. Application of molecular markers can solve this problem, then rapid and accurate selection of individuals containing several targent genes can be achieved. Marker assisted selection was used in this research for screening individuals possessing disease resistance genes and later bolting gene in traditional cross and backcross progenies, which was aimed to provide a basis for gene pyramiding breeding and to obtain excellent cabbage germplasm.
This experiment was mainly composed of two parts:
(1) A trail group and two selecting populations were constructed with materials A21 (with high resistance to TuMV and black rot) and P02 (with later bolting gene). BC1F1 population derived from F1 and P02 backcross was used to verify the linkage relation between RAPD marker N1 and later bolting gene with reference to bolting trait in the field. And then converted RAPD marker into SCAR marker.
(2) Markers linked to later bolting gene, black rot resistance gene and TuMV resistance gene were analyzed and used to selecte cultivars possessing multiple gene of interes, which provided a basis for marker assisted breeding in headed cabbage.
The main results are summarized as follows:
(1) The linkage relation between N1 marker and later bolting gene was verified accroding to the result that the identification of phenotype in the field is identicial to those of N1 marker.
(2) RAPD specific fragment was recovered, cloned and sequenced, and a pair of SCAR markers was designed based on the obtained sequence. An stable SCAR marker system was established after the optimization of SCAR-PCR reaction. SCAR amplification result were fully identicial with that by RAPD marker in 166 BC1F1 individuals, which demonstrated the accuracy of SCAR marker.
(3) Based on the hybridzation, it has become available to choose target genes plants using MAS combined with inoculation and odservation in early generation. Using the SCAR markers linked to later bolting gene and TuMV resistance gene previously recovered and the SSR marker linked to black rot resistance gene identified,38 pyramiding individuals containing later bolting gene, black rot resistance gene and TuMV resistance gene,132 pyramiding individuals containing later bolting gene and black rot resiatance gene, and 60 pyramiding individuals containing later bolting gene and TuMV resistance gene were selected from the progenies of BC1F2 and F3. And the pyramiding plants can be used as excellent germplasm in cabbage quality and disease resistance breeding.
(4) The system of later bolting, black rot resistance and TuMV resistance marker assisted breeding has been established preliminarily. The result showed that MAS pyramiding breeding can not only increase the accuricy and efficiency of progeny selection but also save time required during breeding cycles to a large extent.
引文
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