Influence of the timing of periodontal intervention on periapical/periodontal repair in endodontic-periodontal lesions: a systematic review.

INTRODUCTION: This study is aimed at answering the following question: "Does the timing of periodontal intervention influence the periapical/periodontal repair in endodontic-periodontal lesions?". MATERIAL AND METHODS: Six electronic databases were systematically searched for studies published up to April 2022, without restriction of language or year of publication, following the PIOS strategy: (P) adult patients with a diagnosis of endodontic-periodontal lesions, (I) endodontic and periodontal treatment, (O) periapical and periodontal healing, and (S) clinical studies. Risk of bias assessment was performed with the revised Cochrane risk of bias tools for randomized trials (RoB 2) and non-randomized interventions (ROBINS-I). The overall quality of evidence was assessed through the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) tool. RESULTS: Three studies (one prospective, one retrospective, and one randomized clinical trial) were included in the present review. Non-randomized studies had a critical and serious risk of bias. The randomized clinical trial had some concerns risk of bias. Non-randomized studies reported that the endodontic intervention should be performed previous to the periodontal intervention. Randomized clinical trial reported improvements when endodontic and periodontal interventions were performed simultaneously. GRADE analysis showed a very low quality of evidence for both randomized and nonrandomized studies. CONCLUSIONS: Based on the evidence from the included studies, although it is suggested that the endodontic treatment should be performed prior to periodontal treatment, it is not possible to assure the best treatment sequence for endodontic-periodontal lesions. CLINICAL RELEVANCE: Evidences suggests that although the endodontic intervention should be the first therapy of choice, it was not possible to specify the best time to perform the periodontal intervention.

Gene duplication and transposition of mobile elements drive evolution of the Rpv3 resistance locus in grapevine.

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC-content in their proximal 5'-intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Genomic tools for durum wheat breeding: de novo assembly of Svevo transcriptome and SNP discovery in elite germplasm.

BACKGROUND: The tetraploid durum wheat (Triticum turgidum L. ssp. durum Desf. Husnot) is an important crop which provides the raw material for pasta production and a valuable source of genetic diversity for breeding hexaploid wheat (Triticum aestivum L.). Future breeding efforts to enhance yield potential and climate resilience will increasingly rely on genomics-based approaches to identify and select beneficial alleles. A deeper characterisation of the molecular and functional diversity of the durum wheat transcriptome will be instrumental to more effectively harness its genetic diversity. RESULTS: We report on the de novo transcriptome assembly of durum wheat cultivar 'Svevo'. The transcriptome of four tissues/organs (shoots and roots at the seedling stage, reproductive organs and developing grains) was assembled de novo, yielding 180,108 contigs, with a N50 length of 1121 bp and mean contig length of 883 bp. Alignment against the transcriptome of nine plant species identified 43% of transcripts with homology to at least one reference transcriptome. The functional annotation was completed by means of a combination of complementary software. The presence of differential expression between the A- and B-homoeolog copies of the durum wheat tetraploid genome was ascertained by phase reconstruction of polymorphic sites based on the T. urartu transcripts and inferring homoeolog-specific sequences. We observed greater expression divergence between A and B homoeologs in grains rather than in leaves and roots. The transcriptomes of 13 durum wheat cultivars spanning the breeding period from 1969 to 2005 were analysed for SNP diversity, leading to 95,358 non-rare, hemi-SNPs shared among two or more cultivars and 33,747 locus-specific (diploid inheritance) SNPs. CONCLUSIONS: Our study updates and expands the de novo transcriptome reference assembly available for durum wheat. Out of 180,108 assembled transcripts, 13,636 were specific to the Svevo cultivar as compared to the only other reference transcriptome available for durum, thus contributing to the identification of the tetraploid wheat pan-transcriptome. Additionally, the analysis of 13 historically relevant hallmark varieties produced a SNP dataset that could successfully validate the genotyping in tetraploid wheat and provide a valuable resource for genomics-assisted breeding of both tetraploid and hexaploid wheats.

Single primer enrichment technology as a tool for massive genotyping: a benchmark on black poplar and maize.

BACKGROUND AND AIMS: The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. METHODS: Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. KEY RESULTS: Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. CONCLUSION: The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.

The build-up of osmotic stress responses within the growing root apex using kinematics and RNA-sequencing.

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth.

A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

Three distinct mutational mechanisms acting on a single gene underpin the origin of yellow flesh in peach.

Peach flesh color (white or yellow) is among the most popular commercial criteria for peach classification, and has implications for consumer acceptance and fruit nutritional quality. Despite the increasing interest in improving cultivars of both flesh types, little is known about the genetic basis for the carotenoid content diversity in peach. Here we describe the association between genotypes at a locus encoding the carotenoid cleavage dioxygenase 4 (PpCCD4), localized in pseudomolecule 1 of the Prunus persica reference genome sequence, and the flesh color for 37 peach varieties, including two somatic revertants, and three ancestral relatives of peach, providing definitive evidence that this locus is responsible for flesh color phenotype. We show that yellow peach alleles have arisen from various ancestral haplotypes by at least three independent mutational events involving nucleotide substitutions, small insertions and transposable element insertions, and that these mutations, despite being located within the transcribed portion of the gene, also result in marked differences in transcript levels, presumably as a consequence of differential transcript stability involving nonsense-mediated mRNA decay. The PpCCD4 gene provides a unique example of a gene for which humans, in their quest to diversify phenotypic appearance and qualitative characteristics of a fruit, have been able to select and exploit multiple mutations resulting from a variety of mechanisms.

Mozambican Coffea accessions from Ibo and Quirimba Islands: identification and geographical distribution.

Mozambique does not have a tradition of farming Coffea arabica or Coffea canephora, the two species that dominate the worldwide coffee market. However, native coffee plants have been growing spontaneously and in some cases cultivated in the Ibo and Quirimba islands in the north of the country and Inhambane province in the south. Historically there has been confusion over the precise taxonomic classification of these indigenous coffee plants, with different botanists identifying the species as C. racemosa, C. zanguebariae or various synonyms of both. The present research aims to clarify the subject and provide new information on these little-described coffee species which may prove valuable as new breeding material for future cultivars, something that is sorely needed to face the present and future challenges of coffee production. Leaf samples were collected from 40 accessions from Ibo Island, Quirimba Island and Inhambane province. The samples were sequenced by whole-genome technology and WGS reads were filtered to identify relevant SNP variants. Diversity among the samples was assessed by PCA, and a phylogenetic tree including several Coffea species was built using additional data available in public databases. Experimental data confirm the presence of C. zanguebariae as the only coffee species present in both Ibo and Quirimba Islands, while it appears that C. racemosa is exclusive to the southern Inhambane province. The present research provides the most detailed analysis so far on the genetic identity of the traditional Mozambican coffee crops. This is the prerequisite for undertaking further scientific studies on these almost unknown coffee species and for starting agronomic development programs for the economic revival of Ibo and Quirimba islands based on coffee cultivation. Furthermore, these species could provide much-needed genetic material for the breeding of new hybrids with the two main commercial coffee species.

A chromosome-scale assembly reveals chromosomal aberrations and exchanges generating genetic diversity in Coffea arabica germplasm.

In order to better understand the mechanisms generating genetic diversity in the recent allotetraploid species Coffea arabica, here we present a chromosome-level assembly obtained with long read technology. Two genomic compartments with different structural and functional properties are identified in the two homoeologous genomes. The resequencing data from a large set of accessions reveals low intraspecific diversity in the center of origin of the species. Across a limited number of genomic regions, diversity increases in some cultivated genotypes to levels similar to those observed within one of the progenitor species, Coffea canephora, presumably as a consequence of introgressions deriving from the so-called Timor hybrid. It also reveals that, in addition to few, early-occurring exchanges between homoeologous chromosomes, there are numerous recent chromosomal aberrations including aneuploidies, deletions, duplications and exchanges. These events are still polymorphic in the germplasm and could represent a fundamental source of genetic variation in such a lowly variable species.

GAM-NGS: genomic assemblies merger for next generation sequencing.

BACKGROUND: In recent years more than 20 assemblers have been proposed to tackle the hard task of assembling NGS data. A common heuristic when assembling a genome is to use several assemblers and then select the best assembly according to some criteria. However, recent results clearly show that some assemblers lead to better statistics than others on specific regions but are outperformed on other regions or on different evaluation measures. To limit these problems we developed GAM-NGS (Genomic Assemblies Merger for Next Generation Sequencing), whose primary goal is to merge two or more assemblies in order to enhance contiguity and correctness of both. GAM-NGS does not rely on global alignment: regions of the two assemblies representing the same genomic locus (called blocks) are identified through reads' alignments and stored in a weighted graph. The merging phase is carried out with the help of this weighted graph that allows an optimal resolution of local problematic regions. RESULTS: GAM-NGS has been tested on six different datasets and compared to other assembly reconciliation tools. The availability of a reference sequence for three of them allowed us to show how GAM-NGS is a tool able to output an improved reliable set of sequences. GAM-NGS is also a very efficient tool able to merge assemblies using substantially less computational resources than comparable tools. In order to achieve such goals, GAM-NGS avoids global alignment between contigs, making its strategy unique among other assembly reconciliation tools. CONCLUSIONS: The difficulty to obtain correct and reliable assemblies using a single assembler is forcing the introduction of new algorithms able to enhance de novo assemblies. GAM-NGS is a tool able to merge two or more assemblies in order to improve contiguity and correctness. It can be used on all NGS-based assembly projects and it shows its full potential with multi-library Illumina-based projects. With more than 20 available assemblers it is hard to select the best tool. In this context we propose a tool that improves assemblies (and, as a by-product, perhaps even assemblers) by merging them and selecting the generating that is most likely to be correct.

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

A 3,000-loci transcription map of chromosome 3B unravels the structural and functional features of gene islands in hexaploid wheat.

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

Genotypic and tissue-specific variation of Populus nigra transcriptome profiles in response to drought.

Climate change is one of the most important challenges for mankind in the far and near future. In this regard, sustainable production of woody crops on marginal land with low water availability is a major challenge to tackle. This dataset is part of an experiment, in which we exposed three genetically differentiated genotypes of Populus nigra originating from contrasting natural habitats to gradually increasing moderate drought. RNA sequencing was performed on fine roots, developing xylem and leaves of those three genotypes under control and moderate drought conditions in order to get a comprehensive dataset on the transcriptional changes at the whole plant level under water limiting conditions. This dataset has already provided insight in the transcriptional control of saccharification potential of the three Populus genotypes under drought conditions and we suggest that our data will be valuable for further in-depth analysis regarding candidate gene identification or, on a bigger scale, for meta-transcriptome analysis.

Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar.

BACKGROUND: Most of the grapevine (Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project. RESULTS: We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome. CONCLUSIONS: We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm.

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors.

Automated FingerPrint Background removal: FPB.

BACKGROUND: The construction of a whole-genome physical map has been an essential component of numerous genome projects initiated since the inception of the Human Genome Project. Its usefulness has been proved for whole-genome shotgun projects as a post-assembly validation and recently it has also been used in the assembly step to constrain on BACs positions. Fingerprinting is usually the method of choice for construction of physical maps. A clone fingerprint is composed of true peaks representing real fragments and background peaks, mainly composed of E. coli genomic DNA, partial digestions, star activity by-products, and machine background. High-throughput fingerprinting leads to the production of thousands of BAC clone fingerprints per day. That is why background peaks removal has become an important issue and needs to be automatized, especially in capillary electrophoresis based fingerprints. RESULTS: At the moment, the only tools available for such a task are GenoProfiler and its descendant FPMiner. The large variation in the quality of fingerprints that is usually present in large fingerprinting projects represents a major difficulty in the correct removal of background peaks that has only been partially addressed by the methods so far adopted that all require a long manual optimization of parameters. Thus, we implemented a new data-independent tool, FPB (FingerPrint Background removal), suitable for large scale projects as well as mapping of few clones. CONCLUSION: FPB is freely available at http://www.appliedgenomics.org/tools.php. FPB was used to remove the background from all fingerprints of three grapevine physical map projects. The first project consists of about 50,000 fingerprints, the second one consists of about 70,000 fingerprints, and the third one consists of about 45,000 fingerprints. In all cases a successful assembly was built.

Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of 'Lito' Chromosome 1 and Analysis of Candidate Genes.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which-the cultivar 'Lito' heterozygous for the resistance-has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

Genetic Mapping of the Incompatibility Locus in Olive and Development of a Linked Sequence-Tagged Site Marker.

The genetic control of self-incompatibility (SI) has been recently disclosed in olive. Inter-varietal crossing confirmed the presence of only two incompatibility groups (G1 and G2), suggesting a simple Mendelian inheritance of the trait. A double digest restriction associated DNA (ddRAD) sequencing of a biparental population segregating for incompatibility groups has been performed and high-density linkage maps were constructed in order to map the SI locus and identify gene candidates and linked markers. The progeny consisted of a full-sib family of 229 individuals derived from the cross 'Leccino' (G1) × 'Dolce Agogia' (G2) varieties, segregating 1:1 (G1:G2), in accordance with a diallelic self-incompatibility (DSI) model. A total of 16,743 single nucleotide polymorphisms was identified, 7,006 in the female parent 'Leccino' and 9,737 in the male parent 'Dolce Agogia.' Each parental map consisted of 23 linkage groups and showed an unusual large size (5,680 cM in 'Leccino' and 3,538 cM in 'Dolce Agogia'). Recombination was decreased across all linkage groups in pollen mother cells of 'Dolce Agogia,' the parent with higher heterozygosity, compared to megaspore mother cells of 'Leccino,' in a context of a species that showed exceptionally high recombination rates. A subset of 109 adult plants was assigned to either incompatibility group by a stigma test and the diallelic self-incompatibility (DSI) locus was mapped to an interval of 5.4 cM on linkage group 18. This region spanned a size of approximately 300 Kb in the olive genome assembly. We developed a sequence-tagged site marker in the DSI locus and identified five haplotypes in 57 cultivars with known incompatibility group assignment. A combination of two single-nucleotide polymorphisms (SNPs) was sufficient to predict G1 or G2 phenotypes in olive cultivars, enabling early marker-assisted selection of compatible genotypes and allowing for a rapid screening of inter-compatibility among cultivars in order to guarantee effective fertilization and increase olive production. The construction of high-density linkage maps has led to the development of the first functional marker in olive and provided positional candidate genes in the SI locus.

A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance.

BACKGROUND: Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. RESULTS: The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. CONCLUSION: Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.