What makes a giant fruit? Assembling a genomic toolkit underlying various fruit traits of the mammoth group of Cucurbita maxima.

Since their introduction in Europe, pumpkins (Cucurbita maxima Duch.) have rapidly dispersed throughout the world. This is mainly because of their wide genetic diversity and Plasticity to thrive in a wide range of geographical regions across the world, their high nutritional value and suitability to integrate with local cuisines, and their long shelf life. Competition for growing the showy type or mammoth-sized pumpkins that produce the largest fruit of the entire plant kingdom has drawn attention. In this study, we used genome-wide single nucleotide polymorphisms to resolve admixture among different pumpkin groups. Also, to resolve population differentiation, genome-wide divergence and evolutionary forces underlying the evolution of mammoth-sized pumpkin. The admixture analysis indicates that the mammoth group (also called Display or Giant) evolved from the hubbard group with genome-wide introgressions from the buttercup group. We archived a set of private alleles underlying fruit development in mammoth group, and resolved haplotype level divergence involved in the evolutionary mechanisms. Our genome-wide association study identified three major allelic effects underlying various fruit-size genes in this study. For fruit weight, a missense variant in the homeobox-leucine zipper protein ATHB-20-like (S04_18528409) was significantly associated (false discovery rate = 0.000004) with fruit weight, while high allelic effect was consistent across the 3 years of the study. A cofactor (S08_217549) on chromosome 8 is strongly associated with fruit length, having superior allelic effect across the 3 years of this study. A missense variant (S10_4639871) on translocation protein SEC62 is a cofactor for fruit diameter. Several known molecular mechanisms are likely controlling giant fruit size, including endoreduplication, hormonal regulation, CLV-WUS signaling pathway, MADS-box family, and ubiquitin-proteasome pathway. This study provides a general framework for the evolutionary relationship among horticulture groups of C. maxima and elucidates the origins of rare variants contributing to the giant pumpkin fruit size.

Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids.

KEY MESSAGE: Transcriptome landscape reveals the molecular mechanisms involved in the improvement of fruit traits by the grafting of watermelon and bottle gourd. Grafting has been used as a sustainable alternative for watermelon breeding to control soil-borne pathogens and to increase tolerance to various abiotic stresses. However, some reports have shown that grafting can negatively affect the quality of fruits. Despite several field studies on the effects of grafting on fruit quality, the regulation of this process at the molecular level has not been revealed. The aim of this study was to elucidate various molecular mechanisms involved in different tissues of heterografted watermelon and bottle gourd plants. Grafting with bottle gourd rootstock increased the size and rind thickness of watermelon fruits, whereas that with watermelon rootstock produced bottle gourd fruits with higher total soluble solid content and thinner rinds. Correspondingly, genes related to ripening, softening, cell wall strengthening, stress response and disease resistance were differentially expressed in watermelon fruits. Moreover, genes associated mainly with sugar metabolism were differentially expressed in bottle gourd fruits. RNA-seq revealed more than 400 mobile transcripts across the heterografted sets. More than half of these were validated from PlaMoM, a database for plant mobile macromolecules. In addition, some of these mobile transcripts contained a transfer RNA-like structure. Other RNA motifs were also enriched in these transcripts, most with a biological role based on GO analysis. This transcriptome study provided a comprehensive understanding of various molecular mechanisms underlying grafted tissues in watermelon.

Genome-Wide Differentiation of Various Melon Horticultural Groups for Use in GWAS for Fruit Firmness and Construction of a High Resolution Genetic Map.

Melon (Cucumis melo L.) is a phenotypically diverse eudicot diploid (2n = 2x = 24) has climacteric and non-climacteric morphotypes and show wide variation for fruit firmness, an important trait for transportation and shelf life. We generated 13,789 SNP markers using genotyping-by-sequencing (GBS) and anchored them to chromosomes to understand genome-wide fixation indices (Fst) between various melon morphotypes and genomewide linkage disequilibrium (LD) decay. The FST between accessions of cantalupensis and inodorus was 0.23. The FST between cantalupensis and various agrestis accessions was in a range of 0.19-0.53 and between inodorus and agrestis accessions was in a range of 0.21-0.59 indicating sporadic to wide ranging introgression. The EM (Expectation Maximization) algorithm was used for estimation of 1436 haplotypes. Average genome-wide LD decay for the melon genome was noted to be 9.27 Kb. In the current research, we focused on the genome-wide divergence underlying diverse melon horticultural groups. A high-resolution genetic map with 7153 loci was constructed. Genome-wide segregation distortion and recombination rate across various chromosomes were characterized. Melon has climacteric and non-climacteric morphotypes and wide variation for fruit firmness, a very important trait for transportation and shelf life. Various levels of QTLs were identified with high to moderate stringency and linked to fruit firmness using both genome-wide association study (GWAS) and biparental mapping. Gene annotation revealed some of the SNPs are located in ß-D-xylosidase, glyoxysomal malate synthase, chloroplastic anthranilate phosphoribosyltransferase, and histidine kinase, the genes that were previously characterized for fruit ripening and softening in other crops.

Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits.

A genome-wide scan of selective sweeps and association mapping of fruit traits using microsatellite markers in watermelon.

Our genetic diversity study uses microsatellites of known map position to estimate genome level population structure and linkage disequilibrium, and to identify genomic regions that have undergone selection during watermelon domestication and improvement. Thirty regions that showed evidence of selective sweep were scanned for the presence of candidate genes using the watermelon genome browser (www.icugi.org). We localized selective sweeps in intergenic regions, close to the promoters, and within the exons and introns of various genes. This study provided an evidence of convergent evolution for the presence of diverse ecotypes with special reference to American and European ecotypes. Our search for location of linked markers in the whole-genome draft sequence revealed that BVWS00358, a GA repeat microsatellite, is the GAGA type transcription factor located in the 5' untranslated regions of a structure and insertion element that expresses a Cys2His2 Zinc finger motif, with presumed biological processes related to chitin response and transcriptional regulation. In addition, BVWS01708, an ATT repeat microsatellite, located in the promoter of a DTW domain-containing protein (Cla002761); and 2 other simple sequence repeats that association mapping link to fruit length and rind thickness.

High-resolution genetic map for understanding the effect of genome-wide recombination rate on nucleotide diversity in watermelon.

We used genotyping by sequencing to identify a set of 10,480 single nucleotide polymorphism (SNP) markers for constructing a high-resolution genetic map of 1096 cM for watermelon. We assessed the genome-wide variation in recombination rate (GWRR) across the map and found an association between GWRR and genome-wide nucleotide diversity. Collinearity between the map and the genome-wide reference sequence for watermelon was studied to identify inconsistency and chromosome rearrangements. We assessed genome-wide nucleotide diversity, linkage disequilibrium (LD), and selective sweep for wild, semi-wild, and domesticated accessions of Citrullus lanatus var. lanatus to track signals of domestication. Principal component analysis combined with chromosome-wide phylogenetic study based on 1563 SNPs obtained after LD pruning with minor allele frequency of 0.05 resolved the differences between semi-wild and wild accessions as well as relationships among worldwide sweet watermelon. Population structure analysis revealed predominant ancestries for wild, semi-wild, and domesticated watermelons as well as admixture of various ancestries that were important for domestication. Sliding window analysis of Tajima's D across various chromosomes was used to resolve selective sweep. LD decay was estimated for various chromosomes. We identified a strong selective sweep on chromosome 3 consisting of important genes that might have had a role in sweet watermelon domestication.

Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon.

BACKGROUND: A large single nucleotide polymorphism (SNP) dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. The marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium (LD) within a population. Use of genotyping by sequencing reveals large numbers of SNPs that in turn generate opportunities in genome-wide association mapping and marker-assisted selection, even in crops such as watermelon for which few genomic resources are available. In this paper, we used genome-wide genetic diversity to study LD, selective sweeps, and pairwise FST distributions among worldwide cultivated watermelons to track signals of domestication. RESULTS: We examined 183 Citrullus lanatus var. lanatus accessions representing domesticated watermelon and generated a set of 11,485 SNP markers using genotyping by sequencing. With a diverse panel of worldwide cultivated watermelons, we identified a set of 5,254 SNPs with a minor allele frequency of ≥ 0.05, distributed across the genome. All ancestries were traced to Africa and an admixture of various ancestries constituted secondary gene pools across various continents. A sliding window analysis using pairwise FST values was used to resolve selective sweeps. We identified strong selection on chromosomes 3 and 9 that might have contributed to the domestication process. Pairwise analysis of adjacent SNPs within a chromosome as well as within a haplotype allowed us to estimate genome-wide LD decay. LD was also detected within individual genes on various chromosomes. Principal component and ancestry analyses were used to account for population structure in a genome-wide association study. We further mapped important genes for soluble solid content using a mixed linear model. CONCLUSIONS: Information concerning the SNP resources, population structure, and LD developed in this study will help in identifying agronomically important candidate genes from the genomic regions underlying selection and for mapping quantitative trait loci using a genome-wide association study in sweet watermelon.