Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology. Here we report the draft genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next-generation Illumina GA sequencing technologies to obtain 72.2-fold genome coverage. The absence of recent whole-genome duplication, along with the presence of few tandem duplications, explains the small number of genes in the cucumber. Our study establishes that five of the cucumber's seven chromosomes arose from fusions of ten ancestral chromosomes after divergence from Cucumis melo. The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and 'fresh green' odor. We also identify 686 gene clusters related to phloem function. The cucumber genome provides a valuable resource for developing elite cultivars and for studying the evolution and function of the plant vascular system.

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.

A set of 171 recombinant inbred lines (RIL) were developed from a narrow cross in cucumber ( Cucumis sativus L.; 2n = 2 x = 14) using the determinate ( de), gynoecious ( F), standard-sized leaf line G421 and the indeterminate, monoecious, little-leaf ( ll) line H-19. A 131-point genetic map was constructed using these RILs and 216 F(2) individuals to include 14 SSRs, 24 SCARs, 27 AFLPs, 62 RAPDs, 1 SNP, and three economically important morphological [ F (gynoecy), de (determinate habit), ll (little leaf)] markers. Seven linkage groups spanned 706 cM with a mean marker interval of 5.6 cM. The location of F and de was defined by genetic linkage and quantitative trait locus (QTL) analysis to be associated with SSR loci CSWCT28 and CSWCTT14 at 5.0 cM and 0.8 cM, respectively. RIL-based QTL analysis of the number of lateral branches in three environments revealed four location-independent factors that cumulatively explained 42% of the observed phenotypic variation. QTLs conditioning lateral branching (mlb1.1), fruit length/diameter ratio (ldr1.2) and sex expression (sex1.2) were associated with de. Sex expression was influenced by three genomic regions corresponding to F and de both on linkage Group 1, and a third locus (sex6.1) on linkage Group 6. QTLs conditioning the number of fruit per plant (fpl1.2), the number of lateral branches (mlb1.4) and fruit length/diameter ratio (ldr1.3) were associated with ll. The potential value of these marker-trait associations (i.e., yield components) for plant improvement is portended by the relatively high LOD scores (2.6 to 13.0) and associated R(2) values (1.5% to 32.4%) that are affiliated with comparatively few genetic factors (perhaps 3 to 10).

BackgroundA number of molecular marker linkage maps have been developed for melon (Cucumis melo L.) over the last two decades. However, these maps were constructed using different marker sets, thus, making comparative analysis among maps difficult. In order to solve this problem, a consensus genetic map in melon was constructed using primarily highly transferable anchor markers that have broad potential use for mapping, synteny, and comparative quantitative trait loci (QTL) analysis, increasing breeding effectiveness and efficiency via marker-assisted selection (MAS).ResultsUnder the framework of the International Cucurbit Genomics Initiative (ICuGI, http://www.icugi.org), an integrated genetic map has been constructed by merging data from eight independent mapping experiments using a genetically diverse array of parental lines. The consensus map spans 1150 cM across the 12 melon linkage groups and is composed of 1592 markers (640 SSRs, 330 SNPs, 252 AFLPs, 239 RFLPs, 89 RAPDs, 15 IMAs, 16 indels and 11 morphological traits) with a mean marker density of 0.72 cM/marker. One hundred and ninety-six of these markers (157 SSRs, 32 SNPs, 6 indels and 1 RAPD) were newly developed, mapped or provided by industry representatives as released markers, including 27 SNPs and 5 indels from genes involved in the organic acid metabolism and transport, and 58 EST-SSRs. Additionally, 85 of 822 SSR markers contributed by Syngenta Seeds were included in the integrated map. In addition, 370 QTL controlling 62 traits from 18 previously reported mapping experiments using genetically diverse parental genotypes were also integrated into the consensus map. Some QTL associated with economically important traits detected in separate studies mapped to similar genomic positions. For example, independently identified QTL controlling fruit shape were mapped on similar genomic positions, suggesting that such QTL are possibly responsible for the phenotypic variability observed for this trait in a broad array of melon germplasm.ConclusionsEven though relatively unsaturated genetic maps in a diverse set of melon market types have been published, the integrated saturated map presented herein should be considered the initial reference map for melon. Most of the mapped markers contained in the reference map are polymorphic in diverse collection of germplasm, and thus are potentially transferrable to a broad array of genetic experimentation (e.g., integration of physical and genetic maps, colinearity analysis, map-based gene cloning, epistasis dissection, and marker-assisted selection).

Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact.

Sex determination in cucumber (Cucumis safivus 1.) is controlled largely by three genes: F, m, and a. l h e F and m loci interact to produce monoecious (M-f_) or gynoecious (M-F-) sex phenotypes. Ethylene and factors that induce ethylene biosynthesis, such as 1 -aminocyclopropane-1 -carboxylate (ACC) and auxin, also enhance female sex expression. A genomic sequence (CS-ACSI) encoding ACC synthase was amplified from genomic DNA by a polymerase chain reaction using degenerate oligonucleotide primers. Expression of CS-ACSI is induced by auxin, but not by ACC, in wounded and intact shoot apices. Southern blot hybridization analysis of near-isogenic gynoecious (MMFF) and monoecious (MMff) lines derived from diverse genetic backgrounds revealed the existence of an additional ACC synthase (CS-ACSIC) genomic sequence in the gynoecious lines. Sex phenotype analysis of a segregating F, population detected a 100% correlation between the CS-ACSIG marker and the presence of the F locus. The CS-ACSIG gene is located in linkage group B coincident with the F locus, and in the population tested there was no recombination between the CS-ACSIG gene and the F locus. Collectively, these data suggest that CS-ACSIC is closely linked to the F locus and may play a pivotal role in the determination of sex in cucumber flowers.Sex determination in flowering plants is a developmentally regulated process that has been the topic of much research (Dellaporta and Calderon-Urrea, 1993;Grant et al., 1994). Dioecious and monoecious species of flowering plants present an excellent opportunity to study the diverse, developmental pathways that give rise to unisexual flowers. In monoecious wild-type cucumber (Cucumis sativus L. var sativus), flowers are produced in a preset, developmental sequence along the main stem, with a first phase of staminate flowers, followed by a mixed phase of staminate and pistillate flowers, and terminated by a pistillate flower phase (Galun, 1961;Shifriss, 1961). The develop-

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to characterize genetic relationships among 46 accessions in two C. melo L. subsp. melo (Cantalupensis, Inodorus) and subsp. agrestis (Conomon and Flexuosus) groups. Genetic distance (GD) estimates were made among and between accessions in four melon market classes [Galia, Ogen, Charentais, and Shipper (European and U.S. types)] of Cantalupensis, one market class of Inodorus (Cassaba and Honey Dew), one accession of Conomon, and one accession of Flexuosus by employing three GD estimators; simple matching coefficient, Jaccard's coefficient, and Nei's distance-D. Differences detected among 135 RAPD bands and 54 SSR bands (products of 17 SSR primers) were used to calculate GD. Band polymorphisms observed with 21 RAPD primers and 7 SSR primers was important in the detection of genetic differences. Estimators of GD were highly correlated (P > 0.0001; rs = 0.64 to 0.99) when comparisons were made between estimation methods within a particular marker system. Lower correlations (P > 0.001; rs = 0.17 to 0.40) were detected between marker systems using any one estimator. The GD of the Conomon and Flexuosus accessions was significantly different from the mean GD of all the market classes examined, and market classes were distinguishable from each other. Although lower coefficients of variation can be attained in the estimation of GD when using RAPDs compared to SSRs, the genetic relationships identified using these markers were generally similar. Results of RAPD marker analysis suggest that 80 marker bands were adequate for assessing the genetic variation present in the accessions examined.

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T ( n >/= 7) mononucleotide repeats. Each SSR sequence motif, along with +/-200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber ( Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.