Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Genetic dissection of fruit maturity date in apricot (P. armeniaca L.) through a Single Primer Enrichment Technology (SPET) approach.

BACKGROUND: Single primer enrichment technology (SPET) is an emerging and increasingly popular solution for high-throughput targeted genotyping in plants. Although SPET requires a priori identification of polymorphisms for probe design, this technology has potentially higher reproducibility and transferability compared to other reduced representation sequencing (RRS) approaches, also enabling the discovery of closely linked polymorphisms surrounding the target one. RESULTS: The potential for SPET application in fruit trees was evaluated by developing a 25K target SNPs assay to genotype a panel of apricot accessions and progenies. A total of 32,492 polymorphic sites were genotyped in 128 accessions (including 8,188 accessory non-target SNPs) with extremely low levels of missing data and a significant correlation of allelic frequencies compared to whole-genome sequencing data used for array design. Assay performance was further validated by estimating genotyping errors in two biparental progenies, resulting in an overall 1.8% rate. SPET genotyping data were used to infer population structure and to dissect the architecture of fruit maturity date (MD), a quantitative reproductive phenological trait of great agronomical interest in apricot species. Depending on the year, GWAS revealed loci associated to MD on several chromosomes. The QTLs on chromosomes 1 and 4 (the latter explaining most of the phenotypic variability in the panel) were the most consistent over years and were further confirmed by linkage mapping in two segregating progenies. CONCLUSIONS: Besides the utility for marker assisted selection and for paving the way to in-depth studies to clarify the molecular bases of MD trait variation in apricot, the results provide an overview of the performance and reliability of SPET for fruit tree genetics.

ddRAD-seq reveals the genetic structure and detects signals of selection in Italian brown trout.

BACKGROUND: Brown trout is one of the most widespread fresh-water fish species in Europe. The evolutionary history of and phylogenetic relationships between brown trout populations are complex, and this is especially true for Italian populations, which are heavily influenced in different ways by stocking practices. The characterization of the genetic structure of Italian brown trout populations may give information on the risk of losing endemic Italian populations due to lack of genetic diversity or to admixture with stocking populations. The identification of signatures of selection, and the information deriving from dense genotyping data will help genotype-informed breeding programs. We used a ddRAD-seq approach to obtain more than 100,000 single nucleotide polymorphisms (SNPs), and to characterize the population structure and signatures of selection in 90 brown trout samples. RESULTS: Italian brown trout populations are genetically differentiated, although the stocking practices have introduced strong admixture in endemic Italian trout, especially with the Atlantic lineage. Most of the analysed populations showed high levels of kinship and inbreeding. We detected putative signatures of selection using different approaches, and investigated if the regions were enriched for functional categories. Several regions putatively under selection and characterized by a reduction in heterozygosity across all the studied populations are enriched for genes involved in the response to viral infections. CONCLUSIONS: Our results, which show evidence of admixture with the Atlantic lineage (commonly used for stocking), confirm the need for controlling stocking practices, in order to avoid the erosion of the endemic gene pool; given the apparently high levels of kinship and inbreeding in local populations, our results also show the need to take action for increasing gene diversity. In addition, we used the genetically-distinct lineages to detect signatures of selection and we identified putative signatures of selection in several regions associated with resistance to infectious diseases. These constitute candidate regions for the study of resistance to infections in wild and farmed trout.

Molecular diversity and landscape genomics of the crop wild relative Triticum urartu across the Fertile Crescent.

Modern plant breeding can benefit from the allelic variation that exists in natural populations of crop wild relatives that evolved under natural selection in varying pedoclimatic conditions. In this study, next-generation sequencing was used to generate 1.3 million genome-wide single nucleotide polymorphisms (SNPs) on ex situ collections of Triticum urartu L., the wild donor of the Au subgenome of modern wheat. A set of 75 511 high-quality SNPs were retained to describe 298 T. urartu accessions collected throughout the Fertile Crescent. Triticum urartu showed a complex pattern of genetic diversity, with two main genetic groups distributed sequentially from west to east. The incorporation of geographical information on sampling points showed that genetic diversity was correlated to the geographical distance (R2  = 0.19) separating samples from Jordan and Lebanon, from Syria and southern Turkey, and from eastern Turkey, Iran and Iraq. The wild emmer genome was used to derive the physical positions of SNPs on the seven chromosomes of the Au subgenome, allowing us to describe a relatively slow decay of linkage disequilibrium in the collection. Outlier loci were described on the basis of the geographic distribution of the T. urartu accessions, identifying a hotspot of directional selection on chromosome 4A. Bioclimatic variation was derived from grid data and related to allelic variation using a genome-wide association approach, identifying several marker-environment associations (MEAs). Fifty-seven MEAs were associated with altitude and temperature measures while 358 were associated with rainfall measures. The most significant MEAs and outlier loci were used to identify genomic loci with adaptive potential (some already reported in wheat), including dormancy and frost resistance loci. We advocate the application of genomics and landscape genomics on ex situ collections of crop wild relatives to efficiently identify promising alleles and genetic materials for incorporation into modern crop breeding.

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.

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Characterisation and cleaning of biogas from sewage sludge for biomethane production.

This study investigates the conversion of sewage sludge from wastewater treatment plants (WWTP) into biomethane for automotive fuel or grid injection. A prototype plant was monitored in Northern Italy, based on vacuum swing adsorption (VSA) on synthetic zeolite 13×: this biogas upgrading method is similar to pressure swing adsorption (PSA) and commonly used for other kinds of biomass. Measurements of biogas inlet, biomethane outlet and off-gas were performed including CH4, CO2, CO, H2, O2, N2, HCl, HF, NH3, H2S and volatile organic compounds (VOCs). Critical levels were observed in the biogas for of H2S and HCl, whose concentrations were 1570 and 26.8 mg m-3, respectively. On the other hand, the concentration of halogenated VOCs (including tetrachloroethylene and traces of perfluoroalkilated substances, PFAS) and mercaptans were relatively low. A simultaneous and reversible adsorption on 13× zeolite was achieved for H2S and CO2, and carbon filters played a minor role in desulfurisation. The presence of HCl is due to clarifying agents, and its removal is necessary in order to meet the required biomethane characteristics: an additional carbon-supported basic adsorbent was successfully used to remove this contaminant. This study also highlights the interference of CO2 towards HCl if sampling is performed in compliance with the new EU standard for biomethane. High total volatile silicon (TVS) was confirmed in sewage sludge biogas, with a major contribution of siloxane D5: the suitability of this compound as an indicator of total siloxanes is discussed. Results demonstrate that volatile methyl siloxanes (VMS) do not represent a critical issue for the VSA upgrading methodology.

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.

Mapping taste and flavour traits to genetic markers in lettuce Lactuca sativa.

Lettuce is the most highly consumed raw leafy vegetable crop eaten worldwide, making it nutritionally important in spite of its comparatively low nutrient density in relation to other vegetables. However, the perception of bitterness caused by high levels of sesquiterpenoid lactones and comparatively low levels of sweet tasting sugars limits palatability. To assess variation in nutritional and taste-related metabolites we assessed 104 members of a Lactuca sativa cv. Salinas x L. serriola (accession UC96US23) mapping population. Plants were grown in three distinct environments, and untargeted NMR and HPLC were used as a rapid chemotyping method, from which 63 unique Quantitative Trait Loci (QTL) were identified. We were able to identify putative regulatory candidate genes underlying the QTL for fructose on linkage group 9, which accounted for up to 36 % of our population variation, and which was stable across all three growing environments; and for 15-p-hydroxyyphenylacetyllactucin-8-sulfate on linkage group 5 which has previously been identified for its low bitterness, while retaining anti-herbivory field effects. We also identified a candidate gene for flavonoid 3',5'- hydroxylase underlying a polyphenol QTL on linkage group 5, and two further candidate genes in sugar biosynthesis on linkage groups 2 and 5. Collectively these candidate genes and their associated markers can inform a route for plant breeders to improve the palatability and nutritional value of lettuce in their breeding programmes.

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.

Development and application of Single Primer Enrichment Technology (SPET) SNP assay for population genomics analysis and candidate gene discovery in lettuce.

Single primer enrichment technology (SPET) is a novel high-throughput genotyping method based on short-read sequencing of specific genomic regions harboring polymorphisms. SPET provides an efficient and reproducible method for genotyping target loci, overcoming the limits associated with other reduced representation library sequencing methods that are based on a random sampling of genomic loci. The possibility to sequence regions surrounding a target SNP allows the discovery of thousands of closely linked, novel SNPs. In this work, we report the design and application of the first SPET panel in lettuce, consisting of 41,547 probes spanning the whole genome and designed to target both coding (~96%) and intergenic (~4%) regions. A total of 81,531 SNPs were surveyed in 160 lettuce accessions originating from a total of 10 countries in Europe, America, and Asia and representing 10 horticultural types. Model ancestry population structure clearly separated the cultivated accessions (Lactuca sativa) from accessions of its presumed wild progenitor (L. serriola), revealing a total of six genetic subgroups that reflected a differentiation based on cultivar typology. Phylogenetic relationships and principal component analysis revealed a clustering of butterhead types and a general differentiation between germplasm originating from Western and Eastern Europe. To determine the potentiality of SPET for gene discovery, we performed genome-wide association analysis for main agricultural traits in L. sativa using six models (GLM naive, MLM, MLMM, CMLM, FarmCPU, and BLINK) to compare their strength and power for association detection. Robust associations were detected for seed color on chromosome 7 at 50 Mbp. Colocalization of association signals was found for outer leaf color and leaf anthocyanin content on chromosome 9 at 152 Mbp and on chromosome 5 at 86 Mbp. The association for bolting time was detected with the GLM, BLINK, and FarmCPU models on chromosome 7 at 164 Mbp. Associations were detected in chromosomal regions previously reported to harbor candidate genes for these traits, thus confirming the effectiveness of SPET for GWAS. Our findings illustrated the strength of SPET for discovering thousands of variable sites toward the dissection of the genomic diversity of germplasm collections, thus allowing a better characterization of lettuce collections.

RAD tag sequencing as a source of SNP markers in Cynara cardunculus L.

BACKGROUND: The globe artichoke (Cynara cardunculus L. var. scolymus) genome is relatively poorly explored, especially compared to those of the other major Asteraceae crops sunflower and lettuce. No SNP markers are in the public domain. We have combined the recently developed restriction-site associated DNA (RAD) approach with the Illumina DNA sequencing platform to effect the rapid and mass discovery of SNP markers for C. cardunculus. RESULTS: RAD tags were sequenced from the genomic DNA of three C. cardunculus mapping population parents, generating 9.7 million reads, corresponding to ~1 Gbp of sequence. An assembly based on paired ends produced ~6.0 Mbp of genomic sequence, separated into ~19,000 contigs (mean length 312 bp), of which ~21% were fragments of putative coding sequence. The shared sequences allowed for the discovery of ~34,000 SNPs and nearly 800 indels, equivalent to a SNP frequency of 5.6 per 1,000 nt, and an indel frequency of 0.2 per 1,000 nt. A sample of heterozygous SNP loci was mapped by CAPS assays and this exercise provided validation of our mining criteria. The repetitive fraction of the genome had a high representation of retrotransposon sequence, followed by simple repeats, AT-low complexity regions and mobile DNA elements. The genomic k-mers distribution and CpG rate of C. cardunculus, compared with data derived from three whole genome-sequenced dicots species, provided a further evidence of the random representation of the C. cardunculus genome generated by RAD sampling. CONCLUSION: The RAD tag sequencing approach is a cost-effective and rapid method to develop SNP markers in a highly heterozygous species. Our approach permitted to generate a large and robust SNP datasets by the adoption of optimized filtering criteria.

Large-scale transcriptome characterization and mass discovery of SNPs in globe artichoke and its related taxa.

Cynara cardunculus (2n = 2× = 34) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. The species includes two cultivated varieties, globe artichoke and cardoon, which are grown mainly for food. Cynara cardunculus is an orphan crop species whose genome/transcriptome has been relatively unexplored, especially in comparison to other Asteraceae crops. Hence, there is a significant need to improve its genomic resources through the identification of novel genes and sequence-based markers, to design new breeding schemes aimed at increasing quality and crop productivity. We report the outcome of cDNA sequencing and assembly for eleven accessions of C. cardunculus. Sequencing of three mapping parental genotypes using Roche 454-Titanium technology generated 1.7 × 106 reads, which were assembled into 38,726 reference transcripts covering 32 Mbp. Putative enzyme-encoding genes were annotated using the KEGG-database. Transcription factors and candidate resistance genes were surveyed as well. Paired-end sequencing was done for cDNA libraries of eight other representative C. cardunculus accessions on an Illumina Genome Analyzer IIx, generating 46 × 106 reads. Alignment of the IGA and 454 reads to reference transcripts led to the identification of 195,400 SNPs with a Bayesian probability exceeding 95%; a validation rate of 90% was obtained by Sanger-sequencing of a subset of contigs. These results demonstrate that the integration of data from different NGS platforms enables large-scale transcriptome characterization, along with massive SNP discovery. This information will contribute to the dissection of key agricultural traits in C. cardunculus and facilitate the implementation of marker-assisted selection programs.

Genome-wide diversity and global migration patterns in dromedaries follow ancient caravan routes.

Dromedaries have been essential for the prosperity of civilizations in arid environments and the dispersal of humans, goods and cultures along ancient, cross-continental trading routes. With increasing desertification their importance as livestock species is rising rapidly, but little is known about their genome-wide diversity and demographic history. As previous studies using few nuclear markers found weak phylogeographic structure, here we detected fine-scale population differentiation in dromedaries across Asia and Africa by adopting a genome-wide approach. Global patterns of effective migration rates revealed pathways of dispersal after domestication, following historic caravan routes like the Silk and Incense Roads. Our results show that a Pleistocene bottleneck and Medieval expansions during the rise of the Ottoman empire have shaped genome-wide diversity in modern dromedaries. By understanding subtle population structure we recognize the value of small, locally adapted populations and appeal for securing genomic diversity for a sustainable utilization of this key desert species.

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.

Ontology and diversity of transcript-associated microsatellites mined from a globe artichoke EST database.

BACKGROUND: The globe artichoke (Cynara cardunculus var. scolymus L.) is a significant crop in the Mediterranean basin. Despite its commercial importance and its both dietary and pharmaceutical value, knowledge of its genetics and genomics remains scant. Microsatellite markers have become a key tool in genetic and genomic analysis, and we have exploited recently acquired EST (expressed sequence tag) sequence data (Composite Genome Project - CGP) to develop an extensive set of microsatellite markers. RESULTS: A unigene assembly was created from over 36,000 globe artichoke EST sequences, containing 6,621 contigs and 12,434 singletons. Over 12,000 of these unigenes were functionally assigned on the basis of homology with Arabidopsis thaliana reference proteins. A total of 4,219 perfect repeats, located within 3,308 unigenes was identified and the gene ontology (GO) analysis highlighted some GO term's enrichments among different classes of microsatellites with respect to their position. Sufficient flanking sequence was available to enable the design of primers to amplify 2,311 of these microsatellites, and a set of 300 was tested against a DNA panel derived from 28 C. cardunculus genotypes. Consistent amplification and polymorphism was obtained from 236 of these assays. Their polymorphic information content (PIC) ranged from 0.04 to 0.90 (mean 0.66). Between 176 and 198 of the assays were informative in at least one of the three available mapping populations. CONCLUSION: EST-based microsatellites have provided a large set of de novo genetic markers, which show significant amounts of polymorphism both between and within the three taxa of C. cardunculus. They are thus well suited as assays for phylogenetic analysis, the construction of genetic maps, marker-assisted breeding, transcript mapping and other genomic applications in the species.

Genetic mapping and annotation of genomic microsatellites isolated from globe artichoke.

Cynara cardunculus includes three taxa, the globe artichoke (subsp. scolymus L. Hegi), the cultivated cardoon (var. altilis) and their progenitor, the wild cardoon (var. sylvestris). Globe artichoke is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Here, we report the isolation of 61 new microsatellite loci which amplified a total of 208 alleles in a panel of 22 C. cardunculus genotypes. Of these, 51 were informative for linkage analysis and 39 were used to increase marker density in the available globe artichoke genetic maps. Sequence analysis of the 22 loci associated with genes showed that 9 are located within coding sequence, with the repetitive domain probably being involved in DNA binding or in protein-protein interactions. The expression of the genes associated with 9 of the 22 microsatellite loci was demonstrated by RT-PCR.

Two-stage granular sludge partial nitritation/anammox process for the treatment of digestate from the anaerobic digestion of the organic fraction of municipal solid waste.

The increasing amount of source separated organic fraction of municipal solid wastes (OFMSW) treated by anaerobic digestion for energy recovery requires the implementation of cost-efficient processes for the treatment of the produced digestate, especially in terms of nitrogen removal. The autotrophic nitrogen removal process, based on the coupling of two biological processes, partial nitritation (PN) and anammox (A), appears as a suitable solution due to important savings in operational costs compared to conventional treatment processes. However, its application could be hampered by the high salinity and inhibitory potential of this kind of digestate. In this contribution, two lab-scale granular sludge reactors performing the PN and anammox processes, respectively, were used to treat (opportunely diluted) real OFMSW digestate originating from full-scale biogas plants with the aim of assessing their treatment feasibility in a two-stage PN/A configuration. The PN process was implemented in an air-lift granular sludge reactor and was able to treat a nitrogen loading rate of about 1 g N L-1 d-1 at 30 ±â€¯0.5 °C; moreover, its effluent was suitable for the subsequent anammox treatment, with an appropriate effluent NO2-/NH4+ ratio and marginal inhibiting effects. In the anammox granular sludge reactor, the anammox activity was affected by high salinity levels, nonetheless a stable reactor performance at a nitrogen removing rate of 0.83 ±â€¯0.20 and 0.31 ±â€¯0.04 g N L-1 d-1 at 35 ±â€¯0.5 °C, were achieved when treating 50% and 30% diluted real wastewaters at a conductivity in the reactor of 9.1 and 11.2 mS cm-1, respectively.