Isozyme and seed protein phylogeny of the genusCitrullus (Cucurbitaceae)

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.     

Comparative analysis of chloroplast DNA variability in wild and cultivated<Emphasis Type="Italic"> Citrullus</Emphasis> species

PCR amplification and restriction site analysis of chloroplast (cp) DNA regions was used to detect inter- and intraspecific differences in the genus Citrullus. More than 55 C. lanatus and 15 C. colocynthis accessions collected from diverse geographical areas, C. ecirrhosus and C. rehmii were used. Most of the cpDNA variation within Citrullus was the result of large indels and transitions and transversions. Indels at the ndhA, trnS-trnfM and trnC-trnD regions and several substitutions at restriction enzyme sites can be used to separate C. colocynthis from the other Citrullus species. A nucleotide substitution at a restriction enzyme site at the 3 flanking region of ndhF provided a diagnostic haplotype for C. lanatus var. lanatus, the cultivated watermelon. Similarly, a nucleotide substitution at an intergenic spacer region of the trnC-trnD region resulted in a diagnostic haplotype for citron, C. lanatus var. citroides. Several C. lanatus var. citroides accessions showed the var. lanatus haplotype. C. rehmii showed almost the same haplotype as C. lanatus var. citroides with the exception of a unique insertion at a cpSSR site. Since C. ecirrhosus lacks the derived diagnostic nucleotide substitutions of C. lanatus, it is probably the progenitor of the cultivated watermelon. Intraspecific haplotypes detected within C. colocynthis were associated with geographic origin.Communicated by J. Dvorak     

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.     

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>

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 F₂ 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 F₂ 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 genetic linkage map for watermelon derived from a testcross population: ( Citrullus lanatus var. citroides x C. lanatus var. lanatus) x Citrullus colocynthis

A genetic linkage map was constructed for watermelon using a testcross population [Plant Accession Griffin 14113 (Citrullus lanatus var. citroides) 2 New Hampshire Midget (NHM; C. lanatus var. lanatus)] 2 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.     

An archaeobotanical contribution to the history of watermelon,<Emphasis Type="Italic"> Citrullus lanatus</Emphasis> (Thunb.) Matsum. &; Nakai (syn.<Emphasis Type="Italic"> C. vulgaris</Emphasis> Schrad.)

The discovery of several 5000-year old seeds of wild watermelon, Citrullus lanatus, at an archaeological site Uan Muhuggiag in southwest Libya, re-opens the debate on the origin, wild distribution and domestication history of this species. The seeds were found within a plant assemblage of wild seeds and fruits, associated with pottery and bones of domestic animals belonging to Neolithic pastoralists. The presumed wild progenitor of the modern cultivar C. lanatus is today found exclusively in a region centring on the Kalahari Desert. This new archaeobotanical record raises the possibility that this distribution was much more extensive in the past.     

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

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

Sequence variation at cpDNA regions of watermelon and related wild species: implications for the evolution of Citrullus haplotypes

Sequencing analysis of one coding and four noncoding cpDNA regions was conducted to infer biogeographic and evolutionary relationships in the genus Citrullus. Eighteen taxa from diverse geographical areas were included. A low number of parsimony informative characters (1.1%) was observed at the ～4 kb section of cpDNA. Variability within Citrullus was detected primarily at noncoding regions of high A + T content. Substitution rates varied from 0-0.48% for ndhF with A + T content of 68.4% to 0.39- 1.69% for the intergenic region of atpA with A + T content of 82.8%, mainly resulting in indels and transversions. Indels at several regions acted as valuable parsimony informative markers. Citrullus lanatus var. lanatus, the cultivated watermelon, and C. ecirrhosus and C. rehmii from Namibia, lacked molecular variability. The genus Citrullus is supported monophyletically and shows two main clades, one of which contains C. colocynthis. In the other clade, C. rehmii is sister to a clade containing C. ecirrhosus and C. lanatus. Two clades were recovered within C. lanatus, consisting of domesticated watermelon and wild citron, var. citroides. Five haplotypes within C. colocynthis were used to deduce colonization routes of the species. Biogeographic patterns point to separate colonization events into Africa and the Far East.     

Variation studies on seed storage proteins and phylogenetics of the genus Cucumis

Seed storage proteins were analyzed for variation in polypeptide patterns and proportion of four protein fractions in Cucumis melo and for interrelationships of different taxa of the genus Cucumis. SDS-PAGE of total protein extracts of 11 lines of C. melo representing different geographic regions showed considerable variation in their polypeptides in the range of molecular weights 50–55, 35–39, 20–26 and 16–19 kDa. As compared to one subunit pair reported earlier, two-dimensional gel electrophoresis revealed subunit pairs of molecular weights 53, 52, 50, 42, 39 and 23 kDa, each consisting of a large and a small subunit like those of legumin-like 11S proteins of leguminous seeds. The salt-soluble globulins represented the major protein fraction (44.8–48.1%), followed by glutelins and albumins with prolamins being the lowest in the seed. Based on the protein profiles of seed protein extracts of 28 Cucumis taxa, similarity matrix indices and UPGMA dendrogram showed that eight taxa of C. melo were clustered in two subclusters of four taxa each. Five taxa of C. sativus along with C. zeyheri belonged to another cluster, sister to the third cluster of remaining taxa. In this third cluster, C. anguria var. longaculeatus showed 100% similarity with C. myriocarpus and C. myriocarpus ssp. leptodermis as compared to only 64% similarity with C. anguria; C. myriocarpus ssp. myriocarpus occurred along with C. prophetarum in the same group rather than with C. myriocarpus. The accession PI-299570 representing an undescribed taxon of Cucumis exhibited 100% similarity with C. pustulatus, and C. sagittatus was the most distant from all Cucumis taxa.     

Citrullus lanatus ‘sentinel’ (watermelon) extract reduces atherosclerosis in LDL receptor-deficient mice

Watermelon (Citrullus lanatus or C. lanatus) has many potentially bioactive compounds including citrulline, which may influence atherosclerosis. In this study, we determined the effects of C. lanatus, provided as an extract of the cultivar ‘sentinel,’ on hypercholesterolemia-induced atherosclerosis in mice. Male low-density lipoprotein receptor-deficient mice at 8 weeks old were given either C. lanatus ‘sentinel’ extract (2% vol/vol; n= 10) or a mixture of matching carbohydrates (2% vol/vol; n= 8) as the control in drinking water while being fed a saturated fat-enriched diet for 12 weeks ad libitum. Mice consuming C. lanatus ‘sentinel’ extract had significantly increased plasma citrulline concentrations. Systolic blood pressure was comparable between the two groups. Consumption of C. lanatus ‘sentinel’ extract led to lower body weight and fat mass without influencing lean mass. There were no differences in food and water intake and in urine output between the two groups. C. lanatus ‘sentinel’ extract administration decreased plasma cholesterol concentrations that were attributed to reductions of intermediate-/low-density lipoprotein cholesterol. Plasma concentrations of monocyte chemoattractant protein-1 and interferon-gamma were decreased and those of interleukin-10 were increased in mice consuming C. lanatus ‘sentinel’ extract. Intake of C. lanatus ‘sentinel’ extract resulted in reductions of atherosclerosis in both aortic arch and thoracic regions. In conclusion, consumption of C. lanatus ‘sentinel’ extract led to reduced body weight gain, decreased plasma cholesterol concentrations, improved homeostasis of pro- and anti-inflammatory cytokines, and attenuated development of atherosclerosis without affecting systolic blood pressure in hypercholesterolemic mice.     

Georeferenced phylogenetic analysis of a global collection of wild and cultivated Citrullus species

The geographical origin of watermelon (Citrullus lanatus) remains debated. While a first hypothesis suggests the center of origin to be West Africa, where the endemic sister species C. mucosospermus thrives, a second hypothesis suggests northeastern Africa where the white‐fleshed Sudanese Kordophan melon is cultivated. In this study, we infer biogeographical and haplotype genealogy for C. lanatus, C. mucosospermus, C. amarus, and C. colocynthis using noncoding cpDNA sequences (trnT‐trnL and ndhF‐rpl32 regions) from a global collection of 135 accessions. In total, we identified 38 haplotypes in C. lanatus, C. mucosospermus, C. amarus, and C. colocynthis; of these, 21 were found in Africa and 17 appear endemic to the continent. The least diverse species was C. mucosospermus (5 haplotypes) and the most diverse was C. colocynthis (16 haplotypes). Some haplotypes of C. mucosospermus were nearly exclusive to West Africa, and C. lanatus and C. mucosospermus shared haplotypes that were distinct from those of both C. amarus and C. colocynthis. The results support previous findings that revealed C. mucosospermus to be the closest relative to C. lanatus (including subsp. cordophanus). West Africa, as a center of endemism of C. mucosospermus, is an area of interest in the search of the origin of C. lanatus. This calls for further historical and phylogeographical investigations and wider collection of samples in West and northeastern Africa.     

Moisture content,mould invasion and seed germinability of stored melon

The variation in seed moisture, mould content and seed germination of two species of melon,Citrullus lanatus L. andCitrullus vulgaris Shrad. stored in jute bags was monitored at intervals of 2 months for one year. Seed moisture decreased in the first 6 months of storage followed by a gradual increase in the rainy months. The field fungi predominated immediately after harvest and disappeared completely after 6 months of storage. The storage fungi increased in both species and abundance throughout the storage period.Citrullus lanatus maintained high germination percentage for the first 4 months,C. vulgaris for the first 2 months, thereafter seed germinability decreased with prolonged storage.     

Autotoxic potential of cucurbit crops

Soil sickness is often observed in cucurbit crops such as Citrullus lanatus, Cucumis melo and Cucumis sativus, but not in cucurbit crops such as Cucurbita moschata, Lagenaria leucantha and Luffa cylindrica. Results showed that root aqueous extracts of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic, but those of Cucurbita moschata, Momordica charantia and Luffa cylindrica were less autotoxic to the radicle elongation of respective species. Plant growth of Citrullus lanatus, Cucumis melo and Cucumis sativus were greatly inhibited by autotoxic substances released from powered root tissue at a rate of 1 g per seedling. Root exudates of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic to radicle elongation and seedling growth of respective species. However, root exudates of Citrullus lanatus did not inhibit radicle elongation of Cucurbita ficifolia, which is commonly used as rootstock for the grafting of Citrullus lanatus, Cucumis melo and Cucumis sativus to decrease soil-borne diseases in commercial production. It seems possible to overcome autotoxicity in cucurbit crops by grafting on Cucurbita ficifolia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Essential oil constituents and RAPD markers to establish species relationship in Cymbopogon Spreng. (Poaceae)

Nineteen Cymbopogon taxa belonging to 11 species, two varieties, one hybrid taxon and four unidentified species were analysed for their essential oil constituents and RAPD profiles to determine the extent of genetic similarity and thereby the phylogenetic relationships among them. Remarkable variation was observed in the essential oil yield ranging from 0.3% in Cymbopogon travancorensis Bor. to 1.2% in Cymbopogon martinii (Roxb.) Wats var. motia. Citral, a major essential oil constituent, was employed as the base marker for chemotypic clustering. Based on genetic analysis, elevation of Cymbopogon flexuosus var. microstachys (Hook. F.) Soenarko to species status and separate species status for C. travancorensis Bor., which has been merged under C. flexuosus (Steud.) Wats were suggested towards resolving some of the taxonomic complexes in Cymbopogon. The separate species status for the earlier proposed varieties of C. martinii (motia and sofia) is further substantiated by these analyses. The unidentified species of Cymbopogon have been observed as intermediate forms in the development of new taxa.     

Linkage relationships of 19 protein coding genes in watermelon

Summary Segregation of seed proteins and isozymes was analysed in two Citrullus crosses. In the first cross an F1 hybrid between C. lanatus and the wild species C. colocynthis was used as a female parent in a backcross to C. lanatus. In this interspecific cross the segregation of 17 markers was analysed. Four linkage groups were identified: linkage group 1 includes the genes Est-2, Skdh-2, Tpi-1, Fdp-1, Sod-1 and Prx-1; linkage group 2 — Got-1, Got-2 and Sp-4; linkage group 3 — Pgm-1 and Gdh-2; linkage group 4 with Pgi-1 and Pgi-2. In the second cross an F1 hybrid between two C. colocynthis accessions was backcrossed to one of its parents. Seven loci were scored and no new linkages were found.     

Pollen morphology of Carthamus L. species in Anatolian flora

In flora of Turkey, there are six species of Carthamus L: Carthamus dentatus (Forssk.) Vahl., C. glaucus M. Bieb. subsp. glaucus, C. lanatus L., C. tenuis (Boiss. & Blanche) Bornm., C. persicus Desf. ex Willd. and C. tinctorius L. Within these species, C. tinctorius L. is an alien species for the Turkish flora. The pollen grains belonging to six taxa collected from different locations of Anatolia and examined both under light microscope (LM) and under scanning electron microscope (SEM). As a result of these examinations and measurements, pollen grains of taxa are radially symmetrical, isopolar, oblate-spheroid, spheroid, tricolporate rarely tetracolporate, echinate. There are high similarities among taxa but some differences are recorded in size and spin length. According to cluster analyses of Carthamus species’ pollens, C. glaucus and C. tenuis have the most similar pollens. C. dentatus grouped with these two. C. persicus placed as the closest relative of C. tinctorius in the dendrogram.