Source: AGRICULTURAL RESEARCH SERVICE submitted to NRP
GENETIC AND MOLECULAR MECHANISMS OF UVB-DEPENDENT HYPOCOTYL ELONGATION IN CUCUMBER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1011920
Grant No.
2017-67013-26195
Cumulative Award Amt.
$410,000.00
Proposal No.
2016-10467
Multistate No.
(N/A)
Project Start Date
Feb 15, 2017
Project End Date
Feb 14, 2022
Grant Year
2017
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
1815 N University
Peoria,IL 61604
Performing Department
USDA-ARS
Non Technical Summary
In cucumber, hypocotyl elongation is inhibited by low-dosage UVB (LDUVB). The hypocotyl tends to show excessive growth in greenhouse environments due to UV exclusion or high temperature. Proper control of hypocotyl elongation is important in large-scale greenhouse production settings where strong seedlings are required for transplantation. In this project, we aim to understand the genetic control of hypocotyl elongation in cucumber by working with two recessive mutants, short hypocotyl1 (sh1) and long hypocotyl1 (lh1) that modify hypocotyl elongation in a LDUVB-independent manner. We will investigate the genetic and molecular mechanisms by which Sh1- and Lh1-regulate hypocotyl elongation and explore the use of the sh1 allele in cucumber production. In particular, we plan to clone the gene Lh1, and investigate the structure and function of the Sh1 and Lh1 candidate genes. We are also interested in understanding the regulatory network that Sh1-/Lh1-mediated in LDUVB-dependent hypocotyl elongation. Finally, we want to explore the potential use of the sh1 gene in cucumber production by development and evaluation of germplasm carrying the sh1 allele for use in cucumber breeding. Our long term goals is to use untapped genetic diversity to improve cucumber productivity in various environments, thus contributing to a more sustainable and profitable cucumber industry.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20114211080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1421 - Cucumber;

Field Of Science
1080 - Genetics;
Goals / Objectives
In cucumber, hypocotyl elongation is inhibited by low-dosage UVB (LDUVB). The hypocotyl tends to show excessive growth in greenhouse environments due to UV exclusion or high temperature. Proper control of hypocotyl elongation is important in large-scale greenhouse production settings where strong seedlings are required for transplantation. Little is known about the genetic control of hypocotyl elongation in cucumber. We identified two recessive mutants, short hypocotyl1 (sh1) and long hypocotyl1 (lh1) that modify hypocotyl elongation in a LDUVB-independent manner. In this project, we will investigate the genetic and molecular mechanisms by which Sh1- and Lh1-regulate hypocotyl elongation and explore the use of the sh1 allele in cucumber production. We hypothesize that: 1) Sh1 regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway via its interaction with CsHY5, a homolog of the Arabidopsis transcription factor HY5. 2) The two genes Lh1 and Sh1 share a common signaling pathway in regulating hypocotyl elongation. 3) A better understanding of the genetic basis of hypocotyl elongation will contribute to the development of new strategies for increasing cucumber productivity. Thus, we have four specific objectives in this project: 1) Positional cloning of the Lh1 gene; 2) Functional characterization of Sh1 and Lh1 candidate genes; 3) Characterization of the regulatory network of Sh1-/Lh1-mediated, LDUVB-dependent hypocotyl elongation; and 4) Development and evaluation of germplasm carrying the sh1 allele for use in cucumber breeding. This project serves our long-term goal of using untapped genetic diversity to improve cucumber productivity in various environments, thus contributing to a more sustainable and profitable cucumber industry.
Project Methods
Objective 1. Positional cloning of Lh1Three segregating populations will be used for cloning of Lh1 including AM149L (lh1lh1) × Gy14 (Lh1Lh1) F2, AM149S F2, and AM149S × AM149L BC1. We will use the AM149S × Gy14 F2 for genetic mapping and cloning of Lh1. The AM149S-derived NIL F2 will be used for BSA RNA-Seq. For mapping of Lh1, BSA will be used to locate Lh1 on a chromosomal region with a set of 240 highly polymorphic SSR markers. We will re-sequence the AM149 genome (~15x coverage) to identify polymorphisms. Fine mapping will be conducted with ~2,000 F2 plants, which should be sufficient to narrow down the target region to <30 kb with 1-3 candidate genes in cucumber.Objective 2. Validation and functional characterization of Sh1 and Lh1 candidate genesOnce the Lh1 candidate gene is identified, genomic DNA and cDNA sequences including the promoter region will be cloned from AM149L and AM149S NILs and aligned with Gy14 reference genome to reveal causal polymorphisms and the candidate gene structure. We will examine allelic diversity at the Lh1 locus among natural cucumber populations with more than 550 lines. The association of hypocotyl length under LDUVB and UVB-free light conditions and nucleotide sequence polymorphisms in the target region will help validate candidate gene functions, and reveal the origin and evolution of the mutant allele and its selection history during cucumber crop evolution. We will investigate the phylogenetic relationships of the cucumber Lh1 candidate gene with its homologs in other species. The neighbor-joining tree will be constructed. We will study the spatial and temporal expression dynamics of Sh1 and Lh1 candidate genes with qPCR using NILs of both Sh1 and Lh1, and sh1lh1 doubt mutant. Samples will be collected from multiple developmental stages and organs of each treatment for qPCR. The spatial expression patterns of Lh1 and Sh1 will also be examined with RNA insitu hybridization as well as β-Glucuronidase (GUS) assay. For GUS assays, transgenic Arabidopsis and cucumber plants expressing GUS driven by the Sh1 and Lh1 promoters will be generated. We will perform ectopic expression of Sh1 and Lh1 candidate genes in Arabidopsis. For the Lh1 transformation experiment, if the Lh1 has multiple copies of homologs in Arabidopsis, Arabidopsis plants carrying multi-mutant loci will also be generated for complementation tests with the Lh1 candidate gene. We will also develop transgenic cucumber plants for Sh1 and Lh1 candidate genes with both overexpression and RNAi constructs.Objective 3: Understanding the Lh1- and Sh1-mediated gene network(s) in regulating LDUVB-dependent hypocotyl elongationWe plan to explore the Sh1- and Lh1-mediated hypocotyl elongation regulatory network(s) at both the mRNA and protein levels. First, we will conduct transcriptome analysis to identify major pathways involved in hypocotyl elongation regulated by the Sh1 and Lh1 using the BSA RNA-Seq (BSR-Seq) strategy. We will examine expression patterns of selected genes in light signaling pathways to reveal their roles in Sh1 and Lh1-mediated hypocotyl elongation including genes that are known to be important players such as CsHY5, CsCOP1, CsUVR8, PIFs, SPAs, or genes regulated by them. The materials used will include NILs for Sh1 and Lh1, as well as sh1lh1 double mutant grown under both UVB-free and LDUVB conditions. We will use yeast two-hybrid (Y2H) and BiFC assays to investigate protein-protein interactions in the Sh1- and Lh1-regulated network(s). The cDNA sequences of Sh1 and Lh1 candidate genes will be subcloned into the PGEM-T Easy vector. Insert DNA will then be digested with Xba I and Kpn I, and inserted into the pUC-GFP vector, which will be verified by restriction and sequencing analysis. Transient expression of the fusion proteins in onion epidermal cells. The fluorescence of GFP will be visualized under a confocal laser scanning microscope. Vectors expressing RFP targeted to the nucleus, cytoplasm, and plasma membrane will also be used as co-transformation controls in order to provide reference points for the various cellular compartments. Additional constructs with RFP targeted to other cellular compartments will also be used if the pattern of GFP localization does not match these common compartments.Objective 4: Germplasm developmentWe will introduce sh1 allele into a gynoecious, parthenocarpic line WI7204 using marker-assisted backcrossing scheme. The resulting WI7204SH will be an isogenic line of WI7204 but carries the homozygous sh1 allele. At each BC generation, we will select heterozygous (Sh1sh1) plants for backcrossing. The performance of WI7204SH and WI7204 will be grown in greenhouse and field conditions in replicated trials to evaluate their potential in cucumber breeding. Yield and fruit quality data will be collected including flowering time, fruit length and diameter, fruit weight, fruit number, internal fruit quality (cavity size, placental hollow) and sex expression.

Progress 02/15/21 to 02/14/22

Outputs
Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Through marker-assisted backcrossing, the sh1 allele has been introgressed into a bait alpha type greenhouse cucumber (WI7204) and pickling type cucumber Gy14. In 2021, the two pairs of near isogenic lines were grown in both growth chambers and field trials to evaluate possible effects of the introgression of sh1 on horticulture performance.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Rutter W, Hajihassani A, Wang YH, Wang Y (2022) Phenotypic characterization and molecular mapping of recessive resistance to Meloidogyne javanica in cucumber, Cucumis sativus. Theor Appl Genet (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Zhang HJ, Wang YH, Tan JY, Weng Y (2022) Functional copy number variation of CsSHNINE1 is associated with fruit skin netting intensity in cucumber. Theor Appl Genet (submitted)
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Zhao JY, Bo KL, Pan YP, Li YH, Yu DL, Chang J, Yu DL, Li C, Chang J, Wu S, Wang ZY, Zhao XL, G XF, Weng Y (2022) CsPhyB-CsPIF3-CsPARF18 module regulates gibberellin- and auxin-dependent hypocotyl elongation in cucumber (in preparation)
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Weng Y, Zhao JY, Bo KL, Seufzer A (2022) WI7694sh1 cucumber inbred line with environmental insensitive hypocotyl elongation (in preparation).


Progress 02/15/17 to 02/11/22

Outputs
Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Positional cloning of Lh1 and lh2 genes Hypocotyl elongation in near isogenic lines (NILs) of lh1, lh2 and lh1lh2 long-hypocotyl mutants were examined under different light conditions including dark, natural sunlight, LDUVB, UVB-free, and monochromic blue, red, and far-red LED lights. The data indicated that lh1 is involved in red light- and LDUVB-dependent hypocotyl elongation. Lh1 is epistatic to lh2 in controlling hypocotyl elongation. Microscopic observations suggested that the long hypocotyl in lh1 and lh1lh2 is due to increased cell elongation. Genetic analysis indicated that the extremely long hypocotyl in the mutant line WI7284 is controlled by the recessive locus lh1. Bulked segregant analysis (BSA) with molecular marlers placed the lh1 gene onto cucumber chromosome 3. Initial linkage analysis in 214 recessive homozygous F2 plants delimited lh1 into a 795 kb region. Fine mapping with 3200 F2 plants narrowed down the lh1 gene into a 36kb region. Multiple lines of evidence suggested that the cucumber phytochrome B (CsPhyB) gene is the candidate gene for lh1. The mutant allele carries a 7-bp deletion in the first exon of lh1 resulting in premature termination of the protein. The cucumber inbred line AM149L is a long hypocotyl mutant. Inheritance studies revealed AM149L as a double mutant carrying both lh1 and lh2. Map-based cloning identified CsPhyB and CsGA2oox-2 as the candidate genes the lh1 and lh2, respectively. Lh1 in AM149L has a 4-bp deletion in the second exon of CsPhyB. There is a 1-bp deletion in CsGA20ox-2 of AM149L as compared with the wild type, which would produce a premature protein with only 7 amino acid residues in CsGA20ox-2 that is a critical enzyme for GA biosynthesis. Objective 2. Functional characterization of Lh1, Lh2 candidate genes The spatiotemporal expression of CsGA20ox-2 in the hypocotyls of Gy14, lh1, and lh1lh2 NILs was examined with qPCR, and RNA in situ hybridization. CsGA20ox-2 had much higher expression in the hypocotyl of lh1 and lh1lh2 mutants than in Gy14 with peak expression at 3d after germination in the vascular tissues of the hypocotyl. Subcellular localization analysis indicated that CsGA20ox-2 was located in both the nucleus and the cyto-membrane. There are five GA20ox members in cucumber. CsGA20ox-2 was the only one with higher expression in the hypocotyls of lh1 and lh1lh2 mutants than in Gy14 and lh2 NIL supporting its important role in hypocotyl growth. The GA20ox-2 gene plays an important role in GA biosynthesis. Hypocotyl growth in four NILs grown in MS media supplied with varying concentrations of bioactive GA3 or PAC (GA biosynthesis inhibitor). Under both LDUVB and UVB-free lights, hypocotyl elongation in Gy14, lh2, and lh1lh2 NILs was positively correlated with GA concentrations. Transgenic cucumber plants overexpressing (OX) the WT allele of CsGA20ox-2 were developed. All OX plants had significantly longer hypocotyl than the control under both LD-UVB and UVB-free white lights. The longer hypocotyl in OX lines was positively correlated with the expression level of CsGA20ox-2 supporting the identity between GA20ox-2 and lh2. Objective 3. Understanding the regulatory network of Lh1-, Lh2- and Sh1-mediated hypocotyl elongation with transcriptome and protein-protein interaction analyses To identify CsPhyB- and GA-regulated gene network for hypocotyl elongation, transcriptome profiling with RNA-Seq was performed on hypocotyls of 7d-old seedlings of WT, lh1 and lh1lh2 NILs under LDUVB. In total, 2,651 differentially expressed genes (DEGs) were identified from three comparisons. Detailed analysis of RNA-Seq data supported the critical roles of CsPIF3 in connecting GA and auxin signaling/biosynthesis pathways for hypocotyl elongation. Among the DEGs, CsPIF3 was up-regulated in both lh1 and lh1lh2 mutants. There are five Phy and five PIF homologs in the cucumber genome. Protein-protein-interactions (PPIs) of CsPhyB with all five CsPIFs were investigated with Y2H and BiFC assays. Positive interactions of CsPhyB with CsPIF1a, CsPIF1b, CsPIF3 and CsPIF4 were observed. Among the DEGs, 9 of the 16 GA-related DEGs were directly involved in GA biosynthesis. Further dual-LUC and Y1H assays supported the critical role of CsPIF3 in linking CsPhyB and GA biosynthesis for hypocotyl growth, in which CsPIF3 binds to the promoter of CsGA20ox-2 to regulate its expression and GA biosynthesis. In addition, there are 73 auxin-related DEGs supporting the involvement of auxin biosynthesis/signaling in hypocotyl growth in cucumber. Y1H and DUAL-LUC assays between CsPIF3 and auxin signaling/biosynthesis pathways genes found that CsPIF3 was able to bind to the G-box of CsARF18 to regulate its expression in auxin signaling/biosynthesis for hypocotyl growth in cucumber. RNA-Seq data also support CsPIF3 mediating UVR8-regulated hypocotyl elongation that is independent of UV-B signaling for defense/acclimation. In the transcriptomes examined, many DEGs are UVB responsive genes or genes involved in UVB-defense or acclimation which included DNA damage repair, and biosynthesis of secondary metabolites. For example, almost all genes in the flavonoid pathway for anthocyanin biosynthesis were up-regulated in lh1, lh1lh2 mutants. RNA-Seq, Y2H and BIFC assays supported a critical role of CsPIF3 in the crosstalk between CsPhyB- and CsUVR8-dependent light signaling for hypocotyl growth in cucumber. Objective 4. Marker-assisted development of germplasm carrying the sh1 allele and evaluating its use in cucumber breeding. Through marker-assisted backcrossing, the sh1 allele has been introgressed into a bait alpha type greenhouse cucumber (WI7204) and pickling type cucumber Gy14. The introgression size in each resulting line was estimated with genotyping-by-sequencing. Hypocotyl elongation of introgressions was evaluated in growth chamber tests under different temperature and light conditions. The results indicated that hypocotyl growth was not affected by low light or high temperature, or UVB-free conditions. Horticulture performance of the introgression lines was also evaluated in replicated field trials. No negative effects were found on overall performance by the introgression.

Publications


    Progress 02/15/20 to 02/14/21

    Outputs
    Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. One postdoc research associate was trained through this project in both theory and practice in the field of Plant Breeding and Genetics. 2. One visiting scientist participated in this project. How have the results been disseminated to communities of interest?1. Giving talks on national and local annual meetings of commondity groups. 2. Presntations in professional meetings. 3. Publication of manuscripts. 4. Collaborative research with seed companies. What do you plan to do during the next reporting period to accomplish the goals?This project will end in February 14, 2021. We are requesting one year non-cost extension to accomplish the following tasks, which were delayed by the pandemic. 1. Field trial to evaluate horticulture performances of sh1 NILs in WI7204 and Gy14 backgrounds. 2. Additional work on gene regulatory network on Sh1-dependent hypocotyl elongation.

    Impacts
    What was accomplished under these goals? 1. Expression of CsGA20ox-2 is positively correlated with hypocotyl elongation We show that lh2 encodes CsGA20ox-2, a key enzyme in GA biosynthesis. We found much higher expression of CsGA20ox-2 in hypocotyls of lh1 and lh1lh2 than in Gy14. RNA in situ hybridization revealed its strong expression in SAM. We developed transgenic cucumber plants overexpressing (OX) the WT allele of CsGA20ox-2 in Poinsett 76. All OX plants had significantly longer hypocotyl than the control under both LD-UVB and UVB-free white lights. The longer hypocotyl in OX lines was positively correlated with the expression level of CsGA20ox-2. 2. CsPIF1b bridges CsPhyB and GA signaling for hypocotyl elongation Lh1 encodes the red-light receptor phytochrome B (CsPhyB). We hypothesized that upon absorption of red light, CsPhyB will be converted into the Pfr active form then translocated into the nucleus to interact with phytochrome interacting factors (CsPIFs) to control hypocotyl growth through regulation of CsGA20ox-2 expression and GA biosynthesis/signaling. We examined protein-protein-interactions using Y2H and BiFC assays. The data suggested that physical interactions among CsPhyB, CsPIF1b, CsPIF3, and two DELLA proteins, CsGAI1 and CsGAI2 may play critical roles in GA-dependent hypocotyl elongation in cucumber. From Y1H assays, we further show the critical role of CsPIF1b in linking CsPhyB signaling and GA pathways in which CsPIF1b binds to the promoter of CsGA20ox-2 to regulate its expression and thus GA biosynthesis. These observations were further evidence from transcriptome proofing that reveals Lh1- and lh2-regulated gene network for hypocotyl elongation in cucumber. 3. CsPIF4 mediates UVR8-regulated hypocotyl elongation that is independent of UV-B signaling for defense/acclimation. Although lh1 and lh1lh2 mutants are LDUVB insensitive, RNA-Seq analysis indicated that the CsUVR8-mediated signaling for UVB protection and/or acclimation is working in the two mutants. We examined protein-protein interactions key players in CsPhyB and CsUVR8 signaling pathways. We confirmed known physical interactions of CsCOP1 with CsUVR8 and CsHY5 in cucumber. We also found a physical interaction between CsUVR8 and CsPIF4. These data supported a critical role of CsPIF4 in the crosstalk between CsPhyB- and CsUVR8-dependent light signalings for hypocotyl growth in cucumber. 4. Development of introgression lines. Through marker-assisted backcrossing, the sh1 allele has been introgressed into a bait alpha type greenhouse cucumber (WI7204) and pickling type cucumber Gy14. In 2020, 2e evaluated hypocotyl growth of the two NIL pairs in growth chambers at two temperature settings. Adiditonal field evaluation will be conducted in 2021 field season.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang Y, Jiang B, Dymerski R, Xu XW, Weng Y (2020) Quantitative trait loci for horticulturally important traits defining the Sikkim cucumber, Cucumis sativus var. sikkimensis. Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-020-03693-y
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Pan YP, Wen CL, Han YH, Wang YH, Li YH, Li S, Cheng XM, Weng Y (2020) QTL for horticulturally important traits associated with pleiotropic andromonoecy and carpel number loci, and a paracentric inversion in cucumber. Theoretical and Applied Genetics 133: 2271-2290
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Weng Y (2021) Cucumis sativus chromosome evolution, domestication, and genetic diversity  Implications for cucumber breeding. Plant Breeding Review 49: 77-111
    • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Zhao JY, Bo KL, Pan YP, Li YH, Yu DL, Chang J, Wu S, Wang ZY, Zhao XL, G XF, Weng Y (2021) CsPIF1b, CsPIF3 and CsPIF4 integrate CsPhyB and CsUVR8 signaling pathways to regulated gibberellin- and auxin-dependent hypocotyl elongation in cucumber. Submitted


    Progress 02/15/19 to 02/14/20

    Outputs
    Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. One postdoc is being trained through this project in both theory and practice in the field of Plant Breeding and Genetics. 2. One undergraduate student and one high school student were trained in learning basic molecular biological research tools. 3. Three visiting scientists also participated in this project. How have the results been disseminated to communities of interest?1. Giving talks on national and local annual meetings of commondity groups. 2. Presntations in professional meetings. 3. Publication of manuscripts. 4. Collaborative research with seed companies. What do you plan to do during the next reporting period to accomplish the goals?We will conduct tehr eserch in this project as planned. We expect to wrap up the project on time by Feb 2021.

    Impacts
    What was accomplished under these goals? 1. The cucumber line WI7284 exhibits extremely long hypocotyl that is UVB insensitive and is controlled by the recessive locus lh1. Segregation populations from the cross between WI7284 and Gy14 (wild type) were developed. Under LDUVB condition, the F1 showed short hypocotyl as Gy14, and the F2 was segregating at 3 short to 1 long. Bulked segregant analysis (BSA) allowing placing the lh1 locus on Chromosome 3. Linkage analysis in 214 recessive homozygous F2 plants delimited lh1 into a 795 kb region. Fine mapping in a total of 3200 F2 plants delimit the lh1 locus into a 36kb region. The cucumber phytochrome B (CsPhyB) gene was proved to be the candidate gene for lh1. The mutant allele carries a 7-bp deletion in the first exon of CsPhyB)resulting in premature termination of the protein. 2. The cucumber line AM149 is a long hypocotyl mutant. Inheritance studies revealed AM149L as a double mutant carrying both lh1 and lh2. Lh1 is epistatic to lh2. Map-based cloning identified CsPhyB and CsGA2oox-2 as the candidate genes the lh1 and lh2, respectively. Lh1 in AM149L has a 4-bp deletion in the second exon of CsPhyB. The mutant allele of lh2 has a SNP resulting in atruncated protein. CsGA20ox-2 is a critical enzyme for GA biosynthesis. 3. The lh1 locus is ~2Mbp away from sh1 on Chr 3. Through marker-assisted selection, we developed near isogenic lines for lh1, lh2, sh1, and their combinations in the same Gy14 (wild type) genetic background. We conducted extensive phenotypic characterization of these NILs under LDUVB and UVB-free conditions. Microscopic examination revealed that The mutants lh1, lh1lh2 and sh1 are all LDUVB insensitive. The long hypocotyl in lh1 and lh2 is due to extended longitudinal cell growth in the hypocotyl. 4. The functions of lh1, lh2 and sh1 candidate genes were validated with multiple approaches including allelic analysis in natural populations, spatiotemporal gene expression in different light conditions, and development of transgenic cucumber plants (overexpression). 5. To understand the regulatory network, RNA-Seq was conducted with samples collected from lh1, lh1lh2, and sh1 NILs under white light UVB-free and LDUVB conditions. Analysis of the transcriptomes revealed the GA pathway and the auxin pathway plays important roles in lh1/lh2-dependent and sh1-depent hypocotyl elongation, respectively. 6. Subcellular localization of the SH1, LH1 and LH2 proteins were conducted, which show that all are located on cell membranes. However, LH1 will move to the nucleus upon red light treatment. Yeast two-hybrid (Y2H) and BiFC assays were conducted to identify interacting partners of CsPHYB and CsGA20ox-2 in regulation of LDUVB-dependent hypocotyl elongation in cucumber. A model was proposed in which CsPHYB and CsUVR8 interact with phytochrome interacting factors (PIFs) and regulators of the GA signaling pathway (DELLAb and GID) to modulate GA biosynthesis in control of hypocotyl elongation. Additional work on SH1-regulated hypocotyl elongation is underway. 7. Through marker-assisted backcrossing, the sh1 allele has been introgressed into a bait alpha type greenhouse cucumber (WI7204) and pickling type cucumber Gy14. Homologous BC4F2 NILs were evaluated in greenhouses and open fields for horticultural traits in 2019.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhao JZ, Bo KL, Pan YP, Gu XF, Weng (2020) CsphyB and CsGA20ox-2 coordinate hypocotyl elongation in cucumber. Abstract for Plant and Animal Genome Conference XXVIII (Jan 13-18, 2020, San Diego, CA)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Liu HQ, Zhao JY, Weng YQ (2020) A modified protocol improves efficiency of Agrobacterium-mediated transformation in cucumber (Cucumis sativus L.).Abstract for Plant and Animal Genome Conference XXVIII (Jan 13-18, 2020, San Diego, CA)
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Pan YP, Wang YH, McGregor C, Liu S, Luan FS, Gao ML, Weng Y (2020) Genetic architecture of fruit size and shape variation in cucurbits: a comparative perspective. Theor Appl Genet 133: 1-21
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang YH, Bo KL, Gu XF, Pan JS, Li YH, Chen JF, Wen CL, Ren ZH, Ren HZ, Chen XH, Grumet G, Weng Y (2019) Molecularly tagged genes and quantitative trait loci in cucumber with recommendations for QTL nomenclature. Horticulture Research 7: 3.
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Li Z, Han YH, Niu HH, Wang YH, Jiang B, Weng Y (2020) Gynoecy instability in cucumber (Cucumis sativus L.) is due to unequal crossover at the copy number variation-dependent femaleness (F) locus. Horticulture Research 7:32. doi: 10.1038/s41438-020-0251-2
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sheng YS, Pan YP, Li YH, Yang LM, Weng Y (2019) Quantitative trait loci for fruit size and flowering time-related traits under domestication and diversifying selection in cucumber (Cucumis sativus L.). Plant Breeding https://doi.org/10.1111/pbr.12754


    Progress 02/15/18 to 02/14/19

    Outputs
    Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. One postdoc is being trained through this project in both theory and practice in the field of Plant Breeding and Genetics. 2. One undergraduate student and one high school student were trained in learning basic molecular biological research tools. 3. One visiting scientist participated in this project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Will condcut various experiments as planned.

    Impacts
    What was accomplished under these goals? 1. The cucumber line WI7284 exhibits extremely long hypocotyl that is UVB insensitive and is controlled by the recessive locus lh1. Segregation populations from the cross between WI7284 and Gy14 (wild type) were developed. Under LDUVB condition, the F1 showed short hypocotyl as Gy14, and the F2 was segregating at 3 short to 1 long. Bulked segregant analysis (BSA) allowing placing the lh1 locus on Chromosome 3. Linkage analysis in 214 recessive homozygous F2 plants delimited lh1 into a 795 kb region. Fine mapping in 2018 further narrowed this region to ~50 kb with 6 predicted genes. 2. The lh1 locus is ~2Mbp away from sh1 on Chr 3. Through marker-assisted selection, we developed double mutant for the two loci (lh1lh1sh1lsh1) which showed extra-long hypocotyl under LDUVB condition suggesting Lh1 is epistatic to Sh1. The single and double mutants in the same genetic background (NILs) were phenotypically characterized at various light conditions. The results suggest possibly different pathways employed by the Sh1 and Lh1 for regulation of hypocotyl elongation. 3. Near-isogenic lines (NILs) for both sh1 and lh1 loci were developed in the 9930 cucumber genetic background. Development of the lh1 NILs is alos underway. To understand the regulatory network, RNA-Seq was conducted with samples collected from two NILs under white light UVB-free and LDUVB conditions. The same set of materials were also subjected to DNase-Seq to reveal possible epigenetic regulation mediated by the sh1 locus. Genetic transformation for the sh1 locus is underway. 4. subcellular localization of the SH1 and LH1 proteins were conducted, which show that both are located on cell membranes. Based on transcriptome data, yeast two hybrid (Y2H) assays will be performed to infer relevant regulator pathways involved in LH1 and SH1 signaling. 5. Through marker-assisted backcrossing, the sh1 allele has been introgressed into a bait alpha type greenhouse cucumber (WI7204), which is now at BC3F1. Homologous NILs will be phenotyped for horticultural traits in greenhouse conditions.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bo KL, Pan YP, Li YH, Zhang WL, Krysan PJ, Weng Y (2018) Genetic and molecular mechanisms of UVB-dependent hypocotyl elongation in cucumber. . Abstract for Plant and Animal Genome Meeting XXVII (Jan 12-16, 2018, San Diego, CA)
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yagcioglu M. Jiang B. Wang P. Wang YH, Ellialtioglu SS, Weng Y. 2019. QTL mapping of low temperature germination ability in cucumber. Euphytica 215: 84
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhao JY, Jiang L, Che G, Pan YP, Li YQ, Hou Y, Zhao WS, Zhong YT, Ding L, Yan SS, Sun CZ, Liu RY, Yan LY, Wu T, Li XX, Weng Y, Zhao XL. 2019. A functional allele of CsFUL1 regulates fruit length through repressing CsSUP and inhibiting auxin transport in cucumber. Plant Cell, doi:10.1105/tpc.18.00905


    Progress 02/15/17 to 02/14/18

    Outputs
    Target Audience:The immediate target audience is the stakeholders of the cucumber industry (seed companies, public/private sector researchers, government and university) that will utilize the information and resources to develop better cucumber for the consumers. Additional audiences include the cucurbit research community, and the biological sciecne reserch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. One postdoc is being trained through this project in both theory and practice in the field of Plant Breeding and Genetics. 2. One undergraduate student and one high school student are bring trained in learning basic molecular biological research tools. How have the results been disseminated to communities of interest?Weng Y (2017) Novel Players in Plant Low-dosage UVB Signaling Revealed from Natural Variation of Hypocotyl Elongation in Cucumber. An invited talk at The First International Symposium on Horticulture Biology (4/24-25, 2017, Fuzhou, China). What do you plan to do during the next reporting period to accomplish the goals?All activities will be perfomred as planned. 1. Continue fine mapping of lh1 to clone the candidate gene. Perform functinal validation of the candidate gene with various tools (qPCR, alleleci diversity). 2. Initiate genetic transforamtion of sh1 and lh1 candidate genes. 3. Data anlaysis for RNA-Seq and DNase-Seq to understand teh sh1-mediated regulatory network. 4. Perform RNA-Seq with NILs of lh1 and sh1-lh1 double mutants.

    Impacts
    What was accomplished under these goals? 1. The cucumber line WI7284 exhibits extremely long hypocotyl. We show this long hypocotyl elongation is UVB insensitive and is controlled by the recessive locus lh1. Segregation populations from the cross between WI7284 and Gy14 (wild type) were developed. Under LDUVB condition, the F1 showed short hypocotyl as Gy14, and the F2 was segregating at 3 short to 1 long. Bulked segregant analysis (BSA) placed the lh1 locus on Chromosome 3. Linkage analysis in 214 recessive homozygous F2 plants delimited lh1 into a 795 kb region. Fine mapping further narrowed this region to ~100 kb with 12 predicted genes. 2. The lh1 locus is ~2Mbp away from sh1 on Chr 3. Through marker-assisted selection, we developed double mutant for the two loci (lh1lh1sh1lsh1) which showed extra-long hypocotyl under LDUVB condition suggesting Lh1 is epistatic to Sh1. 3. Near-isogenic lines (NILs) for the sh1 locus were developed in the 9930 cucumber genetic background. Development of the lh1 NILs is alos underway. To understand the regulatory network, RNA-Seq was conducted with samples collected from two NILs under white light UVB-free and LDUVB conditions. The same set of materials were also subjected to DNase-Seq to reveal possible epigenetic regulation mediated by the sh1 locus. Genetic transformation for the sh1 locus was also initiated. 4. Marker-assisted backcross was initiated aiming to introgressing the sh1 locus into a bait alpha type greenhouse cucumber (WI7204). The F1 plant from sh1-NILxWI7204 was backcrossed with WI7204. Now it has been adavnced to BC2.

    Publications