摘要
由于耕地面积和水资源的限制,今后水稻产量的增加主要依赖于单产的提高。水稻杂种优势利用对提高水稻产量发挥了巨大的作用,继三系法杂交水稻技术在我国取得巨大成功后,科研人员又经过多年研究,使两系法杂交水稻技术也趋向于成熟,并且已经大面积应用于生产。
两系法杂交水稻是利用光温敏雄性不育系为技术基础的。根据光、温敏不育系对影响育性转换的光温条件的反应,可将它们分为光敏、温敏和光温互作不育系三大类。根据光照反应条件又可将光敏不育系分为长光敏不育系和短光敏不育系。长光敏感型不育系的基础与应用研究较多,而短光敏感型不育系的遗传生理基础研究相对较少,对此类不育系进行基础研究对丰富两系法杂交水稻的理论基础和资源类型、探讨新的利用途径是必要的。本研究以短光敏感雄性不育水稻D52S为研究对象,该材料具有在长光高温条件下高度可育、短光低温条件下完全败育的特性,因此可以在特定的光温条件下达到一系两用的目的。
本研究利用短光敏雄性不育水稻D52S与常规水稻桂99、蜀恢527杂交的F2代群体为材料,以花粉可育度为指标,以自然结实率为参考对该基因的遗传模式进行了分析；利用本实验室开发的SSR分子标记,采用分离群体混合分析法(BSA法)找到与该不育基因最邻近的SSR分子标记,运用极大似然法计算标记位点与目标基因间的重组值,绘制出了D52S短光敏雄性不育基因在染色体上的遗传连锁图,从而为精细定位和克隆该基因,为研究水稻短光敏雄性不育相关基因以及进行分子标记辅助选择选育新型光敏不育系打下基础。主要研究结果如下：
1.F2群体花粉育性鉴定。从2008年9月10日起,在大田光温条件使D52S短光敏不育性开始表达的情况下,对D52S/桂99和D52S/蜀恢527杂交组合的两个F2群体进行花粉育性鉴定,制作了两个F2群体的花粉育性分布图。分析表明：D52S/桂99和D52S/蜀恢527杂交组合的两个F2群体花粉可育度数值集中在“0～5%”和“80%～100%”两个区间,大体上呈现双峰分布。
2.两个F2群体自然结实率的考察。2008年10月底,考察成熟期两个F2群体的自然结实率,作为参考指标,制作了两个F2群体单株自然结实率分布图。分析表明：在田间自然生长条件下,两F2群体的自然结实率数值主要集中在“0～10%”和“60%～100%”两个区间,呈双峰分布。
3.D52S短光敏不育基因的遗传分析。根据花粉育性鉴定结果,确定可育度5%作为两个F2群体单株可育不育的分界线。分析表明：D52S/桂99的F2群体中,可育株有164株,不育株有50株,χ2测验表明该群体的可育株与不育株的分离比例符合3：1。同样,D52S/蜀恢527的F2群体中,可育株有252株,不育株有76株,χ2测验表明该群体的可育株与不育株的分离比例符合3：1。因此可以断定,控制D52S短光敏雄性不育性状的是1对主效不育基因。
4.利用SSR标记对D52S短光敏不育基因进行定位。在所有的357个SSR标记中,D52S和蜀恢527之间表现多态性的是124个。构建极端可育不育DNA池分析,得到稳定多态性标记6个。利用这些SSR标记对F2群体全体单株进行分析,所得各单株带型数据用于构建遗传连锁图。运用极大似然法计算标记位点与目标基因间的重组值,利用Mapdraw软件绘制出该基因在水稻染色体上的局部遗传连锁图。将D52S的短光敏雄性不育基因初步定位于第10染色体的SSR标记RM5271和RM244之间,距离两分子标记的遗传图距分别是：5.3cM、4.6cM,并且暂时命名为rpms3(t)。
As the restrict factors are exist in farmland and water resources,the yield increase in rice will mainly based on the increase of individuals yield in the future. The use of heterosis to raising the production of rice has played an important role. Following the great success of three-line hybrid rice technology,after years of study by researchers,the two-line hybrid rice technology has also tend to mature,and begin to used in large-scale rice production.
The two-line hybrid rice is based on the technology of photo(thermo)sensitive male sterile rice. According to the fertility conversion reaction of light and temperature conditions of light and temperature-sensitive male sterile lines,it can be divided into three kinds:photo sensitive male sterile line、thermo sensitive male sterile line and photo-thermo interactive sensitive male sterile line. According to the fertility conversion reaction of light conditions,it can be divided into long day-light photo sensitive male sterile line and short day-light photo sensitive male sterile line.Now there are more basic and applied research in long day-light photo sensitive male sterile line,but very little physiological and genetic researchment are developed in short day-light photo sensitive male sterile line. We can see that now it is necessary to research and study the short day-light line,from this we can rich the basic theory of two-line hybrid rice and develop new kind of resource type. This study is to research the short photoperiod sensitive male sterile rice D52S,this kind of rice is absolutely fertile when it is long day light and sterile when short day light. So we can used it into two purposes when at suitable time.
In this study,we use the materials of F2 generation which are made from D52S/Gui99 and D52S/Shuhui527,use pollen sterility as an quota,use the seed-setting rate as reference to analysis the genetic model of this gene. We use the SSR molecular markers and BSA method to find the nearest SSR markers of the sterile gene and to calculated the reorganization data between markers and the gene through maximum likelihood method. At last we should draw the genetic linkage map of D52S'sterile gene on the chromosome. Through this we can fine mapping and cloning the gene in the future,we can also use this result to study the relative gene of rice short photoperiod sensitive male sterile gene and use MAS to choice the new photoperiod sensitive male sterile materials.Major results are as follows:
1.Identification of pollen sterility of F2 generation. From Sep 10th of 2008,at the condition that the D52S has began to turn to sterile,we began to identify the pollen sterility of F2 generation,and made two distribution chart of pollen sterility. It shows that the two F2 generation's pollen sterility data are mainly focus on two intervals:"0～5%" & " 80%～100%",generally presented as a bimodal distribution.
2.Investigate the seed-setting rate of two F2 generation. By the end of October 2008, we began to investigate the seed-setting rate of two F2 generation as a reference indicator,and made two distribution chart of IV the seed-setting rate of two F2 generation. It shows that under natural growth conditions in the field,the data mainly focus on wo intervals:"0～10%"&"60%～100%",presented as a bimodal distribution.
3.The genetic analysis of D52S'S sterile gene. According to the results of pollen fertility identification,we choice fertility data 5% as the line of fertile and sterile. It shows that:in F2 of D52S/Gui99,there have 164 fertile individuals and 50 sterile individuals; in F2 of D52S/Shuhui527,there have 252 fertile individuals and 76 sterile individuals. Chi-square test shows that the separation ratio of F and S are equal to 3:1.So we can make sure that the sterile gene are controlled by a main-effect sterile gene.
4.Use SSR markers to map the sterile gene of D52S.In all totally 357 SSR markers, there have 124 markers showed polymorphism between D52S and Shuhui527. After construct DNA pools and used it to analysis the parents'polymorphism markers,we got 6 markes which are polymorphism between two pools. And then use this 6 markers to analysis all the 328 F2 individuals,the band data are used to construct the genetic map.We calculated the reorganization data between the markers and the gene through maximum likelihood method,and use Mapdraw software to draw the genetic map of the sterile gene on the chromosome. The results show that:the sterile gene is located on NO.10 chromosome,and the distence between the gene and SSR markers RM5271、RM244 are 5.3cM and 4.6cM respectively.
引文
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