【作者】 洪登峰；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2006， 博士

【摘要】 1985年，李树林等报道了一批新型的甘蓝型油菜细胞核雄性不育材料，它具有败育彻底、不育性稳定、无不良胞质效应等特点；在遗传分析的基础上，认为这些核不育材料的育性是受两对显性基因互作控制的，一对为显性不育基因，一对为显性恢复基因，不育基因单独存在时能够导致不育，但若恢复基因存在时，则育性恢复。因此，显性核不育有保持系和恢复系，可以类似于细胞质雄性不育“三系”法来配制杂交种。最近，宋来强对其发现的显性核不育材料609AB经过深入、系统的研究后，认为甘蓝型油菜显性核不育(包括Rs1046AB)更符合一对复等位基因遗传的假设，使核不育的遗传陷入争论。因此，对显性核不育的深入研究显得非常必要。本研究的主要内容包括：利用Rs1046AB为基础材料，通过杂交、回交和测交的方法对两种遗传假设进行了初步的判别；利用与不育基因连锁的特异标记，展开了核不育纯合两型系的分子标记辅助转育；利用近等基因系Rs1046AB及衍生的F2群体定位了核不育相关的基因，并将结果与前人的相关研究进行了整合。主要的实验结果如下：1．通过不育系Rs1046AB杂交后代的育性分离情况，从16份品系(品种)中，筛选到10份核不育系的临保系，5份核不育系的恢复系；另有一份在恢复基因位点杂合的材料，它能恢复部分单株的育性。2．不育系可育株(Rs1046B)与临保系杂交，F1代可育株自交并继续回交，其F2和BC1代单株全部可育，表明Rs1046B的不育基因与恢复基因在减数分裂中发生分离，因此不育基因与恢复基因为等位基因。不育株(Rs1046A)与恢复系195A-14杂交F2代的20个不育株再与临保系7-5测交，后代育性全部表现不育，证明了该恢复系的恢复基因与不育系的不育基因等位。因此确认核不育系Rs1046AB符合复等位基因的遗传模式，即Rs1046AB不育株的基因型为MsMs，可育株的基因型为MsMf，而临保系的基因型为msms，恢复系195A-14的基因型为MfMf。3．前期按照两对基因互作遗传假设，利用两个与不育基因连锁的SCAR标记，开展了不育基因的标记辅助转育研究。两个受体亲本中，7-5由于在BC1代全部表现为可育，没有分离出预期的不育株而放弃。另一个材料195A是核不育的恢复系，通过标记跟踪和背景选择，在BC2F2代中出现了背景与195A比较一致的核不育株，表明标记辅助转育的过程有效。但是由于获得的不育株存在严重的雌性不孕现象，最终很难得到纯合型不育系。4．利用近等基因系并结合BSA法，对Rs1046AB的137个单株进行了标记分析。在筛选2048对AFLP引物后，获得了6个与恢复基因连锁较为紧密的标记，遗传距离分别为0.7 cM(HDA、HDC和HDF)、1.4 cM(HDB)和3.6 cM(HDE和HDG)，且全部位于目标基因同一侧。将遗传距离较近的4个标记中的2个(HDF和HDB)直接转化成稳定的SCAR标记(SCHDF和SCHDB)；而通过侧翼序列分离后，亦将另外2个标记(HDA和HDC)成功转化(SCWA和SCWC)。这些SCAR标记与原始AFLP标记定位到连锁图同一位置。5．利用1017个单株的较大近等基因系群体对彼此共分离的3个SCAR标记(SCWA、SCWC和SCHDF)进行了区分，结果表明：标记SCHDF与恢复基因的遗传距离比标记SCWA和SCWC更近，前者的遗传距离为1.1 cM；但三个标记之间的遗传距离仍然很小。6．为了获得两侧及更近的标记，又利用192个单株的F2群体(Rs1046A／195A-14)构建了显性核不育基因／恢复基因的局部遗传连锁图，它包括20个AFLP标记和2个SCAR标记，一共覆盖10.4 cM的遗传区间。其中14个标记位于Ms／Mf一侧，7个标记位于另外一侧，一个标记与Ms／Mf共分离。Ms／Mf位点两侧最近标记的遗传距离分别为0.1 cM(E3M10-580)和1.0 cM(E1M13-260)。7．根据作图结果，将遗传距离最近的6个标记(S8T13-160、S10T2-90、E3M10-580、S5T5-480、E1M13-260及E14M1-80)中的3个成功转化为SCAR标记，分别是SCDG1(E3M10-580)、SCDG2(S5T5-480)和SCDG3(E1M13-260)。群体分析显示，3个SCAR标记与Ms／Mf位点的遗传距离与原始AFLP标记一致。8．通过对上述两个定位群体中部分标记序列(或侧翼序列)的BLASTn分析，在拟南芥第一染色体短臂顶端鉴定出一个同源区。标记在连锁图上的排列顺序与其同源区在拟南芥上的排列顺序基本一致，表明甘蓝型油菜中核不育相关基因(Ms／Mf)所在区域与拟南芥同源区之间存在着较好的共线性关系，但进化过程中，该区域中亦存在少量的染色体倒位和易位事件。与陆光远和宋来强的标记比对结果进行整合后发现，核不育相关基因的标记同源区，覆盖拟南芥从AT1g04950到AT1g14340之间的大约3.4Mb的物理区间。9．利用相同的SCAR标记或拟南芥同源区的信息，将陆光远的BC1定位群体、宋来强的BC1定位群体及本研究中的两个不同定位群体的定位结果进行了整合。在本研究的F2定位群体的基础上，将宋来强定位群体上的2个SCAR标记整合到的标记ELM2-310和E4M9-150之间；该区域包含核不育相关基因(Ms／Mf)，其界定的遗传图距为4.6 cM，共有11个标记(包括5个SCAR标记)。更多还原

【Abstract】 A new kind of genie male sterility (GMS) in Brassica napus was reported by Li et alin 1985. Due to its complete and stable sterility, and almost no negative cytoplasmiceffect, this mutant is considered as a promising alternative to CMS in heterosis utilization.A hypothesis of two dominant genes with interaction was subsequently proposed tointerpret it, that the sterility is conditioned by a dominant male sterility gene (Ms) and arestorer gene (Mf), and Ms can result in male sterility alone, but Mf can restore it. Basedon this model, a three-line hybrid production system just like CMS was then presented.However, after a systematic investigation on a natural dominant GMS mutant (609AB),Song et al. regarded that the dominant GMS is prone to be explained by one gene withmultiple alleles, which makes the mechanism of dominant GMS involved in a debate.Consequently, a further research on dominant GMS is fairly necessary.Towards this goal, our research was developed in three main fields. First, wedifferentiated the two genetic hypotheses of dominant GMS by cross, backcross and testcross, Rs1046AB used as a fundamental material. Then, a marker-assisted backcrossbreeding program to transfer the Ms gene to two elite inbred lines was carried out, usingtwo SCAR markers developed earlier. Finally, two mapping population were adopted tolocalize genes responsible for dominant GMS phenotype, and the results from differentpopulations were integrated together according to the same SCAR markers andcollinearity relation between Arabidopsis and Brassica napus. Main results of the presentresearch are as follow:1. By observing the fertility segregation of cross progenies, 10 temporary maintainers ofdominant GMS were identified from a total of 16 inbred lines. Other 5 inbred lines,which can restore the fertility of GMS, were selected as the restorers. Additional, oneinbred line which can restore the fertility of about a half progenies was concluded tocarry a heterozygous restorer gene.2. From the 1:1 segregation F1 population produced by crossing Rs1046B withmaintainers, fertile plants were selected to be selfed and baekcross to maintainers. Thesubsequent progenies were all fertile plants, suggesting that Ms and Mf werecompleted segregated in meiosis, so in Rs1046AB Mf is allelic to Ms. 20 sterile plantsfrom F2 progenies generated by crossing restorer 195A-14 with Rs1046A wererandomly selected to test crossing with maintainer 7-5, and no fertile individual wasobserved in test cross F1, indicating Mf from 195A-14 is allelic to Ms from Rs1046A,which confirmed the multiple allelism hypothesis again. Therefore, the genotypes of Rs1046A and Rs1046B are MsMs and MsMf and inbred line 195A-14 and 7-5 are ofgenotypes MfMf and msms, respectively.3. Two SCAR markers developed earlier were used to transfer Ms to two elite inbredlines, 7-5 and 195A-14, through a backcross breeding program. However, because nosterile plant was observed as expected in BC1 progenies, the program about 7-5 had tobe cancelled. By successive foreground selection and background selection for twotimes, Ms was successfully introgressed into 195A-14, and male sterile plants withsimilar appearance with 195A-14 were isolated, showing the MAS process is effective.Regretfully, due to the severely weak fecundity of female in sterile plants, thehomozygous dominant GMS line can’t be obtained.4. Combining comparing NILs and BSA, 137 individuals from Rs1046AB were used formarker analysis. From the survey of 2, 048 pairs of AFLP primers, six fragments onthe same side were identified to be tightly linked to Mf, with a genetic distancevarying from 0.7 cM to 3.6 cM. Two of the four nearer AFLP fragments were thenconverted to SCAR markers directly. For the other two markers, after isolating theadjacent sequences by suppressed PCR, corresponding SCAR marker wassuccessfully obtained as well. All the SCAR markers have a same genetic distance toMf with original AFLP marker.5. By PCR analysis in a population of 1,017 individuals, three SCAR markerscosegregated with each other were differentiated, that SCHDF was mapped closer toMf than other two markers, with a distance of 1.1 cM.6. A local linkage map of Mf/Ms genomic region was constructed based on 192individuals of the F2 population from the Rs1046A/195A-14 cross. 20 AFLP markersand two SCAR markers, spanning a genetic distance of 10.4 cM, were identified to beassociated with the Mf/Ms locus. On this map, 14 markers and 8 markers reside oneach side of target gene. The Mf/Ms locus, cosegregated with marker S5T5-480, wasbracketed by E3M10-580 and ELM13-260, with a genetic distance of 0.1 cM and 1.0cM, respectively. Six markers with a nearer genetic distance were chosen for SCARconversion, and three of them were successfully converted, i.e. SCDG1 (E3M10-580),SCDG2 (S5T5-480) and SCDG3 (ELM13-260), which have a same genetic distanceto Mf/Ms with respect to original AFLP markers.7. BLASTn analysis of some markers’ sequence (or flanking sequence) from above twopopulations on NCBI website can identify many homologues, most of whichdistribute on the short arm of Arabidopsis chromosomeⅠ. Comparison of the markerloci in linkage map and the homologous loci in Arabidopsis indicated that there was a collinearity relation between the region flanking Ms/Mf and Arabidopsis homologues,though it was likely disrupted by some chromosome inversion and translocationevents. Integrating the BLASTn result of earlier researches, the homologous region inArabidopsis chromosomeⅠcovered a physical distance of 3.4 Mb, betweenAT1g04950 and AT1g 14340.8. Utilization of the same SCAR markers or information of Arabidopsis homologues, weintegrated the mapping results of two earlier BC1 populations and two maps in thepresent research. Finally, the Ms/Mf gene can be positioned in an interval restricted bymarker E1M2-310 and E4M9-150, which contains 11 markers (including five SCARmarkers) and covered a genetic distance of 4.6 cM.更多还原