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Sources of resistance to the watermelon strain of Papaya ringspot virus (PRSV-W) have been identified within the watermelon (Citrullus lanatus) germplasm collection. Inheritance of the resistance to PRSV-W was studied in three Citrullus amarus (formerly C. lanatus var. citroides) plant introduction (PI) accessions: PI 244017, PI 244019, and PI 485583. Three susceptible parent lines, 'Allsweet', 'Calhoun Gray', and 'New Hampshire Midget', were crossed with resistant PI accessions to develop F1, F2, and BC1 progenies in six families. A single recessive gene was found to control the resistance to PRSV-W in all three resistant PI accessions. Allelism tests indicated that the three PI accessions carry the same resistance allele to PRSV-W. The gene symbol 'prv' is proposed for PRSV-W resistance in PI 244017, PI 244019, and PI 485583 in watermelon.
Bacterial fruit blotch (BFB) caused by the bacterium Acidovorax avenae subsp. citrulli (Aac) is a seed-borne disease that threatens most cucurbit crops. Although limited resistance has been found in a small number of Plant introductions (PIs) in watermelon (Citrullus spp.), there are no reports of high levels of resistance in germplasm lines of Cucumis spp. In this study, 332 Cucumis spp. PIs were screened for resistance to Aac using a newly developed seed vacuum-infusion assay. Significant differences in the reaction of the PI to BFB were observed. The majority of lines were found to be extremely susceptible to the disease. However, several PIs with lower levels of resistance were also identified. Variability in the reaction of plants within each PI was also observed. Of the 332 PI tested, 16 were selected for additional evaluation using a standard spray inoculation tests. PI 353814, PI 381171, PI 536573, and PI 614401, all belonging to C. melo, and PI 504558 (C. ficifolius) were found to have significantly greater levels of resistance than susceptible control cultivars or other PIs in two independent spray inoculation tests. Germplasm lines developed from these PIs may be useful as sources of resistance to BFB in Cucumis breeding programs.
Pigweeds (genus Amaranthus) are problematic weeds in crop production throughout the world and are responsible for significant yield losses in many crops (2). Members of this genus can produce hundreds of thousands of seeds per plant and are also capable of supporting populations of important crop pathogens including viruses, nematodes, fungi, and oomycetes. Thirty-one pigweed samples (tentatively identified as Amaranthus lividus L. based on leaf notch and growth habit) were collected in November and December of 2009 from a watermelon field near Immokalee, FL, previously found to contain watermelon plants infected with three whitefly-transmitted viruses: Cucurbit yellow stunting disorder virus (CYSDV), Cucurbit leaf crumple virus (CuLCrV), and Squash vein yellowing virus (SqVYV). Although no obvious virus symptoms were observed on any of the pigweed plants, whiteflies (Bemisia tabaci), a known vector of CYSDV, CuLCrV, and SqVYV, were observed on leaves. Consequently, replica tissue blots were made from all...
Genes that encode eukaryotic initiation factors (eIF) 4E and iso(4E) have been associated with the recessively inherited resistance to potyviruses in a number of plant species. Using previously developed degenerate primers, partial eIF4E and eIF(iso)4E gene sequence regions were obtained through polymerase chain reaction of the genomic DNA templates prepared from the Zucchini yellow mosaic virus (ZYMV)-resistant U.S. Plant Introduction (PI) 595203, and the ZYMV-susceptible 'New Hampshire Midget' (NHM). A single nucleotide polymorphism (SNP) was identified in the intron II region of the eIF4E sequence between the resistant and susceptible parental genotypes. F2 and BC1 populations were developed using PI 595203 and NHM and each plant was phenotyped for resistance to ZYMV and resistance was found to be conferred by a single recessive gene. A codominant Cleaved Amplified Polymorphic Sequence (CAPS) marker was created using the identified SNP in the eIF4E gene. This marker was linked to the ZYMV resistance locus (zym) at a linkage distance of 7 cM as estimated with the F2 and BC1 populations. A marker in the eIF(iso)4E gene was created and was not linked to the ZYMV resistance locus.
Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is a major vegetable crop in the world, accounting for 6.8% of the world area devoted to vegetable crops. Watermelon is a useful vegetable crop for genetic research because of its small genome size, and the many available gene mutants. The watermelon genes were originally organized and summarized in 1944, and have been expanded and updated periodically. However, the action of some watermelon genes has not been described clearly in some cases. Also, the interaction of multiple gene loci that control similar traits needs to be described more clearly. Finally, it is necessary to identify the inbred lines having each published gene mutant, for use as type lines in studies of gene action, allelism, and linkage. The objective of this work was to update the gene list, identify the cultivar or line having each gene mutant, and collect seeds of the lines for use by interested researchers. In addition, the gene descriptions were expanded and clarified, information on gene interactions was added, and errors in naming or citing previously described genes were corrected. New genes that have not previously been described (cr, Ctr, dw-3, ms-2, Ti, ts and zym-FL) were added to the list, for a total of 163 watermelon gene mutants.
How sugar transporters regulate sugar accumulation in fruits is poorly understood and particularly so for species storing high concentration sucrose. Accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a major quality trait, had been selected during domestication. Still, the molecular mechanisms controlling this quantitative trait are unknown. We resequenced 96 recombinant inbred lines, derived from crossing sweet and unsweet accessions, to narrow down the size of a previously described sugar content quantitative trait locus, which contains a putative Tonoplast Sugar Transporter gene (ClTST2). Molecular and biochemical analyses indicated that ClTST2 encodes a vacuolar membrane gene, whose expression is associated with tonoplast uptake and accumulation of sugars in watermelon fruit flesh cells. We measured fruit sugar content and resequenced the genomic region surrounding ClTST2 in 400 watermelon accessions and associated the most sugar-related significant SNP to the ClTST2 promoter. Large-scale population analyses strongly suggests increased expression of ClTST2 as a major molecular event in watermelon domestication associated with a selection sweep around the ClTST2 promoter. Further molecular analyses explored the binding of a sugar induced transcription factor (SUSIWM1) to a sugar responsive cis element within the ClTST2 promoter, which contains the QTL causal SNP. The functional characterization of ClTST2 and its expression regulation by SUSIWM1 provide novel tools to increase sugar sink potency in watermelon and possibly in other vegetable and fruit crops.
The common flesh color of commercially grown watermelon is red due to the accumulation of lycopene. However, natural variation in carotenoid composition that exists among heirloom and exotic accessions results in a wide spectrum of flesh colors. We previously identified a unique orange flesh watermelon accession (NY0016) that accumulates mainly β-carotene and no lycopene. We hypothesized this unique accession could serve as a viable source for increasing provitamin A content in watermelon. Here we characterize the mode of inheritance and genetic architecture of this trait. Analysis of testcrosses of NY0016 with yellow and red fruited lines indicated a codominant mode of action as F1 fruits exhibited a combination of carotenoid profiles from both parents. We combined visual color phenotyping with genotyping-by-sequencing of an F2:3 population from a cross of NY0016 by a yellow fruited line, to map a major locus on chromosome 1, associated with β-carotene accumulation in watermelon fruit. The QTL interval is approximately 20 cM on the genetic map and 2.4 Mb on the watermelon genome. Trait-linked marker was developed and used for validation of the QTL effect in segregating populations across different genetic backgrounds. This study is a step toward identification of a major gene involved in carotenoid biosynthesis and accumulation in watermelon. The codominant inheritance of β-carotene provides opportunities to develop, through marker-assisted breeding, β-carotene-enriched red watermelon hybrids.
Root-knot nematodes [Meloidogyne arenaria (Neal) Chitwood, Meloidogyne incognita (Kofoid & White) Chitwood, and Meloidogyne javanica (Treub) Chitwood] are serious pests of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in the southern United States and worldwide. Watermelon cultivars with resistance to any of these nematode pests are not available. Therefore, we evaluated all accessions of Citrullus colocynthis (L.) Schrad.(21) and Citrullus lanatus (Thunb.) Matsum. & Nakai var. citroides (L.H. Bailey) Mansf.(88), and about 10% of C. lanatus var. lanatus (156) accessions from the U.S. Plant Introduction (PI) Citrullus germplasm collection for resistance to M. arenaria race 1 in greenhouse tests. Only one C. lanatus var. lanatus accession exhibited very low resistance [root gall index (GI) = 4.9] and 155 C. lanatus var. lanatus accessions were susceptible (GI ranged from 5.0 to 9.0, where 1 = no galls and 9 = ≥81% root system covered with galls). All C. colocynthis accessions were highly susceptible (GI range = 8.5 to 9.0). However, 20 of 88 C. lanatus var. citroides accessions were moderately resistant with a GI range of 3.1 to 4.0; overall GI range for the C. lanatus var. citroides accessions was 3.1 to 9.0. Resistance to M. arenaria race 1 identified in the C. lanatus var. citroides accessions was confirmed on a subset of accessions in a replicated greenhouse test. The results of our evaluations demonstrated that there is significant genetic variability within the U.S. PI Citrullus germplasm collection for resistance to M. arenaria race 1 and also identified C. lanatus var. citroides accessions as potential sources of resistance.
The B-biotype sweetpotato whitefly, Bemisia tabaci (Gennadius), feeds on and damages numerous vegetable crops including watermelon (Citrullus sp.). Seven watermelon cultivars, a triploid line, and 16 U.S. Plant Introduction accessions (PIs) of C. lanatus var. lanatus; 10 PIs of C. lanatus var. citroides; and eight PIs of C. colocynthis, were evaluated for resistance to B. tabaci. Bioassays were based on nonpreference and performance of the whiteflies on the 42 Citrullus genotypes. Most of the watermelon cultivars and C. lanatus PIs tested were highly susceptible to whitefly infestation, while the C. colocynthis accessions exhibited whitefly resistance. Among the C. colocynthis accessions tested, PI 386015, PI 386018, and PI 386024 were most resistant to B. tabaci. This study identified useful sources of germplasm that can be used for the improvement of watermelon for resistance to whiteflies.
A genetic linkage map was constructed for watermelon using 117 recombinant inbred lines (RILs) (F2S7) descended from a cross between the high quality inbred line 97103 [Citrullus lanatus var. lanatus (Thunb.) Matsum. & Nakai] and the Fusarium wilt (races 0, 1, and 2) resistant U.S. Plant Introduction (PI) 296341 (C. lanatus var. citroides). The linkage map contains 87 randomly amplified polymorphic DNA (RAPD) markers, 13 inter simple sequence repeat (ISSR) markers, and four sequenced characterized amplified region (SCAR) markers. The map consists of 15 linkage groups. Among them are a large linkage group of 31 markers covering a mapping distance of 277.5 cM, six groups each with 4 to 12 markers covering a mapping distance of 51.7 to 172.2 cM, and eight small groups each with 2-5 markers covering a mapping distance of 7.9 to 46.4 cM. The map covers a total distance of 1027.5 cM with an average distance of 11.7 cM between two markers. The map is useful for the further development of quantitative trait loci (QTLs) affecting fruit qualities and for identification of genes conferring resistance to Fusarium wilt (races 0, 1 and 2). The present map can be used for further construction of a reference linkage map for watermelon based on an immortalized mapping population with progenies homozygous for most gene loci.
The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), often causes serious damage to watermelon ( Citrullus spp . ), and there is a need to evaluate and identify watermelon germplasm resistant to T. urticae . Watermelon cultivars ( Citrullus lanatus var. lanatus ), and U.S. plant introduction (PI) accessions of C. lanatus var. citroides and C. colocynthis, were evaluated for preference by T. urticae (number of adults and eggs on leaves). In open-choice experiments in the greenhouse and in laboratory rearing cages, there was a significant preference by T . urticae for watermelon cultivars, Citrullus lanatus var. lanatus PIs, and C. lanatus var. citroides PIs over C. colocynthis PIs . All watermelon cultivars and PIs were infested, but the C. colocynthis PIs were significantly less infested with T. urticae . The C. colocynthis PIs may be useful sources for enhancing T. urticae resistance in cultivated watermelon.
Powdery mildew has been reported on Citrullus lanatus in Africa and Europe for the past 9 years, and in the United States for the past 6 years. During this time, it has occurred in the main watermelon production areas in the U.S. and has been documented in nine states (South Carolina, Georgia, Florida, Oklahoma, Texas, Maryland, New York, Arizona, and California). This is of great concern to the watermelon industry since powdery mildew is difficult to control and can have a severe impact on yield and fruit quality due to loss of photosynthetic area and sunscald. Finding resistant C. lanatus germplasm is needed for the development of commercial varieties containing this resistance. This report summarized the status of an ongoing project to screen the entire USDA–ARS C. lanatus germplasm collection. Currently, the collection is being screened for race 1 and race 2 Podosphaera xanthii (syn. Sphaerotheca fuliginea auct. p.p.), the causal agent of powdery mildew in C. lanatus in the United States. Resistance genes appear to exist for both races and the genes conferring resistance to race 1 appear to be different than race 2 resistance genes. Allelism tests are currently in process to determine the number of resistance genes present.
Differential modulation of 127 2x-ESTs was confirmed by Q-PCR using two additional biological replicates. One hundred twenty-seven 2x-ESTs that show differential modulation in the microarray (Biorep 1) were confirmed by Q-PCR (Biorep 2 & Biorep 3). 2x-ESTs induced at least two-fold were highlighted in red whereas 2x-ESTs repressed at least two-fold were highlighted in green. Q-PCR fold inductions are shown for each 2x-EST for green, pink, and red flesh as compared to leaf.
The flowering pattern of watermelon species (Citrullus spp.) is either monoecious or andromonoecious. Ethylene is known to play a critical role in floral sex determination of cucurbit species. In contrast to its feminizing effect in cucumber and melon, in watermelon ethylene promotes male flower development. In cucumber, the rate-limiting enzyme of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development, we isolated four genomic sequences of ACS from watermelon (CitACS1-4). Both CitACS1 and CitACS3 are expressed in floral tissue. CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate precursor of ethylene. No discernible differential floral sex-dependent expression pattern was observed for this gene. The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite), but not in female flowers. CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development. The expression of CitACS2 was not detected in vegetative or reproductive organs but was up-regulated by auxin. CitACS4 transcript was not detected under our experimental conditions. Restriction fragment length polymorphism (RFLP) and sequence tagged site (STS) marker analyses of the CitACS genes showed polymorphism among and within the different Citrullus groups, including watermelon cultivars, Citrullus lanatus var. lanatus, the central subspecies Citrullus lanatus var. citroides, and the desert species Citrullus colocynthis (L).
Powdery mildew (Podosphaera xanthii) can cause severe damage to cucurbit crops grown in open fields and greenhouses. In recent years, there has been an increased interest in the United States in grafting watermelon plants onto various cucurbit rootstocks. Bottle gourd plants (Lagenaria siceraria) are being used throughout the world as rootstocks for grafting watermelon. Although gourd plants are beneficial, they may still be susceptible hosts to various soilborne and foliar diseases. Bottle gourd plant introductions (PI) resistant to diseases and pests can be a valuable source of germplasm in rootstock breeding programs. We evaluated 234 U.S. PIs of L. siceraria for tolerance to powdery mildew in two greenhouse tests. Young seedlings were inoculated by dusting powdery mildew spores of melon race 1 on the cotyledons. Plants were rated 2 weeks after inoculation using a 1 to 9 scale of increasing disease severity. Although none of the L. siceraria PIs were immune to powdery mildew, several PIs had significantly lower levels of powdery mildew severity compared with susceptible watermelon cultivar Mickey Lee. The experiment was repeated with 26 select PIs on whole seedlings and cotyledon disks. Significant variability in the level of resistance to powdery mildew on plants within PI was observed. Moderate resistance in several PIs to powdery mildew was confirmed. PI 271353 had consistently lower ratings in the various tests and can be considered the most resistant to P. xanthii race 1 among the L. siceraria accessions evaluated in this study. A few other PIs with moderate resistance to powdery mildew included PI 271357, PI 381840, and PI 273663. These results suggest that novel sources of resistance could be developed by careful selection and screening of several of the PIs with moderate resistance described in our study.
The primary purpose of grafting vegetables worldwide has been to provide resistance to soilborne diseases. The potential loss of methyl bromide as a soil fumigant combined with pathogen resistance to commonly used pesticides will make resistance to soilborne pathogens even more important in the future. The major disease problems addressed by grafting include fusarium wilt, bacterial wilt, verticillium wilt, monosporascus root rot, and nematodes. Grafting has also been shown in some instances to increase tolerance to foliar fungal diseases, viruses, and insects. If the area devoted to grafting increases in the future, there will likely be a shift in the soil microbial environment that could lead to the development of new diseases or changes in the pathogen population of current diseases. This shift in pathogen populations could lead to the development of new diseases or the re-emergence of previously controlled diseases. Although grafting has been demonstrated to control many common diseases, the ultimate success will likely depend on how well we monitor for changes in pathogen populations and other unexpected consequences.
Vegetable grafting began in the 1920s using resistant rootstock to control soilborne diseases. This process is now common in Asia, parts of Europe, and the Middle East. In Japan and Korea, most of the cucurbits and tomatoes (Lycopersicon esculentum Mill.) grown are grafted. This practice is rare in the United States, and there have been few experiments to determine optimal grafting production practices for different geographical and climatic regions in America. This is beginning to change as a result of the phase out of methyl bromide. The U.S. cucurbit and tomato industries are evaluating grafting as a viable option for disease control. Because reports indicate that type of rootstock alters yield and quality attributes of the scion fruit, some seed companies are investigating grafting as a means to improve quality. It has been reported that pH, flavor, sugar, color, carotenoid content, and texture can be affected by grafting and the type of rootstock used. Reports vary on whether grafting effects are advantageous or deleterious, but it is usually agreed that the rootstock/scion combination must be carefully chosen for optimal fruit quality. Additionally, it is important to study rootstock/scion combinations under multiple climatic and geographic conditions because many rootstocks have optimal temperature and moisture ranges. This report gives an overview of the effect of grafting on vegetable quality.
Thirty-eight melon accessions, which are of primary breeding importance in the Ukraine, were analysed for diversity. These collections represent a major non-US and non-western Europe source of melon germplasm that have not yet been subjected to molecular characterization. Molecular diversity was estimated based on a robust set of 465 polymorphisms gathered by amplified fragment length polymorphisms and simple sequence repeats (SSR). In this paper, we report 12 newly developed polymorphic SSR primer pairs, and their use for molecular characterization in the Ukrainian melon collections. Based on these polymorphisms, we estimated similarity indices that ranged from 0.70 to 1.00 among various accessions. The phylogenetic tree based on the similarity indices and a three-dimensional plot of the first three vectors of the principal component analysis corresponded fairly well with the existing three classical morphotypes namely aestivalis, europeus and hiemalis, under the convar Europeus, which is also known as adana. The polymorphisms generated in the current study, which are specific to the grouping of fruit types and days to maturity will be very useful for further genetic studies and marker-assisted selections.
Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar 'New Hampshire Midget' ('NHM') showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F(2) and 114 BC(1R) progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F(2) and BC(1R) populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.
A study was conducted to evaluate the use of reflective mulch and host plant resistance for the management of the sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), in watermelon [Citrullus lanatus var. lanatus (Thunberg) Matsum & Nakai]. Whitefly abundance data were collected under both greenhouse (caged and uncaged) and field conditions. Consistently, a reflective mulch (also called silver or metallic) treatment resulted in a lower incidence of adult whiteflies as compared with a standard black mulch treatment. Moreover, two whitefly resistant Citrullus colocynthis (L.) Schrad genotypes, which are wild relatives of cultivated watermelon, reduced whitefly populations as compared with standard watermelon. There was generally no interaction between the mulch and genotype variables. No effect of mulch color was observed on sticky card capture of Delphastus catalinae (Horn), a whitefly predator, or on capture of an Eretmocerus sp. whitefly parasitoid in caged trials, which suggests no adverse effect on these natural enemies when using reflective mulch. Overall whitefly populations were relatively low during four seasons of field trials (2006–2009). Results from this study suggest that a combination of using reflective mulch and host plant resistance can additively suppress whitefly infestations, which have particular importance in the fast-growing organic vegetable production industry.
We did fluorescent in situ hybridization (FISH) in cultivated watermelon var. lanatus (PI 270306) and its wild counterpart var. citroides (PI 244018), using 18S-28S rDNA and 5SrDNA probes. Well separated somatic chromosomes were prepared from root meristems, using enzyme digestion technique for in situ hybridization following the standard protoplast technique to prepare somatic chromosome spread. We have observed two different organizational features in these two species. In lanatus, we have identified two major 18S-28S rDNA sites and these are located on two different homologous pairs of chromosomes. One 5S rDNA site, on the other hand, was observed and located on a pair of homologous pairs of chromosomes. A two-color FISH (dual FISH) showed the 5S rDNA site was located interstitially and was syntenic to one of the 18S-28S rDNA sites . As revealed by the interphase FISH , the 18S-28S rDNA and the 5S rDNA loci are spaced out and may not tightly linked to each other. In contrary, only one 18S-28S rDNA site and two 5S rDNA sites were observed in Citroides accession (PI 244018). To our knowledge, it is unusual in plant species, where the number of 18S-28S rDNA sites is always higher than the 5S rDNA site. A dual FISH showed that all three rDNA sites were on three different pairs of homologous chromosomes . During the evolutionary process the 18S-28S rDNA site has been duplicated in lanatus and one of the 5S rDNA sites might have lost in this species. These results indicated that some structural rearrangements might have occurred during the evolution of lanatus. Further, meiocytes analysis of pollen mother cell involving the rDNA FISH with 18S-28S rDNA and 5S rDNA probes would shed light on structural rearrangements in lanatus. The current research also explored additional insights such as extent of diversity at the methylation level among the watermelon cultivars. DNA profiles were generated using Methylation-Sensitive AFLP Assay for 47 watermelon heirlooms. Results indicated that methylation specific diversity (43%) in US watermelon heirlooms is higher than the diversity (19.8%) as estimated by several investigators using conventional DNA markers.
Key message: A major quantitative trait locus (QTL) for Fusarium oxysporum Fr. f. sp. niveum race 1 resistance was identified by employing a "selective genotyping" approach together with genotyping-by-sequencing technology to identify QTLs and single nucleotide polymorphisms associated with the resistance among closely related watermelon genotypes. Fusarium wilt is a major disease of watermelon caused by the soil-borne fungus Fusarium oxysporum Schlechtend.:Fr. f. sp. niveum (E.F. Sm.) W.C. Snyder & H.N. Hans (Fon). In this study, a genetic population of 168 F3 families (24 plants in each family) exhibited continuous distribution for Fon race 1 response. Using a "selective genotyping" approach, DNA was isolated from 91 F2 plants whose F3 progeny exhibited the highest resistance (30 F2 plants) versus highest susceptibility (32 F2 plants), or moderate resistance to Fon race 1 (29 F2 plants). Genotyping-by-sequencing (GBS) technology was used on these 91 selected F2 samples to produce 266 single nucleotide polymorphism (SNP) markers, representing the 11 chromosomes of watermelon. A major quantitative trait locus (QTL) associated with resistance to Fon race 1 was identified with a peak logarithm of odds (LOD) of 33.31 and 1-LOD confidence interval from 2.3 to 8.4 cM on chromosome 1 of the watermelon genetic map. This QTL was designated "Fo-1.1" and is positioned in a genomic region where several putative pathogenesis-related or putative disease-resistant gene sequences were identified. Additional independent, but minor QTLs were identified on chromosome 1 (LOD 4.16), chromosome 3 (LOD 4.36), chromosome 4 (LOD 4.52), chromosome 9 (LOD 6.8), and chromosome 10 (LOD 5.03 and 4.26). Following the identification of a major QTL for resistance using the "selective genotyping" approach, all 168 plants of the F 2 population were genotyped using the SNP nearest the peak LOD, confirming the association of this SNP marker with Fon race 1 resistance. The results in this study should be useful for further elucidating the mechanism of resistance to Fusarium wilt and in the development of molecular markers for use in breeding programs of watermelon.
We used genotyping by sequencing to identify a set of 10,480 single nucleotide polymorphism (SNP) markers for constructing a high-resolution genetic map of 1,096 cM for watermelon. We assessed the genome-wide variation in recombination rate (GWRR) across the map and found an association between GWRR and genome-wide nucleotide diversity. Colinearity between the map and the genome-wide reference sequence for watermelon was studied to identify inconsistency and chromosome rearrangements. We assessed genome-wide nucleotide diversity, linkage disequilibrium (LD) and selective sweep for wild, semi-wild and domesticated accessions of Citrullus lanatus var. lanatus to track signals of domestication. Principal component analysis combined with chromosome-wide phylogenetic study based on 1,563 SNPs obtained after LD pruning with minor allele frequency = 0.05 resolved the differences between semi-wild and wild accessions as well as relationships among worldwide sweet watermelon. Population structure analysis revealed predominant ancestries for wild, semi-wild and domesticated watermelons as well as admixture of various ancestries that were important for domestication. Sliding window analysis of Tajima's D across various chromosomes was used to resolve selective sweep. LD decay was estimated for various chromosomes. We identified a strong selective sweep on chromosome 3 consisting of important genes that might have had a role in sweet watermelon domestication.
Selection for and incorporation of host plant resistance into cultivars is a fundamental strategy to control insects and diseases and may help reduce reliance on synthetic pesticides. The sweetpotato whitefly, Beinisia tabaci (Gennadius), is an important pest of watermelon [Citrullus lanatus (Thunberg) Matsum. and Nakai var. lanatus] and is among the most damaging pests in many agricultural systems worldwide. Citrullus colocynthis L., a perennial melon species indigenous to arid regions of northern Africa, the Mediterranean region, and southwestern Asia, is a valuable source of resistance to insect pests and diseases of watermelon. Laboratory and greenhouse experiments were conducted to evaluate selected C. colocynthis genotypes for sources of resistance to B. tabaci. Thirty genotypes of C. colocynthis, collected in different geographic regions, were evaluated against the heirloom cultivar Calhoun Gray using first a horizontal Y-tube olfactometer in the laboratory. A selected subset of the genotypes was evaluated in a second experiment in the laboratory using a vertical monitoring assay. In this assay, whiteflies could freely move upward to feed and oviposit on leaves placed in the upper portion of a Y-tube. In a third experiment, a choice assay was conducted on selected genotypes in cages in the greenhouse. Of the 30 C. colocynthis genotypes evaluated, PI 346082 (collected in Afghanistan) exhibited the highest level of resistance against B. tabaci based on all three experiments. PI 537277 (collected in Pakistan) exhibited a significantly high level of whitefly resistance based on low survival of adult whiteflies and a low ratio of nymphs to eggs. P1346082 and PI 537277 should be a useful source for breeding projects aiming to improve whitefly resistance in watermelon cultivars.
Additional index words. Carya illinoinensis, nucleic acid, embryos Abstract. A simple and efficient protocol is reported for the isolation of RNA from embryos and leaves of pecan [ Carya illinoinensis (Wangenh.) K. Koch]. The method relies on suppression of the polyphenols from interaction with the RNA and their rapid removal from the homogenate by chloroform extraction. This method produced abundant amounts of high-quality RNA. This protocol is likely to be useful for Juglanda-ceous species and other recalcitrant plants with high levels of phenolic compounds.
Methods were developed for the production of somatic embryos of asparagus (Asparagus officinalis L.) in suspension culture and subsequent conversion to plantlets on solidified medium. Stem-derived callus that was subcultured twice on Murashige and Skoog (MS) medium + 0.54 M naphthaleneacetic acid (NAA) and 1.4 M 2-isopentenyladenine (2iP) was used to initiate suspension cultures. Six out of 15 such cell suspensions (MS medium with 54 to 107 M NAA) had a high embryogenic capacity. These cell suspensions consisted primarily of single elongated cells (about 90% of all single cells), embryogenic cell clusters (2571/ml), and globular translucent embryos (32/ml). The latter converted to plantlets within four weeks on embryo development medium (EDM), which was solidified MS medium containing 0.54 M NAA and 0.98 M 2iP. Suspension-derived embryos formed secondary globular embryos at high frequencies (251 to 258/g callus) when placed on EDM with a low carbohydrate (sucrose, glucose or fructose) level (2%). In contrast, EDM with a high carbohydrate level (10%) caused a reduction in the frequency of secondary embryos (30 to 85/g callus), while resulting in the promotion of embryo growth and conversion, 3.6 to 8.5 times higher than 2% carbohydrates. Transfer of globular somatic embryos from cell suspension to EDM with high carbohydrate levels (4 to 10%) for two weeks followed by transfer to EDM with a low carbohydrate level (2%) resulted in a 2 to 4 times higher conversion rate to plantlets than those that remained at the 4 to 10% levels.
Root-knot nematode-resistant rootstock lines (designated RKVL for Root-Knot Vegetable Laboratory) derived from wild watermelon (Citrullus lanatus var. citroides) were compared with wild tinda (Praecitrullus fistulosus) lines and commercial cucurbit rootstock cultivars for grafting of seedless triploid watermelon 'Tri-X 313' (C. lanatus var. lanatus) in a field infested with the southern root-knot nematode (RKN) (Meloido-gyne incognita) in Charleston, SC, during 2009 and 2010. In both years, RKN infection was severe in 'Emphasis' bottle gourd, 'Strong Tosa' hybrid squash, and wild tinda rootstocks with galling of the root system ranging from 86% to 100%. In 2009, the RKVL wild watermelon rootstocks exhibited lower (P < 0.05) percentages of root galling (range 9% to 16%) than non-grafted 'Tri-X 313' (41%), 'Emphasis', 'Strong Tosa', and the wild tinda rootstocks. The grafted wild watermelon rootstock RKVL 318 produced more (P £ 0.05) fruit (12 per plot) than all other entries (mean = five per plot), and it produced a heavier (P £ 0.05) fruit yield (29.5 kg per plot) than all entries except self-grafted 'Tri-X 313' (21.5 kg per plot). In 2010, soil in half of the plots was treated with methyl bromide (50%):chloropicrin (50%) (392 kg · ha –1) before planting. The RKVL wild watermelon rootstocks exhibited resistance to RKN with percentages of root system galled ranging from 11% for RKVL 316 to 56% for RKVL 301 in the untreated control plots. Fruit yields in the untreated plots were 21.9, 25.6, and 19.9 kg/plot for RKVL 301, RKVL 316, and RKVL 318, respectively. Yields were greater (P £ 0.05) for the three RKVL rootstocks than for 'Strong Tosa' (3.0 kg) and 'Ojakkyo' wild watermelon rootstock (2.8 kg) in the untreated plots. Yields of watermelon grafted on 'Strong Tosa' were nearly seven times greater (P £ 0.05) in the methyl bromide-treated plots than in the untreated plots. In contrast, yields of RKVL 316 and RKVL 318 were similar in both treatments and the yield of RKVL 301 was less (P £ 0.05) in the methyl bromide-treated plots than in the untreated plots. The three RKVL wild watermelon rootstock lines exhibited resistance to RKN. RKVL 316 had low root galling and produced the heaviest fruit yield and greatest numbers of fruit of any rootstock evaluated in 2010. The RKVL lines should be useful sources of RKN resistance for rootstocks for grafted watermelon. The southern RKN (Meloidogyne incog-nita) is a serious constraint to U.S. watermelon production and can significantly reduce watermelon yields in the southern
Genetic diversity and relatedness were assessed among 46 American cultivars of watermelon (Citrullus lanatus var. lanatus), and 12 U.S. Plant Introduction accessions (PIs) of Citrullus sp. using 25 randomly amplified polymorphic DNA (RAPD) primers. These primers produced 288 distinct reproducible bands that could be scored with high confidence among cultivars and PIs. Based on the RAPD data, genetic similarity coefficients were calculated and a dendrogram was constructed using the unweighted pair-group method with arithmetic average (UPGMA). The cultivars and C. lanatus var. lanatus PIs differentiated at the level of 92% to 99.6% and 88% to 95% genetic similarity, respectively. In contrast, the C. lanatus var. citroides, and C. colocynthis PIs were more divergent and differentiated at the level of 65% to 82.5% and 70.5% genetic similarity, respectively. The low genetic diversity among watermelon cultivars in this study emphasizes the need to expand the genetic base of cultivated watermelon.
Citrullus colocynthis (CC) is a viable source of genes for enhancing disease and pest resistance in common cultivated watermelon [Citrullus lanatus var. lanatus (CLL)] cultivars. However, there is little information about genetic diversity within CC or the relationship of CC accessions to C. lanatus. In this study, we examined genetic diversity and relationships among 29 CC accessions collected in northern Africa, the Middle East, and Asia, and their relationships to 3 accessions and 3 cultivars of CLL, 12 accessions of citron melon [C. lanatus ssp. lanatus var. citroides (CLC)], and 1 accession representing the desert perennial Citrullus ecirrhosus (CE). Twenty-three high-frequency oligonucleotides-targeting active gene (HFO–TAG) primers were used to produce a total of 431 polymorphic fragments that target coding regions of the genome. Cluster and multidimensional scaling plot analysis, separated the CC into five groups, in general agreement with their geographic origins. CC genotypes admixed with CLL and CLC also were identified. Major reproductive barriers resulted in significantly reduced fertility in CC · CLL hybridizations. However, several of the U.S. PIs of CC were successfully crossed with watermelon cultivars using traditional breeding procedures, and the seeds produced from these crosses were viable. This suggests that CC can be a viable source to introduce biotic and abiotic stress resistance genes into cultivated watermelon. © 2017, American Society for Horticultural Science. All rights reserved.
A genetic linkage map was constructed for watermelon using a testcross population [Plant Accession Griffin 14113 ( Citrullus lanatus var. citroides) x New Hampshire Midget (NHM; C. lanatus var. lanatus)] x U.S. Plant Introduction (PI) 386015 ( Citrullus colocynthis). The map contains 141 randomly amplified polymorphic DNA (RAPD) markers produced by 78 primers, 27 inter-simple sequence repeat (ISSR) markers produced by 17 primers, and a sequence-characterized amplified region (SCAR) marker that was previously reported as linked (1.6 cM) to race-1 Fusarium wilt [incited by Fusarium oxysporum Schlechtend.:Fr. f. sp. niveum (E.F.Sm.) W.C. Synder & H.N. Hans] resistance in watermelon. The map consists of 25 linkage groups. Among them are a large linkage group that contains 22 markers covering a mapping distance of 225.6 cM and six large groups each with 10-20 markers covering a mapping distance of 68.8 to 110.8 cM. There are five additional linkage groups consisting of 3-7 markers per group, each covering a mapping distance of 36.5 to 57.2 cM. The 13 remaining linkage groups are small, each consisting of 2-11 markers covering a mapping distance of 3.5-29.9 cM. The entire map covers a total distance of 1,166.2 cM with an average distance of 8.1 cM between two markers. This map is useful for the further development of markers linked to disease resistance and watermelon fruit qualities.
Genetic diversity was estimated among 42 U.S. PlantIntroduction (PI) accessions of the genusCitrullus (of these, 34 PIs are reported tohave disease resistance), and 5 watermelon cultivars, using 30RAPD primers. These primers produced 662 RAPD markers that could berated with high confidence. Based on these markers, geneticsimilarity coefficients were calculated and a dendrogram wasconstructed using the unweighted pair-group method witharithmetic average (UPGMA). The analysis delineated threemajor clusters. The first cluster consisted of a group of fivewatermelon cultivars, a group of C.lanatus var. lanatusaccessions, and a group of C.lanatus var. lanatusaccessions that contained some C.lanatus var. citroidesgenes. The second cluster consisted of the C.lanatus var. citroidesaccessions, while the third cluster consisted of theC. colocynthis accessions.The two C. lanatus clustersdifferentiated from each other and from the C.colocynthis cluster at the level of 58.8%and 38.9% genetic similarity, respectively. Assessment ofgenetic diversity among accessions that have been reported to havedisease resistance indicated that resistance to either anthracnose,downy mildew, powdery mildew, or watermelon mosaic virus is foundamong all major groups of Citrullus PIs.Additionally, resistance to gummy stem blight or Fusarium wilt mayexist among C. lanatus var.citroides PIs. This study demonstrates thatmolecular markers can be useful in assessing genetic diversity, andin sorting Citrullus PIs into phylogeneticgroups prior to their evaluation for disease or pestresistance.
Key message: A major QTL for resistance to Fusarium oxysporum f. sp. niveum race 2 was mapped to a narrow 1.2 Mb interval using a high-density GBS-SNP linkage map, the first map of Citrullus lanatus var. citroides. Fusarium wilt, a fungal disease caused by Fusarium oxysporum f. sp. niveum (Fon), devastates watermelon crop production worldwide. Several races, which are differentiated by host range, of the pathogen exist. Resistance to Fon race 2, a particularly virulent strain prevalent in the United States, does not exist in edible cultivars of the sweet cultivated watermelon Citrullus lanatus var. lanatus (Cll) and has been well described in a few plant introductions of the wild subspecies of watermelon, C. lanatus var. citroides (Clc). Clc provides a vital source of genetic diversity, as well as resistance to numerous diseases. Unfortunately, both genetic diversity and disease resistance are lacking in Cll due to the narrow genetic base. Despite the importance of Clc to continued watermelon improvement, intra-variety genetic studies are lacking. Here, we present the first Clc genetic linkage map, generated with 2495 single nucleotide polymorphisms developed through genotyping-by-sequencing, and use it to identify quantitative trait loci associated with Fon race 2 resistance. Multiple QTL mapping in a Clc F2:3 population (N = 173) identified one major and four minor QTL. The major QTL explained 43% of the variation in Fon race 2 resistance and was delimited to a 1.2-Mb interval on chromosome 9, a region spanning 44 genes.
There is a continuous need to enhance watermelon cultivars for disease and pest resistance. Different U.S. Plant Introductions (PIs) of Citrullus lanatus subsp. lanatus var. lanatus [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) collected in southern Africa are a useful source for enhancing disease or pest resistance in watermelon cultivars. They are also valuable as rootstocks for grafted watermelon, particularly in fields infested with root-knot nematodes or Fusarium wilt. However, there is little information about genetic relationships among these PIs. In this study, genetic diversity was examined among 74 CLC PIs collected from their center of origin in southern Africa. Also, 15 Citrullus lanatus subsp. lanatus (CLL) PIs and the American heirloom cultivars Charleston Gray and Black Diamond (Citrullus lanatus subsp. vulgaris (Schrader ex Eckl. et Zeyh.) Fursa) (CLV) and five Citrullus colocynthis (L.) Schrader (CC) PIs collected in different locations throughout the world were used as out-groups in the phylogenetic analysis for the CLC PIs. Twenty-three high frequency oligonucleotides—targeting active gene (HFO-TAG) primers were used in polymerase chain reaction (PCR) experiments to produce a total of 562 polymorphic markers among the Citrullus PIs and cultivars. Cluster and multidimensional scaling plot analysis produced distinct groups of CLC, CLL, and CC PIs. Several PIs that were designated as CLC or CLL were in transitional positions, indicating that they are the result of gene flow between the major Citrullus groups or subgroups. Population structure analysis indicated that CLC comprises two subgroups; each containing a set of unique alleles. Also, unique alleles exist in the CLL and the CC genotypes. Overall, broad genetic diversity exists among the Citrullus PIs. The data in this study should be useful for identifying PIs with a wide genetic distance between them that could be used in breeding programs aiming to develop heterotic F1 hybrid rootstock lines for grafted watermelon.