叶桑, 崔翠, 郜欢欢, 雷维, 王刘艳, 王瑞莉, 陈柳依, 曲存民, 唐章林, 李加纳, 周清元^,西南大学农学与生物科技学院,重庆400715

QTL Identification for Fatty Acid Content in Brassica napus Using the High Density SNP Genetic Map

YE Sang, CUI Cui, GAO HuanHuan, LEI Wei, WANG LiuYan, WANG RuiLi, CHEN LiuYi, QU CunMin, TANG ZhangLin, LI JiaNa, ZHOU QingYuan^,College of Agronomy and Biotechnology, Southwest University, Chongqing 400715

通讯作者: 周清元,Tel：13883388890;E-mail： zhouqy2005@163.com

收稿日期:2019-05-28接受日期:2019-07-11网络出版日期:2019-11-08

基金资助:

国家重点研发计划.2018YFD0100500 农业部现代农业产业技术体系.CARS-12 重庆市技术创新与应用发展.cstc2019jscx-msxmX0383

Received:2019-05-28Accepted:2019-07-11Online:2019-11-08
作者简介 About authors
叶桑,Tel：13068300612;E-mail： 837276825@qq.com。

崔翠,Tel：13883787860;E-mail： cuigreeny@163.com。叶桑和崔翠为同等贡献作者。












摘要
目的 菜籽油在烹饪、食品加工及工业生产中广泛应用,因此,根据生产需要改善菜籽油脂肪酸组分是油菜育种的重要目标。通过对2种环境下甘蓝型油菜主要脂肪酸组成进行QTL定位分析,寻找甘蓝型油菜脂肪酸组分的QTL及影响本群体脂肪酸组分的候选基因。方法以人工合成甘蓝型油菜10D130和甘蓝型油菜常规品种中双11构建高世代重组自交系（RIL）为研究材料,分别于2016-2017年和2017-2018年2个年度在重庆市北碚区2个不同的环境中设置田间试验,收获自交种子,采用气相色谱法3次重复对种子的脂肪酸组分进行分析。利用油菜6K SNP芯片对该RIL群体进行基因分型,DNA样品预处理及芯片处理严格按照Illumina Inc 公司Infinium HD Assay Ultra操作说明进行。取最小阈值LOD 2.0利用JoinMap4.0软件构建高密度遗传连锁图谱。通过QTL IciMapping V4.1完备区间作图法对油菜主要脂肪酸组成进行QTL定位。结果 2种环境中,两亲本各性状间差异及RIL群体各性状在株系间差异均达到显著或极显著水平,且6种脂肪酸含量在2个环境中均表现为连续分布,适合进行QTL检测。构建用于QTL定位的遗传图谱包含1 897个多态性SNP标记,覆盖甘蓝型油菜基因组3 214.19 cM,平均图距1.69 cM。利用此图谱,在2个环境共检测到位于8条染色体上的23个控制脂肪酸组分QTL位点,与硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸和芥酸含量相关的QTL位点分别为6、3、4、5、2和3个,其中在A05、A08和C03染色体上发现多种脂肪酸含量的QTL“富集区”。在A05染色体上检测到亚油酸和亚麻酸含量重叠的主效QTL,亚油酸与亚麻酸表现加性效应相同;在A08和C03上都检测到油酸、廿碳烯酸和芥酸含量重叠的主效QTL,油酸与廿碳烯酸及芥酸表现加性效应相反。与拟南芥脂肪酸代谢基因进行同源性比对分析,在17个QTL置信区间内筛选到22个候选基因,主要通过编码脂肪酸去饱和酶、全羧化酶合酶、碳链延长酶和参与酰基辅酶A生物合成等途径调控脂质的生物合成和代谢。结论 利用甘蓝型油菜6K SNP芯片准确定位了2种环境条件脂肪酸组成的QTL位点,筛选到位于A05、A08和C03染色体上多种脂肪酸QTL的“富集区”,并与拟南芥脂肪酸代谢基因比对出该群体油菜脂肪酸代谢基因,可作为改善油菜籽脂肪酸组成的重要区段及候选基因。
关键词： 甘蓝型油菜;脂肪酸;遗传图谱;QTL;候选基因

Abstract
【Objective】 Rapeseed oil is widely used in cooking, food processing and industrial production. Therefore, improving the fatty acid composition of rapeseed oil according to the specific production objective is an important goal of rapeseed breeding. In this study, QTL mapping of main fatty acid composition in Brassica napus under two environments was conducted, which was designed to search for the QTL and related candidate genes of fatty acid components in Brassica napus.【Method】 The high generation recombinant inbred lines (RILs) were used as experiment materials, which were derived from synthetic 10D130 and conventional variety Zhongshuang11, and field experiments were conducted in 2016-2017 and 2017-2018 with two different environments in Beibei District of Chongqing City, respectively. After self-pollinated seeds were harvested each year, fatty acid components of seeds were measured by GC with three technical repeats. Then, the RIL population genotype was analyzed with the rapeseed 6K SNP chip array. The DNA preparation and the chip preparation were processed strictly according to Infinium HD Assay Ultra manual of Illumina Inc. The SNP linkage map was constructed by using JoinMap 4.0 program with minimum LOD 2.0. QTL mapping of main fatty acid composition was conducted by composite interval mapping using software Windows QTL IciMapping V4.1.【Result】 In the two environments, the differences of parents’ traits and RILs population’s traits reached significant or extremely significant levels, and the contents of six fatty acids showed continuous distribution, which were suitable for the detection of QTLs. The reference SNP genetic map contains 1 897 polymorphic SNP markers, covering 3 214.19 cM of Brassica napus genome with an average map distance of 1.69 cM. Twenty-three QTLs loci of fatty acid components on 8 chromosomes were detected in two environments. The QTLs loci related to stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid and erucic acid contents were 6, 3, 4, 5, 2 and 3, respectively. And the “enrichment regions” of multiple fatty acid contents were found on chromosomes A05, A08 and C03. Meantime, the main QTL with overlapping linoleic acid and linolenic acid content was detected on A05 chromosome, showing the same additive effect. And the main effect QTLs, oleic acid with overlapping contents of eicosenic acid and erucic acid were detected on A08 and C03, which was opposite to the additive effect of eicosenic acid and erucic acid. Twenty-two candidate genes of fatty acid metabolic were found underlying confidence intervals of seventeen QTLs by comparing with homologous genes in Arabidopsis thaliana. These genes regulate lipid biosynthesis and metabolism through encoding fatty acid desaturase, total carboxylase synthase, carbon chain lengthening enzyme and participating in acyl coenzyme A biosynthesis. 【Conclusion】 The fatty acid composition QTL under two environments were mapped accurately with the 6K SNP chip of rapeseed, and the “enrichment regions” of multiple fatty acid QTLs on chromosomes A05, A08 and C03 were screened. Compared with Arabidopsis thaliana fatty acid metabolic genes, candidate genes for fatty acid metabolism in this population were detected, which could be used for improving fatty acid composition in rapeseed.
Keywords：Brassica napus;fatty acid;genetic map;QTL;candidate gene


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本文引用格式
叶桑, 崔翠, 郜欢欢, 雷维, 王刘艳, 王瑞莉, 陈柳依, 曲存民, 唐章林, 李加纳, 周清元. 基于SNP遗传图谱对甘蓝型油菜部分脂肪酸 组成性状的QTL定位[J]. 中国农业科学, 2019, 52(21): 3733-3747 doi:10.3864/j.issn.0578-1752.2019.21.002
YE Sang, CUI Cui, GAO HuanHuan, LEI Wei, WANG LiuYan, WANG RuiLi, CHEN LiuYi, QU CunMin, TANG ZhangLin, LI JiaNa, ZHOU QingYuan. QTL Identification for Fatty Acid Content in Brassica napus Using the High Density SNP Genetic Map[J]. Scientia Agricultura Sinica, 2019, 52(21): 3733-3747 doi:10.3864/j.issn.0578-1752.2019.21.002

0 引言

【研究意义】油菜是中国主要的油料作物,其种植面积和总产量居世界首位^[1],目前,已经成为继水稻、小麦、玉米之后的第四大作物,是中国的优势油料作物^[2]。菜籽油是中国主要的食用油之一,其主要脂肪酸组成包括硬脂酸（C18﹕0）、油酸（C18﹕1）、亚油酸（C18﹕2）、亚麻酸（C18﹕3）、廿碳烯酸（C20﹕1）和芥酸（C22﹕1）等。食用油中油酸具有极高的营养和应用价值,高油酸菜籽油不仅有利于人体心血管健康^[3],还因其热稳定性高^[4],利于纯化储存而被用于煎炸行业^[5]以及生产、生活用品的原料^[6]。亚油酸是一种必需脂肪酸,可防止动脉粥样硬化,但存在氧化稳定性差、储存时间短的缺点。亚麻酸属多烯类不饱和脂肪酸,因存在3个不饱和键而易氧化变质,故亚麻酸含量较高的菜籽油不耐储存^[7]。芥酸因其碳链较长,在人体内不易被消化吸收,不适用于食品加工^[8],然而芥酸是重要的工业原料,被广泛应用于众多工业领域^[7]。因此,根据生产需要改善油菜脂肪酸的组成成分是油菜育种的重要目标,定位油菜脂肪酸组分的QTL和筛选影响脂肪酸组分的候选基因在油菜育种中都具有重要的指导作用。【前人研究进展】前人研究表明,在油菜主要脂肪酸的形成过程中,首先由棕榈酸在碳链延长酶的作用下合成硬脂酸^[9],硬脂酸在质体基质中再被硬脂酰-ACP脱氢酶催化形成油酸^[10],而油酸包含2条代谢途径：其一,油酸在碳链延长酶的作用下合成廿碳烯酸,廿碳烯酸进一步延长碳链生成芥酸^[11];其二,油酸在△¹²减饱和酶作用下生成亚油酸^[12],亚油酸在△¹⁵减饱和酶作用下进一步减饱和生成亚麻酸^[13]。了解主要脂肪酸的代谢途径是改善油菜脂肪酸组成的前提,通过分子标记定位相关性状QTL并筛选出候选基因有利于更准确了解脂肪酸各组分受到影响的遗传因素。迄今为止,利用分子标记技术的QTL定位分析在油菜相关性状包括抗性^[14,15,16]、育性^[17,18]和品质^[19,20,21]等方面广泛发展,为油菜分子的育种改良奠定了基础。油菜脂肪酸含量具有较高的遗传力,对其相关分子标记的确定是改善油菜脂肪酸组成的关键。BURNS等^[22]利用RFLP分子标记进行油菜脂肪酸组成的QTL定位,获得4个与油酸含量相关的QTL,其中效应较大的QTL位于N3、N8、N18染色体,效应较小的位于N11染色体;获得5个与亚油酸含量相关的QTL,效应相对较大QTL位于N8和N14染色体;获得5个与亚麻酸含量相关的QTL,分别位于N6、N7、N18和N11染色体。张洁夫等^[23]采用RAPD、SSR和SRAP 3种分子标记对油菜脂肪酸组成进行QTL定位,获得3个与硬脂酸含量相关的QTL,分别位于N1、N8和N16染色体;获得2个与油酸含量相关的主效QTL,分别位于N8和N13染色体;获得3个与亚油酸含量相关的QTL,其中2个位于N8染色体,1个位于N13染色体;获得3个与亚麻酸含量相关的微效QTL;获得4个与廿碳烯酸含量相关的QTL,分别位于N8、N13和N15染色体;在N8和N13染色体获得2个与芥酸含量相关的主效QTL。ZHAO等^[24]利用SSR标记构建的遗传图谱找到了7个控制油酸含量的QTL,其中,主效QTL位于N18染色体;找到7个控制亚油酸含量的QTL,其中主效QTL位于N9染色体;并在N14染色体找到1个控制亚麻酸含量的QTL。与上述分子标记技术相比,SNP标记具有数量多、分布广和规模化等优点,已成为大家认可的第三代遗传标记,近几年被广泛应用于油菜产量、品质等性状的分子标记辅助育种及遗传定位等诸多领域。例如,LIU等^[25]利用油菜SNP芯片技术构建高密度遗传图谱,对油菜籽木质素、纤维素和半纤维素等性状进行QTL定位。SHEN等^[26]采用SNP分子标记技术定位了17个油菜分枝角度QTL,并在QTL区间获得27个候选基因。QU等^[27]利用520份油菜资源群体对油菜籽脂肪酸组成进行全基因组关联分析,确定了62个与7种脂肪酸组成显著相关的基因组区域,并鉴定了24个参与脂肪酸生物合成的功能候选基因的同源基因。【本研究切入点】前人对油菜脂肪酸含量的定位研究主要是利用SSR、AFLP、RFLP和SRAP等分子标记,存在标记的多位点现象,给不同连锁图谱之间QTL位点的比较分析带来困难。另外,前人在QTL作图方法上大多采用区间作图法和复合区间作图法,致使QTL效应可能会被侧连标记区间之外的标记变量所吸收^[28]。【拟解决的关键问题】本研究利用油菜6K SNP芯片构建高密度遗传连锁图谱,通过完备区间作图法对油菜主要脂肪酸组成进行QTL定位。结合2年的表型鉴定,发掘在不同环境条件下稳定表达的主效QTL,并利用甘蓝型油菜基因组序列,与拟南芥脂肪酸相关代谢基因进行同源性分析,筛选可能的候选基因,旨在探讨油菜主要脂肪酸之间的内在联系,为改善油菜脂肪酸组成进行分子标记辅助选择、克隆相关候选基因奠定基础。

1 材料与方法

1.1 材料

重组自交系群体的母本为人工合成甘蓝型油菜新品系10D130,其芥酸含量50%左右,父本为高产优质的天然甘蓝型油菜常规品种中双11号（简称ZS11）,其芥酸含量＜1%。两亲本杂交F₂通过单粒法连续自交7代,构建重组自交系群体,以其中的186个材料进行SNP标记分析,构建高密度SNP遗传连锁图谱。所有试验材料均由西南大学重庆市油菜工程技术研究中心提供。

1.2 田间试验

2016-2017年和2017-2018年2个年度,在重庆市北碚区歇马镇油菜实验基地种植重组自交系群体及其亲本,2个环境分别记为17Cq和18Cq。田间播种采用随机区组试验设计,每个小区5行,每行10株。田间管理遵循常规生产方式,在油菜初花期逐株套袋自交,种子完全成熟后每家系收取5株正常植株的种子,自然风干后保存。

1.3 脂肪酸组成的测定

根据文献[29]的方法,利用气相色谱仪GC-2010（Shimazu,JAP）测定2个环境下重组自交系群体种子中硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸及芥酸的含量,每份材料重复测定3株,取其平均值。

1.4 遗传连锁图谱构建及QTL分析

参考刘列钊等^[30]方法,分别从每个株系的5个幼嫩植株叶片取混合样0.15 g,利用DNA提取试剂盒DP321-03（天根,中国北京）提取DNA并稀释至50 ng·μL^-1用于SNP标记分析。严格按照Infinium HD Assay Ultra操作说明书（Illumina Inc 公司）进行DNA样品的预处理（等位扩增、片段化及富集）、与芯片杂交、洗涤、安装流动室、单碱基延伸、染色及包埋。芯片准备好后运用Illumina HiScan扫描仪的iScan Control Software软件扫描,然后利用GenomeStudio genotyping software v2011软件分析扫描结果,获取各个样本的SNP基因型数据,并为获得的SNP标记命名,命名方法以S-95505568为例,S表示SNP,95505568代表GenomeStudio genotyping software生成的相应SNP位点索引号。

连锁图谱的构建采用JoinMap4.0软件^[31],选用Kosambi^[32]函数将重组值转换为图距单位（centiMorgan,cM）,取最小阈值LOD2.0对所有标记进行分组,每个染色体上标记顺序通过两两标记之间最小重组频率计算,构建用于QTL定位的遗传连锁图谱。

采用软件QTL IciMapping V4.1^[33]完备区间作图（inclusive composite interval mapping,ICIM）法,进行2个环境下硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸及芥酸含量的QTL定位和检测。进行ICIM分析时,选用1 cM的步长（walking speed）,作1 000次排列检验和显著水平0.01确定LOD临界值。软件运行结果可同时给出性状QTL的加性效应和表型贡献率。QTL命名时以q加上性状的英文缩写再加上染色体编号及QTL序号表示,字体斜体,如qC03LI-1代表亚油酸含量位于C03染色体上的第一个QTL。同一性状在染色体相同的位置检测到重复的QTL,且加性效应方向一致,认为是同一QTL。

1.5 候选基因筛选

为筛选出与脂肪酸组成相关的候选基因,首先将检测到的QTL置信区间在甘蓝型油菜基因组上对应的序列查询到,然后与BOTELLA等^[34]、QU等^[27]搜索出的拟南芥脂肪酸代谢相关基因进行BLASTN比对,将E值设定为E^-20,最后筛选出每个QTL置信区间内匹配E值小于阈值的候选基因。

2 结果

2.1 重组自交系群体脂肪酸含量的分析

两亲本和RIL群体的表型分析见表1。RIL群体的表型在2个环境中表现一致：6个性状的均值都介于两亲本之间,最大值和最小值都超过亲本值,存在明显的超亲分离现象。另外,两亲本各性状间差异及RIL群体各性状在株系间差异都达到显著或极显著水平,结合变异系数值反映出这几个性状在该群体中具有较广泛的遗传变异。由性状频率分布图（图1）可知,硬脂酸、亚油酸和亚麻酸呈现偏态分布的特征,油酸、廿碳烯酸和芥酸含量则表现为明显的多峰分布,结果表明,6种脂肪酸含量在2个环境中均表现为连续分布,适合进行QTL检测。

Table 1
表1
表12个环境下油菜主要脂肪酸含量的变化
Table 1Changes of fatty acids content under two different environments

环境 Environment	性状 Trait	亲本 Parents		RIL群体 RIL populations
		10D130	ZS11		最大值 Max	最小值 Min	均值 Average	标准差 SE	变异系数 CV (%)
17Cq	C18:0	1.21a	2.84b		6.17	1.00	1.77**	0.61	34.47
	C18:1	18.83A	64.27B		83.15	13.17	38.25**	19.11	49.95
	C18:2	10.86A	23.27B		31.18	6.15	14.49**	4.50	31.04
	C18:3	7.68a	9.55b		12.79	4.04	8.65*	1.59	18.35
	C20:1	8.08A	0.07B		19.33	0.00	10.26**	5.86	57.12
	C22:1	53.34A	0.00B		57.96	0.00	26.56**	17.70	66.62
18Cq	C18:0	1.52a	2.68b		4.73	0.47	1.98**	0.56	28.13
	C18:1	17.91A	63.03B		78.79	12.69	39.14**	18.03	46.07
	C18:2	12.08A	22.94B		26.41	9.27	15.47**	3.94	25.46
	C18:3	8.92a	10.44b		12.20	6.14	8.89*	1.36	15.27
	C20:1	8.86A	0.91B		19.99	0.70	11.20**	6.43	57.44
	C22:1	50.71A	0.00B		51.62	0.00	23.02**	15.79	68.59

^a、^b和^A、^B分别代表在0.05和0.01水平上差异显著,*和**分别代表在0.05和0.01水平上差异显著。C18:0、C18:1、C18:2、C18:3、C22:1和C22:2分别代表硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸和芥酸。下同
^a, ^b and ^A, ^B indicate significance at P＜0.05 and P＜0.01, * and ** indicate significance at P＜0.05 and P＜0.01. C18:0, C18:1, C18:2, C18:3, C22:1 and C22:2 represent stearic acid, oleic acid, linoleic acid, linolenic acid, eicosenoic acid and erucic acid, respectively. The same as below

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由表2可知,在2个环境下油酸含量与硬脂酸及亚油酸含量均表现为极显著正相关（P＜0.01）,与廿碳烯酸及芥酸含量均表现为极显著负相关（P＜0.01）,与亚麻酸含量相关性不强。芥酸含量与廿碳烯酸含量在2个环境中均表现极显著正相关（P＜0.01）,相关系数分别为0.56和0.64;与亚油酸含量在2个环境中均表现极显著负相关（P＜0.01）,相关系数分别为-0.52和-0.54。结果表明,油酸转化为亚油酸的途径受到芥酸途径的掣肘,即油酸和亚油酸的含量受芥酸含量的变化影响较大。而亚麻酸受油酸和芥酸变化的影响较小,在遗传上具有相对独立的稳定性。

图1

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图1RIL群体硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸及芥酸含量的频率分布

P₁、P₂和P’₁、P’₂分别标出了17Cq和18Cq环境下2个亲本的脂肪酸含量
Fig. 1Frequency distribution of stearic acid, oleic acid, linoleic acid, linolenic acid, eicosenoic acid and erucic acid content from the RIL population in two different environments

P₁, P₂ and P'₁, P'₂ indicated the fatty acid content of two parents under 17Cq and 18Cq environment respectively


Table 2
表2
表22个环境下主要脂肪酸含量相关分析
Table 2Correlation analysis of fatty acids content in two different environments

测定指标 Index	C18:0	C18:1	C18:2	C18:3	C20:1	C22:1
C18:0		0.58**	0.18*	-0.32**	-0.28**	-0.59**
C18:1	0.65**		0.36**	-0.13	-0.68**	-0.95**
C18:2	0.03	0.43**		0.45**	-0.50**	-0.52**
C18:3	-0.29**	0.11	0.49**		-0.12	-0.01
C20:1	-0.31**	-0.77**	-0.57**	-0.28**		0.56**
C22:1	-0.64**	-0.96**	-0.54**	-0.20**	0.64**

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右上部代表17Cq环境,左下部代表18Cq环境The upper right represents the 17Cq environment and the lower left represents the 18Cq environment

2.2 遗传连锁图谱

利用包含5 058个标记的6K油菜芯片对186个RIL材料进行基因型鉴定,从中筛选出1 897个高质量多态性的SNP标记,约占37.5%,基于这些标记构建遗传连锁图谱。获得用于QTL定位的图谱覆盖甘蓝型油菜基因组3 214.19 cM,平均图距为1.69 cM。每条染色体长度在86.51-298.72 cM,平均长度为169.17 cM。染色体标记数目在24-153,平均数目为99.84个。但是从标记分布来看,各染色体分布不均,其中染色体A03、C03和C04上标记分布较多,分别为139、147和153个;而染色体C05、C08和C09上的标记数目较少,分别只有48、26和24个。此外各染色体标记密度也有较大差异,C04密度最大,平均间距仅1.10 cM,而密度最小的C08平均间距达6.13 cM。

2.3 主要脂肪酸含量的QTL分析

2.3.1 硬脂酸含量的QTL 在2个环境中,硬脂酸含量共定位到6个QTL,解释表型变异5.08%-25.49%,其中主效QTL位点位于A08和C03上。C03上的主效位点在2个环境中能被重复检测到,分别解释表型变异的11.59%和9.12%;在A08上的2个QTL位点分别解释硬脂酸含量表型变异的14.00%和25.49%。且A08和C03上的QTL位点在2个环境中的加性效应均小于0（表3和图2）,即加性效应均来源于亲本ZS11。

Table 3
表3
表3油菜主要脂肪酸含量在2个环境中的QTL位点
Table 3QTLs for main fatty acids content in two different environments

性状 Trait	环境 Envi.	QTL	染色体 Chr.	标记区间 Position	置信区间1) Confidence interval	LOD	加性效应 Additive effect	贡献率 R2(%)
C18:0	17Cq	qA08ST-1	A08	S-95505568-S-95506507	43.27-48.50	6.13	-0.22	14.00
		qC03ST	C03	S-177827612-S-95636886	216.25-219.73	5.11	-0.20	11.59
	18Cq	qA01ST	A01	S-95637833-S-95664701	116.00-118.62	4.94	0.13	5.08
		qA05ST	A05	S-177830309-S-86232724	107.59-110.16	6.07	0.15	6.27
		qA08ST-2	A08	S-95506569-S-95506120	29.90-31.07	20.45	-0.29	25.49
		qA09ST	A09	S-182142581-S-182167880	224.50-229.51	8.76	-0.18	9.45
		qC03ST	C03	S-95665809-S-95636886	215.70-219.73	8.31	-0.17	9.12
C18:1	17Cq	qA05OL	A05	S-182087654-S-86232724	106.33-110.16	4.59	3.81	3.80
		qA08OL	A08	S-95507415-S-95663297	26.79-30.00	31.31	-11.42	34.39
		qC03OL	C03	S-105305847-S-182158964	218.48-222.84	29.65	-10.95	31.80
	18Cq	qA05OL	A05	S-182087654-S-86232724	106.33-110.16	8.40	3.91	5.70
		qA08OL	A08	S-95507415-S-95663297	26.79-30.00	31.83	-8.66	28.28
		qC03OL	C03	S-105338742-S-182138971	219.51-224.50	42.44	-10.75	43.68
C18:2	17Cq	qA05LI	A05	S-182087654-S-86232724	106.33-110.16	18.43	-2.37	22.67
		qA08LI	A08	S-177633794-S-95506569	25.61-29.50	8.45	-1.48	8.91
		qC03LI-1	C03	S-105306222-S-95637726	235.80-239.65	14.65	-2.06	17.00
		qC03LI-2	C03	S-105309588-S-105307276	250.03-253.95	6.28	1.28	6.69
	18Cq	qA05LI	A05	S-182087654-S-86232724	106.33-110.16	19.71	-1.84	25.25
		qA08LI	A08	S-95507415-S-95663297	26.79-30.00	9.44	-1.18	10.49
		qC03LI-1	C03	S-95637910-S-105307365	237.50-239.83	11.09	-1.30	12.56
C18:3	17Cq	qA02LN	A02	S-95666343-S-95638378	55.50-64.85	4.96	-0.44	8.49
		qC07LN	C07	S-105339086-S-179307020	6.50-11.97	5.84	0.51	11.66
	18Cq	qA05LN	A05	S-182087654-S-86232724	106.33-110.16	33.12	-1.12	23.66
		qA09LN	A09	S-182142581-S-182167880	224.50-229.51	8.28	0.47	4.24
		qA10LN	A10	S-177910390-S-95636447	59.94-63.16	4.90	0.35	2.37
C20:1	17Cq	qC03EI	C03	S-105338742-S-182138971	219.51-224.50	7.45	2.27	17.23
	18Cq	qA08EI	A08	S-95507415-S-95663297	26.79-30.00	6.07	2.14	11.18
		qC03EI	C03	S-105338742-S-182138971	219.51-224.50	11.44	3.05	22.69
C22:1	17Cq	qA08ER-1	A08	S-95506362-S-95664454	10.05-12.58	11.30	5.00	9.39
		qA08ER-2	A08	S-95507415-S-95663297	26.79-30.00	27.90	9.13	32.21
		qC03ER	C03	S-105305847-S-182158964	218.48-222.84	32.17	9.62	36.01
	18Cq	qA08ER-2	A08	S-95507415-S-95663297	26.79-30.00	96.68	17.53	67.26
		qC03ER	C03	S-105305847-S-182158964	218.48-222.84	50.41	8.64	16.45

¹⁾: Linkage location of markers on the genetic linkage map
¹⁾：标记在遗传连锁图谱上的连锁位置

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图2

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图2甘蓝型油菜主要脂肪酸含量QTL在SNP连锁群上的分布

Fig. 2Putative QTL locations of fatty acid content on the SNP genetic map



2.3.2 油酸含量的QTL 在2个环境中共检测到3个与油酸含量相关的 QTL,且3个位点在2个环境中均能被重复检测到,其中位于A08和C03染色体上的是主效位点,可解释油酸含量表型变异的28.28%-43.68%。在2个环境中,位于A05上的QTL位点加性效应分别为3.81和3.91,而A08和C03染色体上主效位点的效应值在-11.42--8.66（表3和图2）,即前者的加性效应来源于亲本10D130,后两者的加性效应则来自亲本ZS11。

2.3.3 亚油酸含量的QTL 在2个环境中,亚油酸含量共检测到4个QTL,除了C03染色体上的qC03ST-2以外,其余3个QTL均能在2个环境中被重复检测到。其中主效QTL位于A05和C03上,可解释亚油酸含量表型变异的12.56%-25.25%;A08染色体上重复检测到的QTL位点在2个环境中的遗传贡献率分别为8.91%和10.49%。同时,在2个环境中重复检测到的3个QTL位点的效应值在-2.37--1.18（表3和图2）,即加性效应均来自亲本ZS11。

2.3.4 芥酸含量的QTL 在2个环境中共定位到3个与芥酸含量相关的QTL,其中位于A08和C03上的主效QTL位点能够在2个环境中被重复检测到,可解释表型变异16.45%-67.26%。芥酸含量的3个QTL位点的加性效应均为正值,加性效应均来源于亲本10D130,其中,qA08ER的效应值最大,为17.53（表3和图2）,属主基因座位。其他主要脂肪酸的QTL定位结果见表3和图2。

2.4 主要脂肪酸代谢候选基因筛选

将23个QTL置信区间序列与拟南芥脂肪酸代谢基因进行比对,分别在A01、A02、A05、A08、A09、A10和C03染色体上的17个QTL区间内检测到22个候选基因,匹配E值介于0-4E^-17（表4）。位于A01染色体上的基因BnaA01g27100D和BnaA01g27120D分别与拟南芥基因AT3G18030和AT1G48605同源,参与辅酶A的生物合成。A02染色体上的基因BnaA02g13270D、BnaA02g13310D和BnaA02g13630D分别与拟南芥基因AT1G77590、AT1G67730和AT2G25710同源,分别编码长链酰基辅酶A合成酶、β-酮酰辅酶A还原酶和全羧化酶合酶。A05染色体上的基因BnaA05g26900D、BnaA05g25110D、BnaA05g25210D、BnaA05g25220D和BnaA05g26460D分别与拟南芥基因AT3G12120、AT3G14360、AT1G53990、AT3G14225和AT1G06800同源,第一个编码脂肪酸脱氢酶,后4个参与脂质代谢过程。在A09染色体上的基因BnaA09g39290D与拟南芥基因AT3G61580同源,编码脂肪酸去饱和酶。A10染色体上的基因BnaA10g10590D和BnaA10g10730D分别与拟南芥基因AT5G56350和AT5G56480同源,前者是丙酮酸激酶家族蛋白成员,后者是脂质转运蛋白家族成员。A08和C03染色体上存在多个同源基因,其中BnaA08g11130D和BnaC03g65980D与拟南芥基因AT4G34520同源,BnaA08g11140D和BnaC03g66040D与拟南芥基因AT4G34510同源,两者同属3-酮酰辅酶A合成酶家族;BnaA08g11440D和BnaC03g66380D与拟南芥基因AT4G33790同源,编码一种脂肪酰辅酶A还原酶。

Table 4
表4
表4在甘蓝型油菜脂肪酸含量QTL置信区间比对拟南芥相关基因获得的候选基因
Table 4Candidate genes in QTL confidence interval of fatty acid content in B. uapus by alignment with related genes in Arabidopsis thaliana

染色体 Chr.	预测基因 Gene prediction	物理位置 Physical position (Mb)	拟南芥相关基因 Related genes in A. thaliana
			基因名称 Gene name	基因登录号 Gene accession	E值 E-value	描述 Description
A01	BnaA01g27100D	18.94	HAL3A	AT3G18030	E-60	辅酶A生物合成过程 Coenzyme A biosynthetic process
	BnaA01g27120D	18.94	HAL3B	AT1G48605	5E-23	辅酶A生物合成过程 Coenzyme A biosynthetic process
A02	BnaA02g13270D	7.29	LACS9	AT1G77590	2E-52	长链酰基辅酶A合成酶9 Long chain acyl-CoA synthetase 9
	BnaA02g13310D	7.31	KCR1	AT1G67730	2E-74	β-酮酰辅酶A还原酶1 Beta-ketoacyl-CoA reductase 1
	BnaA02g13630D	7.49	HCS1	AT2G25710	2E-58	全羧化酶合成酶1 Holocarboxylase synthase 1
A05	BnaA05g26430D	19.37	ALIS1	AT3G12740	2E-93	参与磷脂转运 Involved in phospholipid transport
	BnaA05g26900D	19.54	FAD2	AT3G12120	0	不饱和脂肪酸生物合成过程 Unsaturated fatty acid biosynthetic process
	BnaA05g25110D	18.65	ATOBL1	AT3G14360	E-156	脂质代谢过程 Lipid metabolic process
	BnaA05g25210D	18.70	GLIP3	AT1G53990	4E-17	脂质分解过程 Lipid catabolic process
	BnaA05g25220D	18.70	GLIP4	AT3G14225	E-117	脂质分解过程 Lipid catabolic process
	BnaA05g26460D	19.38	DALL4	AT1G06800	2E-27	脂质代谢过程 Lipid metabolic process
A08	BnaA08g09510D	9.09	OGOX2	AT4G20840	0	FAD-结合小檗碱家族蛋白 FAD-binding Berberine family protein
	BnaA08g11130D	10.19	FAE1	AT4G34520	0	3-酮酰辅酶A合成酶18 3-ketoacyl-CoA synthase 18
	BnaA08g11140D	10.19	KCS17	AT4G34510	0	3-酮酰辅酶A合成酶17 3-ketoacyl-CoA synthase 17
	BnaA08g11440D	10.39	FAR3	AT4G33790	8E-46	脂肪酸还原酶3 FATTY ACID REDUCTASE 3
	BnaA08g11650D	10.51	MCCB	AT4G34030	E-119	3-甲基巴豆酰辅酶A羧化酶 3-methylcrotonyl-CoA carboxylase
	BnaA08g12370D	11.04	WIN2	AT4G31750	3E-71	编码HopW1-1-相互作用蛋白2 Encodes HopW1-1-Interacting protein 2
A09	BnaA09g39290D	27.87	SLD1	AT3G61580	0	脂肪酸去饱和酶 Fatty acid desaturase
A10	BnaA10g10590D	9.01	未知Unknown	AT5G56350	0	丙酮酸激酶家族蛋白 Pyruvate kinase family protein
	BnaA10g10730D	9.08	END2	AT5G56480	4E-62	脂质转移蛋白 Lipid-transfer protein
C03	BnaC03g63920D	53.41	OGOX2	AT4G20840	0	FAD-结合小檗碱家族蛋白 FAD-binding Berberine family protein
	BnaC03g63930D	53.42	BBE22	AT4G20860	5E-23	FAD-结合小檗碱家族蛋白 FAD-binding Berberine family protein
	BnaC03g64130D	53.56	SPHK2	AT4G21534	4E-55	二酰基甘油激酶家族蛋白 Diacylglycerol kinase family protein
	BnaC03g65980D	55.68	FAE1	AT4G34520	0	3-酮酰辅酶A合成酶18 3-ketoacyl-CoA synthase 18
	BnaC03g66040D	55.81	KCS17	AT4G34510	0	3-酮酰辅酶A合成酶17 3-ketoacyl-CoA synthase 17
	BnaC03g66380D	56.21	FAR3	AT4G33790	3E-49	脂肪酸还原酶3 FATTY ACID REDUCTASE 3
	BnaC03g67410D	57.10	WIN2	AT4G31750	2E-73	编码HopW1-1-相互作用蛋白2 Encodes HopW1-1-Interacting protein 2

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3 讨论

3.1 QTL定位结果的比较分析

油菜主要脂肪酸QTL定位研究前人已有报道,本研究的部分结果与已有研究结果一致性较高,但也发现了一些新的QTL区域。例如,本研究通过完备区间作图法发现了控制芥酸含量的3个QTL,其中位于A08和C03染色体上的2个主效QTL在2个环境下被重复检测到,第3个微效QTL也位于A08染色体,与前人将控制芥酸含量的QTL主要集中在A08和C03上的结果一致^{[23,30,35-36]}。油酸的遗传比较复杂,本研究在2个环境中重复检测到位于A08和C03染色体上油酸含量的2个主效QTL,和芥酸的主效QTL重叠,加性效应相反,这与刘列钊等^[30]的研究结果相同。不同的是,本研究亲本并非高油酸材料,却在2个环境中重复定位到位于A05染色体上油酸含量的1个微效QTL,该位点同时还是亚油酸的主效QTL位点,这在前人研究中未发现。本研究在该位点上筛选到候选基因FAD2,同时检测到该群体（RIL）中的4个高油酸材料的FAD2第722位碱基A突变成T导致酪氨酸变成苯丙氨酸,从而影响了蛋白质的空间结构,导致油酸含量提高,这与张宏军等^[37]的研究结果一致。而亲本ZS11的FAD2正常,结合A05染色体上QTL位点的加性效应来源于亲本10D130,猜测FAD2的变异来自亲本10D130。亚麻酸含量是典型的数量性状,易受环境影响^[30]。在17Cq环境中,亚麻酸含量检测到1个位于C07染色体上的主效QTL,解释表型变异的11.66%,而在18Cq环境中,检测到一个主效QTL位于A05染色体,解释亚麻酸含量表型变异的23.66%,这与前人主要将亚麻酸含量主效QTL定位在A04和C04染色体不同^[12,24,38],可能的原因是受环境因素影响较大。本研究采用完备区间作图法对甘蓝型油菜RIL群体的一系列油菜籽脂肪酸含量进行QTL定位,一方面进一步验证了已有研究的可靠性,另一方面也说明这些能够被多次重复定位的主效区段和新发现的基因组区段均是控制油菜籽主要脂肪酸含量的重要区域,对剖析油菜籽脂肪酸含量的遗传具有重要意义。

3.2 主要脂肪酸含量QTL之间的相关性

研究主要对甘蓝型油菜主要脂肪酸,包括硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸及芥酸含量进行QTL定位分析。结果表明,控制这些脂肪酸含量的QTL之间存在明显的相关性,主要表现为A05、A08和C03染色体上出现QTL的“富集区”。在A05染色体的相同位置同时存在硬脂酸、油酸、亚油酸和亚麻酸4种脂肪酸的QTL,其中对于亚油酸和亚麻酸是主效QTL,且加性效应方向一致,可解释二者在2个环境中所表现的0.45和0.49的极显著正相关性。在A08和C03染色体上相同的置信区间内同时检测出油酸、廿碳烯酸和芥酸的主效QTL位点,即控制油酸含量的主效QTL同时也是控制廿碳烯酸和芥酸含量的主效QTL,油酸的2个主效位点加性效应值都为负,而廿碳烯酸和芥酸在此位点上的加性效应则表现为正值,充分解释了油酸和廿碳烯酸含量之间、油酸和芥酸含量之间在2个环境中都表现的极显著负相关性以及廿碳烯酸和芥酸含量在2个环境中的极显著正相关性。

根据表型相关性分析和QTL相关性分析认为,油酸的2条代谢途径并不均衡,其“加碳”途径比“脱氢”途径更具优势。一方面表现在A08和C03上芥酸的2个主效QTL对于油酸也是主效位点,可解释油酸表型变异的28.28%-43.68%,而A05上亚油酸的主效QTL对于油酸是微效位点,只解释油酸表型变异的3.8%-5.7%;另一方面,A08染色体上芥酸的主效QTL同时也是亚油酸的QTL,可解释亚油酸表型变异的8.91%,而亚油酸在A05上的主效QTL并不能解释芥酸含量的表型变异,这就表明,“加碳”途径制约着亚油酸的含量,而“脱氢”途径则对芥酸的含量影响不大。在育种工作中,对亚油酸含量的提高,需要阻断油酸向芥酸转化的途径;而高油酸的获得,则需要对2条代谢途径同时进行抑制。所以针对这些区段的深入研究和挖掘为改善油菜脂肪酸组成奠定基础。

3.3 脂肪酸代谢候选基因

本研究在油菜籽脂肪酸组分QTL置信区间内发现22个脂肪酸代谢相关基因。这些候选基因主要集中在A05、A08和C03染色体上,其中位于A05染色体上的基因FAD2（BnaA05g26900D）编码脂肪酸脱氢酶,是调控多不饱和脂肪酸合成的关键基因^[39]。其中坐落于A08和C03染色体上的同源基因FAE1（BnaA08g11130D和BnaC03g65980D）是参与长链脂肪酸合成的关键基因,已被很多****研究报道^[40,41,42],这表明我们的QTL定位是可靠的。在A08和C03染色体上筛选的另外2个同源基因KCS17（BnaA08G11140D和BNAC03G66040D）和FAR3（BnaA08G11440D和BNAC03G66380D）,通过各自编码一种醇形成的脂肪酰辅酶A还原酶参与饱和脂肪酸的生物合成^[43,44]。位于A01染色体上的基因HAL3A（BnaA01g27100D）和HAL3B（BnaA01g27120D）参与辅酶A的生物合成,有研究^[45]表明,辅酶A可与醋酸盐结合为脂肪酸从头合成的前体物质乙酰辅酶A。在A02染色体上的候选基因BnaA02g13270D与拟南芥基因LACS9同源,LACS9已被证实通过催化脂肪酸、ATP和辅酶A形成酰基辅酶A,参与拟南芥籽油的生物合成^[46]。此外,本研究还在QTL置信区间内发现许多未知功能基因,可能存在油菜特有的脂肪酸代谢功能基因,有待进一步挖掘。

4 结论

在2个环境中共检测到位于A01、A02、A05、A08、A09、A10、C03和C07染色体上的23个脂肪酸组分QTL位点,与硬脂酸、油酸、亚油酸、亚麻酸、廿碳烯酸和芥酸含量相关的QTL位点分别为6、3、4、5、2和3个,其中在A05、A08和C03染色体上发现多种脂肪酸含量的QTL“富集区”,可作为改善油菜籽脂肪酸组成的重要区段。通过与拟南芥脂肪酸代谢基因的比对分析,在QTL置信区间内筛选到22个脂肪酸代谢相关候选基因。

（责任编辑 李莉）

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