Carlo Pozzi

Cloning, mapping and expression analysis of barley MADS-box genes.

Six MADS-box cDNA clones were isolated by heterologous screening from a barley inflorescence cDNA library. Based on sequence comparison to known MADS-box genes, the barley MADS-box (BM) genes were grouped into three distinct phylogenetic subclasses of the MADS-box gene family. The three MADS-box genes BM3, BM5 and BM8 share similarities with genes of the SQUAMOSA (SQUA) subgroup, while BM7 and BM9 belong to the AGAMOUS-LIKE 2 (AGL2) subgroup. BM1 resembles MADS-box genes described as solitary sequences or orphan genes. Expression analysis of the barley MADS-box genes revealed expression patterns that are not characteristic of the barley MADS-box genes of the SQUA subgroup, while expression of BM7 and BM9 was largely as expected for the AGL2 subgroup. BM1 is mainly expressed in vegetative tissues and its primary transcript undergoes alternative splicing such that the corresponding mRNAs differ by two codons. The genes BM1, BM3 and BM8 were mapped by analysis of single-nucleotide polymorphisms onto barley chromosomes 4, 2 and 7, respectively.

A reconsideration of the domestication geography of tetraploid wheats

The domestication of tetraploid wheats started from their wild progenitor Triticum dicoccoides. In this paper, the geographical distribution of this progenitor is revised to include more sampling locations. The paper is based on a collection of wild and domesticated lines (226 accessions in total) analyzed by AFLP at 169 polymorphic loci. The collection includes the 69 wild lines considered by Mori et al. (2003) in their study on chloroplast DNA haplotypes of T. dicoccoides. The goal of the experiment was to reconsider which location thought to have generated the domesticated germplasm has the highest chance of being the actual site from which wild progenitors were sampled during domestication. Phylogenetic analysis of the nuclear AFLP databases indicates that two different genetic taxa of T. dicoccoides exist, the western one, colonizing Israel, Syria, Lebanon and Jordan, and the central-eastern one, which has been frequently sampled in Turkey and rarely in Iran and Iraq. It is the central-eastern race that played the role of the progenitor of the domesticated germplasm. This is supported by the cumulative results of the AFLP data from the collections of Ozkan et al. (2002) and of Mori et al. (2003), which indicate that the Turkish Karacadag population, intermixed with some Iraq-Iran lines, has a tree topology consistent with that of the progenitor of domesticated genotypes. The Turkish Kartal population belongs genetically to the central-eastern T. dicoccoides race but at the nuclear DNA level is less related to the domesticated gene pool. A general agreement between published work on tetraploid wheat domestication emerges from these results. A disagreement is nevertheless evident at the local geographical scale; the chloroplast DNA data indicate the Kartal mountains while AFLP fingerprinting points to the Karacadag Range as the putative site of tetraploid wheat domestication.

Genetic dissection of aroma volatile compounds from the essential oil of peach fruit: QTL analysis and identification of candidate genes using dense SNP maps

Volatile organic compounds (VOCs) in plants are involved in aroma and pest resistance. These compounds form a complex mixture whose composition is specific to species and often to varieties. Despite their importance as essential factors that determine peach fruit quality, understanding of molecular, genetic, and physiological mechanisms underlying aroma formation is limited. The aim of this study was the identification in peach of quantitative trait loci (QTLs) for fruit VOCs to understand their genetic basis using an F1 population of 126 seedlings deriving from the cross between “Bolero” (B) and “OroA” (O), two peach cultivars differing in their aroma profile. Dense single nucleotide polymorphism (SNP) and SSR maps covering the eight linkage groups of the peach genome were constructed by genotyping with the International Peach SNP Consortium peach SNP array v1, and data for 23 VOCs with high or unknown “odor activity value” were obtained by gas chromatography–mass spectrometry analysis of fruit essential oil in the years 2007 and 2008. A total of 72 QTLs were identified, most consistent in both years. QTLs were identified for the 23 VOCs studied, including three major QTLs for nonanal, linalool, and for p-menth-1-en-9-al stable in both years. Collocations between candidate genes and major QTLs were identified taking advantage of the peach genome sequence: genes encoding two putative terpene synthases and one lipoxygenase (Lox) might be involved in the biosynthesis of linalool and p-menth-1-en-9-al, and nonanal, respectively. Implications for marker-assisted selection and future research on the subject are discussed.

ESTree db: a Tool for Peach Functional Genomics

BackgroundThe ESTree db http://www.itb.cnr.it/estree/ represents a collection of Prunus persica expressed sequenced tags (ESTs) and is intended as a resource for peach functional genomics. A total of 6,155 successful EST sequences were obtained from four in-house prepared cDNA libraries from Prunus persica mesocarps at different developmental stages. Another 12,475 peach EST sequences were downloaded from public databases and added to the ESTree db. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts and data were collected in a MySQL database. A php-based web interface was developed to query the database.ResultsThe ESTree db version as of April 2005 encompasses 18,630 sequences representing eight libraries. Contig assembly was performed with CAP3. Putative single nucleotide polymorphism (SNP) detection was performed with the AutoSNP program and a search engine was implemented to retrieve results. All the sequences and all the contig consensus sequences were annotated both with blastx against the GenBank nr db and with GOblet against the viridiplantae section of the Gene Ontology db. Links to NiceZyme (Expasy) and to the KEGG metabolic pathways were provided. A local BLAST utility is available. A text search utility allows querying and browsing the database. Statistics were provided on Gene Ontology occurrences to assign sequences to Gene Ontology categories.ConclusionThe resulting database is a comprehensive resource of data and links related to peach EST sequences. The Sequence Report and Contig Report pages work as the web interface core structures, giving quick access to data related to each sequence/contig.

Version VI of the ESTree db: an improved tool for peach transcriptome analysis

BackgroundThe ESTree database (db) is a collection of Prunus persica and Prunus dulcis EST sequences that in its current version encompasses 75,404 sequences from 3 almond and 19 peach libraries. Nine peach genotypes and four peach tissues are represented, from four fruit developmental stages. The aim of this work was to implement the already existing ESTree db by adding new sequences and analysis programs. Particular care was given to the implementation of the web interface, that allows querying each of the database features.ResultsA Perl modular pipeline is the backbone of sequence analysis in the ESTree db project. Outputs obtained during the pipeline steps are automatically arrayed into the fields of a MySQL database. Apart from standard clustering and annotation analyses, version VI of the ESTree db encompasses new tools for tandem repeat identification, annotation against genomic Rosaceae sequences, and positioning on the database of oligomer sequences that were used in a peach microarray study. Furthermore, known protein patterns and motifs were identified by comparison to PROSITE. Based on data retrieved from sequence annotation against the UniProtKB database, a script was prepared to track positions of homologous hits on the GO tree and build statistics on the ontologies distribution in GO functional categories. EST mapping data were also integrated in the database. The PHP-based web interface was upgraded and extended. The aim of the authors was to enable querying the database according to all the biological aspects that can be investigated from the analysis of data available in the ESTree db. This is achieved by allowing multiple searches on logical subsets of sequences that represent different biological situations or features.ConclusionsThe version VI of ESTree db offers a broad overview on peach gene expression. Sequence analyses results contained in the database, extensively linked to external related resources, represent a large amount of information that can be queried via the tools offered in the web interface. Flexibility and modularity of the ESTree analysis pipeline and of the web interface allowed the authors to set up similar structures for different datasets, with limited manual intervention.

Cross-Talk between the KNOX and Ethylene Pathways Is Mediated by Intron-Binding Transcription Factors in Barley

In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.

Comparative analysis of expressed sequence tags from tissues in ripening stages of peach ( Prunus persica L. Batsch)

Expressed sequence tag (EST) represents a resource for gene discovery, genome annotation and comparative genomics in plants. ESTs were derived by sequencing clones from five libraries created from two different fruit tissues (skin and mesocarp), at four ripening stages (from post-allegation to post-climacteric) in three different genotypes of peach (OroA, Bolero and Suncrest). A total of 10,847 EST sequences were produced (dataset A); in addition, 21,857 peach ESTs (dataset B) were obtained from public databases. Clustering and assembly of both datasets gave 17,858 unigenes. Analysis of the sequences allowed the assignment of a putative function to 70.8% of the ESTs. In order to define the relationship among fruit tissues transcriptome, a gene ontology analysis was performed. Differences among organs and among different maturation stages of the same organs were identified in organelle, signal transducer and antioxidant activity. A distance matrix of pairwise correlation coefficients analysis was applied between the libraries. Shoot appeared to outgroup and our analysis proved to be an efficient tool to parallel and complement gene expression studies (for example, based on microarray analysis). We conducted an analysis of the frequency of genes putatively involved in the metabolism of some volatiles, which pointed to a predominant presence of those transcripts in the skin. The metabolic pathways of esters and lactones were selected for further isolation and cloning of key genes. The EST database is available at the web site www.itb.cnr.it/estree.

The Genetic Basis of Composite Spike Form in Barley and ‘Miracle-Wheat’

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. ‘Compositum-Barley’ and tetraploid ‘Miracle-Wheat’ (T. turgidum convar. compositum (L.f.) Filat.) display noncanonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation ‘Miracle-Wheat’ produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in ‘Compositum-Barley,’ i.e., compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched headt (bht) locus regulating spike branching in tetraploid ‘Miracle-Wheat.’ Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bht locus in a collection of mutant and wild-type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of ‘Miracle-Wheat.’ mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative downstream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat’s yield potential.

Genetics of barley Hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.

AFLP-based bulk segregant analysis for tagging the slow-ripening trait in peach [Prunus persica (L.) Batsch].

Summary Flesh texture is an important quality trait in peach, affecting both fruit firmness and the ripening process. An interesting variation, “slow ripening” (SR), described in some progeny from ‘Fantasia’ nectarine, interferes with the completion of ripening. In these plants, fruit development appears to cease before the end of the cell expansion phase. The SR process was studied at the genetic level using amplified fragment length polymorphisms (AFLPs) combined with bulked segregant analysis (BSA).A marker linked to the SR trait was found at 5.6 cM in a segregating ‘BO 95021’ selfed progeny related to the cultivars ‘Fantasia’ and ‘Fairlane’.The original F1 parent tree was not available at the time of this trial which prevented traditional gene mapping and required the use of BSA. The F1 plant was derived by crossing ‘Fantasia’, a normal melting-flesh (M) nectarine, and ‘BO 7803302’, an SR selection generated by open-pollination of ‘Fairlane’, another M-flesh nectarine, putatively heterozygous for the SR trait. The generally high level of inbreeding in peach could explain the low level of genetic variability observed in our AFLP patterns.