ELONGATED HYPOTCOTYL5 and SPINE BASE SIZE1 together mediate light-regulated spine expansion in cucumber.

Plant trichome development is influenced by diverse developmental and environmental signals, but the molecular mechanisms involved are not well understood in most plant species. Fruit spines (trichomes) are an important trait in cucumber (Cucumis sativus L.), as they affect both fruit smoothness and commercial quality. Spine Base Size1 (CsSBS1) has been identified as essential for regulating fruit spine size in cucumber. Here, we discovered that CsSBS1 controls a season-dependent phenotype of spine base size in wild-type plants. Decreased light intensity led to reduced expression of CsSBS1 and smaller spine base size in wild-type plants, but not in the mutants with CsSBS1 deletion. Additionally, knockout of CsSBS1 resulted in smaller fruit spine base size and eliminated the light-induced expansion of spines. Overexpression of CsSBS1 increased spine base size and rescued the decrease in spine base size under low light conditions. Further analysis revealed that ELONGATED HYPOTCOTYL5 (HY5), a major transcription factor involved in light signaling pathways, directly binds to the promoter of CsSBS1 and activates its expression. Knockout of CsHY5 led to smaller fruit spine base size and abolished the light-induced expansion of spines. Taken together, our study findings have clarified a CsHY5-CsSBS1 regulatory module that mediates light-regulated spine expansion in cucumber. This finding offers a strategy for cucumber breeders to develop fruit with stable appearance quality under changing light conditions.

Identification of candidate genes that regulate the trade-off between seedling cold tolerance and fruit quality in melon (Cucumis melo L.).

Trade-offs between survival and growth are widely observed in plants. Melon is an annual, trailing herb that produces economically valuable fruits that are traditionally cultivated in early spring in China. Melon seedlings are sensitive to low temperatures, and thus usually suffer from cold stress during the early growth period. However, little is known about the mechanism behind the trade-offs between seedling cold tolerance and fruit quality in melon. In this study, a total of 31 primary metabolites were detected from the mature fruits of eight melon lines that differ with respect to seedling cold tolerance; these included 12 amino acids, 10 organic acids, and 9 soluble sugars. Our results showed that concentrations of most of the primary metabolites in the cold-resistant melons were generally lower than in the cold-sensitive melons; the greatest difference in metabolite levels was observed between the cold-resistant line H581 and the moderately cold-resistant line HH09. The metabolite and transcriptome data for these two lines were then subjected to weighted correlation network analysis, resulting in the identification of five key candidate genes underlying the balancing between seedling cold tolerance and fruit quality. Among these genes, CmEAF7 might play multiple roles in regulating chloroplast development, photosynthesis, and the ABA pathway. Furthermore, multi-method functional analysis showed that CmEAF7 can certainly improve both seedling cold tolerance and fruit quality in melon. Our study identified an agriculturally important gene, CmEAF7, and provides a new insight into breeding methods to develop melon cultivars with seedling cold tolerance and high fruit quality.

Melon yellow-green plant (Cmygp) encodes a Golden2-like transcription factor regulating chlorophyll synthesis and chloroplast development.

KEY MESSAGE: A SNP mutation in CmYGP gene encoding Golden2-like transcription factor is responsible for melon yellow-green plant trait. Chlorophylls are essential and beneficial substances for both plant and human health. Identifying the regulatory network of chlorophyll is necessary to improve the nutritional quality of fruits. At least six etiolation genes have been identified in different melon varieties, but none of them have been cloned, and the molecular mechanisms underlying chlorophyll synthesis and chloroplast development in melon remain unclear. Here, the NSL73046, a yellow-green plant (Cmygp) mutant, enabled the map-based cloning of the first etiolation gene in melon. CmYGP encodes a Golden2-like transcription factor. Spatiotemporal expression analyses confirmed the high CmYGP expression in all green tissues, particularly in young leaves and fruit peels. Virus-induced gene silencing and the development of near-isogenic line by marker-assisted selection further confirmed that downregulation of CmYGP can reduce chloroplast number and chlorophyll content, thereby resulting in yellow-green leaves and fruits in melon, and overexpression of CmYGP in tomatoes also led to dark-green leaves and fruits. RNA-seq analysis revealed that CmYGP greatly affected the expression of key genes associated with chloroplast development. Taken together, these findings demonstrated that CmYGP regulate chlorophyll synthesis and chloroplast development thus affect fruit development in melon. This study also offers a new strategy to enhance fruit quality in melon.

Chromosomal fragment deletion in APRR2-repeated locus modulates the dark stem color in Cucurbita pepo.

KEY MESSAGE: Cp4.1LG15g03420 (CpDsc-1), which encodes a two-component response regulator-like protein (APRR2) in the nucleus, influences dark green stem formation in Cucurbita pepo by regulating the chlorophyll content. Stem color is an important agronomic trait in zucchini (Cucurbita pepo) for robust seeding and high yield. However, the gene controlling the stem color has not been characterized. In this study, we identified a single locus accounting for the dark green stem color of C. pepo (CpDsc-1). Genetic analysis of this trait in segregated populations derived from two parental lines (line 296 with dark green stems and line 274 with light green stems) revealed that stem color was controlled by a single dominant gene (dark green vs. light green). In bulked segregant analysis, CpDsc-1 was mapped to a 2.09-Mb interval on chromosome 15. This region was further narrowed to 65.2 kb using linkage analysis of the F2 population. Sequencing analysis revealed a 14 kb deletion between Cp4.1LG15g03420 and Cp4.1LG15g03360; these two genes both encoded a two-component response regulator-like protein (APRR2). The incomplete structures of the two APRR2 genes and abnormal chloroplasts in line 274 might be the main cause of the light green phenotype. Gene expression pattern analysis showed that only Cp4.1LG15g03420 was upregulated in line 296. Subcellular localization analysis indicated that Cp4.1LG15g03420 was a nuclear gene. Furthermore, a co-dominant marker, G4563 (93% accuracy rate), and a co-segregation marker, Fra3, were established in 111 diverse germplasms; both of these markers were tightly linked with the color trait. This study provided insights into chlorophyll regulation mechanisms and revealed the markers valuable for marker-assisted selection in future zucchini breeding.

The branchless gene Clbl in watermelon encoding a TERMINAL FLOWER 1 protein regulates the number of lateral branches.

KEY MESSAGE: A SNP mutation in Clbl gene encoding TERMINAL FLOWER 1 protein is responsible for watermelon branchless. Lateral branching is one of the most important traits, which directly determines plant architecture and crop productivity. Commercial watermelon has the characteristics of multiple lateral branches, and it is time-consuming and labor-costing to manually remove the lateral branches in traditional watermelon cultivation. In our present study, a lateral branchless trait was identified in watermelon material WCZ, and genetic analysis revealed that it was controlled by a single recessive gene, which named as Clbl (Citrullus lanatus branchless). A bulked segregant sequencing (BSA-seq) and linkage analysis was conducted to primarily map Clbl on watermelon chromosome 4. Next-generation sequencing-aided marker discovery and a large mapping population consisting of 1406 F2 plants were used to further map Clbl locus into a 9011-bp candidate region, which harbored only one candidate gene Cla018392 encoding a TERMINAL FLOWER 1 protein. Sequence comparison of Cla018392 between two parental lines revealed that there was a SNP detected from C to A in the coding region in the branchless inbred line WCZ, which resulted in a mutation from alanine (GCA) to glutamate (GAA) at the fourth exon. A dCAPS marker was developed from the SNP locus, which was co-segregated with the branchless phenotype in both BC1 and F2 population, and it was further validated in 152 natural watermelon accessions. qRT-PCR and in situ hybridization showed that the expression level of Cla018392 was significantly reduced in the axillary bud and apical bud in branchless line WCZ. Ectopic expression of ClTFL1 in Arabidopsis showed an increased number of lateral branches. The results of this study will be helpful for better understanding the molecular mechanism of lateral branch development in watermelon and for the development of marker-assisted selection (MAS) for new branchless watermelon cultivars.

Long-read sequencing and de novo assembly of the Luffa cylindrica (L.) Roem. genome.

Sponge gourd (Luffa cylindrica (L.) Roem.) or luffa is a diploid herbaceous plant with 26 chromosomes (2n = 26) and belongs to the family Cucurbitaceae. To address the limited knowledge of the genome of Luffa species, the chromosome-level genome of L. cylindrica was assembled and analysed using PacBio long reads and Hi-C data. We combined Hi-C data with a draft genome assembly to generate chromosome-length scaffolds. Thirteen scaffolds corresponding to the 13 chromosomes were assembled from 1,156 contigs to a final size of 669 Mb with a contig N50 size of 5 Mb and a scaffold N50 size of 53 Mb. After removing redundant sequences, 416.31 Mb (62.18% of the genome) of repeat sequences was detected. Subsequently, 31,661 protein-coding genes with an average of 5.69 exons per gene were identified in the L. cylindrica genome using de novo methods, transcriptome data and homologue-based approaches. In addition, 27,552 protein-coding genes (87.02%) were annotated in five databases. According to the phylogenetic analysis, L. cylindrica is closely related to Cucurbita and Cucumis species and diverged from their common ancestor ~28.6-67.1 million years ago. Genome collinearity analysis was performed in Cucurbita moschata, Cucumis sativus and L. cylindrica, and it demonstrated a high degree of conserved gene order in these three species. The completeness of the genome will provide high-quality genomic knowledge on breeding and reveal genetic variation in L. cylindrica.

A Single Nucleotide Deletion in an ABC Transporter Gene Leads to a Dwarf Phenotype in Watermelon.

Dwarf habit is one of the most important traits in crop plant architecture, as it can increase plant density and improved land utilization, especially for protected cultivation, as well as increasing lodging resistance and economic yield. At least four dwarf genes have been identified in watermelon, but none of them has been cloned. In the current study, the Cldw-1 gene was primary-mapped onto watermelon chromosome 9 by next-generation sequencing-aided bulked-segregant analysis (BSA-seq) of F2 plants derived from a cross between a normal-height line, WT4, and a dwarf line, WM102, in watermelon. The candidate region identified by BSA-seq was subsequently validated and confirmed by linkage analysis using 30 simple sequence repeat (SSR) markers in an F2 population of 124 plants. The Cldw-1 gene was further fine-mapped by chromosome walking in a large F2 population of 1,053 plants and was delimited into a candidate region of 107.00 kb. Six genes were predicted to be in the candidate region, and only one gene, Cla010337, was identified to have two single nucleotide polymorphisms (SNPs) and a single nucleotide deletion in the exons in the dwarf line, WM102. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed from the single nucleotide deletion, co-segregated with the dwarf trait in both the F2 population and a germplasm collection of 165 accessions. Cla010337 encoded an ATP-binding cassette transporter (ABC transporter) protein, and the expression levels of Cla010337 were significantly reduced in all the tissues tested in the dwarf line, WM102. The results of this study will be useful in achieving a better understanding of the molecular mechanism of the dwarf plant trait in watermelon and for the development of marker-assisted selection (MAS) for new dwarf cultivars.

Microsatellite Markers Reveal Genetic Diversity and Relationships within a Melon Collection Mainly Comprising Asian Cultivated and Wild Germplasms.

Melon, Cucumis melo L., is an important horticultural crop with abundant morphological variability, but the genetic diversity and relationships within wild and cultivated melons remain unclear to date. In this study, thick-skinned (TC) (cultivated subspecies melo), thin-skinned (TN) (cultivated subspecies agrestis), and wild accessions were analyzed for genetic diversity and relationships using 36 microsatellite markers. A total of 314 alleles were detected with a mean allelic number of 8.72 and polymorphism information content of 0.67. Cluster analysis of the accessions resulted in four distinct clusters (I, II, III, and IV) broadly matching with the TC, TN, and wild groups. Cluster I contained only two Indian wild accessions. Cluster II was consisted of 49 South Asian accessions, 34 wild accessions, and 15 TN accessions. Cluster III was a typical TC group including 51 multiorigin TC accessions and one wild accession. The remaining 88 accessions, including 75 TN accessions, 6 wild accessions, and 7 TC accessions, formed the cluster IV, and all the TN and wild accessions in this cluster were from China. These findings were also confirmed by Principal component analysis and STRUCTURE analysis. The South Asian subspecies agrestis accessions, wild and cultivated, had close genetic relationships with a distinctive genetic background. Chinese wild melons showed closeness to cultivated subspecies agrestis landraces and could be a return from the indigenous cultivated melons. The AMOVA and pairwise F statistics (F ST) presented genetic differentiation among the three groups, with the strongest differentiation (F ST = 0.380) between TC and TN melons. These results offer overall information on genetic diversity and affiliations within a variety of melon germplasms and favor efficient organization and utilization of these resources for the current breeding purpose.

Dissecting the Genetic Architecture of Melon Chilling Tolerance at the Seedling Stage by Association Mapping and Identification of the Elite Alleles.

Low temperature is an important abiotic stress that negatively affects morphological growth and fruit development in melon (Cucumis melo L.). Chilling stress at the seedling stage causes seedling injury and poor stand establishment, prolonging vegetative growth and delaying fruit harvest. In this study, association mapping was performed for chilling tolerance at the seedling stage on an expanded melon core collection containing 212 diverse accessions by 272 SSRs and 27 CAPSs. Chilling tolerance of the melon seedlings was evaluated by calculating the chilling injury index (CII) in 2016 and 2017. Genetic diversity analysis of the whole accession panel presented two main groups, which corresponded to the two subspecies of C. melo, melo, and agrestis. Both the subspecies were sensitive to chilling but with agrestis being more tolerant. Genome-wide association study (GWAS) was conducted, respectively, on the whole panel and the two subspecies, totally detecting 51 loci that contributed to 74 marker-trait associations. Of these associations, 35 were detected in the whole panel, 21 in melo, and 18 in agrestis. About half of the associations identified in the two subspecies were also observed in the whole panel, and seven associations were shared by both the subspecies. CMCT505_Chr.1 was repeatedly detected in different populations with high phenotypic contribution and could be a key locus controlling chilling tolerance in C. melo. Nine loci were selected for evaluation of the phenotypic effects related to their alleles, which identified 11 elite alleles contributing to seedling chilling tolerance. Four such alleles existed in both the subspecies and six in either of the two subspecies. Analysis of 20 parental combinations for their allelic status and phenotypic values showed that the elite alleles collectively contributed to enhancement of the chilling tolerance. Tagging the loci responsible for chilling tolerance may simultaneously favor dissecting the complex adaptability traits and elevate the efficiency to improve chilling tolerance using marker-assisted selection in melon.

GLABROUS (CmGL) encodes a HD-ZIP IV transcription factor playing roles in multicellular trichome initiation in melon.

KEY MESSAGE: Map-based cloning identified CmGL that encodes a HD-ZIP type IV transcription factor that controls multicellular trichome initiation in melon. Trichomes are small hairs covering the aerial parts of plants that originate from the epidermal cells, which can protect plants against the damage by insects and pathogens. The regulatory pathway of unicellular trichomes has been well studied in the model plant Arabidopsis. Little is known about the genetic control and regulation of trichome development in melon (Cucumis melo L.) which has multicellular trichomes. In this study, we identified a melon mutant, cmgl, which showed completely glabrous on all aerial organs. A bulked segregant analysis was conducted to identify polymorphic markers for linkage analysis in a population with 256 F2 plants, which allowed to locate the cmgl locus in melon chromosome VIII. Next-generation sequencing-aided marker discovery and fine mapping in a large population with 1536 F2 plants narrowed the candidate gene region to 12 kb that harbored only one candidate gene for cmgl, which encoded a class IV homeodomain-associated leucine zipper transcription factor. Four SNPs in the coding region of the CmGL gene were identified between the two parental lines; a single base substitution from C to A resulted in a premature termination codon and a truncated protein in the cmgl. The SNP was converted into a dCAPS marker, which showed co-segregation in the F2 population and 564 melon accessions. Result of this study will be helpful for better understanding of genetic control of trichome development in melon and marker-assisted selection in developing new cultivars.

Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis.

BACKGROUND: Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. RESULTS: A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these cross-species SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis clustered in another group. Furthermore, structure analysis was consistent with the dendrogram indicating the 134 watermelon accessions were classified into two populations. CONCLUSION: The large number of genome wide SSR markers developed herein from the watermelon genome provides a valuable resource for genetic map construction, QTL exploration, map-based gene cloning and marker-assisted selection in watermelon which has a very narrow genetic base and extremely low polymorphism among cultivated lines. Furthermore, the cross-species transferable SSR markers identified herein should also have practical uses in many applications in species of Cucurbitaceae family whose whole genome sequences are not yet available.