Radiation hybrid mapping of two members of the Toll-like receptor gene family in pigs.
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Animals↗
Biomedical subjects
E Giuffra
Publications and source records attributed to E Giuffra.
Identification of quantitative trait loci for production traits in commercial pig populations.
The aim of this study was to investigate methods for detecting QTL in outbred commercial pig populations. Several QTL for back fat and growth rate, previously detected in experimental resource populations, were examined for segregation in 10 different populations. Two hundred trait-by-population-by-chromosome tests were performed, resulting in 20 tests being significant at the 5% level. In addition, 53 QTL tests for 11 meat quality traits were declared significant, using a subset of the populations. These results show that a considerable amount of phenotypic variance observed in these populations can be explained by major alleles segregating at several of the loci described. Thus, despite a relatively strong selection pressure for growth and back fat traits in these populations, these alleles have not yet reached fixation. The approaches used here demonstrate that it is possible to verify segregation of QTL in commercial populations by limited genotyping of a selection of informative animals. Such verified QTL may be directly exploited in marker-assisted selection (MAS) programs in commercial populations and their molecular basis may be revealed by positional candidate cloning.
Adipose Tissue↗
Full pedigree quantitative trait locus analysis in commercial pigs using variance components.
In commercial livestock populations, QTL detection methods often use existing half-sib family structures and ignore additional relationships within and between families. We reanalyzed the data from a large QTL confirmation experiment with 10 pig lines and 10 chromosome regions using identity-by-descent (IBD) scores and variance component analyses. The IBD scores were obtained using a Monte Carlo Markov Chain method, as implemented in the LOKI software, and were used to model a putative QTL in a mixed animal model. The analyses revealed 61 QTL at a nominal 5% level (out of 650 tests). Twenty-seven QTL mapped to areas where QTL have been reported, and eight of these exceeded the threshold to claim confirmed linkage (P < 0.01). Forty-two of the putative QTL were detected previously using half-sib analyses, whereas 46 QTL previously identified by half-sib analyses could not be confirmed using the variance component approach. Some of the differences could be traced back to the underlying assumptions between the two methods. Using a deterministic approach to estimate IBD scores on a subset of the data gave very similar results to LOKI. We have demonstrated the feasibility of applying variance component QTL analysis to a large amount of data, equivalent to a genome scan. In many situations, the deterministic IBD approach offers a fast alternative to LOKI.
Analysis of Variance↗
Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.
Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.
Carotenoids↗
Mapping loci causing susceptibility to anal atresia in pigs, using a resource pedigree.
BACKGROUND/PURPOSE: Very little information on the genetic background for anal atresia (anorectal malformations; AA) in humans has been described. A strikingly similar natural anomaly occurs in piglets. The authors have used this as an animal model for various research purposes. The affected piglets were treated surgically soon after birth, raised, and used for breeding. The authors have generated a resource pedigree segregating for this naturally occurring nonsyndromal AA and describe here the first attempt to map susceptibility loci by marker analysis. METHODS: A pig pedigree with a high incidence of AA has been established by selective breeding using 3 probands from the Landrace and Large White breeds. It has been maintained by intrafamilial crossing for more than 15 years. A backcross pedigree has now been generated by mating 4 AA females to an unaffected male from the Chinese Meishan breed. F(1) animals were both intercrossed and backcrossed to affected AA animals. A genome scan was carried out using the F(0), F(1), and affected backcross progeny. Ninety-two microsatellite loci were analyzed using fluorescently labelled primers and an ABI377 sequencer. Linkage analysis was done with the CRI-MAP 2.4 software. RESULTS: Crossing affected parents increased the incidence of abnormalities from 30% to 61.9%. All 39 F(1) pigs were unaffected. In the F(1) intercross, only 3 of 205 (1.5%) were affected, whereas 42 of 523 (8.0%) backcross progeny were affected. The marked difference in the incidence of affected progeny in the F(1) intercross and in the backcross indicates the presence of multiple genes causing AA. The genome scan showed suggestive evidence for the presence of a susceptibility locus on pig chromosome 15 (lod score 2.7 for a pig microsatellite marker SW2072). CONCLUSIONS: The results clearly show that AA has a oligogenic or polygenic background. The genome scan showed one suggestive locus causing AA on pig chromosome 15. The long-term goal is to identify causative genes for this malformation by comparative positional candidate cloning. This study provides, for the first time, linkage mapping of nonsyndromal anorectal malformations with a polygenic inheritance.
Animals↗
Molecular phylogenetics relationship between six Chinese native pig breeds and three Swedish pig breeds from mitochondrial DNA.
Mitochondrial DNA genetic polymorphisms were used to evaluate the relationship between 6 Chinese indigenous pig breeds and 3 Swedish domestic pig breeds. A total of 440 bp of the control region and 798 bp of cytochrome b (cyt b) gene of mtDNA were determined from 140 pigs of 9 different breeds. The results of phylogenetics analysis showed that 6 Chinese native pig breeds originated from the Asian wild boar. The pairwise nucleotide sequences divergence suggested that they might occur about 413,000-875,000 years before present (YBP) between Chinese native pig breeds and European wild boar, and approximately 7,500-15,600 YBP between Chinese native pig breeds and Asian wild boar. Three Swedish pig breeds are present both European clade and Asian clade, which is due to the documented introgression of Chinese pig breeds into European domestic pig breeds 2 thousand years ago or during early 18th century.
Animals↗
The origin of the domestic pig: independent domestication and subsequent introgression.
The domestic pig originates from the Eurasian wild boar (Sus scrofa). We have sequenced mitochondrial DNA and nuclear genes from wild and domestic pigs from Asia and Europe. Clear evidence was obtained for domestication to have occurred independently from wild boar subspecies in Europe and Asia. The time since divergence of the ancestral forms was estimated at approximately 500,000 years, well before domestication approximately 9,000 years ago. Historical records indicate that Asian pigs were introduced into Europe during the 18th and early 19th centuries. We found molecular evidence for this introgression and the data indicated a hybrid origin of some major "European" pig breeds. The study is an advance in pig genetics and has important implications for the maintenance and utilization of genetic diversity in this livestock species.
Animals↗
The Belt mutation in pigs is an allele at the Dominant white (I/KIT) locus.
A white belt is a common coat color phenotype in pigs and is determined by a dominant allele (Be). Here we present the result of a genome scan performed using a Hampshire (Belt)/Pietrain (non-Belt) backcross segregating for the white belt trait. We demonstrate that Belt maps to the centromeric region of pig Chromosome (Chr) 8 harboring the Dominant white (I/KIT) locus. Complete cosegregation between Belt and a single nucleotide polymorphism in the KIT gene was observed. Another potential candidate gene, the endothelin receptor type A gene (EDNRA), was excluded as it was assigned to a different region (SSC8q21) by FISH analysis. We argue that Belt is a regulatory KIT mutation on the basis of comparative data on mouse KIT mutants and our previous sequence analysis of the KIT coding sequence from a Hampshire pig. Quantitative PCR analysis revealed that Belt is not associated with a KIT duplication, as is the case for the Patch and Dominant white alleles. Thus, Belt is a fourth allele at the Dominant white locus, and we suggest that it is denoted I(Be).
Alleles↗
A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus.
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Animals↗
Analysis of some optical properties of a native and reconstituted photosystem II antenna complex, CP29: pigment binding sites can be occupied by chlorophyll a or chlorophyll b and determine spectral forms.
The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.
Apoproteins↗
A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29.
Energy-dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein-modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light-harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild-type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.
Amino Acid Sequence↗
Reconstitution and pigment-binding properties of recombinant CP29.
The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.
Apoproteins↗
Mitochondrial control region and protein coding genes sequence variation among phenotypic forms of brown trout Salmo trutta from northern Italy.
The Pô River basin of northern Italy is the home of distinctive and endemic morphological forms of brown trout Salmo trutta. We used PCR-direct sequencing and RFLP techniques to study variation in the mitochondrial control region of 225 trout in order to assess genetic relatedness among 18 populations from that region. The distribution analysis of these genotypes among north Italian populations confirmed the phylogenetic differentiation of marbled trout Salmo trutta marmoratus populations and the postglacial origin of S. t. carpio. Extensive genetic heterogeneity was observed among morphologically identical S. t. fario populations. Introgression with domestic strains of Atlantic basin origin was detected in all forms. In order to assess the phylogenetic congruence detected in coding and noncoding regions of the mitochondrial genome, we also analysed sequence variation in segments of the cytochrome b and ATPase subunit VI genes among representatives of all variants detected in the analysis of the control region. Variation in protein coding genes was only slightly less than that observed in the control region of the same individuals, both in terms of number of variants detected and of pairwise sequence divergence estimates among variants. Phylogenetic analysis based on protein coding genes sequences identified the same phylogenetic groupings defined by the control region analysis and also allowed a partial resolution of their phyletic relationships that was previously unresolved. However, coding and noncoding segments differed substantially in the transition-transversion ratio (17:0 in coding segments vs. 17:6 in control region segments).
Animals↗
Genetic diversity of eleven European pig breeds.
A set of eleven pig breeds originating from six European countries, and including a small sample of wild pigs, was chosen for this study of genetic diversity. Diversity was evaluated on the basis of 18 microsatellite markers typed over a total of 483 DNA samples collected. Average breed heterozygosity varied from 0.35 to 0.60. Genotypic frequencies generally agreed with Hardy-Weinberg expectations, apart from the German Landrace and Schwäbisch-Hällisches breeds, which showed significantly reduced heterozygosity. Breed differentiation was significant as shown by the high among-breed fixation index (overall F(ST)= 0.27), and confirmed by the clustering based on the genetic distances between individuals, which grouped essentially all individuals in 11 clusters corresponding to the 11 breeds. The genetic distances between breeds were first used to construct phylogenetic trees. The trees indicated that a genetic drift model might explain the divergence of the two German breeds, but no reliable phylogeny could be inferred among the remaining breeds. The same distances were also used to measure the global diversity of the set of breeds considered, and to evaluate the marginal loss of diversity attached to each breed. In that respect, the French Basque breed appeared to be the most "unique" in the set considered. This study, which remains to be extended to a larger set of European breeds, indicates that using genetic distances between breeds of farm animals in a classical taxonomic approach may not give clear resolution, but points to their usefulness in a prospective evaluation of diversity.
Journal Article↗