Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication

Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison

<p>Abstract</p> <p>Background</p> <p>The cultivated olive (<it>Olea europaea </it>L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of <it>Olea </it>subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.</p> <p>Results</p> <p>The complete plastid genome of the olive cultivar Frantoio was determined by direct sequence analysis using universal and novel PCR primers designed to amplify all overlapping regions. The chloroplast genome of the olive has an organisation and gene order that is conserved among numerous Angiosperm species and do not contain any of the inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses that have been found in the chloroplast genomes of the genera <it>Jasminum </it>and <it>Menodora</it>, from the same family as <it>Olea</it>.</p> <p>The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive.</p> <p>Conclusions</p> <p>In addition to identifying mutations that may play a functional role in modifying the metabolism and adaptation of olive cultivars, the new chloroplast markers represent a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking.</p

Characterization of the plastome of Physalis cordata and comparative analysis of eight species of Physalis sensu stricto

In this study, we sequenced, assembled, and annotated the plastome of Physalis cordata Mill. and compared it with seven species of the genus Physalis sensu stricto. Sequencing, annotating, and comparing plastomes allow us to understand the evolutionary mechanisms associated with physiological functions, select possible molecular markers, and identify the types of selection that have acted in different regions of the genome. The plastome of P. cordata is 157,000 bp long and presents the typical quadripartite structure with a large single-copy (LSC) region of 87,267 bp and a small single-copy (SSC) region of 18,501 bp, which are separated by two inverted repeat (IRs) regions of 25,616 bp each. These values are similar to those found in the other species, except for P. angulata L. and P. pruinosa L., which presented an expansion of the LSC region and a contraction of the IR regions. The plastome in all Physalis species studied shows variation in the boundary of the regions with three distinct types, the percentage of the sequence identity between coding and non-coding regions, and the number of repetitive regions and microsatellites. Four genes and 10 intergenic regions show promise as molecular markers and eight genes were under positive selection. The maximum likelihood analysis showed that the plastome is a good source of information for phylogenetic inference in the genus, given the high support values and absence of polytomies. In the Physalis plastomes analyzed here, the differences found, the positive selection of genes, and the phylogenetic relationships do not show trends that correspond to the biological or ecological characteristics of the species studied

Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles

The papers included in this Special Issue address a variety of important aspects of Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles, including new advances in the sequencing of both mitochondria and chloroplasts’ genomes using Next-Generation Sequencing technology in plant species and algae including important crop and tree species, in vitro culture protocol, and identification of a core module of genes involved in plastid development. In particular, the published studies focus on the description of adaptive evolution, elucidate mitochondrial mRNA processing, highlight the effect of domestication process on plastome variability and report the development of molecular markers. A meta-analysis of recently published genome-wide expression studies allowed the identification of novel nuclear genes, involved in the complex and still unrevealed mechanisms at the basis of communication between chloroplast and nucleus (retrograde signalling) during plastid development (biogenic control). Finally, an optimized regeneration protocol useful in plastid transformation of recalcitrant species, such as sugarcane, has been reported

Determination Of Evolutionary History Of Big Bluestem Populations Through Chloroplast DNA Analysis

Andropogon gerardii Vitman (big bluestem) is one of the most dominant and widely distributed grasses of the North American prairie. It is widely used in restoration projects for the recovery of grassland ecosystems. A. gerardii demonstrates genetic and adaptive variation among populations across the prairie. With the objective to understand the evolutionary relationship between the A. gerardii populations, two noncoding chloroplast DNA (cpDNA) spacers (rpl32-trnL(UAG) and trnQ(UUG)-rps16) were studied. Similarly, genetic differentiation among the populations was also calculated based on the spacers. The trnQ(UUG)-rps16 spacer had more polymorphic sites than the rpl32-trnL(UAG) spacer. A phylogenetic tree based on combined cpDNA spacers generated a monophyletic tree for A. gerardii with a Colorado population and sand bluestem (Andropogon hallii) as the outgroups. The monophyletic tree was further resolved into two sub-clades. Most of the branches and nodes were well supported, with more than 70% of posterior probability values. However, the grouping of populations did not support the resolution of the phylogenetic tree with geological distribution. Analysis of molecular variance suggests there is a low level of genetic differentiation among the populations, with 90% of variation within the populations and 10% of variation among the populations. The observed high genetic variation within populations could be the result of potential gene flow, polyploidy, and the outcrossing nature of big bluestem

Complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera, and comparative analyses with other grass genomes

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19-37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae

The chloroplast genome sequence of bittersweet (Solanum dulcamara): plastid genome structure evolution in Solanaceae

Bittersweet (Solanum dulcamara) is a native Old World member of the nightshade family. This European diploid species can be found from marshlands to high mountainous regions and it is a common weed that serves as an alternative host and source of resistance genes against plant pathogens such as late blight (Phytophthora infestans). We sequenced the complete chloroplast genome of bittersweet, which is 155,580 bp in length and it is characterized by a typical quadripartite structure composed of a large (85,901 bp) and small (18,449 bp) single-copy region interspersed by two identical inverted repeats (25,615 bp). It consists of 112 unique genes from which 81 are protein-coding, 27 tRNA and four rRNA genes. All bittersweet plastid genes including non-functional ones and even intergenic spacer regions are transcribed in primary plastid transcripts covering 95.22% of the genome. These are later substantially edited in a post-transcriptional phase to activate gene functions. By comparing the bittersweet plastid genome with all available Solanaceae sequences we found that gene content and synteny are highly conserved across the family. During genome comparison we have identified several annotation errors, which we have corrected in a manual curation process then we have identified the major plastid genome structural changes in Solanaceae. Interpreted in a phylogenetic context they seem to provide additional support for larger clades. The plastid genome sequence of bittersweet could help to benchmark Solanaceae plastid genome annotations and could be used as a reference for further studies. Such reliable annotations are important for gene diversity calculations, synteny map constructions and assigning partitions for phylogenetic analysis with de novo sequenced plastomes of Solanaceae.Peer reviewe

Production and transformation of tobacco and Brassica containing macrochloroplasts

Plastid division, sustained by the equilibrium expression and coordination of plastid division genes is vital for the maintenance of plastid populations in dividing plant cells. Macrochloroplasts (MCP), the occurrence of one or a few chloroplasts per cell is due to the imbalance in the expression of plastid division genes. Because of the MCP size and number it was proposed that they may provide better targets for the plastid transformation than the normal (WT) chloroplasts and result in better plastid transformation frequencies. The objective of this research was to produce transgenic plants containing macrochloroplasts by nuclear transformation and then to use these plants as a model for the development of plastid transformation of crop species. By using AtFtsZ1-1 and AtMinD1 as query sequences in the TIGR (U.S.A) and ASTRA (Australia) Brassica oleracea EST databases, this project resulted in the isolation of cauliflower FtsZ1-1 (EU684588) and MinD (EU684589) genes. In addition, AtFtsZ1-1 was used as a control gene for comparison to the cauliflower FtsZ1-1. Binary vectors were constructed to express these genes in tobacco and cauliflower either by Agrobacterium tumefaciens-mediated or PEG-mediated transformation methods. Transgenic tobacco and cauliflower plants with abnormal chloroplasts (MCP, minichloroplasts, honeycomb or doughnut shaped chloroplasts, uneven surface membrane chloroplasts) were developed. Furthermore, the transgenic tobacco and cauliflower plants were examined by PCR, RT-PCR and Southern blotting. In addition, th ese plants were also analysed for the different abnormal chloroplast phenotypes by fluorescence microscopy. This project also generated the first plastid transformants from macrochloroplast bearing tobacco plants via biolistics. After one round of regeneration homoplasmic plastid transformants were obtained from both WT chloroplast and MCP tobacco plants. The homoplasmic nature of plastid transformants were confirmed by PCR and Southern blotting. Plastid expression of GFP in WT and MCP was confirmed by fluorescence/confocal microscopy and western blot analysis. This project showed for the first time the characterisation of cauliflower FtsZ1-1 and MinD plastid division genes in homologous and heterologous systems (cauliflower and tobacco). Moreover, obtaining homoplasmic plastid transformant shoots from one round of regeneration from the MCP containing tobacco plants is reported for the first time in this study. In addition this study explored the effect of transgene expression level on the chloroplast abnormality, highlighting the importance of analysing transgenic tobacco and cauliflower plants at the protein lev el specifically with regard to plastid division genes. The maintenance of MCP phenotype in the regenerated shoots and the requirement of standardisation of MCP containing plants via biolistics for increasing the plastid transformation frequency were also examined