María José Díez

Application of genomic tools in plant breeding.

Plant breeding has been very successful in developing improved varieties using conventional tools and methodologies. Nowadays, the availability of genomic tools and resources is leading to a new revolution of plant breeding, as they facilitate the study of the genotype and its relationship with the phenotype, in particular for complex traits. Next Generation Sequencing (NGS) technologies are allowing the mass sequencing of genomes and transcriptomes, which is producing a vast array of genomic information. The analysis of NGS data by means of bioinformatics developments allows discovering new genes and regulatory sequences and their positions, and makes available large collections of molecular markers. Genome-wide expression studies provide breeders with an understanding of the molecular basis of complex traits. Genomic approaches include TILLING and EcoTILLING, which make possible to screen mutant and germplasm collections for allelic variants in target genes. Re-sequencing of genomes is very useful for the genome-wide discovery of markers amenable for high-throughput genotyping platforms, like SSRs and SNPs, or the construction of high density genetic maps. All these tools and resources facilitate studying the genetic diversity, which is important for germplasm management, enhancement and use. Also, they allow the identification of markers linked to genes and QTLs, using a diversity of techniques like bulked segregant analysis (BSA), fine genetic mapping, or association mapping. These new markers are used for marker assisted selection, including marker assisted backcross selection, ‘breeding by design’, or new strategies, like genomic selection. In conclusion, advances in genomics are providing breeders with new tools and methodologies that allow a great leap forward in plant breeding, including the ‘superdomestication’ of crops and the genetic dissection and breeding for complex traits.

A cytochrome P450 regulates a domestication trait in cultivated tomato.

Significance This study reports the cloning of a tomato gene, SlKLUH, that controls fruit mass by increased cell layers and delayed fruit ripening. In addition, we identified a potential regulatory SNP in the promoter of SlKLUH that is significantly associated with the fruit mass. Altogether, our study encompasses several genetic analyses, as well as association mapping, plant transformation experiments, and phenotypic evaluations to offer insights into the molecular basis of the regulation of tomato fruit mass, a critical trait in the domestication of fruit and vegetable crops. Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SlKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SlKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SlKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SlKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops.

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.

Development and validation of an automated high-throughput system for zebrafish in vivo screenings.

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.

Genetics of tomato spotted wilt virus resistance coming from Lycopersicon peruvianum

New resistance sources coming from Lycopersicon peruvianum, especially those introgressed in UPV 32 line, are studied. UPV 32 resistance is controlled by a single gene. Resistance and dominance levels of this gene are conditioned by thrips transmission and isolate aggressiveness. A partial overcoming of resistance occurs due to the incomplete penetrance of the gene. Incomplete dominance of resistance also happens, which suggests gene dosage dependence. UPV 32 gene segregates independently of both Sw-5 gene and UPV 1 resistance gene, also coming from Lycopersicon peruvianum. It is proposed to name Sw-6 this new locus from UPV 32. Sw-5 gene and UPV 1 resistance gene show higher resistance than Sw-6. Heterozygotes for UPV 1 resistance gene were more resistant than heterozygotes for Sw-5. The lower dependence of UPV 1 resistance gene on the gene dosage effect makes it very useful for the development of commercial hybrids.

Effect of Temperature Regime and Growth Stage Interaction on Pattern of Virus Presence in TSWV-Resistant Accessions of Capsicum chinense

We studied the resistance to tomato spotted wilt virus in plant introduction accession (PI)-151225 and PI-159236 under Mediterranean climatic conditions. Two temperature regimes were utilized, corresponding to early and late cultivation cycles. Inoculations were made at 2- and 4-leaf stages to determine the effect of early infection. The existence of interaction between temperature regime and developmental stage was also studied. When plants of both PIs were maintained at 30/18°C (day/night), all plants at both growth stages when inoculated developed systemic infection. At 25/18°C, only those plants inoculated at 2-leaf stage became systematically infected; however, those inoculated at the 4-leaf stage behaved as resistant. Thus, there was an interaction between temperature regime and growth stage. There is potential for using this type of resistance in areas with mild climates, providing seedling infections are avoided.

Identification of a CAPS marker tightly linked to the Tomato yellow leaf curl disease resistance gene Ty-1 in tomato

During the process of breeding programmes, several resistance genes have been introgressed into tomato (Solanum lycopersicum) cultivars from different wild tomato relatives. A number of these resistance genes have been mapped to chromosome 6. Among them, Ty-1 and Mi, which confer resistance to Tomato yellow leaf curl disease and to Meloidogyne spp., respectively, are in most cases incorporated in commercial hybrids. Several molecular markers tightly linked to Mi have been identified. This study was conducted in order to find an informative molecular marker linked to Ty-1. Six markers mapped in the same region as Ty-1 were analysed in plant material carrying different combinations of Ty-1 and Mi alleles. Three of the six markers revealed polymorphism among the assayed accessions. One allele of JB-1 marker showed association with Ty-1. Furthermore, the presence of Mi did not interfere with the results. The analysis of several accessions of wild tomato relatives with the three polymorphic markers allowed the establishment of the origin of the alleles found in cultivated plant material, showing that introgressions from S. lycopersicum, S. pimpinellifolium and S. habrochaites will not interfere with the results of this marker which tags Ty-1. Furthermore this analysis enabled the location of CT21, the RFLP marker from which JB-1 was designed.

Genomic variation in tomato, from wild ancestors to contemporary breeding accessions

BackgroundDomestication modifies the genomic variation of species. Quantifying this variation provides insights into the domestication process, facilitates the management of resources used by breeders and germplasm centers, and enables the design of experiments to associate traits with genes. We described and analyzed the genetic diversity of 1,008 tomato accessions including Solanum lycopersicum var. lycopersicum (SLL), S. lycopersicum var. cerasiforme (SLC), and S. pimpinellifolium (SP) that were genotyped using 7,720 SNPs. Additionally, we explored the allelic frequency of six loci affecting fruit weight and shape to infer patterns of selection.ResultsOur results revealed a pattern of variation that strongly supported a two-step domestication process, occasional hybridization in the wild, and differentiation through human selection. These interpretations were consistent with the observed allele frequencies for the six loci affecting fruit weight and shape. Fruit weight was strongly selected in SLC in the Andean region of Ecuador and Northern Peru prior to the domestication of tomato in Mesoamerica. Alleles affecting fruit shape were differentially selected among SLL genetic subgroups. Our results also clarified the biological status of SLC. True SLC was phylogenetically positioned between SP and SLL and its fruit morphology was diverse. SLC and “cherry tomato” are not synonymous terms. The morphologically-based term “cherry tomato” included some SLC, contemporary varieties, as well as many admixtures between SP and SLL. Contemporary SLL showed a moderate increase in nucleotide diversity, when compared with vintage groups.ConclusionsThis study presents a broad and detailed representation of the genomic variation in tomato. Tomato domestication seems to have followed a two step-process; a first domestication in South America and a second step in Mesoamerica. The distribution of fruit weight and shape alleles supports that domestication of SLC occurred in the Andean region. Our results also clarify the biological status of SLC as true phylogenetic group within tomato. We detect Ecuadorian and Peruvian accessions that may represent a pool of unexplored variation that could be of interest for crop improvement.

Inheritance of Tomato yellow leaf curl virus Resistance Derived from Solanum pimpinellifolium UPV16991

Resistance to tomato yellow leaf curl disease (TYLCD) in accession UPV16991 Solanum pimpinellifolium has been previously reported by our group. A breeding program was developed from an initial S. lycopersicum × S. pimpinellifolium UPV16991 cross. This first cross was followed by several selfing generations. Selection for resistance to Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) was carried out for plants of each generation. One partially resistant F6 plant (L102) was chosen to form the family to study the genetic control of resistance to TYLCV. Crosses between four breeding lines susceptible to TYLCD and L102 were also performed to study the dominance of the resistance in S. lycopersicum genetic backgrounds. Response to TYLCV infection of P1, P2, F1, F2, BC1, and BC2 generations fitted, for this line, a monogenic control with partial recessiveness and incomplete penetrance. The percentage of homozygotic plants with partial resistance was 72.75. Among the four hybrids developed, the highest levels of resistance were found in the hybrid formed from the most vigorous S. lycopersicum line. These results must be considered for breeding purposes. Partial resistance derived from UPV16991 will be useful in homozygosis or combined with resistance genes from other sources.

Resistance to Tomato spotted wilt virus introgressed from Lycopersicon peruvianum in line UPV 1 may be allelic to Sw-5 and can be used to enhance the resistance of hybrids cultivars

The breeding line UPV 1 developed from the PE-18 accession of Lycopersicon peruvianum collected in Huallanca, Ancash, Peru, shows resistance to TSWV. Mechanical inoculation and thrips transmission were used to study the inheritance of TSWV resistance of this line. UPV 1resistance is controlled by a dominant gene. The penetrance of this resistance gene was complete in mechanical inoculation and incomplete when thrips transmission was used. Linkage tests between the resistance genes of lines UPV 1 and RDD (Sw-5), indicated allelism. A molecular analysis using a SCAR marker tightly linked to Sw-5 also supported this hypothesis. In heterozygotes the level of resistance expressed in UPV 1 is higher than that expressed in RDD (Sw-5), indicating that the resistance from UPV 1 may be of higher value for the development of commercial hybrids.