Two populations [an F(2) and a set of 77 double haploid lines (DHLs)] developed from a cross between a 'Piel de Sapo' cultivar (PS) and the exotic Korean accession PI 161375 were used to detect QTLs involved in melon fruit quality traits: earliness (EA), fruit shape (FS), fruit weight (FW) and sugar content (SSC); and loci involved in the colour traits: external colour (ECOL) and flesh colour (FC). High variation was found, showing transgressive segregations for all traits. The highest correlation among experiments was observed for FS and the lowest for FW and SSC. Correlations among traits within experiments were, in general, not significant. QTL analysis, performed by Composite Interval Mapping, allowed the detection of nine QTLs for EA, eight for FS, six for FW and five for SSC. Major QTLs ( R(2)>25%) were detected for all traits. QTLs for different traits were no clearly co-localised, suggesting low pleiotropic effects at QTLs. Sixty-one per cent of them were detected in two or more experiments. QTLs for FS were detected in more trials than QTLs for FW and SSC, confirming that FS is under highly hereditable polygenic control. ECOL segregated as yellow:green in both experimental populations. The genetic control of ECOL was found to be complex, probably involving more than two loci with epistatic interactions. One of these loci was mapped on linkage group 9, but the other loci could not be clearly resolved. FC segregated as white:green:orange. The locus responsible for the green FC was mapped on linkage group 1, and it was proposed to correspond to the previously described locus gf. The genetic control of orange FC was complex: two loci in linkage groups 2 and 12 were associated with orange flesh, but larger population sizes would be necessary to elucidate completely the genetic control of orange flesh in this cross. Exotic alleles from PI161375 showed beneficial effects on EA, FW and SSC, indicating the usefulness of PI 161375 as a new source of genetic variability to improve European and American cultivars.

A set of 118 simple sequence repeat (SSR) markers has been developed in melon from two different sources: genomic libraries (gSSR) and expressed sequence-tag (EST) databases (EST-SSR). Forty-nine percent of the markers showed polymorphism between the 'Piel de Sapo' (PS) and PI161375 melon genotypes used as parents for the mapping populations. Similar polymorphism levels were found in gSSR (51.2%) and EST-SSR (45.5%). Two populations, F2 and a set of double haploid lines (DHLs), developed from the same parent genotypes were used for map construction. Twenty-three SSRs and 79 restriction fragment length polymorphisms (RFLPs), evenly distributed through the melon genome, were used to anchor the maps of both populations. Ten cucumber SSRs, 41 gSSRs, 16 EST-SSR, three single nucleotide polymorphism (SNP) markers, and the Nsv locus were added in the DHL population. The maps developed in the F2 and DHL populations were co-linear, with similar lengths, except in linkage groups G1, G9, and G10. There was segregation distortion in a higher proportion of markers in the DHL population compared with the F2, probably caused by selection during the construction of DHLs through in vitro culture. After map merging, a composite genetic map was obtained including 327 transferable markers: 226 RFLPs, 97 SSRs, three SNPs, and the Nsv locus. The map length is 1,021 cM, distributed in 12 linkage groups, and map density is 3.11 cM/marker. SSR markers alone cover nearly 80% of the map length. This map is proposed as a basis for a framework melon map to be merged with other maps and as an anchor point for map comparison between species of the Cucurbitaceae family.

Loci on LGIV, VI, and VIII of melon genome are involved in the control of fruit domestication-related traits and they are candidate to have played a role in the domestication of the crop. The fruit of wild melons is very small (20-50 g) without edible pulp, contrasting with the large size and high pulp content of cultivated melon fruits. An analysis of quantitative trait loci (QTL) controlling fruit morphology domestication-related traits was carried out using an in vitro maintained F population from the cross between the Indian wild melon "Trigonus" and the western elite cultivar 'Piel de Sapo'. Twenty-seven QTL were identified in at least two out of the three field trials. Six of them were also being detected in BC1 and BC3 populations derived from the same cross. Ten of them were related to fruit morphological traits, 12 to fruit size characters, and 5 to pulp content. The Trigonus alleles decreased the value of the characters, except for the QTL at andromonoecious gene at linkage group (LG) II, and the QTL for pulp content at LGV. QTL genotypes accounted for a considerable degree of the total phenotypic variation, reaching up to 46%. Around 66% of the QTL showed additive gene action, 19% exhibited dominance, and 25% consisted of overdominance. The regions on LGIV, VI, and VIII included the QTL with more consistent and strong effects on domestication-related traits. QTLs on those regions were validated in BC2S1, BC2S2, and BC3 families, with "Trigonus" allele decreasing the fruit morphological traits in all cases. The validated QTL could represent loci involved in melon domestication, although further experiments as genomic variation studies across wild and cultivated genotypes would be necessary to confirm this hypothesis.

BackgroundMelon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited.ResultWe constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences.ConclusionThe collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.

A new and simple protocol for androgenesis in bell pepper is described. The initial medium, a modification of Nitsch and Nitsch's H medium, consisted of a two-phase system of semi-solid and liquid medium and contained maltose as carbon source. The total number of embryos formed was greater with maltose at 40 g·L-1, but embryos developed better at 10 to 20 g·L-1. Depending on the genotype, the number of embryos and plants recovered ranged from 3 to 750 and 0.25 to 8, respectively, per 100 flowers. Further increases in the number of embryos (up to 3561 per 100 flowers) and plants (up to 23 per 100 flowers) could be attained by flushing cultures with air enriched with CO2 at 900 μL·L-1. The ploidy level and the microspore origin of the recovered plants were determined by flow cytometry and zymograms for isocitrate dehydrogenase. Nearly 65% of the acclimated plants had undergone spontaneous doubling of the chromosome number, as confirmed by flow cytometry of leaf nuclei. Isocitrate dehydrogenase zymograms demonstrated that plants originated from microspores and that the two parental alleles were equally represented among the haploid and dihaploid plants.

Appropriate micropropagation regimes were determined for four Pyrus species (P. amygdaliformis Vill., P. betulaefolia Bunge, P. calleryana Dcne., and P. communis L.) and Cydonia oblonga L. Shoot multiplication was optimal at 10 or 20/~M N6-benzyladenine and high light intensity (135 #E m-2s-l). Root formation of the Pyrus species was stimulated by exposure of shoots to high levels (10 or 32/~M) of/~-indolebutyric acid (IBA) for 7 days or a dip in 10 mM IBA for 15 s, followed by a passage on auxin-free medium. 0~-Naphthaleneacetic acid was more effective than IBA in stimulating rooting of C. oblonga. The effects of Fe-limiting conditions in vitro were determined by comparing the responses of shoots and rooted plantlets to medium containing FeEDTA or FeSO4, with or without bicarbonate. Symptoms of Fe deficiency were genotypedependent and most severe in the presence of FeSO4 and bicarbonate. Chlorosis was pronounced in Cydonia, absent from P. amygdaliformis and P. communis, and intermediate inP. betulaefolia and P. calleryana, indicating a good correlation between in vitro and field responses. Similar responses were obtained with rooted and unrooted shoots. These findings suggest that in vitro cultures may be used for studies of Fe-chlorosis as well as screening for tolerant rootstocks.

ADDITIONAL INDEX WORDS. Cucumis sativus, Cucurbitaceae, parthenogenesis, haploid, DHL, pure line, hybrid ABSTRACT. Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co 60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identifi ed by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by fl ow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfi ng of selected lines. Codominant markers and fl ow cytometry confi rmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μM colchicine. Rescue of diploid or chimeric plants was shown by fl ow cytometry, prior to their acclimation and planting in the greenhouse. Selfi ng of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.

Melon (Cucumis melo) is one of the principal vegetable crops for fresh market, for which a large number of breeding programs, oriented to generate inbred pure lines and hybrids, is established worldwide. The process to obtain and select these lines has been highly accelerated by the use of biotechnological techniques such as the generation of doubled haploid line (DHL) populations and molecular markers. Moreover, the use of DHLs in genetic studies is a useful tool because of their complete homozygosity and the permanent availability of plant material perpetuated by seed. In this work, the parthenogenetic response of 17 melon genotypes and the F1 hybrid PI 161375 × Spanish cultivar Piel de Sapo (PS) was studied considering three stages along the in vitro DHL generation process. The response of the analyzed melon cultivars was heterogeneous through the DHL generation with different limiting steps for each genotype. The response of the PI 161375 × PS hybrid was more similar to the male (PS) than the female parent (PI 161375), although the response of the maternal genotype was higher for some stages. This points to the important role of alleles from both parents in the different steps of the DHL generation process, and it could explain the identification of six genomic regions with distorted allelic segregation skewed toward PS or PI 161375. This hybrid was used to generate a population of 109 DHLs, the gametophytic origin of which was confirmed by flow cytometry and molecular markers.