萝卜胞质雄性不育(Ogura CMS)芥菜材料的创制

doi:10.3969/j.issn.1004-1524.2023.05.09

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1058-1068.DOI: 10.3969/j.issn.1004-1524.2023.05.09

萝卜胞质雄性不育(Ogura CMS)芥菜材料的创制

李崇娟¹^,²(), 吕凤仙¹^,², 杨鼎¹^,², 张丽琴²^,³, 兰梅²^,³, 杨红丽²^,³, 徐学忠²^,³, 胡靖锋²^,³, 申时品⁴, 吴毓飞⁴, 和江明²^,³^,^*(), 董相书¹

1.云南大学资源植物研究院,云南昆明 650504
2.云南省农业科学院园艺作物研究所,云南昆明 650205
3.国家蔬菜改良分中心云南分中心,云南昆明 650205
4.镇雄县农业科技推广中心,云南镇雄 657200

收稿日期:2022-04-13 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:*和江明,E-mail: hejiangming666@qq.com
李崇娟(1997—),女,彝族,云南昆明人,硕士研究生,研究方向为十字花科蔬菜育种。E-mail: lcj649954517lcj@163.com
通讯作者: 和江明
基金资助:
国家大宗蔬菜产业技术体系(CARS-23-G33);云南省“万人计划”产业技术领军人才(YNWR-CYJS-2018-040);云南种子种业联合实验室项目(202205AR070001)

Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea

LI Chongjuan¹^,²(), LYU Fengxian¹^,², YANG Ding¹^,², ZHANG Liqin²^,³, LAN Mei²^,³, YANG Hongli²^,³, XU Xuezhong²^,³, HU Jingfeng²^,³, SHEN Shipin⁴, WU Yufei⁴, HE Jiangming²^,³^,^*(), DONG Xiangshu¹

1. Resource Plant Research Institute, Yunnan University, Kunming 650504, China
2. Horticulture Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China
3. Yunnan Branch of National Vegetable Improvement Center, Kunming 650205, China
4. Agricultural Science and Technology Promotion Center of Zhenxiong County, Zhenxiong 657200, Yunnan, China

Received:2022-04-13 Online:2023-05-25 Published:2023-06-01
Contact: HE Jiangming

摘要/Abstract

摘要：

为获得萝卜胞质雄性不育芥菜,利用远缘杂交将芥菜的B基因组导入白菜,通过连续回交法结合胚挽救技术,创制出保留白菜雄性不育基因的芥菜。用萝卜胞质雄性不育白菜CCR17001与可育叶用芥菜JC17000杂交,并以叶用芥菜为轮回亲本连续进行5代回交,获得萝卜胞质雄性不育芥菜材料,对其进行形态学、细胞学、分子生物学鉴定。结果表明:该材料植株形态与轮回亲本相似,蜜腺发育良好,雄蕊高度退化成花瓣,不育度和不育株率均为100%,且不育性稳定,有正常结籽能力,可作为芥菜优势育种的可靠材料。

关键词: 芥菜, 远缘杂交, 萝卜胞质雄性不育, 胚挽救

Abstract:

In order to obtain Ogura cytoplasmic male sterility Brassica juncea, the B genome of Brassica juncea was introduced into Chinese cabbage by hybridization, and the male sterility gene of Chinese cabbage was created by backcrossing combined with embryo rescue technique. With CCR17001 Ogura cytoplasmic male sterility Chinese cabbage and leaf mustard JC17000 hybridization, and leaf mustard as recurrent parent for 5 consecutive backcross generations, the Ogura cytopathic male sterile mustard material was obtained and identified by morphology, cytology and molecular biology. The results showed that the plant morphology of this material was similar to that of its recurrent parent, with well-developed nectaries, highly degenerated stamens into petals, 100% sterility and sterility rate, stable sterility, normal seed setting ability, which could be used as a reliable material for breeding advantage of mustard.

Key words: Brassica juncea, interspecies cross, Ogura cytoplasmic male sterility, embryo rescue

中图分类号:

S637.2

李崇娟, 吕凤仙, 杨鼎, 张丽琴, 兰梅, 杨红丽, 徐学忠, 胡靖锋, 申时品, 吴毓飞, 和江明, 董相书. 萝卜胞质雄性不育(Ogura CMS)芥菜材料的创制[J]. 浙江农业学报, 2023, 35(5): 1058-1068.

LI Chongjuan, LYU Fengxian, YANG Ding, ZHANG Liqin, LAN Mei, YANG Hongli, XU Xuezhong, HU Jingfeng, SHEN Shipin, WU Yufei, HE Jiangming, DONG Xiangshu. Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1058-1068.

图/表 13

图1 萝卜胞质雄不育芥菜转育模式

Fig.1 Breeding model for the transfer of Ogura CMS in mustard

表1 胚挽救不同培养基成分

Table 1 Components of embryo rescue medium for interspecific hybrid

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

表2 鉴定萝卜胞质不育的引物信息

Table 2 Information of primers for identifying Ogura cytoplasm male sterility

引物名称 Primer name	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')
ogu-1	ATGATTACCTTTTTCGAAAAAATTGC	TTTATTTTCTCGGTCCATTTTC
ogu-2	TAGCGCTATCTTTCGGCCCT	ACCCGAAGGTCCTGGTCTCT
ogu-3	GCCTCAAACTGGATAAATCT	CAAGGATCTCGTTCATTC
ogu-4	CCATATTTGGCTAAGCTGGTTTTCT	TATCATCTCGGTCCATTGTCCAC

图2 不同成分培养基的出胚率 M1~M8为不同培养基,具体成分见表1。柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.2 Embryogenesis rate of different culture mediums M1-M8 were different culture mediums, their components were the same as in Table 1. Data on the bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图3 F1×JC17000种间杂种胚挽救情况 a,胚珠萌发;b,成活胚增殖分化;c和d为胚挽救幼苗。

Fig.3 Embryo rescue situation of interspecific hybrid of F1×JC17000 a, Ovule germination; b, Proliferation and differentiation of survival embryos; c and d, Embryo rescued seedling.

表3 不同取材时间的出胚率

Table 3 Embryogenesis rate at different sampling time

授粉后时间 Time after pollination/d		取胚数 Number of ovules	出胚数 Number of embryogenesis	出胚率 Embryogenesis rate/%
第1次回交	11	169	17	10.06±0.05 c
The first backcross	12	263	40	15.21±0.11 b
	13	121	32	26.44±0.03 a
	14	101	16	15.84±0.04 b
	15	166	14	8.36±0.06 d
	16	109	4	4.73±1.55 e
第2次回交	11	71	8	11.07±0.16 d
The second backcross	12	91	16	17.58±0.07 b
	13	150	49	32.66±0.16 a
	14	204	33	16.71±0.02 c
	15	139	15	10.89±0.08 d
	16	87	5	5.75±0.11 e

图4 亲本与杂交回交后代的植株形态 a,轮回亲本芥菜JC17000;b,萝卜胞质不育白菜CCR17001;c,种间杂种F1;d,BC5。

Fig.4 Plant morphology of parents, hybrid and backcross progeny a, Recurrent parent mustard JC17000; b, Ogura CMS Chinese cabbage CCR17001; c, Interspecific hybridition F1; d, BC5.

图5 BC5与轮回亲本的花器官形态比较 a,JC17000的花;b,CCR17001的花;c,BC5的花;d,JC17000花的解剖;e,CCR17001花的解剖;f,BC5花的解刨。标尺=1 cm。

Fig.5 Comparison of flower morphology between BC5 and recurrent parents a, The flower of JC17000; b, The flower of CCR17001; c, The flower of BC5; d, Dissection of JC17000 flower; e, Dissection of CCR17001 flower; f, Dissection of BC5 flower. Scale bar=1 cm.

表4 萝卜胞质不育芥菜与亲本花器官结构特征的比较

Table 4 Comparison of floral organ structure characteristics between cytoplasmic male sterility of Brassica juncea and its parents cm

材料 Material	花蕾长 Bud length	花蕾直径 Bud diameter	花瓣长 Corolla length	花瓣宽 Corolla diameter	花柱长 Nectar length	雄蕊长 Stamens length
JC17000	0.66 ab	0.34 ab	1.17 a	0.56 b	0.55 b	0.77 ab
CCR17001	0.67 a	0.38 a	1.24 a	0.79 a	0.54 b	0.70 b
BC₅	0.58 b	0.27 b	0.70 b	0.26 c	0.76 a	—

表5 亲本与杂交及回交后代的结实性比较

Table 5 Comparison of fruitfulness between parents, cross and backcross generations

材料 Material	授粉花数 Number of flowers	结籽数 Number of seeds	平均单花结籽数 Average number of seeds per fiower
JC17000	243	2 638	10.85
F1	109	394	3.61
BC₁	321	-	-
BC₂	298	-	-
BC₃	466	2 319	4.98
BC₄	267	1 623	7.08
BC₅	352	2 923	10.30

图6 亲本与杂交及回交后代的染色体观察 a,萝卜胞质不育白菜CCR17001的有丝分裂细胞,2n=20;b,轮回亲本芥菜JC17000的有丝分裂细胞,2n=36;c,种间杂交后代F1的有丝分裂细胞,2n=28;d,BC5的有丝分裂细胞,2n=36。标尺=10 μm。

Fig.6 Chromosome observation of cross and backcross generations a, Mitotic cells of ogura CMS Chinese cabbage CCR17001, 2n=20; b, Mitotic cells of the recurrent parent mustard JC17000, 2n=36; c, Mitotic cells of interspecific hybrid progeny F1, 2n=28; d, Mitotic cells of BC5, 2n=36. Scale bar=10 μm.

图7 亲本与杂交及回交后代流式细胞图 a,萝卜胞质不育白菜CCR17001;b,轮回亲本芥菜JC17000;c,种间杂种F1;d,BC5。

Fig.7 Flow cytometry of parent, cross and backcross progeny a, Ogura CMS Chinese cabbage CCR17001; b, Recurrent parent mustard JC17000; c, Interspecific hybrid progeny F1; d, BC5.

图8 利用Ogura胞质雄性不育分子鉴定BC5及其亲本材料 M,DL2000 marker;1,萝卜胞质不育白菜CCR17001;2,轮回亲本芥菜JC17000;3,BC5;4,可育白菜CCR21001。

Fig.8 Ogura cytoplasmic male sterility molecular identification BC5 and its parents M, DL2000 marker; 1, Ogura CMS Chinese cabbage CCR17001; 2, Recurrent parent mustard JC17000; 3, BC5; 4, Fertile Chinese cabbage CCR21001.

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萝卜胞质雄性不育(Ogura CMS)芥菜材料的创制

Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea

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编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

[1]	吕凤仙, 和江明, 李崇娟, 杨鼎, 胡靖锋, 杨红丽, 兰梅, 徐学忠, 张丽琴. 菜心和芥蓝种间杂交创制异源四倍体蔬菜种质[J]. 浙江农业学报, 2022, 34(8): 1638-1647.
[2]	孟秋峰, 胡美华, 王洁, 任锡亮, 高天一, 陈建明. 浙江省芥菜研究进展[J]. 浙江农业学报, 2020, 32(9): 1732-1740.
[3]	郑永利1，汪恩国2，蔡建军2，李国均1，姚晓明1. 鲜食小芥菜烟粉虱空间分布型及抽样技术研究[J]. 浙江农业学报, 2015, 27(4): 589-.
[4]	王泽翻;韩倩;林毅雁;崔娇鹏;刘恒星;赵世伟;赵惠恩;*. 几种蔷薇和‘月月粉’与现代月季杂交亲和性评价[J]. , 2012, 24(6): 0-1032.
[5]	张富仙;章心惠;陈润兴;徐建祥;项小敏. 美洲南瓜与印度南瓜远缘杂交亲和性研究[J]. , 2010, 22(1): 0-29.

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1