摘要
【目的】创制Ogura细胞质不育(Ogura CMS)的恢复系是利用Ogura CMS种质资源的有效途径。本研究基于已获得的BC2代育性恢复单株15Q23,继续用芥蓝进行回交,通过分析育性恢复基因Rfo的传递效率,及BC3代Rfo阳性株的遗传背景、育性表现、结实性和倍性,从而加速甘蓝类蔬菜Ogura CMS恢复材料的创制,获得甘蓝类蔬菜Ogura CMS恢复系。【方法】以BC2代育性较好的单株15Q23为父本,Ogura CMS芥蓝15Y102为母本进一步回交,利用Rfo特异分子标记对所有回交后代进行筛选,计算BC3代Rfo传递效率,并调查BC3代Rfo阳性单株的形态特征、育性恢复情况、结实性、倍性;选取遗传背景近于芥蓝、结实较好的重点单株继续回交获得BC4代,分析BC4代Rfo传递效率和阳性单株倍性。【结果】在开花的不同时期,单株15Q23花粉活力差异明显,以不同时期的花粉进行回交的结实性也存在极显著差异(P<0.01),平均结实率为0.07粒/授粉花蕾(7%)。利用Rfo特异标记对获得的BC3代单株进行苗期筛选,结果表明,Rfo可稳定传递,遗传背景分析结果表明,BC3代单株与芥蓝15Y102遗传相似系数在0.81—0.92,遗传背景比BC2代单株15Q23(0.73)更近于芥蓝,形态标记聚类结果与遗传背景标记聚类结果一致。形态观察发现,BC3单株形态特征都近于芥蓝,但生长势较亲本芥蓝更强;倍性鉴定结果表明,多数BC3后代接近于四倍体。开花后观察34个BC3后代单株的育性,有Rfo标记的单株育性均得到恢复。但不同单株间花粉活力存在差异,单株16Q1-4、16Q1-7、16Q1-10在整个花期花粉活力一直在75%以上。利用亲本芥蓝对花粉活力较好的Rfo阳性单株(花粉活力>50%)进行回交,以单株16Q1-4和单株16Q1-10当父本时结实性最好,结实率分别为15%和9%,显著高于BC2代阳性单株15Q23(7%,P<0.05)。BC4代单株中Rfo可以稳定传递,Rfo传递效率接近33%;对以16Q1-4为父本回交获得的24株阳性株进行倍性检测,不同单株间倍性差异较大,其中3株的荧光峰值和亲本芥蓝相近,倍性近于芥蓝。【结论】利用芥蓝对种间杂交六倍体Rfo阳性株进行第3、第4代回交,Rfo可稳定传递,成功获得了遗传背景、形态特征近于亲本芥蓝,结实性较BC2代单株15Q23显著提升的BC3代Ogura CMS育性恢复材料16Q1-4和16Q1-10。
【Objective】 Development of Ogura cytoplasmic male sterility（Ogura CMS） restorer lines is an effective way to utilize Ogura CMS germplasm resources. In order to obtain the Ogura CMS fertility-restored lines in Brassica oleracea, BC2 fertility-restored individuals 15 Q23 were backcrossed with Chinese kale parent, and the transmission rate（TR） of Rfo restorer gene, genetic background, seed setting and ploidy were investigated in the BC3 Rfo-positive progenies, which will accelerate the creation the Ogura CMS fertility restorer lines in B. oleracea. 【Method】 The BC2 fertility-restored individual 15 Q23 with better fertility performance was chosen as male parent to backcross with Ogura CMS Chinese kale 15 Y102. The BC3 progenies were produced and screened by the Rfo specific markers. The TR of Rfo in BC3 generations was calculated. Morphological identification, fertility observation, seed setting ability and ploidy evaluation were performed to select BC3 Rfo-positive individuals with better seed setting and genetic background similar to the parent Chinese kale; these individuals were then used as pollen donors to develop the BC4 generation. The TR of Rfo and ploidy of the Rfo-positive individuals in BC4 generations were evaluated. 【Result】 The pollen viability and seed setting of 15 Q23 differed at different flowering periods, with an average of 0.07 seeds per pod（7%）. The BC3 generations individuals were screened by Rfo-specific marker and the Rfo could be transmitted stably. The genetic similarity coefficients between BC3 individuals and Chinese kale 15 Y102 were 0.81-0.92, higher than that of 15 Q23（0.73）, indicating that the genetic background of BC3 individuals was closer to the parent Chinese kale. These results were further confirmed by the clustering result of morphological markers. Morphological observation revealed that the BC3 individuals were very similar to the parent Chinese kale 15 Y102, whereas the growth vigor of BC3 individual was higher than that of the parent Chinese kale. Ploidy identification showed that most of the BC3 individuals were still close to tetraploid. During the flowering stage, all of the BC3 Rfo-positive individuals were fertility-restored and the pollen viability of them was different among different BC3 individuals. The individuals 16 Q1-4, 16 Q1-7, 16 Q1-10 showed better fertility performance during the whole flowering period, and their pollen viability was above 75%. The individuals with〉 50% pollen viability were chosen as pollen donors to backcross with Chinese kale and their seed setting was calculated. Compared with other BC3 Rfo-positive individuals, the seed setting of individual 16 Q1-4 and 16 Q1-10 was 15% and 9%, respectively, much higher than other BC3 Rfo-positive individuals, and significantly higher than that of 15 Q23（7%, P〈0.05）. Furthermore, the Rfo could be transmitted in BC4 generation and the TR of Rfo was nearly 33%. Ploidy identification was performed among the 24 BC4 Rfo-positive individuals, which were derived from 16 Q1-4. The results indicated that the ploidy differed in the 24 BC4 Rfo-positive individuals and the peaks of G0/G1 period in three individuals were close to that of parent Chinese kale. 【Conclusion】 The interspecific hexaploid hybrid was further backcrossed with Chinese kale to produce the BC3 and BC4 generations. The results indicated that the Rfo can be stably transmitted. Two individuals, 16 Q1-4 and 16 Q1-10, morphologically similar to parent Chinese kale 15 Y102, have been successfully created with closer genetic background to Chinese kale compared with BC2 pollen donor 15 Q23, and significantly improved seed setting.
作者
于海龙
李志远
杨丽梅
刘玉梅
庄木
吕红豪
李占省
方智远
张扬勇
YU HaiLong;LI ZhiYuan;YANG LiMei;LIU YuMei;ZHUANG Mu;LU HongHao;LI ZhanSheng;FANG ZhiYuan;ZHANG YangYong(College of Horticulture, Northwest A ＆F University, Yangling 712100, Shaanxi;1nstitute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081)
出处
《中国农业科学》
CAS
CSCD
北大核心
2018年第9期1746-1757,共12页
Scientia Agricultura Sinica
基金
国家重点研发计划(2016YFD0101702)
国家自然科学基金(31572141)
国家现代农业产业技术体系建设专项(CARS-25)
农业部园艺作物生物学与种质创制重点实验室项目
关键词
芥蓝
育性恢复
Rfo传递效率
遗传背景
Chinese kale
fertility restoration
transmission rate （TR） of Rfo
genetic background
