s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Prevalence and Bacteria Associated with White Band Disease on Acropora sp. from Gili Labak Island Sumenep District Indonesia

Coral reefs have many problems including coral diseases. One of the coral diseases that infect Acropora sp. from Gili Labak Island is the White Band Disease (WBD). Their prevalence and bacteria associated with the WBD on Acropora sp. is warranted further investigation. This study aims to determine the disease prevalence and bacteria associated with WBD on Acropora sp. from the Gili Labak Island. The samples were collected from coral infected. The laboratory analysis includes bacterial isolation, DNA extraction, DNA amplification, electrophoresis, sequencing, and phylogenetic analysis. The results showed that the disease prevalences of WBD on Acropora sp. were between 0 to 23%, in which the highest prevalence was found in the site where marine tourism activities and boat mooring occurred. Four bacterial isolates were identified based on morphological features. These four isolates were then analyzed using the Mega 5.2 program and resulted in a complete nucleotide sequence and had similarities to the bacteria present in the Gen Bank through BLAST analysis. The result of BLAST analysis showed that ACWB2A isolate had 99% similarity with Vibrio alginolyticus bacteria; ACWB 6 had 99% similarity with Vibrio owensii; and isolates 5 and 8 had 99% similarity with Pseudoalteromonas rubra bacteria. These are the first record that Pseudoalteromonas rubra bacterium is associated with White Band Disease.

Introduction
Coral reefs are benthic animals that live in the bottom of the waters. The animal is largely colonized in its life composed of calcium carbonate (CaCO3) as a result of secretion from Zooxanthellae. Coral reefs are an area where various important economic fishes (Lutjanidae, Haemulidae, Lethrinidae, Scaridae, and Siganidae) [1][2][3], invertebrates (mollusks, crustaceans, and sea cucumbers), and seaweeds spawn and live [4]. Among the world's total fish catches in the 1970s, more than 10% was contributed by reef-related fisheries [5]. Coral reefs also play an important role to protect shoreline and preserve species diversity [6]. Furthermore, coral reefs also give various contributions to the social and cultural aspect of communities such as tourism activities [4] and cultural and religious values [6].
Indonesian coral reefs are in serious threat as more than 50% of reefs are in poor condition. Damage to coral reefs can be caused by human activities such as the use of bombs and cyanide poison, anchor drainage, walking on the reef, using muroami fishing gear, mining of rocks, and sand mining [7]. In addition, the cause of coral damage can be caused by coral diseases. One of the diseases that occurred on coral reefs in Gili Labak is White Band Disease. Gili Labak Island is a small island in Sumenep District with vibrant marine tourist activities.
White Band Disease (WBD) is a disease that infects Acropora sp. coral species from Gili Labak Island Indonesia. It is described by Raymundo et al. [8] that this WBD disease can only be observed on Acropora corals. Meanwhile, WBD is also found on Acropora cervicornis dan Acropora palmate in the Caribbean [9], Venezuela and Columbia [10]. The bacteria associated with WBD were varied in different places and coral. WBD prevalence and bacteria associated with WBD that infects Acropora sp. coral in Gili Labak Island waters are still unknown, so what bacteria associated with WBD is warranted further investigation. This research will add the information of the WBD disease associated with the bacteria that could be used for further management of coral disease in the area.

Methods
The survey and sample collection were conducted on 27-

Data Collection Procedure
In this research, field data were collected by identifying corals infected with WBD. Identification of corals infected with WBD was performed using the Coral Finder based on Kelley [11] and Veron [12], while the identification of WBD was based on Raymundo et al. [8].
Survey for disease prevalence was conducted using underwater visual survey using 2 × 20 m transect. Three transects were used in each site. Four sites were observed to represent all places of the island, namely north, east, south, and west side. The data were then subjected to Equation 1.

Number of diseased colonies
Coral disease prevalence = 100% Total number of colonies  A sample of the diseased colony was also taken and stored into ziplock and then transported to the laboratory using a cool box with dry ice.

Isolation and Purification
The sample was then isolated to grow bacterial WBD disease. The isolation stage began with a dilution of the sample up to 10 -10 dilutions, and the dilutions of 10 -5 , 10 -7 , and 10 -10 dilutions were taken as 100 μL by micropipette and isolated on Zobell 2216E media then incubated upside down for 2×24 hours. The isolation and purification of bacterial isolates were performed based on morphological appearance by streak method until pure culture obtained [13].

DNA Amplification Extraction
After obtaining the pure culture then the DNA was extracted to attain the DNA template of the bacteria. The extraction was done by taking bacterial cells from the agar plate and transferred into a tube and added with chelex.
DNA amplification was done by inserting a 27F and 1492R primer into a sterile tube of 2 μL each and added with 6 mL of ddH2O. Then it was added with 2.5μL of extraction sample and finally combined with 12.5 μL of Kapa/Promega that made the total volume in tube was 25 μL. PCR analysis was carried out for 30 cycles, which is the pre-denaturation stage for 3 min with temperature at 95 °C, 1 minute denaturation stage at 95 °C, 1 minute annealing stage at 55 °C, 1 minute pre-extension at 72 °C and 7 minutes extension stage at 72 °C [14].

Sequencing and Phylogenetic Analysis
The 16 srDNA sequence results from the sample were analyzed using the Mega 5 program. This program was also used to construct phylogenetic trees. The DNA sequences were compared with Gen Bank data from NCBI website (http://www.ncbi.nlm.gov) using BLAST in Mega 5.

Results and Discussion
The means of White Band Disease prevalence on Acropora sp. in the Gili Labak Island were between 0 and 23.4%, with the highest prevalence found in the north side ( Figure 2). The north side is a location used for boat mooring and place where tourist does the activities such as snorkeling and kayaking. Meanwhile, the East and South side of the island does not have a high intensity of human activities. Our finding confirms that tourist activities could increase the prevalence of coral disease. Lamb et al. [15] found that coral disease prevalence increased 3-fold at high tourist used area.  Four isolates were found based on the morphological observation of bacterial colonies. These bacterial isolates are identified by morphological appearance such as color, shape, and texture ( Figure 3 and Table 1). The amplification process was carried out as much as 30 cycles to increase the amount of bacterial DNA so it could be analyzed for sequencing. Four isolates amplification showed that they had about 1500 bp which was suitable for sequencing ( Figure 4).

Phylogenetic Analysis
The BLAST analysis showed that ACWB2A isolate had 99% homology with Vibrio alginolyticus (ATCC 17749 strain), ACWB 6 isolate had 99% homology with Vibrio owensii bacteria (DY05 strain), while ACWB 5 and ACWB 8 isolates had 99% homology with Pseudoalteromonas rubra bacteria (ATCC 29570 strain). Hagstrom [16] explains that if the likeness is ≥ 97% then it can be considered as the same species. Phylogenetic analysis is a continuation of the BLAST analysis. This analysis makes it possible to create a phylogenetic tree (a phylogenetic tree) that can be used to see the proximity between the sample DNA and the DNA in the Bank Genes. The phylogenetic analysis confirms that ACWB2A isolate similar to Vibrio alginolyticus (ATCC 17749 strain), ACWB 6 isolate similar Vibrio owensii bacteria (DY05 strain) ( Figure 5).  Vibrio bacteria are aerobic, but there is also anaerobic facultative. Vibrio bacteria including motile bacteria because of its movement using flagella, in addition, are classified as gram-negative bacteria with the shape resembling a curved stem like a coma. Gignoux-Wolfsohn and Vollmer [17] reported that bacteria causing White Band Disease (WBD) might be species from Vibrio and Rickettsia. Previous studies showed that Vibrio bacteria found on coral Acropora affected by White Band Disease had bacteria Vibrio carchariae [18].
Vibrio alginolyticus bacteria are also found in another coral disease such as Yellow Band Disease (YBD) occurred in Montastrea sp. [19]. Cunning et al. [20] also found Vibrio fortis and Vibrio harveyi dominate in the healthy and diseased YBD coral Montastrea faveolata. Vibrio alginolyticus bacteria are also suspected as a pathogenic bacterium on Porites andrewsi infected with White syndrome disease [21]. Vibrio alginolyticus bacteria are scattered in the ocean and estuary; these bacteria can survive under high salinity conditions. Several studies have shown that the Vibrio alginolyticus bacteria are considered a species that lives freely in water and sediment and can survive in nutrientpoor seawater [22]. Therefore, there is a high possibility that the Vibrio alginolyticus bacteria are associated with WBD in the Gili Labak Island. Ritchie and Smith [19] explained that the diseased corals contain a higher proportion of Vibrio bacteria than healthy corals. In addition, Vibrio alginolyticus bacteria are in high concentrations during summer, when the temperature becomes hotter than usual [22]. Gili Labak Island waters might become a habitat that is very suitable for the growth of Vibrio alginolyticus bacteria.
Vibrio owensii bacteria are found on Acropora hyacinthus coral affected by White Syndrome [23]. Ushijima et al. [24] explained that the Vibrio owensii bacteria potentially lead to White Syndrome disease on the coral Montipora capitata. Vibrio owensii bacteria, including gram-negative bacteria, are facultatively anaerobic, have a slightly curved rod shape with a width of 1.0 μm and a length of 3.1 μm [25]. Tout et al. [26] explain that Vibrio bacteria are most commonly found on coral reefs. This research found Pseudoalteromonas rubra bacterium was associated with Acropora sp. coral infected with White Band Disease. These are the first time that Pseudoalteromonas rubra bacterium found is associated with White Band Disease in Acropora sp. Tout et al. [26] stated that Pseudoalteromonas bacteria in the marine environment are most often associated with coral reefs. These bacteria were also found to be symbiotic with soft corals Sarcophyton sp. [27][28]. Pseudoalteromonas rubra bacteria are Gram-negative bacteria and can be found in marine waters attached to the surface of rocks, algae, coral reefs, marine animals, sediments, and can be found in coastal waters. Pseudoalteromonas rubra bacteria are aerobic, having a straight or slightly curved stem shape of 0.2-1.5 μm × 1.8-4.0 μm [29]. These findings contribute to the body of knowledge that Pseudoalteromonas rubra bacterium was found also to be associated with Acropora sp. coral infected with White Band Disease.

Conclusion
Based on the research results, it can be concluded that the WBD prevalences on Acropora sp. were between 0 and 23.4%, with the highest prevalence at the north side used for boat mooring and marine tourist activities. The bacteria associated with WBD were also found. The similarity between ACWB 2A and Vibrio alginolyticus bacteria was 99%, ACWB 6 isolate with Vibrio owensii bacteria was 99%, and ACWB 5 and ACWB 8 both had similarity of 99% with Pseudoalteromonas rubra bacteria. These three bacteria provide new information about the bacteria associated with White Band Disease on Acropora sp. from Gili Labak Island, Madura, Indonesia.


extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Genome of ‘Charleston Gray’, the principal American watermelon cultivar, and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection

Summary Years of selection for desirable fruit quality traits in dessert watermelon (Citrullus lanatus) has resulted in a narrow genetic base in modern cultivars. Development of novel genomic and genetic resources offers great potential to expand genetic diversity and improve important traits in watermelon. Here, we report a high‐quality genome sequence of watermelon cultivar ‘Charleston Gray’, a principal American dessert watermelon, to complement the existing reference genome from ‘97103’, an East Asian cultivar. Comparative analyses between genomes of ‘Charleston Gray’ and ‘97103’ revealed genomic variants that may underlie phenotypic differences between the two cultivars. We then genotyped 1365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping‐by‐sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25 000 high‐quality single nucleotide polymorphisms (SNPs) were derived from the GBS data using the ‘Charleston Gray’ genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome‐wide association studies. The high‐quality ‘Charleston Gray’ genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.

Introduction
Watermelon (Citrullus lanatus) is an important crop consumed throughout the world and is a rich source of lycopene, citrulline and other human health-promoting compounds (Perkins-Veazie et al., 2007). Nearly six per cent of all land used for growing vegetables is planted with watermelon, which had an annual global production of 117 million tonnes in 2016 (FAOSTAT, http://www.fao.org/faostat/en/). China is the largest producer and consumer of watermelon with 70 million tonnes produced per year. China and the United States both have intensive watermelon breeding programmes to meet varied consumer preferences. For example, cultivars of East Asian ancestry mainly produce small to mid-sized globular fruits with a thin rind, while American cultivars tend to produce large, oblong fruits with a thick rind. Differential selection pressures in the two regions have led to distinct East Asian and American watermelon ecotypes (Guo et al., 2013;Sheng et al., 2012). Currently, the only published watermelon reference genome was constructed using the Chinese elite line '97103', representing the East Asian type (Guo et al., 2013). Recent comparative genomic and pangenome studies have highlighted the importance of structural variations (SVs), particularly presence-absence variations (PAVs) and copy number variations (CNVs) (Gao et al., 2019;Zhang et al., 2015). Quantitative trait locus (QTL) and association mapping studies with American germplasm using the '97103' genome as the reference would be limited to the genetic makeup of a single East Asian cultivar, potentially missing causal variants.
A large number of watermelon cultivars have been developed in the United States since the mid-19th century (Levi et al., 2001), including 'Charleston Gray' which is considered the principal American dessert watermelon cultivar. 'Charleston Gray' was released in 1954 and was the most popular commercially grown watermelon in the United States for more than a decade. It produces large oblong fruits with a light green exterior, pink-red flesh, and a thick and tough rind, as required for long-distance shipping. It also harbours resistance to the soil-borne disease Fusarium wilt and the foliar disease anthracnose. 'Charleston Gray' has been used in numerous watermelon breeding programmes (Wehner, 1999) and in the development of many improved cultivars. Therefore, unlocking the genome of 'Charleston Gray' would further facilitate watermelon breeding.
Many years of cultivation and selection for desirable fruit qualities have resulted in an overall narrowing of the genetic base among sweet dessert watermelon cultivars, and consequently contributed to their susceptibility to a large number of diseases and pests (Levi et al., 2001. As a result, there is a continual need to expand the genetic base of watermelon cultivars and enrich them with alleles conferring resistance to biotic stresses such as diseases, insects and nematodes in addition to alleles associated with desirable fruit quality. The Citrullus genus, whose centre of origin is Africa, includes seven recognized species (Chomicki and Renner, 2015). Two of these species, C. amarus (also known as citron) and C. mucosospermus (also known as egusi melon), were until recently considered to be subspecies of C. lanatus (Chomicki and Renner, 2015), and they can cross readily with cultivated watermelon. Citrullus lanatus is thought to be native to northern Africa, C. mucosospermus to sub-Saharan western Africa, and C. amarus to southern Africa (Jarret et al., 1997;Levi et al., 2017;Paris, 2015). Unlike C. lanatus, C. mucosospermus is primarily cultivated for oil-rich seeds. Citrullus mucosospermus and C. amarus are valuable sources of resistance to many diseases including Phytophthora fruit rot, powdery mildew, Fusarium wilt, gummy stem blight, anthracnose and various viruses . Therefore, these highly interfertile species have the potential to provide valuable alleles to broaden the genetic base of watermelon and increase disease and pest resistance in elite cultivars.
The U.S. National Plant Germplasm System (NPGS; https:// www.ars-grin.gov/npgs/index.html) maintains a large collection of Citrullus accessions as Plant Introductions (PIs) collected/ acquired from different geographic regions in Africa and throughout the world. The PIs within this Citrullus collection exhibit great phenotypic and genetic diversity Nimmakayala et al., 2014) and thus represent a valuable resource for discovery of unique qualities useful for improving fruit nutritional contents and resistance to biotic and abiotic stresses in watermelon cultivars. Elucidating the genetic diversity and relationships among these PIs is an important step towards the identification of QTLs associated with beneficial attributes.
In this study, we sequenced and de novo assembled a highquality genome of the principal American watermelon cultivar 'Charleston Gray', as a complement to the genome of '97103', an East Asian cultivar. Comparative genomic analyses of 'Charleston Gray' and '97103' identified a number of genome variations overlapping with QTLs of important traits such as fruit weight and shape. We then genotyped 1365 Citrullus accessions from the NPGS (including C. lanatus, C. amarus and C. mucosospermus) using genotyping-by-sequencing (GBS; Elshire et al., 2011). Single nucleotide polymorphisms (SNPs) were called from the GBS data using the 'Charleston Gray' genome as the reference and were subsequently used to assess genetic diversity, phylogenetic relationships and population structure among these Citrullus accessions. The SNP set was also used to perform genome-wide association studies (GWAS) for the identification of genomic regions associated with several important fruit quality and disease resistance traits.

Results
Genome sequencing, assembly, anchoring and quality evaluation The 'Charleston Gray' genome was sequenced using the Illumina technology, which produced a total of 95.5 Gb of high-quality cleaned sequences from paired-end and mate-pair libraries with insert sizes ranging from 400 bp to 20 kb (Table S1). These sequences represented approximately a 2289 coverage of the 'Charleston Gray' genome with an estimated size of 419.2 Mb based on the k-mer depth distribution analysis of the sequencing reads ( Figure S1). De novo assembly yielded a draft genome of 396.4 Mb, representing 94.6% of the 'Charleston Gray' genome. The assembly consisted of 21 498 contigs and 2034 scaffolds (>500 bp), with N50 sizes of 36.7 kb and 7.47 Mb, respectively, and 90% of the genome draft was assembled into only 60 scaffolds (Table 1). The 'Charleston Gray' assembly had higher contiguity compared with that of the Chinese elite watermelon line '97103' (Guo et al., 2013), as indicated by increased N50 and N90 scaffold lengths (Table 1).
Using an integrated high-density genetic map (Ren et al., 2014), 90.3% (378.7 Mb) of the 'Charleston Gray' assembly were anchored to 11 linkage groups, and 90.5% of the anchored scaffolds (342.8 Mb) were oriented. We assigned orientation to an additional of 9.9 Mb of scaffolds, according to two other recently developed genetic maps (Branham et al., 2017;Reddy et al., 2014). Furthermore, based on the genome synteny between '97103' and 'Charleston Gray', an additional 26.6 Mb of the 'Charleston Gray' scaffolds were oriented, and 3.8 Mb of scaffolds were anchored. One misassembled scaffold was identified according to the genetic map and mate-pair read alignments, and was broken into two scaffolds. Finally, we obtained a chromosome-scale assembly of the 'Charleston Gray' genome, of which 382.5 Mb (96.2% of the total assembly) in  (Table S2 and Figure 1).
To evaluate the quality of the assembly, we first aligned RNA-Seq reads and ESTs (expressed sequence tags; Levi et al., 2006) to the 'Charleston Gray' genome. Up to 94% of the RNA-Seq reads were mapped to the genome, and more than 95% of the ESTs were covered by the genome (Table S3). The completeness of the 'Charleston Gray' assembly was further assessed with BUSCO (Simão et al., 2015), which showed that 91.8% of the core conserved plant genes were completely covered by the assembly, while another 1.7% were partially covered (Table S4). Together, these results confirmed the high quality of the 'Charleston Gray' assembly.

Comparative genomic analysis of 'Charleston Gray' and '97103'
We identified 194.9 Mb of repeat sequences in the 'Charleston Gray' genome, contributing to 51.7% of the genome (Table S5), which was higher than the 45.2% repetitive content in the '97103' genome (Guo et al., 2013), mainly attributed to a better assembly of repeat regions in the 'Charleston Gray' genome. A total of 22 546 protein-coding genes were predicted in 'Charleston Gray', among which 18 982 genes (84.2%) were assigned with biological functions. Comparison between orthologous genes in 'Charleston Gray' and '97103' revealed a chromosome-level syntenic relationship ( Figure S2). Most differences between the two assemblies were observed around the centromeric regions ( Figure S3). For example, the 'big inversion' on chromosome 1 might not be an actual SV between the two genomes but was likely caused by incorrect anchoring in the '97103' genomes as a result of uncertainty in ordering short scaffolds within a close genetic distance ( Figure S4a). In this case, the assembly of the 'Charleston Gray' scaffolds 15 and 19 spanning the 'inversion' breakpoints was well supported by the mate-pair reads ( Figure S4b), which suggested that, with a higher contiguity, the 'Charleston Gray' assembly provided more reliable pseudomolecule structures of the watermelon genome for facilitating QTL mapping and molecular genetic studies.
A total of 214 113 SNPs and 16 052 small indels were identified between the 'Charleston Gray' and '97103' genomes, of which 184 567 SNPs and 13 664 small indels were located in the intergenic regions, and 29 546 SNPs and 2388 small indels were located in the introns of genes (23 947 SNPs and 2275 indels) or coding regions (5599 SNPs and 113 indels) (Table S6). Among genes with SNPs in the coding regions, 1810 had nonsynonymous mutations, 80 lost start or stop codons, and 1506 had synonymous mutations. We further identified a total of 3766 high-confidence large SVs (≥50 bp) between the 'Charleston Gray' and '97103' genomes, including 2913 indels and 853 tandem/repeat contractions/expansions (Table S7). The majority of these SVs (88.8%) were less than 1 kb in size ( Figure S5). About 81.5% (3069) of the SVs were located in intergenic regions, 518 in introns of genes, and 179 affected the coding sequences (Table S7). A total of 815 SVs were found to overlap with 52 QTLs known to affect watermelon fruit traits (Table S8), and 400 were located within fruit shape and fruit weight QTLs (Table S8), which might be associated with the more elongated fruit shape and larger fruit size of 'Charleston Gray' as compared to '97103', such as the 159-bp deletion in the ClFS1 gene (ClCG03G016090) of 'Charleston Gray' known to be associated with elongated fruit shape (Dou et al., 2018).

Genotyping of the watermelon germplasm collection and variation identification
To facilitate the utilization of the Citrullus spp. germplasm stored in the NPGS for watermelon improvement, we performed GBS on 1365 Citrullus PIs collected throughout the world (Figure 2), including 1211 C. lanatus, 52 C. mucosospermus and 102 C. amarus PIs (Table S9). Most C. lanatus PIs are modern cultivars and are the result of many years of domestication and selection for desirable fruit qualities that do not exist in their closely related citron or egusi type watermelons. The C. lanatus and C. mucosospermus PIs in this study were collected in East Asia, North and South America, Europe, Central and West Asia, Oceania and different regions in Africa, while the majority of C. amarus PIs were collected in their natural surroundings in southern Africa ( Figure 2 and Table S9).
A total of~1.0 billion reads of 101 bp in length were generated using GBS, from which 388 293 unique tags with at least 10 read counts were obtained, corresponding to~0.8 billion reads, and used for SNP calling. About 49.5% and 16.5% of these reads were aligned to unique and multiple positions in the 'Charleston Gray' genome, respectively, and the unaligned reads were mainly from the chloroplast and mitochondrion genomes. Approximately 2.4% of the 'Charleston Gray' genome was covered by GBS reads, which is typical for GBS data (Elshire et al., 2011). A total of 61 520 SNPs were identified, among which 25 308 were biallelic with a minor allele frequency (MAF) >0.01 and a missing data rate ≤50%, and were used in the subsequent phylogenetic and population structure analyses. These SNPs were distributed across the 'Charleston Gray' genome with an average of one SNP per 15.7 Kb ( Figure S6). Phylogenetic relationships and population structure of Citrullus accessions To infer phylogenetic relationships among the 1367 Citrullus accessions (including 'Charleston Gray' and '97103' in addition to the 1365 accessions genotyped with GBS), we constructed a maximum-likelihood tree, which revealed that C. lanatus and C. mucosospermus accessions exhibited close genetic relationships and were distant from C. amarus ( Figure 3a). Within C. lanatus and C. mucosospermus, four major clades were identified ( Figure 3a and Table S9). Group 1 included C. mucosospermus along with a subset of C. lanatus accessions collected in southern Africa, primarily Zimbabwe and Zambia.
The remaining groups were all comprised of C. lanatus accessions primarily collected in Central and West Asia (Group 2), North America and East Asia (Group 3) and Europe (Group 4). For C. lanatus collected in northern Africa, four Sudanese accessions collected in Khartoum (PI 254622, PI 254623 and PI 260733; white flesh and beige seeds) and Darfur (PI 481871; white flesh and black/beige seeds) were closely related to C. mucosospermus in Group 1 (Table S9). Eight other accessions collected in northern Africa were in Group 2 (Central and West Asia) in one of the deepest branches of C. lanatus outside Group 1, including six collected in Egypt (PI 525090, PI 525086, PI 525087, PI 525084, PI 525091 and PI 525088), one in Sudan (PI 270545, red flesh and black seeds) and one in Algeria (PI 542617; white flesh and black The principal American watermelon cultivar 2249 seeds) (Table S9). For accessions collected in East Africa, five out of seven accessions collected in Ethiopia were closely related to accessions collected in Europe (Group 4), and five out of six accessions collected in Somalia were in Group 1 (Table S9). Similarly, accessions collected in Central and West Africa were placed in all four groups (Table S9), with more accessions (mostly collected in Central Africa) clustered with cultivars from North America and East Asia (Group 3). Despite the geographic distance, the North American C. lanatus accessions (including 'Charleston Gray') and the East Asian cultivars (including '97103') were closely related, although they formed two distinguishable clades in Group 3, reflecting the two major cultivated ecotypes. Population structure of the 1367 watermelon accessions was investigated. DK analysis showed that K = 2 ancestral types best explained the structure of this population and separated C. amarus from C. lanatus/C. mucosospermus ( Figure S7). At K = 3 or 4, three primary clusters could be observed representing C. amarus, the C. mucosospermus/southern African C. lanatus group and the remaining C. lanatus accessions (Figure 3b). At K = 5, a new subgroup emerged, which included North American and East Asian cultivars, while most C. lanatus from Central and West Asia and Europe had similar backgrounds (Figure 3b). Principal component analysis (PCA) using all the accessions also revealed a clear separation of C. amarus from C. lanatus and C. mucosospermus (Figure 3c). According to the phylogenetic relationship and genetic background, a few accessions were likely misclassified or had ambiguous identity, including 26 accessions classified as C. lanatus, among which 11 might be C. amarus and 15 had mixed genetic background, and 14 accessions labelled as C. amarus but did not have a typical C. amarus ancestry pattern (Table S9). These accessions were excluded from the following genetic diversity and population divergence analyses. PCA using the remaining C. lanatus and C. mucosospermus accessions illustrated a pattern further separating C. mucosospermus, C. lanatus collected in southern Africa and the rest of the C. lanatus accessions, while the small portion of the total variance explained by the first two principal components further supported the close genetic relationship between C. lanatus and C. mucosospermus ( Figure 3d). Together, the phylogeny, population structure and PCA results all indicated a distant relationship between C. amarus and C. lanatus/C. mucosospermus, and a similar genetic background in C. mucosospermus and a group of C. lanatus mostly from southern Africa.

Genetic diversity and population divergence in watermelons
The genetic diversity within C. amarus estimated by the average value of genome-wide nucleotide diversity (p) was 3.375 9 10 À4 , higher than that within C. mucosospermus (p = 2.561 9 10 À4 ) and those within different C. lanatus groups based on geographic locations, ranging from 2.405 9 10 À4 (Europe) to 3.035 9 10 À4 (Southern Africa) ( Figure S8). Population divergence among different C. lanatus groups and C. mucosospermus was evaluated by pairwise fixation index (F ST ). Multidimensional scaling visualization of pairwise F ST values clearly showed the distinction between C. mucosospermus and C. lanatus, and between C. lanatus collected in southern Africa and the rest of C. lanatus groups, and much less divergence among the C. lanatus groups outside southern Africa ( Figure S8). The weighted pairwise F ST values between C. mucosospermus and different C. lanatus groups, and between the southern Africa C. lanatus group and the other groups ranged from 0.286 to 0.428, and from 0.238 to 0.385, respectively, while the values among the other C. lanatus groups were much lower, ranging from 0.020 to 0.149 (Table S10). The F ST values were largely consistent with relationships of different groups of Citrullus accessions inferred from phylogenetic and population structure analyses.

GWAS for fruit quality and disease resistance traits
We performed GWAS for several fruit quality and disease resistance traits to identify QTLs underlying these important traits. We collected historical phenotypic data from the NPGS for fruit flesh colour, fruit shape and rind stripe pattern for 788, 864 and 695 accessions, respectively, that were genotyped in this study. We also evaluated the resistance to bacterial fruit blotch (BFB), powdery mildew race 2W and Papaya ringspot viruswatermelon strain (PRSV-W) on 1125, 1147 and 908 PIs, respectively (Table S11). A group of SNPs on chromosome 4 (at 15.3-15.8 Mb) were associated with red coloration of fruit flesh (Figure 4a and Table S12), and this genome region contained the LCYB gene (chromosome 4: 15 694 446-15 696 571) that converts lycopene to downstream carotenoids (Bang et al., 2010). In addition, 14 additional SNPs on other chromosomes were also associated with red flesh colour (Table S12). A peak associated with rind stripe pattern was identified on chromosome 6 (at~30.2 Mb) (Figure 4a), overlapping with the 'S' locus that controls the foreground stripe pattern (Park et al., 2016). Nine additional SNPs were also significantly associated with rind stripe pattern. Five SNPs on chromosome 3 (at~31.1 Mb) were associated with elongated fruit shape (Figure 4a and Table S12), which were located near the ClFS1 gene (~22.6 Kb distance; chromosome 3: 31 086 958-31 090 579) known to control watermelon fruit elongation (Dou et al., 2018). Two other SNPs associated with fruit shape were on chromosomes 2 and 6. Two peaks on chromosomes 10 (~32.6-32.9 Mb) and 6 (~10.9 Mb), respectively, were associated with resistance to BFB in fruit (Figure 4b and Table S12). A group of SNPs on chromosome 2 (~29.2-31.0 Mb) were associated with powdery mildew race 2W resistance in stems, and an additional 21 SNPs on other chromosomes were also associated ( Figure 4b and Table S12). The same peak on chromosome 2 was also associated with powdery mildew race 2W resistance in leaves, and an overall similar association pattern was observed. For PRSV-W resistance, 15 significantly associated SNPs were identified on multiple chromosomes (Figure 4b and Table S12).

Discussion
Watermelon is among the first cucurbit crops to have its genome sequenced, assembled and annotated (Zheng et al., 2019). Genome sequence of the East Asian watermelon cultivar, '97103', was released in 2013 (Guo et al., 2013). To complement the '97103' reference genome, and to capture genome information specifically present in the American watermelon ecotype, we assembled and annotated the genome of the principal American cultivar 'Charleston Gray', performed detailed characterization of sequence variations between 'Charleston Gray' and '97103', and identified those overlapping with known watermelon fruit trait QTLs, which may underlie the phenotypic differences between the two cultivated ecotypes. Comparative analyses suggested that the assembled 'Charleston Gray' genome had higher continuity and quality than that of '97103'. The highquality 'Charleston Gray' assembly will facilitate its use as a reference for integrative genetic-genomic studies aiming to identify QTLs associated with important agronomical traits.
We characterized the genetic composition of 1365 watermelon PIs maintained in the NPGS, belonging to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. The latter two are of particular interest because of the presence of potential genes/ alleles associated with fruit quality or resistance to major cultivated watermelon diseases . In agreement with the known narrow genetic background in dessert watermelon, all C. lanatus groups from different geographic regions displayed low genetic diversity, with that of the southern Africa group being slightly higher, consistent with southern Africa being the likely centre of origin of Citrullus (Chomicki and Renner, 2015;Paris, 2015).
Our sampling included a large number of accessions collected in major dessert watermelon production areas and geographic regions where wild and primitive watermelons exist. This enabled us to reveal phylogenetic relationships among watermelon accessions collected in different geographic regions that may shed light on the domestication and dispersal of dessert watermelons. The distant relationship between C. amarus and C. lanatus/C. mucosospermus and their distinct genetic backgrounds revealed in the current study are consistent with previous genetic characterization of a few resequenced accessions (Guo et al., 2013). This result indicates that the dessert watermelon is unlikely to be descended from C. amarus in southern Africa, which is further supported by the evidence of genome organization differences based on rDNA chromosome landmarks (Guo et al., 2013;Reddy et al., 2013) and the finding that the emergence of dessert watermelon in Egypt predated the time when farming began in southern Africa (Paris, 2015). It has been hypothesized that the dessert watermelon was domesticated in northeastern Africa and a wild population of 'cordophanus' watermelons in Sudan with non-bitter fruits and white pulp may be the living representatives of the progenitor of the cultivated dessert watermelon (Paris, 2015;Renner et al., 2017). Four Sudanese C. lanatus accessions bearing such fruit phenotypes, including two (PI 481871 and PI 254622) considered as dessert watermelon (Paris, 2015), are closely related to C. mucosospermus collected from Liberia, Ghana, Nigeria and Zaire, suggesting that C. lanatus accessions from Sudan and C. mucosospermus from central/West Africa could be derived from the same ancestral population; one selected for seed traits, and the other domesticated for fruit flesh characteristics. The placement of eight northern African C. lanatus accessions (six from Egypt) in one of the deepest branches of cultivated dessert watermelon groups on the phylogenetic tree ( Figure 3a) further supports the hypothesis that modern dessert watermelons originated in northeastern Africa. Accessions from Central/West Asia are also located in relatively deep branches on the phylogenetic tree, suggesting that dessert watermelons might have been introduced from Africa to Central/West Asia, and then dispersed to the rest of the world. The accessions from East Asia and North America are closely related and yet clearly distinguishable, reflecting the differences in recent breeding efforts in the two areas. East Asian and North American accessions share a unique genetic background different from that of most current accessions in Central/West Asia and Europe, and a small group of European accessions (mostly from Hungary) were found in the deepest branch in the East Asia/North America clade, suggesting that watermelons from East Asia and North America might be derived from a single ancestral group and could be distributed through Europe. By including a large collection of C. lanatus accessions collected in southern Africa, our analyses showed a close relationship between C. lanatus collected in southern Africa and C. mucosospermus in Mali and Senegal. The C. lanatus accessions collected in southern Africa display various flesh colours and sweetness levels (https://npgsweb.ars-grin.gov/grin global/descriptors.aspx) and have a unique genetic background different from the other C. lanatus accessions collected in regions other than southern Africa, suggesting parallel evolution for increased flesh sugar content and coloration in different C. lanatus populations, or introduction of alleles controlling these trait from more improved cultivars to the southern African accessions. Knowledge of causal genes underlying domestication traits and distribution of functionally diverse alleles in different watermelon populations is needed to better demonstrate the evolutionary route of dessert watermelons.
The enhancement of disease resistance has been a major focus of current watermelon improvement programmes. Using the GBS SNP makers combined with GWAS, potential QTLs for resistance to BFB, powdery mildew and PRSV-W were identified. BFB, caused by Acidovorax citrulli, is a devastating disease of most cucurbit crops including watermelon. Numerous BFB outbreaks have occurred in watermelon growing regions in the United States and in countries throughout the world. Recently, using a recombinant inbred line population, Branham et al. (2019) identified six QTLs significantly associated with foliar BFB resistance. Three of the QTLs (qAc-1.1, qAc-2.1 and qAc-8.1, on chromosomes 1, 2 and 8, respectively) were consistent in Given the low heritability and large environmental component of disease response to A. citrulli, the three QTLs (qAc-1.1, qAc-2.1 and qAc-8.1) identified in C. amarus and the two additional QTLs identified in this study on chromosomes 10 (~32.6-32.9 Mb) and 6 (~10.9 Mb) in C. lanatus/C. mucosospermus can be further explored for marker development and used to pyramid genes/loci for enhanced BFB resistance in elite watermelon cultivars.
Powdery mildew is a major disease of cucurbit crops, caused by the fungus Podosphaera xanthii. In the United States, two distinct powdery mildew races, 1W and 2W, are known for watermelon, and several cultivars and wild species highly resistant to these races have been identified (Davis et al., 2007;Tetteh et al., 2010). Recently, Kim et al. (2015) identified a major QTL (pmr2.1) for resistance to powdery mildew race 1W on chromosome 2 that explained 80.0% of the phenotypic variation. In the present study, GWAS analysis identified a major peak associated with powdery mildew race 2W resistance also on chromosome 2 (~29.2-31.0 Mb) overlapping with QTL pmr2.1, and additional SNPs on chromosomes 3, 6, 8, 9 and 10 associated with race 2W resistance (Figure 4b and Table S12). These results indicate the possibility that the QTL on chromosome 2 might be associated with resistance to both powdery mildew races 1W and 2W.
PRSV-W is an important potyvirus causing serious economic damage to the watermelon crop. Resistance to PRSV-W has been identified, and genetic inheritance mode of this important potyvirus in three C. amarus accessions (PI 244017, PI 244019 and PI 485583) has been determined, suggesting that the resistance is controlled by a single recessive gene (Guner et al., 2008). In contrast, a genetic inheritance study of PRSV-W resistance in C. lanatus PI 595201 indicated that the inheritance of PRSV-resistance is complex with additive genetic effects, and is controlled by multiple genes (de Azevedo et al., 2012). The GWAS analysis here, using the C. lanatus/C. mucosospermus PIs, Figure 4 Genome-wide association studies of fruit quality (a) and disease resistance (b) traits. BFBFruit, resistance to bacterial fruit blotch in fruits; PM2Stem, resistance to powdery mildew race 2W in stem; PM2Leaf: resistance to powdery mildew race 2W in leaf; PRSV: resistance to Papaya ringspot virus-watermelon strain. Gray horizontal dashed lines on the Manhattan plots indicate the Bonferroni significance thresholds of GWAS (Àlog10(P) of 5.68 and 6.38, corresponding to a = 0.05 and a = 0.01, respectively). identified SNPs on multiple chromosomes with significant associations with PRSV-W resistance (Figure 4b and Table S12). These results suggest that resistance to PRSV-W in C. lanatus might be controlled by several genes with additive effects while in C. amarus the resistance is controlled by a single gene.
GWAS analyses in the present study also successfully identified genomic regions known to underlie fruit quality traits such as flesh colour, fruit shape and rind pattern, and additional novel SNPs highly associated with these traits. The QTLs identified in this study together with QTLs identified in recent genetic inheritance studies provide a useful platform for the development of molecular markers for use in breeding programmes aiming to enhance disease resistance and fruit quality in elite watermelon cultivars.

Plant materials
For genome and transcriptome sequencing of 'Charleston Gray', seeds were obtained from a line descended from the original 'Charleston Gray' through self-pollination over several generations at the U.S. Vegetable Laboratory (USVL) and germinated in a greenhouse at the USVL. For GBS, a total of 1365 PIs, including 1211 C. lanatus, 52 C. mucosospermus and 102 C. amarus, were obtained from self-pollinated plants (one generation) and grown in the greenhouse at North Carolina State University.

Construction and sequencing of genomic and RNA-Seq libraries
Genomic DNA was extracted from young fresh leaves of 'Charleston Gray' using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Valencia, CA) following the manufacturer's instructions. Two paired-end genomic libraries with insert sizes of 400 bp and 1 kb, respectively, were prepared using the Genomic DNA Sample Prep kit (Illumina, San Diego, CA) according to the manufacturer's protocol, and sequenced on an Illumina GAIIx platform. The 400-bp library was sequenced additionally on a HiSeq 2500 system. Another library with insert size of 400 bp was constructed with the DNA Library Preparation kit (Kapa Biosystems, Wilmington, MA) without PCR amplification, and sequenced on an Illumina MiSeq system. Four mate-pair libraries with insert sizes of 3-5, 8-10 (2 libraries) and 15-20 kb were prepared with the Nextera Mate Pair Sample Preparation kit (Illumina), and sequenced on a HiSeq 2500 system. All libraries were sequenced with the paired-end mode.
Total RNA was extracted from fruit flesh tissues of 'Charleston Gray' at 11, 20, 30 and 40 days after pollination, with two biological replicates for each stage, using the QIAGEN RNeasy Plant Mini Kit (QIAGEN). RNA-Seq libraries were constructed using the NEB Next Ultra TM RNA Library Prep Kit (NEB, Beverly, MA) and sequenced on the Illumina HiSeq 2500 system with the paired-end mode.

De novo genome assembly
Duplicated read pairs, defined as having identical bases at positions of 14-90 in both left and right reads, were collapsed into unique read pairs. The non-redundant reads were processed with Trimmomatic (Bolger et al., 2014) to remove adaptors and low-quality sequences. The mate-pair reads were processed with the ShortRead package (Morgan et al., 2009) to remove junction adaptors. The resulting reads were assembled into scaffolds using SOAPdenovo2 (Luo et al., 2012). Gaps in the scaffolds were filled with GapCloser (Luo et al., 2012). Pilon (Walker et al., 2014) was used to correct base errors, fix mis-assemblies and fill additional gaps. The assembly was then aligned to NCBI non-redundant nucleotide database using BLASTN with an E-value cut-off of 1e-5. Scaffolds with more than 95% of their length similar to sequences of microorganisms, mitochondria or chloroplasts were considered contaminants and removed. Redundant scaffolds with sequence identity higher than 99% and more than 95% of their length covered by other scaffolds were removed.

Transposable element annotation and gene prediction
Long terminal repeat retrotransposon (LTR-RT) and miniature inverted-repeat transposable element (MITE) libraries were de novo constructed by screening the 'Charleston Gray' genome using LTRharvest (Ellinghaus et al., 2008) and MITE-Hunter (Han and Wessler, 2010), respectively. After masking the assembly with these libraries using RepeatMasker (http://www.repeatmasker. org), we further searched for repeat elements in the unmasked sequences using RepeatModeler (http://www.repeatmasker.org/ RepeatModeler.html). All the identified repetitive sequences were combined into a single repeat library and compared against the Swiss-Prot database (Magrane and UniProt Consortium, 2011). Sequences that matched non-TE proteins in the database were removed. TEs were classified using REPCLASS (Feschotte et al., 2009). The classified repeat library was then used to identify TEs in the 'Charleston Gray' genome with RepeatMasker.
The repeat-masked 'Charleston Gray' assembly was used for gene prediction with MAKER (Cantarel et al., 2008) by integrating evidences from ab initio gene prediction, transcript mapping and protein homology to define confident gene models. SNAP (Korf, 2004) and AUGUSTUS (Stanke et al., 2006) were used for ab initio gene predictions. Two transcriptome assemblies from fruit RNA-Seq data were obtained using Trinity (Grabherr et al., 2011) with the de novo mode and the genome-guided mode, respectively, and aligned to the 'Charleston Gray' assembly using PASA2 (Haas, 2003). The resulting alignments were used as the transcript evidence. Protein sequences from Arabidopsis, watermelon, cucumber and melon, as well as the UniProt (Swiss-Prot plant division) database, were aligned to the 'Charleston Gray' genome using Spaln (Iwata and Gotoh, 2012) to provide protein homology evidence. For gene annotation, protein sequences of the predicted 'Charleston Gray' genes were compared against the Arabidopsis protein and UniProt (Swiss-Prot/TrEMBL) databases using BLAST, as well as the InterPro database using InterProScan (Jones et al., 2014). Blast2GO (Conesa et al., 2005) was used to obtain gene ontology (GO) annotations.

Synteny analysis
To identify syntenic regions between the 'Charleston Gray' and '97103' genomes, protein sequences from the two cultivars were aligned using BLASTP, and high-confidence collinear blocks were determined using MCScanX with an E-value cut-off of 1e-10 (Wang et al., 2012). Ks values of orthologous gene pairs were calculated using the Yang-Nielsen algorithm implemented in PAML (Yang, 1997). LAST (v869;Kielbasa et al., 2011) was used to identify unique best alignments between the genomes of 'Charleston Gray' and '97103'.

Identification of SNPs and SVs
To detect SNPs and small indels between '97103' and 'Charleston Gray', paired-end genomic reads of '97103' (Guo et al., 2013) were mapped to the 'Charleston Gray' genome using BWA (version 0.6.2) (Li and Durbin, 2009). Uniquely mapped reads were kept for variant detection. Genotypes were assigned to each genomic position based on the alignment mpileup files generated by SAMtools . SNPs and small indels were then identified if they were supported by at least four mapped reads. To detect large SVs (≥50 bp), genome sequences of '97103' and 'Charleston Gray' were aligned using Minimap2 (Li, 2018), and based on the alignment indels and tandem/repeat expansions/ contractions were identified using Assemblytics (Nattestad and Schatz, 2016). To confirm the identified SVs, reads generated from one genome were aligned to the opposite genome using bowtie (Langmead et al., 2009) allowing one mismatch. SVs with junction sites at both ends having no more than five spanning reads were considered highly confident. Meantime, indels (≥50 bp) were also detected with SpeedSeq (Chiang et al., 2015) based on '97103' genomic reads mapped to the 'Charleston Gray' genome and 'Charleston Gray' reads mapped to the '97103' genome. Sequences flanking the identified indels (five kb on each side) from one genome were aligned to the other genome using BLAST with E-value <1e-10 and sequence identity >90%. Indels with flanking sequences aligned to the proper positions on the other genome were considered highly confident. The two highconfidence sets of SVs by reference sequence comparison and read mapping, respectively, were integrated, and those spanning any gap regions in the assembled genomes were removed.

DNA extraction for GBS
About 100 mg fresh leaf tissue was collected from a young seedling representing each PI. The leaf tissue was freeze-dried and then ground to a fine powder using 5/32" stainless steel balls (AbbottBall, West Hartford, CT) in a Retsch Mixer Mill (Retsch, Newtown, PA). DNA was isolated using the Plant DNA DS Kit (Omega Bio-Tek, Norcross, GA). The DNA was quantified with the Quant-iT PicoGreen dsDNA Kit (Invitrogen, Carlsbad, CA), and its quality was checked by electrophoresis of undigested and HindIIIdigested DNA on agarose gels.

GBS analysis and SNP calling
DNA samples of all PI accessions were subjected to GBS analysis following the protocol described in Elshire et al. (2011) using the ApeKI restriction enzyme (NEB). GBS libraries were sequenced on a HiSeq 2500 system to obtain reads with lengths of 101 bp. The TASSEL 5.0 GBS Discovery Pipeline (Glaubitz et al., 2014) was used for SNP identification using the 'Charleston Gray' genome as the reference. Tags were identified from raw reads possessing a barcode and a restriction enzyme cut site using GBSSeqToTagDB-Plugin with parameters '-kmerLength 90 -minKemrL 30 -mnQS 10 -c 10 -maKmerNum 200000000'. Tags supported by at least ten reads were retrieved and reformatted using TagExportTo-FastqPlugin, and mapped to the 'Charleston Gray' genome using BWA (v0.7.13; with default parameters. Based on the alignments, positions of aligned tags were determined using SAMtoGBSdbPlugin, and SNPs were identified from the aligned tags using DiscoverySNPCallerPluginV2 with default parameters. SNPs were filtered using VCFtools (Danecek et al., 2011) to keep those that were biallelic and had missing rate ≤ 50% and MAF ≥1%.
PCA was performed using Plink (v1.9; Purcell et al., 2007). Population structure analysis was performed using STRUCTURE (v2.3.4;Falush et al., 2003). To determine the most likely cluster number, STRUCTURE analyses were run 20 times for each K value ranging from 2 to 20, using 3715 SNPs filtered by LD information using SNPhylo described above, with an admixture model. After the best K (K = 2) was determined, population structure of the watermelon accessions was inferred using fastStructure (v1.0; Raj et al., 2014) with all SNPs for each K (K = 2-5).
Nucleotide diversity (p) and population fixation index (F ST ) were calculated using vcftools (v 0.1.15; Danecek et al., 2011) using unfiltered raw SNPs. The average nucleotide diversity was calculated as the sum of nucleotide diversity at each site divided by the total bases covered by GBS reads in the genome.

Phenotypic data collection and disease symptom rating
Historical phenotypic data for watermelon fruit quality traits were downloaded from the GRIN database (https://npgsweb.arsgrin.gov/gringlobal/descriptors.aspx). Fruit flesh colour was rated as red (red and pink) and non-red (green, white, yellow and orange). Fruit shape was rated as round and elongated (oblate, oblong and elongated). Rind stripe pattern was rated as striped and solid. For PRSV-W disease screening, young watermelon seedlings were rated at 14, 21 and 28 days post-inoculation (DPI), and the average of the three ratings was used. BFB disease data were collected from young fruits at 21 DPI. Powdery mildew race 2W disease data were collected from young seedlings at 14 and 28 DPI, and the average of the two ratings was used. All three diseases were rated using a visual scale of 0-9, where 0 = no symptoms and 9 = dead plant. Phenotypic data for accessions genotyped in the present study were used for GWAS.

Data availability
The genome sequence of 'Charleston Gray' has been deposited at DDBJ/ENA/GenBank under the accessions CP026477-CP026488. Raw genome and transcriptome sequencing reads have been deposited into the NCBI sequence read archive (SRA) under accessions SRP183199 and SRP183523, respectively. The genome sequence of 'Charleston Gray' and the GBS SNPs are also available at the Cucurbit Genomics Database (http://cucurb itgenomics.org; Zheng et al., 2019).

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Figure S1 K-mer distribution of Illumina genomic sequencing reads of 'Charleston Gray'. Figure S2 Syntenic orthologous gene blocks between 'Charleston Gray' and '97103'. Figure S3 Collinearity between the 'Charleston Gray' and '97103' genomes. Figure S4 Collinearity between 'Charleston Gray' and '97103' chromosome 1. Figure S5 Size distribution of indels between genomes of 'Charleston Gray' and '97103'. Figure S6 SNP density across the 11 watermelon 'Charleston Gray' chromosomes. Figure S7 Plot of DK values with K from 2 to 20 in the STRUCTURE analysis for the 1367 watermelon accessions using GBS SNPs. Figure S8 Multidimensional scaling of pairwise F ST between C. mucosospermus (CM) and C. lanatus accessions from different geographic regions.  Table S11 Phenotypic data for GWAS. Table S12 SNPs significantly associated with agronomic traits identified through GWAS. Table S13 Evaluation of the SNP imputation using KNN under the optimal parameters 'w = 50, k = 7, p = -9, r = 0.8'.


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Effect of various antioxidants and their combinations on bull semen cryopreservation

* Correspondence: sukrugungor@mehmetakif.edu.tr

Chemicals
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Bulls
Semen was collected by artificial vagina from three Holstein bulls (1-3 years old) and pooled (30 ejaculates and six replicates). All activities involving bulls were reviewed and approved by the Burdur Mehmet Akif Ersoy University Animal Care Committee (Protocol 31/192).
Based on previous experiments [9,[24][25][26], we used 50 mM TRE, 1 mM CYS, and 400 µg CAT individually or in combinations. Pooled semen was divided into six equal portions and put into a Tris-based extender supplemented with no additive (control), TRE, CYS, CAT, TRE + CYS, or TRE + CYS + CAT. For all groups, semen was diluted and cooled from 37 to 4 °C over 2.5 h, and equilibrated semen was loaded in to 0.25-mL straws (20 × 10 6 spermatozoa/ straw) that were exposed to nitrogen liquid vapor (5 cm above liquid nitrogen) for 12 min before being plunged into liquid nitrogen (-196 °C). Straws were stored for at least 3 months and samples were analyzed after thawing in a water bath at 37 °C for 30 s.

Sperm evaluation 2.4.1. General
Motility evaluation was done soon after semen was collected and extended (fresh), after cooling and equilibration (equilibrated), and after freezing and thawing (postthaw), although assessment of the hypoosmotic swelling (HOS) test, sperm morphology, high mitochondrial membrane potential (HMMP), and plasma membrane and acrosomal integrity (PMAI) were done only for frozen-thawed sperm.

Motility
A wet mount was prepared by placing 1 drop (~5 µL) of diluted sample on a warmed slide and covering it with a cover slip. Total sperm motility was subjectively estimated using a phase-contrast microscope [400× magnification, Nikon Eclipse E600 (Tokyo, Japan)] at 37 °C. Motility estimates were done by viewing at least five fields for each sample and the mean was recorded.

Membrane integrity (HOS test)
The HOS test was used to evaluate sperm membrane integrity. For this, 10 µL of semen was placed in 1 mL of 100 mOsm buffer (1.35 g fructose, 0.735 g tri-sodium citrate/100 mL distilled water) at 37 °C for 1 h. Then 5 µL was placed on a slide, cover-slipped, and evaluated with bright-field microscopy [400× magnification, Nikon Eclipse E600 (Tokyo, Japan)]. At least 200 sperms were assessed and the percentage with curled and swollen tails was recorded [29].

Sperm morphology
Abnormal spermatozoon rate was determined by fixation liquid method. Spermatozoon were fixed with Hancock solution. At least 200 sperms per slide were examined under phase-contrast microscopy at 1000× magnification (Nikon Eclipse E600, Tokyo, Japan). Sperm abnormalities were defined as major or minor, as previously described [30,31].

Statistical analyses
Data for sperm motility, PMAI, HMMP, HOS test, and major, minor, and major + minor sperm defects were analyzed to detect differences among groups. First, normality of data distribution was confirmed with a Shapiro-Wilk test; then data were analyzed by ANOVA, with a Tukey test used to locate differences. Independent statistical analyses were conducted for each end point for fresh, equilibrated, and postthaw sperm. Pearson correlations between HMMP and PMAI, motility, HOS tests, and sperm abnormalities were also calculated.
All data analyses were performed with SPSS 22.0 for Windows, with P < 0.05 considered significant. All data were reported as arithmetic mean ± standard error of the mean (SEM).

Results
There was no significant effect of group on motility of fresh sperm (Table 1). In chilled-equilibrated semen, motility was highest in the CAT and lowest in the control group (mean ± SEM, 75.7 ± 2.3% vs. 68.6 ± 1.4%, respectively). In postthaw semen, motility was lowest (P < 0.05) in CAT (27.1%), but not significantly different among the remaining five groups. For PMAI and HMMP, values were highest in TRE and TRE + CYS (no significant difference between these two groups for either end point) and were lowest (P < 0.05) in the control group (Table 2). There was a lower (P < 0.05) percentage of HOS test-positive sperm in the control and CAT groups compared to the other four groups (Table 3), whereas major, minor, and major + minor defects were lowest (P < 0.05) in the TRE group. HMMP was positively correlated with PMAI (r = 0.66; P < 0.01) and HOS test (r = 0.342; P < 0.05) and negatively correlated with major + minor abnormalities (r = -0.349, P < 0.05; Table 4).

Discussion
Sperm function and the fertility of frozen-thawed semen can be decreased in a variety of ways, especially due to the actions of free radicals [32]. In the present study, bull sperm was cryopreserved with various antioxidant compounds (TRE, CYS, and CAT) individually or in combination. The addition of TRE or TRE + CYS to the extender was generally favorable with regards to PMAI, HMMP, HOS tests, and sperm morphology. Previous studies showed that TRE had positive effects on sperm morphology during the freezing process and this was similar to our findings [29].
Based on motility, there was no evidence that any of the extenders had deleterious effects on fresh semen. Addition    of 1 mM CYS to semen extender resulted in the highest postthaw total motility, although it was only significantly greater than that of CAT (P < 0.05). CYS has a potential positive role on antioxidants that scavenge reactive oxygen species (ROS) by enhancing glutathione synthesis during cryopreservation [33,34]. Although addition of 400 µg/mL CAT to the sperm medium resulted in the highest motility for chilled sperm, it also resulted in the significantly lowest motility after the freezing process. Compared to other groups, CAT decreased postthaw motility, as well as plasma membrane and acrosome integrity and morphological integrity, consistent with previous studies [24,35]. Similarly, 200 or 400 µg CAT also decreased postthawed sperm motility in ram semen [24]. Although 50-100 µg CAT failed to improve motility in fresh extended or cooled monkey semen [36], conversely, 400 µg CAT had positive effects on the postthaw motility of bull [22,37], goat [23], boar [38], and ram [39] sperm.
Despite ample evidence that excessively high ROS concentrations are deleterious, physiological ROS concentrations have critical roles in sperm physiology, acrosome reaction, capacitation, hyperactivation, and signaling to ensure fertilization [40]. Strategic use of antioxidants can mitigate excessive ROS concentrations and enhance sperm quality and function, whereas excessive antioxidant activity could be very detrimental. Although it was suggested that since CAT is an endogenous product, it does not need to be supplemented [36], perhaps apparent differences among studies are related to the species and/or concentrations of CAT used.
In the present study, PMAI was highest with the combination of TRE + CYS, although it was not significantly different than that of TRE. PMAI is important for fertilization, as essential enzymes are stored in the acrosome. Similar to our study, the addition of TRE to the extender of semen from red deer [41] and goats [42] improved postthaw PMAI. Furthermore, TRE significantly increased the viability and acrosome integrity of ram sperm [21] and promoted the integrity of mouse sperm [43].
HMMP was highest in the TRE group (but not significantly different from the TRE + CYS group) and lowest in the control group. Mitochondria are the main source of ROS, with actions of TRE to reduce oxidative stress occurring in the mitochondria [44]. Lee et al. [45] accounted for why TRE, despite having similar cryoprotective effects compared to other sugars, resulted in a higher rate of sperm with high membrane fluidity. Sperm motility is mostly dependent on mitochondria, located in the midpiece and producing energy essential for propagation and generation of flagellar waves [46]. Sperm membrane integrity and viability are critical aspects of sperm quality and function, including maintenance of homeostasis and the ability to move, interact with the environment, and achieve fertilization. A motile spermatozoon should be considered viable and viable spermatozoa must have an intact plasma membrane, as the latter is essential for the sperm's interactions with the environment and other cells [47]. Consequently, there is an association between plasma membrane integrity and HMMP [1]. Dense fibrils in the mitochondrial axoneme produce intracellular ATP [8], required for sperm motility [48]. Furthermore, mitochondrial membrane potential evaluation not only reflects mitochondrial function but is also evidence of early apoptosis, as impaired mitochondrial function may lead to increased apoptosis [49]. In the present study, HMMP was positively correlated with PMAI (r = 0.66, P < 0.01) and negatively correlated with the HOS test (r = -0.342, P < 0.05) and total abnormality (r = -0.349, P < 0.05). This finding was similar to results for frozenthawed human [50] and bovine [2] sperm.
In conclusion, the addition of 50 mM TRE alone or in combination with 1 mM CYS to Tris-based freezing medium improved the postthaw quality of bull semen. Further studies with a wider range of concentrations, combinations of both compounds, and effects on fertility of frozen-thawed semen are warranted.


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Antiulcerogenic activities of Agelanthus dodoneifolius leaf against acetyl salicylate and ethanol induced ulceration in rats

Antiulcer and antioxidant activities of crude methanol (800 and 1600 mg/kg bw), n-hexane and ethyl acetate fractions (800 mg/kg bw) of Agelanthus dodoneifolius were investigated against acetylsalicylate and ethanol induced ulcer in rats. The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66 ± 6.66 %) in ethanol model, however, in acetyl salicylate model, crude extract exhibited the highest (54.16 ± 4.16 %) and the lowest (33.33 ± 4.16 %) protective effect at 1600 and 800 mg/kgbw respectively. The methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. Treatment with crude and fractions of A. dodenofolius significantly (p<0.05) increased the activities of SOD and Catalase and decreases the MDA concentration when compared with ulcerative untreated rats in both ulcer models. In conclusion, A. dodoneifolius could serve as a sources of new plant based antiulcer agent.

Introduction
Ulcer disease is a condition that affects the skin around the lining of human body either externally or around its internal viscera: typically characterized by the presence of an open sore in the lining of epithelial cells or deep lesion in the body [1]. Ulcer disease is seen as one of the world's most common illnesses, which is a global health problem affecting the youths [2]. The major aggressive factors that cause ulcer are pepsin, gastric acid, non-steroidal anti-inflammation drugs (NSAID), bile salts, abnormal motility, alcohol and a bacterial infection known as Helicobacter pylori. Although, there are ample factors that could be protective in function which safeguard the stomach from ulcer formation and these include among others; bicarbonate production, mucus secretion, gastro-protective prostaglandin synthesis and normal tissue microcirculation [3]. The proposed strategies put in place for the prevention of peptic ulcer disease are those of attenuating gastric acid production and increasing gastric mucosal protection [4]. Evaluation of therapeutic potential of medicinal plant against gastric ulcer is attested by employing the use of ethanol to induce gastric injury [5]. Ethanol elicit its effects by causing intense lesions in the sub-mucosa by penetrating through the membrane [6], elevating Reactive Oxygen Species (ROS) formation, and reduces the mucus membrane formation [7] which can cause death of cells around the gastric mucosa. Ethanol inhibits endogenous prostaglandins formation and also suppresses cyclooxygenase enzyme activitiues [8].
Herbal medicine has witnessed widespread attention globally in recent years because of their use in the field of medicine for treating many diseases. Loranthaceae also known as is a parasitic plant growing on a large number of tree species such as Parkia biglobosa, Tamarindus indica, acacia, kola, citrus, orange and many other trees as a host plants. Presence of tannins, flavonoids, alkaloids and saponins were revealed in the methanol and chloroform extracts of the plant [9] Incidence of ulcer was discovered in about 87.4 million people worldwide in 2015 and about 10% of people develop peptic ulcer at some point in their life time. Peptic ulcer also accounted for the deaths of 267,500 people in 2015 alone [10]. It is known to be the most predominant of the gastrointestinal diseases with a global prevalence of around 4% of the population [11].
Liver toxicity also known as hepatotoxicity is a common disease resulting into serious debilities such as serious metabolic disorders to even death. Hepatotoxicity in often due to increase free radical. Free radicals produced by toxicant metabolism initiate what is termed toxicant cascade [12]. The free radical scavengers known to be endogenous in the body includes among others; catalase, super oxide dismutase, reduced glutathione, vitamin C and vitamin E, are present in the body. Cell necrosis is initiated by inhibited free radicals thus leading to hepatotoxicity [13]. Synthetic drugs have little to offer for alleviation of these disorders. Natural occurring antioxidants have better antioxidant activity from their natural source and this is why they are preferred to synthetic antioxidants [14]. The use of NSAIDs is crucial for the treatment of inflammatory diseases, it however presented some adverse effects such as gastric/ duodenal ulceration and renal failure which pose a serious threat in their medical use for treatment [15] The objectives of the present research were to evaluate the antiulcer activities of the methanol extract of the leaves of Agelanthus dodoneifolius using various animal models. Therefore, the present study was undertaken for the purpose of verifying its use by the locals in the treatment of ulcer.

Material and methods
The present work was designed to determine the anti-ulcer activities of the medicinal plant Agelanthus dodoneifolius. This was performed at the Department of Biochemistry, Federal University of Technology, Minna, Nigeria. In order to ascertain the anti-ulcer activities activities of Agelanthus dodoneifolius, the following steps were involved.

Animals
Wistar rats of about 180 ± 2 g weights were obtained, and are in the range of 6 weeks old from Animal House of the Federal University of Technology Minna were used for the experiments. The animals were conveniently housed under standard environmental conditions Temperature (27 ± 2) °C; 70% relative humidity; 12h daylight/night cycle) and had free access to commercial feed pellets and water.

Fractionation of the extract
Solubilization of the extract was done in methanol: water in 80:20 and the resulting solution was subjected to successive extractions in separating funnel with the solvents: n-hexane, ethyl-acetate (AcOEt) and methanol (MeOH). Three fractions were obtained from the extracts.

Antiulcer and Cytoprotective Studies
Three (3) animals for each group: Oral administration of drugs or saline was achieved at 9:00am daily for 8 days. Treatment was conducted for 8 days, the rats to be experimented were kept fasting for 36 hours in separate cages with raise wide mesh wire bottom to ensure complete emptying of the stomach and to prevent coprophagia (eating their faeces) before subjecting them to ulcerogen, and allowed water ad-libitum Same design was used for ethanol model using ethanol.

Ethanol (EtOH) and aspirin (ASP) ulcer induction
The Ethanol induced ulcer was carried in rats by administering Ethanol as described by Hollander et al. [16] and Aspirin Induced ulcer was carried out as described by Rajkapoor et al. [17].

Induced ulcers
Aspirin was administered orally on the day of experiment at about 10 AM with the help of an orogastric tube in the form of an aqueous water suspension (200 mg/kg, per oral) and animals were sacrificed after 4 hours of administration. The stomach was incised along with the greater curvature and examined for ulcers as described earlier.

Biochemical parameters
A 50 dilution of the stomach homogenate was prepared and the activities of superoxide dismutase and catalase were evaluated as described by Ekanem & Yusuf, [18]. The levels of Malonaldehyde as an index of lipid peroxidation was estimated as described previously [19] while method described by [20] was used for total proteins estimation. Histological examination was conducted following the procedure of [21].

Statistical analysis
Data obtained in this study were analysed using the statistical software SPSS 16.0, 2016. Numerical data were presented as Mean ± SD. The significance of the mean difference between two independent groups was determined using Student's t-test and one-way analysis of variance (ANOVA) while multiple comparisons were used when comparing more than two groups. A p-value<0.05 was considered significant.

Results and discussion
The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66±6.66 %) in ethanol model, however, in acetylsalicylate model, crude extract exhibited the highest (54.16±4.16 %) and the lowest (33.33±4.16 %) protective effect at 1600 and 800 mg/kg bw respectively. The methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. Treatment with crude and fractions of A. dodenofolius significantly (p<0.05) increased the activities of SOD and Catalase and decreases the MDA concentration when compared with ulcerative untreated rats in both ulcer models.
Methanol crude extracts and fractions of A. dodoneifolius significantly (p<0.05) decrease the Malonaldehyde (MDA) content, compare to the control group of both models. An increase in MDA content is correlated to an increase in tissue damage. The control group of both the acetylsalicylate model and the ethanol model were elevated compared to the treated groups which had significantly (p<0.05) reduced MDA content. The MDA is the functional product of lipid peroxidation and it is used to determine lipid peroxidation levels [22]. Increasing lipid peroxidation production is an important cause of NSAID's damaging effects on stomach tissue [22]. Earlier studies have shown that gastric damage caused an increased MDA level [23] The result indicates marked increase in the catalase level when administered with methanol crude extract and fractions for the two models (ethanol and acetylsalicylate) as compared to the control. Catalase involvement in gastric injury caused by ethanol is seen when catalase functions in the conversion of hydrogen peroxide to water. Catalase activity is indicative of oxidative stress and significantly (p<0.05) increased upon induction by the ethanol and acetylsalicylate models [24]. These findings were in accordance with the main experimental studies which showed that catalase activity in stomach tissue increased gastric damage [25] In intact rat stomach tissue superoxide dismutase (SOD) activity was significantly (p<0.05) lower than that of the control rats in the ethanol model and there were no significant (p<0.05) difference in SOD content of the acetylsalicylate model. SOD activity is least in damage tissues and it is highest in healthy tissues [26] The results revealed that the mean ulcer score of the two models (acetylsalicylate and ethanol) in the untreated groups were 8.00 ± 0.52 and 5.00 ± 0.57 respectively. The mean ulcer scores indicated the severity of the ulcer induction and the acetylsalicylate has more ulceration as compared with the ethanol model. . The higher mean ulcer score of the acetylsalicylate indicated higher epithelial damage than the ethanol model during ulceration and this findings can be compared to the earlier studies [27]. The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66±6.66 %) in ethanol model, however, in acetylsalicylate model, crude extract exhibited the highest (54.16±4.16 %) and the lowest (33.33±4.16 %) protective effect at 1600 and 800 mg/kg bw respectively.
The histological findings revealed that the methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. The result may not be unconnected with the earlier report of the acetylsalicylate damaging more epithelium, hence low level of protection as compared to the ethanol model in this study. The negative control without treatment shows discontinuity in mucosal epithelium in acetylsalicylate model of rat ulceration. A standard drug (cimetidine) of 50g/kg bw showed no ulcer formation and a similar result was gotten from the treated group when a crude extract of A. dodoneifolius with a dose of 1600mg/kg bw was administered.

Conclusion
The study showed that the crude extract and fractions (methanol, n-haxane and ethylacetate) of Agelanthus dodoneifolius have significant anti-ulcer activity with high safety margin. The crude methanolic extract of the leaves and its various fractions possess significant phytochemicals and antioxidant activities demonstrates potent anti-ulcer activities on the Wister rats. The evidence obtained supported the use of the plant for treatment of ailment as traditional herb by the natives, particularly Africans. Therefore, the crude extract and fractions of Agelanthus dodoneifolius are potential sources of new plant based antiulcer agent.

Statement of ethical approval
The principles governing the use of laboratory animals as laid out by the Federal University of Technology, Minna Committee on Ethics for Medical and Scientific Research and also existing internationally accepted principles for laboratory animal use and care as contained in the Canadian Council on Animal Care Guidelines and Protocol Review were duly observed.