利用野生甘蓝改良油菜Ogu CMS恢复材料的菌核病抗性

doi:10.3864/j.issn.0578-1752.2020.10.003

摘要/Abstract

摘要：

【目的】油菜生产受菌核病危害严重,但油菜中缺乏抗源,严重限制了油菜抗菌核病育种进程。萝卜胞质不育系（Ogu cytoplasmic male sterility,Ogu CMS）是生产油菜杂交种最好利用的授粉控制系统之一,但目前还没有抗菌核病油菜Ogu CMS恢复系选育的报道。野生甘蓝中存在高抗菌核病的资源,以抗病野生甘蓝为抗源,拟将野生甘蓝的菌核病抗病遗传成分转入Ogu CMS材料中,发展优质抗菌核病的油菜Ogu CMS恢复材料,为进一步发展抗菌核病的油菜Ogu CMS恢复系提供资源。【方法】首先采用抗菌核病野生甘蓝Brassica incana与白菜种间杂交,经胚挽救及染色体加倍技术发展人工合成甘蓝型油菜;然后将人工合成甘蓝型油菜与含Ogu CMS的油菜杂交,后代自交,并利用与甘蓝抗病QTL连锁的marker进行分子标记辅助选择（marker-assisted selection,MAS）,筛选携带主效抗病位点C1-QTL和C9-QTL的材料;MAS选中的材料通过侧枝离体接种鉴定评价其菌核病抗性,选择相对感病度S（susceptibility）显著低于甘蓝型油菜中双9号（耐病对照）的材料进行籽粒硫苷和芥酸含量测定,选择籽粒品质相对较好的材料进行恢保测定,筛选含抗病位点、抗菌核病、且结实率较好的Ogu CMS恢复材料。【结果】成功获得抗病甘蓝与白菜杂交发展的人工合成甘蓝型油菜AC1-4,其菌核病抗性显著高于甘蓝型油菜中双9号;AC1-4与Ogu CMS甘蓝型油菜4Q292杂交产生了146个F₁单株,从中选择到含有甘蓝C1-QTL和C9-QTL、结实正常、相对感病度S分别为0.66和0.40的2个单株4C99和4C115发展F₂世代;并从124个F₂植株中筛选出8份材料自交发展了982个F_2:3单株,并从中筛选到50株含有抗病位点C1-QTL和C9-QTL的植株,其相对感病度S介于0.34—0.89;其中有2份籽粒品质接近单低的材料5X82-2（硫苷含量为45.38 μmol·g^-1饼粕）和5X82-7（芥酸含量为6.5%）。【结论】改良了油菜Ogu CMS恢复材料的菌核病抗性。

关键词: 甘蓝型油菜, 菌核病抗性, 萝卜胞质不育, 恢复系

Abstract:

【Objective】Sclerotinia stem rot (SSR) is a devastating disease in rapeseed (Brassica napus L.). The resistance breeding is greatly limited due to the lack of resistance resources in rapeseed. Ogu CMS is one of the most effective pollination control systems in Brassica. Up to date, however, there has no Ogu CMS restorer with high resistance against SSR reported in rapeseed. High resistant source was reported in a wild B. oleracea which carried two major resistant loci, C1-and C9-QTL. The objective of this study is to transfer the resistance loci from the resistant B. oleracea and develop resistant Ogu CMS rapeseed restorer.【Method】Resynthesized rapeseed line was developed via interspecies cross between a SSR-resistant wild B. oleracea (namely B. incana) and B. rapa by embryo rescue and chromosome doubling. The hybridization was carried out between the resynthesized line and a rapeseed Ogu CMS line, followed by successive self-pollination. Marker assisted selection (MAS) was carried out in each generation to select individual plants carrying resistant loci from B. incana. These plants were screened for resistance level to SSR by inoculation test on detached branches. The plants which exhibited significant lower susceptibility (S) than rapeseed cultivar Zhongshuang 9 (the tolerant control) were then chosen for seed quality analysis. Fertile plants with relatively lower erucic acid and glucosinolate content were self-pollinated for the next generation development. Finally, self-pollinated lines carrying resistant QTL, exhibiting high resistant to SSR, performing good seed quality and yield were considered as SSR-resistant rapeseed Ogu CMS restorer.【Result】Resynthesized rapeseed line AC1-4 was successfully developed from the resistant B. oleracea and B. rapa, which showed significantly higher resistance level to SSR than the tolerant control Zhongshuang 9. From 146 F₁ plants derived from AC1-4 and a rapeseed Ogu CMS line 4Q292, two individual plants (4C99 and 4C115) were selected to develop F₂ generation, which carried resistant QTLs (C1- and C9-QTL) and exhibited normal seed setting and low susceptibility values (0.66 and 0.40, respectively). Among 124 F₂ plants, eight individual plants with good self-fertility were selected to develop 982 F_2:3 plants, from which a total of 50 individual plants were identified to carry resistant QTL C1-QTLand C9-QTL. Especially, two fertile plants (5X82-2 and 5X82-7) exhibited ‘single low’ seed quality (6.5% erucic acid in 5X82-7, and 45.38 μmol·g^-1 meal glucosinolate in 5X82-7) and low susceptibility to SSR (S = 0.48 and 0.33, respectively).【Conclusion】It successfully improved SSR-resistance in Ogu CMS rapeseed restorer using wild B. oleracea as donor, which will be helpful for developing Ogu CMS restorer with high resistance against SSR and good seed quality in rapeseed.

Key words: Brassica napus, sclerotinia resistance, Ogu CMS, restorer

万华方,丁一娟,陈致富,梅家琴,钱伟. 利用野生甘蓝改良油菜Ogu CMS恢复材料的菌核病抗性[J]. 中国农业科学, 2020, 53(10): 1950-1958.

WAN HuaFang,DING YiJuan,CHEN ZhiFu,MEI JiaQin,QIAN Wei. Improvement of the Resistance Against Sclerotinia sclerotiorum in Ogu CMS Restorer in Brassica napus Using Wild B. oleracea as Donor[J]. Scientia Agricultura Sinica, 2020, 53(10): 1950-1958.

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序号 No.	标记 Marker	抗病QTL* Resistant QTL	连锁群 Linkage group	R²(%)	引物序列 Primer sequence (5′-3′)
1	Na10-H06	qLR09-3/qLR10-1	C1	13.6	F：AGAATGAGACCCAGAAACCG R：GCCACACTCTCTCTTACTAGGGC
2	SWUC150	qLR09-3/qLR10-1	C1	13.6	F：AGATCCTTGCAATTTCGCAC R：TCCTGCAAAGCTTCATCCTC
3	SWUC455	qLR09-4/qLR10-2	C3	10.8	F：GACAACACAACAGACGCA R：GCATTTCCCATTACTTCCA
4	SWUC427	qSR10-2	C7	3.4	F：CCTCTGTTTCTTTGCTCTTTG R：GATTCATTGTGTGTGTGATGT
5	SWUC260	qSR09-1/qSR10-3	C9	16.1	F：AACGGCGCTCTAGAGGAAAT R：AGCTGAACAGCTTGATTGGG
6	SWUC663	qSR09-1/qSR10-3	C9	16.1	F：TGCAGTCGGAATAACCATCA R：TTCCTATTCTCCAACCGCAG
7	SWUC934	qLR09-2	C9	24.1	F：TTCACACTCCCGATCTCTCC R：AGATTCCCTCCCCAACAAGT

编号 Code	家系大小（株） Size of family (plants)	含/不含QTL植株数 Amount of plants inherited/non-inherited QTL		同时含有C1和C9 QTL植株数/百分 Amount/percentage of plants inherited both QTL on C1 and C9
编号 Code	家系大小（株） Size of family (plants)	C1	C9
5X77	72	20/52	5/67	1/1.3
5X78	46	21/20	27/25	20/43.5
5X79	70	23/47	0/70	0/0.0
5X80	184	96/88	99/85	68/37.0
5X81	129	30/98	7/122	3/2.3
5X82	144	70/74	74/70	58/40.3
5X83	184	34/150	26/158	16/8.7
5X84	153	81/72	72/81	56/36.6
共计Total	982	-	-	222/22.6

序号 No.	编号 Code	相对感病度 Relative susceptibility	芥酸 Erucic acid (%)	硫苷 Glucosinolate (μmol·g^-1 meal)
1	5X82-2	0.33±0.07	25.62±1.02	45.38±2.04
2	5X80-29	0.43±0.27	20.69±1.21	96.85±0.82
3	5X80-77	0.47±0.04	19.27±0.63	92.66±1.53
4	5X82-7	0.48±0.10	6.50±1.41	74.88±0.25
5	5X84-89	0.48±0.22	30.37±1.59	107.99±2.35
6	5X82-6	0.5±0.24	20.22±2.79	105.30±0.72
7	5X78-18	0.51±0.01	46.43±0.66	122.93±0.88
8	5X82-136	0.58±0.00	25.26±1.00	96.80±2.52
9	5X82-65	0.63±0.31	28.26±0.94	96.90±1.01
10	5X80-50	0.65±0.19	32.44±2.00	101.39±2.19
11	5X82-43	0.67±0.10	25.33±1.13	79.09±0.68
12	5X84-28	0.71±0.06	16.41±2.24	94.41±0.61
13	5X80-7	0.74±0.10	3.03±1.77	78.74±0.94
14	5X80-161	0.81±0.00	22.39±1.60	114.42±1.11
15	5X80-61	0.82±0.05	23.99±1.76	87.40±1.18
16	5X80-28	0.84±0.02	17.49±1.17	76.48±1.17
17	5X80-110	0.89±0.05	20.08±1.11	68.34±2.01