大豆细胞质雄性不育恢复基因的SSR标记
摘要
大豆是世界上重要的油料作物之一，同时也是重要的工业生产原料。针对我国对大豆需求的逐年增加，及大豆遗传改良进度缓慢的事实，在耕种面积不断减少的情况下，只能从提高大豆的单位面积产量上来实现大豆的增产。而利用杂种优势是挖掘大豆潜力、显著提高大豆产量的重要途径。
大豆细胞质雄性不育细胞质的发现和利用，标志着我国在大豆杂种优势研究上取得了突破性进展。“三系”是大豆杂交种选育的基础材料。其中，强恢复系的选育非常繁琐，且恢复力只能通过与不育系的测交来鉴定，既耗时又费力。利用DNA分子标记技术筛选与育性恢复基因连锁的标记，可对恢复基因进行精确定位。利用分子标记辅助育种可以缩短恢复系育种年限，同时也为育性恢复分子机理的研究和最终克隆育性恢复基因打下基础。因此，定位细胞质雄性不育恢复基因具有重要意义。
本研究以单基因配子体遗传的RN型细胞质雄性不育系JLCMS9A导入恢复系JLR2的恢复基因构建的回交导入系的BC9F
2代分离群体为材料，开花期对F2单株的花粉育性进行观察，确定F2单株的育性。根据F2单株花粉育性观察结果，确定F2群体中可育单株和半不育单株比例为1:1，这一结果表明RN型大豆细胞质雄性不育符合单基因配子体不育的遗传模式。提取F2分离群体的单株及父、母本单株的基因组DNA，根据F2单株育性鉴定结果，随机取20株完全可育单株DNA等量混合构建典型可育基因池，取20株典型半不育单株DNA等量混合构建典型半不育基因池，取20株不育系母本单株DNA构建完全不育基因池；选用200对均匀分布在大豆20个连锁群上的经典SSR引物，筛选三个典型基因池，获得了具有多态性的引物Sctt011，并将恢复基因定位在J连锁群上。进一步在J连锁群上选择和设计了93对SSR引物进一步筛选基因池。最终获得3个与恢复基因紧密连锁的分子标记，分别为JS22、JS30和JS51，与恢复基因的遗传距离分别为7.6cM、10.7cM和5.2cM。
通过本研究所获得的连锁标记，有望加速RN型恢复系的选育速度，间接推进杂交大豆新品种育种进程，这对于增强我国大豆育种的自主创新能力，促进大豆产业可持续发展，具有重要的理论和实践意义。同时，也为今后克隆恢复基因和解析RN型细胞质雄性不育育性恢复机制奠定基础。
Soybean is an important grain and oil crop, also it is an important industrial rawmaterials. Domestic demand for soybeans increased year by year, but the production ofsoybean was grown slowly. To increasing the production of soybean, that the only way is toimprove the unit output. And useing heterosis is one important way to diging latent capacityand improving the production of soybean.
The discovery of cytoplasmic male sterile cytoplasm in soybean and the utilization ofsoybean hybrids are the breakthrough developments. Cytoplasmic male sterility"three–lines” are the basic materials for soybean hybrid breeding. Breeding for strong restorer linesis very complicated and restoring ability can only be identified by testcross with malesterile lines, which is time-consuming and labor-intensive.Through screening makers linkedwith restore gene closely by DNA molecular maker technology, we can accurately locaterestore genes. Molecular assisted breeding can shorten the breeding time, and it also lay thefoundation for studing molecular mechanism of fertility restoration and eventually cloningof restorer gene. Therefore, carried out the research of cytoplasmic male sterility restoregenes location has important significance.
F2segregating population, which was obtained by crossing between the gametophytegenetically RN-cytoplasmic male sterile line and backcrossing lines with strong restorergene.Then it will be used for the Rf gene mapping through determination of the inheritancepattern by sterility identification of each generation and further polymorphism analysis byhigh density of SSR markers and fine mapping of the restorer gene.
Firstly，the RN type soybean cytoplasm sterile lines9A cross with strong restorer linesJLR2to get F2segregating population. According to the results of microscope detection, theF1is semi-sterility, ratio of fertile to semi–sterility in F2separation population is1:1.Theresults of this study show that RN type soybean cytoplasmic male sterile lines is controlledby single gene gametophyte sterility.
Gene pools from F2segregating population was screened with200SSR primers, andSctt011showed polymorphism among the three gene pools, which was located in J linkagegroups. Then,93additional SSR primers from J linkage groups were chosen to screen genepolls. Finally, three primers are linked with restorer gene which are JS22，JS30and JS51, and the genetic distances are7.6cM,10.7cM and5.2cM,respectively.The restorer genewas located in J linkage groups.
The linked markers with restorer gene identified in this study will accelerate thebreeding process of RN-restorer lines, and lay the foundation for Rf gene cloning andRN-CMS fertility restoration mechanism exploration in future.
大豆细胞质雄性不育细胞质的发现和利用，标志着我国在大豆杂种优势研究上取得了突破性进展。“三系”是大豆杂交种选育的基础材料。其中，强恢复系的选育非常繁琐，且恢复力只能通过与不育系的测交来鉴定，既耗时又费力。利用DNA分子标记技术筛选与育性恢复基因连锁的标记，可对恢复基因进行精确定位。利用分子标记辅助育种可以缩短恢复系育种年限，同时也为育性恢复分子机理的研究和最终克隆育性恢复基因打下基础。因此，定位细胞质雄性不育恢复基因具有重要意义。
本研究以单基因配子体遗传的RN型细胞质雄性不育系JLCMS9A导入恢复系JLR2的恢复基因构建的回交导入系的BC9F
2代分离群体为材料，开花期对F2单株的花粉育性进行观察，确定F2单株的育性。根据F2单株花粉育性观察结果，确定F2群体中可育单株和半不育单株比例为1:1，这一结果表明RN型大豆细胞质雄性不育符合单基因配子体不育的遗传模式。提取F2分离群体的单株及父、母本单株的基因组DNA，根据F2单株育性鉴定结果，随机取20株完全可育单株DNA等量混合构建典型可育基因池，取20株典型半不育单株DNA等量混合构建典型半不育基因池，取20株不育系母本单株DNA构建完全不育基因池；选用200对均匀分布在大豆20个连锁群上的经典SSR引物，筛选三个典型基因池，获得了具有多态性的引物Sctt011，并将恢复基因定位在J连锁群上。进一步在J连锁群上选择和设计了93对SSR引物进一步筛选基因池。最终获得3个与恢复基因紧密连锁的分子标记，分别为JS22、JS30和JS51，与恢复基因的遗传距离分别为7.6cM、10.7cM和5.2cM。
通过本研究所获得的连锁标记，有望加速RN型恢复系的选育速度，间接推进杂交大豆新品种育种进程，这对于增强我国大豆育种的自主创新能力，促进大豆产业可持续发展，具有重要的理论和实践意义。同时，也为今后克隆恢复基因和解析RN型细胞质雄性不育育性恢复机制奠定基础。
Soybean is an important grain and oil crop, also it is an important industrial rawmaterials. Domestic demand for soybeans increased year by year, but the production ofsoybean was grown slowly. To increasing the production of soybean, that the only way is toimprove the unit output. And useing heterosis is one important way to diging latent capacityand improving the production of soybean.
The discovery of cytoplasmic male sterile cytoplasm in soybean and the utilization ofsoybean hybrids are the breakthrough developments. Cytoplasmic male sterility"three–lines” are the basic materials for soybean hybrid breeding. Breeding for strong restorer linesis very complicated and restoring ability can only be identified by testcross with malesterile lines, which is time-consuming and labor-intensive.Through screening makers linkedwith restore gene closely by DNA molecular maker technology, we can accurately locaterestore genes. Molecular assisted breeding can shorten the breeding time, and it also lay thefoundation for studing molecular mechanism of fertility restoration and eventually cloningof restorer gene. Therefore, carried out the research of cytoplasmic male sterility restoregenes location has important significance.
F2segregating population, which was obtained by crossing between the gametophytegenetically RN-cytoplasmic male sterile line and backcrossing lines with strong restorergene.Then it will be used for the Rf gene mapping through determination of the inheritancepattern by sterility identification of each generation and further polymorphism analysis byhigh density of SSR markers and fine mapping of the restorer gene.
Firstly，the RN type soybean cytoplasm sterile lines9A cross with strong restorer linesJLR2to get F2segregating population. According to the results of microscope detection, theF1is semi-sterility, ratio of fertile to semi–sterility in F2separation population is1:1.Theresults of this study show that RN type soybean cytoplasmic male sterile lines is controlledby single gene gametophyte sterility.
Gene pools from F2segregating population was screened with200SSR primers, andSctt011showed polymorphism among the three gene pools, which was located in J linkagegroups. Then,93additional SSR primers from J linkage groups were chosen to screen genepolls. Finally, three primers are linked with restorer gene which are JS22，JS30and JS51, and the genetic distances are7.6cM,10.7cM and5.2cM,respectively.The restorer genewas located in J linkage groups.
The linked markers with restorer gene identified in this study will accelerate thebreeding process of RN-restorer lines, and lay the foundation for Rf gene cloning andRN-CMS fertility restoration mechanism exploration in future.
引文
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