So far in this course we have dealt entirely with the evolution of characters that are controlled by simple Mendelian inheritance at a single locus. There are notes on the course website about gametic disequilibrium and how allele frequencies change at two loci simultaneously, but we didn’t discuss them. In every example we’ve considered we’ve imagined that we could understand something about evolution by examining the evolution of a single gene. That’s the domain of classical population genetics. For the next few weeks we’re going to be exploring a field that’s actually older than classical population genetics, although the approach we’ll be taking to it involves the use of population genetic machinery. If you know a little about the history of evolutionary biology, you may know that after the rediscovery of Mendel’s work in 1900 there was a heated debate between the “biometricians” (e.g., Galton and Pearson) and the “Mendelians” (e.g., de Vries, Correns, Bateson, and Morgan). Biometricians asserted that the really important variation in evolution didn’t follow Mendelian rules. Height, weight, skin color, and similar traits seemed to

Human biochemical genetics

Fatty acid-binding proteins (FABPs) are members of the superfamily of lipid-binding proteins (LBP). So far 9 different FABPs, with tissue-specific distribution, have been identified: L (liver), I (intestinal), H (muscle and heart), A (adipocyte), E (epidermal), Il (ileal), B (brain), M (myelin) and T (testis). The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. The structure of all FABPs is similar — the basic motif characterizing these proteins is β-barrel, and a single ligand (e.g. a fatty acid, cholesterol, or retinoid) is bound in its internal water-filled cavity. Despite the wide variance in the protein sequence, the gene structure is identical. The FABP genes consist of 4 exons and 3 introns and a few of them are located in the same chromosomal region. For example,A-FABP, E-FABP andM-FABP create a gene cluster. Because of their physiological properties some FABP genes were tested in order to identify mutations altering lipid metabolism. Furthermore, the porcineA-FABP andH-FABP were studied as candidate genes with major effect on fatness traits.

The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism.

Alterations in the nuclear positioning of chromosomes and specific genes during differentiation and development have suggested strongly the existence of a relationship between non-random organization of the genome and its function. In this study, we have examined the genome organization in interphase nuclei during adipogenesis, using the pig as a model organism. We hypothesized that changes in the gene expression profile and chromatin remodeling which occur during cellular differentiation would elicit repositioning of whole chromosomes, moving specific genes on them to different regions of the nucleus. We established an in vitro adipogenesis differentiation system using mesenchymal stem cells, derived from porcine bone marrow. The nuclear position of seven adipogenesis genes (PPARG, SREBF1, FABP4, CEBPA, CEBPB, CREB, and GATA2), two control genes (SOX9 and MYL1), and six chromosomes carrying these gene loci (SSC4, SSC6, SSC12, SSC13, SSC15, and SSC17) was determined. We found that during adipogenesis, using the in vitro stem cell model system, in contrast to our original hypothesis, the nuclear position of genes involved in adipogenesis was altered radically with the up-regulation of gene expression correlating with these genes becoming more internally located within nuclei. Chromosome territories, containing these genes, were also found to alter their nuclear position during the in vitro adipogenesis model, with the most dramatic repositioning being SSC4 that moved from the nuclear periphery towards the nuclear interior. We found that during in vitro adipogenesis chromosome territories decondensed and the genes were found on loops and projections of chromatin, away from the main body of the chromosomes. From our data, it appears that the temporal repositioning of genes, emanating away from chromosomes, during adipogenesis is correlated with gene activity, supporting models of the involvement of spatial genome repositioning in regulating gene expression and the nuclear interior being an important region of the nucleus for transcription.

The spatial repositioning of adipogenesis genes is correlated with their expression status in a porcine mesenchymal stem cell adipogenesis model system

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h(2)) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 and FABP4), melanocortin receptor type 4 (MC4R), and the FTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of the IGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of the FTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.

Genetics of fat tissue accumulation in pigs: a comparative approach.

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of ≈ 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency A polymorphism (P(nominal) G polymorphism (P(nominal) = 8.0E-05). The third top SNP (P(nominal) = 6.2E-04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P(nominal) < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKKβ/NFκB program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.

Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach.

Summary

Three non-synonymous single nucleotide polymorphisms (T221C, T232A and C233T) were detected in exon 4 of the porcine leptin receptor (LEPR) gene. The T232A substitution could be identified as a (Tsp509I) restriction fragment length polymorphism. The frequency of genotype TT varied in six genetic groups from 0.62 (Duroc) to 0.99 (Polish Large White). Sequencing of exon 4, performed for 30 animals, revealed that only two intragenic haplotypes (TC and AT at nucleotide position 232–233) were present. The phenotypic effect of the Tsp509I polymorphism was tested for the Polish Landrace (n = 241) and a synthetic line 990 (n = 243). There was no statistical evidence for the direct effect of the LEPR polymorphisms on fatness traits. However, in Polish Landrace allele A at position 232 was associated with thicker backfat over shoulder.

Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits.

. Pedigree and molecular data were used to evaluate genetic diversity in the Polish populations of the Polish primitive horse (also known as Polish Konik) and Hucul breeds over the time period of 30 years (1980–2011). Based on genotypes in 12 microsatellite loci (for 3865 Polish primitive horses and 1627 Huculs), as well as on pedigree data derived from over 7000 individuals (both breeds), several indices describing structure of the analysed populations were estimated. For both analysed breeds, we observed an increasing trend of inbreeding since 1980 which seems to be much more stable (oscillating around 10 % in the Polish primitive horse and 5 % in Hucul) since the beginning of 2000s when they were included in conservation programs in Poland. We observed that generally, indices related to genetic diversity are higher in the Hucul breed. Our study indicated that genetic diversity in the Polish primitive horse and Hucul breeds in Poland is still relatively high and conservation programs should be continued to keep it on the "safe" level in the future.

/pdf/genetic-diversity-in-hucul-and-polish-primitive-horse-breeds-x6q9ksovaq.pdf

Genetic diversity in Hucul and Polish primitive horse breeds

Summary

Porcine leptin gene (LEP) and its association with production traits was analysed in the Polish Large White (PLW, n = 135), Polish Landrace (PL, n = 120) and synthetic line 990 (L990, n = 184). Two fragments of exon 3 of LEP were studied with the use of RFLP and SSCP techniques. The frequencies of C allele for the T3469C polymorphic site were 0.11, 0.10 and 0.11 in PLW, PL and L990, respectively. Phenotypic data were collected for the average daily weight gain, the feed conversion ratio, the weight of abdominal fat, backfat thickness (five measurements), intramuscular fat, meat content, loin weight, loin muscle area, ham weight and ham cut weight. The contrasts between TT and TC genotypes at the T3469C polymorphic site were estimated in the unitrait Animal Model with genotype at the RYR1 locus included. The lowest p values occurred for association test between T3469C polymorphism and intramuscular fat content in PLW (0.26 ± 0.14%, p = 0.05) and loin weight in L990 (−0.32 ± 0.13 kg, p = 0.01). We conclude that the detected associations are population-specific and the analysed polymorphism of the LEP gene does not contribute directly to genetic variability of growth and carcass traits in pigs.

Zusammenfassung

Das porcine Leptin-Gen (LEP) und Assoziationen mit Produktionsmerkmalen wurden in der Polnischen Large White (PLW, n = 135), Polnischen Landrasse (PL, n = 120) und der Synthetischen Linie 990 (L990, n = 184) analysiert. Dazu wurden zwei Fragmente von Exon 3 des LEP mittels RFLP- und SSCP-Techniken untersucht. Die Frequenzen des C-Alleles fur die polymorphe Stelle T3469C betrugen 0,11, 0,10 beziehungsweise 0,11 in PWL, PL und L990. Phanotypische Daten wurden gesammelt fur die durchschnittliche Tageszunahme, die Futterverwertung, das Gewicht des abdominalen Fetts, die Ruckenspeckdicke (5 Messpunkte), das intramuskulare Fett, den Fleischanteil, das Lendengewicht, die Lendenmuskelflache, das Schinkengewicht (abgespeckt) und das Gesamtschinkengewicht. Die Gegensatze zwischen dem TT- und dem TC-Genotyp an der polymorphen Stelle T3469C wurden in einem Ein-Merkmal-Tier-Modell unter Einbeziehung des Genotyps am RYR1-Locus geschatzt. Die niedrigsten P-Werte traten beim Assoziationstest zwischen dem T3469C-Polymorphismus und dem intramuskularem Fettgehalt bei PWL (0,26 ± 0,14 %, p = 0,05) sowie dem Lendengewicht bei L990 (−0,32 ± 0,13 kg, p = 0,01) auf. Wir schliesen daraus, dass die nachgewiesenen Assoziationen populationsspezifisch sind und der analysierte Polymorphismus des LEP-Gens nicht direkt zur genetischen Variabilitat der Wachstums- und Schlachtkorpermerkmale in Schweinen beisteuert.

No major effect of the leptin gene polymorphism on porcine production traits

In the present study we show FISH localization of 4 porcine BAC clones harbouring potential candidate genes for fatness traits:DGAT1 (SSC4p15),PPARA (SSC5p15),ADIPOR1 (SSC10p13) andCREB (SSC15q24). Until now theCREB andADIPOR1 genes are considered to be monomorphic,DGAT1 is highly polymorphic, while for thePPARA gene only 1 SNP was identified. Assignment of the studied genes in relation to QTL chromosome regions for meat quality in pig chromosomes SSC4, SSC5, SSC10 and SSC15 is discussed.

Cytogenetic mapping of DGAT1, PPARA, ADIPOR1 and CREB genes in the pig.

We describe for the first time a 245 bp fragment of the porcine leptin gene promoter in the proximity of the transcription start site. Altogether, 720 pigs were screened with the PCR-SSCP technique for polymorphism in this region. Four SNPs, segregating as two haplotypes, have been identified, one of them (C113G) in the putative consensus site for the AP-2 transcription factor. This polymorphism was evenly distributed in the Duroc breed (n=21) and was absent in the Polish Landrace (n=248) and Pietrain breed (n=12). In the Polish Large White (n=191) and synthetic line 990 (n=243), allele G occurred with a very low frequency. The investigation was performed to test if the C113G SNP affects leptin mRNA level in subcutaneous fat and leptin protein concentration in serum. Additionally, the effect of this polymorphism on fatness traits was statistically analyzed. Although there was a trend toward decreased expression in GG animals, the differences were not significant between genotypes. We also found no evidence for an association of the LEP promoter genotype with the analyzed fatness traits.

M. Mackowski

Papers

Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits.

Genetic diversity in Hucul and Polish primitive horse breeds

No major effect of the leptin gene polymorphism on porcine production traits

Cytogenetic mapping of DGAT1, PPARA, ADIPOR1 and CREB genes in the pig.

Polymorphism of the Porcine Leptin Gene Promoter and Analysis of Its Association with Gene Expression and Fatness Traits