Positive selection in the leucine-rich repeat domain of Gro1 genes in Solanum species

In pathogen resistant plants, solvent-exposed residues in the leucine-rich repeat (LRR) proteins are thought to mediate resis- tance by recognizing plant pathogen elicitors. In potato, the gene Gro1-4 confers resistance to Globodera rostochiensis. The investigation of variablity in different copies of this gene represents a good model for the verification of positive selection mechanisms. Two datasets of Gro1 LRR sequences were constructed, one derived from the Gro1-4 gene, belonging to dif- ferent cultivated and wild Solanum species, and the other belonging to paralogues of a resistant genotype. Analysis of non- synonymous to synonymous substitution rates (Ka/Ks) highlighted 14 and six amino acids with Ka/Ks >1 in orthologue and paralogue datasets, respectively. Selection analysis revealed that the leucine-rich regions accumulate variability in a very specific way, and we found that some combinations of amino acids in these sites might be involved in pathogen recognition. The results confirm previous studies on positive selection in the LRR domain of R protein in Arabidopsis and other model plants and extend these to wild Solanum species. Moreover, positively selected sites in the Gro1 LRR domain show that coevolution mainly occurred in two regions on the internal surface of the three-dimensional horseshoe structure of the domain, albeit with different evolutionary forces between paralogues and orthologues

‘Omics’ approaches in tomato aimed at identifying candidate genes for ascorbic acid accumulation in the fruit

Tomato (Solanum lycopersicum) is one of the most important vegetables in the world with significant importance for human health and nutrition. This species has long served as model system for plant genetics, development, physiology, pathology, and fleshy fruit ripening, resulting in the accumulation of many genetic and genomic resources. In addition, the tremendous development of high-throughput technologies, such as transcriptomics, metabolomics and proteomics, collectively denoted as ‘omics’ technologies, has led to a huge collection of data and platforms today available on the net. Nowadays, identifying all the components of a single biological system is within our means; however, assigning function to genes, proteins and metabolites is still a daunting task. Major challenges include interpretation and integration of large datasets to understand the principles underlying the regulation of genes, metabolites and proteins, and how their combined interactions associate with variation in phenotype. In this review, we will focus on the role of the different high-throughput technologies in enhancing tomato breeding particularly for fruit quality traits. We also describe how two ‘omics’ approaches could be combined in order to identify candidate genes for the genetic control of ascorbic acid accumulation in tomato fruit. We report the example of transcriptomic and genomic approaches established on the use of different high-throughput platforms available for tomato.Key words: Tomato, introgression lines, quality trait, genomics, transcriptomics, candidate gene, single nucleotide polymorphism (SNPs)

An improved melon reference genome with single-molecule sequencing uncovers a recent burst of transposable elements with potential impact on genes

The published melon (Cucumis melo L.) reference genome assembly (v3.6.1) has still 41.6 Mb (Megabases) of sequences unassigned to pseudo-chromosomes and about 57 Mb of gaps. Although different approaches have been undertaken to improve the melon genome assembly in recent years, the high percentage of repeats (~40%) and limitations due to read length have made it difficult to resolve gaps and scaffold's misassignments to pseudomolecules, especially in the heterochromatic regions. Taking advantage of the PacBio single- molecule real-time (SMRT) sequencing technology, an improvement of the melon genome was achieved. About 90% of the gaps were filled and the unassigned sequences were drastically reduced. A lift-over of the latest annotation v4.0 allowed to re-collocate protein-coding genes belonging to the unassigned sequences to the pseudomolecules. A direct proof of the improvement reached in the new melon assembly was highlighted looking at the improved annotation of the transposable element fraction. By screening the new assembly, we discovered many young (inserted less than 2Mya), polymorphic LTR-retrotransposons that were not captured in the previous reference genome. These elements sit mostly in the pericentromeric regions, but some of them are inserted in the upstream region of genes suggesting that they can have regulatory potential. This improved reference genome will provide an invaluable tool for identifying new gene or transposon variants associated with important phenotypes.info:eu-repo/semantics/publishedVersio

Phenotypic and molecular selection of a superior Solanum pennellii introgression sub-line suitable for improving quality traits of cultivated tomatoes

The Solanum pennellii Introgression Line (IL) population can be exploited to identify favorable alleles that can improve yield and fruit quality traits in commercial tomato varieties. Over the past few years, we have selected ILs that exhibit increased content of antioxidant compounds in the fruit compared to the cultivar M82, which represents the genetic background in which the different wild regions of the S. pennellii ILs were included. Recently, we have identified seven sub-lines of the IL7-3 accumulating different amounts of antioxidants in the ripe fruit. Since the wild region carried on chromosome 7 induces a low fruit production in IL7-3, the first aim of the present work was to evaluate yield performances of the selected sub-lines in three experimental fields located in the South of Italy. Another aim was to confirm in the same lines the high levels of antioxidants and evaluate other fruit quality traits. On red ripe fruit, the levels of soluble solids content, firmness, and ascorbic acid (AsA) were highly variable among the sub-lines grown in three environmental conditions, evidencing a significant genotype by environment interaction for soluble solids and AsA content. Only one sub-line (coded R182) exhibited a significantly higher firmness, even though no differences were observed for this trait between the parental lines M82 and IL7-3. The same sub-line showed significantly higher AsA content compared to M82, thus resembling IL7-3. Even though IL7-3 always exhibited a significantly lower yield, all the sub-lines showed yield variability over the three trials. Interestingly, the sub-line R182, selected for its better performances in terms of fruit quality, in all the trials showed a production comparable to that of the control line M82. A group of species-specific molecular markers was tested on R182 and on the parental genotypes in order to better define the wild genomic regions carried by the elite line R182. In these regions three candidate genes that could increase the level of AsA in the fruit were identified. In the future, the line R182 could be used as pre-breeding material in order to obtain new varieties improved for nutritional traits.info:eu-repo/semantics/publishedVersio

Exploiting Genetic and Genomic Resources to Enhance Heat-Tolerance in Tomatoes

High temperature is one of the most detrimental abiotic stresses in tomatoes. Many studies highlighted that even small increases in temperature can alter the plant reproductive system, causing a significant reduction in tomato yield. The aim of this study was to exploit the phenotypic and genomic variations of a tomato landrace collection grown at high temperatures. Fifteen genotypes were selected as the best performing in two experimental fields. The selection was based on six yield-related traits, including flower earliness, number of flowers per inflorescence, fruit set, number of fruit per plant, fruit weight and yield per plant. In order to identify markers targeting traits that could be highly influenced by adverse climate conditions, such as flowering and fruit setting, an association mapping approach was undertaken exploiting a tomato high-throughput genomic array. The phenotypic variability observed allowed us to identify a total of 15 common markers associated with the studied traits. In particular, the most relevant associations co-localized with genes involved in the floral structure development, such as the style2.1 gene, or with genes directly involved in the response to abiotic stresses. These promising candidate genes will be functionally validated and transferred to a cultivated tomato to improve its performance under high temperatures

Dissecting a QTL into Candidate Genes Highlighted the Key Role of Pectinesterases in Regulating the Ascorbic Acid Content in Tomato Fruit

Tomato (Solanum lycopersicum) is a crucial component of the human diet because of its high nutritional value and the antioxidant content of its fruit. As a member of the Solanaceae family, it is considered a model species for genomic studies in this family, especially since its genome has been completely sequenced. Among genomic resources available, Solanum pennellii introgression lines represent a valuable tool to mine the genetic diversity present in wild species. One introgression line, IL12-4, was previously selected for high ascorbic acid (AsA) content, and a transcriptomic analysis indicated the involvement of genes controlling pectin degradation in AsA accumulation. In this study the integration of data from different "omics" platforms has been exploited to identify candidate genes that increase AsA belonging to the wild region 12-4. Thirty-two genes potentially involved in pathways controlling AsA levels were analyzed with bioinformatic tools. Two hundred-fifty nonsynonymous polymorphisms were detected in their coding regions, and 11.6% revealed deleterious effects on predicted protein function. To reduce the number of genes that had to be functionally validated, introgression sublines of the region 12–4 were selected using species-specific polymor phic markers between the two Solanum species. Four sublines were obtained and we demonstrated that a subregion of around 1 Mbp includes 12 candidate genes potentially involved in AsA accumulation. Among these, only five exhibited structural delete rious variants, and one of the 12 was differentially expressed between the two Solanum species. We have highlighted the role of three polymorphic pectinesterases and inhibitors of pectinesterases that merit further investigation

Exploiting the great potential of Sequence Capture data by a new tool, SUPER-CAP

The recent development of Sequence Capture methodology represents a powerful strategy for enhancing data generation to assess genetic variation of targeted genomic regions. Here, we present SUPER-CAP, a bioinformatics web tool aimed at handling Sequence Capture data, fine calculating the allele frequency of variations and building genotype-specific sequence of captured genes. The dataset used to develop this in silico strategy consists of 378 loci and related regulative regions in a collection of 44 tomato landraces. About 14,000 high-quality variants were identified. The high depth (>40×) of coverage and adopting the correct filtering criteria allowed identification of about 4,000 rare variants and 10 genes with a different copy number variation. We also show that the tool is capable to reconstruct genotype-specific sequences for each genotype by using the detected variants. This allows evaluating the combined effect of multiple variants in the same protein. The architecture and functionality of SUPER-CAP makes the software appropriate for a broad set of analyses including SNP discovery and mining. Its functionality, together with the capability to process large data sets and efficient detection of sequence variation, makes SUPER-CAP a valuable bioinformatics tool for genomics and breeding purposes

Genotyping-by-sequencing of a melon (Cucumis melo L.) germplasm collection from a secondary center of diversity highlights patterns of genetic variation and genomic features of different gene pools

Background: Melon (Cucumis melo L.) is one of the most important horticultural species, which includes several taxonomic groups. With the advent of next-generation sequencing, single nucleotide polymorphism (SNP) markers are widely used in the study of genetic diversity and genomics. Results: We report the first successful application of genotyping-by-sequencing (GBS) technology in melon. We detected 25,422 SNPs by the analysis of 72 accessions collected in Apulia, a secondary centre of diversity in Southern Italy. Analyses of genetic structure, principal components, and hierarchical clustering support the identification of three distinct subpopulations. One of them includes accessions known with the folk name of 'carosello', referable to the chate taxonomic group. This is one of the oldest domesticated forms of C. melo, once widespread in Europe and now exposed to the risk of genetic erosion. The second subpopulation contains landraces of 'barattiere', a regional vegetable production that was never characterized at the DNA level and we show was erroneously considered another form of chate melon. The third subpopulation includes genotypes of winter melon (C. melo var. inodorus). Genetic analysis within each subpopulation revealed patterns of diversity associated with fruit phenotype and geographical origin. We used SNP data to describe, for each subpopulation, the average linkage disequilibrium (LD) decay, and to highlight genomic regions possibly resulting from directional selection and associated with phenotypic variation. Conclusions: We used GBS to characterize patterns of genetic diversity and genomic features within C. melo. We provide useful information to preserve endangered gene pools and to guide the use of germplasm in breeding. Finally, our findings lay a foundation for molecular breeding approaches and the identification of genes underlying phenotypic traits

Exploiting genomics resources to identify candidate genes underlying antioxidants content in tomato fruit

The tomato is a model species for fleshy fruit development and ripening, as well as for genomics studies of others Solanaceae. Many genetic and genomics resources, including databases for sequencing, transcriptomics and metabolomics data, have been developed and are today available. The purpose of the present work was to uncover new genes and/or alleles that determine ascorbic acid and carotenoids accumulation, by exploiting one Solanum pennellii introgression lines (IL7-3) harboring quantitative trait loci (QTL) that increase the content of these metabolites in the fruit. The higher ascorbic acid and carotenoids content in IL7-3 was confirmed at three fruit developmental stages. The tomato genome reference sequence and the recently released S. pennellii genome sequence were investigated to identify candidate genes (CGs) that might control ascorbic acid and carotenoids accumulation. First of all, a refinement of the wild region borders in the IL7-3 was achieved by analyzing CAPS markers designed in our laboratory. Afterward, six CGs associated to ascorbic acid and one with carotenoids metabolism were identified exploring the annotation and the Gene Ontology terms of genes included in the region. Variants between the sequence of the wild and the cultivated alleles of these genes were investigated for their functional relevance and their potential effects on the protein sequences were predicted. Transcriptional levels of CGs in the introgression region were extracted from RNA-Seq data available for the entire S. pennellii introgression lines collection and verified by Real-Time qPCR. Finally, seven IL7-3 sub-lines were genotyped using 28 species-specific markers and then were evaluated for metabolites content. These analyses evidenced a significant decrease in transcript abundance for one 9-cis-epoxycarotenoid dioxygenase and one L-ascorbate oxidase homolog, whose role in the accumulation of carotenoids and ascorbic acid is discussed. Comprehensively, the reported results demonstrated that combining genetic and genomic resources in tomato, including bioinformatics tools, was a successful strategy to dissect one QTL for the increase of ascorbic acid and carotenoids in tomato fruit

Metabolic and molecular changes of the phenylpropanoid pathway in tomato (Solanum lycopersicum) lines carrying different Solanum pennellii wild Chromosomal regions

Solanum lycopersicum represents an important dietary source of bioactive compounds including the antioxidants flavonoids and phenolic acids. We previously identified two genotypes (IL7-3 and IL12-4) carrying loci from the wild species Solanum pennellii, which increased antioxidants in the fruit. Successively, these lines were crossed and two genotypes carrying both introgressions at the homozygous condition (DHO88 and DHO88-SL) were selected. The amount of total antioxidant compounds was increased in DHOs compared to both ILs and the control genotype M82. In order to understand the genetic mechanisms underlying the positive interaction between the two wild regions pyramided in DHO genotypes, detailed analyses of the metabolites accumulated in the fruit were carried out by colorimetric methods and LC/MS/MS. These analyses evidenced a lower content of flavonoids in DHOs and in ILs, compared to M82. By contrast, in the DHOs the relative content of phenolic acids increased, particularly the fraction of hexoses, thus evidencing a redirection of the phenylpropanoid flux towards the biosynthesis of phenolic acid glycosides in these genotypes. In addition, the line DHO88 exhibited a lower content of free phenolic acids compared to M82. Interestingly, the two DHOs analyzed differ in the size of the wild region on chromosome 12. Genes mapping in the introgression regions were further investigated. Several genes of the phenylpropanoid biosynthetic pathway were identified, such as one 4-coumarate:CoA ligase and two UDP-glycosyltransferases in the region 12-4 and one chalcone isomerase and one UDP-glycosyltransferase in the region 7-3. Transcriptomic analyses demonstrated a different expression of the detected genes in the ILs and in the DHOs compared to M82.These analyses, combined with biochemical analyses, suggested a central role of the 4-coumarate:CoA ligase in redirecting the phenylpropanoid pathways towards the biosynthesis of phenolic acids in the pyramided lines. Moreover, analyses here carried out suggest the presence in the introgression regions of novel regulatory proteins, such as one Myb4 detected on chromosome 7 and one bHLH detected in chromosome 12. Overall our data indicates that structural and regulatory genes identified in this study might have a key role for the manipulation of the phenylpropanoid metabolic pathway in tomato fruit