BackgroundGenome Wide Association Studies (GWAS) have been recently used to dissect complex quantitative traits and identify candidate genes affecting phenotype variation of polygenic traits. In order to map loci controlling variation in tomato marketable and nutritional fruit traits, we used a collection of 96 cultivated genotypes, including Italian, Latin American, and other worldwide-spread landraces and varieties. Phenotyping was carried out by measuring ten quality traits and metabolites in red ripe fruits. In parallel, genotyping was carried out by using the Illumina Infinium SolCAP array, which allows data to be collected from 7,720 single nucleotide polymorphism (SNP) markers.ResultsThe Mixed Linear Model used to detect associations between markers and traits allowed population structure and relatedness to be evidenced within our collection, which have been taken into consideration for association analysis. GWAS identified 20 SNPs that were significantly associated with seven out of ten traits considered. In particular, our analysis revealed two markers associated with phenolic compounds, three with ascorbic acid, β-carotene and trans-lycopene, six with titratable acidity, and only one with pH and fresh weight. Co-localization of a group of associated loci with candidate genes/QTLs previously reported in other studies validated the approach. Moreover, 19 putative genes in linkage disequilibrium with markers were found. These genes might be involved in the biosynthetic pathways of the traits analyzed or might be implied in their transcriptional regulation. Finally, favourable allelic combinations between associated loci were identified that could be pyramided to obtain new improved genotypes.ConclusionsOur results led to the identification of promising candidate loci controlling fruit quality that, in the future, might be transferred into tomato genotypes by Marker Assisted Selection or genetic engineering, and highlighted that intraspecific variability might be still exploited for enhancing tomato fruit quality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0337-9) contains supplementary material, which is available to authorized users.

Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2-to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.

BackgroundKnowledge on population structure and genetic diversity in vegetable crops is essential for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Herein we used the GBS approach for the genome-wide identification of SNPs in a collection of Capsicum spp. accessions and for the assessment of the level of genetic diversity in a subset of 222 cultivated pepper (Capsicum annum) genotypes.ResultsGBS analysis generated a total of 7,568,894 master tags, of which 43.4% uniquely aligned to the reference genome CM334. A total of 108,591 SNP markers were identified, of which 105,184 were in C. annuum accessions. In order to explore the genetic diversity of C. annuum and to select a minimal core set representing most of the total genetic variation with minimum redundancy, a subset of 222 C. annuum accessions were analysed using 32,950 high quality SNPs. Based on Bayesian and Hierarchical clustering it was possible to divide the collection into three clusters. Cluster I had the majority of varieties and landraces mainly from Southern and Northern Italy, and from Eastern Europe, whereas clusters II and III comprised accessions of different geographical origins. Considering the genome-wide genetic variation among the accessions included in cluster I, a second round of Bayesian (K = 3) and Hierarchical (K = 2) clustering was performed. These analysis showed that genotypes were grouped not only based on geographical origin, but also on fruit-related features.ConclusionsGBS data has proven useful to assess the genetic diversity in a collection of C. annuum accessions. The high number of SNP markers, uniformly distributed on the 12 chromosomes, allowed the accessions to be distinguished according to geographical origin and fruit-related features. SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3297-7) contains supplementary material, which is available to authorized users.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5 0 -UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5 0 -UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5 0 -UTR construct, described above, and another containing the same terminator, but with the promoter and 5 0 -UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1(v) gene) or a synthetic sequence optimized for expression in plant plastids (L1(pt) gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5'-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy.

Diploid (2n ‫؍‬ 2x ‫؍‬ 24) Solanum species with endosperm balance number (EBN) ‫؍‬ 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n ‫؍‬ 4x ‫؍‬ 48, 4EBN) and haploid (2n ‫؍‬ 2x ‫؍‬ 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F 1 hybrids between an in vitro-doubled clone of S. commersonii (2n ‫؍‬ 4x ‫؍‬ 48, 2EBN) and diploid 2EBN clones were successfully used in 3x ؋ 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid͞near pentaploid BC 1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC 1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC 2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC 2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.The cultivated potato Solanum tuberosum Group Tuberosum (Tbr) (2n ϭ 4x ϭ 48) is a vegetatively propagated species with tetrasomic inheritance and a high level of heterozygosity (1). Hence, several important characteristics are strongly dependent on intralocus interactions, and genotypes with multiallelic loci are superior to those that are mono-or diallelic. Unfortunately, due to the close relationship between cultivars, the genetic basis of modern varieties is rather limited. By contrast, wild Solanum species, most of which are diploid (2n ϭ 2x ϭ 24), represent a significantly greater source of allelic diversity than that present in the standard cultivars. They also possess many valuable traits, including resistances to biotic and abiotic stresses and superior tuber quality (2).To introgress genes from diploid species into the cultivated potato, Tbr-4x can be crossed directly with 2n gameteproducing species to synthesize a tetraploid progeny. Alternatively, Tbr-4x can be crossed with hybrids between wild species and Tbr haploids. This second approach is preferable to reduce the number of wild genomes present in the tetraploid progeny. However, crosses between certain 24-chromosome species and Tbr repeatedly fail due to strong sexual isolating mechanisms. The most common underlying barrier to interspecific hybridization in Solanum spp...

This report describes the integration and expression of foreign genes into the plastid genome of a commercial cultivar of potato. Plastid transformation of potato was achieved using two tobacco specific plastid transformation vectors, pZS197 (Prrn/aadA/ psbA3 0 ) and pMSK18 (trc/gfp/Prrn/aadA/ psbA3 0 ). Selection was for spectinomycin resistance after biolistic delivery of plasmid DNA into leaf cells of Solanum tuberosum cv. Desiree. Ten transplastomic lines were obtained from 179 bombarded samples with vector pZS197 and four transplastomic lines selected out of 103 bombarded samples with vector pMSK18. Southern blot and PCR analyses confirmed homoplasmy in the primary regenerants, and incorporation of the aadA and gfp genes into the potato plastid genome by two homologous recombination events via the flanking plastid DNA sequences. Fluorometric measurements confirmed GFP expression in leaves and tubers of pMSK18 lines. No transformants were obtained with a third tobacco vector, pNtcZ7 (Prrn/gfp/ psbA3 0 /trc/aadA/rrnB-ter) in which the selectable marker gene is driven by a bacterial (trc) promoter, which does permit selection of plastid transformants in tobacco, and allows low level expression of the reporter gene, gfp, in potato. #