ALBERT

Description: Background: The genome-wide association (GWA) approach represents an alternative to biparental linkage mapping for determining the genetic basis of trait variation. Both approaches rely on recombination to re-arrange the genome, and seek to establish correlations between phenotype and genotype. The major advantages of GWA lie in being able to sample a much wider range of the phenotypic and genotypic variation present, in being able to exploit multiple rounds of historical recombination in many different lineages and to include multiple accessions of direct relevance to crop improvement. Results: A 191 accessions eggplant (Solanum melongena L.) association panel, comprising a mixture of breeding lines, old varieties and landrace selections originating from Asia and the Mediterranean Basin, was SNP genotyped and scored for anthocyanin pigmentation and fruit color at two locations over two years. The panel formed two major clusters, reflecting geographical provenance and fruit type. The global level of linkage disequilibrium was 3.4 cM. A mixed linear model appeared to be the most appropriate for GWA. A set of 56 SNP locus/phenotype associations was identified and the genomic regions harboring these loci were distributed over nine of the 12 eggplant chromosomes. The associations were compared with the location of known QTL for the same traits. Conclusion: The GWA mapping approach was effective in validating a number of established QTL and, thanks to the wide diversity captured by the panel, was able to detect a series of novel marker/trait associations.

Description: 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.

Description: 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.

Description: Background The Asteraceae species Cynara cardunculus (2n = 2x = 34) includes the two fully cross-compatible domesticated taxa globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC). As both are out-pollinators and suffer from marked inbreeding depression, linkage analysis has focussed on the use of a two way pseudo-test cross approach. Results A set of 172 microsatellite (SSR) loci derived from expressed sequence tag DNA sequence were integrated into the reference C. cardunculus genetic maps, based on segregation among the F1 progeny of a cross between a globe artichoke and a cultivated cardoon. The resulting maps each detected 17 major linkage groups, corresponding to the species’ haploid chromosome number. A consensus map based on 66 co-dominant shared loci (64 SSRs and two SNPs) assembled 694 loci, with a mean inter-marker spacing of 2.5 cM. When the maps were used to elucidate the pattern of inheritance of head production earliness, a key commercial trait, seven regions were shown to harbour relevant quantitative trait loci (QTL). Together, these QTL accounted for up to 74% of the overall phenotypic variance. Conclusion The newly developed consensus as well as the parental genetic maps can accelerate the process of tagging and eventually isolating the genes underlying earliness in both the domesticated C. cardunculus forms. The largest single effect mapped to the same linkage group in each parental maps, and explained about one half of the phenotypic variance, thus representing a good candidate for marker assisted selection.

Description: Background The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds. Results Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps. Conclusion A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus.

Description: Background The eggplant (Solanum melongena L.) genome is relatively unexplored, especially compared to those of the other major Solanaceae crops tomato and potato. In particular, no SNP markers are publicly available; on the other hand, over 1,000 SSR markers were developed and publicly available. We have combined the recently developed Restriction-site Associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of both SNP and SSR markers for eggplant. Results RAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 contigs. The resulting non-redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were putative coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb. Over 2,000 of the SNPs are likely to be mappable via the Illumina GoldenGate assay. A subset of 384 SNPs was used to successfully fingerprint a panel of eggplant germplasm, producing a set of informative diversity data. The RAD sequences also included nearly 2,000 putative SSRs, and primer pairs were designed to amplify 1,155 loci. Conclusion The high throughput sequencing of the RAD tags allowed the discovery of a large number of DNA markers, which will prove useful for extending our current knowledge of the genome organization of eggplant, for assisting in marker-aided selection and for carrying out comparative genomic analyses within the Solanaceae family.

Description: Background Eggplant (Solanum melongena L.) is a member of the Solanaceae family. In spite of its widespread cultivation and nutritional and economic importance, its genome has not as yet been extensively investigated. Few analyses have been carried out to determine the genetic diversity of eggplant at the DNA level, and linkage relationships have not been well characterised. As for the other Solanaceae crop species (potato, tomato and pepper), the level of intra-specific polymorphism appears to be rather limited, and so it is important that an effort is made to develop more informative DNA markers to make progress in understanding the genetics of eggplant and to advance its breeding. The aim of the present work was to develop a set of functional microsatellite (SSR) markers, via an in silico analysis of publicly available DNA sequence. Results From 〉3,300 genic DNA sequences, 50 SSR-containing candidates suitable for primer design were recovered. Of these, 39 were functional, and were then applied to a panel of 44 accessions, of which 38 were cultivated eggplant varieties, and six were from related Solanum species. The usefulness of the SSR assays for diversity analysis and taxonomic discrimination was demonstrated by constructing a phylogeny based on SSR polymorphisms, and by the demonstration that most were also functional when tested with template from tomato, pepper and potato. As a results of BLASTN analyses, several eggplant SSRs were found to have homologous counterparts in the phylogenetically related species, which carry microsatellite motifs in the same position. Conclusion The set of eggplant EST-SSR markers was informative for phylogenetic analysis and genetic mapping. Since EST-SSRs lie within expressed sequence, they have the potential to serve as perfect markers for genes determining variation in phenotype. Their high level of transferability to other Solanaceae species can be used to provide anchoring points for the integration of genetic maps across species.