Lanatus Var Research Articles

Genetic analyses of Turkish watermelons based on SRAP markers

Genetic studies of watermelons (Citrullus lanatus (Thunb.) Matsum. & Nakai) sampled from Turkey, the second leading producer, may provide valuable information for breeding and research programs. The objectives of this study were to estimate the level of genetic diversity, population structure, and optimum genome sampling size among the Turkish and several introduced watermelons. From the collection, 256 watermelon lines representing all watermelon-growing areas of Turkey along with two accessions of a related genus (Praecitrullus fistulosus (Stocks) Pangalo) and a few popular cultivars were genotyped using sequence-related amplified polymorphism (SRAP) markers. Twenty-seven primers generated 210 molecular markers for genetic analyses. The unweighted pair group method with arithmetic average analysis produced no clear-cut pattern, while principal component analysis indicated three subgroups among the lines studied. They had a narrow genetic base within C. lanatus var. lanatus and were mostly distinguished from each other. Model-based structure analysis indicated that the number of subpopulations in watermelons was four. Among the 258 lines, only 20% (51 lines) had 0.80 or greater membership coefficients to one subpopulation, and therefore were not admixed. The remaining 207 were admixed by at least two subpopulations. The similarity matrix of genome sampling size of 40 randomly selected markers was highly correlated (r = 0.915) with the matrix of simple matching coefficients based on 100 markers, implying that 40 markers were detected to be sufficient for this species. Overall, this study concluded that the majority of the Turkish watermelons were distinguished from each other, difficult to classify, and admixed, with narrow genetic variation.

Analyzing Lycopene Content in Fruits

Abstract Lycopene is an antioxidant carotenoid compound occurring in plants. This research determined lycopene content in fresh fruits, including tomatoes ( Solanum lycopersicum ), watermelons ( Citrullus lanatus var. lanatus ), jackfruits ( Artocarpus heterophyllus ), bananas ( Musa acuminata ), grapes ( Vitis vinifera ), oranges ( Citrus reticulata ) and papayas ( Carica papaya ). Lycopene in fresh fruit samples was extracted using hexane:ethanol:acetone (2:1:1). The results showed that lycopene in tomatoes ( Solanum lycopersicum ), watermelons ( Citrullus lanatus var. lanatus ), jackfruits ( Artocarpus heterophyllus ), bananas ( Musa acuminata ), grapes ( Vitis vinifera ), oranges ( Citrus reticulata ) and papayas ( Carica papaya ) were 104.699±0.000, 144.27 ±0.001, 4.122±0.000, 31.189±0.001, 10.028±0.000, 13.1904±0.000 and 45.342±0.000 mg/kg, respectively. The highest lycopene amount was found in watermelons ( Citrullus lanatus var. lanatus ) while the lowest lycopene quantity was observed in jackfruits ( Artocarpus heterophyllus ).

Genetic diversity within Turkish watermelon [Citrullus lanatus (Thunb.)Matsumura & Nakai] accessions revealed by SSR and SRAP markers

The genetic diversity of Turkish watermelon genotypes selected from the watermelon genetic resource collection was evaluated by simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) markers. Most of the accessions were collected from various geographical regions of Turkey. Different Citrullus species, wild relatives, foreign landraces, open pollinated (OP), and commercial hybrid cultivars were also assessed for genetic relatedness. Fourteen SSR primers and 31 SRAP primer combinations were used in the experiment. Both SSR markers (100%) and SRAP markers (97.3%) generated high polymorphisms. Based on the SSR and SRAP data, the genetic similarity coefficients were calculated, and dendrograms were constructed using the unweighted pair-group method with arithmetic average (UPGMA). Cluster and principle coordinate analyses indicated that Citrullus lanatus var. Lanatus subspecies genotypes collected from the different regions of Turkey were closely genetically related. Overall, our results displayed low genetic variability within the Turkish watermelon germplasm in contrast with their high morphological diversity.

An integrative morphological and molecular approach to identification of three Australian cucurbitaceous invasive weeds: Citrullus colocynthis, C. lanatus and Cucumis myriocarpus

Camel melon (Citrullus lanatus), colocynth (Citrullus colocynthis) and prickly paddy melon (Cucumis myriocarpus) are summer-growing invasive weeds distributed throughout Australia. We used DNA-sequence information from samples collected across Australia and morphological data from glasshouse-grown plants to determine diagnostic features of these species, and to determine the infraspecific identity of Australian Citrullus lanatus and Cucumis myriocarpus. All species possessed distinct genotypes and haplotypes at nuclear G3pdh and chloroplast ycf6–psbM gene regions and could be easily identified on the basis of molecular phylogenetic analysis. A combination of vegetative, floral, fruit and seed characters also allowed for species identification at all developmental stages. On the basis of our morphological and molecular analyses, Australian camel melon and prickly paddy melon populations were identified as Citrullus lanatus var. citroides and Cucumis myriocarpus subsp. myriocarpus respectively.

Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus).

An intraspecific genetic map for watermelon was constructed using an F2 population derived from ‘Arka Manik’ × ‘TS34’ and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits.

FISH Karyotype Analysis of Four Wild Cucurbitaceae Species Using 5S and 45S rDNA Probes and the Emergence of New Polyploids in Trichosanthes kirilowii Maxim.

Wild relative species of domesticated crops are useful genetic resources for improving agronomic traits. Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that can be exploited in a breeding program. Here, we used FISH analysis to characterize the ploidy level, chromosome constitution, and genomic distribution o f 5S and 4 5S r ibosomal DNA ( rDNA) in f our wild C ucurbitaceae s pecies, namely, Citrullus lanatus (Thunb.) Mansf. var. citroides L. H. Bailey (2n = 22), Melothria japonica Maxim. (2n = 22), Sicyos angulatus L. (2n = 24), and Trichosanthes kirilowii Maxim. (2n = 66, 88, 110 cytotypes), collected in different areas of Korea. All species were diploids, except for T. kirilowii, which included hexa-, octa-, and decaploid cytotypes (2n = 6x = 66, 8x = 88, and 10x = 110). All species have small metaphase chromosomes in the range of 2–5 μm. The 45S rDNA signals were localized distally compared to the 5S rDNA. C. lanatus var. citroides and M. japonica showed one and two loci of 45S and 5S rDNA, respectively, with co-localization of rDNA signals in one M. japonica chromosome. S. angulatus showed two co-localized signals of 5S and 45S rDNA loci. The hexaploid T. kirilowii cytotype showed five signals each for 45S and 5S rDNA, with three being co-localized. This is the first report of hexaploid and decaploid cytotypes in T. kirilowii. These results will be useful in future Cucurbitaceae breeding programs.

Inheritance of Resistance to Powdery Mildew Race 1W in Watermelon.

Powdery mildew caused by Podosphaera xanthii is a major disease of watermelon in Israel. In this study, 291 accessions of Citrullus spp. were evaluated for resistance against P. xanthii race 1W. Only eight accessions exhibited high level of resistance. Inheritance of resistance against P. xanthii race 1W was studied by crossing three resistant accession of Citrullus lanatus var. citroides BIU 119, PI 189225, or PI 482312 with the susceptible cultivar 'Malali' or 'Sugar Baby'. Parents, F1, F2, and back cross progenies were evaluated for resistance in growth chambers at the cotyledon stage and the 4-leaf stage and in the field, at the 15-leaf stage. Resistance at the cotyledon stage was controlled by a single, partially dominant gene, whereas at the 4-leaf stage or the 15-leaf stage resistance was controlled by three complimentary, partially dominant genes. Crosses made among these resistant accessions revealed that BIU 119 and PI 189225 carry the same genes for resistance, whereas PI 482312 shares two out of three genes with both BIU 119 and PI 189225. A breeding line with high resistance level and good fruit qualities was developed from BIU 119 × HA5500.

Growth, photosynthesis and adaptive responses of wild and domesticated watermelon genotypes to drought stress and subsequent re-watering

The growth and morphophysiological responses of wild watermelon (C. lanatus var. citroide) M20 and Chinese domesticated watermelon (C. lanatus var. lanatus) Y34 to drought stress and subsequent re-watering were compared. Wild watermelon is drought-tolerant, whereas the domesticated watermelon is susceptible. Irrigation was withheld from seedlings for 10 days and the seedlings were then allowed to recover for 1 day. Drought treatment resulted in the wilting and yellowing of leaves in both genotypes, but symptoms occurred earlier and more visibly in Y34. Drought stress inhibited the growth of both genotypes but increased the root/shoot ratio more pronouncedly in M20 than in Y34. Under drought conditions, M20 maintained a higher leaf water status than Y34 due to its denser trichomes and more sensitive stomatal control, which minimized the transpiration rate. Y34 was more vulnerable to drought, resulting in larger decreases in photosystem II efficiency, initial Rubisco activity and chlorophyll concentration. H2O2, O2 −, and MDA contents were significantly increased in both genotypes; however, these increases were smaller in M20, possibly due to a greater enhancement of antioxidant enzyme activities and related-gene expression levels. Moreover, M20 accumulated soluble sugars and proline to greater levels to counter reduced soil moisture. These adaptive mechanisms enabled M20 to recover more rapidly after re-watering. Our findings provide guidance for improving the drought tolerance of Chinese watermelon cultivars.

ESTABLISHMENT OF COTYLEDON EXPLANTS REGENERATION SYSTEM IN WILD WATERMELON

Wild watermelon is ideal rootstock for cultivated watermelon grafting because of its graft compatibility, tolerance to environmental stresses, and less effect on the fruit quality of the scion. However, it is hard to acquire the excellent wild watermelon genotype with multiple resistances through traditional breeding programs. Genetic transformation is an efficient approach for genetic improvement. In the study, we analyzed the effect of plant growth regulators on the cotyledon regeneration efficiency with the aim of establishing genetic transformation system for wild watermelon. Wild watermelon (Citrullus lanatus var. citroides) seeds were germinated under aseptic conditions. Cotyledons were cut from five day seedlings and cultured on MS medium supplemented with different plant growth regulators. The results showed that explants cultured in MS + BA (1.0 mg/L) achieved the highest regeneration rate, and 6.3 shoots were regenerated from each explant on an average. Adventitious shoots were able to elongate on MS medium with KT concentrations ranging from 0.1 to 0.3 mg/L. On an average 62% adventitious shoots were successfully rooted on MS medium supplemented with 0.05 mg/L NAA. Rooted seedlings were successfully acclimatized and gradually hardened-off to green-house conditions and subsequently cultivated in soil with a mean survival rate of 80%. The cotyledon regeneration system for wild watermelon was successfully established in the study, which is needed for genetic transformation of wild watermelon in the future.

Flowering Stage Resistance to Bacterial Fruit Blotch in the Watermelon Germplasm Collection

ABSTRACTCucurbit bacterial fruit blotch caused by Acidovorax avenae subsp. citrulli is a significant threat to watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] production worldwide. In the United States, seedless cultivars are primarily used in watermelon production, which now relies largely on transplant production in greenhouses to ensure a high germination rate. Unfortunately, the warm and humid greenhouse environment provides ideal conditions for the spread of bacterial fruit blotch. Treatments designed to remove bacteria from the surface of the seed coat were investigated previously, but none eliminated the bacteria despite significant reductions reported in research studies. Resistant cultivars offer a solution to the problem if genetic resistance can be identified. The objectives of this study were to (i) identify germplasm resistant to bacterial fruit blotch using the available PI accessions in the USDA germplasm collection and (ii) improve the methods for screening in the field. Field evaluations on the basis of foliar disease symptoms at the flowering stage were conducted at Clinton, NC in 2011 to 2013. The field experiment was a randomized complete block with 1699 cultigens, 3 yr, and two replications of single‐plant plots. Disease rating was on a 0 to 9 scale when the disease was uniformly distributed throughout the field (0 = no symptoms, 1–2 = trace, 3–4 = slight, 5–6 = moderate, 7–8 = severe, and 9 = dead). Plots were rated multiple times each year. Significant differences were found for disease resistance among accessions (P = 0.05). Rating dates having the greatest F ratio for differences among accessions were identified as best ratings for each year–block combination. Resistant accessions have the best ratings (<4.5), low standard deviation across replications, and multiple replication (≥4). The 23 most resistant cultigens originated from Africa, mainly Zimbabwe, Zambia, Republic of South Africa, and Nigeria, and they were either cultivated watermelon (C. lanatus var. lanatus) or citron (C. lanatus var. citroides).

Interspecific Transferability of Watermelon EST-SSRs Assessed by Genetic Relationship Analysis of Cucurbitaceous Crops

본 연구는 수박의 EST-SSR 마커를 이용하여, 네 개의 주요 박과(Cucurbitaceae) 작물인 수박, 호박, 오이, 멜론의 유연 관계를 분석하고 마커의 타 박과 작물에 활용 가능성을 알아보기 위해 수행되었다. Cucurbit Genomics Initiative(ICuGI) database로부터 선발된 120 EST-SSR 프라이머 중 51(49.17%)가 PCR이 성공하였고, 49(40.8%)가 8개 박과 유전자원에서 다형성을 보였다. 총 24개 박과 유전자원을 24개 EST-SSR 프라이머로 분석한 결과 총 382개 대립유전자 특이적 PCR 밴드를 얻었으며, 이를 토대로 짝유사행렬과 계통도를 작성하였다. 짝유사행렬의 범위는 0.01-0.85였으며, 작성된 계통도에서 24개 유전자원이 두 개의 주요그룹(Clade I, II)으로 분류되었다. Clade I은 다시 수박으로 구성된 하위집단 I-1[I-1a, I-1b-2: 각 1개와 2수박 야생종(Citrullus lanatus var. citroides Mats. & Nakai)으로 구성, I-1b-1: 6개수박 재배종(Citrullus lanatus var. vulgaris Schrad.)로 구성]과 멜론과 오이로 구성된 하위집단I-2[I-2a-1: 4개 멜론 재배종(Cucumis melo var. cantalupensis Naudin.), I-2a-2: 2개 참외 재배종(Cucumis melo var. conomon Makino.), I-2b: 5개 오이 재배종(Cucumis sativus L.)]로 분류되었다. 호박으로 구성된 Clade II는 다시 Cucurbita moschata(Duch. ex Lam.) Duch. & Poir와 Cucurbita maxima Duch.로 구성된 하위집단 II-1과 Cucurbita pepo L.과 Cucurbita ficifolia Bouche로 구성된 하위집단 II-2로 나누어졌다. 이러한 결과는 기존의 종명법에 따른 분류와 일치하며, 따라서 수박 EST-SSR 마커를 이용한 타 박과 작물의 비교 유전체 등 연구분야에 적용 가능성을 확인하였다. This study was performed to analyze genetic relationships of the four major cucurbitaceous crops including watermelon, melon, cucumber, and squash/pumpkin. Among 120 EST-SSR primer sets selected from the International Cucurbit Genomics Initiative (ICuGI) database, PCR was successful for 51 (49.17%) primer sets and 49 (40.8%) primer sets showed polymorphisms among eight Cucurbitaceae accessions. A total of 382 allele-specific PCR bands were produced by 49 EST-SSR primers from 24 Cucurbitaceae accessions and used for analysis of pairwise similarity and dendrogram construction. Assessment of the genetic relationships resulted in similarity indexes ranging from 0.01 to 0.85. In the dendrogram, 24 Cucurbitaceae accessions were classified into two major groups (Clade I and II) and 8 subgroups. Clade I comprised two subgroups, Clade I-1 for watermelon accessions [I-1a and I-1b-2: three wild-type watermelons (Citrullus lanatus var. citroides Mats. & Nakai), I-1b-1: six watermelon cultivars (Citrullus lanatus var. vulgaris S chrad.)] a nd C lade I -2 for melon and cucumber accessions [I-2a-1 : 4 melon cultivars(Cucumis melo var. cantalupensis Naudin.), I-2a-2: oriental melon cultivars (Cucumis melo var. conomon Makino.), and I-2b: five cucumber cultivars (Cucumis sativus L.)]. Squash and pumpkin accessions composed Clade II {II-1: two squash/ pumpkin cultivars [Cucurbita moschata (Duch. ex Lam.)/Duch. & Poir. and Cucurbita maxima Duch.] and II-2: two squash/pumpkin cultivars, Cucurbita pepo L./Cucurbita ficifolia Bouche.}. These results were in accordance with previously reported classification of Cucurbitaceae species, indicating that watermelon EST-SSRs show a high level of marker transferability and should be useful for genetic study in other cucurbit crops.

Influence of Citrullus lanatus var. citroides Rootstocks and Their F1 Hybrids on Yield and Response to Root-knot Nematode, Meloidogyne incognita, in Grafted Watermelon

Southern root-knot nematodes (Meloidogyne incognita) are an important re-emerging pest of watermelon in the United States and worldwide. The re-emergence of root-knot nematodes (RKNs) in watermelon and other cucurbits is largely the result of the intensive cultivation of vegetable crops on limited agricultural lands coupled with the loss of methyl bromide for pre-plant soil fumigation, which has been the primary method for control of RKNs and many soilborne diseases of cucurbits and other vegetable crops for several decades. One alternative for managing RKN in watermelon is the use of resistant rootstocks for grafted watermelon. We have developed several RKN-resistant Citrullus lanatus var. citroides lines (designated RKVL for Root-Knot Vegetable Laboratory), which have shown promise as rootstocks for grafted watermelon. In 2011 and 2012, we demonstrated that F1 hybrids derived from our selected RKVL lines exhibited resistance to RKN that was equal to or greater than that of the parental RKVL lines when grown in fields highly infested with M. incognita. In 2011, although significant differences were not observed among rootstocks, the F1 hybrids produced slightly higher yields compared with the selected parental lines. Among the selected parental lines, RKVL 318 produced high yields in both years. In 2011, three of four RKVL parental lines and all four of their F1 hybrids produced greater (P < 0.05) fruit yields than self-grafted ‘Tri-X 313’, ‘Emphasis’ bottle gourd, and ‘Strong Tosa’ squash hybrid. In 2012, three RKVL F1 hybrid lines produced higher yields than the selected parents. Overall, these F1 hybrids were vigorous and should provide useful genetic material for selection and development of robust RKN-resistant C. lanatus var. citroides rootstock lines.

Accessions of Citrullus lanatus var. citroides Are Valuable Rootstocks for Grafted Watermelon in Fields Infested with Root-knot Nematodes

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) (Meloidogyne 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.

High-resolution genetic map for understanding the effect of genome-wide recombination rate on nucleotide diversity in watermelon.

We used genotyping by sequencing to identify a set of 10,480 single nucleotide polymorphism (SNP) markers for constructing a high-resolution genetic map of 1096 cM for watermelon. We assessed the genome-wide variation in recombination rate (GWRR) across the map and found an association between GWRR and genome-wide nucleotide diversity. Collinearity between the map and the genome-wide reference sequence for watermelon was studied to identify inconsistency and chromosome rearrangements. We assessed genome-wide nucleotide diversity, linkage disequilibrium (LD), and selective sweep for wild, semi-wild, and domesticated accessions of Citrullus lanatus var. lanatus to track signals of domestication. Principal component analysis combined with chromosome-wide phylogenetic study based on 1563 SNPs obtained after LD pruning with minor allele frequency of 0.05 resolved the differences between semi-wild and wild accessions as well as relationships among worldwide sweet watermelon. Population structure analysis revealed predominant ancestries for wild, semi-wild, and domesticated watermelons as well as admixture of various ancestries that were important for domestication. Sliding window analysis of Tajima’s D across various chromosomes was used to resolve selective sweep. LD decay was estimated for various chromosomes. We identified a strong selective sweep on chromosome 3 consisting of important genes that might have had a role in sweet watermelon domestication.

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullusspp.).

Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon.

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

BackgroundA large single nucleotide polymorphism (SNP) dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. The marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium (LD) within a population. Use of genotyping by sequencing reveals large numbers of SNPs that in turn generate opportunities in genome-wide association mapping and marker-assisted selection, even in crops such as watermelon for which few genomic resources are available. In this paper, we used genome-wide genetic diversity to study LD, selective sweeps, and pairwise FST distributions among worldwide cultivated watermelons to track signals of domestication.ResultsWe examined 183 Citrullus lanatus var. lanatus accessions representing domesticated watermelon and generated a set of 11,485 SNP markers using genotyping by sequencing. With a diverse panel of worldwide cultivated watermelons, we identified a set of 5,254 SNPs with a minor allele frequency of ≥ 0.05, distributed across the genome. All ancestries were traced to Africa and an admixture of various ancestries constituted secondary gene pools across various continents. A sliding window analysis using pairwise FST values was used to resolve selective sweeps. We identified strong selection on chromosomes 3 and 9 that might have contributed to the domestication process. Pairwise analysis of adjacent SNPs within a chromosome as well as within a haplotype allowed us to estimate genome-wide LD decay. LD was also detected within individual genes on various chromosomes. Principal component and ancestry analyses were used to account for population structure in a genome-wide association study. We further mapped important genes for soluble solid content using a mixed linear model.ConclusionsInformation concerning the SNP resources, population structure, and LD developed in this study will help in identifying agronomically important candidate genes from the genomic regions underlying selection and for mapping quantitative trait loci using a genome-wide association study in sweet watermelon.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-767) contains supplementary material, which is available to authorized users.

Analysis of the Citrullus colocynthis transcriptome during water deficit stress.

Citrullus colocynthis is a very drought tolerant species, closely related to watermelon (C. lanatus var. lanatus), an economically important cucurbit crop. Drought is a threat to plant growth and development, and the discovery of drought inducible genes with various functions is of great importance. We used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the C. colocynthis leaf transcriptome under drought treatment. Leaf samples at four different time points (0, 24, 36, or 48 hours of withholding water) were used for RNA extraction and Illumina sequencing. qRT-PCR of several drought responsive genes was performed to confirm the accuracy of RNA sequencing. Leaf transcriptome analysis provided the first glimpse of the drought responsive transcriptome of this unique cucurbit species. A total of 5038 full-length cDNAs were detected, with 2545 genes showing significant changes during drought stress. Principle component analysis indicated that drought was the major contributing factor regulating transcriptome changes. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling and citrulline metabolism occurred under the water deficit conditions. The C. colocynthis transcriptome data highlight the activation of a large set of drought related genes in this species, thus providing a valuable resource for future functional analysis of candidate genes in defense of drought stress.

First Report of Cucumber green mottle mosaic virus on Melon in the United States

In July 2013, a melon (Cucumis melo var. Saski) field in Yolo County, California, was inspected as part of a phytosanitary inspection for seed production. The leaves of the plants showed mosaic, green mottle, and blotches. When plant sap was examined using a transmission electron microscope, rigid rod-shaped particles were observed. Melon plant samples were analyzed by both CDFA and USDA APHIS PPQ laboratories and tested positive using DAS-ELISA against Cucumber green mottle mosaic virus (CGMMV) (Agdia, Elkhart, IN). To confirm the presence of CGMMV, total RNA was analyzed by RT-PCR using primers CGMMV-F5370 5'-CTAATTATTCTGTCGTGGCTGCGGATGC-3' and CGMMV-R6390 5'-CTTGCAGAATTACTGCCCATA-3' designed by PPQ based on 21 genomic sequences of CGMMV found worldwide. The 976-bp amplicon was sequenced (GenBank Accession No. KJ453559) and BLAST analysis showed the sequence was 95% identical to MP and CP region of CGMMV isolates reported from Russia (GQ495274, FJ848666), Spain (GQ411361), and Israel (KF155231), and 92% to the isolates from China (KC852074), Korea (AF417243), India (DQ767631), and Japan (D12505). These analyses confirm the virus was CGMMV. To our knowledge, this is the first report of CGMMV in the United States. Based on our sequence data, a second set of primers (CGMMV-F5796 5'-TTGCGTTTAGTGCTTCTTATGT-3' and CGMMV-R6237 5'-GAGGTGGTAGCCTCTGACCAGA-3'), which amplified a 440-bp amplicon from CGMMV CP region, was designed and used for testing all the subsequent field and seed samples. Thirty-seven out of 40 randomly collected Saski melon samples tested positive for CGMMV, suggesting the virus was widespread in the field. All the melon samples also tested positive for Squash mosaic virus (SqMV) using DAS-ELISA (Agdia). Therefore, the symptoms observed likely resulted from a mixed infection. The melon field affected by CGMMV was immediately adjacent to fields of cucumber (Cucumis sativus var. Marketmore 76) and watermelon (Citrullus lanatus var. Sugar Baby) crops, both for seed production with no barrier between the crops. CGMMV was also detected from symptomatic plants from both fields. Seed lots used for planting all three crops were tested and only the melon seed was positive for CGMMV, suggesting the seed as the source of infection. The sequenced 440-bp RT-PCR amplicons from CGMMV-infected cucumber and watermelon plants and melon seeds were 99% identical to the CGMMV from the field melon. A cucumber plant infected with CGMMV but not SqMV was used for mechanical inoculation at the Contained Research Facility at University of California, Davis. Inoculated cucumber, melon, and watermelon plants showed green mottle and mosaic similar to that observed in the field. CGMMV is a highly contagious virus and damage by this virus on cucurbit crops has been reported in regions where CGMMV is present (2). CGMMV was detected on cucumber grown in greenhouses in Canada with 10 to 15% yield losses reported due to this virus (1). The three cucurbit crops in Yolo County were planted in an isolated area with no other cucurbits nearby. Measures, including destroying all the cucurbit plant material, have been taken to eradicate the virus. Use of CGMMV free cucurbit seed is necessary for prevention of this disease.

Statistical genetics in traditionally cultivated crops

Traditional farming systems have attracted a lot of attention over the past decades as they have been recognized to supply an important component in the maintenance of the genetic diversity worldwide. A broad spectrum of traditionally managed crops has been studied to investigate how reproductive properties in combination with husbandry characteristics shape the genetic structure of the crops over time. However, traditional farms typically involve populations of small size whose genetic evolution is overwhelmed with statistic fluctuations inherent to the stochastic nature of the crossings. Hence there is generally no one-to-one mapping between crop properties and measured genotype data, and claims regarding crop properties on the basis of the observed genetic structure must be stated within a confidence level to be estimated by means of a dedicated statistical analysis. In this paper, we propose a comprehensive framework to carry out such statistical analyses. We illustrate the capabilities of our approach by applying it to crops of C. lanatus var. lanatus oleaginous type cultivated in Côte d׳Ivoire. While some properties such as the effective field size considerably evade the constraints from experimental data, others such as the mating system turn out to be characterized with a higher statistical significance. We discuss the importance of our approach for studies on traditionally cultivated crops in general.

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

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