芥蓝BC3代Ogura CMS育性恢复材料的创制及Rfo基因传递和背景分析

doi:10.3864/j.issn.0578-1752.2018.09.012

摘要/Abstract

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。

关键词: 芥蓝, 育性恢复, Rfo传递效率, 遗传背景

Abstract: 【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 15Q23 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 15Q23 with better fertility performance was chosen as male parent to backcross with Ogura CMS Chinese kale 15Y102. The BC₃progenies 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 15Q23 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 15Y102 were 0.81-0.92, higher than that of 15Q23 (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 15Y102, 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 16Q1-4, 16Q1-7, 16Q1-10 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 16Q1-4 and 16Q1-10 was 15% and 9%, respectively, much higher than other BC₃ Rfo-positive individuals, and significantly higher than that of 15Q23 (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 16Q1-4. 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, 16Q1-4 and 16Q1-10, morphologically similar to parent Chinese kale 15Y102, have been successfully created with closer genetic background to Chinese kale compared with BC₂ pollen donor 15Q23, and significantly improved seed setting.

Key words: Chinese kale, fertility restoration, transmission rate (TR) of Rfo, genetic background

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. 芥蓝BC₃代Ogura CMS育性恢复材料的创制及Rfo基因传递和背景分析[J]. 中国农业科学, 2018, 51(9): 1746-1757.

YU HaiLong, LI ZhiYuan, YANG LiMei, LIU YuMei, ZHUANG Mu, Lü HongHao, LI ZhanSheng, FANG ZhiYuan, ZHANG YangYong. Development of Fertility-Restored BC₃ Progenies in Ogura CMS Chinese Kale and Analysis on Gene Transmission Rate of Rfo and Genetic Background[J]. Scientia Agricultura Sinica, 2018, 51(9): 1746-1757.

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芥蓝BC₃代Ogura CMS育性恢复材料的创制及Rfo基因传递和背景分析

Development of Fertility-Restored BC₃ Progenies in Ogura CMS Chinese Kale and Analysis on Gene Transmission Rate of Rfo and Genetic Background

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