大白菜抗根肿病DH系的创制与鉴定

doi:10.7668/hbnxb.20194742

摘要/Abstract

摘要：

为了快速获得纯合稳定的抗根肿病大白菜DH系,为大白菜抗根肿病育种提供基础材料,以5个大白菜抗根肿病基因型为试材进行游离小孢子培养,并结合分子标记、形态观察和根肿菌人工接种对获得的小孢子再生植株进行了表型和抗性鉴定。结果表明,5个基因型均诱导出胚,出胚率变异在0.02~1.72个胚/蕾,3个基因型20aCR12、21aCR6、21aCR12获得再生植株,再生植株率分别为27.09%,1.45%,26.07%。抗性标记鉴定表明,与CRa、CRb^kato连锁的分子标记在获得的50个小孢子再生植株均扩增出了抗性条带。表型调查表明,50个小孢子株系生殖期基生叶片形状、颜色、抽薹早晚、育性等差异很大,自交获得的29个DH系营养期株型、叶片性状及结球性状表现出多样性。抗性接种表明,11份DH系对根肿菌4号和1号生理小种侵染病情指数均小于33.33,表现为抗病或耐病,其中2种菌侵染病情指数均小于5.0的高抗DH系有3份,分别为20aCR12-23、20aCR12-29和21aCR12-39。研究表明,单倍体培养结合分子标记辅助鉴定可快速获得纯合的抗根肿病DH系。

关键词: 大白菜, 根肿病抗性, 小孢子培养, 分子标记, 双单倍体

Abstract:

In order to quickly obtain homozygous and stable DH lines of Chinese cabbage resistant to clubroot disease,and to provide basic materials for breeding Chinese cabbage resistant varieties to clubroot disease,5 resistant genotypes of Chinese cabbage were used as materials for isolated microspore culture in this study,and phenotype and resistance of the obtained microspore regenerated plants were identified by combining molecular markers,morphological observation,and artificial inoculation of P.brassicae.The results showed that all 5 genotypes were induced to produce embryos,with a variation of 0.02 to 1.72 embryos per bud.Three genotypes,20aCR12,21aCR6,and 21aCR12,obtained regenerated plants,with regeneration rates of 27.09%,1.45%,and 26.07%,respectively.Resistance marker identification showed that molecular markers linked to CRa and CRb^kato all amplified the resistant bands in 50 obtained microspore plants.Phenotypic investigation showed that there were significant differences in the shape and color of basal leaves,bolting timing,and fertility among 50 microspore plants at reproductive growth stage,and 29 DH lines obtained through self-pollination also showed exhibited diversity in plant type,leaf traits,and heading traits at nutritional growth stage.Resistance inoculation showed that the disease index of 11 DH lines to races 4 and 1 of P.brassicae was less than 33.33,indicating resistance or tolerance to clubroot disease.Among them,there were 3 highly resistant DH lines with a disease index less than 5.0 for two races of P.brassicae,namely 20aCR12-23,20aCR12-29,and 21aCR12-39.The research results have shown that the homozygous DH lines resistant to clubroot can be obtained quickly by microspore culture combined with molecular marker assisted identification.

Key words: Chinese cabbage, Clubroot resistance, Microspore culture, Molecular marker, Double haploid

张帅宇, 常玉璀, 郝广华, 王彦华, 顾爱侠, 罗双霞, 马利松, 轩淑欣, 申书兴. 大白菜抗根肿病DH系的创制与鉴定[J]. 华北农学报, 2024, 39(3): 187-194. doi: 10.7668/hbnxb.20194742.

ZHANG Shuaiyu, CHANG Yucui, HAO Guanghua, WANG Yanhua, GU Aixia, LUO Shuangxia, MA Lisong, XUAN Shuxin, SHEN Shuxing. Creation and Identification of DH Lines for Chinese Cabbage Resistant to Clubroot Disease[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 187-194. doi: 10.7668/hbnxb.20194742.

http://www.hbnxb.net/CN/Y2024/V39/I3/187

图/表 7

表1 抗性基因连锁分子标记信息

Tab.1 Molecular markers information linked with resistance genes

抗性基因 Resistant genes	分子标记 Molecular markers	正向引物序列(5'—3') Forward primer sequences(5'—3')	反向引物序列(5'—3') Reverse primer sequence(5'—3')
CRa	GC2360	AGTTTTGTAATTTTCACCCAAAGTATCA	CAGCTGGAGGAGCACTGCAACGGAGAGA
	GC3070	AGAAATAGCAAAGCTCAAACGG	TTTGGGTTTCCACAAAACAGATTACTTTA
CRb^kato	B1321	AGTGAAACAGACTATGATTAATGTTTT	GTTTCAACTTTGGTATAAGCGTTGAGA
	B1210	ATTGAAAAGTTGACTCCGTTGA	GTTTCTTCCTTGAAGTTGCTTCAGCTCTTC

图1 小孢子培养胚发生及植株再生过程 A.小孢子胚;B.胚状体成苗或愈伤化;C.小孢子再生植株;D.小孢子苗根系。

Fig.1 Embryogenesis and plant regeneration in microspore culture A.Microspore embryoids;B.Embryoid seedlings or callus;C.Microspore regenerated plants;D.The roots of microspore seedling.

表2 不同基因型小孢子出胚率及植株再生率

Tab.2 Embryo production rate and plant regeneration rate of microspores from different genotype

基因型 Genotype	接种花蕾个数 Number of buds inoculated	出胚个数 Number of embryoids	出胚率/(个胚/蕾) Embryogenesis rate	再生植株数 Number of regenerated plants	再生植株率/% Rate of regenerated plants
20aCR4	90	2	0.02	0	0.00
20aCR6	70	3	0.04	0	0.00
20aCR12	90	155	1.72	42	27.09
21aCR6	90	138	1.53	2	1.45
21aCR12	90	23	0.26	6	26.07

图2 小孢子再生植株抗性分子标记PCR鉴定

Fig.2 PCR identification of resistant molecular markers in microspore plantlets A.GC2360;B.GC3070;C.B1210;D.B1321。 M.DNA Marker;L1.139;L5.252;1—42.20aCR12-1~20aCR12-42;43,44.21aCR6-1,21aCR6-2;45—50.21aCR12-1~21aCR12-6.

图3 小孢子植株表型性状 A—J.生殖生长期;K—U.营养生长结球期。A.20aCR12-3;B.20aCR12-4;C.20aCR12-6;D.20aCR12-7;E.20aCR12-9;F.20aCR12-19;G.20aCR12-41;H.21aCR6-2;I.二倍体植株;J.单倍体植株;K.20aCR12-23;L.20aCR12-16;M.20aCR12-11;N.21aCR6-1;O.20aCR12-29;P.20aCR12-5;Q.20aCR12-31;R.20aCR12-8;S.20aCR12-34;T.20aCR12-41;U.21aCR12-6.

Fig.3 Phenotypic traits of microspore plants A—J.Reproductive growth stage;K—U.Nutritional growth stage.A.20aCR12-3;B.20aCR12-4;C.20aCR12-6;D.20aCR12-7;E.20aCR12-9;F.20aCR12-19;G.20aCR12-41;H.21aCR6-2;I.Diploid plant;J.Haploid plant;K.20aCR12-23;L.20aCR12-16;M.20aCR12-11;N.21aCR6-1;O.20aCR12-29;P.20aCR12-5;Q.20aCR12-31;R.20aCR12-8;S.20aCR12-34;T.20aCR12-41;U.21aCR12-6.

表3 11份DH系接种2个根肿菌小种抗性鉴定结果

Tab.3 Identification results of resistance in 11 DH lines inoculated with 2 races of P.brassicae

DH系编号 No. of DH lines	结球期性状 Traits at heading stage	根肿菌4号生理小种 Race 4 of P. brassicae			根肿菌1号生理小种 Race 1 of P. brassicae
DH系编号 No. of DH lines	结球期性状 Traits at heading stage	发病率/% Incidence	病情指数 Disease index	抗性水平 Resistance level	发病率/% Incidence	病情指数 Disease index	抗性水平 Resistance level
20aCR12-1	舒心,轻微干叶	16.67	11.11	T	43.48	23.19	T
20aCR12-4	不结球,干叶,腐烂	47.06	23.53	T	11.11	6.17	R
20aCR12-8	结球,合抱	28.57	19.05	T	20.00	6.67	R
20aCR12-9	结球,叠抱	79.17	31.94	T	41.67	13.89	T
20aCR12-23	轻微干叶,舒心不实	4.76	3.17	R	9.52	3.17	R
20aCR12-29	轻微干叶,不结球	0.00	0.00	I	5.00	1.67	R
20aCR12-34	结球,拧抱	16.67	9.26	R	16.67	11.11	T
20aCR12-39	结球,舒心	14.29	4.76	R	0.00	0.00	I
20aCR12-41	结球,舒心	16.67	9.26	R	12.50	4.17	R
21aCR12-3	结球,合抱	27.59	11.49	T	0.00	0.00	I
21aCR12-6	结球,叠抱	21.05	14.04	T	27.27	15.15	T
油绿3号 Youlü 3	结球,叠抱	100.00	97.44	HS	100.00	95.83	HS

图4 11份DH系接种2个根肿菌小种抗性鉴定结果 A—D.根肿菌4号生理小种;E—H.根肿菌1号生理小种。A—D.Race 1 CQ9-5;E—H.Race 4 TS-1.

Fig.4 Identification results of resistance in 11 DH lines inoculated with 2 races of P.brassicae A.Youlü 3;B.20aCR12-29;C.20aCR12-34;D.21aCR12-6;E.20aCR12-8;F.20aCR12-9;G.20aCR12-23;H.21aCR12-3.

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