【作者】 何俊平；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 9012AB是甘蓝型油菜隐性细胞核不育材料中的一种,其具有败育彻底、不育性稳定、恢复源广泛及无不良胞质效应等特点。遗传分析研究表明,该不育材料的育性受两对隐性重叠不育基因(ms3ms3、ms4ms4)和一对隐性上位抑制基因(rfrf)互作控制;两对不育基因隐性纯合时可导致雄性不育,而上位抑制基因隐性纯合时则可抑制不育基因的表达,使育性恢复正常(陈凤祥等,1998)。依照此遗传模式,9012AB不育系可以实现三系配套,从而解决核不育制种时需拔除50%可育株的问题。因此,近年来该系统引起了研究者的广泛关注;目前,利用该系统的育种工作已取得突破,对其不育控制机理的初步研究也已展开。以9012AB为主要研究材料,柯丽萍(2005)已将其中一个不育基因(ms3)初步定位在0.5cM的遗传区间。以此为基础,本研究进一步开展了隐性核不育基因ms3的精细定位研究,取得的主要结果和结论如下:1.以9012AB近等基因系为材料,应用重组单株BSA法,结合AFLP技术,继续筛选了2048对引物组合,获得了21个与目标基因更为紧密连锁的AFLP分子标记。其中有2个标记(AH15、AH17)在所用群体中与目标基因共分离,14个标记(AH1、AH2、AH3、AH4、AH6、AH8、AH9、AH10,AH11、AH13,AH18、AH19,AH20、AH21)位于目标基因的一侧,5个标记(AH5、AH7、AH12、AH14、AH16)位于目标基因的另一侧。两侧最近分子标记在1506株的近等基因系群体中将ms3基因限定在0.14cM的遗传区间.2.将AFLP标记AH2、AH6、AH15、AH9、AH16和AH22、AH23(王贵春博士提供)成功转化为稳定的SCAR标记(依次命名为SW0、SW1、SW2、SW3、SW4、SW5、SW6)。分别利用6,407、4,768和8,041个单株的9012AB分离群进一步分析上述SCAR标记与ms3基因的连锁关系。结果显示SCAR标记SW2、SW5、SW6依然与目标基因共分离,两侧最近标记SW1和SW4与目标基因间的遗传距离均为0.029cM,ms3基因被界定在0.058cM的遗传区段,实现了目标基因的精细遗传定位。3.Blastn分析表明:标记AH2、AH6、AH9、AHl5、AH16、AH22、AH23序列及其侧翼序列与拟南芥第5染色体上的部分序列具有较高的同源性,且标记在甘蓝型油菜基因组中的排列顺序与其同源区在拟南芥基因组中的排列顺序基本一致,覆盖拟南芥同源区约576kb的物理距离。据此推断,甘蓝型油菜中ms3基因所在区段在拟南芥基因组中存在一个共线性较好的区域。4.根据拟南芥同源区中已定位的油菜、甘蓝和白菜的ESTs、GSSs序列及拟南芥的基因组序列,设计引物开发出了6个与ms3基因紧密连锁的ACGM标记(AR23、AR44、AR45、AR48、AR52、AR53)。群体验证及重组单株分析发现.AR23与SW4同侧且与其共分离,另外5个ACGM标记与SW1同侧且与目标位点均有唯一的交换单株;至此,目标基因被进一步缩小至0.034cM的遗传范围内。5.以目标基因两侧标记AH9、AH16的侧翼序列,及共分离标记AH15的SCAR标记SW2和AH17的侧翼序列为探针,筛选Tapidor BAC文库,得到86个具有强杂交信号的BAC克隆。PCR分析从中鉴定出14个阳性克隆,但它们之间并不能形成一个完整的跨叠克隆群。6.以上述阳性克隆中BAC克隆o的质粒酶切产物为探针,再次筛选TapidorBAC文库,得到48个阳性克隆。PCR检测表明与BAC克隆o可能存在物理重叠关系的克隆为7个,且它们与上述14个克隆可以形成覆盖ms3区段的完整克隆重叠群。7.对21个阳性BAC质粒的Southern杂交分析进一步证明了克隆之间的相互重叠关系。结合PCR分析的结果,发现ms3区间至少可由6个BAC克隆构成一个完整的重叠群。利用亚克隆方法构建了一个可能含有目标基因的BAC克隆的测序文库,经测序已经获得了该克隆约150kb的序列。更多还原

【Abstract】 9012AB is one type of the recessive genic male sterility(RGMS) in Brassica napus L.It has some advantages:such as complete and stable male sterility,widely spread of restorers and no negative cytoplasmic effect on yield as CMS might do.Genetic analysis indicated that the male sterility of 9012AB is controlled by two paris of recessive duplicate sterile genes(ms3ms3 and ms4ms4) interacting with one pair of a recessive epistatic inhibitor gene(rfrf)(Chen et al.,1998).Homozygous recessive sterile gene at both loci can result in male sterility,but homozygousity at the rf locus(rfrf) inhibits the expression of the recessive male sterility trait and restores the male fertility of plants. Based on this genetic model,a three-line system for hybrid seed production can be developed in this RGMS system.The three-line system can resolve the difficulty that 50%male fertile plants must be removed from the female lines during hybrid seed production.So,many researchers have put their eyes on the system in recent years.A three-line hybrid production on this RGMS system has been well-documented and the study about molecular mechanism has been started.Previous study had mapped the ms3 gene of 9012AB to a region of 0.5cM(Ke et al.,2005).In this study,9012AB was used to further fine map the ms3 gene based on the result above.Main results of the present study are as follows:1.AFLP technology combined with bulked segregant analysis(BSA) of recombinants was used to identify the genetic markers more tightly linked to ms3 in the 9012AB NIL population.Additional 2048 primer combinations were screened and 21 AFLP markers were obtained.Among them,2 markers(AH15 and AH17) co-segregated with the target gene in the tested population,14 markers(AH1、AH2、AH3、AH4、AH6、AH8、AH9、AH10、AH11、AH13、AH18、AH19、AH20、AH21) located at one side of the target locus,and other 5 markers(AH5、AH7、AH12、AH14、AH16) located at the other side of the target locus.The ms3 gene was mapped to a region of 0.14cM by the nearest flanking markers in a NIL population of 1506 plants.2.Seven AFLP markers(AH2,AH6,AH15,AH9,AH16,AH2 and AH23) were converted into SCAR markers successfully(designated as SW0,SW1,SW2,SW3,SW4, SW5 and SW6).These SCAR markers were used to analysis in three populations derived from 9012AB NIL including 6,407;4,768 and 8,041 individuals,respectively. As a result,three SCAR markers(SW2,SW5 and SW6) still co-segregated with the target gene;the ms3 gene was mapped between the nearest SCAR markers SW1 and SW4 with the distance of 0.029 cM each.Then the ms3 gene was delimited in a region of 0.058cM. 3.BLASTn analysis of seven markers’ sequences above and their flanking squences on NCBI website can identify many homologues,most of which distribute on Arabidopsis chromosome v.And the order of their homologous loci in Arabidopsis chromosome v is the same as the marker loci in linkage map in B.napus;the homologous region spanning a physical distance of about 576kb in Arabidopsis chromosome v.The result of alignment above indicates that there is a good collinearity between the region flanking ms3 and Arabidopsis homologues.4.Specific PCR primers developed from the published EST and GSS of B.napus,B. rapa and B.oleracea which are comparatively located in the Arabidopsis syntenic region and Arabidopsis genomic sequences.Six ACGM markers which more tightly linked to ms3 locus were obtained(designated as AR23,AR44,AR45,AR48,AR52 and AR53). Subsequently,these ACGM markers were used to analysis in a small size population and recombinants derived from 9012AB NIL.As a result,AR23 cosegregated with the marker SW4 and owned the same recombinants;the other five ACGM markers shared only one recombinants and located at the same side with the marker SW1.The target gene was further delimited in a region of 0.034cM.5.Four probes(SW2 and flanking squences of AH9,AH16 and AH17) screen the Tapidor BAC library,and 86 BAC clones were obtained.14 positive BAC clones were identified by primers from probes.However,the 14 positive BAC clones can not construct a whole contigs spanning ms3 gene region.6.Based on the result above,the probe(o BAC clone digested with HindⅢ) was used to further screen the Tapidor BAC library,and 48 positive BAC clones were obtained.7 positive BAC clones which could be overlap with o BAC were identified by PCR analysis.Then,an overlap contigs of ms3 genomic region were constucted by the 7 positive BACs and 14 positive BACs obtained above.7.The BAC contigs covering the ms3 region was further confirmed by Southern hybridization of 21 BACs digested with restrction enzymes.Combining the PCR analysis with Southern analysis,the physical map of the ms3 gene region including 6 BACs at least was identified.Subsequently,a sequenced library which could be contains the target gene was constucted by subclone method.Now,about 150kb sequences were obtained from the subclone library.更多还原