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.

Identification of miRNAs and Their Targets Involved in Flower and Fruit Development across Domesticated and Wild Capsicum Species.

MicroRNAs (miRNAs) are regulators of the post-transcription stage of gene activity documented to play central roles in flower and fruit development in model plant species. However, little is known about their roles and differences in domesticated and wild Capsicum species. In this study, we used high-throughput sequencing to analyze the miRNA content at three developmental stages (flower, small fruit, and middle fruit) from two cultivated (C. baccatum and C. annuum) and two wild (C. chacoense and C. eximium) pepper species. This analysis revealed 22 known and 27 novel miRNAs differentially expressed across species and tissues. A number of stage- and species-specific miRNAs were identified, and Gene Ontology terms were assigned to 138 genes targeted by the miRNAs. Most Gene Ontology terms were for the categories "genetic information processing", "signaling and cellular processes", "amino acid metabolism", and "carbohydrate metabolism". Enriched KEGG analysis revealed the pathways amino acids, sugar and nucleotide metabolism, starch and sucrose metabolism, and fructose-mannose metabolism among the principal ones regulated by miRNAs during pepper fruit ripening. We predicted miRNA-target gene interactions regulating flowering time and fruit development, including miR156/157 with SPL genes, miR159 with GaMYB proteins, miR160 with ARF genes, miR172 with AP2-like transcription factors, and miR408 with CLAVATA1 gene across the different Capsicum species. In addition, novel miRNAs play an important role in regulating interactions potentially controlling plant pathogen defense and fruit quality via fructokinase, alpha-L-arabinofuranosidase, and aromatic and neutral amino acid transporter. Overall, the small RNA-sequencing results from this study represent valuable information that provides a solid foundation for uncovering the miRNA-mediated mechanisms of flower and fruit development between domesticated and wild Capsicum species.

Genome-Wide Association Study of Natural Variation in Arabidopsis Exposed to Acid Mine Drainage Toxicity and Validation of Associated Genes with Reverse Genetics.

Acid mine drainage (AMD) is a huge environmental problem in mountain-top mining regions worldwide, including the Appalachian Mountains in the United States. This study applied a genome-wide association study (GWAS) to uncover genomic loci in Arabidopsis associated with tolerance to AMD toxicity. We characterized five major root phenotypes-cumulative root length, average root diameter, root surface area, root volume, and primary root length-in 180 Arabidopsis accessions in response to AMD-supplemented growth medium. GWAS of natural variation in the panel revealed genes associated with tolerance to an acidic environment. Most of these genes were transcription factors, anion/cation transporters, metal transporters, and unknown proteins. Two T-DNA insertion mutants, At1g63005 (miR399b) and At2g05635 (DEAD helicase RAD3), showed enhanced acidity tolerance. Our GWAS and the reverse genetic approach revealed genes involved in conferring tolerance to coal AMD. Our results indicated that proton resistance in hydroponic conditions could be an important index to improve plant growth in acidic soil, at least in acid-sensitive plant species.

Population demographic history and population structure for Pakistani Nili-Ravi breeding bulls based on SNP genotyping to identify genomic regions associated with male effects for milk yield and body weight.

The domestic Nili-Ravi water buffalo (Bubalus bubalis) is the best dairy animal contributing 68% to total milk production in Pakistan. In this study, we identified genome-wide single nucleotide polymorphisms (SNPs) to estimate various population genetic parameters such as diversity, pairwise population differentiation, linkage disequilibrium (LD) distribution and for genome-wide association study for milk yield and body weight traits in the Nili-Ravi dairy bulls that they may pass on to their daughters who are retained for milking purposes. The genotyping by sequencing approach revealed 13,039 reference genome-anchored SNPs with minor allele frequency of 0.05 among 167 buffalos. Population structure analysis revealed that the bulls were grouped into two clusters (K = 2), which indicates the presence of two different lineages in the Pakistani Nili-Ravi water buffalo population, and we showed the extent of admixture of these two lineages in our bull collection. LD analysis revealed 4169 significant SNP associations, with an average LD decay of 90 kb for these buffalo genome. Genome-wide association study involved a multi-locus mixed linear model for milk yield and body weight to identify genome-wide male effects. Our study further illustrates the utility of the genotyping by sequencing approach for identifying genomic regions to uncover additional demographic complexity and to improve the complex dairy traits of the Pakistani Nili-Ravi water buffalo population that would provide the lot of economic benefits to dairy industry.

Haplotype Networking of GWAS Hits for Citrulline Variation Associated with the Domestication of Watermelon.

Watermelon is a good source of citrulline, a non-protein amino acid. Citrulline has several therapeutic and clinical implications as it produces nitric oxide via arginine. In plants, citrulline plays a pivotal role in nitrogen transport and osmoprotection. The purpose of this study was to identify single nucleotide polymorphism (SNP) markers associated with citrulline metabolism using a genome-wide association study (GWAS) and understand the role of citrulline in watermelon domestication. A watermelon collection consisting of 187 wild, landraces, and cultivated accessions was used to estimate citrulline content. An association analysis involved a total of 12,125 SNPs with a minor allele frequency (MAF) >0.05 in understanding the population structure and phylogeny in light of citrulline accumulation. Wild egusi types and landraces contained low to medium citrulline content, whereas cultivars had higher content, which suggests that obtaining higher content of citrulline is a domesticated trait. GWAS analysis identified candidate genes (ferrochelatase and acetolactate synthase) showing a significant association of SNPs with citrulline content. Haplotype networking indicated positive selection from wild to domesticated watermelon. To our knowledge, this is the first study showing genetic regulation of citrulline variation in plants by using a GWAS strategy. These results provide new insights into the citrulline metabolism in plants and the possibility of incorporating high citrulline as a trait in watermelon breeding programs.

Genome-wide divergence, haplotype distribution and population demographic histories for Gossypium hirsutum and Gossypium barbadense as revealed by genome-anchored SNPs.

Use of 10,129 singleton SNPs of known genomic location in tetraploid cotton provided unique opportunities to characterize genome-wide diversity among 440 Gossypium hirsutum and 219 G. barbadense cultivars and landrace accessions of widespread origin. Using the SNPs distributed genome-wide, we examined genetic diversity, haplotype distribution and linkage disequilibrium patterns in the G. hirsutum and G. barbadense genomes to clarify population demographic history. Diversity and identity-by-state analyses have revealed little sharing of alleles between the two cultivated allotetraploid genomes, with a few exceptions that indicated sporadic gene flow. We found a high number of new alleles, representing increased nucleotide diversity, on chromosomes 1 and 2 in cultivated G. hirsutum as compared with low nucleotide diversity on these chromosomes in landrace G. hirsutum. In contrast, G. barbadense chromosomes showed negative Tajima's D on several chromosomes for both cultivated and landrace types, which indicate that speciation of G. barbadense itself, might have occurred with relatively narrow genetic diversity. The presence of conserved linkage disequilibrium (LD) blocks and haplotypes between G. hirsutum and G. barbadense provides strong evidence for comparable patterns of evolution in their domestication processes. Our study illustrates the potential use of population genetic techniques to identify genomic regions for domestication.

Genetic diversity and association mapping of bacterial blight and other horticulturally important traits with microsatellite markers in pomegranate from India.

This genetic diversity study aimed to estimate the population structure and explore the use of association mapping strategies to identify linked markers for bacterial resistance, growth and fruit quality in pomegranate collections from India. In total, 88 accessions including 37 cultivated types were investigated. A total of 112 alleles were amplified by use of 44 publicly available microsatellites for estimating molecular genetic diversity and population structure. Neighbor-joining analysis, model-based population structure and principal component analysis corroborated the genetic relationships among wild-type and cultivated pomegranate collections from India. Our study placed all 88 germplasm into four clusters. We identified a cultivated clade of pomegranates in close proximity to Daru types of wild-type pomegranates that grow naturally near the foothills of the Himalayas. Admixture analysis sorted various lineages of cultivated pomegranates to their respective ancestral forms. We identified four linked markers for fruit weight, titratable acidity and bacterial blight severity. PGCT001 was found associated with both fruit weight and bacterial blight, and the association with fruit weight during both seasons analyzed was significant after Bonferroni correction. This research demonstrates effectiveness of microsatellites to resolve population structure among the wild and cultivar collection of pomegranates and future use for association mapping studies.

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.