María Belén Picó

Genetic diversity of some accessions of Cucurbita maxima from Spain using RAPD and SBAP markers

Increasing the knowledge of the molecular diversity of a crop is essential for extending its genetic base, identifying cultivars and selecting parental varieties for breeding programs. In this sense, Cucurbita maxima Duch. is poorly characterised. Nineteen accessions of this species and 8 related Cucurbita accessions were included in a genetic diversity analysis. For this purpose, Random Amplified Polymorphic DNA markers (RAPDs), which analyse neutral variability, and Sequence-Based Amplified Polymorphism (SBAPs), which preferentially amplify coding regions of the genome, were used. While the UPGMA cluster and the principal coordinates analysis obtained using RAPDs did not group the different accessions according either to fruit morphological criteria or to passport data (origin and agro-climatic conditions), the principal coordinates analysis obtained using SBAPs grouped the different pumpkin accessions fundamentally according to the type of use (human consumption, animal fodder or ornamental). This passport trait is reported to be associated with agronomic breeding characters of interest. The usefulness of both types of markers for discriminating accessions of breeding interest is discussed.

Melon transcriptome characterization: Simple Sequence Repeats and Single Nucleotide Polymorphisms discovery for high throughput genotyping across the species.

Melon (Cucumis melo L.) ranks among the highest‐valued fruit crops worldwide. Some genomic tools are available for this crop, including a Sanger transcriptome. We report the generation of 689,054 C. melo high‐quality expressed sequence tags (ESTs) from two 454 sequencing runs, using normalized and nonnormalized complementary DNA (cDNA) libraries prepared from four genotypes belonging to the two C. melo subspecies and the main commercial types. 454 ESTs were combined with the Sanger available ESTs and de novo assembled into 53,252 unigenes. Over 63% of the unigenes were functionally annotated with Gene Ontology (GO) terms and 21% had known orthologs of Arabidopsis thaliana (L.) Heynh. Annotation distribution followed similar tendencies than that reported for Arabidopsis thaliana, suggesting that the dataset represents a fairly complete melon transcriptome. Furthermore, we identified a set of 3298 unigenes with microsatellite motifs and 14,417 sequences with single nucleotide variants of which 11,655 single nucleotide polymorphism met criteria for use with high‐throughput genotyping platforms, and 453 could be detected as cleaved amplified polymorphic sequence (CAPS). A set of markers were validated, 90% of them being polymorphic in a number of variable C. melo accessions. This transcriptome provides an invaluable new tool for biological research, more so when it includes transcripts not described previously. It is being used for genome annotation and has provided a large collection of markers that will allow speeding up the process of breeding new melon varieties.

Genetic diversity of Spanish Cucurbita pepo landraces: an unexploited resource for summer squash breeding

Cucurbita pepo is a worldwide cultivated vegetable of American origin. Most of the widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). These forms were developed in Europe after the arrival of the first American landraces through a process of selection and fixation that led to a loss of genetic diversity. Part of the genetic variability of the first American cultigens remains intact in diverse landraces that are still cultivated for self-consumption and sale in local markets. Using the first collection of genomic and EST-derived microsatellites that has just become available for the species, we compared the natural variation present in a collection of Spanish landraces with that of a set of commercial varieties and hybrids, representing current summer squash market offerings. A total of 194 alleles allowed us to distinguish all the genotypes, even those that were closely related. In general, Cocozelle and Vegetable marrow, groups with considerably long histories, were more variable than the Zucchini group, of more recent origin. We found significant genetic diversity among landraces. The variation present among landraces belonging to the Zucchini group was larger than that of the commercial cultivars. Cluster, principal coordinate and population structure results suggested that the variation of the Spanish landraces has not been extensively used in breeding. Commercial summer squashes can therefore benefit from this underexploited variability, especially from certain landraces that already display favourable commercial traits.

Mechanical transmission of Tomato leaf curl New Delhi virus to cucurbit germplasm: selection of tolerance sources in Cucumis melo

Cucurbits are major crop species, including fruits and vegetables cultivated worldwide that supply essential vitamins and minerals to current diets in developed and developing countries. Viral diseases are main factors affecting cucurbits cultivation. The most widespread and damaging have been aphid-borne viruses belonging to the Potyviridae family. Whitefly-transmitted begomoviruses (Geminiviridae) have been identified more recently in different cucurbit species. A severe outbreak of Tomato leaf curl New Delhi virus (ToLCNDV) occurred in pumpkins and melons in the main production area of Southern Spain in 2012–2014. We developed a mechanical inoculation method to facilitate the screening of germplasm against this virus. Mechanical transmission with this method was confirmed in 4 genera and 13 species of the family, including the main crops, cucumber, melon, watermelon and pumpkins, and also crop-related exotic germplasm (landraces and wild species) used for cucurbits breeding. Diversity in the response was observed within and among species. Tolerance to mechanical transmission of ToLCNDV was identified in melon, within Cucumis melo subsp. agrestis var. momordica and in wild agrestis accessions. All the tolerant accessions came from India, the country in which this virus was firstly reported. Some of these accessions have been previously reported to be tolerant or resistant to other viruses and as they are fully crossable to commercial melons, they are good sources to develop new melon varieties with tolerance to ToLCNDV.

Screening a variable germplasm collection of Cucumis melo L. for seedling resistance to Macrophomina phaseolina

We evaluate the seedling resistance to charcoal rot caused by Macrophomina phaseolina in ninety-seven Cucumis melo accessions, from different geographical origins and five F1 generations, derived from crosses of five accessions selected for their resistance. Artificial inoculations with the toothpick method, previously reported to be useful for predicting shoot resistance, were performed, and plants were scored using a scale of disease severity. The average disease severity was calculated for each accession and was used to cluster the accession in five reaction classes. The screening revealed that sources of natural resistance to this fungus are limited. However, seedlings of seven accessions of different botanic groups displayed a resistant response to the stem inoculation, one cantaloup from Israel, one conomon accession from Korea, two wild agrestis and one acidulus from Africa, and two dudaim accessions from Middle East. The response of the F1 progenies varied from susceptibility to high resistance, the latter in progenies from the two agrestis wild types. These results suggest differences in the genetic basis of the resistance in the different selected sources. The resistant accessions are suggested to be screened under field conditions to confirm the level of resistance at adult plant stage and under stressful conditions.

Fruit quality assessment of watermelons grafted onto citron melon rootstock

BACKGROUND The grafting of watermelons (Citrullus lanatus) is a common technique that increases yield under stressful soil conditions. The most common rootstocks for watermelons are Cucurbita hybrids. However, they often have a negative impact on fruit quality. Exploiting novel Citrullus germplasm such as citron melon (Citrullus lanatus var. citroides) is an alternative to avoid these quality problems. RESULTS Citron melon has been validated as watermelon rootstock, comparing its effects on watermelon quality to those of Cucurbita hybrids. Larger fruits with thicker rinds were observed in fruits from plants grafted onto both citron and Cucurbita rootstocks. The citron melon had no significant effect on flesh sugars or acid profiles compared to non-grafted watermelons, except for an increase in glucose and malic acid content, which also occurred in the Cucurbita rootstocks. The aroma profile of fruits produced on citron melon was similar to that of the non-grafted and self-grafted controls. The citron rootstock did not display the increased levels of (Z)-6-nonen-1-ol (a compound associated with pumpkin-like odors) found in fruits produced with Cucurbita hybrids. CONCLUSION The low impact of citron melon rootstock on fruit quality, along with the enhanced resistance against nematodes, make the citron a promising alternative to Cucurbita rootstocks.

Brazilian melon landraces resistant to Podosphaera xanthii are unique germplasm resources

Podosphaera xanthii is the most important causal agent of powdery mildew in melon, a crop ranked within the most economically important species worldwide. The best strategy to face this fungus disease, which causes important production losses, is the development of genetically resistant cultivars. Genetic breeding programmes require sources of resistance, and a few ones have been reported in melon, mostly in Momordica and Acidulus horticultural groups. However, the existence of many races that reduces the durability of the resistance makes necessary to find new resistant genotypes with different genetic backgrounds. In this work, Brazilian germplasm, together with a set of Indian landraces, and the COMAVs (Institute for the Conservation and Breeding of Agricultural Biodiversity) melon core collection, representing the whole variability of the species, were assessed for resistance against some common races in Spain and Brazil and genotyped with a 123-SNP (single nucleotide polymorphisms) genotyping platform to study the molecular relationships of the resistant accessions. In the first experiment, carried out in Valencia (Spain) in 2013, seventy-nine melon accessions were evaluated using artificial inoculation. Five accessions selected as resistant were also evaluated against races 1, 3, and 5 in Mossoro (Brazil, 2015) and against race 3.5 in Valencia (2016) under greenhouse conditions, and under four field conditions in Brazil. The accessions, AL-1, BA-3, CE-3, and RN-2, within the Brazilian collection, presented resistance against all the races of P. xanthii assayed in all conditions tested. AL-1, CE-3 and RN-2 were molecularly more similar to wild agrestis and Acidulus melons from Asia and Africa, while BA-3 grouped with Momordica types. Molecular analysis also confirmed that these new Brazilian sources of resistance differ from those previously reported, constituting interesting materials to encourage genetic breeding programmes, especially in Brazil and Spain.