【作者】 刘玉霞；

【导师】 赵坚义；

【作者基本信息】 浙江师范大学 ， 植物学， 2011， 硕士

【摘要】 本课题组借助分子标记和QTL作图等生物学技术在9个环境中利用282个DH群体对甘蓝型油菜的19对染色体进行了千粒重的初定位,其中有7个环境都一致地在第7号染色体(A7)上相同的区域检测到一个效应值较大的QTL,这个QTL的LOD值范围大致在3.35到16.75之间,其遗传变异的贡献率达到4.90%～17.90%,加性效应在0.071-0.205之间,平均加性效应为0.148g；其QTL的标记区间在EST12a到ZAAS864之间,遗传距离有40.3cM。A7-千粒重QTL的初定位结果,为后续的精细定位和图位克隆打下了良好的基础。本研究在此基础上进行,通过对A7-目标QTL区间标记辅助选育,发展含供体目标基因组片段的近等基因系BC3F1/BC3F2和BC4F1/BC4F2世代,分别利用1138株BC3F1和594株BC4F1单株群体进行标记基因型分析,并用其相应单株以及自交后代株系(BC3F2和BC4F2)群体进行两重复的田间试验和千粒重表型测定。并借助白菜基因组第7连锁群的序列信息以及与拟南芥基因序列进行同源比对针对A7-千粒重QTL区间发展位置特异性标记。从而对A7-千粒重QTL进行确认和精细定位,主要结果如下：1.A7-目标QTL区间标记加密根据甘蓝型油菜A7-千粒重QTL的初定位结果,借助白菜A7连锁群基因组序列信息针对甘蓝型油菜A7-千粒重主效QTL区域发展了位置特异性标记上百个,其中定位上了44个。标记从原来的19个增加到现在的63个,并使其标记间平均距离由2.12cM减小到0.60cM。2.A7-目标QTL的确认利用1138株BC3F1世代的单株基因型和相应的自交后代BC3F2株系表现型对甘蓝型油菜A7-千粒重QTL进行确认和缩小区间。通过关联分析以及近等替换系间的比对分析,发现在A7-千粒重QTL区间内大粒和小粒等位基因间确实存在显著的差异(P=0.001),其均值差为0.190g；QTL区间由初定位的40.3cM缩小到9.0cM,位于ESPSC2和ZAAS864之间。3.A7-目标QTL的精细定位利用594株BC4F1世代的单株和BC4F2株系对甘蓝型油菜A7-千粒重主效QTL进行精细定位。通过BC4F1世代单株基因型和BC4F2株系表型的关联分析以及其近等基因系间的比对分析之后,将A7-千粒重QTL区间进一步缩小到了sr0282R和ZAAS864两标记之间,其遗传距离为6.9cM。6.9cM的目标区域内含纯合大粒等位基因(Sollux)片段的株系对千粒重的贡献相对于含纯合小粒等位基因(Gaoyou)片段的株系平均要高0.231g,差异达到极显著(P=0.009),说明A7上控制油菜千粒重的QTL很可能就位于这个6.9cM的QTL区间内。但是目前这个QTL区间的遗传距离还比较大,所以需要进一步加密区间标记以及培育更高世代的回交群体,才能进一步地缩小目标区间的遗传距离。本结果表明在A7-千粒重QTL目标区间内,当中国品种’Gaoyou"中控制小粒等位基因被欧洲品种’Sollux"中控制大粒等位基因纯合替代时,可显著提高千粒重约0.231g,所以有必要对A7-目标区间作更进一步的精细定位和图位克隆控制该QTL的关键基因。更多还原

【Abstract】 Based on the initial QTL mapping for 1000-seed weight using SG-DH population (282 lines) over 9 environments, one major QTL located on linkage group 7 (A7) was selected for further fine mapping. This QTL was stably detected in 7 of 9 environments with LOD value from 3.35 to 16.75 and explained the phenotypic variation in population from 4.90% to 17.90%. This QTL showed an average additive effect of 0.148g and was flanked by markers EST12a and ZAAS864 (40.3cM).In present study, the major QTL for seed weight on A7 was validated and further fined mapped through the following procedures.1) QTL near isogonics lines including BC3F1/BC3F2 and BC4F1/BC4F2 generations covering target genomic region were developed by marker-assistant backcross; 2) 1138 and 594 single plants from BC3F1 and BC4F1 populations were individually isolated and subsequently analyzed by the markers within QTL region to get marker genotypes. For increasing the marker density, the positional specific markers were developed by using the sequence information from A7 of Brassica rape; 3) the corresponding selffing progenies of BC3F2 and BC4F2 families were employed for field trials with replications to obtain the trait phenotypes. The main results were as follows:1. Increasing markers in the target QTL intervalBased on the initial QTL mapping for 1000-seed weight on A7, hundreds of positional specific markers were developed in the QTL target interval by using the sequences information of A7 in Brassica rape. Of them,44 markers could be mapped within the QTL region and therefore, greatly increased the marker density from 19 to 63 markers with the average distance between two markers from 2.12cM to 0.60cM.2. The validation of the target QTL on A7The QTL for 1000-seed weight on A7 was validated by using the marker genotypes from BC3F1 single plants (n=1138) and trait phenotypes from their corresponding selfing progenies BC3F2. The results showed clearly linkages between three marker genotypes and trait phenotypes in each of marker loci within QTL region. Further, a significant difference of 0.190 g (P=0.001) for 1000-seed weight between homozygous BC3F2 sister sub-NILs carrying "Sollux" fragment (n=101, 3.169g) and NILs containing "Gaoyou" segment (n=85,2.979g) in the target region were observed. By comparison analysis among near isogonics lines in this step, the QTL region was narrowed from 40.cM to 9.0cM3. The further fine mapping of A7-QTL for seed weightThe further fine mapping for A7-QTL of 1000-seed weight was carried out by using the BC4F1/BC4F2 plants/families. Marker genotypes were analyzed with BC4F1 single plants (n=594), and trait phenotypes were obtained from their corresponding selfing progenies BC4F2. The same strategy as used in the populations of BC3F1/BC3F2 was adopted to conduct the linkage and comparative analysis. The results further narrowed the QTL from 9.0cM to 6.9cM region and fixed it between markers sR0282R and ZAAS864. Notably, the larger difference with 0.231g (P=0.009) for 1000-seed weight between two homologous genotypes of NILs carrying "Sollux" fragment (n=44,3.234g) and NILs containing "Gaoyou" segment (n=51,3.003g) in the 6.9cM region than identified in populations of BC3F1/BC3F2 were observed. This shows a trend when the genetic background are more identical between two QTL-NILs, the additive effect of the QTL might increase and therefore to further indicate the potential for map-based cloning and for breeding purpose of this QTL.更多还原