荐红举^,1,2,**, 霍强^,1,2,**, 高玉敏^1,2, 李阳阳^1,2, 谢玲^1,2, 魏丽娟^1,2, 刘列钊^1,2, 卢坤^1,2, 李加纳^,1,2,*1 西南大学农学与生物科技学院, 重庆 400715
² 西南大学现代农业科学研究院, 重庆 400715

Selection of candidate genes for chlorophyll content in leaves of Brassica napus using genome-wide association analysis

JIAN Hong-Ju^,1,2,**, HUO Qiang^,1,2,**, GAO Yu-Min^1,2, LI Yang-Yang^1,2, XIE Ling^1,2, WEI Li-Juan^1,2, LIU Lie-Zhao^1,2, LU Kun^1,2, LI Jia-Na^{,1,2,* 1} College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
² Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China

通讯作者: * 李加纳, E-mail: ljn1950@swu.edu.cn, Tel: 023-68250701

** 同等贡献(Contributed equally to this work)
收稿日期:2020-01-12接受日期:2020-04-15网络出版日期:2020-04-27

基金资助:

国家重点研发计划项目.2018YFD0100504-05 重庆市民生工程主题专项.cstc2016shms-ztzx80020

Received:2020-01-12Accepted:2020-04-15Online:2020-04-27

Fund supported:

National Key Research and Development Program of China.2018YFD0100504-05 Special Project of Chongqing People’s Livelihood.cstc2016shms-ztzx80020

作者简介 About authors
荐红举, E-mail: hjjian518@swu.edu.cn;

霍强, E-mail: 354011524@qq.com










摘要
提高油菜产量是保障国家粮油安全的重要举措。作物“源”“流”“库”理论表明, 充足的光合产物(源)是高产的前提, 而叶绿素是直接参与光合作用的物质, 因此, 选育高叶绿素含量的甘蓝型油菜是提高产量的重要途径。本课题组前期对全球收集的588份优异油菜种质资源进行5X重测序, 获得385,692个高质量SNP标记。利用SPAD-502叶绿素仪于2018—2019连续2年测定苗期完全伸展的叶片叶绿素总量, 结合获得的SNP标记进行全基因组关联分析(genome-wide association study, GWAS), 筛选与叶绿素含量显著关联的SNP位点。结果表明, 2018年鉴定到5个显著关联的SNP位点, 贡献率为5.51%~7.89%, 其中S6_3493805位点贡献率最大; 2019年检测到46个SNP位点, 贡献率为7.29%~10.34%, 其中S13_11413088位点贡献率最大。将显著关联SNP位点上下游各500 kb区间内的基因与参考基因组比对, 初步筛选出2022个油菜基因。将其基因序列在拟南芥基因组内进行BLAST比对, 结合前人已报道的拟南芥同源基因功能, 筛选到23个候选基因, 其中5个属于叶绿素合成途径同源基因。本研究结果为后续油菜叶片叶绿素含量的遗传改良奠定了基础。
关键词： 甘蓝型油菜;叶绿素含量;全基因组关联分析;候选基因

Abstract
Increasing rapeseed production is important to ensure the national food and oil security. According to the theory of crop source and sink, sufficient photosynthate (sources) is the premise of high yield, and chlorophyll is the important substance for photosynthesis. Therefore, breeding high chlorophyll content Brassica napus is an important way to ensure high yield. In our previous study, 588 excellent germplasm resources collected worldwide were re-sequenced with 5x and 385,692 high-quality SNP markers were obtained. SPAD-502 chlorophyll meter was used to measure the total chlorophyll content of mature leaves in 2018-2019. Genome wide association study (GWAS) was conducted to screen SNP sites significantly related to chlorophyll content. Five SNP loci were identified in 2018, with a contribution rate of 5.51%-7.89%, of which S6_3493805 had the largest contribution. 46 SNP loci were detected in 2019, with a contribution rate of 7.29%-10.34%, of which S13_11413088 had the largest contribution. In total, 2022 rapeseed genes were screened out by comparing the reference genome with genes in the regions of the 500 kb before and after the SNP. Based on the function of Arabidopsis homologous genes previously reported, screened 23 candidate genes, among which five were homologous genes in chlorophyll synthesis pathway. These results lay a foundation for the genetic improvement of chlorophyll content in leaves of B. napus in future.
Keywords：Brassica napus;chlorophyll content;GWAS;candidate genes


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本文引用格式
荐红举, 霍强, 高玉敏, 李阳阳, 谢玲, 魏丽娟, 刘列钊, 卢坤, 李加纳. 用全基因组关联分析筛选甘蓝型油菜叶片叶绿素含量候选基因[J]. 作物学报, 2020, 46(10): 1557-1565. doi:10.3724/SP.J.1006.2020.04007
JIAN Hong-Ju, HUO Qiang, GAO Yu-Min, LI Yang-Yang, XIE Ling, WEI Li-Juan, LIU Lie-Zhao, LU Kun, LI Jia-Na. Selection of candidate genes for chlorophyll content in leaves of Brassica napus using genome-wide association analysis[J]. Acta Agronomica Sinica, 2020, 46(10): 1557-1565. doi:10.3724/SP.J.1006.2020.04007


油菜是中国四大油料作物之一, 也是产油效率最高的油料作物, 而菜籽作为我国食用植物油的最大来源, 自给率严重不足, 因此, 提高油菜产量和含油量是油菜育种的迫切目标。叶绿素是光合作用的重要色素, 筛选并研究控制油菜叶绿素含量的基因对提高油菜产量具有重要意义。

叶绿素在光合作用中能够吸收光能并参与能量传递^[1]。在生物学和遗传学上已经对叶绿素代谢进行了广泛的研究^[2,3,4]。叶绿素的生物合成首先是叶绿素前体L-谷氨酰tRNA (ALA)到原卟啉IX的合成^[4,5], 然后通过ALA脱水酶(ALAD)将ALA的2个分子缩合成吡咯分子, 即胆色素原。再通过酶的连续转化和原卟啉IX的金属螯合反应来划分通路, 从而指导最终产物叶绿素和血红素的形成^[4]。在黑暗中, 叶绿素的生物合成分支在中间原叶绿素酸酯(Pchlide)被阻断, 因为原叶绿素酸酯向叶绿素的转化是由依赖光的NADPH催化的^[6]。然而, 过量的原叶绿素酸酯游离和其他吡啶中间体在黑暗中积累可能在光照射下产生活性氧(ROS), 从而导致子叶光漂白甚至细胞死亡^[7]。因此, 叶绿素的生物合成在植物生长发育过程中扮演着重要的角色。叶绿素生物合成相关基因的表达差异造成了叶绿素含量的不同。

叶片是植物光合作用的主要场所, 也是植物源到库的重要源场所。而植物叶色突变体的叶色主要表现在苗期。甘蓝型油菜中的叶色黄化突变体BnaC.ygl是由于血红素加氧酶(HO1)的活性降低导致血红素的积累, 从而抑制了叶绿素合成前体ALA的合成, 造成叶绿素生物合成受到抑制导致叶色变黄^[8]; 玉米中5个黄化突变体是由于ZmPTAC2、ZmMurE、ZmPTAC10、ZmPTAC12和ZmPRIN2缺失所引起, 这5个基因与磷酸烯醇式丙酮酸(PEP)合成相关, 相关基因的缺失导致细胞中含有较少的质体核糖体和光合复合物, 表明PEP在叶绿素生物合成中起到关键作用^[9]。植物光合作用可以通过叶绿素将CO₂和H₂O转化为可储存的有机物, 因此叶绿素是植物有机物积累的重要物质。通过降低叶绿素降解的时间可以增加产量^[10]。另外, 叶片中叶绿素的含量也与作物粒饱满程度、有机物积累及单株产量显著相关^[11]。

近年来在高粱^[12]、玉米^[13]、小麦^[14]、水稻^[15]、大豆^[16]等植物中进行了叶绿素相关研究, 但关于甘蓝型油菜叶片叶绿素含量相关基因鉴定的报道较少^[17,18]。Ge等^[19]利用白菜一个F_2:3群体2年的叶绿素a和叶绿素b含量数据进行QTL定位, 一共检测到10个QTL, 单个QTL解释表型率为7%~17%。Wang等^[17]利用一个甘蓝型油菜叶绿素缺失突变体cde1和中双11构建的F₂群体及620个F_2:3群体将CDE1基因定位在一个长度为311 kb区间内, 该定位区间包括22个候选基因; Qian等^[18]探究了甘蓝型油菜中bnnye1-A01的缺失对光系统(LHC)相关基因表达及光合反应中心蛋白PSI和PSII表达的影响以及对籽粒油脂积累的影响, 同时也发现携带bnnye1-A01基因也会影响株高。

随着高通量测序技术的成熟以及基因型分型成本的降低, 全基因组关联分析(genome-wide association)已经成为植物基因探究以及复杂性状解析的重要方法^[20]。全基因组关联分析已经在棉花^[21,22]、杨树^[23]、大豆^[24]等植物中广泛应用。本研究对588份甘蓝型油菜自然群体的叶片进行2年叶绿素含量测定并进行全基因组关联分析, 筛选出控制甘蓝型油菜叶片叶绿素含量相关的候选基因, 为油菜叶绿素含量的遗传改良奠定基础。

1 材料方法

1.1 试验材料

用于关联分析的588份甘蓝型油菜是由全球各地征集的油菜品种组成, 其中大部分来自中国重庆、湖北、湖南、江苏、陕西等地, 部分来自加拿大、德国、瑞典、丹麦、澳大利亚等国家。所有材料由重庆市西南大学油菜工程中心提供。

1.2 田间试验与叶绿素测定

该群体于2017—2018年, 2018—2019年连续种植于重庆市北碚区歇马镇油菜基地。采用随机区组设计, 2个重复, 以育苗移栽方式每小区种植3行, 每行15株, 行距40 cm, 株距20 cm。按照常规生产方式进行田间管理。在植株苗期用SPAD-502Plus叶绿素仪测定完全伸展的叶片中间部位叶绿素含量, 对每个品种测定5株。利用IBM SPSS Statistics 19.0统计分析软件进行描述性统计分析并制作正态分布图。

1.3 全基因组关联分析的方法

参考文献进行基因型数据测定与分析、群体结构、亲缘关系分析与连锁不平衡分析^[25]。全基因组关联分析采用naive、Q、PCA、K、Q+K和PCA+K六种统计模型来评估群体结构和亲缘关系的影响(Q: 群体结构; PCA: 主成分; K: 亲缘关系), 进行表型和SNP关联分析。运用TASSEL 5.0软件进行这6种模型的关联分析, 其中使用一般线性模型(GLM)分析naive、Q和PCA模型, 使用混合线性模型(MLM)分析K、Q+K和PCA+K模型。利用SAS软件检测这6种模型的运算结果, 对其lg(P)的观测值和lg(P)期望值绘制Quantile-quantile散点图, 通过QQ图比较后确定最佳模型, 在最佳模型下进行叶片叶绿素含量的全基因组关联分析, 利用R软件作Manhattan图。本试验使用的SNP数据为385,692个, P值小于阈值(1/385,692 = 2.593E-06)的位点为显著关联位点。

1.4 候选基因的挖掘

根据甘蓝型油菜“Darmor-bzh”参考基因组^[26], 提取分析得到的叶片叶绿素含量显著关联的SNP标记各位点500 kb内的基因^[27]; 将需要功能分析的基因序列与拟南芥所有基因序列进行BLASTn比对, e-value阈值为1e-10, 并以同源性最高的拟南芥基因作为待分析基因进行功能注释来筛选与叶绿素含量相关的候选基因。

1.5 候选基因验证

对叶绿素含量极端材料在苗期取3个生物学重复的叶片并提取RNA。根据前期所取材料的叶片提取的RNA, 使用TaKaRa公司的PrimeScript RT reagent Kit with gDNA Eraser反转录合成cDNA, 用于qRT-PCR验证。以油菜UBC21作为内参基因, 使用TaKaRa公司的SYBR Premix Ex Taq II试剂盒进行qRT-PCR验证候选基因的相对表达量。设置每个样品3次技术重复。在BIO-RAD定量PCR仪上进行qRT-PCR扩增。在网站http://biodb.swu.edu.cn/ qprimerdb上查找设计候选基因的qRT-PCR引物(表1), 由上海生工生物工程有限公司合成。

Table 1
表1
表1候选基因引物
Table 1List of primer sequences for candidate genes

基因Gene	引物序列Primer sequence (5°-3°)
BnaA04g00600D	F: AAATGTCGTTGAGGAATTACGC; R: TGAATGATATACGTCAGCACCA
BnaC09g54390D	F: AAGGAGCTAACACAATGACTGA; R: GGTATCATCACAGGTAACCCAT
BnaC03g18820D	F: AATCATGTTTCCATTAGCCACG; R: CATTACCACCGTTGAAAACCAA
BnaC06g24160D	F: AATGTTGCGCATTATCTTCCTC; R: TGTTTGTAGTGTTTCCAGGAGT
BnaC07g39280D	F: AGAAAACGATGCTTATGCTGAC; R: CATAGCTGCAGAGATATCGCTA
BnaA04g00500D	F: AGCTCATATACATTGTGGAGCA; R: TGCTGCTAGATTGGTCTAAGAG
BnaA02g24670D	F: CTAATCGAATCAAGCCCTGTTG; R: GGTGAAAACGCAAAGAAATTGG
BnaC02g01240D	F: CTCAAGGAGCTATTGGAATTGC; R: TATCCAGCAATAGGTGTACGAC
BnaC03g20950D	F: CTTTATGGTGTTGGCCAAGTAC; R: AACTTTACCACGCCGTATTTTC
BnaC05g38600D	F: GACTCTCTGAAGATACTGAGGC; R: ACTCTTGATCACCGTATACGTC
BnaA10g02050D	F: GAGCTGGAACAATAACTCCTCT; R: CACAGTTTCTTTGCCTTCTCTG
BnaC05g10770D	F: GAGTGTTCCTGATTGTATGCAC; R: AGATGTGCCAATCCAAAAGAAC
BnaC04g01850D	F: GATAGGAACGGTGATGGTTTTG; R: TTCCGTCACGAAATTAAAACCC
BnaC02g25030D	F: GTAAATGTGCCTGATGAGGTTG; R: TGAACAATCTCTTCGTCAAGGA
BnaA06g06000D	F: GTCGCTGCTTAAACAGTTACAA; R: GAAACCCAACAAAGAGTGAACA
BnaA05g01720D	F: GTTCACAACGTCAATGATCACA; R: GTGGCCCAAGAATCAAAGATTT
BnaC08g27000D	F: GTTTCTGAACGAACAGGTTGAA; R: ATTTAAGCAGCAGCAGAAGTTC
BnaC09g05970D	F: TTACTAGCTTGGCTCACACTAC; R: CGTTCTCATCAACAACACTCAG
BnaC09g05970D	F: TTACTAGCTTGGCTCACACTAC; R: CGTTCTCATCAACAACACTCAG
BnaA01g20460D	F: TTATCAAGCGTGCGAAGTAAAG; R: CGCGCTGATCTTAACCTAGTTA
BnaC08g27310D	F: TTTGCTACCTGTTGAAAACGAC; R: TTATTCAACTGTGACAAAGCCG

新窗口打开|下载CSV

2 结果与分析

2.1 叶绿素含量表型分析

自然群体中, 叶绿素含量在2018年和2019年的变异系数分别为11.27%和12.73%, 2年间的变异系数较为稳定(表2)。自然群体2年叶绿素含量表现出连续正态分布(图1), 说明该性状符合由多基因控制的数量性状特点, 适合全基因组关联分析。

Table 2
表2
表2甘蓝型油菜叶绿素含量的表型数据统计分析
Table 2Phenotypic variation of chlorophyll content in natural population

性状 Trait	环境 Environment	均值±标准差 Mean ± SD	范围 Range	变异系数 CV (%)
叶绿素含量	2018	47.79±5.39	33.44-65.82	11.27
Chlorophyll content	2019	46.37±5.90	32.92-72.04	12.73

SD: standard deviation; CV: coefficient of variation.

新窗口打开|下载CSV

图1

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图1甘蓝型油菜叶片叶绿素含量的频次分布

Fig. 1Frequency distribution of chlorophyll content in leaves of B. napus

2.2 甘蓝型油菜叶片叶绿素含量全基因组关联分析

对叶绿素含量关联分析6种模型下的结果绘制Quantile-quantile散点图(图2), 由图2可知, 最佳模型为P+K模型。在最佳模型下, 利用385,692个SNPs, 以P值小于阈值(1/385,692 = 2.593E-06)确定显著关联SNP位点(表3), 并绘制Manhattan图(图3)。

图2

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图2甘蓝型油菜叶绿素含量在各模型中的QQ图

Fig. 2Quantile-quantile plot for six models of chlorophyll contents

图3

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图3甘蓝型油菜叶绿素含量在最佳模型中的曼哈顿图

Fig. 3Manhattan plots of association analysis using the optimal model of chlorophyll contents



Table 3
表3
表3最佳模型下叶绿素含量显著位点表
Table 3Summary of significant SNPs for chlorophyll content by using the best model

环境Environment		位点 SNP		染色体 Chr.		位置 Position		阈值 lg (P-value)	贡献率 R2 (%)
2018		S1_12845378		A03		12,845,378		5.73	5.51
		S6_3493805		A06		3,493,805		7.49	7.89
		S6_3812220		A06		3,812,220		6.05	6.10
		S10_4969863		A10		4,969,863		6.25	6.23
		S14_37741561		C04		37,741,561		5.69	5.67
2019		S1_12688640		A01		12,688,640		6.39	8.42
		S1_12540615		A01		12,540,615		6.10	8.24
		S1_10570293		A01		10,570,293		5.93	8.51
		S1_12628926		A01		12,628,926		5.86	8.28
		S1_12540545		A01		12,540,545		5.84	7.67
		S1_12540622		A01		12,540,622		5.83	7.66
环境Environment	位点 SNP			染色体 Chr.		位置 Position		域值 lg (P-value)	贡献率 R2 (%)
2019	S1_12628563		A01		12,628,563		5.79		9.76
	S1_12568566		A01		12,568,566		5.78		7.55
	S2_17839296		A02		17,839,296		6.91		9.73
	S3_13514793		A03		13,514,793		5.73		7.72
	S4_547942		A04		547,942		5.93		8.08
	S8_16108365		A08		16,108,365		6.46		8.44
	S8_16192480		A08		16,192,480		5.88		7.80
	S8_16181282		A08		16,181,282		5.78		7.85
	S8_16103637		A08		16,103,637		5.61		7.97
	S8_16181355		A08		16,181,355		5.60		7.29
	S9_10532790		A09		10,532,790		6.18		8.70
	S10_14050715		A10		14,050,715		5.61		7.72
	S12_20495602		C02		20,495,602		5.95		9.35
	S12_22225349		C02		22,225,349		5.74		8.76
	S13_11413088		C03		11,413,088		7.01		10.34
	S13_11423629		C03		11,423,629		6.95		9.31
	S13_11415175		C03		11,415,175		6.61		8.92
	S13_11433444		C03		11,433,444		6.39		8.88
	S13_11433450		C03		11,433,450		6.38		8.87
	S13_9816561		C03		9,816,561		6.06		8.70
	S13_48351338		C03		48,351,338		6.00		8.06
	S13_11249216		C03		11,249,216		5.97		8.22
	S14_7018412		C04		7,018,412		6.81		9.39
	S14_1487936		C04		1,487,936		5.73		7.96
	S14_5798300		C04		5,798,300		5.68		8.45
	S14_36521995		C04		36,521,995		5.60		7.95
	S15_6002527		C05		6,002,527		6.06		8.65
	S16_24042352		C06		24,042,352		6.55		9.94
	S16_14397036		C06		14,397,036		6.31		8.84
	S16_25797577		C06		25,797,577		6.25		8.34
	S16_25797598		C06		25,797,598		6.14		8.15
	S16_25233735		C06		25,233,735		6.14		8.09
	S16_25797558		C06		25,797,558		6.00		8.46
	S16_25234451		C06		25,234,451		5.97		8.45
	S16_25234262		C06		25,234,262		5.92		8.11
	S16_25797570		C06		25,797,570		5.89		8.06
	S16_25234230		C06		25,234,230		5.88		8.31
	S18_31711591		C08		31,711,591		6.32		8.85
	S18_28228959		C08		28,228,959		5.77		8.36
	S19_32818852		C09		32,818,852		5.62		8.15

新窗口打开|下载CSV

2018年检测到5个SNP位点, 分别位于A01 (1个)、A06 (2个)、A10 (1个)和C04 (1个)染色体, 它们的贡献率为5.51%~7.89%, 其中S6_3493805位点贡献率最大; 2019年检测到46个SNP位点, 分别位于A01 (8个)、A02 (1个)、A03 (1个)、A04 (1个)、A08 (5个)、A09 (1个)、A10 (1个)、C02 (2个)、C03 (8个)、C04 (4个)、C05 (1个)、C06 (10个)、C08 (2个)和C09 (1个)染色体, 贡献率为7.29%~10.34%, 其中S13_11413088位点贡献率最大。

2.3 甘蓝型油菜叶片叶绿素含量候选基因筛选

初步筛选出23个与油菜叶片叶绿素含量显著关联的候选基因, 与拟南芥基因序列进行Blastn比对, 结合前人已报道的拟南芥同源基因功能, 筛选可能在本研究中发挥作用的候选基因(表4)。

Table 4
表4
表4叶绿素含量候选基因
Table 4Candidate genes for chlorophyll content

基因名字 Gene name	物理位置 Physical position	拟南芥同源基因 Homologs in A. thaliana	功能注释 Functional annotation
BnaA01g20460D	A01:12331065-1233745	AT3G48740	Nodulin MtN3 family protein
BnaA02g24670D	A02:17989410-17990090	AT5G46780	VQ motif-containing protein
BnaA04g00500D	A04:365464-366530	AT3G61890	Homeobox 12 (HB-12)
BnaA04g00600D	A04:460722-461017	AT3G61640	Arabinogalactan protein 20 (AGP20)
BnaA05g01720D	A05:984996-986020	AT2G40490	HEME2
BnaA06g06000D	A06:3306811-3307795	AT1G10200	WLIM1
BnaA10g02050D	A10:1025298-1027102	AT1G03630	Protochlorophyllide oxidoreductase C (POR C)
BnaC02g01240D	C02:511615-513517	AT5G08280	Hydroxymethylbilane synthase (HEMC)
BnaC02g25030D	C02:22402218-22403498	AT1G07440	NAD(P)-binding Rossmann-fold superfamily protein
BnaC03g18820D	C03:9634933-9635479	AT2G33830	Dormancy/auxin associated family protein
BnaC03g20950D	C03:11192271-11192876	AT2G37970	SOUL-1
BnaC04g01850D	C04:1467437-1468078	AT2G41410	Calcium-binding EF-hand family protein
BnaC05g10770D	C05:6194068-6195531	AT1G14480	Ankyrin repeat family protein
基因名字 Gene name	物理位置 Physical position	拟南芥同源基因 Homologs in A. thaliana	功能注释 Functional annotation
BnaC05g38600D	C05:37280225-37281849	AT3G14930	HEME1
BnaC06g24160D	C06:25958767-25959390	AT1G72610	Germin-like protein 1 (GER1)
BnaC07g39280D	C07:40238482-40239858	AT4G25080	Magnesium-protoporphyrin IX methyltransferase (CH LM)
BnaC08g27000D	C08:28191454-28192970	AT3G56090	Ferritin 3 (FER3)
BnaC08g27310D	C08:28357417-28358091	AT3G56360	Unknown protein
BnaC09g05970D	C09:3640560-3642915	AT5G63570	Glutamate-1-semialdehyde-2,1-aminomutase (GSA1)
BnaC03g18980D	chrC03:9831772-9832572	AT2G34430	Light-harvesting chlorophyll-protein complex II subunit B1 (LHB1B1)
BnaC02g25060D	chrC02:22446772-22448037	AT1G78600	Light-regulated zinc finger protein 1 (LZF1)
BnaA04g00660D	chrA04:488057-489279	AT3G61470	Photosystem I light harvesting complex gene 2 (LHCA2)
BnaA08g22200D	chrA08:16221799-16222764	AT1G19150	Photosystem I light harvesting complex gene 6 (LHCA6)

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2.4 候选基因的验证

挑选20个候选基因进行荧光定量验证, 检测其在2类材料中的表达情况表明, 相比叶绿素含量高的材料, 在叶绿素含量低的材料中, 11个基因上调表达, 9个基因下调表达(图4)。上调基因包括BnaC05g38600D, 其编码HEME1蛋白, 下调基因包括BnaC09g54390D, 其编码HEMG2, 这些基因均参与叶绿素合成途径。

图4

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图4甘蓝型油菜叶片叶绿素含量候选基因定量验证

Fig. 4Expression profiles of chlorophyll content candidate genes in leaves of B. napus using qRT-PCR

3 讨论

利用数量性状座位(quantitative trait loci, QTL)或者全基因组关联分析可以快速鉴定与性状关联的分子标记和基因, 是非常有效的定位候选基因的方法。定位甘蓝型油菜重要农艺性状QTL已经在产量性状如千粒重^[27]、角果长度^[28]和农艺性状如开花期^[29]、株高^[30]等方面取得重要进展。利用GWAS定位甘蓝型油菜复杂的数量性状同样取得重要进展^[31,32,33]。但是, 影响甘蓝型油菜叶片叶绿素含量相关QTL或者候选基因的研究相对较少, 仅有1篇通过突变体鉴定到的一个候选基因BnaC.YGL^[8]。

本研究中, 利用588份全球搜集的优质种质资源, 具有较为丰富的表型变异, 叶绿素含量变异范围广(表2); 课题组前期已经对该资源材料进行重测序, 挖掘出385,692个高质量的SNP位点^[25], 这些为通过GWAS分析筛选叶绿素含量显著关联位点奠定基础。植物合成的叶绿素含量不仅受到遗传因素的调控, 也会受到外界环境的影响, 如外界光照、温度和营养^[34]。而当植物叶绿素含量发生变化, 细胞内叶绿素的超微结构、叶片生理生化特征以及叶色调控机制也都会发生改变^[17]。油菜叶片叶绿素含量受年度间水肥条件和光温条件差异影响, 基因型与环境互作较大, 不同环境下有不同的基因群发挥作用, 进而造成关联位点的差异。通过测定甘蓝型油菜苗期叶片叶绿素含量, 2018和2019年分别筛选出与叶绿素显著关联的SNP位点5个和46个。通过对SNP位点上下游各500 kb范围内候选基因的筛选, 共得到2022个候选基因, 其中, BnaA10g02050D (BnaPOR C)、BnaC02g01240D (BnaHEMC)、Bna C05g38600D (BnaHEME2)和BnaC09g54390D (BnaHEMG2) 4个基因的同源基因在拟南芥中参与叶绿素合成途径(表4)。这些将作为重要候选基因进一步分析。

另外, 4个基因参与叶绿体发育和光合作用过程, 如BnaC03g18980D, 编码叶绿素a/b结合蛋白(LIGHT-HARVESTING CHLOROPHYLL-PROTEIN COMPLEX II SUBUNIT B1, LHB1B1), 该蛋白定位在叶绿体膜、类囊体膜上参与光捕获, 响应光刺激^[35]。BnaA04g00660D和BnaA08g22200D分别编码光合系统I的光捕获复合体基因2 (PHOTOSYSTEM I LIGHT HARVESTING COMP LEX GENE 2, LHCA2)和LHCA6, 都定位在叶绿体类囊体膜, 参与光捕获过程^[36,37]。BnaC02g25060D编码光调控的锌指蛋白1 (light-regulated zinc finger protein 1, LZF1), 又名B-box domain protein 22 (BBX22和DBB3), 其定位在细胞核, 参与含花青素的复合生物合成过程、叶绿素生物合成过程、叶绿体组织等调控过程^[38,39]。

4 结论

2年分别鉴定到5个和46个显著关联的SNP标记, SNP位点上下游500 kb区间内共筛选出23个与叶绿素合成途径相关的候选基因, 并进行了定量验证, 本研究为油菜后续叶片叶绿素含量的遗传改良奠定了基础。

参考文献 View Option 原文顺序
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[12] Kassahun B, Bidinger F R, Hash C T, Kuruvinashetti M S. Stay-green expression in early generation sorghum [Sorghum bicolor (L.) Moench] QTL introgression lines
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2F3/BC1F4 generations) of four stable stay-green QTLs (StgB, Stg1, Stg3 and Stg4) from donor parent B35 to senescent variety R 16. The majority of the introgression lines had higher leaf chlorophyll levels at flowering (a distinctive trait of the donor parent) and a greater percentage green leaf area during the latter part of grain filling, than did R 16, indicating that the stay-green QTLs were expressed phenotypically in the R 16 background. None of the QTL introgression lines achieved the same level of stay-green as B35, however. Maintenance of a greater relative green leaf area during the latter half of grain filling was related to a greater relative grain yield in two of three post-flowering moisture deficit environments in which the materials were evaluated (r 2=0.34 in 2004–2005 and r 2=0.76 in 2005–2006), as was a direct measure of leaf chlorophyll in one of the post-flowering stress environments in which this was measured (r 2=0.42, P<0.05). Thus the study provided useful evidence that the marker-assisted backcross transfer of stay-green QTLs from B35 into an adapted, but senescent background has the potential to enhance tolerance of post-flowering drought stress in sorghum.]]>

[13] Cai H G, Chu Q, Yuan L X, Liu J C, Chen X H, Chen F J, Mi G H, Zhang F S. Identification of quantitative trait loci for leaf area and chlorophyll content in maize (Zea mays) under low nitrogen and low phosphorus supply
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[14] Czyczylo Mysza I, Tyrka M, Marcinska I, Skrzypek E, Karbarz M, Dziurka M, Hura T, Dziurka K, Quarrie S A. Quantitative trait loci for leaf chlorophyll fluorescence parameters, chlorophyll and carotenoid contents in relation to biomass and yield in bread wheat and their chromosome deletion bin assignments
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DOI:10.1007/s11032-013-9862-8URLPMID:23794940 [本文引用: 1]
Relatively little is known of the genetic control of chlorophyll fluorescence (CF) and pigment traits important in determining efficiency of photosynthesis in wheat and its association with biomass productivity. A doubled haploid population of 94 lines from the wheat cross Chinese Spring x SQ1 was trialled under optimum glasshouse conditions for 4 years to identify quantitative trait loci (QTL) for CF traits including, for the first time in wheat, JIP-test parameters per excited cross section (CSm): ABS/CSm, DIo/CSm, TRo/CSm, RC/CSm and ETo/CSm, key parameters determining efficiency of the photosynthetic apparatus, as well as chlorophyll and carotenoid contents to establish associations with biomass and grain yield. The existing genetic map was extended to 920 loci by adding Diversity Arrays Technology markers. Markers and selected genes for photosynthetic light reactions, pigment metabolism and biomass accumulation were located to chromosome deletion bins. Across all CF traits and years, 116 QTL for CF were located on all chromosomes except 7B, and 39 QTL were identified for pigments on the majority of chromosomes, excluding 1A, 2A, 4A, 3B, 5B, 1D, 2D, 5D, 6D and 7D. Thirty QTL for plant productivity traits were mapped on chromosomes 3A, 5A, 6A, 7A, 1B, 2B, 4B, 6B, 7B, 3D and 4D. A region on chromosome 6B was identified where 14 QTL for CF parameters coincided with QTL for chlorophyll content and grain weight per ear. Thirty-five QTL regions were coincident with candidate genes. The environment was shown to dominate in determining expression of genes for those traits.

[15] Jiang S, Zhang X, Zhang F, Xu Z, Chen W, Li Y. Identification and fine mapping of qCTH4, a quantitative trait loci controlling the chlorophyll content from tillering to heading in rice (Oryza sativa L.)
J Hered, 2012,103:720-726.
DOI:10.1093/jhered/ess041URL [本文引用: 1]
The chlorophyll content is one of the most important traits selected by breeders, and it is controlled by quantitative trait loci (QTLs) derived from natural variations in rice. We analyzed the QTL controlling chlorophyll content by using 94 RILs derived from a cross between 2 japonica rice cultivars, Lijiangxintuanheigu (LTH) and Shennong265 (SN265). Twenty-two QTLs controlling chlorophyll content at tillering stage, heading stage, and maturity stage were detected, respectively. Among them, Rice cv. LTH had a positive allele only at 1 locus (qCTH4) on chromosome 4. Further analysis indicated that the genetic effect of qCTH4 was the net effects within the period from tillering to heading. The QTL qCTH4 controlling chlorophyll content from tillering to heading locates between RM255 and RM349 on chromosome 4 with a LOD score 19.41, and the QTL qCTH4 explains 61.42% of phenotypic variation. In order to eliminate the influence of other QTLs, 1 single residual heterozygous plant, RH-qCTH4, was selected based on the genotypes of 114 Simple Sequence Repeat (SSR) markers. Using the segregating population derived from RH-qCTH4 by self-crossing, this region was narrowed down to an interval between RM3276 and RM17494 in an approximately 771 kb target region. These results are useful for map-based cloning of qCTH4 and for marker-assisted selection of high photosynthetic efficiency variety.

[16] Dhanapal A P, Ray J D, Singh S K, Hoyos Villegas V, Smith J R, Purcell L C, Fritschi F B. Genome-wide association mapping of soybean chlorophyll traits based on canopy spectral reflectance and leaf extracts
BMC Plant Biol, 2016,16:174.
DOI:10.1186/s12870-016-0861-xURLPMID:27488358 [本文引用: 1]
BACKGROUND: Chlorophyll is a major component of chloroplasts and a better understanding of the genetic basis of chlorophyll in soybean [Glycine max (L.) Merr.] might contribute to improving photosynthetic capacity and yield in regions with adverse environmental conditions. A collection of 332 diverse soybean genotypes were grown in 2 years (2009 and 2010) and chlorophyll a (eChl_A), chlorophyll b (eChl_B), and total chlorophyll (eChl_T) content as well as chlorophyll a/b ratio (eChl_R) in leaf tissues were determined by extraction and spectrometric determination. Total chlorophyll was also derived from canopy spectral reflectance measurements using a model of wavelet transformed spectra (tChl_T) as well as with a spectral reflectance index (iChl_T). RESULTS: A genome-wide associating mapping approach was employed using 31,253 single nucleotide polymorphisms (SNPs) to identify loci associated with the extract based eChl_A, eChl_B, eChl_R and eChl_T measurements and the two canopy spectral reflectance-based methods (tChl_T and iChl_T). A total of 23 (14 loci), 15 (7 loci) and 14 SNPs (10 loci) showed significant association with eChl_A, eChl_B and eChl_R respectively. A total of 52 unique SNPs were significantly associated with total chlorophyll content based on at least one of the three approaches (eChl_T, tChl_T and iChl_T) and likely tagged 27 putative loci for total chlorophyll content, four of which were indicated by all three approaches. CONCLUSIONS: Results presented here show that markers for chlorophyll traits can be identified in soybean using both extract-based and canopy spectral reflectance-based phenotypes, and confirm that high-throughput phenotyping-amenable canopy spectral reflectance measurements can be used for association mapping.

[17] Wang Y K, He Y J, Yang M, He J B, Xu P, Shao M Q, Chu P, Guan R Z. Fine mapping of a dominant gene conferring chlorophyll-deficiency in Brassica napus
Sci Rep, 2016,6:31419. doi: 10.1038/srep31419.
DOI:10.1038/srep31419URLPMID:27506952 [本文引用: 3]
Leaf colour regulation is important in photosynthesis and dry material production. Most of the reported chlorophyll-deficient loci are recessive. The dominant locus is rarely reported, although it may be more important than the recessive locus in the regulation of photosynthesis efficiency. During the present study, we mapped a chlorophyll-deficient dominant locus (CDE1) from the ethyl methanesulfonate-mutagenized Brassica napus line NJ7982. Using an F2 population derived from the chlorophyll-deficient mutant (cde1) and the canola variety 'zhongshuang11', a high-density linkage map was constructed, consisting of 19 linkage groups with 2,878 bins containing 13,347 SNP markers, with a total linkage map length of 1,968.6 cM. Next, the CDE1 locus was mapped in a 0.9-cM interval of chromosome C08 of B. napus, co-segregating with nine SNP markers. In the following fine-mapping of the gene using the inherited F2:3 populations of 620 individuals, the locus was identified in an interval with a length of 311 kb. A bioinformatics analysis revealed that the mapping interval contained 22 genes. These results produced a good foundation for continued research on the dominant locus involved in chlorophyll content regulation.

[18] Qian L W, Voss Fels K, Cui Y X, Jan H U, Samans B, Obermeier C, Qian W, Snowdon R J. Deletion of a stay-green gene associates with adaptive selection in Brassica napus
Mol Plant, 2016,9:1559-1569.
DOI:10.1016/j.molp.2016.10.017URLPMID:27825945 [本文引用: 2]
Chlorophyll levels provide important information about plant growth and physiological plasticity in response to changing environments. The stay-green gene NON-YELLOWING 1 (NYE1) is believed to regulate chlorophyll degradation during senescence, concomitantly affecting the disassembly of the light-harvesting complex and hence indirectly influencing photosynthesis. We identified Brassica napus accessions carrying an NYE1 deletion associated with increased chlorophyll content, and with upregulated expression of light-harvesting complex and photosynthetic reaction center (PSI and PSII) genes. Comparative analysis of the seed oil content of accessions with related genetic backgrounds revealed that the B. napus NYE1 gene deletion (bnnye1) affected oil accumulation, and linkage disequilibrium signatures suggested that the locus has been subject to artificial selection by breeding in oilseed B. napus forms. Comparative analysis of haplotype diversity groups (haplogroups) between three different ecotypes of the allopolyploid B. napus and its A-subgenome diploid progenitor, Brassica rapa, indicated that introgression of the bnnye1 deletion from Asian B. rapa into winter-type B. napus may have simultaneously improved its adaptation to cooler environments experienced by autumn-sown rapeseed.

[19] Ge Y, Wang T, Wang N, Wang Z, Liang C, Ramchiary N, Choi S R, Lim Y P, Piao Z Y. Genetic mapping and localization of quantitative trait loci for chlorophyll content in Chinese cabbage (
Sci Hortic-Amsterdam, 2012,147:42-48.
DOI:10.1016/j.scienta.2012.09.004URL [本文引用: 1]

[20] Luo J. Metabolite-based genome-wide association studies in plants
Curr Opin Plant Biol, 2015,24:31-38.
DOI:10.1016/j.pbi.2015.01.006URLPMID:25637954 [本文引用: 1]
The plant metabolome is the readout of plant physiological status and is regarded as the bridge between the genome and the phenome of plants. Unraveling the natural variation and the underlying genetic basis of plant metabolism has received increasing interest from plant biologists. Enabled by the recent advances in high-throughput profiling and genotyping technologies, metabolite-based genome-wide association study (mGWAS) has emerged as a powerful alternative forward genetics strategy to dissect the genetic and biochemical bases of metabolism in model and crop plants. In this review, recent progress and applications of mGWAS in understanding the genetic control of plant metabolism and in interactive functional genomics and metabolomics are presented. Further directions and perspectives of mGWAS in plants are also discussed.

[21] Su J J, Li L B, Zhang C, Wang C X, Gu L J, Wang H T, Wei H L, Liu Q B, Huang L, Yu S X. Genome-wide association study identified genetic variations and candidate genes for plant architecture component traits in Chinese upland cotton
Theor Appl Genet, 2018,131:1299-1314.
DOI:10.1007/s00122-018-3079-5URLPMID:29497767 [本文引用: 1]
KEY MESSAGE: Thirty significant associations between 22 SNPs and five plant architecture component traits in Chinese upland cotton were identified via GWAS. Four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits. A candidate gene, Gh_D03G0922, might be responsible for plant height in upland cotton. A compact plant architecture is increasingly required for mechanized harvesting processes in China. Therefore, cotton plant architecture is an important trait, and its components, such as plant height, fruit branch length and fruit branch angle, affect the suitability of a cultivar for mechanized harvesting. To determine the genetic basis of cotton plant architecture, a genome-wide association study (GWAS) was performed using a panel composed of 355 accessions and 93,250 single nucleotide polymorphisms (SNPs) identified using the specific-locus amplified fragment sequencing method. Thirty significant associations between 22 SNPs and five plant architecture component traits were identified via GWAS. Most importantly, four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits, and these SNPs were harbored in one linkage disequilibrium block. Furthermore, 21 candidate genes for plant architecture were predicted in a 0.95-Mb region including the four peak SNPs. One of these genes (Gh_D03G0922) was near the significant SNP D03_31584163 (8.40 kb), and its Arabidopsis homologs contain MADS-box domains that might be involved in plant growth and development. qRT-PCR showed that the expression of Gh_D03G0922 was upregulated in the apical buds and young leaves of the short and compact cotton varieties, and virus-induced gene silencing (VIGS) proved that the silenced plants exhibited increased PH. These results indicate that Gh_D03G0922 is likely the candidate gene for PH in cotton. The genetic variations and candidate genes identified in this study lay a foundation for cultivating moderately short and compact varieties in future Chinese cotton-breeding programs.

[22] Li T G, Ma X F, Li N Y, Zhou L, Liu Z, Han H Y, Gui Y J, Bao Y M, Chen J Y, Dai X F. Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)
Plant Biotechnol J, 2017,15:1520-1532.
DOI:10.1111/pbi.12734URLPMID:28371164 [本文引用: 1]

[23] Fahrenkrog A M, Neves L G, Resende M F R, Vazquez A I, de los Campos G, Dervinis C, Sykes R, Davis M, Davenport R, Barbazuk W B, Kirst M. Genome-wide association study reveals putative regulators of bioenergy traits in Populus deltoides
New Phytol, 2017,213:799-811.
DOI:10.1111/nph.14154URLPMID:27596807 [本文引用: 1]
Genome-wide association studies (GWAS) have been used extensively to dissect the genetic regulation of complex traits in plants. These studies have focused largely on the analysis of common genetic variants despite the abundance of rare polymorphisms in several species, and their potential role in trait variation. Here, we conducted the first GWAS in Populus deltoides, a genetically diverse keystone forest species in North America and an important short rotation woody crop for the bioenergy industry. We searched for associations between eight growth and wood composition traits, and common and low-frequency single-nucleotide polymorphisms detected by targeted resequencing of 18 153 genes in a population of 391 unrelated individuals. To increase power to detect associations with low-frequency variants, multiple-marker association tests were used in combination with single-marker association tests. Significant associations were discovered for all phenotypes and are indicative that low-frequency polymorphisms contribute to phenotypic variance of several bioenergy traits. Our results suggest that both common and low-frequency variants need to be considered for a comprehensive understanding of the genetic regulation of complex traits, particularly in species that carry large numbers of rare polymorphisms. These polymorphisms may be critical for the development of specialized plant feedstocks for bioenergy.

[24] Wei W, Mesquita A C O, Figueiro A D, Wu X, Manjunatha S, Wickland D P, Hudson M E, Juliatti F C, Clough S J. Genome-wide association mapping of resistance to a Brazilian isolate of Sclerotinia sclerotiorum in soybean genotypes mostly from Brazil
BMC Genomics, 2017,18:849.
DOI:10.1186/s12864-017-4160-1URLPMID:29115920 [本文引用: 1]

[25] Lu K, Wei L J, Li X L, Wang Y T, Wu J, Liu M, Zhang C, Chen Z Y, Xiao Z C, Jian H J, Cheng F, Zhang K, Du H, Cheng X C, Qu C M, Qian W, Liu L Z, Wang R, Zou Q Y, Ying J M, Xu X F, Mei J Q, Liang Y, Chai Y R, Tang Z L, Wan H F, Ni Y, He Y J, Lin N, Fan Y H, Sun W, Li N N, Zhou G, Zheng H K, Wang X W, Paterson A H, Li J N. Whole-genome resequencing reveals Brassica napus origin and genetic loci involved in its improvement
Nat Commun, 2019,10:1154.
DOI:10.1038/s41467-019-09134-9URLPMID:30858362 [本文引用: 2]
Brassica napus (2n = 4x = 38, AACC) is an important allopolyploid crop derived from interspecific crosses between Brassica rapa (2n = 2x = 20, AA) and Brassica oleracea (2n = 2x = 18, CC). However, no truly wild B. napus populations are known; its origin and improvement processes remain unclear. Here, we resequence 588 B. napus accessions. We uncover that the A subgenome may evolve from the ancestor of European turnip and the C subgenome may evolve from the common ancestor of kohlrabi, cauliflower, broccoli, and Chinese kale. Additionally, winter oilseed may be the original form of B. napus. Subgenome-specific selection of defense-response genes has contributed to environmental adaptation after formation of the species, whereas asymmetrical subgenomic selection has led to ecotype change. By integrating genome-wide association studies, selection signals, and transcriptome analyses, we identify genes associated with improved stress tolerance, oil content, seed quality, and ecotype improvement. They are candidates for further functional characterization and genetic improvement of B. napus.

[26] Chalhoub B, Denoeud F, Liu S Y, Parkin I A P, Tang H B, Wang X Y, Chiquet J, Belcram H, Tong C B, Samans B. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome
Science, 2014,345:950-953.
DOI:10.1126/science.1253435URLPMID:25146293 [本文引用: 1]
Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72x genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

[27] Fu Y, Wei D Y, Dong H L, He Y J, Cui Y X, Mei J Q, Wan H F, Li J, Snowdon R, Friedt W, Li X R, Qian W. Comparative quantitative trait loci for silique length and seed weight in Brassica napus
Sci Rep, 2015,5:14407.
DOI:10.1038/srep14407URLPMID:26394547 [本文引用: 2]
Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1-74.3% and 24.4-62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection.

[28] Wang X D, Chen L, Wang A N, Wang H, Tian J H, Zhao X P, Chao H B, Zhao Y J, Zhao W G, Xiang J, Gan J P, Li M T. Quantitative trait loci analysis and genome-wide comparison for silique related traits in Brassica napus
BMC Plant Biol, 2016,16:71.
DOI:10.1186/s12870-016-0759-7URLPMID:27000872 [本文引用: 1]
BACKGROUND: Yield of rapeseed is determined by three components: silique number, seed number per silique and thousand seed weight. Seed number per silique and thousand seed weight are influenced by silique length, seed density, silique breadth, silique thickness and silique volume. Some QTLs for silique traits have been reported in B. napus, however, no studies have focused on the six agronomic traits (seed number per silique, silique length, silique breadth, silique thickness, seed density and silique volume) simultaneously, and the genetic determinism of such complex traits have not been fully elucidated. RESULTS: In this study, the six silique traits were evaluated using 348 lines of a doubled haploid population, the KN population. The results showed that 2, 4, 1, 1 and 2 QTLs explaining > 10 % of phenotypic variation were obtained for silique length, silique breadth, silique thickness, seed number per silique and silique volume, respectively. Notably, three major effect QTLs (cqSB-C6-1, cqSB-C6-2 and cqSV-C6-3) were identified in at least three environments, and 17 unique QTLs controlling at least two traits were obtained. A high-density consensus map containing 1225 markers was constructed for QTL comparison by combining the KN map with other five published maps. The comparative results revealed that 14, 13 and 11 QTLs for silique breadth, silique thickness and silique volume might be the potential new QTLs because few QTLs for these traits were reported in B. napus. In addition, potential new QTLs for silique length (11), seed number per silique (6) and seed density (5) were also identified. Twenty-five candidate genes underlying 27 QTLs for silique related traits were obtained. CONCLUSIONS: This study constructed QTL analysis in B. napus, and obtained 60 consensus QTLs for six silique related traits. The potential new QTLs will enhance our understanding of the genetic control of silique traits, and the stable QTLs provided the targets for improving seed yield in future. These findings provided comprehensive insights into the genetic network affecting silique traits at QTL level in B. napus.

[29] Jian H J, Zhang A X, Ma J Q, Wang T Y, Yang B, Shuang L S, Liu M, Li J N, Xu X F, Paterson A H, Liu L Z. Joint QTL mapping and transcriptome sequencing analysis reveal candidate flowering time genes in Brassica napus L
BMC Genomics, 2019,9:390.
DOI:10.1186/1471-2164-9-390URLPMID:18713468 [本文引用: 1]
BACKGROUND: DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. RESULTS: By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. CONCLUSION: The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results.

[30] Shen Y S, Xiang Y, Xu E S, Ge X H, Li Z Y. Major co-localized QTL for plant height, branch initiation height, stem diameter, and flowering time in an alien introgression derived Brassica napus DH population
Front Plant Sci, 2018,9:390.
DOI:10.3389/fpls.2018.00390URLPMID:29643859 [本文引用: 1]
Plant height (PH), branch initiation height (BIH), and stem diameter (SD) are three stem-related traits that play crucial roles in plant architecture and lodging resistance. Herein, we show one doubled haploid (DH) population obtained from a cross between Y689 (one Capsella bursa-pastoris derived Brassica napus intertribal introgression) and Westar (B. napus cultivar) that these traits were significantly positively correlated with one another and with flowering time (FT). Based on a high-density SNP map, a total of 102 additive quantitative trait loci (QTL) were identified across six environments. Seventy-two consensus QTL and 49 unique QTL were identified using a two-round strategy of QTL meta-analysis. Notably, a total of 19 major QTL, including 11 novel ones, were detected for these traits, which comprised two QTL clusters on chromosomes A02 and A07. Conditional QTL mapping was performed to preliminarily evaluate the genetic basis (pleiotropy or tight linkage) of the co-localized QTL. In addition, QTL by environment interactions (QEI) mapping was performed to verify the additive QTL and estimate the QEI effect. In the genomic regions of all major QTL, orthologs of the genes involved in phytohormone biosynthesis, phytohormone signaling, flower development, and cell differentiation in Arabidopsis were proposed as candidate genes. Of these, BnaA02g02560, an ortholog of Arabidopsis GASA4, was suggested as a candidate gene for PH, SD, and FT; and BnaA02g08490, an ortholog of Arabidopsis GNL, was associated with PH, BIH and FT. These results provide useful information for further genetic studies on stem-related traits and plant growth adaptation.

[31] Lu K, Xiao Z C, Jian H J, Peng L, Qu C M, Fu M L, He B, Tie L M, Liang Y, Xu X F, Li J N. A combination of genome-wide association and transcriptome analysis reveals candidate genes controlling harvest index-related traits in Brassica napus
Sci Rep, 2016,6:36452.
DOI:10.1038/srep36452URLPMID:27811979 [本文引用: 1]
Harvest index (HI), the ratio of seed mass to total biomass of the aboveground plant parts, is an important trait for harvestable yield of crops. Unfortunately, HI of Brassica napus is lower than that of other economically important crops. To identify candidate genes associated with high HI, a genome-wide association study of HI and four HI-related traits was conducted with 520 B. napus accessions cultivated in both Yunnan and Chongqing. We detected 294 single nucleotide polymorphisms significantly associated with the abovementioned traits, including 79 SNPs that affected two or more traits. Differentially expressed genes between extremely high- and low-HI accessions were identified in 8 tissues at two cultivated regions. Combination of linkage disequilibrium and transcriptome analyses revealed 33 functional candidate genes located within the confidence intervals of significant SNPs associated with more than one trait, such as SHOOT GRAVITROPISM 5 (Bna.SGR5), ATP-CITRATE LYASE A-3 (Bna.ACLA-3) and CAROTENOID CLEAVAGE DIOXYGENASE 1 (Bna.CCD1), their orthologs in the Arabidopsis thaliana have been shown to play key roles in photosynthesis, inflorescence, and silique development. Our results provide insight into the molecular mechanisms underlying establishment of high-HI B. napus and lay a foundation for characterization of candidate genes aimed at developing high-HI B. napus varieties.

[32] Wang J, Xian X H, Xu X F, Qu C M, Lu K, Li J N, Liu L Z. Genome-wide association mapping of seed coat color in Brassica napus
J Agric Food Chem, 2017,65:5229-5237.
DOI:10.1021/acs.jafc.7b01226URLPMID:28650150 [本文引用: 1]
Seed coat color is an extremely important breeding characteristic of Brassica napus. To elucidate the factors affecting the genetic architecture of seed coat color, a genome-wide association study (GWAS) of seed coat color was conducted with a diversity panel comprising 520 B. napus cultivars and inbred lines. In total, 22 single-nucleotide polymorphisms (SNPs) distributed on 7 chromosomes were found to be associated with seed coat color. The most significant SNPs were found in 2014 near Bn-scaff_15763_1-p233999, only 43.42 kb away from BnaC06g17050D, which is orthologous to Arabidopsis thaliana TRANSPARENT TESTA 12 (TT12), an important gene involved in the transportation of proanthocyanidin precursors into the vacuole. Two of eight repeatedly detected SNPs can be identified and digested by restriction enzymes. Candidate gene mining revealed that the relevant regions of significant SNP loci on the A09 and C08 chromosomes are highly homologous. Moreover, a comparison of the GWAS results to those of previous quantitative trait locus (QTL) studies showed that 11 SNPs were located in the confidence intervals of the QTLs identified in previous studies based on linkage analyses or association mapping. Our results provide insights into the genetic basis of seed coat color in B. napus, and the beneficial allele, SNP information, and candidate genes should be useful for selecting yellow seeds in B. napus breeding.

[33] Xiao Z C, Zhang C, Tang F, Yang B, Zhang L Y, Liu J S, Huo Q, Wang S F, Li S T, Wei L J, Du H, Qu C M, Lu K, Li J N, Li N N. Identification of candidate genes controlling oil content by combination of genome-wide association and transcriptome analysis in the oilseed crop Brassica napus
Biotechnol Biofuels, 2019,12:216.
DOI:10.1186/s13068-019-1557-xURLPMID:31528204 [本文引用: 1]
Background: Increasing seed oil content is one of the most important targets for rapeseed (Brassica napus) breeding. However, genetic mechanisms of mature seed oil content in Brassica napus (B. napus) remain little known. To identify oil content-related genes, a genome-wide association study (GWAS) was performed using 588 accessions. Results: High-throughput genome resequencing resulted in 385,692 high-quality single nucleotide polymorphism (SNPs) with a minor allele frequency (MAF) > 0.05. We identified 17 loci that were significantly associated with seed oil content, among which 12 SNPs were distributed on the A3 (11 loci) and A1 (one loci) chromosomes, and five novel significant SNPs on the C5 (one loci) and C7 (four loci) chromosomes, respectively. Subsequently, we characterized differentially expressed genes (DEGs) between the seeds and silique pericarps on main florescences and primary branches of extremely high- and low-oil content accessions (HO and LO). A total of 64 lipid metabolism-related DEGs were identified, 14 of which are involved in triacylglycerols (TAGs) biosynthesis and assembly. Additionally, we analyzed differences in transcription levels of key genes involved in de novo fatty acid biosynthesis in the plastid, TAGs assembly and lipid droplet packaging in the endoplasmic reticulum (ER) between high- and low-oil content B. napus accessions. Conclusions: The combination of GWAS and transcriptome analyses revealed seven candidate genes located within the confidence intervals of significant SNPs. Current findings provide valuable information for facilitating marker-based breeding for higher seed oil content in B. napus.

[34] Ferreira V D S, Anna C S. Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications
World J Microbiol Biotechnol, 2017,33:20.
DOI:10.1007/s11274-016-2181-6URLPMID:27909993 [本文引用: 1]
Chlorophyll is a commercially important natural green pigment responsible for the absorption of light energy and its conversion into chemical energy via photosynthesis in plants and algae. This bioactive compound is widely used in the food, cosmetic, and pharmaceutical industries. Chlorophyll has been consumed for health benefits as a nutraceutical agent with antioxidant, anti-inflammatory, antimutagenic, and antimicrobial properties. Microalgae are photosynthesizing microorganisms which can be extracted for several high-value bioproducts in the biotechnology industry. These microorganisms are highly efficient at adapting to physicochemical variations in the local environment. This allows optimization of culture conditions for inducing microalgal growth and biomass production as well as for changing their biochemical composition. The modulation of microalgal culture under controlled conditions has been proposed to maximize chlorophyll accumulation. Strategies reported in the literature to promote the chlorophyll content in microalgae include variation in light intensity, culture agitation, and changes in temperature and nutrient availability. These factors affect chlorophyll concentration in a species-specific manner; therefore, optimization of culture conditions has become an essential requirement. This paper provides an overview of the current knowledge on the effects of key environmental factors on microalgal chlorophyll accumulation, focusing on small-scale laboratory experiments.

[35] Peltier J B, Ytterberg A J, Sun Q, van Wijk K J. New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy
J Biol Chem, 2004,279:49367-49383.
DOI:10.1074/jbc.M406763200URLPMID:15322131 [本文引用: 1]
Identification of membrane proteomes remains challenging. Here, we present a simple, fast, and scalable off-line procedure based on three-phase partitioning with butanol to fractionate membrane proteomes in combination with both in-gel and in-solution digestions and mass spectrometry. This should help to further accelerate the field of membrane proteomics. Using this new strategy, we analyzed the salt-stripped thylakoid membrane of chloroplasts of Arabidopsis thaliana. 242 proteins were identified, at least 40% of which are integral membrane proteins. The functions of 86 proteins are unknown; these include proteins with TPR, PPR, rhodanese, and DnaJ domains. These proteins were combined with all known thylakoid proteins and chloroplast (associated) envelope proteins, collected from primary literature, resulting in 714 non-redundant proteins. They were assigned to functional categories using a classification developed for MapMan (Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P., Selbig, J., Muller, L. A., Rhee, S. Y., and Stitt, M. (2004) Plant J. 37, 914-939), updated with information from primary literature. The analysis elucidated the likely location of many membrane proteins, including 190 proteins of unknown function, holding the key to better understanding the two membrane systems. The three-phase partitioning procedure added a new level of dynamic resolution to the known thylakoid proteome. An automated strategy was developed to track possible ambiguous identifications to more than one gene model or family member. Mass spectrometry search results, ambiguities, and functional classifications can be searched via the Plastid Proteome Database.

[36] Wientjes E, Croce R. The light-harvesting complexes of higher-plant Photosystem I: Lhca1/4 and Lhca2/3 form two red-emitting heterodimers
Biochem J, 2011,433:477-485.
DOI:10.1042/BJ20101538URLPMID:21083539 [本文引用: 1]
The outer antenna of higher-plant PSI (Photosystem I) is composed of four complexes [Lhc (light-harvesting complex) a1-Lhca4] belonging to the light-harvesting protein family. Difficulties in their purification have so far prevented the determination of their properties and most of the knowledge about Lhcas has been obtained from the study of the in vitro reconstituted antennas. In the present study we were able to purify the native complexes, showing that Lhca2/3 and Lhca1/4 form two functional heterodimers. Both dimers show red-fluorescence emission with maxima around 730 nm, as in the intact PSI complex. This indicates that the dimers are in their native state and that LHCI-680, which was previously assumed to be part of the PSI antenna, does not represent the native state of the system. The data show that the light-harvesting properties of the two dimers are functionally identical, concerning absorption, long-wavelength emission and fluorescence quantum yield, whereas they differ in their high-light response. Implications of the present study for the understanding of the energy transfer process in PSI are discussed. Finally, the comparison of the properties of the native dimers with those of the reconstituted complexes demonstrates that all of the major properties of the Lhcas are reproduced in the in vitro systems.

[37] Otani T, Yamamoto H, Shikanai T. Stromal loop of lhca6 is responsible for the linker function required for the NDH-PSI supercomplex formation
Plant Cell Physiol, 2017,58:851-861.
DOI:10.1093/pcp/pcx009URLPMID:28184910 [本文引用: 1]
The light-harvesting complex I (LHCI) proteins in Arabidopsis thaliana are encoded by six genes. Major LHCI proteins (Lhca1-Lhca4) harvest light energy and transfer the resulting excitation energy to the PSI core by forming a PSI supercomplex. In contrast, the minor LHCI proteins Lhca5 and Lhca6 contribute to supercomplex formation between the PSI supercomplex and the chloroplast NADH dehydrogenase-like (NDH) complex, although Lhca5 is also solely associated with the PSI supercomplex. Lhca6 was branched from Lhca2 during the evolution of land plants. In this study, we focused on the molecular evolution involved in the transition from a major LHCI, Lhca2, to the linker protein Lhca6. To elucidate the domains of Lhca6 responsible for linker function, we systematically swapped domains between the two LHCI proteins. To overcome problems due to the low stability of chimeric proteins, we employed sensitive methods to evaluate supercomplex formation: we monitored NDH activity by using Chl fluorescence analysis and detected NDH-PSI supercomplex formation by using protein blot analysis in the form of two-dimensional blue-native (BN)/SDS-PAGE. The stromal loop of Lhca6 was shown to be necessary and sufficient for linker function. Chimeric Lhca6, in which the stromal loop was substituted by that of Lhca2, was not functional as a linker and was detected at the position of the PSI supercomplex in the BN-polyacrylamide gel. The stromal loop of Lhca6 is likely to be necessary for the interaction with chloroplast NDH, rather than for the association with the PSI supercomplex.

[38] Chang C S J, Li Y H, Chen L T, Chen W C, Hsieh W P, Shin J, Jane W N, Chou S J, Choi G, Hu J M, Somerville S, Wu S H. LZF1, a HY5-regulated transcriptional factor, functions in Arabidopsis de-etiolation
Plant J, 2008,54:205-219.
DOI:10.1111/j.1365-313X.2008.03401.xURLPMID:18182030 [本文引用: 1]
We surveyed differential gene expression patterns during early photomorphogenesis in both wild-type and mutant Arabidopsis defective in HY5, an influential positive regulator of the responses of gene expression to a light stimulus, to identify light-responsive genes whose expression was HY5 dependent. These gene-expression data identified light-regulated zinc finger protein 1 (LZF1), a gene encoding a previously uncharacterized C2C2-CO B-box transcriptional regulator. HY5 has positive trans-activating activity toward LZF1 and binding affinity to LZF1 promoter in vivo. HY5 is needed but not sufficient for the induction of LZF1 expression. Anthocyanin content is significantly diminished in lzf1 under far red, which is the most efficient light for the induction of LZF1. The expression of PAP1/MYB75 is elevated in plants overexpressing LZF1, which leads to the hyperaccumulation of anthocyanin in transgenic Arabidopsis. The transition from etioplast to chloroplast and the accumulation of chlorophyll were notably compromised in the lzf1 mutant. We provide molecular evidence that LZF1 influences chloroplast biogenesis and function via regulating genes encoding chloroplast proteins. In the absence of HY5, mutation of LZF1 leads to further reduced light sensitivity for light-regulated inhibition of hypocotyl elongation and anthocyanin and chlorophyll accumulation. Our data indicate that LZF1 is a positive regulator functioning in Arabidopsis de-etiolation.

[39] Chang C S J, Maloof J N, Wu S H. COP1-mediated degradation of BBX22/LZF1 optimizes seedling development in Arabidopsis
Plant Physiol, 2011,156:228-239.
DOI:10.1104/pp.111.175042URL [本文引用: 1]
Light regulates multiple aspects of growth and development in plants. Transcriptomic changes govern the expression of signaling molecules with the perception of light. Also, the 26S proteasome regulates the accumulation of positive and negative regulators for optimal growth of Arabidopsis (Arabidopsis thaliana) in the dark, light, or light/dark cycles. BBX22, whose induction is both light regulated and HY5 dependent, is a positive regulator of deetiolation in Arabidopsis. We found that during skotomorphogenesis, the expression of BBX22 needs to be tightly regulated at both transcriptional and posttranslational levels. During photomorphogenesis, the expression of BBX22 transiently accumulates to execute its roles as a positive regulator. BBX22 protein accumulates to a higher level under short-day conditions and functions to inhibit hypocotyl elongation. The proteasome-dependent degradation of BBX22 protein is tightly controlled even in plants overexpressing BBX22. An analysis of BBX22 degradation kinetics shows that the protein has a short half-life under both dark and light conditions. COP1 mediates the degradation of BBX22 in the dark. Although dispensable in the dark, HY5 contributes to the degradation of BBX22 in the light. The constitutive photomorphogenic development of the cop1 mutant is enhanced in cop1BBX22ox plants, which show a short hypocotyl, high anthocyanin accumulation, and expression of light-responsive genes. Exaggerated light responsiveness is also observed in cop1BBX22ox seedlings grown under short-day conditions. Therefore, the proper accumulation of BBX22 is crucial for plants to maintain optimal growth when grown in the dark as well as to respond to seasonal changes in daylength.