GWAS Analysis of Hypocotyl Length and Candidate Gene Mining in Cucumber Seedlings
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| HeXu CAI,KaiLiang BO,Qi ZHOU,Han MIAO,ShaoYun DONG,XingFang GU,ShengPing ZHANG |
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Scientia Agricultura Sinica
2020,53(1
):122
-132. DOI:10.3864/j.issn.0578-1752.2020.01.012
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【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.
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Genome-Wide Identification of Cucumber ERF Gene Family and Expression Analysis in Female Bud Differentiation
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| Jian PAN,HaiFan WEN,HuanLe HE,HongLi LIAN,Gang WANG,JunSong PAN,Run CAI |
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Scientia Agricultura Sinica
2020,53(1
):133
-147. DOI:10.3864/j.issn.0578-1752.2020.01.013
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【Objective】The objectives of this research were to identify the Ethylene Response Factor (ERF) family genes from cucumber (Cucumis sativus L.) genome and to know the profile of ERF family such as gene number, gene structure and expression characters in cucumber, so as to provide a theoretical basis for exploring what roles the ERF transcription factors played in female flowers development. 【Method】 The ERF genes in cucumber genome were identified by BLAST software in the 9930_V2 genome database based on Arabidopsis ERF genes. EMBOSS, MEME, TBtools, ExPASy and MEGA 7.0 software were used for carrying out various bioinformatics analysis of ERF. The qRT-PCR was used to detect the expression of cucumber ERF gene family in female buds’ differentiation. 【Result】 Total of 138 ERF genes were identified from cucumber genome, which could be divided into 10 classes. These 138 ERF genes were named from CsERF1 to CsERF138, and the number of amino acids of these ERF genes was between 126 and 745. Multiple sequence alignments and Motif analysis showed that the CsERF gene family had two conserved domains, namely AP2/ERF domain and B3 domain. There were 19 ERF genes showed different expression between FFMMAA and ffMMAA genotype, 9 of them up-regulated in FFMMAA genotype and the other 10 up-regulated in ffMMAA genotype. The expression trend analysis showed 31 ERF genes up-regulated and 30 genes down-regulated in the initial-phase of female buds’ differentiation. Furthermore, it was demonstrated that CsERF9 and CsERF31 could bind the GCC-box. 【Conclusion】 138 ERF gene family members were identified from the cucumber 9930_V2 genome. These entire ERF gene shared AP2/ERF domain. Some of the ERF family members were related to sex determination and female flower development and could bind GCC-box to regulate downstream genes expression.
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Cloning and Functional Analysis of CsRPL1/2 in Cucumber
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| WeiYuan SONG,Yu HOU,JianYu ZHAO,XiaoFeng LIU,XiaoLan ZHANG |
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Scientia Agricultura Sinica
2020,53(1
):148
-159. DOI:10.3864/j.issn.0578-1752.2020.01.014
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【Objective】AtRPL is an important gene regulating Arabidopsis fruit development and mediating replum formation. Using homologous cloning, the RPL homolog in cucumber was identified, the biological function of CsRPL by expression analysis and ectopic transformation in Arabidopsis was explored.【Method】We cloned the CsRPL gene by performing a BLAST search of AtRPL in cucumber genome. Then we performed amino acid sequence alignment of CsRPL and RPL homologs from other species by MEGA5.2. CsRPL expression pattern in cucumber was detected by real-time quantitative PCR (qRT-PCR) and in situ hybridization. Expression and phenotypic analysis of transgenic Arabidopsis upon ectopic expression of CsRPL were performed as well.【Result】Two RPL genes, named CsRPL1 and CsRPL2, were identified in cucumber with the conserved BELL domain and Homeodomain, and two EAR-Motifs. CsRPL1 was expressed in all organs of cucumber, with the highest expression level in male flowers at anthesis. During early stages of fruit development, the CsRPL1 expression decreased gradually. The expression level of CsRPL2 was significantly lower than that of CsRPL1. In situ hybridization revealed that signals of CsRPL1/2 were detected in the placenta of fruits and the central zone (CZ) of the shoot apical meristem (SAM). Ectopic overexpression of CsRPL1/2 into Arabidopsis resulted in shorter siliques, reduced pollen fertility, and inhibited seed development.【Conclusion】CsRPL1/2 were involved in the development of reproductive organs and might have functional redundancy in cucumber. CsRPL1 might play the primary role under normal growth condition. The function of CsRPL1/2 was not fully conserved as compared to that of AtRPL.
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Inheritance and QTL Mapping for Parthenocarpy in Cucumber
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| ZhiHong NIU,XiaoFei SONG,XiaoLi LI,XiaoYu GUO,ShuQiang HE,LuanJingZhi HE,ZhiHong FENG,ChengZhen SUN,LiYing YAN |
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Scientia Agricultura Sinica
2020,53(1
):160
-171. DOI:10.3864/j.issn.0578-1752.2020.01.015
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【Objective】 Parthenocarpy is an important trait affecting both yield and quality in the protected cucumber production. Genetic analysis of parthenocarpy and its QTL mapping are of great significance for improving breeding efficiency in cucumber.【Method】 Based on three-year phenotypic data, the genetic linkage map of cucumber was constructed by using SSR molecular markers obtained from cucumber genome sequencing, and the QTL-Seq analysis was used to map the parthenocarpy of cucumber, using recombinant inbred line F2:8 constructed from strong parthenocarpic inbred line ‘6457’ and weak parthenocarpic inbred line ‘6426’.【Result】 The inheritance of parthenocarpy in cucumber accorded with quantitative inheritance. A genetic map containing 11 linkage groups was constructed using SSR markers, covering 555.0 cM of the genome with an average distance of 6.8 cM. In the spring of 2016, 2017 and 2018, a single QTL locus related to parthenocarpy was commonly detected between SSR19430 and SSR15419 markers (3.33-5.57 Mb) on chromosome 3, and the genetic distance was 6.6 cM, with 11%, 12.5% and 6.3% contribution rate, respectively. By QTL-Seq analysis, four QTLs related to parthenocarpy were identified on chromosomes 1 (4.38-11.00 Mb), 3 (2.24-10.66 Mb) and 6 (15.67-17.93 Mb; 26.33-27.49 Mb), the QTL on chromosome 3 overlapped with that detected by Map QTL approach. Csa3G047740, Csa3G073810, Csa3G043910 and Csa6G362930 were proposed to be candidate genes associated with parthenocarpic trait in cucumber. 【Conclusion】Four QTLs were identified on chromosome 1, 3 and 6, including one QTL on chromosome 3 that was consistently detected over years with relatively high contribution. The results could facilitate marker-assisted selection and understanding the underlying mechanism of parthenocarpy in cucumber.
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Screening and Identification of Cucumber Mutant Resistant to Powdery Mildew
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| Fei QI,Shu LIN,MengFei SONG,MengRu ZHANG,ShuYan CHEN,NaiXin ZHANG,JinFeng CHEN,QunFeng LOU |
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Scientia Agricultura Sinica
2020,53(1
):172
-182. DOI:10.3864/j.issn.0578-1752.2020.01.016
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【Objective】Powdery mildew is one of the most serious diseases that effects cucumber yield and quality. The discovery and research of materials resistant to powdery mildew can fundamentally solve the disease problem. To screen out materials of cucumber resistant to powdery mildew and enrich the breeding population, the Changchunmici mutants were investigated and analyzed in the study.【Method】Totally, 400 Changchunmici mutant lines were inoculated with powdery mildew at seedling stage, and resistant materials were screened preliminarily based on leaf lesion observation combined with disease index analysis. The selected resistant materials were further observed in the natural environment in the field environment. Physiological indexes of resistant materials in the seedling stages were analyzed, including the activities of superoxide dismutase, peroxidase and catalase activity, and physiological indicators such as the contents of chlorophyll a, chlorophyll b, carotenoids and soluble protein. The resistant materials were screened by natural pathogenesis in the field and were further analyzed for the photosynthetic indexes such as transpiration rate, intercellular carbon dioxide concentration, stomatal conductance and net photosynthetic rate. And the contents of ethylene, jasmonic acid and salicylate were determined, and expression of related genes in defense signaling pathways such as ethylene, jasmonic acid, salicylic acid, lignin, and disease-related proteins in leaves were analyzed by real-time PCR. 【Result】Compared with susceptible materials, the resistant materials had less plaque. The net photosynthetic rate and stomatal conductance of resistant materials were higher than those of wild-type Changchunmici, and the intercellular CO2 concentration of them was lower than that of Changchunmici. In terms of defensive hormones, the contents of ethylene, jasmonic acid and salicylate of resistant materials were higher than those in control. The expression of defense signal related genes in mature leaves of resistant materials was higher compared with susceptible materials. Two powdery mildew resistance materials, Mu-86-2 and Mu-58-9 were screened by inoculation of powdery mildew in seedling stage and natural disease. 【Conclusion】The new materials resistant to powdery mildew could be obtained by screening mutant libraries. The acquisition of these materials had the important value for the genetic research and new varieties breeding of cucumber resistance to powdery mildew.
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