BACKGROUND
watermelon, ranking among the top five most-frequently purchased fruits, is cultivated globally, with a per capita annual consumption of ~7 kg .
narrow genetic diversity is associated with susceptibility to a large number of diseases and pests among the world’s cultivated watermelons.
modern breeding practices have stressed the introgression of new genetic variation, especially for disease resistance, from underutilized germplasm accessions.
one explanation for the narrow genetic diversity in american and european germplasm could be the founder effect, whereby a small number of accessions are brought to a continent or region as people travel  <cit> .
watermelons may have either entered europe around 512 ad, when the moors invaded the iberian peninsula or during the crusades  <cit> .
in india and china, watermelon was introduced around  <dig> and 1100 ad, respectively  <cit> .
the introduction of watermelon cultivars into the americas occurred after the second voyage of columbus and during the slave trade and colonization .
nimmakayala et al.
<cit>  performed the most recent diversity analysis of watermelon with  <dig> single nucleotide polymorphisms  and  <dig> cultivars belonging to africa, asia, europe, and the americas.
the authors found seven different clusters, with no clear distinction of accessions based on the site of collection or geographic identity.
these findings agree with previous studies  concluding that molecular diversity in cultivated watermelon has a range of 2–4%.
the set of polymorphic snps previously used were too limited to address important population genetic questions necessary for association genetics.
in addition to molecular diversity, the extent of linkage disequilibrium  in sweet watermelon collections must be estimated.
the number of markers needed to perform genome-wide association studies  depends largely on the extent of ld in the breeding population  <cit> .
in other words, if the ld is large in a breeding population, a moderate number of snps would suffice for a gwas.
employing genotyping by sequencing  to develop large numbers of snps and mapping them to a reference genome sequence created a unique opportunity to perform a gwas in crops such as watermelon, for which few genomic resources are available .
dense genome-wide snp datasets generated by gbs can be used to estimate chromosome-wide molecular diversity and population structure very precisely.
detecting and accounting for population stratification is essential in gwass to reduce spurious associations  <cit> .
the reduction in nucleotide diversity underlying genetic bottlenecks during domestication causes selective sweeps in genomic areas containing genes of agricultural importance  <cit> .
selection by early farmers and systematic breeding efforts to improve varieties can significantly affect genetic diversity, as has occurred in several crops and domestic animals .
strong selection can fix advantageous large-effect mutations underlying domestication, ecotype characteristics, adaptation, and fruit quality for example  <cit> .
such selection is reflected in chromosomal regions as sweeps, whereby diversity flanking the selected allele is eroded  <cit> .
characterizing the genome-wide distribution of genetic diversity has identified selective sweeps in the genomes of many crop and animal species  <cit> .
detecting selective sweeps can elucidate the identities of genes and mutations with large phenotypic effects, even if they are no longer segregating within any one population.
such areas cannot be detected by forward genetics  <cit> .
the emergence of high-throughput snp datasets has allowed for gwass of crop plants .
most crops other than maize and rice feature extensive ld because of bottlenecks.
in this case, a medium-resolution gwas can still be applied to capture significant genetic effects present in the cultivated gene pool using a few thousand snps with a minor allele frequency  of  <dig> % or greater  <cit> .
in this study, we analyzed genome-wide diversity using a large snp dataset from a collection of watermelon cultivars drawn from representative accessions grown across the world.
we characterized genome-wide ld and explored its genetic effects on soluble solid content .
we also identified and characterized regions of the watermelon genome that have been subjected to selective sweeps.
we selected  <dig> watermelon accessions, including the previously tested  <dig> accessions, with  <dig> snp markers  <cit> .
this collection represents the entire world cultivars, with wide ecotype variation.
RESULTS
snp identification and characterization
of  <dig>  snps genotyped in this study,  <dig>  were filtered with a maf of ≥  <dig> , a call rate of 90%, and biallelic.
chromosomes 1– <dig> contained  <dig> ,  <dig> ,  <dig> ,  <dig>   <dig> ,  <dig> ,  <dig>   <dig>   <dig> ,  <dig> , and  <dig> snps, respectively.
further ld pruning to remove the duplicate or non-informative markers combined with minimal maf =  <dig>  and hardy-weinberg equilibrium   resulted in  <dig>  associations mapping suitable snps.
we characterized  <dig>  snps located in various exons across the whole genome,  <dig>  were from the non-coding part of the genome.
non-coding snp distribution on chromosomes 1– <dig> represented  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> snps, respectively.
exon-specific snps on chromosomes 1– <dig> represented  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> snps, respectively.
molecular diversity and population structure
we used principal component analysis  of the  <dig>  snps to classify sweet watermelons belonging to various countries.
accessions from zimbabwe, zambia, and kenya were grouped in quadrant i and accessions from zaire, mali, and nigeria in quadrant iv.
the rest of the african accessions and those from various asian, european, and american countries clustered in quadrant iii .
a second pca performed to understand the relationships of accessions in africa alone produced three clusters : the accessions from zaire and nigeria ; zimbabwe, kenya, and zambia ; and a mixture of accessions from various african countries , which was shared with the global accessions.
in addition, with  <dig>  snps located in non-coding regions, pca grouped african sweet watermelons into two clusters as in pca i .
to understand the effect of coding snps on clustering, pca iv grouped all subclusters of the african types, so that the exon-specific snps were less discriminative than the non-coding snps .figure  <dig>
principal component analysis  showing the first two components of global watermelon accession collections with  <dig>  single nucleotide polymorphisms  generated by genotyping by sequencing .
pca showing the first two components of africa watermelon accession collections using  <dig>  snps generated by gbs.
pca of global watermelon accession collections with  <dig>  noncoding snps.
pca of global watermelon accession collections with  <dig>  snps located in the exons.
we used a model-based approach to population structure analysis to analyze the entire panel of  <dig> sweet watermelon accessions .
use of structure harvester provided mean lnp and delta k values .
k- <dig> was the most appropriate cluster for this population, with the highest delta k value of  <dig> as compared with the other clusters.
we used population structure analysis rather than clustering to examine ancestry.
ancestry distribution of k- <dig>  showed all ancestries are present in africa , whereas ancestry analysis suggested that large numbers of the watermelon cultivars currently available in africa are not yet used in breeding programs in the rest of the world.
ancestry colored yellow was more predominant in asia and europe.
north and south american accessions were predominantly red.
population structure analysis provided strong evidence for multiple parallel domestications across africa.figure  <dig>
ancestry analysis of global watermelon accession collections by population structure, a model-based approach.
k <dig> had the highest peak  indicating that three clusters sufficiently define watermelon population structure.
characterization of genome-wide ld
we conducted an extensive ld analysis on the entire dataset of  <dig> sweet watermelons, on all adjacent marker pairs within a chromosome or within a haplotype block.
the results provided values for both the expectation-maximization  algorithm and composite haplotype method .
r <dig>  and d'  values for the em and chm methods are given in additional file 2: table s <dig>  additional file 3: table s <dig>  and additional file 4: table s <dig>  we created ld plots using marker-pair associations of adjacent snps within a chromosome, adjacent snps within a haplotype block, and adjacent snps within genes .figure  <dig>
genome-wide linkage disequilibrium  across various watermelon chromosomes when compared using a) individual snps, b) snp haplotypes, and c) within individual genes.
ld decay varied along chromosomes, with regions of high ld interspersed with regions of low.
pairwise ld was estimated by r <dig> and we compared the pattern of decay at different levels.
first, when performing pair-wise analysis considering snps across chromosomes, we noted ld decay on average, with an average block size of  <dig> kb .
second, analysis based on adjacent snps within haplotypes revealed ld decay within  <dig> kb .
third, analysis of the snps located in exons revealed ld decay within  <dig> kb .
on chromosome  <dig>   <dig> genes under ld were identified .
chromosome  <dig> appeared to harbor a large extent of ld, followed by chromosomes  <dig> and  <dig>  when analyzed separately and including various accessions from africa and the rest of the world, lower ld was noted for all chromosomes in african accessions as compared with those from the rest of the world.
narrow genetic diversity increased ld significantly .
on chromosome  <dig>  the ld covered a block of  <dig>  mb in cultivars from the rest of the world, which indicates strong selection in the region, but covered  <dig>  mb in african accessions.
the expansion of this ld block in cultivars from the rest of the world appears to be a hitchhiking effect rather than a selective sweep because of the narrow genetic diversity.
we identified  <dig> haplotypes with  <dig> snps .
a list of the genes across various chromosomes and the extent of ld within genes are given in additional file 2: table s <dig> figure  <dig>
ld distribution within  <dig> genes on chromosome  <dig>
comparison of ld distribution across various chromosomes between watermelon accessions from africa and the rest of the world.
selective sweeps and domestication signature characterization
highly significant pairwise fst  distribution in sweet watermelon accessions from different geographical areas is illustrated in figure 9a and b. the african and american groups showed high genetic diversity relative to asian and european groups.
furthermore, the patterns of fst variation indicated genomic areas with selective sweep signatures and patterns of world watermelon breeding practices.
selection signatures detected loci with large effects under strong selection on chromosomes  <dig> and  <dig> figure  <dig>
genome-wide window based scans of pairwise
f
st
for accessions from asia, europe, and the americas compared with those from africa.
selection signatures can be seen on parts of chromosome  <dig> and  <dig>  where f
st distribution revealed distinct sweeps.
by scanning the chromosome  <dig> genome at the selective sweep location, especially in the  <dig>  mb ld block, we identified potential gene candidates selected during sweet watermelon domestication.
we identified  <dig> candidate genes within  <dig>  mb of the genome; therefore, this region is the most significant for domestication  with important roles in ripening, sugar-mediated signaling and carbohydrate transport, fruit development, nitrate transmembrane transporter, cytochrome p <dig>  pectinesterase/pectinesterase inhibitor, zinc finger  family protein, glyceraldehyde-3-phosphate dehydrogenase, pectate lyase family protein, and catalytic/cation binding/hydrolase.
implementation of a medium-resolution gwas for the fresh juice ssc trait
a set of  <dig> genotypes were grown in controlled conditions and the means for the ssc trait clearly followed a normal distribution.
therefore, the trait is under the control of multiple genes .
we used a gwas with  <dig>  snps to identify alleles that affect total ssc.
results pertaining to the gwas are presented in a manhattan plot .
in manhattan plots, genomic coordinates are displayed along the x-axis with the negative log  <dig> of the association p-value for each single nucleotide polymorphism on the y-axis.
because the strongest associations have the smallest p-values, their negative logarithms will be the greatest.
in this study, four snps were associated with total ssc after bonferroni correction according to the emmax model, which corrects for population structure as well as identity by descent .
the marker s1_ <dig>  is located on chromosome  <dig>  this snp is a synonymous mutation for leucine and is located in the exon of the gene cla <dig>  a ubiquitin-protein ligase with r =  <dig> .
allele a was the minor allele with a frequency of  <dig>  and 100% call rate.
s6_ <dig> is a non-synonymous mutation causing a gln → lys change on cla <dig>  an unknown gene.
this marker was associated with a bonferroni p =  <dig>  and a minor allele frequency  of  <dig> , with a call rate of 97%.
the strength of association was negative .
two other snps  were positively associated with ssc, with r <dig> =  <dig>  and  <dig> , and could withstand bonferroni correction .
the mafs for these two snps  were  <dig>  and  <dig> , with call rates of 99% and 94%, respectively.
s11_ <dig> is located in the intergenic region of cla <dig> and cla <dig>  which code for profilin and ppr repeat protein, respectively.
s10_ <dig> is located in the cla <dig> promoter region, its function is unknown.figure  <dig>
manhattan plot of the genome-wide association study for the soluble solids trait.
chromosome coordinates are displayed along the x-axis with the negative log  <dig> of the association p-value for each single nucleotide polymorphism displayed on the y-axis.
a higher negative log  <dig> indicates stronger association with the trait.
discussion
genotyping by sequencing
many of the challenges posed by complex crop genomes can be overcome by gbs  <cit> .
this protocol is a multiplexed, high-throughput, low-cost method to explore genetic diversity in populations  <cit> .
in this paper, we report a robust set of  <dig>  snps mapped to various chromosomes with a maf of ≥  <dig> .
sandlin et al.
<cit> , ren et al.
<cit> , and nimmakayala et al.
<cit>  developed  <dig> ,  <dig>  and  <dig> snps, respectively, for genetic mapping and diversity studies in watermelon.
guo et al.
<cit>  re-sequenced  <dig> watermelon accessions including sweet, semi-wild, and wild watermelons to identify  <dig> , <dig> candidate snps and  <dig>  small insertions/deletions .
we used the largest set of a cultivated collection of  <dig> accessions representing all of the important ecotypes from around the globe to resolve the diversity of cultivated watermelon, which will allow for incorporating diverse alleles into cultivated watermelons worldwide.
domestication and divergence of sweet watermelon
we identified three sweet watermelons clusters within africa and related one of those to the sweet watermelon accessions from the rest of the world.
the other two african sweet watermelon clusters were not used in watermelon breeding programs in other parts of the world.
therefore, the underutilized accessions from these clusters could be an important resource for widening watermelon cultivar genetic diversity.
founder effects based on relatively few cultivars appear to account for the prevailing narrow genetic diversity in global cultivar germplasm.
this finding agrees with previous results  <cit> , in particular, the lower diversity among american, chinese, and east asian types.
location of selective sweeps across the genome
domestication effects lead to complete fixation of the genomic regions that harbor alleles of importance by purifying selection.
the loss of nucleotide diversity in the flanking regions  <cit>  is known as the hitchhiking effect and a region of the genome in which selection has driven a haplotype to complete fixation is defined as having undergone a selective sweep  <cit> .
such regions may also occur within the genome because of random drift and are not distinguishable from regions that have undergone a selective sweep.
we minimized the errors in locating the regions that have undergone selective sweeps caused by random drift or narrow genetic diversity by including a large diverse panel of accessions derived from different ancestral populations, e.g., asia, europe, and america as well as a wide range of ancestral populations from africa.
the selective sweep approach is a type of reverse genetic tool that begins with a selection signature and attempts to infer the selected mutation and its associated phenotype  <cit> .
in contrast, a gwas is a forward genetics approach that progresses from a phenotype to the identification of underlying causal genes and mutations.
the snps discovered by gbs allowed to compare the pairwise fst for accessions from asia, europe, and the americas to those from africa.
by scanning a moving window of these pair-wise fst values across all chromosomes, we could identify selective sweep regions to assess the effects of selection during the breeding history of cultivated watermelon.
the selection of individuals with favorable mutations during domestication and by breeding practices has led to reduced genetic diversity in crop species  <cit> .
pairwise fst distributions on numerous chromosomes reflected the breeding history patterns across several continents.
ld is a key factor in determining the number of markers needed for a gwas and genomic selection .
using genome-wide snps, we evaluated the genome-wide ld pattern for a diverse collection of sweet watermelon.
genomes with high ld will require low marker density for gwass or gs, therefore our snps may be of immense use for gwass of watermelon breeding.
in the current research, we characterized extensive ld in the watermelon collections of asia, europe, and the americas as compared with those of africa, possibly because of less divergent cultivar pools.
gwass can be implemented with moderate marker density in barley and alfalfa because these populations contain extensive admixture ld, as does watermelon  <cit> .
by selecting subsets of genotypes from globally cultivated watermelons, breeders can modulate ld to acceptable levels and build suitable association mapping panels for genome-wide marker-based breeding projects.
a complete understanding of ld within genes is important for realizing the impact of marker-assisted selection.
if a particular gene is under high ld, marker-assisted selection can be efficiently performed with a single marker irrespective of its location in or near the gene.
in contrast, if a gene is under low ld, several markers at various positions of the gene must be used to transfer a complete allele because recombination can occur within the gene.
in our study, we characterized genic ld across all of the chromosomes.
usefulness of the current diversity panel for future association mapping studies
gwass based on ld provide a promising tool for detecting and fine-mapping quantitative trait loci underlying complex traits.
in this study, we explored the genetic basis of variation for the total ssc trait in a diverse watermelon collection of global origin.
despite the advantages of gwas in pinpointing genetic polymorphisms underlying traits, this approach may incur inflated false-positive findings because of population structure  <cit> .
imprecise gwas modeling would result in spurious marker associations if the model cannot precisely correct for population stratification  <cit> .
variance component approaches, such as efficient mixed-model association , can correct for a wide range of sample structures by explicitly accounting for pairwise relatedness between individuals, using high-density snp markers to model the phenotype distribution  <cit> .
in this study, we used a more efficient emma, expedited , which reduces the computational time for analyzing large gwas data sets and includes pca eigen vectors and identity by descent  matrices in correcting for sample structure  <cit> .
CONCLUSIONS
analysis of genetic diversity in world collections of cultivar accessions helps understand ld decay at various levels.
because ld in the watermelon is quite high, the marker set we developed would be sufficient for a low-power gwas of sweet watermelon cultivars; a high-power study might require up to  <dig>  snps with a maf =  <dig> .
from this pilot study, gbs can effectively detect genome-wide snps and provide a powerful tool for the systematic exploration of global watermelon collections.
re-sequencing strategies to develop millions of snps for crops such as maize and rice  have shown what is possible for the watermelon research community  <cit> .
the identification of watermelon snps, as in this and previous studies, will allow for genome-wide association mapping and marker-assisted selection to support breeding programs.

 watermelon, ranking among the top five most-frequently purchased fruits, is cultivated globally, with a per capita annual consumption of ~7 kg. narrow genetic diversity is associated with susceptibility to a large number of diseases and pests among the world’s cultivated watermelons. modern breeding practices have stressed the introgression of new genetic variation, especially for disease resistance, from underutilized germplasm accessions. one explanation for the narrow genetic diversity in american and european germplasm could be the founder effect, whereby a small number of accessions are brought to a continent or region as people travel. watermelons may have either entered europe around 512 ad, when the moors invaded the iberian peninsula or during the crusades. in india and china, watermelon was introduced around and 1100 ad, respectively. the introduction of watermelon cultivars into the americas occurred after the second voyage of columbus and during the slave trade and colonization. nimmakayala et al. performed the most recent diversity analysis of watermelon with single nucleotide polymorphisms and cultivars belonging to africa, asia, europe, and the americas. the authors found seven different clusters, with no clear distinction of accessions based on the site of collection or geographic identity. these findings agree with previous studies concluding that molecular diversity in cultivated watermelon has a range of 2–4%. the set of polymorphic snps previously used were too limited to address important population genetic questions necessary for association genetics. in addition to molecular diversity, the extent of linkage disequilibrium in sweet watermelon collections must be estimated. the number of markers needed to perform genome-wide association studies depends largely on the extent of ld in the breeding population. in other words, if the ld is large in a breeding population, a moderate number of snps would suffice for a gwas. employing genotyping by sequencing to develop large numbers of snps and mapping them to a reference genome sequence created a unique opportunity to perform a gwas in crops such as watermelon, for which few genomic resources are available. dense genome-wide snp datasets generated by gbs can be used to estimate chromosome-wide molecular diversity and population structure very precisely. detecting and accounting for population stratification is essential in gwass to reduce spurious associations. the reduction in nucleotide diversity underlying genetic bottlenecks during domestication causes selective sweeps in genomic areas containing genes of agricultural importance. selection by early farmers and systematic breeding efforts to improve varieties can significantly affect genetic diversity, as has occurred in several crops and domestic animals. strong selection can fix advantageous large-effect mutations underlying domestication, ecotype characteristics, adaptation, and fruit quality for example. such selection is reflected in chromosomal regions as sweeps, whereby diversity flanking the selected allele is eroded. characterizing the genome-wide distribution of genetic diversity has identified selective sweeps in the genomes of many crop and animal species. detecting selective sweeps can elucidate the identities of genes and mutations with large phenotypic effects, even if they are no longer segregating within any one population. such areas cannot be detected by forward genetics. the emergence of high-throughput snp datasets has allowed for gwass of crop plants. most crops other than maize and rice feature extensive ld because of bottlenecks. in this case, a medium-resolution gwas can still be applied to capture significant genetic effects present in the cultivated gene pool using a few thousand snps with a minor allele frequency of % or greater. in this study, we analyzed genome-wide diversity using a large snp dataset from a collection of watermelon cultivars drawn from representative accessions grown across the world. we characterized genome-wide ld and explored its genetic effects on soluble solid content. we also identified and characterized regions of the watermelon genome that have been subjected to selective sweeps. we selected watermelon accessions, including the previously tested accessions, with snp markers. this collection represents the entire world cultivars, with wide ecotype variation.  snp identification and characterization of snps genotyped in this study, were filtered with a maf of ≥ , a call rate of 90%, and biallelic. chromosomes 1– contained,,,,,,, and snps, respectively. further ld pruning to remove the duplicate or non-informative markers combined with minimal maf =  and hardy-weinberg equilibrium resulted in associations mapping suitable snps. we characterized snps located in various exons across the whole genome, were from the non-coding part of the genome. non-coding snp distribution on chromosomes 1– represented and snps, respectively. exon-specific snps on chromosomes 1– represented and snps, respectively. molecular diversity and population structure we used principal component analysis of the snps to classify sweet watermelons belonging to various countries. accessions from zimbabwe, zambia, and kenya were grouped in quadrant i and accessions from zaire, mali, and nigeria in quadrant iv. the rest of the african accessions and those from various asian, european, and american countries clustered in quadrant iii. a second pca performed to understand the relationships of accessions in africa alone produced three clusters: the accessions from zaire and nigeria; zimbabwe, kenya, and zambia; and a mixture of accessions from various african countries, which was shared with the global accessions. in addition, with snps located in non-coding regions, pca grouped african sweet watermelons into two clusters as in pca i. to understand the effect of coding snps on clustering, pca iv grouped all subclusters of the african types, so that the exon-specific snps were less discriminative than the non-coding snps.figure principal component analysis showing the first two components of global watermelon accession collections with single nucleotide polymorphisms generated by genotyping by sequencing. pca showing the first two components of africa watermelon accession collections using snps generated by gbs. pca of global watermelon accession collections with noncoding snps. pca of global watermelon accession collections with snps located in the exons. we used a model-based approach to population structure analysis to analyze the entire panel of sweet watermelon accessions. use of structure harvester provided mean lnp and delta k values. k- was the most appropriate cluster for this population, with the highest delta k value of as compared with the other clusters. we used population structure analysis rather than clustering to examine ancestry. ancestry distribution of k- showed all ancestries are present in africa, whereas ancestry analysis suggested that large numbers of the watermelon cultivars currently available in africa are not yet used in breeding programs in the rest of the world. ancestry colored yellow was more predominant in asia and europe. north and south american accessions were predominantly red. population structure analysis provided strong evidence for multiple parallel domestications across africa.figure ancestry analysis of global watermelon accession collections by population structure, a model-based approach. k had the highest peak indicating that three clusters sufficiently define watermelon population structure. characterization of genome-wide ld we conducted an extensive ld analysis on the entire dataset of sweet watermelons, on all adjacent marker pairs within a chromosome or within a haplotype block. the results provided values for both the expectation-maximization algorithm and composite haplotype method. r and d' values for the em and chm methods are given in additional file 2: table s additional file 3: table s and additional file 4: table s we created ld plots using marker-pair associations of adjacent snps within a chromosome, adjacent snps within a haplotype block, and adjacent snps within genes.figure genome-wide linkage disequilibrium across various watermelon chromosomes when compared using a) individual snps, b) snp haplotypes, and c) within individual genes. ld decay varied along chromosomes, with regions of high ld interspersed with regions of low. pairwise ld was estimated by r and we compared the pattern of decay at different levels. first, when performing pair-wise analysis considering snps across chromosomes, we noted ld decay on average, with an average block size of kb. second, analysis based on adjacent snps within haplotypes revealed ld decay within kb. third, analysis of the snps located in exons revealed ld decay within kb. on chromosome genes under ld were identified. chromosome appeared to harbor a large extent of ld, followed by chromosomes and when analyzed separately and including various accessions from africa and the rest of the world, lower ld was noted for all chromosomes in african accessions as compared with those from the rest of the world. narrow genetic diversity increased ld significantly. on chromosome the ld covered a block of  mb in cultivars from the rest of the world, which indicates strong selection in the region, but covered  mb in african accessions. the expansion of this ld block in cultivars from the rest of the world appears to be a hitchhiking effect rather than a selective sweep because of the narrow genetic diversity. we identified haplotypes with snps. a list of the genes across various chromosomes and the extent of ld within genes are given in additional file 2: table s figure ld distribution within genes on chromosome comparison of ld distribution across various chromosomes between watermelon accessions from africa and the rest of the world. selective sweeps and domestication signature characterization highly significant pairwise fst distribution in sweet watermelon accessions from different geographical areas is illustrated in figure 9a and b. the african and american groups showed high genetic diversity relative to asian and european groups. furthermore, the patterns of fst variation indicated genomic areas with selective sweep signatures and patterns of world watermelon breeding practices. selection signatures detected loci with large effects under strong selection on chromosomes and figure genome-wide window based scans of pairwise f st for accessions from asia, europe, and the americas compared with those from africa. selection signatures can be seen on parts of chromosome and where f st distribution revealed distinct sweeps. by scanning the chromosome genome at the selective sweep location, especially in the  mb ld block, we identified potential gene candidates selected during sweet watermelon domestication. we identified candidate genes within  mb of the genome; therefore, this region is the most significant for domestication with important roles in ripening, sugar-mediated signaling and carbohydrate transport, fruit development, nitrate transmembrane transporter, cytochrome p pectinesterase/pectinesterase inhibitor, zinc finger family protein, glyceraldehyde-3-phosphate dehydrogenase, pectate lyase family protein, and catalytic/cation binding/hydrolase. implementation of a medium-resolution gwas for the fresh juice ssc trait a set of genotypes were grown in controlled conditions and the means for the ssc trait clearly followed a normal distribution. therefore, the trait is under the control of multiple genes. we used a gwas with snps to identify alleles that affect total ssc. results pertaining to the gwas are presented in a manhattan plot. in manhattan plots, genomic coordinates are displayed along the x-axis with the negative log of the association p-value for each single nucleotide polymorphism on the y-axis. because the strongest associations have the smallest p-values, their negative logarithms will be the greatest. in this study, four snps were associated with total ssc after bonferroni correction according to the emmax model, which corrects for population structure as well as identity by descent. the marker s1_ is located on chromosome this snp is a synonymous mutation for leucine and is located in the exon of the gene cla a ubiquitin-protein ligase with r = . allele a was the minor allele with a frequency of and 100% call rate. s6_ is a non-synonymous mutation causing a gln → lys change on cla an unknown gene. this marker was associated with a bonferroni p = and a minor allele frequency of, with a call rate of 97%. the strength of association was negative. two other snps were positively associated with ssc, with r = and, and could withstand bonferroni correction. the mafs for these two snps were and, with call rates of 99% and 94%, respectively. s11_ is located in the intergenic region of cla and cla which code for profilin and ppr repeat protein, respectively. s10_ is located in the cla promoter region, its function is unknown.figure manhattan plot of the genome-wide association study for the soluble solids trait. chromosome coordinates are displayed along the x-axis with the negative log of the association p-value for each single nucleotide polymorphism displayed on the y-axis. a higher negative log indicates stronger association with the trait. discussion genotyping by sequencing many of the challenges posed by complex crop genomes can be overcome by gbs. this protocol is a multiplexed, high-throughput, low-cost method to explore genetic diversity in populations. in this paper, we report a robust set of snps mapped to various chromosomes with a maf of ≥. sandlin et al., ren et al., and nimmakayala et al. developed, and snps, respectively, for genetic mapping and diversity studies in watermelon. guo et al. re-sequenced watermelon accessions including sweet, semi-wild, and wild watermelons to identify, candidate snps and small insertions/deletions. we used the largest set of a cultivated collection of accessions representing all of the important ecotypes from around the globe to resolve the diversity of cultivated watermelon, which will allow for incorporating diverse alleles into cultivated watermelons worldwide. domestication and divergence of sweet watermelon we identified three sweet watermelons clusters within africa and related one of those to the sweet watermelon accessions from the rest of the world. the other two african sweet watermelon clusters were not used in watermelon breeding programs in other parts of the world. therefore, the underutilized accessions from these clusters could be an important resource for widening watermelon cultivar genetic diversity. founder effects based on relatively few cultivars appear to account for the prevailing narrow genetic diversity in global cultivar germplasm. this finding agrees with previous results, in particular, the lower diversity among american, chinese, and east asian types. location of selective sweeps across the genome domestication effects lead to complete fixation of the genomic regions that harbor alleles of importance by purifying selection. the loss of nucleotide diversity in the flanking regions is known as the hitchhiking effect and a region of the genome in which selection has driven a haplotype to complete fixation is defined as having undergone a selective sweep. such regions may also occur within the genome because of random drift and are not distinguishable from regions that have undergone a selective sweep. we minimized the errors in locating the regions that have undergone selective sweeps caused by random drift or narrow genetic diversity by including a large diverse panel of accessions derived from different ancestral populations, e.g., asia, europe, and america as well as a wide range of ancestral populations from africa. the selective sweep approach is a type of reverse genetic tool that begins with a selection signature and attempts to infer the selected mutation and its associated phenotype. in contrast, a gwas is a forward genetics approach that progresses from a phenotype to the identification of underlying causal genes and mutations. the snps discovered by gbs allowed to compare the pairwise fst for accessions from asia, europe, and the americas to those from africa. by scanning a moving window of these pair-wise fst values across all chromosomes, we could identify selective sweep regions to assess the effects of selection during the breeding history of cultivated watermelon. the selection of individuals with favorable mutations during domestication and by breeding practices has led to reduced genetic diversity in crop species. pairwise fst distributions on numerous chromosomes reflected the breeding history patterns across several continents. ld is a key factor in determining the number of markers needed for a gwas and genomic selection. using genome-wide snps, we evaluated the genome-wide ld pattern for a diverse collection of sweet watermelon. genomes with high ld will require low marker density for gwass or gs, therefore our snps may be of immense use for gwass of watermelon breeding. in the current research, we characterized extensive ld in the watermelon collections of asia, europe, and the americas as compared with those of africa, possibly because of less divergent cultivar pools. gwass can be implemented with moderate marker density in barley and alfalfa because these populations contain extensive admixture ld, as does watermelon. by selecting subsets of genotypes from globally cultivated watermelons, breeders can modulate ld to acceptable levels and build suitable association mapping panels for genome-wide marker-based breeding projects. a complete understanding of ld within genes is important for realizing the impact of marker-assisted selection. if a particular gene is under high ld, marker-assisted selection can be efficiently performed with a single marker irrespective of its location in or near the gene. in contrast, if a gene is under low ld, several markers at various positions of the gene must be used to transfer a complete allele because recombination can occur within the gene. in our study, we characterized genic ld across all of the chromosomes. usefulness of the current diversity panel for future association mapping studies gwass based on ld provide a promising tool for detecting and fine-mapping quantitative trait loci underlying complex traits. in this study, we explored the genetic basis of variation for the total ssc trait in a diverse watermelon collection of global origin. despite the advantages of gwas in pinpointing genetic polymorphisms underlying traits, this approach may incur inflated false-positive findings because of population structure. imprecise gwas modeling would result in spurious marker associations if the model cannot precisely correct for population stratification. variance component approaches, such as efficient mixed-model association, can correct for a wide range of sample structures by explicitly accounting for pairwise relatedness between individuals, using high-density snp markers to model the phenotype distribution. in this study, we used a more efficient emma, expedited, which reduces the computational time for analyzing large gwas data sets and includes pca eigen vectors and identity by descent matrices in correcting for sample structure.  analysis of genetic diversity in world collections of cultivar accessions helps understand ld decay at various levels. because ld in the watermelon is quite high, the marker set we developed would be sufficient for a low-power gwas of sweet watermelon cultivars; a high-power study might require up to snps with a maf =. from this pilot study, gbs can effectively detect genome-wide snps and provide a powerful tool for the systematic exploration of global watermelon collections. re-sequencing strategies to develop millions of snps for crops such as maize and rice have shown what is possible for the watermelon research community. the identification of watermelon snps, as in this and previous studies, will allow for genome-wide association mapping and marker-assisted selection to support breeding programs. 
a large single nucleotide polymorphism dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. 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. with a diverse panel of worldwide cultivated watermelons, we identified a set of snps with a minor allele frequency of ≥ , distributed across the genome. we further mapped important genes for soluble solid content using a mixed linear model. 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. principal component and ancestry analyses were used to account for population structure in a genome-wide association study. 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. linkage disequilibriumgwasselective sweeppopulation structuregenotyping by sequencingwatermeloncitrullus lanatus var. pairwise analysis of adjacent snps within a chromosome as well as within a haplotype allowed us to estimate genome-wide ld decay. a sliding window analysis using pairwise fst values was used to resolve selective sweeps. we identified strong selection on chromosomes and that might have contributed to the domestication process. ld was also detected within individual genes on various chromosomes. the marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium within a population.
 a large single nucleotide polymorphism 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 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.  we examined citrullus lanatus var. lanatus accessions representing domesticated watermelon and generated a set of snp markers using genotyping by sequencing. with a diverse panel of worldwide cultivated watermelons, we identified a set of snps with a minor allele frequency of ≥ , 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 and 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.  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 contains supplementary material, which is available to authorized users. keywords linkage disequilibriumgwasselective sweeppopulation structuregenotyping by sequencingwatermeloncitrullus lanatus var. lanatusissue-copyright-statement© the author 2014 