Fernando Nuez

Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers

Cucurbita pepo is a highly polymorphic species. The cultivars can be grouped into eight morphotypes in two subspecies, ssp. pepo and ssp. ovifera. A collection of 69 accessions representative of the morphotypes and some unclassified types was used for analysing the morphological and molecular diversity of this species. This collection includes commercial cultivars and Spanish landraces, which represent the great diversification of types that have arisen in Europe after this species arrived from America. For the molecular variability studies, two PCR-based systems were employed, AFLP and SRAP, which preferentially amplify ORFs. Principal coordinates analysis and cluster analysis using the UPGMA method clearly separate the accessions into the two subspecies through the use of both markers. However, the gene diversity and the genetic identity values among morphotypes and subspecies varied between the two marker systems. The information given by SRAP markers was more concordant to the morphological variability and to the evolutionary history of the morphotypes than that of AFLP markers. In ssp. ovifera, the accessions of the different morphotypes were basically grouped according to the fruit colour. This may indicate different times of development and also the extent of breeding in the accessions used. This study has allowed identification of new types that can be employed for the development of new cultivars. The landraces of the spp. ovifera, used as ornamental in Europe, have proved to be of great interest for preserving the diversity of C. pepo.

The genome of melon (Cucumis melo L.)

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site–leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.

MELOGEN: an EST database for melon functional genomics

BackgroundMelon (Cucumis melo L.) is one of the most important fleshy fruits for fresh consumption. Despite this, few genomic resources exist for this species. To facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags (ESTs) from eight normalized cDNA libraries from different tissues in different physiological conditions.ResultsWe determined over 30,000 ESTs that were clustered into 16,637 non-redundant sequences or unigenes, comprising 6,023 tentative consensus sequences (contigs) and 10,614 unclustered sequences (singletons). Many potential molecular markers were identified in the melon dataset: 1,052 potential simple sequence repeats (SSRs) and 356 single nucleotide polymorphisms (SNPs) were found. Sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. Functional classification of the unigenes was carried out following the Gene Ontology scheme. In total, 9,402 unigenes were mapped to one or more ontology. Remarkably, the distributions of melon and Arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. Bioinformatic analyses primarily focused on potential precursors of melon micro RNAs (miRNAs) in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. Patterns of transcript accumulation were characterised by Real-Time-qPCR for 20 of these genes.ConclusionThe collection of ESTs characterised here represents a substantial increase on the genetic information available for melon. A database (MELOGEN) which contains all EST sequences, contig images and several tools for analysis and data mining has been created. This set of sequences constitutes also the basis for an oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome.

How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis.

Microspore embryogenesis is the most powerful androgenic pathway to produce haploid and doubled haploid plants. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. In this review, we compile the most recent advances in the understanding of the changes undergone by the induced microspore to readapt to the new developmental scenario. We devote special attention to the efforts made to uncover changes in the transcriptome of the induced microspore and microspore-derived embryo (MDE). Finally, we discuss the influence that an in vitro environment exerts over the MDE, as compared with its zygotic counterpart.

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.

Evaluation of whitefly-mediated inoculation techniques to screen Lycopersicon esculentum and wild relatives for resistance to Tomato yellow leaf curl virus

For two consecutive years nine hybrids and three varieties of tomato, four Lycopersicon peruvianum and four Lycopersicon chilense accessions were screened for Tomato yellow leaf curl virus (TYLCV) resistance. Three inoculation techniques using Bemisia tabaci, the vector of TYLCV, were compared: (1) artificial mass inoculation-simultaneous infection of cultivated and wild material in greenhouses; (2) artificial cage inoculation-individual infection in insect-proof cages; (3) natural field infection. Artificial inoculations led to higher levels of infection, but different patterns of response to each inoculation technique were found depending on the resistance level.Tomato varieties showed an important fruit set reduction after both artificial and natural inoculations. In contrast, field infection was milder in tomato hybrids, in which yield was barely affected. These hybrids showed a wide range of reactions with the two artificial inoculation techniques, but infection was always more severe after mass inoculation. Extreme severity of mass infection made it difficult to differentiate among variable degrees of resistance that were more reliably detected with cage inoculation. The hybrids F3524, F3522, Fiona, and Tyking showed the highest level of resistance. F3524 and F3522 had an acceptable yield in field and cage assays, but their resistance collapsed under massive conditions of infection. Tyking and Fiona exhibited the best response in all conditions, although their yield was moderately reduced in mass assays. Mass inoculation was not adequate for the screening of wild Lycopersicon. Some susceptible plants escaped infection, probably as a consequence of non-preference mechanisms and loss of vector infectivity. Individual inoculation in cages prevented the risk of non-infection, ensuring 100% disease incidence. This technique allowed the selection of highly resistant wild sources. L. chilense LA 1969 and LA 1963 had the highest level of resistance with the three inoculation techniques. L. peruvianum PI-126944 and L. chilense LA 1932, which were only tested in mass and field conditions, also exhibited a promising response.The results proved that the inoculation technique influences the response of tomato and wild Lycopersicon spp to TYLCV. It is concluded that artificial cage inoculation, although more time-consuming, is the most efficient, adequate, and reliable technique to screen both cultivated and wild Lycopersicon species for resistance to TYLCV.

Variation Revealed by SNP Genotyping and Morphology Provides Insight into the Origin of the Tomato

Tomato, Solanum lycopersicum, is divided into two widely distributed varieties: the cultivated S. lycopersicum var. lycopersicum, and the weedy S. lycopersicum var. cerasiforme. Solanum pimpinellifolium is the most closely related wild species of tomato. The roles of S. pimpinellifolium and S. l. cerasiforme during the domestication of tomato are still under debate. Some authors consider S. l. cerasiforme to be the ancestor, whereas others think that S. l. cerasiforme is an admixture of S. pimpinellifolium and the cultivated S. l. lycopersicum. It is also not clear whether the domestication occurred in the Andean region or in Mesoamerica. We characterized 272 accessions (63 S. pimpinellifolium, 106 S. l. cerasiforme, 95 S. l. lycopersicum and 8 derived from hybridization processes) were morphologically and genetically using the SolCap platform (7,414 SNPs). The two species were distinguished in a PCA analysis and displayed a rich geographic structure. Solanum lycopersicum var. cerasiforme and S. l. lycopersicum were also differentiated in the PCA and Structure analyses, which supports maintaining them as different varieties. Solanum pimpinellifolium and the Andean S. l. cerasiforme were more diverse than the non-Andean S. lycopersicum. Solanum lycopersicum var. cerasiforme was morphologically and molecularly intermediate between S. pimpinellifolium and tomato. Solanum lycopersicum var. cerasiforme, with the exception of several Ecuadorian and Mexican accessions, is composed of the products of admixture processes according to the Structure analysis. The non-admixtured S. l. cerasiforme might be similar to the ancestral cultivars from which the cultivated tomato originated, and presents remarkable morphological diversity, including fruits of up to 6 cm in diameter. The data obtained would fit a model in which a pre-domestication took place in the Andean region, with the domestication being completed in Mesoamerica. Subsequently, the Spaniards took plants from Mesoamerica to Spain and from there they were exported to the rest of the world.

An oligo-based microarray offers novel transcriptomic approaches for the analysis of pathogen resistance and fruit quality traits in melon (Cucumis melo L.)

BackgroundMelon (Cucumis melo) is a horticultural specie of significant nutritional value, which belongs to the Cucurbitaceae family, whose economic importance is second only to the Solanaceae. Its small genome of approx. 450 Mb coupled to the high genetic diversity has prompted the development of genetic tools in the last decade. However, the unprecedented existence of a transcriptomic approaches in melon, highlight the importance of designing new tools for high-throughput analysis of gene expression.ResultsWe report the construction of an oligo-based microarray using a total of 17,510 unigenes derived from 33,418 high-quality melon ESTs. This chip is particularly enriched with genes that are expressed in fruit and during interaction with pathogens. Hybridizations for three independent experiments allowed the characterization of global gene expression profiles during fruit ripening, as well as in response to viral and fungal infections in plant cotyledons and roots, respectively. Microarray construction, statistical analyses and validation together with functional-enrichment analysis are presented in this study.ConclusionThe platform validation and enrichment analyses shown in our study indicate that this oligo-based microarray is amenable for future genetic and functional genomic studies of a wide range of experimental conditions in melon.

ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence

BackgroundThe possibilities offered by next generation sequencing (NGS) platforms are revolutionizing biotechnological laboratories. Moreover, the combination of NGS sequencing and affordable high-throughput genotyping technologies is facilitating the rapid discovery and use of SNPs in non-model species. However, this abundance of sequences and polymorphisms creates new software needs. To fulfill these needs, we have developed a powerful, yet easy-to-use application.ResultsThe ngs_backbone software is a parallel pipeline capable of analyzing Sanger, 454, Illumina and SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequence reads. Its main supported analyses are: read cleaning, transcriptome assembly and annotation, read mapping and single nucleotide polymorphism (SNP) calling and selection. In order to build a truly useful tool, the software development was paired with a laboratory experiment. All public tomato Sanger EST reads plus 14.2 million Illumina reads were employed to test the tool and predict polymorphism in tomato. The cleaned reads were mapped to the SGN tomato transcriptome obtaining a coverage of 4.2 for Sanger and 8.5 for Illumina. 23,360 single nucleotide variations (SNVs) were predicted. A total of 76 SNVs were experimentally validated, and 85% were found to be real.Conclusionsngs_backbone is a new software package capable of analyzing sequences produced by NGS technologies and predicting SNVs with great accuracy. In our tomato example, we created a highly polymorphic collection of SNVs that will be a useful resource for tomato researchers and breeders. The software developed along with its documentation is freely available under the AGPL license and can be downloaded from http://bioinf.comav.upv.es/ngs_backbone/ or http://github.com/JoseBlanca/franklin.

Evolutionary analysis of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe economic losses in many crops worldwide and often overcomes resistant cultivars used for disease control. Comparison of nucleotide and amino acid sequences suggested that tomato resistance conferred by the gene Sw-5 can be overcome by the amino acid substitution C to Y at position 118 (C118Y) or T120N in the TSWV movement protein, NSm. Phylogenetic analysis revealed that substitution C118Y has occurred independently three times in the studied isolates by convergent evolution, whereas the substitution T120N was a unique event. Analysis of rates of non-synonymous and synonymous changes at individual codons showed that substitution C118Y was positively selected.