Massoud Malek

A molecular genome scan analysis to identify chromosomal regions influencing economic traits in the pig. II. Meat and muscle composition

Genome scans can be employed to identify chromosomal regions and eventually genes (quantitative trait loci or QTL) that control quantitative traits of economic importance. A three-generation resource family was developed by using two Berkshire grand sires and nine Yorkshire grand dams to detect QTL for growth and body composition traits in pigs. A total of 525 F2 progeny were produced from 65 matings. All F2 animals were phenotyped for birth weight, 16-day weight, growth rate, carcass weight, carcass length, back fat thickness, and loin eye area. Animals were genotyped for 125 microsatellite markers covering the genome. Least squares regression interval mapping was used for QTL detection. All carcass traits were adjusted for live weight at slaughter. A total of 16 significant QTL, as determined by a permutation test, were detected at the 5% chromosome-wise level for growth traits on Chromosomes (Chrs) 1, 2, 3, 4, 6, 7, 8, 9, 11, 13, 14, and X, of which two were significant at the 5% genome-wise level and two at the 1% genome-wise level (on Chrs 1, 2, and 4). For composition traits, 20 QTL were significant at the 5% chromosome-wise level (on Chrs 1, 4, 5, 6, 7, 12, 13, 14, 18), of which one was significant at the 5% genome-wise level and three were significant at the 1% genome-wise level (on Chrs 1, 5, and 7). For several QTL the favorable allele originated from the breed with the lower trait mean.

Association of INOS, TRAIL, TGF-β2, TGF-β3, and IgL genes with response to Salmonella enteritidis in poultry

Several candidate genes were selected, based on their critical roles in the hosts response to intracellular bacteria, to study the genetic control of the chicken response to Salmonella enteritidis (SE). The candidate genes were: inducible nitric oxide synthase (INOS), tumor necrosis factor related apoptosis inducing ligand (TRAIL), transforming growth factor β2 (TGF-β2), transforming growth factor β3 (TGF-β3), and immunoglobulin G light chain (IgL). Responses to pathogenic SE colonization or to SE vaccination were measured in the Iowa Salmonella response resource population (ISRRP). Outbred broiler sires and three diverse, highly inbred dam lines produced 508 F1 progeny, which were evaluated as young chicks for either bacterial load isolated from spleen or cecum contents after pathogenic SE inoculation, or the circulating antibody level after SE vaccination. Fragments of each gene were sequenced from the founder lines of the resource population to identify genomic sequence variation. Single nucleotide polymorphisms (SNP) were identified, then PCR-RFLP techniques were developed to genotype the F1 resource population. Linear mixed models were used for statistical analyses. Because the inbred dam lines always contributed one copy of the same allele, the heterozygous sire allele effects could be assessed in the F1 generation. Association analyses revealed significant effects of the sire allele of TRAIL-Sty I on the spleen (P < 0.07) and cecum (P < 0.0002) SE bacterial load. Significant effects (P < 0.04) were found on the cecum bacterial load for TGF-β3-Bsr I. Varied and moderate association was found for SE vaccine antibody response for all genes. This is the first reported study on the association of SNP in INOS, TRAIL, TGF-β2, TGF-β3, and IgL with the chicken response to SE. Identification of candidate genes to improve the immune response may be useful for marker-assisted selection to enhance disease resistance.

New insights into porcine-human synteny conservation

Abstract. Eleven genes were mapped to the porcine genome with the aim of improving the human-porcine comparative gene map. Five of these genes were from regions of the human genome painted by porcine chromosomal probes; of these, two mapped to chromosomes not expected from the painting results. Among the six genes from human regions not painted by porcine chromosomal probes, three genes did not map where expected by the principle of parsimony. Several of the gene assignments indicate the existence of small regions of conserved synteny not detected by heterologous chromosome painting, especially in telomeric regions. We have also detected new rearrangements in gene order within the regions of correspondence between human Chromosome (HSA) 15 and porcine Chromosome (SSC) 1 as well as between HSA4 and SSC8.

Analysis of the mouse high-growth region in pigs

In the mouse, homozygous animals for the high growth mutation show a 30-50% increase in growth without becoming obese. This region is homologous to the distal part of pig chromosome 5 (SSC5). A previous genome scan detected several quantitative trait loci (QTL) in this region for body composition and meat quality using a three generation Berkshire x Yorkshire resource family. In this study, the effects on swine growth, fat and meat quality traits of three genes previously identified within the mouse high growth region were analysed. The genes studied were CASP2 and RIPKI domain containing adaptor with death domain (CRADD), suppressor of cytokine signalling 2 (SOCS2) and plexinC1 (PLXNC1). In addition, the influence of two other genes located very close to this region, namely the plasma membrane calcium-transporting ATPase 1 (ATP2B1) and dual specificity phosphatase 6 (DUSP6) genes, was also investigated. Single nucleotide polymorphisms were identified and used to map these genes to the QTL region on SSC5. Results indicate significant associations between these genes and several phenotypic traits, including fat deposition and growth in pigs. The present study suggests associations of these genes with swine fat and growth related traits, but further studies are needed in order to clearly identify the genes involved in the regulation of the QTL located on SSC5.

Linkage and physical mapping of the porcine prepro-orexin gene.

It is well established that the brain, and specifically the hypothalamus, is a major site where various central nervous system signals are integrated to affect the expression of complex hormonal and neuroendocrine functions, such as food intake and energy homeostasis. Orexin-A and B (also called prepro-orexin), are hypothalamic peptides, encoded by a single mRNA transcript, which are derived from the same precursor. These peptides bind and activate two closely related orphan G protein-coupled receptors (Sakurai et al. 1998). Prepro-orexin has been proposed to have a physiological role in the regulation of food intake in the mouse, rat, pig, and human (Mondal et al. 1999). Edwards et al. (1999) studied the effect of orexin-A and B on feed intake in the rat. Their data indicated that orexin-A consistently stimulated food intake, but orexin-B only occasionally stimulated food intake in the rat. Dyer et al. (1999) showed that cumulative feed intake increased by administration of orexin-B and total feed intake at 24 h was improved by 18% in orexin-treated pigs. Given the role of preproorexin, the present study was designed to characterize porcine prepro-orexin gene structure and chromosomal localization as a prelude to future candidate gene analysis for feed intake traits. In order to sequence the gene, a PCR primer pair (forward 58-AGC GGC AGA CAC CATGAA-38; reverse 58-CGA GGT CAG CCC CCC AGA-38) was developed for porcine preproorexin on the basis of the published cDNA sequence (GenBank accession no. AF075241.1) from Dyer et al. (1999). The PCR reaction was performed with 12.5 ng of porcine genomic DNA, 1× PCR buffer, 1.25 mM MgCl2, 3.2 mM dNTP, 3 pmol of each primer, 5% dimethyl sulfoxide (DMSO), and 0.35 U Taq DNA polymerase (Promega, Madison, Wis) in a 10ml final volume. The PCR profile included 40 cycles with denaturation at 95°–94°C (95°C for 2 min the first cycle, 95°C for 1 min the second cycle, and 94°C for 1 min the remaining cycles), annealing at 62°C for 40 s, and extension at 72°C for 2 min followed by 30 s at 4°C. The PCR product was detected with agarose (Metaphor 2%, FMC Bioproducts, Me., USA) gel electrophoresis, and ethidium bromide staining. Five DNA pools (several animals each) representing five breeds (Meishan, Duroc, Hampshire, Landrace, and Yorkshire) were sequenced, and the sequences were examined for polymorphic sites. If found, such sites were used, when possible, to make PCR-RFLPs for genotyping. The PCR product was digested with the restriction enzyme and incubated overnight at 37°C. The digested fragments were separated by 2% Metaphor gel electrophoresis. For linkage mapping, all members of the PiGMaP families were then genotyped (Archibald et al. 1995), and two-point linkage analysis was performed with the CRI-MAP program (Green et al. 1990). For physical gene mapping, a pig/rodent somatic cell hybrid panel developed by Yerle et al. (1996) was used to physically map this gene in the pig. Primers (forward 5 8-ACG CTG CTG CTT CTG CTA CT -38; and reverse 5 8-AGC GGG CAT CCT GAC CAT -38) were used that produced a 251-bp PCR product that was then used for gene mapping. Amplified products were analyzed by 2% Metaphor gel electrophoresis. The results for sequencing and mapping are as follows. The 1247-bp PCR product was confirmed as prepro-orexin, since it had 99% identity to the previously published porcine orexin sequence in a 389-bp overlap. Our results revealed that the porcine preproorexin gene consisted of two exons and one intron distributed over 1247 bp (Fig. 1). Further sequence analysis revealed three single nucleotide polymorphisms (SNPs), T/C, A/G, and T/C substitutions at position 62 bp, 426 bp, and 974 bp, respectively in our sequence (GenBank AF169352) (Table 1). The first two SNPs occurred in the intron, and the third SNP occurred in exon 2, but did not change the predicted amino acid. The PCR-RFLPs were then redesigned for large-scale testing. The first polymorphic site was detected withBstUI and a 704-bp fragment amplified with the primers 58-AGC GGC AGA CAC CAT GAA TC -38 (forward) and 58-CAG AGG GCA TTG AGC AAA GGC T -38 (reverse). The second polymorphic site was detected with AciI, and a fragment was amplified from bases 361–686 (325 bp) with the primers 58-GTA GGT GGA CAA AGC AGC CTG G -38 (forward) and 58CAG AGG GCA TTG AGC AAA GGC T -38 (reverse). This PCR-RFLP resulting from theAciI digestion of the PCR product produced an undigested PCR product of 325 bp (allele 1) or 65 bp and 260 bp (allele 2) fragments. The third polymorphic site was detected withNlaIII and a fragment amplified from the exon 2 coding region with the primers 5 8-ACG CTG CTG CTT CTG CTA CT -38 (forward) and 58-AGC GGG CAT CCT GAC CAT -38 (reverse). TheNlaIII digestion of the PCR product produced a 251-bp fragment (allele 1) and 137 bp, and 114 bp (allele 2) polymorphic fragments (Fig. 2a). This site was useful for linkage mapping. Allele frequencies for the porcine prepro-orexin NlaIII PCRRFLP were determined in 22 grandparental animals from the European PiGMaP families and in 19 unrelated animals from the Iowa State University swine breeding farm. Allele 1 was observed with a frequency of 1.0 in Meishan (n 4 9), 0.4 in Hampshire (n 4 10), 0.17 in Large White (n4 20), and 0.0 in Wild Boar (n4 2). While these data are interesting, more samples per breed need to be collected before any conclusions can be drawn. The prepro-orexin gene was physically mapped to SSC12 p13p11 with complete concordance with the somatic cell hybrid panel (Yerle et al. 1996). TheNlaIII polymorphic site was genotyped in the PiGMaP reference families (Archibald et al. 1995). Linkage mapping with the PiGMaP families confirmed the physical mapping location of prepro-orexin. The results of two-point analysis showed that prepro-orexin gene was significantly linked to three markers on porcine Chr 12 (SSC12). The linked markers were PRKAR1A(protein kinase, cAMP-dependent, regulatory, type I, The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number AF169352.