【作者】 俎峰；

【导师】 涂金星；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 甘蓝型油菜隐性细胞核雄性不育系7-7365A的育性受两对隐性重叠不育基因(ms3ms3、ms4ms4)和一对隐性上位基因(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。该材料具有败育彻底,不育性稳定,无不良胞质效应,恢复源广泛,任何一个品系均能成为其恢复系,配组相当自由,极易获得强优势组合等特点。同时用该不育系配制杂交种可以免除人工拔除50%不育株的麻烦,因此在油菜育种中的应用将会越来越广泛。本研究以7-7365A衍生材料7-736512AB两型系为材料,围绕雄性不育育性恢复基因(BnMs4)及不育基因(Bnms4)开展了以下工作:1)利用不育材料7-736512A(Bnms3ms3ms4ms4RfRf)与可育材料7-736512B(Bnms3ms3Ms4ms4RfRf)构建Bulk池筛选768对AFLP引物,获得12个与BnMs4连锁的AFLP标记,将标记差异片段回收、克隆、测序并设计引物转化SCAR标记。获得2个SCAR标记(SM25,SM129),位于目标基因的同侧,遗传距离分为0.6cM、0.75cM。2)对获得的12个与BnMs4连锁的AFLP标记中8个标记通过PCR Walking技术进行侧翼序列分离,共计获得12kb的标记侧翼序列信息,依据得到的侧翼序列,设计引物转化SCAR标记。获得2个SCAR标记(SM98,SM113),同上述两个SCAR标记同侧,遗传距离分为0.85cM、1.2cM。3)利用不育材料7-736512A(Bnms3ms3ms4ms4RfRf)与纯合可育材料7-736512B(Bnms3ms3Ms4Ms4RfRf)构建Bulk池筛选512对AFLP引物,获得8个与Bnms4连锁的AFLP标记,将标记差异片段回收、克隆、测序设计引物转SCAR。结果获得2个SCAR标记(Sm163,Sm312)。4)将获得的SCAR标记用于育种实践,发现SM113与Sm163在多种类型的材料中有多态性,能够运用于新的隐性核不育系的转育和恢复系、临保系的筛选。更多还原

【Abstract】 The Brassica napus oilseed rape line,7-7365AB,is a recessive epistatic genic male sterile(RGMS) two-type line.The sterility is controlled by two pairs of recessive duplicate genes(Bnms3 and Bnms4) and one pair of recessive epistatic inhibitor gene (Bnrf).Homozygosity at the Bnrf locus(Bnrfrf) inhibits the expression of the two recessive male sterile genes in homozygous Bnms3ms3ms4ms4 plants and produces male fertile pollens.This fertile plant pollinated to 7-7375A to generate a completely male sterile population which can be used to produe hybrid seeds.Studies by years showed that the RGMS line has complete and stable pollination control system as well as abroad restorers.Moreover,the production of F1 hybrid seeds doesn’t require the removal of 50%of segregating mother plants in the sterile line.So,this line has a good potential for heterosis utilization.In this research,we used two-type line 7-736512AB derived from sterile line 7-7365A as materials and carried out some works about male sterile restorer gene(BnMs4) and male sterile gene(Bnms4) as follows:1) From a survey of 768 primer combinations,12 AFLP markers were identified linked to the BnMs4 gene in two-type line population 7-736512AB through AFLP technology combined with BSA.All the fragments obtained from AFLP markers were cloned and sequenced.Then we designed specific PCR primers based on the sequences. Results showed that M25 and M129 were successfully converted into SCAR markers.2) We separated the flanking sequences of eight markers by PCR Walking.Then M113 and M98 were successfully converted into SCAR markers.3) AFLP aasay combined with BSA was also used to analyze the 7-736512A (Bnms3ms3ms4ms4RfRf) and a homozygous line at BnMs4 lcous (Bnms3ms3Ms4Ms4RfRf) selfing from 7-736512B.Five hundred and twevel primer combinations were used to screen the two bulks and parents,and 8 AFLP markers were identified linked to the Bnms4 gene.All the AFLP markers was cloned and sequenced. Then two SCAR markers m163 and m312 were successfully obtained.4) Six SCAR markers were used for breeding of new male sterile lines and temporary maintaimer lines.Moreover,SCAR markers SM98 and Sm163,showed good polymorphism in many materials.更多还原