Padma Nimmakayala

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

BackgroundA 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.ResultsWe 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.ConclusionsInformation 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.

Characterization of the small RNA component of leaves and fruits from four different cucurbit species

BackgroundMicroRNAs (miRNAs) are a class of non-coding small RNAs involved in post-transcriptional regulation of gene expression critical for plant growth and development, stress responses and other diverse biological processes in plants. The Cucurbitaceae or cucurbit family represents some of economically important species, particularly those with edible and medicinal fruits. Genomic tools for the molecular analysis of members of this family are just emerging. Partial draft genome sequence became available recently for cucumber and watermelon facilitating investigation of the small RNA component of the transcriptomes in cucurbits.ResultsWe generated four small RNA libraries from bottle gourd (Lagenaria siceraria), Cucurbita moschata, Cucurbita pepo, and, watermelon (Citrullus lanatus var. lanatus) in order to identify conserved and novel lineage specific miRNAs in these cucurbits. Deep sequencing of small RNA libraries from these species resulted in 1,597,263, 532,948, 601,388, and 493,384 unique sRNA reads from bottle gourd, moschata, pepo and watermelon, respectively. Sequence analysis of these four libraries resulted in identification of 21 miRNA families that are highly conserved and 8 miRNA families that are moderately conserved in diverse dicots. We also identified 4 putative novel miRNAs in these plant species. Furthermore, the tasiRNAs were identified and their biogenesis was determined in these cucurbits. Small RNA blot analysis or q-PCR analyses of leaf and fruit tissues of these cucurbits showed differential expression of several conserved miRNAs. Interestingly, the abundance of several miRNAs in leaves and fruits of closely related C. moschata and C. pepo was also distinctly different. Target genes for the most conserved miRNAs are also predicted.ConclusionHigh-throughput sequencing of small RNA libraries from four cucurbit species has provided a glimpse of small RNA component in their transcriptomes. The analysis also showed considerable variation within four cucurbit species with regards to expression of individual miRNAs.

High frequency oligonucleotides: targeting active gene (HFO-TAG) markers revealed wide genetic diversity among Citrullus spp. accessions useful for enhancing disease or pest resistance in watermelon cultivars

There is a continuous need to enhance watermelon cultivars for disease and pest resistance. Different U.S. Plant Introductions (PIs) of Citrullus lanatus subsp. lanatus var. lanatus [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) collected in southern Africa are a useful source for enhancing disease or pest resistance in watermelon cultivars. They are also valuable as rootstocks for grafted watermelon, particularly in fields infested with root-knot nematodes or Fusarium wilt. However, there is little information about genetic relationships among these PIs. In this study, genetic diversity was examined among 74 CLC PIs collected from their center of origin in southern Africa. Also, 15 Citrullus lanatus subsp. lanatus (CLL) PIs and the American heirloom cultivars Charleston Gray and Black Diamond (Citrullus lanatus subsp. vulgaris (Schrader ex Eckl. et Zeyh.) Fursa) (CLV) and five Citrullus colocynthis (L.) Schrader (CC) PIs collected in different locations throughout the world were used as out-groups in the phylogenetic analysis for the CLC PIs. Twenty-three high frequency oligonucleotides—targeting active gene (HFO-TAG) primers were used in polymerase chain reaction (PCR) experiments to produce a total of 562 polymorphic markers among the Citrullus PIs and cultivars. Cluster and multidimensional scaling plot analysis produced distinct groups of CLC, CLL, and CC PIs. Several PIs that were designated as CLC or CLL were in transitional positions, indicating that they are the result of gene flow between the major Citrullus groups or subgroups. Population structure analysis indicated that CLC comprises two subgroups; each containing a set of unique alleles. Also, unique alleles exist in the CLL and the CC genotypes. Overall, broad genetic diversity exists among the Citrullus PIs. The data in this study should be useful for identifying PIs with a wide genetic distance between them that could be used in breeding programs aiming to develop heterotic F1 hybrid rootstock lines for grafted watermelon.

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.

Map-based molecular diversity, linkage disequilibrium and association mapping of fruit traits in melon

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.

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.

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

AbstractThis 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.

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 β-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.

Cytomolecular characterization of rDNA distribution in various Citrullus species using fluorescent in situ hybridization

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.

Vitamin C and reducing sugars in the world collection of Capsicum baccatum L. genotypes.

This study aimed to analyze 123 genotypes of Capsicum baccatum L. originating from 22 countries, at two stages of fruit development, for vitamin C content and its relationship with reducing sugars in fruit pericarp. Among the parametric population, vitamin C and reducing sugar concentrations ranged between 2.54 to 50.44 and 41-700mgg(-1) DW of pericarp, respectively. Overall, 14 genotypes accumulated 50-500% of the RDA of vitamin C in each 2g of fruit pericarp on a dry weight basis. Compared with ripened fruits, matured (unripened) fruits contained higher vitamin C and lower reducing sugars. About 44% variation in the vitamin C content could be ascribed to levels of reducing sugars. For the first time, this study provides comprehensive data on vitamin C in the world collection of C. baccatum genotypes that could serve as a key resource for food research in future.