黄瓜幼苗下胚轴长度GWAS分析及候选基因挖掘

doi:10.3864/j.issn.0578-1752.2020.01.012

摘要/Abstract

摘要：

【目的】挖掘与黄瓜幼苗下胚轴长度显著相关的SNP位点及候选基因,为揭示下胚轴长度的遗传基础和分子机制提供理论依据,为短下胚轴分子标记辅助选择育种奠定基础。【方法】以95份黄瓜核心种质为试验材料,分别于2016年春季、2017年春季、2017年秋季和2018年春季在中国农业科学院南口试验基地塑料大棚进行种植,在两叶一心期调查黄瓜幼苗的下胚轴长度;利用Structure 2.3.4软件分析群体结构,Haploview 软件分析连锁不平衡的衰减;基于最优模型对下胚轴长度进行全基因组关联分析（GWAS）,依据关联SNP位点的LD区间序列,预测与下胚轴长度相关的重要关联候选基因,并利用荧光定量PCR对预测基因进行表达模式分析。【结果】共检测到8个显著关联的位点（Hl1.1、Hl1.2、Hl2.1、Hl3.1、Hl3.2、Hl4.1、Hl5.1、Hl6.1）,分别位于1、2、3、4、5、6号染色体,其中,Hl2.1、Hl3.1、Hl3.2、Hl5.1、Hl6.1等5个位点被重复检测到两次以上。通过分析关联SNP位点的LD区间序列,获得Csa1G074930、Csa1G475980、Csa2G381650、Csa3G141820、Csa4G051570、Csa3G627150、Csa5G174640、Csa6G362970 8个与黄瓜下胚轴长度有关的候选基因,其中既有光形态建成、泛素化、激素信号通路等调控基因,也有调控网络下游参与细胞生长发育,调节细胞大小,直接调控黄瓜下胚轴长度的基因。多基因在不同黄瓜材料中的有机分布,形成了具有不同下胚轴长度的黄瓜种质。基因表达分析显示Csa1G074930、Csa1G475980、Csa2G381650、Csa4G051570、Csa5G174640在短下胚轴材料中高表达。Csa3G141820、Csa3G627150在长下胚轴材料中高表达。【结论】检测到Hl1.1、Hl1.2、Hl2.1、Hl3.1、Hl3.2、Hl4.1、Hl5.1、Hl6.1等8个与黄瓜下胚轴长度密切关联的SNP位点,挖掘到Csa1G074930、Csa1G475980、Csa2G381650、Csa3G141820、Csa4G051570、Csa3G627150、Csa5G174640、Csa6G362970等8个调控下胚轴长度的候选基因。

关键词: 黄瓜, 下胚轴长度, 全基因组关联分析, 候选基因

Abstract:

【Objective】The aim of this study was to identify SNP loci and candidate genes significantly correlated with cucumber hypocotyl length trait, which could provide a theoretical basis for revealing the genetic basis and molecular mechanism of cucumber hypocotyl length trait, and lay a foundation for marker-assisted selection breeding of cucumber hypocotyl length trait.【Method】The natural population including 95 cucumber germplasm was employed in this study, and seedlings were grown in the plastic house in Nankou Experimental Field of Chinese Academy of Agricultural Sciences in spring 2016, spring 2017, autumn 2017 and spring 2018, respectively. The hypocotyl length was measured at the two true leaves stage. Structure 2.3.4 software was used to analyze the population structure, and Haploview software was used to analyze the attenuation of linkage imbalance. Then, the whole genome association analysis of hypocotyl length was carried out based on the optimal model. The important candidate genes related to hypocotyl length were predicted according to the LD interval sequence of the associated SNP loci, and the expression pattern of candidate genes were performed by fluorescence quantitative PCR. 【Result】A total of 8 loci, including Hl1.1, Hl1.2, Hl2.1, Hl3.1, Hl3.2, Hl4.1, Hl5.1 and Hl6.1, were detected on Chr. 1, 2, 3, 4 and 5, respectively. Five of them, Hl2.1, Hl3.1, Hl3.2, Hl5.1 and Hl6.1, were detected repeatedly in two or more different environments. By analyzing the LD interval sequences of the associated SNP loci, eight candidate genes, Csa1G074930, Csa1G475980, Csa2G381650, Csa3G141820, Csa4G051570, Csa3G627150, Csa5G174640 and Csa6G362970, were predicted, which were related to cucumber hypocotyl length. Some of the candidate genes involved in regulating plant photomorphogenesis, ubiquitination, and hormone signaling pathway. And some of them were downstream genes regulating cell growth, development and cell size, thus they directly regulated hypocotyl length. Thus, the varied distribution of above genes in different cucumber materials resulted in the different hypocotyl length cucumber germplasm. The organic distribution of polygenes in different cucumber materials formed cucumber germline with different Hypocotyl length. Gene expression analysis showed that Csa1G074930, Csa1G475980, Csa2G381650, Csa4G051570 and Csa5G174640 were highly expressed in short hypocotyl materials and Csa3G141820 and Csa3G627150 were highly expressed in long hypocotyl materials.【Conclusion】Eight SNP loci linked with hypocotyl length, Hl1.1, Hl1.2, Hl2.1, Hl3.1, Hl3.2, Hl4.1, Hl5.1 and Hl6.1, were detected in this study. Eight candidate genes regulating hypocotyl length were predicted, including Csa1G074930, Csa1G475980, Csa2G381650, Csa3G141820, Csa4G051570, Csa3G627150, Csa5G174640 and Csa6G362970.

Key words: cucumber, hypocotyl length, genome-wide association study, candidate gene

蔡和序,薄凯亮,周琪,苗晗,董邵云,顾兴芳,张圣平. 黄瓜幼苗下胚轴长度GWAS分析及候选基因挖掘[J]. 中国农业科学, 2020, 53(1): 122-132.

HeXu CAI,KaiLiang BO,Qi ZHOU,Han MIAO,ShaoYun DONG,XingFang GU,ShengPing ZHANG. GWAS Analysis of Hypocotyl Length and Candidate Gene Mining in Cucumber Seedlings[J]. Scientia Agricultura Sinica, 2020, 53(1): 122-132.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.01.012

https://www.chinaagrisci.com/CN/Y2020/V53/I1/122

图/表 12

表1

表2

表3

表4

图1

图2

图3

表5

图4

表6

图5

图6

参考文献 30

[1]	LIN Y, SCHIEFELBEIN J . Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis. Development, 2001,128(19):3697-3705.
[2]	金正爱, 司龙亭, 李丹丹, 高兴 . 弱光下黄瓜幼苗叶片下胚轴长的遗传分析. 江苏农业科学, 2009(3):158-161.
	JIN Z A, SI L T, LI D D, GAO X . Genetic analysis of hypocotyl length in cucumber seedling leaves under weak light. Jiangsu agricultural science, 2009(3):158-161.(in Chinese)
[3]	李丹丹, 司龙亭, 罗晓梅, 李涛 . 弱光胁迫下黄瓜苗期下胚轴性状的遗传分析. 西北农林科技大学学报(自然科学版), 2009,37(11):113-119.
	LI D D, SI L T, LUO X M, LI T . Genetic analysis of hypocotyl traits in cucumber seedling under low light stress. Journal of Northwestern Agricultural and Forestry University (Natural Science Edition), 2009,37(11):113-119. (in Chinese)
[4]	张冠英, 司龙亭, 李丹丹 . 弱光胁迫下黄瓜幼苗下胚轴性状QTL分析. 园艺学报, 2011,38(2):295-302.
	ZHANG G Y, SI L T, LI D D . QTL analysis for hypocotyl traits of cucumber seedlings under low light stress. Acta Horticulturae Sinica, 2011,38(2):295-302. (in Chinese)
[5]	邹士成, 李丹丹, 孙博华, 张成凤, 于思琦 . 黄瓜幼苗下胚轴响应弱光胁迫的研究. 安徽农学通报, 2015,21(19):29-30.
	ZOU S C, LI D D, SUN B H, ZHANG C F, YU S Q . Response of hypocotyl of cucumber seedlings to light stress. Anhui Agricultural Science Bulletin, 2015,21(19):29-30. (in Chinese)
[6]	SONG J L, CAO K, HAO Y W, SONG S W, SU W, LIU H C . Hypocotyl elongation is regulated by supplemental blue and red light in cucumber seedling. Gene, 2019,707:117-125.
[7]	肖苏琪, 王冰华, 曲梅, 高丽红 . 冬春季节育苗温室补光光强对黄瓜幼苗质量的影响. 中国蔬菜, 2018(10):40-45.
	XIAO S Q, WANG B H, QU M, GAO L H . Effect of supplementary light intensity on quality of winter-spring cucumber seedling in solar greenhouse. Chinese Vegetables, 2018(10):40-45. (in Chinese)
[8]	张子默, 卢俊成, 齐晓花, 许学文, 陈学好, 徐强 . 高温下黄瓜幼苗下胚轴长度遗传效应的研究. 分子植物育种, 2019,17(4):1326-1332.
	ZHANG Z M, LU J C, QI X H, XU X W, CHEN X H, XU Q . Study on genetic effects of hypocotyl length in cucumber seedlings under high temperature. Molecular Plant Breeding, 2019,17(4):1326-1332. (in Chinese)
[9]	董春娟, 曹宁, 王玲玲, 张焕欣, 王红飞, 台连丽, 尚庆茂 . 黄瓜子叶源生长素对下胚轴不定根发生的调控作用. 园艺学报, 2016,43(10):1929-1940.
	DONG C J, CAO N, WANG L L, ZHANG H X, WANG H F, TAI L L, SHANG Q M . Regulatory roles of cotyledon-generated auxin in adventitious root formation on the hypocotyls of cucumber seedling. Acta Horticulturae Sinica, 2016,43(10):1929-1940. (in Chinese)
[10]	LOPEZ-JUEZ E, KOBAYASHI M, SAKURAI A, KAMIYA Y, KENDRICK R E . Phytochrome, gibberellins, and hypocotyl growth (a study using the cucumber (Cucumis sativus L.) long hypocotyl mutant). Plant Physiology, 1995,107(1):131-140.
[11]	DAN H, IMASEKI H, WASTENEYS G O, KAZAMA H . Ethylene stimulates endoreduplication but inhibits cytokinesis in cucumber hypocotyl epidermis. Plant Physiology, 2003,133(4):1726-1731.
[12]	BO K L, WANG H, PAN Y P, BEHERA T K, PANDEY S, WEN C L, WANG Y H, SIMON P W, LI Y H, CHEN J F, WENG Y Q . SHORT HYPOCOTYL 1 encodes a SMARCA3-like chromatin remodeling factor regulating elongation. Plant Physiology, 2016,172:1273-1292.
[13]	苗晗, 顾兴芳, 张圣平, 张忠华, 黄三文, 王烨, 方智远 . 黄瓜苗期主要农艺性状相关QTL定位分析. 园艺学报, 2012,39(5):879-887.
	MIAO H, GU X F, ZHANG S P, ZHANG Z H, HUANG S W, WANG Y, FANG Z Y . Mapping QTLs for seedling-associated traits in cucumber. Acta Horticulturae Sinica, 2012,39(5):879-887. (in Chinese)
[14]	ATWELL S, HUANG Y S, VILHJALMSSON B J, WILLEMS G, HORTON M, LI Y, MENG D, PLATT A, TARONE A M, HU T T, JIANG R, MULIYATI N W, ZHANG X, AMER M A, BAXTER I, BRACHI B, CHORY J, DEAN C, DEBIEU M, DE MEAUX J, ECKER J R, FAURE N, KNISKERN J M, JONES J D, MICHAEL T, NEMRI A, ROUX F, SALT D E, TANG C, TODESCO M, TRAW M B, WEIGEL D, MARJORAM P, BOREVITZ J O, BERGELSON J, NORDBORG M . Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature, 2010,465(7298):627-631.
[15]	BRACHI B, MORRIS G P, BOREVITZ J O . Genome-wide association studies in plants: The missing heritability is in the field. Genome Biology, 2011,12(10):232.
[16]	WANG M, YAN J B, ZHAO J R, SONG W, ZHANG X B, XIAO Y N, ZHENG Y L . Genome-wide association study (GWAS) of resistance to head smut in maize. Plant Science, 2012,196:125-131.
[17]	HUANG X H, WEI X H, SANG T, ZHAO Q A, FENG Q, ZHAO Y, LI C L, ZHU C R, LU T T, ZHANG Z W, LI M, FAN D L, GUO Y L, WANG A H, WANG L, DENG L W, LI W J, LU Y Q, WENG Q J, LIU K Y, HUANG T, ZHOU T Y, JING Y F, LI W, LIN Z, BUCKLER E S, QIAN Q, ZHANG Q F, LI J Y, HAN B . Genome-wide association studies of 14 agronomic traits in rice landraces. Nature Genetics, 2010,42(11):961-967.
[18]	QI J J, LIU X, SHEN D, MIAO H, XIE B Y, LI X X, ZENG P, WANG S H, SHANG Y, GU X F, DU Y C, LI Y, LIN T, YUAN J H, YANG X Y, CHEN J F, CHEN H M, XIONG X Y, HUANG K, FEI Z J, MAO L Y, TIAN L, STADLER T, RENNER S S, KAMOUN S, LUCAS W J, ZHANG Z H, HUANG S W . A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nature Genetics, 2013,45(12):1510-1515.
[19]	YANG J A, LEE S H, GODDARD M E, VISSCHER P M . A tool for genome-wide complex trait analysis. The American Journal of Human Genetics, 2011,88(1):76-82.
[20]	BARRETT J C, FRY B, MALLER J, DALY M J . Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics, 2005,21(2):263-265.
[21]	ZHANG Z W, ERSOZ E, LAI C Q, TODHUNTER R J, TIWARI H K, GORE M A, BRADBURY P J, YU J M, ARNETT D K, ORDOVAS J M, BUCKLER E S . Mixed linear model approach adapted for genome-wide association studies. Nature Genetics, 2010,42(4):355-360.
[22]	BRADBURY P J, ZHANG Z W, KROON D E, CASSTEVENS T M, RAMDOSS Y, BUCKLER E S . TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics, 2007,23(19):2633-2635.
[23]	ZHANG Z W, ERSOZ E, LAI C Q, TODHUNTER R J, TIWARI H K, GORE M A, BRADBURY P J, YU J M, ARNETT D K, ORDOVAS J M, BUCKLER E S . Mixed linear model approach adapted for genome-wide association studies. Nature Genetics, 2010,42(4):355-360.
[24]	DAN H, IMASEKI H, WASTENEYS G O, KAZAMA H . Ethylene stimulates endoreduplication but inhibits cytokinesis in cucumber hypocotyl epidermis1. Plant Physiology, 2003,133(4):1726-1731.
[25]	ZHAO Q X, YUAN S, WANG X, ZHANG Y L, ZHU H, LU C M . Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and egta in vitro. Plant Physiology, 2008,147(4):1874-1885.
[26]	KOEDA S, SATO K, SAITO H, NAGANO A J, YASUGI M, KUDOH H, TANAKA Y . Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum. Theoretical and Applied Genetics, 2019,132(1):65-80.
[27]	ZHAO L M, PENG T, CHEN C Y, JI R J, GU D C, LI T T, ZHANG D D, TU Y S, WU K Q, LIU X C . HY5 interacts with the histone deacetylase HDA15 to repress hypocotyl cell elongation in photomorphogenesis. Plant Physiology, 2019,180(3):1450-1466.
[28]	CHENGE-ESPINOSA M, CORDOBA E, ROMERO-GUIDO C, TOLEDO-ORTIZ G, LEON P . Shedding light on the methylerythritol phosphate (MEP)-pathway: Long hypocotyl 5 (HY5)/phytochrome- interacting factors (PIFs) transcription factors modulating key limiting steps. Plant Journal, 2018,96(4):828-841.
[29]	RAIJMAKERS R, NOORDMAN Y E, VAN VENROOIJ W J, PRUIJN G J M . Protein-protein interactions of hCsl4p with other human exosome subunits. Journal of Molecular Biology, 2002,315(4):809-818.
[30]	BREMBU T, WINGE P, SEEM M, BONES A M . NAPP and PIRP encode subunits of a putative wave regulatory protein complex involved in plant cell morphogenesis. The Plant Cell, 2004,16(9):2335-2349.

反应组分 Reaction Component	浓度 Concentration	体积 Volume (µL)
SybrGreen qPCR Master Mix	2×	10
引物F	10 µmol∙L^-1	0.4
引物R	10 µmol∙L^-1	0.4
ddH₂0		7.2
Template (cDNA)		2

热循环仪器 Thermal Cycler	时间和温度 Times and temperatures
	初始步骤 Initial Steps	45个循环 Each of 45 cycles
	初始步骤 Initial Steps	解旋 Melt	退火 Anneal	延伸 Extend
LightCycler480 Software Setup (罗氏 Roche)	95℃ 3 min	95℃ 15 s	57℃ 15 s	72℃ 30 s

环境 Environment	均值±标准差 Mean±SD	最小值 Minimum	50%分位数 50% quantile	最大值 Max	变异系数 CV (%)
16S	6.02±1.95	1.75	5.84	11.25	32.32
17S	6.19±2.06	0.60	6.16	11.66	33.27
17A	8.53±2.24	3.28	9.02	13.18	26.28
18S	5.65±1.70	1.30	5.59	10.36	30.17

	16S	17S	17A	18S
16S	1
17S	0.660**	1
17A	0.636**	0.746**	1
18S	0.565**	0.680**	0.607**	1

位点 Locus	基因名称 Gene name	同源基因名称 Homologous gene name	功能注释 Functional annotation
Hl1.1	Csa1G074930	SMG7L	端粒酶结合蛋白EST1A,可能在细胞增长和发展中发挥作用 Telomerase binding protein EST1A, may play a role in cell growth and development
Hl1.2	Csa1G475980	SCAR 3	参与肌动蛋白和微管组织的调节,参与植物细胞形态形成 Participate in the regulation of actin and microtubule tissue, and participate in the morphological formation of plant cells
Hl2.1	Csa2G381650	HY5	转录因子,促进光形态发生 Transcription factors to promote photomorphogenesis
Hl3.1	Csa3G141820	RAD5B	调控光形态发生中的低剂量UVB响应影响下胚轴伸长 Effect of low dose UVB response on Hypocotyl extension during Photomorphogenesis
Hl3.2	Csa3G627150	CSL4	参与细胞RNA的加工和降解 Participate in the processing and degradation of cell RNA
Hl4.1	Csa4G051570	ATL21A	参与蛋白质泛素化 Participate in protein ubiquitin
Hl5.1	Csa5G174640	MED36A	参与调控几乎所有RNA聚合酶II依赖基因转录的辅激活因子 Coactivating factors involved in the regulation of almost all RNA polymerase II dependent gene transcription
Hl6.1	Csa6G362970	BIG GRAIN 1-like A	参与生长素转运。调节生长素信号通路 Participate in auxin transport. Regulation of auxin signaling pathway