水稻育性恢复基因的分子标记辅助选择
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摘要
细胞质雄性不育(CMS)及其育性恢复是三系杂交水稻育种和生产应用的首要基础,而恢复基因是杂种优势利用的基础,在生产上应用的种类繁多的CMS类型中,野败型应用最早并且最广泛。
本研究应用珍汕97A/明恢63的F2群体,从中筛选结实率低于5%的个体组成极端不育群体作为定位群体,针对水稻第1染色体短臂Rf3所在染色体的可能区间,应用37个SSR标记检测亲本,从16个多态性标记中挑选出9个检测定位群体。结果表明物理位置连续排列的SSR标记RM10353、RM1195和RM3746各有8个单株与Rf3基因发生了单交换,且重组子数表现最少,据此可将Rf3定位于这3个标记的两侧标记内。因此最终将Rf3定位在相距679.9kb的SSR标记RM10338和RM10376之间。
在育性恢复基因定位研究的基础上,选取与育性恢复基因Rf3连锁的6个分子标记,与Rf1/Rf4连锁的5个分子标记,对103份我国主栽水稻品种及亲本进行多态性检测。结果表明:(1)11个连锁分子标记在103份供试水稻材料中共检测到41个等位基因,每对扩增得到2-7个等位基因,平均每个座位有3.7个等位基因。标记的多态性频率FP值变动范围为0.16-0.68,平均值为0.48;(2)所有粳稻材料在Rf3连锁标记位点表现单态,不育系和恢复系之间没有差异,均与籼型不育系一致,表明粳稻材料中均不含有Rf3;而在Rf1/Rf4连锁标记位点粳稻不育系和恢复系表现多态,分别与相应的籼稻材料带型一致,表明粳稻材料中携带Rf1/Rf4;(3)分别对所有103份供试材料和70份三系杂交稻亲本的聚类分析表明,所选育性恢复基因的连锁标记能有效的区分不育系和恢复系,两种不同类型材料之间相似性系数较小。
针对前期根据Pi25-Pi26(t)连锁标记筛选的106个重组自交系,进行稻瘟病抗性鉴定和育性恢复基因的分子标记辅助选择。在两个不同环境的稻瘟病重复接种结果中,所有单株均表现高抗,两个标记对稻瘟病抗性基因的选择效率为100%。选择与Rf3连锁的分子标记RM10338、RM10353、RM1344和RM10435,与Rf4连锁的分子标记RM6100和RM1108检测106个单株。根据4个分子标记检测的结果,挑选Rf3和Rf4位点在连锁标记间基因型一致的材料共78个,分别同时与中9A,协青早A配组,获得两套F1,考察F2结实率。结果表明:分子标记检测两个育性恢复基因都存在的情况下,后代结实率最高,大于60%,而当两个基因都不存在时,后代结实率只有20%,基因型与表型相关性较高。用STS标记SA7和SSR标记RM3330对Pi25(t)进行选择,RM10338、RM10353、RM1344和RM10435对Rf3进行选择,RM6100和RM1108对Rf4进行选择结果是可靠的。
Cytoplasmic male sterility (CMS) and its restoration are bases for the breeding and commercial application of three-line hybrid rice. Wild abortive CMS (WA-CMS) was firstly used in the commercial production of hybrid rice and has always been most extensively applied.
In present study, an F2 population derived from the cross Zhenshan97A/Minghui63 was used. The mapping was performed using the recessive class consisting of 119 extreme sterile individuals. Parental polymorphism survey was conducted using 37 SSRs located in the probable region for Rf3 on the short arm of rice chromosome 1. Of the 16 polymorphic SSRs detected, 9 were selected to test the extreme sterile individuals. RM10353, RM1195 and RM3746 which were located consecutively in the segmental map each showed 8 single crossover with Rf3, and this number was the lowest among the number of recombinant observed between the 9 SSRs and Rf3. This result indicates that Rf3 was located in the genomic region covered by interval RM10353-RM1195-RM3746 and its flanking markers. Thus, the Rf3 locus was mapped between RM10338 and RM10376 that have a physical distance of 679.9kb.
Based on the mapping of fertility-restoring genes, 11 markers linked to Rf3 or Rf1/Rf4 were selected to test 103 major rice varieties and parents in China. The main results listed as follows: 41 alleles of 11 linkage markers were observed in the 103 varieties and parents; there were 2-7 alleles of each marker the average was 3.7. The FP of the markers ranged from 0.16 to 0.68, and the average is 0.48. All the japonica materials performed the same genotype at the locus of Rf3, and there were no variation between the sterile restorer lines and the CMS lines. However, they performed polymorphism at the locus of Rf1/Rf4. Therefore, Rf3 was contained in the indica materials and Rf4 was contained in the two types. The clustering analysis of the 103 varieties and parents based on the 41 alleles indicated that the primers that linked to Rf genes could distinguish CMS lines from its restorers and the similarity of the two types was low.
The MAS of the identification to blast resistence gene and Rf genes were based on the 106 RILs selected by the markers linked to Pi25-Pi26 (t). The 106 plants were inoculated with mixed M. griseai in two different environments. The result showed that all the plants were resistance. The efficiency of MAS was 100%. The markers linked to Rf3 or Rf4 were selected to test the 106 plants. According to the result of the test, 78 plants which were chosen as the female parents were crossed to Zhong9A and XieqingzaoA, respectively. Two F1 groups were obtained, and then the SF of F2 was inspected. The result shows: the SF were higher when the two Rf genes existing than the absence of them. Therefore, the MAS is efficient.
本研究应用珍汕97A/明恢63的F2群体,从中筛选结实率低于5%的个体组成极端不育群体作为定位群体,针对水稻第1染色体短臂Rf3所在染色体的可能区间,应用37个SSR标记检测亲本,从16个多态性标记中挑选出9个检测定位群体。结果表明物理位置连续排列的SSR标记RM10353、RM1195和RM3746各有8个单株与Rf3基因发生了单交换,且重组子数表现最少,据此可将Rf3定位于这3个标记的两侧标记内。因此最终将Rf3定位在相距679.9kb的SSR标记RM10338和RM10376之间。
在育性恢复基因定位研究的基础上,选取与育性恢复基因Rf3连锁的6个分子标记,与Rf1/Rf4连锁的5个分子标记,对103份我国主栽水稻品种及亲本进行多态性检测。结果表明:(1)11个连锁分子标记在103份供试水稻材料中共检测到41个等位基因,每对扩增得到2-7个等位基因,平均每个座位有3.7个等位基因。标记的多态性频率FP值变动范围为0.16-0.68,平均值为0.48;(2)所有粳稻材料在Rf3连锁标记位点表现单态,不育系和恢复系之间没有差异,均与籼型不育系一致,表明粳稻材料中均不含有Rf3;而在Rf1/Rf4连锁标记位点粳稻不育系和恢复系表现多态,分别与相应的籼稻材料带型一致,表明粳稻材料中携带Rf1/Rf4;(3)分别对所有103份供试材料和70份三系杂交稻亲本的聚类分析表明,所选育性恢复基因的连锁标记能有效的区分不育系和恢复系,两种不同类型材料之间相似性系数较小。
针对前期根据Pi25-Pi26(t)连锁标记筛选的106个重组自交系,进行稻瘟病抗性鉴定和育性恢复基因的分子标记辅助选择。在两个不同环境的稻瘟病重复接种结果中,所有单株均表现高抗,两个标记对稻瘟病抗性基因的选择效率为100%。选择与Rf3连锁的分子标记RM10338、RM10353、RM1344和RM10435,与Rf4连锁的分子标记RM6100和RM1108检测106个单株。根据4个分子标记检测的结果,挑选Rf3和Rf4位点在连锁标记间基因型一致的材料共78个,分别同时与中9A,协青早A配组,获得两套F1,考察F2结实率。结果表明:分子标记检测两个育性恢复基因都存在的情况下,后代结实率最高,大于60%,而当两个基因都不存在时,后代结实率只有20%,基因型与表型相关性较高。用STS标记SA7和SSR标记RM3330对Pi25(t)进行选择,RM10338、RM10353、RM1344和RM10435对Rf3进行选择,RM6100和RM1108对Rf4进行选择结果是可靠的。
Cytoplasmic male sterility (CMS) and its restoration are bases for the breeding and commercial application of three-line hybrid rice. Wild abortive CMS (WA-CMS) was firstly used in the commercial production of hybrid rice and has always been most extensively applied.
In present study, an F2 population derived from the cross Zhenshan97A/Minghui63 was used. The mapping was performed using the recessive class consisting of 119 extreme sterile individuals. Parental polymorphism survey was conducted using 37 SSRs located in the probable region for Rf3 on the short arm of rice chromosome 1. Of the 16 polymorphic SSRs detected, 9 were selected to test the extreme sterile individuals. RM10353, RM1195 and RM3746 which were located consecutively in the segmental map each showed 8 single crossover with Rf3, and this number was the lowest among the number of recombinant observed between the 9 SSRs and Rf3. This result indicates that Rf3 was located in the genomic region covered by interval RM10353-RM1195-RM3746 and its flanking markers. Thus, the Rf3 locus was mapped between RM10338 and RM10376 that have a physical distance of 679.9kb.
Based on the mapping of fertility-restoring genes, 11 markers linked to Rf3 or Rf1/Rf4 were selected to test 103 major rice varieties and parents in China. The main results listed as follows: 41 alleles of 11 linkage markers were observed in the 103 varieties and parents; there were 2-7 alleles of each marker the average was 3.7. The FP of the markers ranged from 0.16 to 0.68, and the average is 0.48. All the japonica materials performed the same genotype at the locus of Rf3, and there were no variation between the sterile restorer lines and the CMS lines. However, they performed polymorphism at the locus of Rf1/Rf4. Therefore, Rf3 was contained in the indica materials and Rf4 was contained in the two types. The clustering analysis of the 103 varieties and parents based on the 41 alleles indicated that the primers that linked to Rf genes could distinguish CMS lines from its restorers and the similarity of the two types was low.
The MAS of the identification to blast resistence gene and Rf genes were based on the 106 RILs selected by the markers linked to Pi25-Pi26 (t). The 106 plants were inoculated with mixed M. griseai in two different environments. The result showed that all the plants were resistance. The efficiency of MAS was 100%. The markers linked to Rf3 or Rf4 were selected to test the 106 plants. According to the result of the test, 78 plants which were chosen as the female parents were crossed to Zhong9A and XieqingzaoA, respectively. Two F1 groups were obtained, and then the SF of F2 was inspected. The result shows: the SF were higher when the two Rf genes existing than the absence of them. Therefore, the MAS is efficient.
引文
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