Santiago Vilanova

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.

Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature

Loss of pollen-S function in Prunus self-compatible mutants has recently been associated with deletions or insertions in S-haplotype-specific F-box (SFB) genes. We have studied two self-compatible cultivars of apricot (Prunus armeniaca), Currot (SCSC) and Canino (S2SC), sharing the naturally occurring self-compatible (SC)-haplotype. Sequence analysis showed that whereas the SC-RNase is unaltered, a 358-bp insertion is found in the SFBC gene, resulting in the expression of a truncated protein. The alteration of this gene is associated with self-incompatibility (SI) breakdown, supporting previous evidence that points to SFB being the pollen-S gene of the Prunus SI S-locus. On the other hand, PCR analysis of progenies derived from Canino showed that pollen grains carrying the S2-haplotype were also able to overcome the incompatibility barrier. However, alterations in the SFB2 gene or evidence of pollen-S duplications were not detected. A new class of F-box genes encoding a previously uncharacterized protein with high sequence similarity (approximately 62%) to Prunus SFB proteins was identified in this work, but the available data rules them out of producing S-heteroallelic pollen and thus the cause of the pollen-part mutation. These results suggest that cv Canino has an additional mutation, not linked to the S-locus, which causes a loss of pollen-S activity when present in pollen. As a whole, these findings support the proposal that the S-locus products besides other S-locus independent factors are required for gametophytic SI in Prunus.

Synteny conservation between two distantly-related Rosaceae genomes: Prunus (the stone fruits) and Fragaria (the strawberry)

BackgroundThe Rosaceae encompass a large number of economically-important diploid and polyploid fruit and ornamental species in many different genera. The basic chromosome numbers of these genera are x = 7, 8 and 9 and all have compact and relatively similar genome sizes. Comparative mapping between distantly-related genera has been performed to a limited extent in the Rosaceae including a comparison between Malus (subfamily Maloideae) and Prunus (subfamily Prunoideae); however no data has been published to date comparing Malus or Prunus to a member of the subfamily Rosoideae. In this paper we compare the genome of Fragaria, a member of the Rosoideae, to Prunus, a member of the Prunoideae.ResultsThe diploid genomes of Prunus (2n = 2x = 16) and Fragaria (2n = 2x = 14) were compared through the mapping of 71 anchor markers – 40 restriction fragment length polymorphisms (RFLPs), 29 indels or single nucleotide polymorphisms (SNPs) derived from expressed sequence tags (ESTs) and two simple-sequence repeats (SSRs) – on the reference maps of both genera. These markers provided good coverage of the Prunus (78%) and Fragaria (78%) genomes, with maximum gaps and average densities of 22 cM and 7.3 cM/marker in Prunus and 32 cM and 8.0 cM/marker in Fragaria.ConclusionOur results indicate a clear pattern of synteny, with most markers of each chromosome of one of these species mapping to one or two chromosomes of the other. A large number of rearrangements (36), most of which produced by inversions (27) and the rest (9) by translocations or fission/fusion events could also be inferred. We have provided the first framework for the comparison of the position of genes or DNA sequences of these two economically valuable and yet distantly-related genera of the Rosaceae.

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.

Diversity and Relationships of Eggplants from Three Geographically Distant Secondary Centers of Diversity

Eggplant (Solanum melongena L.) was domesticated in the Indo-Birmanian region, which is also the primary center of diversity for this crop. From there eggplant spread to other regions, and diversity accumulated in several secondary centers of diversity. We have assessed the diversity and relationships of 52 accessions of eggplant from three geographically distant secondary centers of diversity (China, Spain, and Sri Lanka) using 28 morphological descriptors and 12 highly polymorphic genomic SSRs. A wide variation was found for most morphological traits, and significant differences among the three centers of diversity were detected for 22 of these traits. The PCA analysis showed that eggplants from the three origins were morphologically differentiated, and accessions from each of the three secondary centers of diversity presented a typical combination of morphological characteristics. In this respect, discriminant analysis showed that accessions could be correctly classified to their origin using only six traits. The SSR characterization identified 110 alleles and allowed obtaining a unique genetic fingerprint for each accession. Many alleles were found to be private to each origin, but no universal alleles were found for any of the origins. The PCA analysis showed that the genetic differentiation among origins was less clear than for morphological traits, although the analysis of the population structure shows that accessions mostly group according to the origin, but also provides evidence of migration among the three secondary centers of diversity. The genetic diversity (HT) within each origin was high, ranging between HT = 0.5400 (Sri Lanka) and HT = 0.4943 (China), while the standardized genetic differentiation (G’ST) among origins was moderate (G’ST = 0.2657). The correlation between morphological and SSR distances was non-significant (r = 0.044), indicating that both data are complementary for the conservation of germplasm and breeding of eggplant. These results are relevant for the management of genetic resources, breeding programmes, and evolutionary studies of eggplant.

Analysis of loquat germplasm (Eriobotrya japonica Lindl) by RAPD molecular markers

The loquat’s adaptation to Spain has proved very successful. In the Valencia area, the crop has met with very good environmental conditions for its development. Many new cultivars have been selected by growers and a European loquat germplasm collection has been established in Valencia at IVIA. An efficient sampling as well as implementation of germplasm resources requires the accurate identification of plant material. Molecular markers offer an effective tool for cultivar fingerprinting, estimation of genetic similarity and relationships. In this study, as a tool for germplasm management, RAPD markers were tested. Thirty-six primers were used to screen 33 cultivars. Twenty-three primers proved polymorphic. These primers generated 29 polymorphic amplification fragments that were selected as markers. Twenty-two cultivars out of 33 were identified by unique combinations of RAPD markers. Four different combinations were shared by two or more cultivars each. Cluster analysis based on the similarity matrix obtained from Nei’s coefficient among cultivars showed groupings that agreed according to geographical and genetic origin. RAPD technology was useful in distinguishing those cultivars obtained through hybridization but could not be used to distinguish those obtained by selection of mutations.

Diversity and relationships in key traits for functional and apparent quality in a collection of eggplant: fruit phenolics content, antioxidant activity, polyphenol oxidase activity, and browning.

Eggplant (Solanum melongena) varieties with increased levels of phenolics in the fruit present enhanced functional quality, but may display greater fruit flesh browning. We evaluated 18 eggplant accessions for fruit total phenolics content, chlorogenic acid content, DPPH scavenging activity, polyphenol oxidase (PPO) activity, liquid extract browning, and fruit flesh browning. For all the traits we found a high diversity, with differences among accessions of up to 3.36-fold for fruit flesh browning. Variation in total content in phenolics and in chlorogenic acid content accounted only for 18.9% and 6.0% in the variation in fruit flesh browning, and PPO activity was not significantly correlated with fruit flesh browning. Liquid extract browning was highly correlated with chlorogenic acid content (r = 0.852). Principal components analysis (PCA) identified four groups of accessions with different profiles for the traits studied. Results suggest that it is possible to develop new eggplant varieties with improved functional and apparent quality.

Localization of QTLs for in vitro plant regeneration in tomato

BackgroundLow regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato.ResultsWe developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration.ConclusionsIn this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation.

Diversity for chemical composition in a collection of different varietal types of tree tomato (Solanum betaceum Cav.), an Andean exotic fruit

We evaluated 23 tree tomato (Solanum betaceum) accessions from five cultivar groups and one wild relative (Solanum cajanumense) for 26 composition traits. For all traits we found highly significant differences (P<0.001) among the materials studied. The high diversity found within S. betaceum for composition traits was matched by a high diversity within each of the cultivar groups. We found that sucrose and citric acid were the most important soluble sugar and organic acid, respectively, in tree tomato. Fruit in the anthocyanin pigmented (purple) group had a carotenoid content similar to that in the yellow-orange cultivar groups. Total phenolic content was significantly correlated (r=0.8607) with antioxidant activity. Analyses of mineral content showed that tree tomato is a good source of K, Mg, and Cu. Multivariate principal components analysis (PCA) confirmed that an important diversity exists within each cultivar group. The results we have obtained indicate that the high diversity found within the tree tomato could be exploited for selection and breeding for developing the tree tomato as a commercial crop.

Breeding Vegetables with Increased Content in Bioactive Phenolic Acids

Vegetables represent a major source of phenolic acids, powerful antioxidants characterized by an organic carboxylic acid function and which present multiple properties beneficial for human health. In consequence, developing new varieties with enhanced content in phenolic acids is an increasingly important breeding objective. Major phenolic acids present in vegetables are derivatives of cinnamic acid and to a lesser extent of benzoic acid. A large diversity in phenolic acids content has been found among cultivars and wild relatives of many vegetable crops. Identification of sources of variation for phenolic acids content can be accomplished by screening germplasm collections, but also through morphological characteristics and origin, as well as by evaluating mutations in key genes. Gene action estimates together with relatively high values for heritability indicate that selection for enhanced phenolic acids content will be efficient. Modern genomics and biotechnological strategies, such as QTL detection, candidate genes approaches and genetic transformation, are powerful tools for identification of genomic regions and genes with a key role in accumulation of phenolic acids in vegetables. However, genetically increasing the content in phenolic acids may also affect other traits important for the success of a variety. We anticipate that the combination of conventional and modern strategies will facilitate the development of a new generation of vegetable varieties with enhanced content in phenolic acids.