【作者】 黄镇；

【导师】 傅廷栋； 涂金星；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 杂种优势利用的关键是利用简而易行的方法使不同亲本间的杂交获得具有一定生产潜力的F₁杂交种。这个问题包含两个方面的含义:一是行之有效的授粉控制系统,如植物雄性不育系的利用。目前,在我国油菜杂种优势利用的主要途径是细胞质雄性不育和细胞核雄性不育,细胞质雄性不育系由于有恢复源较窄,育性容易受温度影响等一系列的缺点,使其在生产上的应用受到一定的限制。相反细胞核雄性不育系具有恢复源广泛,育性稳定的优点,目前正受到育种家的关注。隐性细胞核雄性不育系7-7365A（Bnms3ms3ms4ms4RfRf）的育性由两对重叠基因与一对上位基因控制,已经在生产上实现了“三系”配套,但是由于遗传基础复杂,传统的改良方法需要进行大量的测交分析。二是亲本间遗传差异,很多的育种实践表明,亲本间适当的遗传距离有助于杂种F₁代生产潜力的发掘。孟金陵教授课题组利用分子标记辅助选择,创建了新型甘蓝型油菜7-749（A^rA^rC^cC^c）,该材料具有白菜的A^r基因组与埃塞俄比亚芥的C^c基因组,遗传背景与普通的甘蓝型油菜的A、C基因组具有一定的差异,它们产生的杂种具有较强的亚基因组间的杂种优势。为了更好利用这一优良隐性细胞核雄性不育材料与新型甘蓝型油菜,本研究对隐性细胞核雄性不育系7-7365A相关基因进行了分子标记定位,并通过标记辅助选择,将7-7365A的不育基因导入到新型甘蓝型油菜的遗传背景中,以改良7-7365A的配合力。其主要结果如下:1 BnMs3基因的精细定位利用AFLP技术结合集团分析法（BSA）在近等基因系群体7-7365AB中筛选1024对AFLP引物,获得了17个AFLP标记。整合Ke等（2005）发表的7个AFLP标记,有4个标记显示了差异,用2000个单株的大群体对BnMs3基因进行定位分析,21个标记分布于BnMs3基因的两侧,P05MC11₃₅₀与EA01MC12为基因两侧最近的标记,其遗传距离分别为0.5与0.1cM。将所有的标记进行测序,设计引物,有5个AFLP标记(P05MG05、P11MG02、P03MG04、P05MC11₂₅₀、EA09P06)被成功转化成SCAR标记。为了将BnMs3基因定位在公布的甘蓝型油菜遗传连锁图谱上,我们利用了两个DH群体Tapidor×Ningyou7和Quantum×No.2127-17的遗传连锁图谱,结果BnMs3基因被定位到了甘蓝型油菜的N19上,并在图谱上获得了一个共显性的SSR标记sR12384,该标记与BnMs3基因仅相距2.0cM。通过BLASTn分析,将该基因定位到拟南芥的第五条染色体上,对应区间为At5g13020-At5g17020。2 BnMs4基因的定位通过构建近等基因系7-736512AB,利用AFLP技术结合集团分析法（BSA）在近等基因系群体7-736512AB中筛选了1792对AFLP引物,获得了12个AFLP标记,经过1986个单株的大群体分析,所有标记分布于基因的两侧,标记AF6与AF8为两侧最近的标记,遗传距离分别为0.9与0.8 cM,将所有标记测序,设计引物,有3个标记P16MC08、P16MC12、AF6直接转化成了SCAR标记,其中AF6被转化成了一个共显性标记（S1）。经过PCR-walking扩增后,AF8也被成功转化成了一个SCAR标记。3分子标记辅助选择培育新型细胞核雄性不育系MAS与传统表型选择和品质分析相结合,应用杂交—自交—回交的方法,将7-7365A中的雄性不育基因导入到新型甘蓝型油菜7-749中。通过3次背景选择、一次前景选择,结合田间表型以及品质分析,选出了外源基因组含量（指A^r与C^c的含量之和）在44.2%-49.8%之间的不育株以及用于兄妹交的姊妹单株,通过连续2次的兄妹交,获得了稳定的不育系。3.1将育成的不育系与10个恢复系测交,按随机区组设计,对产量及其相关性状进行了配合力分析。改良后的不育系在主花序角果数、全株角果数、单株产量和小区产量四个性状上的一般配合力均表现正效应值,与未改良的不育系相比其差异达到极显著水平。对于每果粒数,改良后的不育系的一般配合力的效应值为负值,其差异达到了显著水平。对于千粒重,改良后的不育系的一般配合力的效应值为正值,但差异未达到显著水平。在配制的10个组合中,在小区产量性状上有7个组合表现正向超标优势,其中与7-1260、7-107、7-138所配组合的超标优势超过10%。3.2亲本分子遗传距离与杂种表现及配合力的分析结果表明:SSR遗传距离在产量及产量相关性状上与F₁的表型值、一般配合力、特殊配合力均没有显著相关性。4分子标记辅助选择新型细胞核雄性不育系的临保系用15对AFLP引物对临保系选育过程中两个世代的每个分离群体的可育单株进行了两次背景选择。结果表明,它们的外源基因组含量分别为14.8%-22.3%与27.2%-31.4%,结合田间表型在两个世代的群体中分别选择了外源基因组含量较高的2个单株进行下一个世代的选择过程。更多还原

【Abstract】 The key problem in utilizing heterosis is that of producing potential hybrid seeds economically,which contains two aspects of meaning.The first one is the effective pollination system,such as male sterility.Currently,the main approaches for rapeseed hybrid production are cytoplasmic male sterility（CMS） and genic male sterility（GMS） in China. There are some disadvantages in CMS system,such as limited availability of restorer lines, sensitivity to temperature.On the other hand,GMS has more advantages,such as rich resources of restorers,stable and complete male sterility.Now GMS is widely accepted and utilized.Although the recessive genic male sterility（RGMS） 7-7365A （Bnms3ms3ms4ms4RfRf） has been used in rapeseed production,yet for its very complex genetic basis,the traditional methods of three-line improvement are involved in a great deal of test cross analyses.The second aspect is genetic difference of parents.Many breeding practices showed that appropriate genetic difference of parents was helpful to F₁ hybrid production.Professor Meng’ lab created the new-typed Brassica napus 7-749（A^rA^rC^cC^c）, which had A^r genome from B.rapa and C^c genome from Brassica carinata.There existed some differences between 7-749 and common Brassica napus,therefore the hybrids of them showed strong intersubgenomic heterosis.In order to take advantage of this RGMS line and the new-typed B.napus,we mapped the recessive genic male sterile genes in 7-7365A,and transferred the genetic background of the new-typed B.napus to 7-7365A,so as to improve the 7-7365A.The main results were as follows:1 Fine mapping of BnMs3 gene1.1 From a survey of 1,024 primer combinations,we identified 17 AFLP markers linked to BnMs3 gene in an NIL（near-isogenic lines） population through AFLP analysis combined with BSA（bulked segregation analysis）.By integrating the previous markers reported by Ke （2005） linked to the BnMs3 gene,four of which showed polymorphlism.The NIL population consisting of 2000 individuals was used to analyse the linkage of all markers,as a result,they were located on either side of the BnMs3 gene.EA01MC12 and P05MC11₃₅₀,the nearest markers,were at a distance of 0.1 and 0.5 cM,respectively.All markers were sequenced and primers were designed,five of which(P05MG05,P11MG02,P03MG04,P05MC11₂₅₀, EA09P06) were converted into SCARs successfully.1.2 In order to determine the location of the BnMs3 gene in the published B.napus genetic maps,we used two DH populations derived from Tapidor×Ningyou7 and Quantum×No.2127-17.Finally the gene was mapped to the N19 of B.napus genetic maps,and a co-dominant marker sR12384 was identified at a distance of 2.0 cM from the BnMs3 gene.1.3 The sequences of all the markers were delivered to NCBI web site for BLASTn analysis and the genes corresponding to the marker sequences were all located on the chromosome 5 of Arabidopsis.The homologous region of BnMs3 in Arabidopsis was between At5g13020 and At5g17020.2 Mapping of BnMs4 geneWe constructed an NIL population 7-736512AB.From a survey of 1,792 primer combinations,we identified 12 AFLP markers linked to BnMs3 gene in the NIL population 7-736512AB through AFLP analysis combined with BSA.All of markers were used to screen the 7-736512AB population including 1986 individuals,as a result,they were located on either side of the BnMs4 gene,AF6 and AF8,the nearest markers,were at a distance of 0.9 and 0.8 cM,respectively.All of markers were sequenced and three markers（P16MC08, P16MC12,AF6） were converted into SCARs successfully,among which AF6 was a co-dominant marker.Through PCR-walking,AF8 was also converted into a SCAR marker.3 Improvement of new recessive genie male sterile lines in B.napus by molecular marker-assisted selection（MAS）By MAS along with phenotype selection and quality analysis,we successfully transferred the genic male sterility genes of 7-7365A into the new-typed B.napus 7-749 using cross-self-backcross methods,Through three times of background selection and one foreground selection,we selected the sterile individuals and their sibmates,of which the exogenous genome content（A^rand C^c） ranged from 44.2%to 49.8%.We obtained a stable sterile line through continuous sibmating for two times.3.1 The combining ability analyses of yeild and yeild-related characters were done in the crosses from the new sterile line and 10 restorers within a random block design.The results demonstrated that the improved sterile line showed positive effects of value in the general combining ability（GCA） of silique numbers on main inflorescence,siliques per plant,seed yield per plant and yield per plot,compared with the original sterile line;the difference was very significant.For seed number per silique,the improved sterile line showed negative GCA value,and the difference reached a significant level.For 1000-seed weight,the improved sterile line showed positive GCA value,but the difference was not significant.In the 10 crosses,seven crosses showed positive heterosis compared with the control in the yield per plot character,and the heterosis advantage of three crosses from the improved sterile line with 7-1260,7-107 and 7-138 was up to more than 10%.3.2 Correlation analysis between GD and heterosis showed no significant correlation between the GD based on SSR markers,F₁ traits performance,SCA and GCA.4 Improvement of new recessive genie male temporary maintainer in B.napus by molecular-marker assisted selectionFifteen AFLP primer combinations were used to perform background selection on the fertile plants of the two segregating populations in two generations for two times, respectively.The results showed that the exogenous genome content of them ranged from 14.8%to 22.3%and 27.2%to 31.4%in two populations,respectively.Combined with agronomic characters and quality analysis,we selected two individuals which had high exogenous genome content in the two populations respectively for the next selection procedure.更多还原