Comparative genome analysis with the human genome reveals chicken genes associated with fatness and body weight.

The selection of meat-type chickens (broilers) for rapid growth has been accompanied by excessive fat deposition. In this study, we analysed 53 candidate genes that are associated with obesity and obesity-related traits in humans, for which we found chicken orthologues by BLAST searches. We have identified single nucleotide polymorphisms (SNPs) with significant differences in allele frequencies between broilers and layers in each of the following six candidate genes: adrenergic, beta-2-, receptor, surface (ADRB2); melanocortin 5 receptor (MC5R); leptin receptor (LEPR), McKusick-Kaufman syndrome (MKKS), milk fat globule-EGF factor 8 protein (MFGE8) and adenylate kinase 1 (AK1). To examine associations with fatness and/or body weight, we used birds of extreme phenotypes in F(2) and backcross populations with varying levels of abdominal fat weight per cent (%AFW) and body weight. We then assessed the level of gene expression by real-time PCR. In two genes, ADRB2 and MFGE8, we found significant association with %AFW. The ADRB2 gene was found to have a significantly higher expression in the liver of lean chickens compared with those of the fat individuals. We believe that this approach can be applied for the identification of other quantitative genes.

Biodiversity of 20 chicken breeds assessed by SNPs located in gene regions.

Twenty-five single nucleotide polymorphisms (SNPs) were analyzed in 20 distinct chicken breeds. The SNPs, each located in a different gene and mostly on different chromosomes, were chosen to examine the use of SNPs in or close to genes (g-SNPs), for biodiversity studies. Phylogenetic trees were constructed from these data. When bootstrap values were used as a criterion for the tree repeatability, doubling the number of SNPs from 12 to 25 improved tree repeatability more than doubling the number of individuals per population, from five to ten. Clustering results of these 20 populations, based on the software STRUCTURE, are in agreement with those previously obtained from the analysis of microsatellites. When the number of clusters was similar to the number of populations, affiliation of birds to their original populations was correct (>95%) only when at least the 22 most polymorphic SNP loci (out of 25) were included. When ten populations were clustered into five groups based on STRUCTURE, we used membership coefficient (Q) of the major cluster at each population as an indicator for clustering success level. This value was used to compare between three marker types; microsatellites, SNPs in or close to genes (g-SNPs) and SNPs in random fragments (r-SNPs). In this comparison, the same individuals were used (five to ten birds per population) and the same number of loci (14) used for each of the marker types. The average membership coefficients (Q) of the major cluster for microsatellites, g-SNPs and r-SNPs were 0.85, 0.7, and 0.64, respectively. Analysis based on microsatellites resulted in significantly higher clustering success due to their multi-allelic nature. Nevertheless, SNPs have obvious advantages, and are an efficient and cost-effective genetic tool, providing broader genome coverage and reliable estimates of genetic relatedness.

Characterization and Inheritance of a New Source of Resistance to Fusarium oxysporum f. sp. melonis Race 1.2 in Cucumis melo.

Four physiological races (0, 1, 2, and 1.2) of Fusarium oxysporum f. sp. melonis, causal agent of Fusarium wilt in melons, have been described. Whereas resistance against each of the races 0, 1, and 2 is encoded by a single dominant R-gene, resistance against the fourth race, FOM 1.2, is polygenically inherited and was recently characterized in the cultivar Isabelle. In the present study, we report on an independently derived source of resistance to race 1.2, the Israeli breeding line BIZ. The disease response of BIZ seedlings was compared with two susceptible genotypes, Line 33 and PI 414723, and the partially resistant genotype, Isabelle, at increasing concentrations of inoculum. BIZ exhibited near-complete resistance to race 1.2, even at inoculum levels of 106 spores per ml and root wounding, indicating that such resistance is stronger than that in Isabelle. Although in previous studies the F1 hybrids between BIZ and the sensitive lines displayed full resistance in the field, in the present study they were susceptible under artificial inoculation conditions that involve high inoculum concentrations of 106 spores per ml and root wounding; under intermediate inoculum levels (105 and 5 × 105 spores per ml) they were partially resistant. Segregation of the resistance response in the F2 and back-cross-1 progeny from a cross between BIZ and PI 414723 supported a model in which two complementary, recessive genes are required to obtain full resistance. While the fungus was shown to colonize root tissue of both resistant and susceptible lines, colonization was markedly lower in the resistant plants, and the fungus was nearly absent from upper stem segments.

Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance.

Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed.

Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber.

Ethylene has been implicated as a sex-determining hormone in cucumber: its exogenous application increases femaleness, and gynoecious genotypes were reported to produce more ethylene. In this study, three full-length ACC oxidase cDNAs were isolated from cucumber floral buds. RFLP analysis of a population that segregates for the F(femaleness) locus indicated that CS-ACO2 is linked to F at a distance of 8.7 cM. Expression of two of the genes, CS-ACO2 and CS-ACO3, was monitored in flowers, shoot tips and leaves of different sex genotypes. In situ mRNA hybridization indicated different patternsof tissue- and stage-specific expression of CS-ACO2 and CS-ACO3 in developing flowers. CS-ACO3 expression in mid-stage female flowers was localized to the nectaries, pistil and in the arrested staminoids, whereas CS-ACO2 transcript levels accumulated later and were found in placental tissue, ovary and staminoids. In male flowers, petals and nectaries expressed both genes, whereas ACO2 expression was strong in pollen of mature flowers. In young buds, strong expression was observed along developing vascular bundles. Four sex genotypes were compared for CS-ACO2 and CS-ACO3 expression in the shoot apex and young leaf. FF genotypes had higher transcript levels in leaves but lower levels in the shoot apex and in young buds, as compared to ff genotypes; the shoot-tip pattern is, therefore, inversely correlated with femaleness, and the possibility of a feedback inhibition mechanism underlying such correlation is discussed. The two CS-ACO genes studied displayed a differential response to ethrel treatment in different organs and sex genotypes, further demonstrating the complexity of the mechanisms controlling ethylene production during cucumber floral development.

Expression of multiple AGAMOUS-like genes in male and female flowers of cucumber (Cucumis sativus L.).

Members of the MADS-box gene family control reproductive development in higher plants. In cucumber, floral development exhibits several interesting features related to a genetically determined sex-expression mechanism, that affects the differentiation of male and female flowers. In this study, three cDNA homologues of the homeotic gene AGAMOUS have been cloned from early-stage floral buds of Cucumis sativus and fully sequenced. Their expression was studied by Northern analysis using two contrasting sex genotypes, an androecious line and a gynoecious one. The three genes are expressed at low levels at earlier bud stages, the levels rising as the bud matures. Two of the clones, CAG1 and CAG3, are expressed in the third and fourth whorl of mature flowers, while CAG2 is restricted to the carpel; none is expressed in leaves. The transcript levels do not appear to be modulated by gibberellin or ethephon, two treatments that alter sex expression in cucumber. While MADS-box genes probably play an essential role in cucumber floral development, as they do in other plants, our findings may imply that the pathway leading to reproductive organ arrest in cucumber unisexual buds acts independently of MADS-box gene expression.

Do Selye's mammalian "GAS" concept and "co-stress" response exist in plants?

Converging data indicate the possible existence of a general adaptation syndrome (GAS) in which different types of stress evoke identical coping mechanisms. In Selyean terms, this implies a "co-stress" response whereby one type of stress resistance may impart co-resistance to others. Common coping denominators may be physiological or morphological. The former include oxy-free radical scavenging, osmoregulation, ABA, jasmonates, chaperones, HSPs, and phytochelatins. Morphological GAS adaptations include leaf pubescence, movements and stance, and rooting characteristics. The feasibility, with certain reservations, of the GAS hypothesis is discussed here.

Enhanced oxidative-stress defense in transgenic potato expressing tomato Cu,Zn superoxide dismutases.

The two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10(-5) M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.

The tomato Cu,Zn superoxide dismutase genes are developmentally regulated and respond to light and stress.

The expression of the two Cu,Zn superoxide dismutase (SOD) genes of tomato was followed in different organs and plant developmental stages at the transcript and enzymatic activity levels. The cDNA clones used as probes code for the chloroplast Cu,Zn SOD (clone T1) and the cytosolic Cu,Zn SOD (clone P31). The two genes were found to display distinct expression patterns. While the T1 transcript was rare or absent from roots, stems and ripening fruits, the P31 transcript was very abundant in these organs. Shoot tips, flower buds, seedlings and young leaves contained high levels of the two mRNAs. During leaf expansion, the levels of both transcripts diminish markedly. Despite the diminished presence of transcripts, SOD activity levels of the corresponding cytosolic and chloroplast isozymes accumulated and were sustained throughout leaf expansion. In non-photosynthetic organs, the SOD-3 (cytosolic) isozyme contained most of the activity, while in the expanded leaf the SOD-1 (chloroplast) isozyme was more abundant. Light-regulated accumulation of both the P31 transcript (1.7-fold) and the T1 transcript (3-fold) was observed upon light exposure of etiolated seedlings. However, only SOD-1 activity was observed to increase, after a lag of a few hours. The levels of both transcripts increased in response to paraquat and mechanical wounding. The level of the cytosolic transcript and the respective isozyme activity increased dramatically during prolonged drought stress while the chloroplast transcript remained unaffected. The expression of both genes was enhanced by spraying tomato plants with ethephon--a compound that releases ethylene. Our data show that the expression of Cu,Zn SOD genes in tomato is modulated in response to a variety of factors and suggest the importance of oxyradical toxicity as well as the role of SOD in the defence mechanism of plants exposed to stress.

Genetic mapping of tomato cDNA clones encoding the chloroplastic and the cytosolic isozymes of superoxide dismutase.

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences.

A cDNA library was derived from the poly(A)(+) RNA of young tomato leaves. The library was cloned in a λgt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5' end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)(+) RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.

The Cucumis plastome: physical map, intrageneric variation and phylogenetic relationships.

A restriction map of the Cucumis melo L. (melon) plastome was constructed by using several mapping approaches: single and double digestions of the chloroplast DNA (chlDNA) with endonucleases (XhoI, SmaI, SacI and PvuII) and hybridization to heterologous chlDNA probes and to isolated melon chlDNA fragments. Four plastome-coded genes were located using heterologous probes. The overall organization and gene position of the melon plastome was found to be similar to that of tobacco and other angiosperm species. Restriction patterns based on digestion of the chlDNA with nine endonucleases were obtained in over 20 wild species and cultivated varieties of Cucumis. These led to mutational analysis of the restiction sites yielding the most parsimonious phylogenetic tree of the Cucumis plastome. Most African species from a compact group ("Anguria group") which is distant from the melon, the cucumber and a few other species (C. sagittatus, C. metuliferus and C. humifructus). All of these are also far apart from each other. The distribution of polymorphic restriction sites along the Cucumis plastome is described and conservative regions as well as "hot spots" are suggested.

Phylogeny of Cucumis based on isozyme variability and its comparison with plastome phylogeny.

An electrophoretic comparison of 29 nuclear-coded enzymes was carried out for 21 Cucumis species, and a phylogeny based on pairwise measurements of the respective genetic distances was computed. This phylogeny was compared to the one based on chlDNA cariation (Perl-Treves and Galun 1985). The two phylogenies were found to share the main dendrogram features; they also agree well with most taxonomic data available on Cucumis. Accordingly, most of the African Cucumis species form a close group ("Anguria group" - "Group A"), which is distant from the melon (C. melo), and from a few other distinct species, all of which are far apart from each other. The cucumber (C. sativus) is the most distant species within the genus. Some specific taxonomic implications as well as some general evolutionary problems related to such a parallel investigation of the nuclear genome and the plastome are evaluated.