A genetic linkage map was previously constructed for watermelon using a wide testcross population [{Plant Accession Griffin 14113; Citrullus lanatus var. citroides (L.H. Baiely) Mansf.} × the watermelon cultivar New Hampshire Midget; NHM {(Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus)} × United States Plant Introduction (PI) 386015 {Citrullus colocynthis (L.) Schrad.}]. One-hundred forty-six markers [randomly amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and sequence-related amplified polymorphism (SRAP) markers] unique to NHM and representing different linkage groups on the map were tested for polymorphism among 24 watermelon cultivars limited in genetic diversity. Five (9.4%) of 53 RAPD, six (40.0%) of 15 ISSR, 30 (81.0%) of 37 AFLP, and 33 (80.5%) of 41 SRAP markers tested produced polymorphism among the 24 cultivars. The polymorphic markers used in this study are scattered throughout the watermelon genome. However, a large number (19 of the 30) of AFLP markers clustered on one linkage group on the map. The SRAP markers proved to be most effective in producing polymorphism and in representing different linkage regions of watermelon genome. The polymorphic markers represent all 10 large linkage groups and five of the nine small linkage groups (altogether 15 of 19 linkage groups) of the genetic linkage map constructed so far for watermelon. These polymorphic markers can be useful in DNA fingerprinting of cultivars, in testing seed purity of breeding lines, and in identifying triploid (seedless) hybrid watermelons derived from crosses between closely related tetraploid and diploid lines.

"Novel Watermelon Breeding Lines Containing Chloroplast and Mitochondrial Genomes derived from the Desert Species Citrullus colocynthis" published on Apr 2006 by American Society for Horticultural Science.

Seventy-one amplified fragment length polymorphism (AFLP), 93 sequence related amplified polymorphism (SRAP), and 14 simple sequence repeat (SSR) markers were used to extend an initial genetic linkage map for watermelon [ Citrullus lanatus (Thunb.) Matsum. & Nakai]. The initial map was based on 151 randomly amplified polymorphic DNA (RAPD) and 30 and inter-simple sequence repeat (ISSR) markers. A testcross population previously used for mapping of RAPD and ISSR markers was used in this study: {plant accession Griffin 14113 [ C. lanatus var . citroide (L.H. Bailey) Mansf.] × the watermelon cultivar New Hampshire Midget ( C. lanatus var. lanatus )} × PI 386015 [ C. colocynthis (L.) Schrad.]. The linkage map contains 360 DNA markers distributed on 19 linkage groups, and covers a genetic distance of 1976 cM with an average distance of 5.8 cM between two markers. A genomic DNA clone representing 1-amino-cyclopropane-1-carboxylic acid (ACC-) synthase gene, involved in ethylene biosynthesis, was also mapped. As in previous mapping studies for watermelon, a large number of AFLP and SRAP markers were skewed away from the 1:1 segregation ratio, and had to be excluded from the final mapping analysis. The stringent mapping criteria (JoinMap 3.0 mapping program) produced linkage groups with marker order consistent with those reported in previous mapping study for watermelon.

A cucurbit species named Praecitrullus fistulosus (Stocks) Pangalo, which thrives in India, is considered to be a distant relative of watermelon. Recent experiments indicated that it has mild resistance to whiteflies (Bemisia tabaci). However, our attempts to cross various US plant introductions (PIs) of P. fistulosus with watermelon or other Citrullus PIs have not been successful. Thus, to determine genetic relatedness among those species, phylogenetic analysis [based on simple sequence repeat (SSR)–anchored (also termed ISSR), and randomly amplified polymorphic DNA (RAPD) markers] was conducted among PIs of P. fistulosus, Citrullus lanatus var. lanatus (watermelon), C. lanatus var. citroides and the wild Citrullus colocynthis. Phylogenetic relationships were also examined with Cucumis melo (melon), Cucumis sativus (cucumber), and wild Cucumis species including C. africanus, C. metuliferus, C. anguria, C. meeusei, and C. zeyheri. Wide genetic distance exists between Citrullus and Cucumis groups (8% genetic similarity). Phylogenetic relationships among Citrullus species and subspecies are closer (25–55% genetic similarity) as compared with those among most Cucumis species (14–68% genetic similarity). P. fistulosus appeared to be distant from both Cucumis and Citrullus species (genetic similarity between P. fistulosus and Cucumis or Citrullus groups is less than 3%). Although wide genetic differences and reproductive barriers exist among cucurbit species examined in this study, they are still considered as potential germplasm source for enhancing watermelon and melon crops using traditional breeding and biotechnology procedures.

Genetic linkage map is being constructed for watermelon based on a testcross population and an F2 population. The testcross map comprises 262 markers (RAPD, ISSR, AFLP, SSR and ASRP markers) and covers 1,350 cM. The map comprises 11 large linkage groups (50.7–155.2 cM), 5 medium-size linkage groups (37.5–46.2 cM), and 16 small linkage groups (4.2–31.4 cM). Most AFLP markers are clustered on two linkage regions, while all other marker types are randomly dispersed on the genome. Many of the markers in this study are skewed from the classical (Mendelian) segregation ratio of1:1 in the testcross or the 3:1 ratio in the F2 population. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and F2 population. A cDNA library was constructed using RNA isolated from watermelon flesh 1 week (rapid cell division stage), 2 weeks (cell growth and storage deposition stage, 4 weeks (maturation stage), and 5 weeks (postmaturation stage) post pollination. Over 1,020 cDNA clones were sequenced, and were analyzed using the Basic Local Alignment Search Tool (BLAST). The sequenced cDNA clones were designated as expressed sequenced tag (EST) markers and will be used in mapping analysis of watermelon genome.

Twelve and six DNA clones representing various parts of chloroplast and mitochondrial genomes, respectively, were used to detect polymorphism among five watermelon cultivars and 21 U.S. Plant Introductions (PIs) collected from diverse geographical locations and representing major groups of Citrullus species. Cluster analysis based on 20 chloroplast DNA (cpDNA) and 10 mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) markers differentiated the accessions into three major phenetic groups: PIs and watermelon cultivars of Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa (also designated as C. lanatus var. lanatus) (group I), PIs of C. lanatus var. citroides (of C. lanatus subsp. lanatus Schrad. ex Eckl. et Zeyh.)(group II), and C. colocynthis (L.) Schrad. PIs (group III). The chloroplast and mitochondrial genomes of watermelon cultivars are distinct, but closely related to those of the C. lanatus var. lanatus PIs. On the other hand, the chloroplast and mitochondrial genomes of the wild species C. colocynthis are more similar to those of C. lanatus var. citroides. Polymorphic cpDNA and mtDNA markers identified in this study can complement isozyme and nuclear DNA data used in earlier phylogenetic and phenetic classifications of Citrullus PIs. These cpDNA and mtDNA markers are being used in experiments designed to enhance watermelon cultivars by replacing the chloroplast and mitochondrial genome of cultivated watermelon with those of the wild species C. colocynthis.

Genetic linkage map is being constructed for watermelon based on a testcross population and an F 2 population. About 51.0% and 31.8% of the markers in the testcross and F 2 populations are skewed form the expected segregation ratios. AFLP markers appeared to be clustered on linkage regions, while ISSR and RAPD markers are randomly dispersed on the genome. AFLP markers also have greater genetic distances as compared with ISSR and RAPD markers, resulting in significant increase of map distance. An initial genetic map (based on the testcross population) that contains 27 ISSR and 141 RAPD markers has a total linkage distance of 1,166.2 cM. The addition of 2 ISSR, 8 RAPD and 77 AFLP markers increased the genetic distance of the map to 2,509.9 cM. Similar results with AFLP markers were also shown in mapping experiments with an F 2 S 7 recombinant inbred line (RIL) population that was recently constructed for watermelon. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and the F2 population. Experiments with SSR, and EST markers are being conducted to saturate the linkage map of watermelon genome.

A genetic linkage map was constructed for watermelon based on a testcross population and an F 2 population. The testcross map includes 312 markers (RAPD, ISSR, AFLP, SSR, and ASRP). This map covered a genetic distance of 1385 cM, and identified 11 large (50.7-155.2 cm), five intermediate (37.5-46.2 cm), and 16 small linkage groups (4.2-31.4 cm). Most AFLP markers are clustered in two linkage regions, while all other markers are randomly dispersed throughout the genome. Many of the markers in this study were skewed from the classical (Mendelian) segregation ratio of 1:1 in the testcross or 3:1 in the F 2 population. The order of the markers within linkage groups was similar in the testcross and F 2 populations. Additionally, a cDNA library was constructed using RNA isolated from watermelon flesh 1 week (rapid cell division stage), 2 weeks (cell growth and storage deposition stage), 4 weeks (maturation stage), and 5 weeks (mature fruit) after pollination. More than 1020 cDNA clones were sequenced, and analyzed using the basic local alignment search Tool (BLAST). The sequenced cDNA clones were designated as expressed sequenced tag (EST). The ESTs were searched for simple sequence repeats. About 7% of the ESTs contained SSR motifs. The ESTs containing SSRs are being used to design PCR primers and the putative markers are being tested for polymorphism among the parental lines of the mapping populations. Polymorphic markers will then be mapped using the mapping populations.

Two hundred sixty-six Citrullus lanatus (Thumb.) Matsum. & Nakai accessions (Plant Introductions and named cultivars) were tested against a race 2 Sphaerotheca fuliginea (Schlechtend.: Fr.) Pollacci isolate to evaluate for resistance to powdery mildew disease. Growth room-grown seedlings were artificially inoculated with. conidia from watermelon host leaves at 2-day intervals from the appearance of the first true leaf until test results data were taken, when the second true leaf was fully expanded. Plants were evaluated on a 1 to 9 scale of increasing disease severity. Disease indices (DIs) were calculated as weighted averages for each entry. All genotypes with resistant plants (powdery mildew rating 1 to 3) were reevaluated in a replicated test of 3 replications of 10 plants each. Disease indices were again calculated. Twenty-two plant introductions (PIs) and one named variety displayed intermediate resistance to powdery mildew in the replicated test with DIs ranging from 5.0 to 6.0.

The soil-borne fungus Fusarium oxysporum f.sp. melonis causes significant losses in the cultivated melon, a key member of the economically important family, the Cucurbitaceae. Here, we report the map-based cloning and characterization of the resistance gene Fom-2 that confers resistance to race 0 and 1 of this plant pathogen. Two recombination events, 75 kb apart, were found to bracket Fom-2 after screening approximately 1324 gametes with PCR-based markers. Sequence analysis of the Fom-2 interval revealed the presence of two candidate genes. One candidate gene showed significant similarity to previously characterized resistance genes. Sequence analysis of this gene revealed clear polymorphisms between resistant and susceptible materials and was therefore designated as Fom-2. Analysis of susceptible breeding lines (BL) presenting a haplotype very similar to the resistant cultivar MR-1 indicated that a gene conversion had occurred in Fom-2, resulting in a significant rearrangement of this gene. The second candidate gene which shared high similarity to an essential gene in Arabidopsis, presented an almost identical sequence in MR-1 and BL, further supporting Fom-2 identity. The gene conversion in Fom-2 produced a truncated R gene, revealing new insights into R gene evolution. Fom-2 was predicted to encode an NBS-LRR type R protein of the non-TIR subfamily. In contrast to most members of this class a coiled-coil structure was predicted within the LRR region rather than in the N-terminal. The Fom-2 physical region contained retroelement-like sequences and truncated genes, suggesting that this locus is complex.

Wide phenotypic diversity exists among American heirloom cultivars of watermelon (Citrullus lanatus var. lanatus). However, in published studies, low or no polymorphism was revealed among those heirlooms using isozyme or randomly amplified polymorphic DNA (RAPD) markers. In this study, experiments with inter-simple sequence repeat (ISSR) [also known as simple sequence repeat-(SSR-) anchored primers] and amplified fragment-length polymorphism (AFLP) markers produced high polymorphisms among watermelon heirloom cultivars. ISSR (111) and AFLP (118) markers (229 total) identified 80.2% to 97.8% genetic similarity among heirloom cultivars. The phylogenetic relations based on ISSR and AFLP markers are highly consistent with the parental records available for some of the heirloom cultivars, providing confidence in the dendogram constructed for heirlooms based on similarity values. As compared with RAPD markers, ISSRs and AFLPs are highly effective in differentiating among watermelon cultivars or elite lines with limited genetic diversity.

Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and I of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.

Downy mildew (Pseudoperonospora cubensis (Berk. and Curt.) Rostov.) is an important disease in most cucumber (Cucumis sativus L.) production areas worldwide. A set of cucumber cultivars and breeding lines (hereafter referred to collectively as cultigens) resistant to downy mildew in particular regions of the world were tested for resistance in geographical regions thought to differ in pathogen virulence or race (US, Poland, China, and India). Cucumber cultigens used in the study were developed in the US, Poland or China, and differed in their resistance to downy mildew. These cultigens were evaluated against local isolates of P. cubensis under field conditions (North Carolina and India) or greenhouse conditions (South Carolina and Poland). Significant differences were observed among cultigens for resistance to P. cubensis at all locations. Individual cultigens differed in their resistance to the pathogen at different geographic locations, providing evidence that the different local isolates represented different races of the pathogen. Cultigens from PR China that were resistant to downy mildew in that country were also resistant in India. However, those same cultigens were intermediate in resistance in the US and Poland. Cultigens from the US and Poland that were resistant in those countries were intermediate in resistance in India. The most resistant cultigens over all locations were ‘Nongchen #4’ (PR China) and M 21 (NC State University).

Genetic diversity was estimated among 42 U.S. PlantIntroduction (PI) accessions of the genus (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 . var. accessions, and a group of . var. accessions that contained some . var. genes. The second cluster consisted of the . var. accessions, while the third cluster consisted of the. accessions.The two . clustersdifferentiated from each other and from the . 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 PIs.Additionally, resistance to gummy stem blight or Fusarium wilt mayexist among . var. PIs. This study demonstrates thatmolecular markers can be useful in assessing genetic diversity, andin sorting PIs into phylogeneticgroups prior to their evaluation for disease or pestresistance.

A genetic linkage [randomly amplified polymorphic DNA (RAPD)-based] map was constructed for watermelon [ Citrullus lanatus (Thunb.) Matsum and Nakai] using a BC 1 population [PI 296341-fusarium wilt resistant × New Hampshire Midget (fusarium susceptible)] × `New Hampshire Midget'. The map contains 155 RAPD markers, and a 700-base pair sequenced characterized amplified region (SCAR) marker that corresponds to a fragment produced by the RAPD primer GTAGCACTCC. This marker was reported previously as linked (1.6 cM) to race 1 fusarium wilt resistance in watermelon. The markers segregated to 17 linkage groups. Of these, 10 groups included nine to 19 markers, and seven groups included two to four markers. The map covers a genetic linkage distance of 1295 cM. Nine of the 10 large linkage groups contained segments with low (or no) level of recombination (0 to 2.6 cM) among markers, indicating that the watermelon genome may contain large chromosomal regions that are deficient in recombination events. The map should be useful for identification of markers linked closely to genes that control fruit quality and fusarium wilt (races 1 and 2) resistance in watermelon.

Downy mildew, incited by the biotrophic fungal parasite, Peronospora parasitica (Pers. Fr.) Fr., is one of the most destructive diseases of broccoli ( Brassica oleracea L., Italica Group) and other related crop species throughout the world. Cultivation of resistant cultivars is the most desirable control method because it provides a practical, long-term, and environmentally benign means of limiting damage from this disease. The commercial hybrid cultivar, Everest, has been shown previously to contain a high level of downy mildew resistance. Doubled-haploid (DH) lines developed from that hybrid were also shown to exhibit a similar, high level of resistance at the three- to four-leaf stage. To determine the mode of inheritance of this true leaf resistance, the resistant DH line was crossed to a susceptible line (derived from `Marathon') to produce an F 1 hybrid. Subsequently, F 2 and backcross (BC) populations were developed from the hybrid. In addition, a DH population of ≈100 lines was developed from the same F 1 used to create the F 2 and BC. All populations were evaluated for response to artificial inoculation with P. parasitica at the three- to four-leaf stage. F 1 plants were resistant like the resistant parent and F 2 populations segregated approximately nine resistant to seven susceptible. Using the resistant parent as recurrent parent, BC populations contained all resistant plants, while the BC to the susceptible parent fit a 1 resistant: 3 susceptible segregation ratio. These results can be explained by a model with two complementary dominant genes. This model was confirmed by the DH population that segregated ≈1:3, resistant to susceptible. Due to the dominant nature of this resistance, controlling genes should be easily incorporated into F 1 hybrids and used commercially to prevent downy mildew.

Genetic diversity was estimated among 42 U.S. PlantIntroduction (PI) accessions of the genus Citrullus (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. lanatus accessions, and a group of C . lanatus var. lanatus accessions that contained some C . lanatus var. citroides genes. The second cluster consisted of the C . lanatus var. citroides accessions, while the third cluster consisted of the C . 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.

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.

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F 2 cross between MR-1 and AY was screened using 240 Pst I/ Mse I and 200 Eco RI/ Mse I primer combinations to identify AFLP markers linked to Fom-2 . Fifteen markers potentially linked to Fom-2 were identified, all with Eco RI/ Mse I primer pairs. These were mapped relative to Fom -2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2 . These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.

Genetic relatedness was estimated among 42 U.S. plant introduction (PI) accessions of the genus Citrullus (37 PIs of which were reported to have disease resistance and five watermelon cultivars) using 30 RAPD primers. These primers produced 662 RAPD markers that could be scored with high confidence. Based on these markers, genetic similarity coefficients were calculated, and a dendrogram was constructed using the unweighted pair-group method with arithmatic average (UPGMA). The analysis delineated three major clusters. The first cluster consisted of a group of five watermelon cultivars, a group of C. lanatus var. lanatus accessions and a group of C. lanatus var. lanatus accessions that contained some C. lanatus var. citroides genes. The second cluster consisted of the C. lanatus var. citroides accessions, while the third cluster consisted of the C. colocynthis accessions. The two C. lanatus clusters differentiated from each other and from the C. colocynthis cluster at the level of 58.8% and 38.9% genetic similarity. Our results indicate that closely related Citrullus PIs may have resistances to the same diseases. Thus, molecular markers may be a useful tool in the development of core collections of Citrullus PIs with resistance to diseases.