Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 122-132.doi: 10.3864/j.issn.0578-1752.2020.01.012

• SPECIAL FOCUS: MOLECULAR BIOLOGY OF CUCUMBER • Previous Articles     Next Articles

GWAS Analysis of Hypocotyl Length and Candidate Gene Mining in Cucumber Seedlings

HeXu CAI,KaiLiang BO,Qi ZHOU,Han MIAO,ShaoYun DONG,XingFang GU(),ShengPing ZHANG()   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-07-24 Accepted:2019-09-16 Online:2020-01-01 Published:2020-01-19
  • Contact: XingFang GU,ShengPing ZHANG E-mail:guxingfang@caas.cn;zhangshengping@caas.cn

RichHTML

27

PDF

300

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

Table 1

Fluorescence quantitative reaction system"

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

Table 2

PCR reaction procedure"

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
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

Table 3

Statistical analysis of hypocotyl length in cucumber"

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

Table 4

Correlation analysis of hypocotyl length in cucumber under four batches"

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

Fig. 1

Frequency distribution of hypocotyl length in cucumber"

Fig. 2

The genome-wide LD decay in cucumber"

Fig. 3

Manhattan plots of genome-wide association study for seedling hypocotyl length in cucumber"

Table 5

Candidate genes and functional annotation"

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

Fig. 4

Electrophoresis analysis of total RNA"

Table 6

The hypocotyl length of qPCR materials at seedling stage"

品种 Variety 时期1 Stage 1 时期2 Stage 2 时期3 Stage 3 时期4 Stage 4 时期5 Stage 5
CG9 5.23±0.25b 7.03±0.21b 9.77±0.25b 11.07±0.25b 12.27±0.21a
CG11 4.4±0.22c 6.27±0.21c 8.07±0.17c 9.2±0.16c 9.97±0.29b
CG19 3.17±0.21d 3.1±0.08d 3.67±0.12f 4.97±0.21f 6.6±0.08d
CG44 2.8±0.08e 3.07±0.25d 4.3±0.16e 6±0.24e 6.57±0.17d
CG54 2.1±0.08f 2.23±0.12e 3.27±0.25f 3.47±0.21g 4.17±0.12e
CG98 1.63±0.12g 3.27±0.17d 3.5±0.16f 4.1±0.29g 4.67±0.12e
CG106 5.43±0.12b 6.03±0.12c 6.87±0.12d 8.2±0.16d 8.97±0.21c
CG112 6.23±0.12a 8.13±0.12a 11.77±0.21a 12.53±0.12a 12.67±0.29a

Fig. 5

Comparison of long hypocotyl materials with short hypocotyl materials CG54 (left) and CG112 (right) in two leaf and one bud period"

Fig. 6

Analysis of relative expression levels of candidate genes CG9, CG11, CG106 and CG112 are long hypocotyl materials. CG19, CG44, CG54 and CG98, are all short hypocotyl materials. The material with the lowest expression level was selected from each candidate gene, and its relative expression level was set as 1"

[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.
[1] HU Sheng,LI YangYang,TANG ZhangLin,LI JiaNa,QU CunMin,LIU LieZhao. Genome-Wide Association Analysis of the Changes in Oil Content and Protein Content Under Drought Stress in Brassica napus L. [J]. Scientia Agricultura Sinica, 2023, 56(1): 17-30.
[2] LI QingLin,ZHANG WenTao,XU Hui,SUN JingJing. Metabolites Changes of Cucumber Xylem and Phloem Sap Under Low Phosphorus Stress [J]. Scientia Agricultura Sinica, 2022, 55(8): 1617-1629.
[3] ZHI Lei,ZHE Li,SUN NanNan,YANG Yang,Dauren Serikbay,JIA HanZhong,HU YinGang,CHEN Liang. Genome-Wide Association Analysis of Lead Tolerance in Wheat at Seedling Stage [J]. Scientia Agricultura Sinica, 2022, 55(6): 1064-1081.
[4] LI Heng,ZI XiangDong,WANG Hui,XIONG Yan,LÜ MingJie,LIU Yu,JIANG XuDong. Screening of Key Regulatory Genes for Litter Size Trait Based on Whole Genome Re-Sequencing in Goats (Capra hircus) [J]. Scientia Agricultura Sinica, 2022, 55(23): 4753-4768.
[5] XIE XiaoYu, WANG KaiHong, QIN XiaoXiao, WANG CaiXiang, SHI ChunHui, NING XinZhu, YANG YongLin, QIN JiangHong, LI ChaoZhou, MA Qi, SU JunJi. Restricted Two-Stage Multi-Locus Genome-Wide Association Analysis and Candidate Gene Prediction of Boll Opening Rate in Upland Cotton [J]. Scientia Agricultura Sinica, 2022, 55(2): 248-264.
[6] KANG Chen,ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing. Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus [J]. Scientia Agricultura Sinica, 2022, 55(19): 3751-3766.
[7] BaoHua CHU,FuGuo CAO,NingNing BIAN,Qian QIAN,ZhongXing LI,XueWei LI,ZeYuan LIU,FengWang MA,QingMei GUAN. Resistant Evaluation of 84 Apple Cultivars to Alternaria alternata f. sp. mali and Genome-Wide Association Analysis [J]. Scientia Agricultura Sinica, 2022, 55(18): 3613-3628.
[8] CHANG LiGuo,HE KunHui,LIU JianChao. Mining of Genetic Locus of Maize Stay-Green Related Traits Under Multi-Environments [J]. Scientia Agricultura Sinica, 2022, 55(16): 3071-3081.
[9] LI Ting,DONG Yuan,ZHANG Jun,FENG ZhiQian,WANG YaPeng,HAO YinChuan,ZHANG XingHua,XUE JiQuan,XU ShuTu. Genome-Wide Association Study of Ear Related Traits in Maize Hybrids [J]. Scientia Agricultura Sinica, 2022, 55(13): 2485-2499.
[10] WANG Juan, MA XiaoMei, ZHOU XiaoFeng, WANG Xin, TIAN Qin, LI ChengQi, DONG ChengGuang. Genome-Wide Association Study of Yield Component Traits in Upland Cotton (Gossypium hirsutum L.) [J]. Scientia Agricultura Sinica, 2022, 55(12): 2265-2277.
[11] CUI ChengQi, LIU YanYang, JIANG XiaoLin, SUN ZhiYu, DU ZhenWei, WU Ke, MEI HongXian, ZHENG YongZhan. Multi-Locus Genome-Wide Association Analysis of Yield-Related Traits and Candidate Gene Prediction in Sesame (Sesamum indicum L.) [J]. Scientia Agricultura Sinica, 2022, 55(1): 219-232.
[12] CHEN Xi,LIU YingJie,DONG YongHao,LIU JinYan,LI Wei,XU PengJun,ZANG Yun,REN GuangWei. Effects of CMV-Infected Tobacco on the Performance, Feeding and Host Selection Behavior of Myzus persicae [J]. Scientia Agricultura Sinica, 2021, 54(8): 1673-1683.
[13] ZHANG PengFei,SHI LiangYu,LIU JiaXin,LI Yang,WU ChengBin,WANG LiXian,ZHAO FuPing. Advance in Genome-Wide Scan of Runs of Homozygosity in Domestic Animals [J]. Scientia Agricultura Sinica, 2021, 54(24): 5316-5326.
[14] YAN YongLiang,SHI XiaoLei,ZHANG JinBo,GENG HongWei,XIAO Jing,LU ZiFeng,NI ZhongFu,CONG Hua. Genome-Wide Association Study of Grain Quality Related Characteristics of Spring Wheat [J]. Scientia Agricultura Sinica, 2021, 54(19): 4033-4047.
[15] SONG ChunHui,CHEN XiaoFei,WANG MeiGe,ZHENG XianBo,SONG ShangWei,JIAO Jian,WANG MiaoMiao,MA FengWang,BAI TuanHui. Identification of Candidate Genes for Waterlogging Tolerance in Apple Rootstock by Using SLAF-seq Technique [J]. Scientia Agricultura Sinica, 2021, 54(18): 3932-3944.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd