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

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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"

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