High-throughput Screening of Transmission Rate for Ogura CMS Fertility Restorer Gene Rfo by HRM Technique in Cabbage

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (11): 2197-2210.doi: 10.16420/j.issn.0513-353x.2020-1078

• Original article • Previous Articles Next Articles

High-throughput Screening of Transmission Rate for Ogura CMS Fertility Restorer Gene Rfo by HRM Technique in Cabbage

REN Wenjing, YU Hailong, CHEN Li, ZHANG Bin, CHEN Wendi, FANG Zhiyuan, YANG Limei, ZHUANG Mu, LÜ Honghao, WANG Yong, JI Jialei, ZHANG Yangyong()

Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Received:2021-02-25 Revised:2021-08-20 Published:2021-12-02

Abstract

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

CLC Number:

S635.1

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.

Figures/Tables 8

References 46

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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	/

High-throughput Screening of Transmission Rate for Ogura CMS Fertility Restorer Gene Rfo by HRM Technique in Cabbage

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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/% TR of Rfo in backcross progenies
5GH1	J34 × 18QR13-23	54	45	634	14.1	360	0
5GH2	J35 × 18QR17-26	69	58	865	14.9	288	3	1.04
5GH3	J34 × 18QR17-116	104	94	1 484	15.8	360	3	0.83
5GH4	J35 × 18QR17-127	131	102	1 536	15.1	360	2	0.56
5GH5	J34 × 18QR17-175	19	14	290	20.7	288	0
5GH6	J35 × 18QR17-175	11	4	87	21.8	72	1	1.39
5GH7	J35 × 18QR17-216	99	87	1 474	16.9	576	3	0.52
5GH8	J35 × 18QR17-253	112	91	1 285	14.1	576	0
5GH9	J35 × 18QR17-303	173	167	2 396	14.3	360	2	0.56
5GH10	J35 × 18QR17-438	94	92	1 428	15.5	360	1	0.28
5GH11	J35 × 18QR19-426	45	39	464	11.9	360	1	0.28
5GH12	J35 × 18QR20-37	193	183	2 945	16.1	360	5	1.39
5GH13	J34 × 18QR20-73	43	26	420	16.2	360	1	0.28
5GH14	J34 × 18QR20-93	92	91	1 394	15.3	360	0
5GH15	J35 × 18QR20-155	117	109	2 029	18.6	360	2	0.56
5GH16	J35 × 18QR20-171	65	64	953	14.9	360	6	1.67
5GH17	J35 × 18QR20-194	348	335	5 720	17.1	360	1	0.28
5GH18	J34 × 18QR21-126	125	117	1 901	16.2	360	5	1.39
5GH19	2177 × 18QR21-126	186	176	3 292	18.7	432	3	0.69
	2183 × 18QR21-126	7	2	28	14.0	0
5GH21	J34 × 18QR21-262	180	278	3 793	13.6	360	1	0.28
5GH22	J34 × 18QR24-311	26	17	330	19.4	288	2	0.69
	J34 × 18QR25-20	8	1	13	13.0	0
5GH24	J35 × 18QR25-20	324	319	5 402	16.9	432	1	0.23
5GH25	J34 × 18QR26-17	338	323	4 587	14.2	360	7	1.94
5GH26	J35 × 18QR26-17	20	19	425	22.4	360	8	2.22
5GH27	J34 × 18QR26-34	64	56	779	13.9	360	0
5GH28	J35 × 18QR26-34	72	61	850	13.9	360	0
5GH29	J34 × 18QR26-104	127	97	1 437	14.8	360	0
5GH30	J35 × 18QR26-104	47	24	485	20.2	360	1	0.28
	J34 × 18QR26-112	38	9	130	14.4	0
5GH32	J34 × 18QR26-135	45	24	338	14.1	288	3	1.04
5GH33	J34 × 18QR26-136	49	21	361	17.2	360	12	3.33
5GH34	J35 × 18QR26-188	31	19	352	18.5	288	0
5GH35	J34 × 18QR31-265	120	82	1 389	16.9	360	0
5GH36	J35 × 18QR33-149	134	69	970	14.1	576	7	1.22
5GH37	J34 × 18QR33-179	39	37	561	15.2	360	2	0.56
5GH38	J34 × 18QR34-410	640	633	11 399	18.0	504	3	0.60
5GH39	2177 × 18QR34-410	28	25	395	15.8	288	1	0.35
5GH40	2183 × 18QR34-410	132	126	2 560	20.3	216	1	0.46
5GH41	2186 × 18QR17-216	244	183	3 358	18.3	504	4	0.79
5GH42	2202 × 18QR17-216	125	119	1 759	14.8	504	2	0.40
5GH43	2147 × 18QR17-216	183	174	2 686	15.4	504	22	4.37
5GH44	2185 × 18QR17-216	133	110	2 193	19.9	504	1	0.20

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