Cristina Esteras

Transcriptome sequencing for SNP discovery across Cucumis melo

BackgroundMelon (Cucumis melo L.) is a highly diverse species that is cultivated worldwide. Recent advances in massively parallel sequencing have begun to allow the study of nucleotide diversity in this species. The Sanger method combined with medium-throughput 454 technology were used in a previous study to analyze the genetic diversity of germplasm representing 3 botanical varieties, yielding a collection of about 40,000 SNPs distributed in 14,000 unigenes. However, the usefulness of this resource is limited as the sequenced genotypes do not represent the whole diversity of the species, which is divided into two subspecies with many botanical varieties variable in plant, flowering, and fruit traits, as well as in stress response. As a first step to extensively document levels and patterns of nucleotide variability across the species, we used the high-throughput SOLiD™ system to resequence the transcriptomes of a set of 67 genotypes that had previously been selected from a core collection representing the extant variation of the entire species.ResultsThe deep transcriptome resequencing of all of the genotypes, grouped into 8 pools (wild African agrestis, Asian agrestis and acidulus, exotic Far Eastern conomon, Indian momordica and Asian dudaim and flexuosus, commercial cantalupensis, subsp. melo Asian and European landraces, Spanish inodorus landraces, and Piel de Sapo breeding lines) yielded about 300 M reads. Short reads were mapped to the recently generated draft genome assembly of the DHL line Piel de Sapo (inodorus) x Songwhan Charmi (conomon) and to a new version of melon transcriptome. Regions with at least 6X coverage were used in SNV calling, generating a melon collection with 303,883 variants. These SNVs were dispersed across the entire C. melo genome, and distributed in 15,064 annotated genes. The number and variability of in silico SNVs differed considerably between pools. Our finding of higher genomic diversity in wild and exotic agrestis melons from India and Africa as compared to commercial cultivars, cultigens and landraces from Eastern Europe, Western Asia and the Mediterranean basin is consistent with the evolutionary history proposed for the species. Group-specific SNVs that will be useful in introgression programs were also detected. In a sample of 143 selected putative SNPs, we verified 93% of the polymorphisms in a panel of 78 genotypes.ConclusionsThis study provides the first comprehensive resequencing data for wild, exotic, and cultivated (landraces and commercial) melon transcriptomes, yielding the largest melon SNP collection available to date and representing a notable sample of the species diversity. This data provides a valuable resource for creating a catalog of allelic variants of melon genes and it will aid in future in-depth studies of population genetics, marker-assisted breeding, and gene identification aimed at developing improved varieties.

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.

Towards a TILLING platform for functional genomics in Piel de Sapo melons

BackgroundThe availability of genetic and genomic resources for melon has increased significantly, but functional genomics resources are still limited for this crop. TILLING is a powerful reverse genetics approach that can be utilized to generate novel mutations in candidate genes. A TILLING resource is available for cantalupensis melons, but not for inodorus melons, the other main commercial group.ResultsA new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1 mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point mutations were found for the seven gene targets, six of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the Phytoene desaturase gene, which is involved in carotenoid biosynthesis.ConclusionsThe TILLING approach was successful at providing new mutations in the genetic background of Piel de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic and breeding studies.

Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.)

BackgroundA collection of 175 melon (Cucumis melo L.) accessions (including wild relatives, feral types, landraces, breeding lines and commercial cultivars) from 50 countries was selected to study the phenotypic variability for ripening behavior and sugar accumulation. The variability of single nucleotide polymorphisms (SNPs) at 53 selected candidate genes involved in sugar accumulation and fruit ripening processes was studied, as well as their association with phenotypic variation of related traits.ResultsThe collection showed a strong genetic structure, defining seven groups plus a number of accessions that could not be associated to any of the groups (admixture), which fitted well with the botanical classification of melon varieties. The variability in candidate genes for ethylene, cell wall and sugar-related traits was high and similar to SNPs located in reference genes. Variability at ripening candidate genes had an important weight on the genetic stratification of melon germplasm, indicating that traditional farmers might have selected for ripening traits during cultivar diversification. A strong relationship was also found between the genetic structure and phenotypic diversity, which could hamper genetic association studies. Accessions belonging to the ameri group are the most appropriate for association analysis given the high phenotypic and molecular diversity within the group, and lack of genetic structure.The most remarkable association was found between sugar content and SNPs in LG III, where a hotspot of sugar content QTLs has previously been defined. By studying the differences in allelic variation of SNPs within horticultural groups with specific phenotypic features, we also detected differential variation in sugar-related candidates located in LGIX and LGX, and in ripening-related candidates located in LGII and X, all in regions with previously mapped QTLs for the corresponding traits.ConclusionsIn the current study we have found an important variability at both the phenotypic and candidate gene levels for ripening behavior and sugar accumulation in melon fruit. By combination of differences in allelic diversity and association analysis, we have identified several candidate genes that may be involved in the melon phenotypic diversity.

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.

De novo assembly of the zucchini genome reveals a whole-genome duplication associated with the origin of the Cucurbita genus

Summary The Cucurbita genus (squashes, pumpkins and gourds) includes important domesticated species such as C. pepo, C. maxima and C. moschata. In this study, we present a high‐quality draft of the zucchini (C. pepo) genome. The assembly has a size of 263 Mb, a scaffold N50 of 1.8 Mb and 34 240 gene models. It includes 92% of the conserved BUSCO core gene set, and it is estimated to cover 93.0% of the genome. The genome is organized in 20 pseudomolecules that represent 81.4% of the assembly, and it is integrated with a genetic map of 7718 SNPs. Despite the small genome size, three independent lines of evidence support that the C. pepo genome is the result of a whole‐genome duplication: the topology of the gene family phylogenies, the karyotype organization and the distribution of 4DTv distances. Additionally, 40 transcriptomes of 12 species of the genus were assembled and analysed together with all the other published genomes of the Cucurbitaceae family. The duplication was detected in all the Cucurbita species analysed, including C. maxima and C. moschata, but not in the more distant cucurbits belonging to the Cucumis and Citrullus genera, and it is likely to have occurred 30 ± 4 Mya in the ancestral species that gave rise to the genus.

Fruit flesh volatile and carotenoid profile analysis within the Cucumis melo L. species reveals unexploited variability for future genetic breeding: Fruit flesh volatile and carotenoid profile variability within the Cucumis melo L. species

BACKGROUND Aroma profile and carotenoids content of melon flesh are two important aspects influencing the quality of this fruit that have been characterized using only selected genotypes. However, the extant variability of the whole species remains unknown. RESULTS A complete view of the volatile/carotenoid profiles of melon flesh was obtained analyzing 71 accessions, representing the whole diversity of the species. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used to analyze 200 volatile compounds and five carotenoids. Genotypes were classified into two main clusters (high/low aroma), but with a large diversity of differential profiles within each cluster, consistent with the ripening behavior, flesh color and proposed evolutionary and breeding history of the different horticultural groups. CONCLUSION Our results highlight the huge amount of untapped aroma diversity of melon germplasm, especially of non-commercial types. Also, landraces with high nutritional value with regard to carotenoids have been identified. All this knowledge will encourage melon breeding, facilitating the selection of the genetic resources more appropriate to develop cultivars with new aromatic profiles or to minimize the impact of breeding on melon quality. The newly characterized sources provide the basis for further investigations into specific genes/alleles contributing to melon flesh quality.

‘MAK-10’: A Long Shelf-life Charentais Breeding Line Developed by Introgression of a Genomic Region from Makuwa Melon

This work has been carried out in the framework of the Programa de Valorizacion y Recursos Conjuntos de I+D+i de VLC/CAMPUS and has been funded by the Ministerio de Educacion, Cultura y Deporte as part of the Programa Campus de Excelencia Internacional. IL generation, genotyping, and phenotyping were supported by SAFQIM project, AGL2012-40130-C02-02 of the Spanish Ministry of Economy and Competitivity (MINECO). We wish to thank the MINECO project AGL2014-53398-C2-2-R, co-funded with FEDER funds.This work has been carried out in the framework of the Programa de Valorizacion y Recursos Conjuntos de I+D+i de VLC/CAMPUS and has been funded by the Ministerio de Educacion, Cultura y Deporte as part of the Programa Campus de Excelencia Internacional. IL generation, genotyping, and phenotyping were supported by SAFQIM project, AGL2012-40130-C02-02 of the Spanish Ministry of Economy and Competitivity (MINECO). We wish to thank the MINECO project AGL2014-53398-C2-2-R, co-funded with FEDER funds.

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.

Genetics and Genomics of Cucurbita spp.

Cucurbita pepo is one of the main crops of the Cucurbitaceae family. Despite its agricultural and biological importance, genomic research on this species has started later and progresses slowerthan in other cucurbits. Here we review the latest genetic and genomic tools developed for C. pepo. A whole-genome shotgun strategy based on pair-end and mate-pair Illumina sequencing,has generated a whole genome draft of 263 Mb into 26,005 scaffolds and 32,754 contigs (contig N50:110 kb and scaffold N50:1.8 Mb). The genome sequence has been annotated using the transcriptome (73,239 unigene clusters, with an average length of 1050 bp) and anchored using a high density genetic map based on a recombinant inbred line (RIL) population (with 6763 SNPs distributed across 21 linkage groups, with a total length of 2635 cM). Further RNA-seq resequencing and GBS genotyping efforts in C.pepohave been conducted to better represent the whole species variation, including the three subspecies (pepo, ovifera and fraterna). Similar information has been generated in related cultivated (C. moschata, C. maxima, C. argyrosperma, C. ficifolia) and wild species of the Cucurbita genus, to provide a valuable insight into the Cucurbita genetic variation. These studies have generated large SNPs collections usefulfor different breeding purposes. All this sequence information along with high throughput reverse genetic tools, such as the first C.pepo TILLING population, and with new mapping populations suitable for complex trait genetic dissection, are allowing the identification of candidate genes underlying the variation of key traits for C. pepo breeding.