油菜高油酸种质的创建及高油酸性状遗传与生理特性的分析

doi:10.3864/j.issn.0578-1752.2021.02.003

摘要/Abstract

摘要：

【目的】创建高油酸（high oleic,HO）油菜新种质,探明HO新种质中高油酸性状的遗传模式,明确HO新种质油酸含量变化的生理特性,为培育HO油菜新品种奠定基础。【方法】采用辐射处理油菜萌动发芽种子,获得初级诱变群体后在后续世代利用极端选择法结合小孢子培养技术筛选油菜HO新种质。以HO新种质分别与3个不同遗传背景的常规油菜品系为亲本组合杂交构建6个世代（P₁、P₂、F₁、BC₁P₁、BC₁P₂和F₂）的遗传群体,测定各群体脂肪酸含量后应用主基因+多基因混合遗传模型联合分析方法对遗传群体的高油酸性状进行遗传分析。测定HO新种质种子发芽过程中子叶、不同温度处理下苗期营养器官以及角果成熟过程中种子的油酸含量,明确其变化规律及生理效应。【结果】通过辐射获得油酸含量显著变化的初级诱变群体,在后续世代持续利用极端选择法得到平均油酸含量升高20个百分点的高世代群体,采用小孢子培养得到纯合稳定的油菜HO双单倍体群体,最终根据品质性状筛选成功得到HO新种质B161,其油脂中油酸含量为85%,亚麻酸含量为3%。以B161为HO亲本和常规品系杂交配置得到3个具有不同遗传背景的遗传群体并测定获得各群体的油酸含量表型数据。脂肪酸含量相关性分析表明,十八碳脂肪酸中油酸含量与亚油酸含量和亚麻酸含量具有显著负相关关系。遗传分析结果表明,该种质中高油酸性状由2对具有加性效应的主效基因控制,并且2对基因对油酸含量的效应值接近。生理分析表明,常温下HO品系营养器官（根、茎、叶和叶柄）的油酸含量均显著高于常规品系,亚麻酸含量显著低于常规品系。低温下HO品系营养器官的油酸含量降低,但仍高于常规品系;常规品系油酸含量在低温下稳定。低温下两类品系营养器官的亚麻酸含量均显著提高,但HO品系亚麻酸含量仍低于常规品系。在种子成熟过程和种子发芽过程中,HOLL品系中油酸含量持续显著高于常规油菜,而亚麻酸含量则持续显著低于常规油菜。【结论】成功创制了油菜HO新品系B161,明确了新种质HO性状的遗传规律和生理特性。获得的HO品系具有潜在育种利用价值。

关键词: 油菜, 辐射诱变, 种质创新, 高油酸性状, 遗传分析, 低温响应

Abstract:

【Objective】 This study is to create the new high oleic (HO) canola germplasm, to explore its genetic mode and the physiological characters of the HO trait, which will lay a foundation for breeding HO canola varieties. 【Method】 The primary mutation population with was obtained by radiation treatment of germinating canola seeds. The new HO germplasm was screened by extreme selection method combined with microspore culture technology in subsequent generations. The genetic populations of six generations (P₁, P₂, F₁, BC₁P₁, BC₁P₂ and F₂) were constructed by crossing the HO Germplasm with three conventional canola lines with different genetic background. After the fatty acid content of each population was determined, the genetic analysis of high oleic acid content in the genetic population was analyzed by the mixed major-gene plus polygene inheritance model. The oleic acid content in cotyledons during seed germination, vegetative organs at seedling stage in different temperature regimes and seeds during silique ripening process of the HO germplasm were detected to explore their change patterns and physiological effects.【Result】 The primary mutation population with significantly increased oleic acid content was obtained by radiation treatment, and then the high generation population with 20-percent-increased oleic acid content was obtained by using extreme selection method in subsequent generations and the double haploid population was obtained by microspore culture. Finally, a new HO germplasm B161 (C18:1=85%, C18:3=3%) was successfully screened according to the quality traits. Three genetic populations with different genetic background were obtained by crossing B161 as HO parent with three other conventional lines. The correlation analysis of fatty acid contents showed that there was a significant negative correlation between oleic acid content, linoleic acid content and linolenic acid content. The results of genetic analysis showed that the high oleic acid content was controlled by two major genes with additive effect, and their effect values were close. Physiological analysis showed that the contents of oleic acid in vegetative organs (root, stem, leaf and petiole) of HO line were significantly higher than those of the conventional strain, and the linolenic acid contents of HO line were significantly lower than those of the conventional line. The contents of oleic acid in vegetative organs of HO line decreased at low temperature, but they were still higher than those of the conventional line. The linolenic acid contents in vegetative organs of the two lines increased significantly at low temperature, but the linolenic acid content of HO line was still lower than that of the conventional line. During the process of seed ripening and seed germination, the oleic acid content of HO line was significantly higher than that of conventional line, while the linolenic acid content was significantly lower than that of conventional line.【Conclusion】The new HO germplasm was successfully created and the genetic mode and physiological characters were confirmed. This HO germplasm has the potential value in breeding.

Key words: Brassica napus L., radiation mutagenesis, germplasm creation, high oleic trait, genetic analysis, response to low temperature

龙卫华,浦惠明,高建芹,胡茂龙,张洁夫,陈松. 油菜高油酸种质的创建及高油酸性状遗传与生理特性的分析[J]. 中国农业科学, 2021, 54(2): 261-270.

LONG WeiHua,PU HuiMing,GAO JianQin,HU MaoLong,ZHANG JieFu,CHEN Song. Creation of High-Oleic (HO) Canola Germplasm and the Genetic and Physiological Analysis on HO Trait[J]. Scientia Agricultura Sinica, 2021, 54(2): 261-270.

图/表 8

表1

表2

表3

表4

表5

表6

图1

图2

参考文献 37

[1]	胡忆雨, 朱颖璇, 杨雨豪, 邹军, 陈阜, 尹小刚. 1951-2015年中国主要粮食与油料作物种植结构变化分析. 中国农业大学学报, 2019,24(11):183-196.
	HU Y Y, ZHU Y X, YANG Y H, ZOU J, CHEN F, YIN X G. Changes of the planting structure of major food and oil crops in China from 1951 to 2015. Journal of China Agriculture University, 2019,24(11):183-196. (in Chinese)
[2]	刘成, 冯中朝, 肖唐华, 马晓敏, 周广生, 黄凤洪, 李加纳, 王汉中. 我国油菜产业发展现状、潜力及对策. 中国油料作物学报, 2019,41(4):485-489.
	LIU C, FENG Z C, XIAO T H, MA X M, ZHOU G S, HUANG F H, LI J N, WANG H Z. Development, potential and adaptation of Chinese rapeseed industry. Chinese Journal of Oil Crop Sciences, 2019,41(4):485-489. (in Chinese)
[3]	王汉中. 以新需求为导向的油菜产业发展战略. 中国油料作物学报, 2018,40(5):613-617.
	WANG H Z. New-demand oriented oilseed rape industry developing strategy. Chinese Journal of Oil Crop Sciences, 2018,40(5):613-617. (in Chinese)
[4]	SCARTH R, MCVETTY P B E. Designer oil canola: A review of food-grade Brassica oils with focus on high oleic, low linolenic types//WRATTEN N, SALISBURY P A (eds). Proceeding of 10th International Rapeseed Congress. Canberra, Australia: GCIRC, 1999: 26-29.
[5]	STANISŁAW S, KATARZYNA M, HANNA C, TERESA P, KRYSTYNA K, MARCIN M, JOANNA N, KRZYSZTOF M, IWONA B. Marker assisted selection of new high oleic and low linolenic winter oilseed rape (Brassica napus L.) inbred lines revealing good agricultural value. PLoS ONE, 2020,15(6):e0233959. doi: 10.1371/journal.pone.0233959 pmid: 32497146
[6]	REZA F, MOHAMMAD H R. Oxidative stability of canola oil by Biarum bovei bioactive components during storage at ambient temperature. Food Science and Nutrition, 2018,6(2):342-347. doi: 10.1002/fsn3.560 pmid: 29564101
[7]	AULD D L, HEIKKINEN M K, ERICKSON D A, SERNYK J L, ROMERO J E. Rapeseed mutants with reduced levels of polyunsaturated fatty acids and increased levels of oleic acid. Crop Sciences, 1992,32:657-662.
[8]	RUCKER B, ROBBELEN G. Development of high oleic acid rapeseed//Proceeding of 9th International Rapeseed Congress. Cambridge, UK: GCIRC, 1995: 389-391.
[9]	DEBONTE L R,, FAN Z G, MIAO G H. Fatty acid desaturases and mutant sequences thereof: US 6967243 B2 [P]. 2001-1-29
[10]	和江明, 王敬乔, 陈薇, 李根泽, 董云松, 寸守铣. 用EMS诱变和小孢子培养快速获得甘蓝型油菜高油酸种质材料的研究. 西南农业学报, 2003,16(2):34-36.
	HE J M, WANG J Q, CHEN W, LI G Z, DONG Y S, CUN S X. Studies on rapidly obtaining high oleic acid germplasm of Brassica napus by mutagen EMS and microspore culture. Southwest China Journal of Agricultural Sciences, 2003,16(2):34-36. (in Chinese)
[11]	HU X, SULLIVAN GILBERT M, GUPTA M, THOMPSON S A. Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.). Theoretical and Applied Genetics, 2006,113:497-507. doi: 10.1007/s00122-006-0315-1 pmid: 16767448
[12]	张宏军, 肖钢, 谭太龙, 李栒, 官春云. EMS处理甘蓝型油菜(Brassica napus)获得高油酸材料. 中国农业科学, 2008,41(12):4016-4022.
	ZHANG H J, XIAO G, TAN T L, LI X, GUAN C Y. High oleate material of rapeseed (Brassica napus) produced by EMS treatment. Scientia Agricultura Sinica, 2008,41(12):4016-4022. (in Chinese)
[13]	黄永娟, 张凤启, 杨甜甜, 刘葛山, 蒋守华, 陈健美, 管荣展. EMS 诱变甘蓝型油菜获得高油酸突变体. 分子植物育种, 2011,9(5):611-616.
	HUANG Y J, ZHANG F Q, YANG T T, LIU G S, JIANG S H, CHEN J M, GUAN R Z. High oleate mutants of Brassica napus produced by EMS inducement. Molecular Plant Breeding, 2011,9(5):611-616. (in Chinese)
[14]	官春云, 刘春林, 陈社员, 彭琦, 李栒, 官梅. 辐射育种获得油菜(Brassica napus)高油酸材料. 作物学报, 2006,32(11):1625-1629.
	GUAN C Y, LIU C L, CHEN S Y, PENG Q, LI X, GUAN M. High oleic acid content materials of rapeseed (Brassica napus) produced by radiation breeding. Acta Agronomica Sinica, 2006,32(11):1625-1629. (in Chinese)
[15]	刘列钊, 王欣娜, 阎行颖, 王瑞, 徐新福, 卢坤, 李加纳. 航天诱变高油酸甘蓝型油菜突变体分子标记的筛选. 中国农业科学, 2012,45(23):4931-4938.
	LIU L Z, WANG X N, YAN X Y, WANG R, XU X F, LU K, LI J N. Molecular marker screen for high oleic acid in space flight mutant Brassica napus. Scientia Agricultura Sinica, 2012,45(23):4931-4938. (in Chinese)
[16]	STOUTJESDIJK P A, HURLESTONE C, SINGH S P, GREEN A G. High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous D12-desaturases. Biochemical Society Transactions, 2000,28:938-940. pmid: 11171263
[17]	陈苇, 李劲峰, 董云松, 李根泽, 寸守铣, 王敬乔. 甘蓝型油菜Fad2基因的RNA干扰及无筛选标记高油酸含量转基因油菜新种质的获得. 植物生理与分子生物学学报, 2006,32(6):665-671. pmid: 17167203
	CHEN W, LI J F, DONG Y S, LI G Z, CUN S X, WANG J Q. Obtaining new germplasm of Brassica napus with high oleic acid content by RNA interference and marker-free transformation of Fad2 gene. Journal of Plant Physiology and Molecular Biology, 2006,32(6):665-671. (in Chinese) pmid: 17167203
[18]	田保明, 廉玉利, 凌华, 杨光圣, 赵珍, 范兴福, 李旭娇, 师恭曜. RNAi干扰油菜Δ¹²脂肪酸脱饱和酶基因的表达效果. 中国油料作物学报, 2009,31(2):132-136.
	TIAN B M, LIAN Y L, LING H, YANG G S, FAN S F, LI X J, SHI G Y. Introduction of FAD2 gene fragment into Brassica napus via RNAi plasmid and enhanced oleic acid composition. Chinese Journal of Oil Crop Sciences, 2009,31(2):132-136. (in Chinese)
[19]	陈松, 浦惠明, 张杰夫, 高建芹, 陈锋, 龙卫华, 胡茂龙, 戚存扣. 转基因高油酸甘蓝型油菜新种质的获得. 江苏农业学报, 2009,25(6):1234-1237.
	CHEN S, PU H M, ZHANG J F, GAO J Q, CHEN F, LONG W H, HU M L, QI C K. Identification of high oleic acid germplasm from the T₂ progency of the transgenic Brassica napus L. Jiangsu Journal of Agriculture Sciences, 2009,25(6):1234-1237. (in Chinese)
[20]	PENG Q, HU Y, WEI R, ZHANG Y, GUAN C, RUAN Y. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds. Plant Cell Report, 2010,29:317-325.
[21]	SCHIERHOLT A, BECKER H C. Environmental variability and heritability of high oleic acid content in winter oilseed rape. Plant Breeding, 2001,120:63-66.
[22]	费维新, 吴新杰, 李强生, 陈凤祥, 侯树敏, 范志雄, 江莹芬, 雷伟侠, 荣松柏, 段晓莉, 胡宝成. 甘蓝型油菜高油酸材料的遗传分析. 中国农学通报, 2012,28(1):176-180.
	FEI W X, WU X J, LI Q S, CHEN F X, HOU S M, FAN Z X, JIANG Y F, LEI W X, RONG S B, DUAN X L. Genetic analysis of high oleic acid mutation materials in Brassica napus. Chinese Agricultural Science Bulletin, 2012,28(01):176-180. (in Chinese)
[23]	YANG Q, FAN C, GUO Z, QIN J, WU J, LI Q. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theoretical and Applied Genetics, 2012,125:715-729. doi: 10.1007/s00122-012-1863-1 pmid: 22534790
[24]	LONG W, HU M, GAO J, CHEN S, ZHANG J, CHENG L, PU H. Identification and functional analysis of two new mutant BnFAD2 Alleles that confer elevated oleic acid content in rapeseed. Frontiers in Genetics, 2018,9:399. doi: 10.3389/fgene.2018.00399 pmid: 30294343
[25]	傅寿仲, 吕忠进, 戚存扣, 陈爱华. 甘蓝型油菜十八碳烯酸的进一步改良. 江苏农业学报, 1995,11(1):16-20.
	FOU S Z, LÜ Z J, QI C K, CHEN A H. Further modification of levels of C18 unsaturated fatty acids in rapeseed. Jiangsu Journal of Agriculture Sciences, 1995,11(1):16-20. (in Chinese)
[26]	徐华军, 贺源辉, 陈秀芳. ⁶⁰Co射线对双低甘蓝型油菜(Brassica napus L.)的辐射效应. 核农学报, 1992,6(4):199-206.
	XU H J, HE Y H, CHEN X F. The effects of gamma irradiation on morphological cytological and biochemical characters of double-low rape (Brassica napus L.). Acta Agriculturae Nucleatae Sinica, 1992,6(4):199-206. (in Chinese)
[27]	高建芹, 浦惠明, 戚存扣, 张洁夫, 陈新军, 龙卫华, 傅寿仲. 应用气相色谱仪分析油菜脂肪酸含量. 江苏农业学报, 2008(5):581-585.
	GAO J Q, PU H M, QI C K, ZHANG J F, CHEN X J, LONG W H, FU S Z. Analysis of fatty acid content in rapeseed by gas chromatography. Jiangsu Journal of Agriculture Sciences, 2008(5):581-585. (in Chinese)
[28]	ZHANG Y M. Segregation Analysis of Quantitative Traits and Its R Software. Beau Bassin, Mauritius: Golden Light Academic Press, 2017.
[29]	WELLS R, TRICK M, SOUMPOUROU E, CLISSOLD L, MORGAN C, WERNER P. The control of seed oil polyunsaturated content in the polypoid crop species Brassica napus. Molecular Breeding, 2014,33:349-362. doi: 10.1007/s11032-013-9954-5 pmid: 24489479
[30]	曾新华. 不同诱变方法对油菜种子诱变效果及突变体的研究[D]. 武汉:华中农业大学, 2010.
	ZENG X H. Comparing effectiveness of different mutagens for seed quality and analysis of mutants in Brassica napus[D]. Wuhan: Huazhong Agriculture University, 2010. (in Chinese)
[31]	ZHANG H Z, SHI C H, WU J G, REN Y L, LI C T, ZHANG D Q, ZHANG Y F. Analysis of genetic and genotype × environment interaction effects from embryo, cytoplasm and maternal plant for oleic acid content of Brassica napus L. Plant Science, 2004,167(1):43-48.
[32]	ZHAO Q, WU J, CAI G, YANG Q, SHAHID M, FAN C, ZHANG C, ZHOU Y. A novel quantitative trait locus on chromosome A9 controlling oleic acid content in Brassica napus. Plant Biotechnology Journal, 2019,17(12):2313-2324. pmid: 31037811
[33]	LI Q, ZHENG Q, SHEN W Y, CRAM D, FOWLER D B, WEI Y D, ZOU J T. Understanding the biochemical basis of temperature-induced lipid pathway adjustments in plants. The Plant Cell, 2015(27):86-103.
[34]	HOU Q C, UFER G, BARTELS D. Lipid signaling in plant responses to abiotic stress. Plant, Cell and Environment, 2016,39:1029-1048.
[35]	STACY D S, ZOU J T, RANDALL J W. Abiotic factors influence plant storage lipid accumulation and composition. Plant Science, 2016,243:1-9. doi: 10.1016/j.plantsci.2015.11.003 pmid: 26795146
[36]	KONG Q, YANG Y Z, GUO L, YUAN L, MA W. Molecular basis of plant oil biosynthesis: insights gained from studying the WRINKLED1 transcription factor. Frontiers in Plant Sciences, 2020,11:24.
[37]	DAR A A, CHOUDHURY A R, KANCHARLA P K, ARUMUGAM N. The FAD2 gene in plants: Occurrence, regulation and role. Frontiers in Plant Sciences, 2017,8:1789.

年度 Year	世代 Generation	样本数 No. of sample	C18:1			C18:3
年度 Year	世代 Generation	样本数 No. of sample	平均值 Mean	变幅 Range (%)	变异系数 CV (%)	平均值 Mean	变幅 Range (%)	变异系数 CV (%)
2004	M₁	201	73.44	68.75—78.81	2.19	5.38	3.20—7.91	20.24
2005	M₂	578	73.97	56.14—84.90	4.50	6.02	3.52—8.97	32.63
2006	M₃	248	75.44	65.50—85.99	5.42	4.30	1.87—8.48	36.91
2007	M₄	217	78.76	69.36—87.19	9.69	3.92	1.57—14.02	45.04
2008	M₅	208	83.30	70.54—86.95	4.99	3.10	1.98—7.95	38.68
2009	M₆	65	81.23	73.51—85.47	2.36	4.00	2.54—6.38	14.20

世代 Generation	籽粒数 No. of seeds	脂肪酸 Fatty acid
世代 Generation	籽粒数 No. of seeds	C16:0	C18:0	C18:1	C18:2	C18:3	C20:1	C22:1
P₁	16	3.10±0.31	2.10±0.32	85.36±0.50	5.03±0.21	2.52±0.11	1.25±0.13	0.20±0.05
P₂	17	3.61±0.35	1.59±0.13	64.53±0.72	21.59±0.59	7.94±0.21	1.22±0.14	0.21±0.03
F₁(P₁×P₂)	38	3.28±0.29	1.72±0.22	73.98±1.29	11.66±0.90	4.93±0.42	1.28±0.17	0.19±0.05
F₁(P₂×P₁)	40	3.43±0.31	1.69±0.19	74.14±1.56	11.41±1.30	4.94±0.54	1.31±0.18	0.20±0.04
BC₁P₁	95	3.36±0.39	1.86±0.20	79.57±4.52	9.30±4.07	3.89±1.01	1.33±0.16	0.29±0.16
BC₁P₂	98	3.40±0.35	1.53±0.18	70.84±3.61	15.11±3.40	6.90±1.24	1.32±0.11	0.27±0.15
F₂	198	3.21±0.43	1.63±0.24	74.95±5.20	12.68±4.48	5.34±1.37	1.38±0.19	0.25±0.20

脂肪酸 Fatty acid	棕榈酸C16:0	硬脂酸C18:0	油酸C18:1	亚油酸C18:2	亚麻酸C18:3	二十碳烯酸C20:1	芥酸C22:1
C16:0	1	-0.106	-0.474**	0.409**	0.222**	-0.371**	0.029
C18:0		1	0.029	-0.037	-0.143*	0.033	-0.010
C18:1			1	-0.943**	-0.541**	0.204**	-0.062
C18:2				1	0.303**	-0.330**	-0.007
C18:3					1	-0.092	-0.005
C20:1						1	0.178*
C22:1							1

组合 Combination	C18:1
组合 Combination	备选模型 Model	极大似然值 Max-likelihood-value	AIC值 AIC value
B161×N137	2MG-ADI	-597.9275	1215.855
	2MG-EA	-607.3177	1220.635
	1MG-A	-607.3178	1220.636
B161×N27	2MG-A	-586.1464	1180.293
	2MG-ADI	-582.3989	1184.798
	1MG-AD	-590.8457	1189.691
B161×N15	2MG-A	-576.5989	1161.198
	1MG-AD	-582.3780	1172.756
	2MG-EAD	-583.5032	1173.006

一阶参数 Univalent parameter	估计值 Estimate value
一阶参数 Univalent parameter	B161×N137	B161×N27	B161×N15
第1对主基因的加性效应da	5.6587	4.7107	4.3096
第2对主基因的加性效应db	4.7263	6.3145	4.2901
第1对主基因的显性效应ha	/	/	/
第2对主基因的显性效应hb	/	/	/