INTRODUCTION

In the past five years advances in molecular genetics and the human genome project have contributed to considerable progress in the area of pig gene mapping. At present the combined number of genes and markers on the pig genetic linkage maps is approximately 1800, of which about 250 are genes (Archibald et al. 1994 and personal communication; Marklund et al. 1996a; Rohrer et al. 1996). In addition, the physical genetic map is over 500 genes and markers (Yerle, personal communication).

The coverage on these maps is now sufficient to allow researchers to conduct quantitative trait loci (QTL) linkage analyses. QTL linkage analyses involve using a genomic scan. Often F2 or backcross families are used and genotypes are obtained for many (>75) markers evenly spaced across the genome. Several such experiments are underway and beginning to produce interesting and useful results. Candidate gene and comparative mapping approaches have also been successful in identifying major genes affecting several traits. Candidate gene analysis is when we choose a gene based on the physiology of the trait. The candidate gene is assumed to affect trait performance. Comparative gene analysis allows us to find "positional candidate genes" in the regions associated with possible QTL. To date, several major genes have been found with the candidate gene approach. The purpose of this invited paper is to review the status of QTL and candidate gene analyses in the pig and to forecast future developments.

RESULTS

Results will be presented for both QTL and candidate gene analyses in terms of growth and performance, meat quality, disease resistance and reproduction traits. Many of the results are relatively new and further analyses are needed to confirm them.

Growth and performance

The first successful large QTL analysis was conducted using a Wild Boar and Large White three generation family and revealed major QTL accounting for 20% of the phenotypic variance for average backfat and abdominal fat on chromosome 4 (Andersson et al. 1994). A QTL for growth was also found on chromosome 13 accounting for 7-12% of the phenotypic variation. Additional confirmation of the chromosome 4 results have been seen in subsequent generations (Marklund et al. 1996b) and by other experiments using Chinese pig crosses (Wang et al. 1997a; Milan et al., 1998; Moser and Cepica, personal communication). Using a candidate gene analysis PIT1 was identified to be associated with backfat and birth weight (Yu et al. 1995) and it maps in the center of the chromosome 13 QTL found by Andersson et al. (1994). Chromosome 13 QTL have also been identified for 21 and 35 day weights in Wild Boar by Pietran crosses in the region of PIT1 (Moser, personal communication). Efforts are underway to resolve whether the chromosome effect is due to PIT1 or to another gene in the region (Yu et al. 1997).

The pig major histocompatibility complex (MHC) spans the centromere of chromosome 7. Associations between MHC haplotypes and several traits have been reported over the years. These have been confirmed, in part, using MHC class I DNA probes (Jung et al. 1989). More recently, QTL scans have identified QTL for growth and backfat traits in Chinese crosses on chromosome 7 (Rothschild et al. 1995; Bidanel et al. 1996; Chevalet et al. 1996; Moser et al. 1997; Wang et al. 1997b). The backfat and birth weight QTL centered near the region of TNFA and S0102. A candidate gene analysis involving AMPEPN, which maps to the same region, also revealed an association with growth rate (Nielsen et al. 1996). The overall results to date suggest that at least one growth and backfat QTL exists in this region. Other results have included a growth trait QTL on chromosome 6, but it seems to be associated with the known effect caused by the RYR1 gene causing malignant hyperthermia (Geldermann et al. 1996) or other unknown genes in the immediate vicinity of RYR1. Some additional associations have been reported for chromosome 3 (Casas-Carrillo et al. 1997b), chromosomes 6 and 8 (Wilkie et al. 1996) and chromosome 14 in a limited QTL scan (Bidanel et al. 1996).

A combination of QTL scans and candidate gene analyses using GH have been performed for chromosome 12 for several performance measures (Nielsen et al. 1995; Casas-Carrillo et al. 1997a; Knorr et al. 1997) but results were not in general agreement. Analysis of the chromosome 5 region near IGF-1 revealed significant effects on average daily gain (Casas-Carrillo et al. 1997a). Additionally, Gerbens et al. (1997) and Tepas et al. (1996) reported associations of the heart fatty acid-binding protein (H-FABP) and the myogenin genes with average daily gain. Other candidate genes including leptin, CCK and CCKAR (Clutter et al. 1996) and leptin receptor (Vincent et al. 1997) have been mapped and may prove to be associated with growth, fatness and appetite traits.

Meat quality

It has been known for some time that pale soft and exudative (PSE) pork is caused by the RYR1 gene on chromosome 6. This has been well demonstrated in a QTL scan for several meat quality traits related to PSE in an F2 population originating from a Pietran background (Geldermann et al. 1996). Focus has also centered on a gene originating in Hampshires called the RN gene which is associated with lower pH and increased gycogen content in the meat. The RN gene has now been mapped to chromosome 15 (Milan et al. 1996; Mariani et al. 1996a; Reinsch et al. 1997) and surrounded by flanking markers. The actual gene has alluded researchers, but a recent report suggests that a DNA test for the RN gene has been found by PIC researchers (De Vries et al. 1997). Andersson and colleagues (Andersson-Eklund et al. 1996) have conducted one of the most complete QTL scans for meat quality using 234 markers on 191 F2 animals. QTL for several meat quality traits (pH, water holding capacity and pigmentation) were found to be on chromosome 2 and chromosome 12. Rothschild and colleagues report some association of meat color and firmness scores with regions on chromosomes 4 and 7 (Rothschild et al. 1995, Wang et al. 1997a; Wang et al. 1997b). Additional associations with meat quality traits have been reported for chromosome 7 (Moser et al. 1997) and for number of muscle fibers on chromosome 3 (Milan et al., 1998). Two other interesting associations related to meat quality have been noted for chromosome 7. The activity of Malic enzyme, a lipogenic enzyme in muscle has been shown to be associated with the SLA complex on chromosome 7 (Renard et al. 1996). Furthermore, a directed QTL scan revealed that there was a major QTL for androstenone level which is associated with boar taint in the region of the SLA complex (Bidanel et al. 1996; Milan et al., 1998).

Among candidate genes investigated for muscle quality is the H-FABP gene which may be associated with intramuscular fat (Gerbens et al. 1997). Other genes mapped and investigated include myogenin (Tepas et al. 1996) and calpastatin (Ernst et al. 1997). Coat color, though not directly associated with meat quality is of interest to the packing industry. White pigs are preferred at slaughter and the cost of a "colored carcass" at some locations may be over $1/pig. Andersson and colleagues (Johansson-Moller et al. 1996) have now identified the KIT gene as that responsible for white coat color and a DNA test is patented and being used in MAS programs (Andersson and Plastow, personal communication). The MSHR gene has also been identified to control red and black color in the pig (Mariani et al. 1996b)

Reproduction

Given the necessity of larger resource families and the difficulty and time required to obtain information on reproduction traits, it is not surprising that results of QTL scans for these traits are limited. Initial scans have revealed promising results on chromosome 8. Wilkie et al. (1996) reported possible QTL for uterine length and ovulation rate, though in different chromosomal positions. Rathje et al. reported a sizable QTL for ovulation rate (+3.07 ova) on chromosome 8 also but some distance from the ovulation QTL observed by Wilkie and colleagues. In the French QTL experiment (Milan et al., 1998) a QTL for increased litter size of one piglet was found in the same location on chromosome 8 as Rathje. The large ovulation rate/litter size QTL on chromosome 8 is of interest as it mapped to the region which is syntenic to the Booroola fecundity gene in sheep. Interestingly, using a single microsatellite marker (OPN) in the same chromosome 8 region, Short et al. (1997b) also found significant effects for litter size in commercial line. Limited chromosome QTL analyses have further suggested other reproductive QTL on chromosomes 4 and 6 (Wilkie et al. 1996), on chromosome 7 (Wilkie et al., 1996; Milan et al., 1998) and chromosomes 4, 13, 15 (Rathje et al. 1997). Both Rathje et al. (1997) and Bidanel et al. (1996) have enlarged their experiments which can be expected to yield additional useful results.

Candidate gene analysis for reproduction has shown considerable merit. Results have clearly demonstrated that the estrogen receptor (ESR) is significantly associated with litter size (Rothschild et al. 1996; Short et al. 1997a). Estimates of allelic effects vary from 1.15 pig/litter in Meishan synthetics to .42 pigs/litter in Large White lines. These results have not been confirmed by QTL scans using divergent crosses involving Meishan and Large White pigs. This may have resulted from small sample sizes or the fact that the ESR B allele was in both parental populations involved in the QTL scan and it was not specifically genotyped in those experiments. Other effects have been reported for retinoic acid receptor gamma (RARG), retinol binding protein 4 (RBP4), and melatonin receptor 1A (MTNRIA) genes (Messer et al. 1996; Ollivier et al. 1997). More recent results have demonstrated that the prolactin receptor (PRLR) locus is significantly associated with litter size (Vincent et al. 1998). Most of the candidate gene analyses have involved considerably more sows and litters than the QTL analyses and this might explain the lack of QTL scan confirmation of the regions in which the candidate gene effects have been reported to date.

Disease resistance

To date, QTL scans for disease resistance or immune response QTL have been limited. An exception is work underway by Andersson and colleagues (Edfors-Lilja and Andersson, personal communication) to study some immune response parameters. Some immune capacity QTL have been identified. Also, a QTL for base cortisol level which may be related to stress and perhaps immune response, has been mapped to the end of chromosome 7 (Milan et al., 1998). Analyses of associations of candidate genes have been more substantial. The general location of the K88 E. coli receptor has been known for some time and fine mapping and candidate gene analysis of the region is underway in many labs. Two alpha (1,2) fucosyltransferase genes (FUT1, FUT2) on porcine chromosome 6q11 have been identified and are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci (Vögeli, personal communication). Vögeli and colleagues have found a polymorphism which is closely linked to ECF18R in Large White, Landrace, Hampshire, Duroc and Pietrain pigs and it could be a good marker for marker assisted selection of E. coli F18 adhesion resistant animals in these breeds. Whether the FUT1 or FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined. The SLA complex on chromosome 7 has recently been associated with resistance to primary infections with Trichinella spiralis but not to resistance to toxoplasmosis (Lunney, personal communication). The gene for Natural Resistance Associated Macrophage Protein 1 (NRAMP1), associated with resistance to Salmonella challenge in mice, has been recently mapped to pig chromosome 15 (Sun and Tuggle, personal communication). Several other candidate genes are being investigated. Genes associated with human disorders, which have been identified and mapped in the pig, include clotting factor IX (Signer et al. 1996) and the familial hypercholesterolaemia gene (Hasler-Rapacz et al. 1996).

DISCUSSION

In addition to more extensive scans of existing populations, many new QTL experiments are underway for performance, reproduction and meat quality traits (Moran, personal communication; Rohrer, personal communication; Rothschild, personal communication; Nezer et al. 1996; van Oers et al. 1996). In the short time it has taken to build a considerable genetic map, several QTL and candidate analyses have yielded interesting results. The QTL scans have identified several chromosomes that are now targets for further confirmation of the chromosomal region, advanced fine mapping of the QTL, and positional comparative candidate gene analysis. Additional experiments will either confirm the regions and lead to the eventual isolation of the gene or genes of interest or will produce conflicting results. Such conflicting results may be the results of haplotype effects, epistasis or background genotype effects, or sampling. Given that we wish to detect not only large but also moderate gene effects, experimental size will need to be increased or several experimental results will need to be pooled. At present, several PiGMaP participants are pooling information on chromosome 4 to obtain additional power in the analysis (C. Haley, personal communication). One approach suggested (Janss et al. 1997) is to conduct segregation analyses in some populations as a first stage prior to molecular QTL scans. The QTL experiments to study disease resistance are still lacking though candidate gene analysis using information from human studies will be quite useful. The relevance of results found in crosses involving the Wild Boar and the Chinese breeds will also have to be evaluated in commercial lines. Several companies are already attempting to use their specialized lines for such research.

SUMMARY

Several quantitative trait loci scans and candidate gene analyses have identified important chromosomal regions and major genes associated with traits of economic interest in the pig. These include chromosomal regions for growth and backfat (chromosomes 3, 4, 5, 6, 7, 8, 13, 14), meat quality traits (chromosomes 2, 3, 4, 6, 7, 12, 15) and reproduction (chromosome 4, 6, 7, 8). Candidate genes for litter size (ESR, PRLR), disease resistance (FUT1, SLA, NRAMP), and coat color (KIT, MSHR) have also been identified. At present, the industry is already using some of the resulting information for DNA tests and marker assisted selection (MAS).

ACKNOWLEDGMENTS

The author apologizes for any research inadvertently not included in this review and gratefully acknowledges the many colleagues around the world who kindly provided information in both published and unpublished forms for inclusion in this review. The invitation to speak at this conference is appreciated. Support for the gene mapping and QTL research and activities was provided by the Iowa Agriculture and Home Economics Experiment Station, the USDA-CSREES Pig Genome Coordination program, BRDC, Dalgety, PIC, IPPA, NPPC and in collaboration with the EEC PiGMaP program.

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