甘蓝Ogura CMS育性恢复基因Rfo的传递效率解析及育种应用

doi:10.16420/j.issn.0513-353x.2020-1078

摘要/Abstract

摘要：

为创制甘蓝Ogura细胞质雄性不育（CMS）育性恢复材料,在芥蓝和甘蓝型油菜远缘杂交后,采用甘蓝进行回交,构建BC₄和BC₅群体;基于Ogura CMS恢复基因Rfo编码区及其启动子序列,设计开发了适用于高分辨率熔解曲线（HRM）分析的特异InDel分子标记Rfo-11F/Rfo-11R;利用该标记对BC₄和BC₅代群体进行Rfo基因分型。从8 036株BC₄回交后代中筛选获得Rfo基因阳性后代41株,平均Rfo基因传递率为0.51%,平均花粉活力为81%。从BC₄代挑选5个重点Rfo阳性单株,细胞学观察结果显示5个单株的花粉母细胞染色体在减数分裂时期正常配对和分离的平均比例分别为84%和69%;进一步回交后,从15 408株BC₅回交后代中获得Rfo阳性后代117株,Rfo基因传递率为0.75%。平均花粉活力为83%,7个重点BC₅代Rfo阳性单株的花粉母细胞染色体在减数分裂时期配对和分离正常的平均比例为81%和84%。经过5代回交,甘蓝Rfo育性恢复系的倍性全部回复为正常的二倍体。所有Rfo阳性单株开花后育性表现为可育,与Rfo基因的分子标记鉴定结果完全一致,由此表明,基于本研究中开发标记构建的快速、准确、高效的Rfo基因高通量分型体系,可用于进一步回交转育后代筛选,大大提高育种效率。

关键词: 甘蓝, 高分辨率熔解曲线（HRM）, Ogura CMS, 育性恢复基因, 高通量

Abstract:

In order to create the cabbage Ogura CMS fertility restorer,backcrosses were conducted after distant hybridization between Brassica oleracea L. var. alboglabra and Brassica napus. BC₄ and BC₅generations were developed. In this study,a Rfo-specific InDel molecular marker Rfo-11F/Rfo-11R suitable for high-resolution melting curve（HRM）analysis was designed and developed based on the Rfo gene coding sequence and promoter sequence. This marker was used for Rfo genotyping of BC₄ and BC₅ generations of the distant hybridization. A total of 41 progeny in BC₄ generation with Rfo gene were successfully selected with the average Rfo transmission rate of 0.51% and pollen viability of 81%. Five key Rfo-positive individuals were selected for further backcross. Cytological observation showed that the average proportion of normal pairing and segregating of pollen mother cells in BC₄ generation were 84% and 69%,respectively. After further backcrossing,117 Rfo-positive individuals were obtained from 15 408 BC₅ backcrossing progeny,and the Rfo gene transmission rate was 0.75%. The mean value of pollen viability was 83%,and the proportion of pollen mother cells with normal pairing and segregating chromosomes were 81% and 84%,respectively. After five successive generations of backcrossing,all the fertility-restored cabbage individuals returned to normal diploid level and exhibited fertility-restored,which was consistent with the molecular marker identification results. This indicated that the fast,accurate and efficient high-throughput genotyping system for Ogura CMS fertility restorer gene Rfo,which was constructed based on the developed InDel marker Rfo-11F/Rfo-11R,can be used for further screen of backcrossing progeny and great improvement of the breeding efficiency.

Key words: cabbage, high-resolution melting（HRM）, Ogura CMS, fertility restorer gene, high-throughput

中图分类号:

S635.1

任文静, 于海龙, 陈立, 张斌, 陈文迪, 方智远, 杨丽梅, 庄木, 吕红豪, 王勇, 季家磊, 张扬勇. 甘蓝Ogura CMS育性恢复基因Rfo的传递效率解析及育种应用[J]. 园艺学报, 2021, 48(11): 2197-2210.

REN Wenjing, YU Hailong, CHEN Li, ZHANG Bin, CHEN Wendi, FANG Zhiyuan, YANG Limei, ZHUANG Mu, LÜ Honghao, WANG Yong, JI Jialei, ZHANG Yangyong. High-throughput Screening of Transmission Rate for Ogura CMS Fertility Restorer Gene Rfo by HRM Technique in Cabbage[J]. Acta Horticulturae Sinica, 2021, 48(11): 2197-2210.

图/表 8

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引物名称 Primer name	引物序列（5′-3′） Primer sequence	片段大小/bp Fragment size	参考文献 Reference
BnRFO-AS2F/BnRFO-NEW-R	F:CATGCTTCGATCTCGTCCTTTA; R:TACGACATTGGGCCTACATGTC	500	Yu et al.,2016
Rfo-6eF/Rfo-6eR	F:AGCGGAGAGAGTTGCGAAGCAGGTT; R:CCATAAGTAATCTGGGTAGGCTGGAG	566
Rfo-8F/Rfo-8R	F:CGGAACTCTACTGGTTTCAATAGCTCGG; R:GCCGCAGACTCAGCAGGAGAAGAAGAAC	320
Rfo-11F/Rfo-11R	F:TATAAGCACGGCTCTACCTATGA; R:GAAACACTCACGAAATGCTACA	300
Rfo-page-4eF/Rfo-page-4eR	F:TGAGTCTGCGGCTAGATTGTTCTGT; R:TTTCATGAAACCCACTTTGCAGCTT	125

引物名称 Primer name	引物序列（5′-3′） Primer sequence	片段大小/bp Fragment size	参考文献 Reference
BnRFO-AS2F/BnRFO-NEW-R	F:CATGCTTCGATCTCGTCCTTTA; R:TACGACATTGGGCCTACATGTC	500	Yu et al.,2016
Rfo-6eF/Rfo-6eR	F:AGCGGAGAGAGTTGCGAAGCAGGTT; R:CCATAAGTAATCTGGGTAGGCTGGAG	566
Rfo-8F/Rfo-8R	F:CGGAACTCTACTGGTTTCAATAGCTCGG; R:GCCGCAGACTCAGCAGGAGAAGAAGAAC	320
Rfo-11F/Rfo-11R	F:TATAAGCACGGCTCTACCTATGA; R:GAAACACTCACGAAATGCTACA	300
Rfo-page-4eF/Rfo-page-4eR	F:TGAGTCTGCGGCTAGATTGTTCTGT; R:TTTCATGAAACCCACTTTGCAGCTT	125

新编号 New code	组合 Combination	授粉花蕾数 Number of pollinated flowers	角果数 Number of siliques	种子数 Number of seeds	结实率 Seed setting rate	成苗数 Number of viable plants	Rfo阳性株 Number of Rfo-positive plants	Rfo传递率/% Transmission rate of Rfo in backcross progenies
	17GL1 × F8-839	19	10	137	13.7	0
18QR13	17GL2 × F9-1118	184	171	2 736	16.0	470	1	0.21
18QR14	17GL2 × F8-514	39	22	284	12.9	0
18QR15	17GL3 × F8-514	26	13	239	18.4	188	0	0
	17GL4 × F8-514	37	10	193	19.3	0
18QR16	17GL4 × F6-316	26	15	263	17.5	188	0	0
18QR17	17GL4 × F8-325	438	410	6 549	16.0	470	10	2.13
	17GL5 × F8-325	29	8	153	19.1	0
18QR18	17GL6 × F9-880	37	18	229	12.7	188	0	0
18QR19	17GL7 × F9-1118	163	156	1 657	10.6	470	1	0.21
18QR20	17GL7 × F8-325	25	17	327	19.2	282	7	2.48
18QR21	17GL8 × F8-325	22	19	384	20.2	282	2	0.71
18QR22	17GL8 × F6-316	175	156	2 658	17.0	470	1	0.21
18QR23	17GL8 × F8-514	44	38	563	14.8	470	1	0.21
18QR24	17GL9 × F8-514	274	211	2 943	13.9	470	1	0.21
18QR25	17GL9 × F8-325	48	11	150	13.6	140	1	0.71
18QR26	17GL9 × F8-839	27	10	210	21.0	188	10	5.32
18QR27	17GL9 × F9-1118	175	134	2 542	19.0	470	1	0.21
18QR28	17GL9 × F6-316	241	229	2 987	13.0	470	0	0
18QR29	17GL10 × F6-316	516	505	8 583	17.0	470	0	0
18QR30	17GL10 × F8-514	94	86	1 530	17.8	470	0	0
18QR31	2383 × F6-316	294	273	4 648	17.0	470	1	0.21
18QR32	2383 × F8-514	582	554	9 422	17.0	470	0	0
18QR33	2383 × F8-839	384	315	4 725	15.0	470	3	0.64
	2383 × F9-880	29	8	132	16.5	0
18QR34	2383 × F9-1118	234	219	4 827	22.0	470	1	0.21

新编号 New code	组合 Combination	授粉花蕾数 Number of pollinated flowers	角果数 Number of siliques	种子数 Number of seeds	结实率 Seed setting rate	成苗数 Number of viable plants	Rfo阳性株 Number of Rfo-positive plants	Rfo传递率/% Transmission rate of Rfo in backcross progenies
	17GL1 × F8-839	19	10	137	13.7	0
18QR13	17GL2 × F9-1118	184	171	2 736	16.0	470	1	0.21
18QR14	17GL2 × F8-514	39	22	284	12.9	0
18QR15	17GL3 × F8-514	26	13	239	18.4	188	0	0
	17GL4 × F8-514	37	10	193	19.3	0
18QR16	17GL4 × F6-316	26	15	263	17.5	188	0	0
18QR17	17GL4 × F8-325	438	410	6 549	16.0	470	10	2.13
	17GL5 × F8-325	29	8	153	19.1	0
18QR18	17GL6 × F9-880	37	18	229	12.7	188	0	0
18QR19	17GL7 × F9-1118	163	156	1 657	10.6	470	1	0.21
18QR20	17GL7 × F8-325	25	17	327	19.2	282	7	2.48
18QR21	17GL8 × F8-325	22	19	384	20.2	282	2	0.71
18QR22	17GL8 × F6-316	175	156	2 658	17.0	470	1	0.21
18QR23	17GL8 × F8-514	44	38	563	14.8	470	1	0.21
18QR24	17GL9 × F8-514	274	211	2 943	13.9	470	1	0.21
18QR25	17GL9 × F8-325	48	11	150	13.6	140	1	0.71
18QR26	17GL9 × F8-839	27	10	210	21.0	188	10	5.32
18QR27	17GL9 × F9-1118	175	134	2 542	19.0	470	1	0.21
18QR28	17GL9 × F6-316	241	229	2 987	13.0	470	0	0
18QR29	17GL10 × F6-316	516	505	8 583	17.0	470	0	0
18QR30	17GL10 × F8-514	94	86	1 530	17.8	470	0	0
18QR31	2383 × F6-316	294	273	4 648	17.0	470	1	0.21
18QR32	2383 × F8-514	582	554	9 422	17.0	470	0	0
18QR33	2383 × F8-839	384	315	4 725	15.0	470	3	0.64
	2383 × F9-880	29	8	132	16.5	0
18QR34	2383 × F9-1118	234	219	4 827	22.0	470	1	0.21

世代 Generation	编号 Code	花粉活力/% Pollen viability	Rfo传递率/% Transmission rate of Rfo	9Ⅱ型染色体配对占比/% Proportion of 9Ⅱ chromosome pairing pattern	9:9正常染色体分离占比/% Proportion of normal 9:9 chromosome separation pattern	倍性荧光峰值 The peaks of G0/G1 peak	结实率/% Seeds per pod
BC₄	18QR17-216	95	1.23	88	90	201	17.0
	18QR19-426	79	0.28	72	66	194	11.9
	18QR21-262	89	0.28	84	67	185	13.6
	18QR33-179	75	0.56	94	70	189	15.2
	18QR34-410	87	0.50	82	52	210	18.3
BC₅	5GH15-169	93	/	96	92	197	/
	5GH25-337	83	/	84	88	192	/
	5GH26-234	75	/	80	76	199	/
	5GH26-247	77	/	74	90	195	/
	5GH33-111	81	/	70	96	210	/
	5GH36-589	90	/	78	72	218	/
	5GH39-211	91	/	82	74	191	/