龙卫华^,, 浦惠明^,, 高建芹, 胡茂龙, 张洁夫, 陈松江苏省农业科学院经济作物研究所/农业部长江下游棉花与油菜重点实验室,南京 210014

Creation of High-Oleic (HO) Canola Germplasm and the Genetic and Physiological Analysis on HO Trait

LONG WeiHua^,, PU HuiMing^,, GAO JianQin, HU MaoLong, ZHANG JieFu, CHEN SongInstitute of the Industrial Crops, Jiangsu Academy of Agriculture Sciences/Key Lab of Cotton and Rapeseed (Nanjing) of Ministry of Agriculture, Nanjing 210014

通讯作者: 浦惠明,Tel：025-84390370;E-mail： puhuiming@126.com

责任编辑: 李莉
收稿日期:2020-07-4接受日期:2020-09-1网络出版日期:2021-01-16

基金资助:

国家重点研发项目.2016YFD0101300 江苏省现代作物生产协同创新中心.JCIC-MCP

Received:2020-07-4Accepted:2020-09-1Online:2021-01-16
作者简介 About authors
龙卫华,Tel：025-84390368;E-mail： long-weihua@163.com。








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.
Keywords：Brassica napus L.;radiation mutagenesis;germplasm creation;high oleic trait;genetic analysis;response to low temperature


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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2021.02.003
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 Acricultura Sinica, 2021, 54(2): 261-270 doi:10.3864/j.issn.0578-1752.2021.02.003


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0 引言

【研究意义】油菜是中国三大主要油料作物之一,菜籽油在中国居民日常生活中具有良好的消费基础^[1]。近年来,菜籽油已成为国产植物油的第一大来源,因此,菜籽油的品质（主要是脂肪酸含量比例）对中国居民健康具有重要影响^[2]。在中国油菜全面实现“双低化”（2003年）后,十八碳脂肪酸含量的改良成为目前油菜品质育种的重要目标^[3]。与普通双低菜籽油相比,高油酸（high oleic,HO）菜籽油^[4,5]具有四大优势：（1）较耐贮藏,货架期较长;（2）可降低低密度脂蛋白含量,减少胆固醇形成,预防人体心血管疾病;（3）甲酯化程度高,燃烧值高,更有利于生产生物柴油;（4）有利于提高菜籽油的氧化稳定性,是菜籽油不容易变质生成反式脂肪酸（或位置异构脂肪酸）和产生异味^[6]。创建HO甘蓝型油菜新种质并以此培育HO油菜品种是提供高品质菜籽油的有效途径。【前人研究进展】截至目前,国内外****通过不同方法获得了HO甘蓝型油菜种质。国际上,最早用甲基磺酸乙酯（ethylmethylsulfone,EMS）诱变油菜种子并在后代筛选出高油酸突变系,油酸含量最高可达88%^[7,8]。此后,有报道用叠氮化钠诱变出高油酸油菜突变体^[9]。国内****也重视HO材料创制并得到HO种质。和江明等^[10]最早用EMS诱变处理油菜小孢子筛选出油酸含量为80.3%的高油酸突变体材料,随后多位****通过化学诱变得到高油酸突变体^[11,12,13]。而物理方法主要是采用放射性射线辐照产生变异。运用这种方法成功获得高油酸种质的共有2例,一是利用⁶⁰Co射线辐照油菜干种子后连续定向选择获得最高油酸含量达93.5%（近红外法测定）的株系;二是通过航天诱变方法获得一个高油酸含量（87.22%）的突变株系^[13,14]。此外,转基因方法也可以获得高油酸种质。多位****利用RNAi技术获得了油酸含量超过80%的株系,但转基因种质因需要经过严格的释放前环境评审而不能较快在育种中得到应用,而且目前还未有成功释放的先例^{[15,16,17,18,19,20]}。综合来看,采用非转基因方法得到的高油酸种质具有可即时应用的比较优势。不同****对油菜高油酸性状的遗传模式进行了探索。品系19782/7531的高油酸性状（C18:1=78.4%）由2个突变位点控制^[21]。对高油酸亲本DMS100（C18:1=77%）与普通品系衍生的DH群体进行QTL定位后发现高油酸性状主要由1个主效位点控制^[11]。HO自交系Y539（C18:1=87.22%）的高油酸性状是由分别位于A05和A01染色体上的2个主效基因控制^[15]。H005的高油酸性状（C18:1=83.10%）由2对具有加性和显性效应的主效基因控制^[22]。HO品系SW Hickory（C18:1=78%）的高油酸性状由位于A5染色体上1个主效QTL控制^[23]。HO品系N1379T（C18:1=85%）的高油酸性状则是由位于A5和C5染色体上的2个位点共同控制^[24]。【本研究切入点】尽管通过不同手段得到了HO种质,但控制其HO性状的遗传模式并非一致,表明这些种质在遗传上具有不同的机制。同时,已有HO种质的生理特性也未有深入研究。21世纪初江苏省农业科学院经济作物研究所油菜研究室利用辐射方法获得油菜脂肪酸组分的变异,并结合多种育种方法,最终成功创建一个全新的HO种质B161。【拟解决的关键问题】本研究通过对B161中HO性状的遗传模式及其油酸含量变化的生理特性进行研究,推进该种质的育种利用进程,进一步改良菜籽油品质,为提供高品质菜籽油奠定基础。

1 材料与方法

1.1 材料

诱变原始材料为高世代油菜自交系L13-306-171,油酸（C18:1）与亚麻酸（C18:3）含量经近红外分析含量分别约为72.21%和4.18%（后期经气相色谱分析C18:1≈68%,C18:3≈7%）,其系谱见傅寿仲等^[25]。常规油菜品系N15、N27和N137为3个不同遗传背景的高世代育种自交系。以上材料均由江苏省农业科学院经济作物研究所提供。

1.2 油菜HO种质的诱变及选育过程

选取L13-306-171干净一致的1 000粒种子（即M₀代）排列在培养皿内湿润滤纸上于25℃暗培养,每皿100粒种子,共10皿。待胚根萌动露白后送至扬州市辐照中心进行800 Gray ⁶⁰Co-?射线辐照（辐照剂量参考徐华军等^[26]）。辐射处理后的萌动种子继续发芽后栽于大田。2004年在辐射当代（即M₁代）选择优势单株套袋自交收获种子。测定脂肪酸组分后按油酸含量从高到低选择30株单株种子作为基础选择群体（即M₂代）。从每个单株自交种子中分别取等量种子混合种植。随后连续3年重复上年度的方法获得M₅代基础群体。在本世代选择质量较好的种子进行半粒法脂肪酸含量测定。2007年秋选取油酸含量>80%的对应半粒种子在室内发芽后栽于大田形成M₆代。2008年继续选择油酸高值单株种植于大田,于2009年春季选择单株花蕾进行小孢子培养得到若干DH株系（M₇代）,选取油酸含量较高且亚麻酸含量较低的DH系作为候选株系。次年经全生育期观察比较后筛选最优HO株系。

1.3 高油酸性状的遗传群体构建

以中选的HO种质B161（P₁,C18:1=（85.36±0.50）%）和常规油菜品系N15、N27和N137亲本（P₂）种植于江苏省农业科学院溧水植物科学基地油菜育种田,2014年春季杂交分别获得3个杂交组合的共6个正反交F₁。2015年将各亲本自交,F₁自交得到F₂分离群体种子,同时F₁与2个亲本分别回交得到BC₁代的足量种子（BC₁P₁和BC₁P₂）。

1.4 低温与常温下HO品系与常规品系的组织样品制备

将B161和N137的干净种子在培养皿中发芽后至下胚轴长约0.5 cm后,移栽至直径为15 cm且装满营养土的盆钵中,每品系6钵,每钵4株苗,置于光照培养箱（25℃ 12 h光照/15℃12 h黑暗,光照强度= 2 000 lx）内生长。于三叶期时将对应品系盆钵分成2部分：一半继续放置在原培养箱;另一半置于另一培养箱（12℃ 12 h光照/6℃ 12 h黑暗,光照强度=2 000 lx）内培养。待7 d后将盆钵取出并选取各培养箱内生长一致的菜苗,迅速洗净根系,用剪刀分离根（下胚轴以下部分）、茎（去掉最后一片叶连接点以上部分）、叶（包括去掉主叶脉后的整张叶片）和叶柄等组织,分别将两品系不同单株的相同组织混合置于牛皮纸袋中编号,烘干备用。

1.5 油菜种子成熟及种子发芽过程的样品制备

于大田油菜开花后选择长势均衡、花期一致的B161和N137植株,用棉线标记主轴及倒一、倒二分枝同一天开放的花朵,每个材料标记约10株,每株标记约100朵花,一周后再标记一次作为重复。由于早期的种子含水量很大,故而自角果龄15日开始每7天取样一次,直至种子成熟。大田中角果置于冰盒中带至实验室后,迅速将幼嫩种子从角果中小心剥出,每品系每个样品至少选取100粒种子,及时烘干编号备用。

发芽试验按照粮油检验发芽试验（GB/T 5520-2011）的方法进行,选取B161和N137的饱满种子自开始之日起每天于同一时间剥出发芽种子中的子叶,直至第5天成苗。取样时迅速用镊子剥取子叶部分,每品系每个样品至少选取50个子叶组织,混合后立即烘干编号备用。

1.6 取样及其脂肪酸含量测定

用于遗传分析的6个世代群体种子,按照亲本（P₁、P₂）、F₁、BC₁P₁、BC₁P₂和F₂分别取出粒数不等的种子,按照高建芹等^[27]的半粒法测定种子脂肪酸含量。各个组织样品按照国标（GB/T17377-1998）的方法在安捷伦GC 6910型气相色谱仪上测定脂肪酸含量。由于根、茎、叶和叶脉组织的含油量低,因此,其干样至少保证在0.5 g以上。在加石油醚-乙醚混合液之前,样品需要充分打样磨碎。

1.7 数据分析、图像绘制以及遗传模型分析

采用Microsoft Excel计算最大值、最小值、平均值、变异系数及相关系数。采用Graphpad Prism7软件进行绘图。根据作物数量性状混合遗传模型及主基因+多基因多世代联合分析方法,采用章元明等^[28]发布的植物数量性状软件包（the R software package of SEgregation analysis,SEA）对3个不同遗传背景的6个世代分析群体的高油酸性状进行24个遗传模型的极大似然分析,采用最小AIC值准则筛选最适遗传模型,用最小二乘法估计相应的遗传参数,并估计主基因和多基因效应等遗传参数。

2 结果

2.1 诱变群体各世代油酸与亚麻酸的含量变化及HO种质B161的获得

采用辐射诱变后发现后续世代油酸和亚麻酸含量变异范围明显增加,筛选效果明显。自诱变当代到脂肪酸含量稳定的7个世代,油酸和亚麻酸含量在定向选择过程中均经历了由集中到分散然后又集中的变化趋势（表1）。对2004年（M₁代）套袋自交收的单株种子进行脂肪酸分析发现,油酸含量变幅为68.75%—78.81%,亚麻酸含量变幅为3%—8%,表明诱变产生效果。经过4个世代（M₂—M₅）对油酸进行极端选择后,油酸含量变幅为70.54%—86.95%,但平均值大幅升高。进一步于M₆代对极端单株进行小孢子培养后获得具有65个株系的DH系群体,根据脂肪酸测定结果筛选得到油酸含量≥85%且亚麻酸含量为3%—4%的多个株系。次年经全生育期生长势及农艺性状比较后获得HO稳定品系B161（C18:1为85.4%,C18:3为2.5%）。

Table 1
表1
表1诱变群体不同世代中C18:1及C18:3含量分布
Table 1Distribution of C18:1 and C18:3 contents in generations after mutagenesis

年度 Year	世代 Generation	样本数 No. of sample	C18:1			C18:3
			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

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2.2 HO种质B161高油酸性状的遗传模式

2.2.1 遗传群体中全脂肪酸组分含量的性状变异 成功利用HO种质B161与3个常规品系构建了6个世代分离群体,其中,组合B161×N137的全脂肪酸含量情况如表2。正反交F₁的油酸与亚麻酸含量值基本相当,表明HO性状不受细胞质效应的影响。3个组合衍生的6个世代分离群体油酸含量呈连续性分布,符合数量性状的特征。另外,油酸含量在3个群体中呈正态分布,表明该性状在遗传上受多基因控制。

Table 2
表2
表2组合B161×N137的6个世代遗传群体脂肪酸含量统计
Table 2Statistic of fatty acid contents in the six-generation genetic populations from B161×N137

世代 Generation	籽粒数 No. of seeds	脂肪酸 Fatty acid
		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

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2.2.2 菜籽油中各脂肪酸含量的相关性分析 对组合B161×N137的F₂分离群体单株的脂肪酸含量进行了相关性分析（表3）,结果表明,在各十八碳脂肪酸中油酸含量除了与亚油酸和亚麻酸含量具有显著负相关以外,还与棕榈酸和二十碳烯酸显著相关,而与硬脂酸含量相关较小。

Table 3
表3
表3组合B161×N137的F₂群体中各脂肪酸含量的相关系数
Table 3Correlation coefficient among the fatty acid contents in F₂ population of B161×N137

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

*表示在0.05水平上差异显著,**表示在0.01水平上差异显著
* means the significance at the 0.05 level, ** means the significance at the 0.01 level

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2.2.3 HO种质B161高油酸含量性状的遗传分析 利用SEA软件对3个遗传分析群体油酸含量数据分别计算所有的遗传模型,获得各个遗传模型的最大似然值以及AIC值。根据最小AIC的遗传模型优选准则,选取各遗传群体中3个最小AIC值的遗传模型作为备选（表4）。结果显示,油酸含量大部分符合2对主效基因的遗传模型,但不同遗传群体估算的基因互作效应略有差别。油酸含量的最佳遗传模型为2MG-A,即2对加性主基因模型。

Table 4
表4
表43个组合的候选遗传模型及其极大对数似然函数值和AIC值
Table 4Candidate genetic models of three populations and their maximum log likelihood values and AIC values

组合 Combination	C18:1
	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

MG：主基因;MX：主基因+多基因;A：加性;D：显性;I：互作;E：相等
MG: Major gene; MX: Mixed major gene and polygene; A: Additive; D: Dominance; I: Interaction; E: Equal

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对筛选得到的最佳遗传模型估计了遗传参数（表5）。结果显示,控制高油酸含量的2对主效基因在3个组合遗传群体中体现出的效应值均较为接近,每个主效基因可提高油酸含量4—6个百分点。

Table 5
表5
表5最适遗传模型下3个杂交组合中油酸含量的遗传参数估计
Table 5Estimates of genetic parameters for oleic acid content under the optimal model

一阶参数 Univalent parameter	估计值 Estimate value
	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	/	/	/

/：无估计值
/: Means no value

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2.3 HO种质B161与常规品系苗期营养器官中油酸与亚麻酸含量的比较

不同温度下HO品系与常规品系苗期组织中油酸与亚麻酸含量具有显著变化（表6）。常规品系根、茎、叶及叶柄等组织中的油酸含量在常温和低温下没有差异;但HO品系各组织在常温下油酸含量均较常规品系高,低温使各组织油酸含量显著降低,以叶柄降幅最大。无论在低温或常温下常规品系各组织的亚麻酸含量均高于HO品系,低温使HO品系各组织的亚麻酸含量显著升高。

Table 6
表6
表6不同温度下HO品系与常规品系苗期不同组织中油酸与亚麻酸的含量
Table 6The C18:1 and C18:3 contents in tissues between HO and normal lines at seedling stage under different temperatures

脂肪酸 Fatty acid	材料 Lines	处理 Treat	根 Root	茎 Stem	叶 Leaf	叶柄 Petiole
C18:1	B161	常温 Normal temperature	47.56±2.77a	37.34±2.37a	29.99±2.21a	55.72±3.08a
		低温 Low temperature	38.87±2.33b	23.72±2.28b	14.28±2.74b	20.37±2.42b
	N137	常温 Normal temperature	12.67±1.90c	15.90±1.53c	10.48±1.52c	7.16±0.85c
		低温 Low temperature	12.34±1.61c	17.25±1.67c	11.69±1.75c	8.61±1.14c
C18:3	B161	常温 Normal temperature	18.89±1.35d	28.17±2.26d	30.55±1.96d	14.41±1.43d
		低温 Low temperature	25.56±2.65c	33.31±2.22c	38.37±2.33c	36.92±1.27c
	N137	常温 Normal temperature	33.53±3.37b	41.92±2.72b	45.86±2.07b	43.02±2.72b
		低温 Low temperature	49.56±3.13a	51.91±3.45a	52.81±1.99a	53.75±2.38a

不同小写字母表示在0.05水平差异显著
The different lowercase letters in the table indicate significance at 0.05 level

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2.4 HO种质B161油酸与亚麻酸含量的变化特征

2.4.1 种子成熟过程中HO种质B161与常规品系油酸与亚麻酸的积累 HO品系B161与常规油菜品系N137在花后至种子成熟过程中油酸和亚麻酸含量的增减趋势一致,但含量具有显著差异。两品系油酸含量在此段时期内均表现为先迅速增长而后缓慢增长的趋势。B161在花后第15天油酸含量即达到约50%,至第35天达到约80%后仍继续缓慢增长;整个过程高于常规品系20个百分点。两品系亚麻酸含量在此过程中均表现为持续降低趋势,在花后15 d时两者差异不大,但在此后差距持续拉大（图1）。

图1

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图1花后HO品系和常规品系种子中油酸与亚麻酸的含量变化

Fig. 1Changes of oleic acid and linolenic acid contents in seeds of HO and normal lines after pollination



2.4.2 B161与常规品系子叶中油酸与亚麻酸在发芽进程中的代谢 HO品系与常规品系子叶中油酸与亚麻酸的相对含量在发芽过程中的变化（图2）具有明显差异。在种子萌发后5 d之内,两品系子叶的油酸含量均呈持续下降趋势,下降速率接近,两品系差异值基本维持在20个百分点左右。相反的,在此进程中,两品系亚麻酸含量均呈持续上升趋势,但两品系差异经历了由小到大再变小的过程。

图2

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图2发芽后HO品系和常规品系子叶中油酸与亚麻酸的含量变化

Fig. 2Changes of oleic acid and linolenic acid contents in cotyledons of HO and normal lines after germination

3 讨论

诱变是创建油菜遗传变异材料的有效方法。目前,已报道的非转基因油菜HO种质均通过诱变获得,但诱变方法以及诱变对象不尽相同。大部分HO材料都是通过化学诱变油菜种子获得的^{[7,9,11-13,20-21,29]},也有通过诱变小孢子获得^[10],而利用辐射处理油菜种子得到高油酸种质的案例较少^[14,15]。本研究亦是采用辐射诱变,但在辐射对象上与前人有所不同。本研究辐射对象是处于萌动露白状态的油菜种子。此种状态下油菜种子中各项生理生化活动刚刚启动,对外界环境极度敏感^[30]。从理论上说辐射处理具有较高的诱变率。本研究中在早期世代（M₂代）即发现了油酸含量显著升高的株系,不但说明创建思路是可行的,而且也表明突变位点位于控制HO性状的主效基因内,同时这些主效基因可能位于相对容易诱变的染色体区域。此外,由于辐射诱变可能造成油菜染色体变异,若持续采用系谱法选择可能多代自交仍不能纯合稳定,因此,本例中采用小孢子培养技术迅速固定高油酸性状,效果很好,这与LORIN等^[9]使用的方法一致。本研究获得的HO品系油酸和亚麻酸含量均与现有报道中的极端值相当,表明所集成的诱变及选择方法在油菜上具有实用性,对其他的类似性状可能也具有借鉴价值。

菜籽油中的油酸含量主要受遗传控制,环境影响较小^[21,31]。本研究也发现HO新种质高油酸性状的遗传贡献率高,与前人结果一致。在控制该性状的基因数目上,研究结果有所不同。HU等^[11]、官春云等^[14]、、YANG等^[23]利用QTL定位或基因克隆等方法发现高油酸性状受一个主效基因位点控制。而SCHIERHOLT等^[15]和刘列钊等^[21]分别利用遗传试验和分子标记筛选表明高油酸性状主要受2个主效位点控制。随后WELLS等^[29]利用EMS诱变获得了一批油菜减饱和酶突变体,其不同位点突变对油酸含量的增量效应不同。ZHAO等^[32]还发现在A9染色体也有一个位点控制油酸含量。这表明油菜中油酸含量的控制位点较为丰富。本研究中的高油酸新种质虽然也受2对基因控制,但其基因位点需要进一步确认。从育种实践而言,本研究创建的新种质高油酸性状遗传模式相对简单,育种利用较为容易。自2010年开始将HO种质的高油酸性状转育进入萝卜质不育系统并进行了杂交组合配置工作,品比试验发现一些组合的全生育期表现和产量与对照品种相当,表明本研究发现的高油酸种质具有育种利用潜力（电子附表1）。

脂肪酸是植物细胞质膜的主要组成成分,是细胞与环境互作的前哨,因此,脂肪酸的合成与代谢对油菜的非生物胁迫抗性至关重要^[33,34,35]。油菜生育期长,无论是冬油菜还是春油菜在营养生长阶段都会受到温度影响。本研究发现,常温下HO种质营养器官以及种子中的油酸含量均高于常规品系,而亚麻酸含量则低于常规品系,表明（1）HO种质中控制油酸的基因是组成型表达,可能是油菜中2个组成型表达的BnFAD2发生了突变。（2）油菜的脂肪酸合成途径中油酸有2个去向,绝大部分进入减饱和途径生成亚油酸和亚麻酸,少部分进入加碳途径生成二十碳烯酸和芥酸^[36]。基于油酸与亚麻酸之间的强烈负相关关系,亚麻酸降低是油酸升高的必然结果。也有可能是HO种质中亚麻酸合成酶也产生突变,导致该酶活性降低,不能合成更多的亚麻酸。本研究还发现,低温下HO种质油酸含量降低和亚麻酸含量增加,但均低于常规品系,这表明（1）低温能够增加突变油酸控制基因的表达,提高油酸的转化率,这与BnFAD2的特性符合^[37]。（2）亚麻酸是高不饱和脂肪酸,易于氧化。其含量的增加,可能是油菜进行呼吸作用时更偏好于将亚麻酸氧化从而为其自身提供足够能量;也有可能是因为亚麻酸合成酶活性的变化所致。另外,发现HO种质与常规品系在种子成熟过程和发芽过程中的亚麻酸含量并非持续保持平行差异,说明在HO种质中除了油酸含量变化影响亚麻酸含量之外,亚麻酸本身的合成控制酶也在起作用。值得提出的是,低温下常规品系较HO种质能合成更多的高不饱和度脂肪酸,这意味着常规品系在低温下细胞膜具有更好的流动性,在表型上则可能具有更强的耐寒性。因此可以推断,在较寒冷的油菜产区,HO品系可能耐寒性较常规品系差,需要关注。

4 结论

获得双低甘蓝型油菜HO新种质B161,其高油酸性状受2个主效基因控制。B161中控制高油酸性状的基因为组成型表达,低温对其有诱导增强作用。高油酸种质B161具有育种利用潜力。

参考文献 View Option 原文顺序
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[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/s00122-006-0315-1URLPMID:16767448 [本文引用: 4]
The quality of canola oil is determined by its constituent fatty acids such as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Most canola cultivars normally produce oil with about 55-65% oleic acid and 8-12% linolenic acid. High concentrations of linolenic acid lead to oil instability and off-type flavor, while high levels of oleic acid increase oxidative stability and nutritional value of oil. Therefore, development of canola cultivars with increased oleic acid and reduced linolenic acid is highly desirable for canola oil quality. In this study, we have mapped one locus that has a major effect and one locus that has a minor effect for high oleic acid and two loci that have major effects for low linolenic acid in a doubled haploid population. The major locus for high C18:1 was proven to be the fatty acid desaturase-2 (fad2) gene and it is located on the linkage group N5; the minor locus is located on N1. One major QTL for C18:3 is the fatty acid desaturase-3 gene of the genome C (fad3c) and it is located on N14. The second major QTL resides on N4 and is the fad3a gene of the A genome. We have sequenced genomic clones of the fad2 and fad3c genes amplified from an EMS-induced mutant and a wild-type canola cultivar. A comparison of the mutant and wild-type allele sequences of the fad2 and fad3c genes revealed single nucleotide mutations in each of the genes. Detailed sequence analyses suggested mechanisms by which both the mutations can cause altered fatty acid content. Based on the sequence differences between the mutant and wild-type alleles, two single nucleotide polymorphism (SNP) markers, corresponding to the fad2 and fad3c gene mutations, were developed. These markers will be highly useful for direct selection of desirable fad2 and fad3c alleles during marker-assisted trait introgression and breeding of canola with high oleic and low linolenic acid.

[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.
[本文引用: 1]

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)
[本文引用: 1]

[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.
[本文引用: 3]

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)
[本文引用: 3]

[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.
URL [本文引用: 3]

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)
URL [本文引用: 3]

[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.
URL [本文引用: 4]

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)
URL [本文引用: 4]

[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.
URLPMID:11171263 [本文引用: 1]
Genetic engineering methods have been used successfully to modify the fatty acid profile of elite Australian germplasm of Brassica napus and B. juncea. Co-suppression plasmids carrying oleate desaturase genes from each species have been constructed and transferred into Australian elite breeding lines of B. napus and B. juncea using Agrobacterium tumifaciens plant-transformation techniques. Modifications to existing Brassica transformation protocols and the use of an intron-interrupted hygromycin-resistance gene as the selectable marker have resulted in improved transformation efficiencies. Silencing of the endogenous oleate desaturase genes has resulted in substantial increases in oleic acid levels, up to 89% in B. napus and 73% in B. juncea.

[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.
URLPMID:17167203 [本文引用: 1]
A seed-specific Fad2 gene expression cassette, which is free-marker gene and with sense and antisense structure, was constructed by using the promoter and terminator of rape seed storage protein cruciferin gene. Transgenic rape plants without selection marker genes were obtained by Agrobacterium-mediated transformation. The oleic acid content of transgenic plant seeds is 83.9%, which is nearly the same as that of Brassica napus with double Fad2 mutation (85%). The result of RT-PCR analysis shows that the raising of oleic acid content may be due to the degradation of Fad2 mRNA induced by co-transformation of sense-antisense Fad2 gene. These transgenic plants with high oleic acid trait grew normally and without the disadvantageous agronomic traits such as weak cold resistance, tardy development, death of buds and low rate of seed setting caused by Fad2 inactivation in mutant Brassica napus plants. This work would serve as a good base for breeding of more lines with high oleic acid content.
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)
URLPMID:17167203 [本文引用: 1]
A seed-specific Fad2 gene expression cassette, which is free-marker gene and with sense and antisense structure, was constructed by using the promoter and terminator of rape seed storage protein cruciferin gene. Transgenic rape plants without selection marker genes were obtained by Agrobacterium-mediated transformation. The oleic acid content of transgenic plant seeds is 83.9%, which is nearly the same as that of Brassica napus with double Fad2 mutation (85%). The result of RT-PCR analysis shows that the raising of oleic acid content may be due to the degradation of Fad2 mRNA induced by co-transformation of sense-antisense Fad2 gene. These transgenic plants with high oleic acid trait grew normally and without the disadvantageous agronomic traits such as weak cold resistance, tardy development, death of buds and low rate of seed setting caused by Fad2 inactivation in mutant Brassica napus plants. This work would serve as a good base for breeding of more lines with high oleic acid content.

[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.
URL [本文引用: 1]

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)
URL [本文引用: 1]

[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.
[本文引用: 1]

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)
[本文引用: 1]

[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.
URL [本文引用: 2]

[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.
[本文引用: 4]

[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.
[本文引用: 1]

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)
[本文引用: 1]

[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/s00122-012-1863-1URLPMID:22534790 [本文引用: 2]
Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with different content of C18:3 in amphidiploid B. napus.

[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.2018.00399URLPMID:30294343 [本文引用: 1]
Rapeseed (Brassica napus L.) is a vital oil crop worldwide. High oleic acid content is a desirable quality trait for rapeseed oil, which makes it more beneficial to human health. However, many germplasm resources with high oleic acid content in rapeseed have not been evaluated with regard to their genotypes, making it difficult to select the best strains with this trait for the breeding of high oleic acid rapeseed variety. This work was to explore the gene-regulation mechanism of this trait using a new super-high oleic acid content ( approximately 85%) line N1379T as genetic material. In this study, the sequences of four homologous fatty acid desaturase (BnFAD2) genes were compared between super-high ( approximately 85%, N1379T) and normal ( approximately 63%) oleic acid content lines. Results showed that there were two single-nucleotide polymorphisms (SNPs) in BnFAD2-1 and BnFAD2-2, respectively, which led to the amino acid changes (E106K and G303E) in the corresponding proteins. Functional analysis of both genes in yeast confirmed that these SNPs were loss-of-function mutations, thus limiting the conversion of oleic acid to linoleic acid and resulting in the considerable accumulation of oleic acid. Moreover, two specific cleaved amplified polymorphic sequences (CAPS) markers for the two SNPs were developed to identify genotypes of each line in the F2 and BC1 populations. Furthermore, these two mutant loci of BnFAD2-1 and BnFAD2-2 genes were positively associated with elevated oleic acid levels and had a similar effect with regard to the increase of oleic acid content. Taken together, these two novel SNPs in two different BnFAD2 genes jointly regulated the high oleic acid trait in this special germplasm. The study provided insight into the genetic regulation involved in oleic acid accumulation and highlighted the use of new alleles of BnFAD2-1 and BnFAD2-2 in breeding high oleic acid rapeseed varieties.

[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.
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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)
URL [本文引用: 1]

[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.
URL [本文引用: 1]
The dormant seeds of three varieties of double-low rape were treated with 0,80,100,120,140 and 160 krad of γ-ray.The dose-effect curves of survival rate,yield,length of pod,rate of bearing pods and oil content in rapeseed fit the equation:y= Exp (-Ax-Bx2).The dose-effect curves of field germination rate,seed bearing rate,number of seeds per pod and chromosome aberration rate fit the equation:y = A + Bx.The dose-effect curve of morphological malformation rate fit the equation:y = AeBx The content's of erucic acid,linolenic acid,glucosino-lates and protein in rapeseed,changed irregularly with the increase of doses of gamma irradiation.Close correlation were found among the effects of irradiation on morphological,cytological and biochemical characters.The radiosensitivities of rape varieties were apparently different.
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)
URL [本文引用: 1]
The dormant seeds of three varieties of double-low rape were treated with 0,80,100,120,140 and 160 krad of γ-ray.The dose-effect curves of survival rate,yield,length of pod,rate of bearing pods and oil content in rapeseed fit the equation:y= Exp (-Ax-Bx2).The dose-effect curves of field germination rate,seed bearing rate,number of seeds per pod and chromosome aberration rate fit the equation:y = A + Bx.The dose-effect curve of morphological malformation rate fit the equation:y = AeBx The content's of erucic acid,linolenic acid,glucosino-lates and protein in rapeseed,changed irregularly with the increase of doses of gamma irradiation.Close correlation were found among the effects of irradiation on morphological,cytological and biochemical characters.The radiosensitivities of rape varieties were apparently different.

[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.
URL [本文引用: 1]
运用Agilent 6890N GC对具有不同脂肪酸组分的油菜样品,采用半粒、单粒和混合样3种测试方法进行分析,建立适合于油菜籽各脂肪酸组分含量的定量测试程序.结果表明:在本研究设定的分析条件下,二阶程序升温条件下对油菜7种主要脂肪酸的检测效果好于恒温条件,而测试效率则恒温条件高于二阶程序升温条件下.重复性和再现性试验结果显示:在二阶程序升温和恒温条件下半粒种子、单粒种子和混合样3种方法测试的脂肪酸含量大于5%的组分和小于5%的组分重复间绝对误差、相对误差均小于国家标准,表明这3种方法重复性、再现性好,能满足不同品质材料、不同检测要求的全脂肪酸组分的定量分析需要.
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)
URL [本文引用: 1]
运用Agilent 6890N GC对具有不同脂肪酸组分的油菜样品,采用半粒、单粒和混合样3种测试方法进行分析,建立适合于油菜籽各脂肪酸组分含量的定量测试程序.结果表明:在本研究设定的分析条件下,二阶程序升温条件下对油菜7种主要脂肪酸的检测效果好于恒温条件,而测试效率则恒温条件高于二阶程序升温条件下.重复性和再现性试验结果显示:在二阶程序升温和恒温条件下半粒种子、单粒种子和混合样3种方法测试的脂肪酸含量大于5%的组分和小于5%的组分重复间绝对误差、相对误差均小于国家标准,表明这3种方法重复性、再现性好,能满足不同品质材料、不同检测要求的全脂肪酸组分的定量分析需要.

[28] ZHANG Y M. Segregation Analysis of Quantitative Traits and Its R Software. Beau Bassin, Mauritius: Golden Light Academic Press, 2017.
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[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/s11032-013-9954-5URLPMID:24489479 [本文引用: 2]
Many important plant species have polyploidy in their recent ancestry, complicating inferences about the genetic basis of trait variation. Although the principal locus controlling the proportion of polyunsaturated fatty acids (PUFAs) in seeds of Arabidopsis thaliana is known (fatty acid desaturase 2; FAD2), commercial cultivars of a related crop, oilseed rape (Brassica napus), with very low PUFA content have yet to be developed. We showed that a cultivar of oilseed rape with lower than usual PUFA content has non-functional alleles at three of the four orthologous FAD2 loci. To explore the genetic basis further, we developed an ethyl methanesulphonate mutagenised population, JBnaCAB_E, and used it to identify lines that also carried mutations in the remaining functional copy. This confirmed the hypothesised basis of variation, resulting in an allelic series of mutant lines showing a spectrum of PUFA contents of seed oil. Several lines had PUFA content of ~6 % and oleic acid content of ~84 %, achieving a long-standing industry objective: very high oleic, very low PUFA rapeseed without the use of genetic modification technology. The population contains a high rate of mutations and represents an important resource for research in B. napus.

[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.
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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)
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[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
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[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
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[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
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[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.2015.11.003URLPMID:26795146 [本文引用: 1]
The demand for plant-derived oils has increased substantially over the last decade, and is sure to keep growing. While there has been a surge in research efforts to produce plants with improved oil content and quality, in most cases the enhancements have been small. To add further complexity to this situation, substantial differences in seed oil traits among years and field locations have indicated that plant lipid biosynthesis is also influenced to a large extent by multiple environmental factors such as temperature, drought, light availability and soil nutrients. On the molecular and biochemical levels, the expression and/or activities of fatty acid desaturases, as well as diacylglycerol acyltransferase 1, have been found to be affected by abiotic factors, suggesting that they play a role in the lipid content and compositional changes seen under abiotic stress conditions. Unfortunately, while only a very small number of strategies have been developed as of yet to minimize these environmental effects on the production of storage lipids, it is clear that this feat will be of the utmost importance for developing superior oil crops with the capability to perform in a consistent manner in field conditions in the future.

[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
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[37] DAR A A, CHOUDHURY A R, KANCHARLA P K, ARUMUGAM N. The FAD2 gene in plants: Occurrence, regulation and role
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