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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.

Nearly 5 000 aphid species damage crops, either by sucking plant sap or as disease-transmitting vectors. Microsatellites are used for understanding molecular diversity and eco-geographical relationships among aphid species. Expressed sequence tag (EST)-microsatellite motifs were identified through an in silico approach using inbuilt simple sequence repeat mining tools in aphid EST dataset. Microsatellite mining revealed one in every five aphid genes as containing a repeat motif, and out of 9 290 EST microsatellites mined from Aphis gossypii Glover and Acyrthosiphon pisum (Harris) (both Hemiptera: Aphididae), 80% were of A and/or T (AT, ATA, AAT, AATA, and ATTT) motifs, and the rest contained G and/or C motifs. All microsatellite sequences were annotated using BLAST. Primers for EST microsatellites were designed using the Primer 3.0 tool. 106 primer pairs of both dinucleotide repeats (DNRs) and trinucleotide repeats (TNRs), representing open reading frames (ORFs) and untranslated regions (UTRs), were synthesized to amplify 15 aphid species belonging to the subfamily Aphidinae, collected from diverse hosts. Four hundred forty-five polymorphic alleles were amplified. Fifty TNR and 23 DNR microsatellites amplified across the species studied. Polymorphism information content values of microsatellites ranged from 0.23 to 0.91, amplifying 2–16 alleles. Genetic similarity indices were estimated using the ‘NTSYS-pc’ software package. Unweighted pair group with arithmetic mean and principal component analysis resolved taxonomic relationships of the aphid species studied. The new aphid microsatellites developed will provide valuable information to researchers to study Indian aphid species diversity and genetic relationships.

Cotton bollworm,Helicoverpa armigera(Hübner) (Lepidoptera: Noctuidae), is a serious pest of several crops throughout the world, representing millions of United States of America dollars worth of damage. This pest can adapt to various cropping systems in a wide geographical range and has high migratory potential. It features high fecundity and can develop resistance to almost all insecticides used for its management. Several investigations to develop microsatellite markers forH. armigerahave not been successful because of the paucity of microsatellites in the lepidopteran genome. As well, collections ofH. armigerafrom cotton fields of southern and western India were not yet studied for molecular genetic diversity. The current study aimed to screen publicly available expressed sequence tag resources for simple sequence repeats and assess their potential as DNA markers for assessment of gene flow between collections of southern and western India. We identified 30 polymorphic microsatellites for potential use in diversity analysis ofH. armigeracollections. Genetic diversity analysis revealed that the collections were widely diverse with population differentiation index (Fst) of 0.17. Furthermore, gene flow analysis revealed a mean frequency of private alleles of 11% within the collections. The microsatellite resources we developed could be widely used for molecular diversity or population genetic research involving this important pest of cotton and food crops.

The genome size for watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cuucmis sativus) and pumpkin (Cucurbita pepo) is 425, 454, 367 and 502 Mbp, respectively, and considered medium size as compared with most other crops. Whole-genome duplication is common in angiosperm plants. Research has revealed a paleohexaploidy (ϒ) event in the common ancestor of eudicots after the divergence of monocotyledons and dicotyledons. While analysis of published whole-genome sequences of cucumber, melon and watermelon showed traces of these ancient duplication events, there was not evidence of more recent whole-genome duplications in these species. Analysis of the syntenic relationships among watermelon, cucumber, melon and grape has identified 3543 orthologous relationships covering the watermelon, melon, and cucumber genomes. Comparison of melon and cucumber genomes synteny to detect shorter regions of rearrangements confirmed previously reported ancestral fusions of five melon chromosome pairs in cucumber, and several inter- and intra-chromosome rearrangements between the two species. Sequenced genomes of cucumber, melon and watermelon revealed a comparable range of genes from 23 k to 27 k protein coding genes with an average of four to six exons per gene. Current progress in gene mapping technologies such as optical mapping, which produces maps of large individual DNA molecules, can improve cucurbit comparative genomics to detect large structural variations and DNA rearrangements across the species.

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 Cys 2 His 2 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.

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.

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.

The 18S–28S and 5S rDNA sites are useful chromosome landmarks and provide valuable evidence about genome organization and evolution. This investigation was the first attempt to study the dynamics, distribution and directionality of rDNA gains and losses, as well as to understand the contribution of site number variation in the speciation of the genus Citrullus. In this study, we employed fluorescent in situ hybridization (FISH), using the18S–28S and 5S rDNA gene loci, to evaluate the differences between the (1) cultivated type watermelon C. lanatus var. lanatus (sweet watermelon), (2) the “bitter” desert watermelon C. colocynthis (colocynth) that is indigenous to the deserts of northern Africa, the Middle East and Asia, (3) the C. lanatus var. citroides (citron) “Tsamma” or “cow watermelon” that is known as and is indigenous to southern Africa, (4) and C. rehmii that thrive in the Namibian Desert. The FISH analyses showed that the sweet watermelon and colocynth have similar rDNA configuration. The sweet watermelon and colocynth genomes contain two 18S–28S rDNA gene loci, each located on a different chromosome, and one 5S rDNA locus which is co-localized with one of the 18S–28S rDNA gene loci. On the other hand, the C. rehmii has one 18S–28S rDNA locus and one 5S rDNA locus positioned on different chromosomes, while the citron has one18S–28S rDNA and two 5S rDNA loci, each located on a different chromosome. A FISH analysis of F1 (citron × sweet watermelon) chromosome spreads revealed uniparental homeologous rDNA gene copies pertaining to the sweet watermelon versus the citron chromosomes, with the sweet watermelon chromosome containing the 18S–28S and 5S rDNA locus versus the citron homologue chromosome that has the 5S rDNA locus, but not the 18S–28S rDNA locus. Genomic in situ hybridization (GISH) analysis, using the entire citron genome as a probe to be differentially hybridized on sweet watermelon chromosome spreads, revealed that the citron genomic probes mainly hybridize to subtelomeric and pericentromeric regions of the sweet watermelon chromosomes, suggesting extensive divergence between the citron and sweet watermelon genomes. The FISH and GISH cytogenetic analysis here indicate major differences in genome organization between the cultivated watermelon type sweet watermelon and its counterpart citron that thrive in southern Africa and considered a useful germplasm source for enhancing disease and pest resistance in watermelon cultivars.

Melon has tremendous fruit diversity, the product of complex interactions of consumer preferences in different countries and a wide range of agro-climatic zones. Understanding footprints of divergence underlying formation of various morphotypes is important for developing sustainable and high-quality melons. Basic understanding of population structure and linkage disequilibrium (LD) is limited in melon and has lagged behind other crops. Characterization of population structure and LD are essential for carrying out association mapping of quantitative trait loci (QTL) underlying various complex traits. Mapped single-locus microsatellite markers are known to be very valuable for resolving the population structure and 268 such markers were used in the current study to resolve population structure and LD pattern using 87 accessions of melons belonging to Eastern European, Euro-North American and Asian types. A mixed linear model was implemented to detect QTL for various fruit traits. Various levels of QTL with high to moderate stringency were detected for fruit shape, fruit weight, soluble solids, and rind pressure and a majority of them was found to be in agreement with the previously published data, indicating that association mapping can be very useful for melon molecular breeding. Minor discrepancies in the position, strength and the variation explained by the QTL present between the methods of association and recombinant mapping approaches can be bridged if more melon groups and larger sets of accessions are involved in future studies, combined with high-throughput marker panels.

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.

Thirty-one accessions ofCitrullusspp. belonging toCitrullus lanatusvar.lanatus,C. lanatusvar.citroidesandCitrullus colocynthiswere subjected to phylogenetic analysis using combined datasets of amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs). Tree topologies inferred by neighbour-joining analysis have resolved the phylogenic relationships among the species with special reference to established taxonomic classification. In this study, we have clearly resolved species boundaries of various taxa ofcitroides,lanatusandcolocynthisinto three well-supported clusters. Clustering pattern of principal component analysis with the shared polymorphisms using the subsets of data between any two taxon combinations helped to elucidate the introgression and interrelationships among the species. We report two major groups ofC. lanatustaxa, one of which has undergone wide introgressions with the taxa ofC. lanatusvar.citroidesandC. colocynthis. In this paper, we identified 583 AFLP bands that are polymorphic within the var.lanatusofC. lanatus, which is the largest set ever reported. The species-specific diagnostic SSRs and polymorphic AFLPs that are informative within and between the taxa reported in this paper would be immensely useful for future studies of these economically important genera.

Thirty-eight melon accessions, which are of primary breeding importance in the Ukraine, were analysed for diversity. These collections represent a major non-US and non-western Europe source of melon germplasm that have not yet been subjected to molecular characterization. Molecular diversity was estimated based on a robust set of 465 polymorphisms gathered by amplified fragment length polymorphisms and simple sequence repeats (SSR). In this paper, we report 12 newly developed polymorphic SSR primer pairs, and their use for molecular characterization in the Ukrainian melon collections. Based on these polymorphisms, we estimated similarity indices that ranged from 0.70 to 1.00 among various accessions. The phylogenetic tree based on the similarity indices and a three-dimensional plot of the first three vectors of the principal component analysis corresponded fairly well with the existing three classical morphotypes namelyaestivalis,europeusandhiemalis, under theconvarEuropeus, which is also known asadana. The polymorphisms generated in the current study, which are specific to the grouping of fruit types and days to maturity will be very useful for further genetic studies and marker-assisted selections.

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. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-767) contains supplementary material, which is available to authorized users.