Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BACKGROUND: Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes. RESULTS: Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome. CONCLUSIONS: Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world's most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line ('HapOB') from Rosa chinensis 'Old Blush' and generated a rose genome assembly anchored to seven pseudo-chromosomes (512 Mb with N50 of 3.4 Mb and 564 contigs). The length of 512 Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

Structure and expression of an asparaginyl-tRNA synthetase gene located on chromosome IV of Arabidopsis thaliana and adjacent to a novel gene of 15 exons.

The gene AtNS1 coding for an asparaginyl-tRNA synthetase and located on chromosome IV of Arabidopsis thaliana has been characterized. AtNS1 is the first asparaginyl-tRNA synthetase gene described in higher plants. The genomic environment of AtNS1 has been studied, as well as a partial cDNA of a second homologous asparaginyl-tRNA synthetase gene, AtNS2. Both AtNS1 and AtNS2 exhibit the highest similarity with prokaryotic homologues. A large novel gene of 15 exons, named AtG2484-1, is located adjacent to AtNS1. AtG2484-1 shows features rarely described in plants including large exons and one 3' non-coding exon. PCR and Northern analyses were carried out to obtain information about the expression of these genes in various A. thaliana tissues.

Estimation of the H+/H- ratio of the reaction catalysed by the nicotinamide nucleotide transhydrogenase in chromatophores from over-expressing strains of Rhodospirillum rubrum and in liposomes inlaid with the purified bovine enzyme.

Two strains of Rhodospirillum rubrum were constructed in which, by a gene dosage effect, the transhydrogenase activity of isolated chromatophores was increased 7-10-fold and 15-20-fold, respectively. The H+/H- ratio (the ratio of protons translocated per hydride ion equivalent transferred from NADPH to an NAD+ analogue, acetyl pyridine adenine dinucleotide), determined by a spectroscopic technique, was approximately 1.0 for chromatophores from the over-expressing strains, but was only approximately 0.6 for wild-type chromatophores. Highly-coupled proteoliposomes were prepared containing purified transhydrogenase from beef-heart mitochondria. Using the same technique, the H+/H- ratio was close to 1.0 for these proteoliposomes. It is suggested that the mechanistic H+/H- ratio is indeed unity, but that a low ratio is obtained in wild-type chromatophores because of inhomogeneity in the vesicle population.

Structure and expression of three src2 homologues and a novel subfamily of flavoprotein monooxygenase genes revealed by the analysis of a 25kb fragment from Arabidopsis thaliana chromosome IV.

Biological and computer-assisted analyses of a 25kb fragment from Arabidopsis thaliana chromosome IV led to the characterization of two multigene families and three novel orphan genes, not previously described. The first gene family named AtMO1-4 encodes monooxygenases, related to the prokaryotic salicylate hydroxylases. The second gene family contains three members, two on the analysed 25kb fragment and one on chromosome I. The latter three genes lack introns and are homologous to the previously studied Glycine max src2 gene which is overexpressed at low temperature. Gene expression and primary structure of the deduced proteins are described and compared. Three genes of unknown function, showing tissue specific expressions, are characterized on the 25kb fragment. Full length or partial cognate cDNAs have been sequenced for all the genes studied.

Structure, organization and putative function of the genes identified within a 23.9-kb fragment from Arabidopsis thaliana chromosome IV.

In the framework of the complete genome sequencing programme of the crucifer Arabidopsis thaliana, a 23.9-kb fragment from the long arm of chromosome IV has been analysed. This paper presents a methodological approach, integrating computerized predictions, database screening, the sequencing of cognate cDNAs and a PCR-based detection of expression that allows the accumulation of an important amount of information from an anonymous sequence. This work revealed the organization of novel genes and the vestige of a copia-like retrotransposon. The gene AtRH1 encodes the first member of a new subfamily of the plant DEAD box RNA helicases. A recurrent and complete search of dbEST has been used to evaluate the number of different RNA helicases expressed in A. thaliana. On the 18 discriminated members of the family, only a small number seems to be expressed at a relatively high level. The putative gene AtTS1 encodes a novel terpene synthase in A. thaliana, and the genes G14587-5 and G14587-6 encode unknown proteins. This study illustrates most of the situations that could be encountered during the analysis of an anonymous sequence from A. thaliana.

Analysis of a 14-kb fragment containing a putative cell wall gene and a candidate for the ARA1, arabinose kinase, gene from chromosome IV of Arabidopsis thaliana.

An Arabidopsis thaliana genomic DNA fragment of 14kb has been characterized in the framework of the E.S.S.A. programme. Computational and molecular approaches identified three novel gene sequences coding, respectively, for a protein of unknown function, a putative membrane-anchored cell wall protein and an arabinose kinase gene corresponding to the locus ARA1. The latter two genes named AtSEB1 and AtISA1 have been characterized in detail. They are very different in their organization, codon usage and level of expression. Homologues of AtSEB1 and AtISA1 have been identified. Sequence comparisons showed that the former genes contained a long 5' extension coding for an N-terminal domain probably specifying subcellular localization. Cloning and sequencing of the cognate cDNA for the AtISA1 homologue in A. thaliana, named GAL1, indicate that it encodes for a galactokinase-like protein. Our results highlight the integrative outcome of a systematic sequencing project in which links between biochemically and genetically characterized mutants, ESTs and genomic sequence data are generated.

Composition of phospholipids of white muscle of six tuna species.

A comparative study of the phospholipids of white muscle of six of the commercially utilized tuna species, including quantitative analyses of phospholipid classes and studies of the acyl composition of the major components. Plasmalogen compounds also were identified and quantified. Choline and ethanolamine glycerophospholipids were the most abundant classes in all the samples, as well as the only molecules containing plasmalogens (16:0, 18:0, and 18:1 alkenylether chains). The patterns of fatty acid distribution within each of the phospholipid classes showed general similarities in the species analyzed. However, ratios between certain saturated and polyunsaturated fatty acids in different phospholipid classes showed remarkable differences. The high content of n-3 polyunsaturated fatty acids in the principal phospholipids, such as the plasmalogens, and taking into account the fatty acids possible importance in human nutrition, indicates that the white muscle of tuna species may be a potentially important dietary item.

Genomic analysis of the terpenoid synthase ( AtTPS) gene family of Arabidopsis thaliana.

A family of 40 terpenoid synthase genes ( AtTPS) was discovered by genome sequence analysis in Arabidopsis thaliana. This is the largest and most diverse group of TPS genes currently known for any species. AtTPS genes cluster into five phylogenetic subfamilies of the plant TPS superfamily. Surprisingly, thirty AtTPS closely resemble, in all aspects of gene architecture, sequence relatedness and phylogenetic placement, the genes for plant monoterpene synthases, sesquiterpene synthases or diterpene synthases of secondary metabolism. Rapid evolution of these AtTPS resulted from repeated gene duplication and sequence divergence with minor changes in gene architecture. In contrast, only two AtTPS genes have known functions in basic (primary) metabolism, namely gibberellin biosynthesis. This striking difference in rates of gene diversification in primary and secondary metabolism is relevant for an understanding of the evolution of terpenoid natural product diversity. Eight AtTPS genes are interrupted and are likely to be inactive pseudogenes. The localization of AtTPS genes on all five chromosomes reflects the dynamics of the Arabidopsis genome; however, several AtTPS genes are clustered and organized in tandem repeats. Furthermore, some AtTPS genes are localized with prenyltransferase genes ( AtGGPPS, geranylgeranyl diphosphate synthase) in contiguous genomic clusters encoding consecutive steps in terpenoid biosynthesis. The clustered organization may have implications for TPS gene evolution and the evolution of pathway segments for the synthesis of terpenoid natural products. Phylogenetic analyses highlight events in the divergence of the TPS paralogs and suggest orthologous genes and a model for the evolution of the TPS gene family.

The DEAD box RNA helicase family in Arabidopsis thaliana.

The numerous genomic sequences and ESTs released by the Arabidopsis thaliana Genome Initiative (AGI) have allowed a systematic and functional study of the DEAD box RNA helicase family. Sequencing and in silico analysis led to the characterization of 28 novel A. thaliana DEAD box RNA helicases forming a family of 32 members, named AtRH. Fourteen AtRH genes with an unexpected heterogeneous mosaic structure are described and compared bringing new information about the genesis of the gene family. The mapping of the AtRH genes shows their repartition on the five chromosomes without clustering and therefore AtRH s have been estimated to 60 genes per A.thaliana haploid genome. Sequence comparisons revealed a very conserved catalytic central domain flanked or not by four classes of extensions in the N- and/or C- extremities. The global amino acid composition of the extensions are tentatively correlated to specific functions such as targeting, protein interaction or RNA binding. The expression of the 32 AtRH genes has been recorded in different tissues. Separate patterns of expression and alternative polyadenylation sites have been shown. Based on the integration of all this information, we propose a classification of the AtRH proteins into subfamilies with associated functions.

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants.

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1-6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.

Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2.

The Arabidopsis Genome Initiative has released up to now more than 80% of the genome sequence of Arabidopsis thaliana. About 70% of the identified genes have at least one paralogue. In order to understand the biological function of individual genes, it is essential to study the structure, expression and organization of the entire multigene family. A systematic analysis of multigene families, made possible by the amount of genomic sequence data available, provides important clues for the understanding of genome evolution and plasticity. In this paper, four multigene families of A. thaliana are characterized, namely LCAD, HD-GL2, LGT and MYST. Members of HD-GL2 and LCAD have already been reported in plants. The LGT genes specify proteins containing motifs of glycosyl transferase. No plant genes similar to the LGT genes have been reported to date. The novel MYST family, most likely plant-specific, encodes proteins with no identified function. Sequencing and in silico analysis led to the characterization of 29 novel genes belonging to these four gene families. The organization, structure and evolution of all the members of the four families are discussed, as well as their chromosome location. Expression data of some of the paralogues of each family are also presented.

A comparison between conventional and fluorescence detection methods of cooking-induced damage to tuna fish lipids.

The damage to tuna fish lipids induced by cooking was investigated in the Thunnus obesus and Th. thynnus varieties, using conventional and fluorescence detection methods, and the results were compared. As a consequence of thermal processing, the peaks at longer wavelengths increased, which correlated with other conventional indices of lipid damage (i.e. carbonyl compound formation, browning and increases in the free fatty acid content). A special significance was given to the fluorescence ratio between the maxima of the excitation emission data at 393/460 nm and 327/415 nm; increases in this ratio as a result of cooking were less dependent on the samples than were other conventional methods of measuring lipid damage.

Changes in free amino acids content in albacore (Thunnus alalunga) muscle during thermal processing.

The effects of cooking and sterilization at several temperatures on the free amino acids (FAA) content in albacore (Thunnus alalunga) muscle were studied during the processing of canned tuna. FAAs were derivatized with o-phthalaldehyde, separated on a C18 column by HPLC and detected by both fluorescence and ultra-violet detectors. After cooking the loss of FAAs was not significant. However, in the final product sterilized at 115 degrees C and 110 degrees C (throughout the whole process) there were significant losses with regard to the start material, but not at 118 degrees C (all temperatures leading to the same lethal F-value). The influence of the thermal process time at 115 degrees C was evaluated for 60 and 100 min. Significant losses were found between both canned products (approximately 25%) and between the raw fish and the final product (approximately 12% and approximately 34%, process time 60 and 100 min, respectively). The determination of the content of FAA present in canned albacore may be a useful indication of the severity of the thermal processing.

Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila.

The DEAD box RNA helicase (RH) proteins are homologs involved in diverse cellular functions in all of the organisms from prokaryotes to eukaryotes. Nevertheless, there is a lack of conservation in the splicing pattern in the 53 Arabidopsis thaliana (AtRHs), the 32 Caenorhabditis elegans (CeRHs) and the 29 Drosophila melanogaster (DmRHs) genes. Of the 153 different observed intron positions, 4 are conserved between AtRHs, CeRHs, and DmRHs, and one position is also found in RHs from yeast and human. Of the 27 different AtRH structures with introns, 20 have at least one predicted ancient intron in the regions coding for the catalytic domain. In all of the organisms examined, we found at least one gene with most of its intron predicted to be ancient. In A. thaliana, the large diversity in RH structures suggests that duplications of the ancestral RH were followed by a high number of intron deletions and additions. The very high bias toward phase 0 introns is in favor of intron addition, preferentially in phase 0. Results from this comparative study of the same gene family in a plant and in two animals are discussed in terms of the general mechanisms of gene family evolution.

Annotation of a 95-kb Populus deltoides genomic sequence reveals a disease resistance gene cluster and novel class I and class II transposable elements.

Poplar has become a model system for functional genomics in woody plants. Here, we report the sequencing and annotation of the first large contiguous stretch of genomic sequence (95 kb) of poplar, corresponding to a bacterial artificial chromosome clone mapped 0.6 centiMorgan from the Melampsora larici-populina resistance locus. The annotation revealed 15 putative genetic objects, of which five were classified as hypothetical genes that were similar only with expressed sequence tags from poplar. Ten putative objects showed similarity with known genes, of which one was similar to a kinase. Three other objects corresponded to the toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant disease resistance genes, of which two were predicted to encode an amino terminal nuclear localization signal. Four objects were homologous to the Ty1/ copia family of class I transposable elements, one of which was designated Retropop and interrupted one of the disease resistance genes. Two other objects constituted a novel Spm-like class II transposable element, which we designated Magali.