Giorgio Graziosi

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis

Coffee, tea, and chocolate converge Caffeine has evolved multiple times among plant species, but no one knows whether these events involved similar genes. Denoeud et al. sequenced the Coffea canephora (coffee) genome and identified a conserved gene order (see the Perspective by Zamir). Although this species underwent fewer genome duplications than related species, the relevant caffeine genes experienced tandem duplications that expanded their numbers within this species. Scientists have seen similar but independent expansions in distantly related species of tea and cacao, suggesting that caffeine might have played an adaptive role in coffee evolution. Science, this issue p. 1181; see also p. 1124 The genetic origins of coffee’s constituents reveal intriguing links to cacao and tea. Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin.

Low variability of the protein species synthesized byDrosophila melanogaster embryos

SummaryStainable proteins as well as newly synthesized polypeptide chains of proteins extracted fromDrosophila melanogaster embryos were analyzed by two-dimensional gel electrophoresis. The following developmental stages were studied: unfertilized eggs, early nuclear multiplication (25 min average age), late nuclear multiplication (105 min), cellular blastoderm (165 min), gastrula (4 h), mesodermal segmentation (6 h) and muscleattachment (8 h). One hundred and fifty stainable spots were present at all developmental stages and were all also synthesized during development, with the exception of 5 unkown proteins and the three yolk proteins. Out of 400 proteins which were labelled by35S-methionine, only 5% showed a reproducible pattern of variable synthesis. Three proteins appeared upon fertilization. The early nuclear multiplication stage showed the largest number of labelled spots while the lowest number of labelled spots was observed at blastoderm formation. The pattern of synthesis of a few specific proteins was also followed. Actin I was synthesized only at 8 h, actin II and actin III were synthesized at all stages. β-tubulin was synthesized at all stages, while we observed a reduction, if not a cessation, of synthesis of α-tubulin at 105 min, 165 min and 4 h of development. Non heat-shock embryos synthesized a large amount of heat-shock protein (hsp) 84 at 25 min while hsp 70 and 68 were first detected after 4 h of development. Though it is generally accepted that the embryonic genome is activated at blastoderm formation we did not observe a parallel increase in protein species. It is possible that protein synthesis on the new transcripts is below the detection limit of the technique. Alternatively the embryonic messages may gradually substitute preexisting maternal messages or only become available for translation some time after they are transcribed.

A new approach in association study of single nucleotide polymorphism of genes for carcass and meat quality traits in commercial pigs

Abstract Six batches of four commercial hybrids of heavy pigs, reared for the production of Italian dry-cured hams, were identified for having homogeneous feeding and farm conditions. For a total of 235 pigs, slaughtered in the same slaughterhouse, carcass traits and muscle composition were measured. The pigs were genotyped for single nucleotide polymorphisms (SNPs) of Na+, K+-ATPase subunit alpha 2 (ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; ATP1A2), cystatin B (CSTB), mitochondrial 2,4-dienoyl-CoA reductase 1 (DECR1), leptin (LEP; 3 SNPs), melanocortin receptor 4 (MC4R), melanocortin receptor 5 (MC5R), sarcolipin (SLN) and titin (TTN) genes. All genes showed biallelic polymorphisms and the alleles were differently distributed between the six batches. Pigs were subsequentely clustered in “lean” and “fat” using either carcass traits (lean percentage, backfat thickness, loin muscle thickness, ham weight and ham cover fat thickness: 100 lean and 135 fat) or meat composition data (dry matter, protein, fat and ash of Biceps femoris and Vastus lateralis and pH after 24 hours: 126 lean and 109 fat). The association of gene polymorphisms with leaness and fatness of pigs was thus investigated using a logistic regression. ATP1A2, LEP (HinfI polymorphism) and MC4R, together with sex and ham weight were, included in the model to screen lean and fat pigs classified according to carcass traits data, yielding a correct classification of 71%. For the lean and fat pigs classified according to muscle composition, sex, CSTB, DECR1, MC5R and LEP (AciI/TaqI polymorphisms) were included in the regression analysis, that yielded a 66% of pigs correctly classified. These preliminary results may indicate that some of the selected candidate genes could be associated to production traits and are worth of further investigations.

S-RNase-like Sequences in Styles of Coffea (Rubiaceae). Evidence for S-RNase Based Gametophytic Self-Incompatibility?

Although RNase-based self-incompatibility (SI) is suspected to operate in a wide group of plant families, it has been characterized as the molecular genetic basis of SI in only three distantly related families, Solanaceae, Plantaginaceae, and Rosaceae, all described over a decade ago. Previous studies found that gametophytic SI, controlled by a multi-allelic S-locus, operates in the coffee family (Rubiaceae). The molecular genetic basis of this mechanism remains unknown, despite the immense importance of coffee as an agricultural commodity. Here, we isolated ten sequences with features of T2-S-type RNases from two Coffea species. While three of the sequences were identified in both species and clearly do not appear to be S-locus products, our data suggest that six sequences may be S-alleles in the self-incompatible C. canephora, and one may be a relict in the self-compatible C. arabica. We demonstrate that these sequences show style-specific expression, display polymorphism in C. canephora, and cluster with S-locus products in a phylogenetic analysis that includes other plant families with RNase-based SI. Although our results are not definitive, in part because the available plant materials were limited and data patterns relatively complex, our results strongly hint that RNase-based SI mechanism operates in the Rubiaceae family.

Evaluation of gene expression profiles of pig skeletal muscle in response to energy content of the diets using human microarrays

Abstract The aim of the research was to compare gene transcription profiles of Musculus longissimus dorsi (MLD) between pigs fed diets with high (HED) or low (LED) energy contents. Two groups of 4 Casertana pigs were reared from 3 to 12 months of age in the same environmental conditions and fed HED or LED. In the HED, the ave rage daily gain and back fat thickness were significantly (P<0.05) higher than in LED pigs. Differential expression of genes in MLD of pigs fed diets with different energy density was assessed by a human high-density complementary DNA (cDNA) muscle microarray consisting of 4670 probes and further confirmed by quantitative real time RT-PCR analysis. Seven of the genes up-regulated in MLD of HED pigs were invo l ved in the glycolytic and oxidative metabolism (phosphoglycerate mutase 2, glyc e ra l d e hyde-3-phosphate dehydrogenase, NADH dehydrogenase ubiquinone1 beta 9, muscle pyruvate kinase, enolase 3, muscle creatine kinase, isocitrate dehydrogenase 3 (NAD+) gamma) and four in the contractile apparatus (tropomyosin 1 alpha, troponin C2, fast, fast skeletal myosin light chain 2, troponin T3, skeletal, fast). Instead, HED diet reduced the level of expression of muscle proteins associated with slow fibre type (troponin T1, skeletal, slow, supervillin, myosin binding protein C, slow type, titin, myosin, heavy polypeptide 7, beta, calponin homology-associated smooth muscle) and signal transduction (SH3-binding domain protein 5-like, hypothetical protein FLJ21438, protein kinase cAMP-dependent, catalytic, rho guanine nucleotide exchange factor 15). The down-regulation of CTSB was also observed for HED group. From the results it can be assumed that high energy content of the diet influence physiological processes in the muscle tissue by switching slow fibres into fast reacting fibres and thus enhancing meat quality.

Genome organization in coffee as revealed by EST PCRRFLP, SNPs and SSR analysis

An EST-based PCR-RFLP method was employed to gain insight into genome organization in eight allopolyploid Coffea arabica cultivars and seven diploid coffee species. The PCR-amplified products at 19 EST loci were digested with 46 different restriction enzymes and size fractioned in agarose gels. Most often, the sum of the fragments length was double or more than the PCR product. In arabica, this condition could be explained by assuming the presence of duplicated loci in paralogous chromosomes and this was supported by considerable evidence of multiple loci SSR amplification. Based on the RFLP analysis, 12 EST loci were polymorphic. The level of polymorphism was higher in different species compared to the arabica varieties. Sequencing of the amplified products revealed a SNP frequency of 0.021 among diploid species and of 0.007 among arabica varieties. We propose that the involvement of two genomes in C. arabica maintains a residual level of heterozygosity in the form of paralogous chromosomes, while the self-fertilization in this species tends to drive of homozygosity. The heterozygosity of paralogous chromosomes in arabica creates valuable polymorphism essential for species diversity and survival in various ecological niches, while self-fertility tends to preserve in homozygosity many genes of functional significance.

MwoI and SmaI RFLPs polymorphisms of porcine obese gene and their association with carcass and meat characteristics of heavy pigs

The obese gene encodes leptin, a 16-kDa protein involved in the regulation of fat deposition and energy consumption. Backfat is one of the peculiar characteristics of Italian ham, and represents a fundamental quality factor. Therefore, the obese gene can be considered as a candidate marker for determining economically important production traits such as backfat thickness, feed intake, and growth rate in swine. The aim was to investigate the relationship between obese gene polymorphisms and carcass and meat characteristics of pigs reared for ham production. In the present research, the analyses of three new RFLPs are reported. An MwoI polymorphism occurs at nucleotide 1792, within the intron. Pigs heterozygous at this position have heavier thighs with a thinner layer of fat. Two SmaI polymorphisms occur at nucleotides 5018 and 5410 within the 3́ UTR of the obese gene. Animals heterozygous at position 5410 have characteristics suitable for the production of San Daniele ham: lower backfat thickness and heavier thighs with a thinner fat layer, relative to other genotypes

A dot immunobinding assay to detect anti-α-gliadin antibodies in celiac disease

A dot immunobinding assay to detect anti-α-gliadin-specific antibodies in the sera or whole blood of enteropathic patients is described here. The method is based on the adsorption of α-gliadin as a spot onto nitrocellulose sheets. After incubation with the patient sample, the detection of specific antibodies is performed with alkaline phosphatase-conjugated goat anti-human (IgA or IgG) antibodies. Twenty-one celiac serum samples together with 18 enteropathic or disease controls and 44 healthy controls were analyzed. The classical ELISA test and the dot test gave comparable results. The dot test gave reliable result even when whole blood was tested. The method proved to be simple and sensitive.

Differential responses to ultraviolet irradiation of the polar cytoplasm ofDrosophila eggs: II. Response to dose

SummaryThe posterior pole cytoplasm of early embryos ofDrosophila melanogaster was irradiated with increasing doses of U.V. light at a wave length specific for nucleic acids 35 and 75 min after ovoposition, when nuclei were still inside the yolk mass. Probit analysis showed that:Sterility was higher in irradiations performed at 35 min.Mortality was higher in the experiment performed at 75 min.After treatment at 35 min, sterility and mortality were independent phenomena.The numbers of targets involved in germ cell determination were of the order of tens and hundreds at 35 and 75 min respectively.The numbers of targets involved in viability were of several orders of magnitude in both developmental stages.The irradiation, even at very low doses, caused a background effect which was probably due to “old” embryos. The sex ratio was distorted in favour of females at high doses and of males at low doses.

Transcriptome of pig muscle assessed by erial analysis of gene expression (SAGE)

Riassunto Analisi del trascrittoma del muscolo di suino mediante Serial Analysis of Gene Expression (SAGE). Lo scopo dello studio è stato di studiare il profilo trascrizionale di mRNA di muscolo suino adattando il metodo SAGE, sviluppato per l’uomo. Allo scopo sono stati prelevati dei campioni di longissimus dorsi da suini di 9 mesi mediante biopsia e, dopo estrazione dell’mRNA e retrotrascrizione in cDNA, sono stati analizzati mediante SAGE. Il metodo utilizzato ha previsto la costruzione di librerie di tag a 17bp (LongSAGE), una variante dell’originale protocollo che produceva tag da 10bp troppo corti per la corretta localizzazione su un genoma ancora poco caratterizzato. Inoltre sono state apportate alcune modifiche alla metodica originale per aumentare il controllo tra le numerose fasi procedurali. Allo stato, sono stati identificati oltre 750 tags corrispondenti a geni espressi nel muscolo, variabili per quantità da 1 a 43 coppie di mRNA trascritto. I risultati preliminari indicano la possibilità di utilizzare il metodo SAGE per gli studi di associazione con i caratteri fenotipici e per confrontare condizioni fisiologiche diverse nel suino.