An Investigation into the Genetic

Controls of Pork Quality

Rebecca Emnett1, Steven Moeller1, Keith Irvin1,
Max Rothschild2, Graham Plastow3, and Rodney Goodwin4

1The Ohio State University, Department of Animal Sciences, Columbus, Ohio
2Iowa State University, Department of Animal Science, Ames, Iowa
3PIC International Group, Abingdon, Oxfordshire, UK
4National Pork Producers Council, Des Moines, Iowa

 

Introduction

Consumers and many sectors of the pork industry are demanding improvements in meat quality. This provides a new challenge for the breeding industry, which is seeking advanced genetic tools that can be practically incorporated in selection schemes for trait improvement. The heightened interest in meat (muscle) quality has led to investigations into the physiological and genetic controls of these economically important traits. Often studies in livestock are modeled after known rodent or human genetic markers associated with diseases, which may also be related to body composition or changes in energy metabolism. Genetic markers characterized in other species serve as guides or “candidate” genes for investigation of the phenotypic differences found in swine.

 

Although several genetic markers and QTLs affecting meat quality and performance traits have been detected in the pig (for review see Rothschild and Plastow, 1999), the quest continues to identify markers that explain significant variation in these traits of economic importance. It is anticipated that the detection of new molecular genetic markers, together with advances in the area of quantitative genetics, will lead to the development of marker assisted selection (MAS) programs for meat quality improvement and practical utilization by swine producers.

 

As more information becomes available in swine molecular genetics, there exists a need to analyze associations between these markers and phenotypes in individual genetic lines or populations. The objective of this study was to determine the association between variation found in performance, carcass, and meat quality traits and several candidate genes of interest in different pig populations.

 

Materials & Methods

Candidate Gene Association Studies

The population utilized for the project consisted of both purebred and crossbred animals from the 1998 National Barrow Show Progeny Test and the 1999 Hampshire Sire Progeny Test. All animals were managed by identical protocols, and performance, carcass, meat quality, and sensory panel measurements were taken on each animal.  Individuals of four major U.S. breeds were chosen for DNA extraction from frozen loin chops. Berkshire (n=180), Duroc (n=77), Hampshire (n=160), and Landrace (n=55) sire breeds were represented. Genotypes were obtained for six candidate genes using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) procedures unique for each gene analyzed. Statistical analyses were performed within each breed separately, and across breeds for the total population. Only pigs classified as free of the Porcine Stress Syndrome mutation were utilized in these analyses. A SAS (1999) Mixed Model analysis was completed for the total population utilizing the fixed effects of  breed, genotype, sex, day off-test group (depending on the trait), and Rendement Napole (RN) gene status, with a sire(breed) random effect. The within breed analysis included a random effect of sire, and fixed effects of genotype, sex, day off-test group (depending on the trait), and RN classification. A second PPARg analysis excluding the underrepresented 22-genotype animals (n=12) was performed to more clearly define the differences between the 11 and 12-animals. Allelic frequencies were calculated within breed and for the total population based upon observed genotype classifications.

 

Mapping Procedures

Two additional genes were chosen as possible candidate genes for meat quality based on their observed physiological functions in other mammalian species. Primers for use in PCR amplification were designed based on porcine sequence available for each gene in GenBank. PCR conditions were optimized for each gene separately. Sequencing and comparisons of breed DNA pools were completed in order to search for sequence variation among the breeds. Physical mapping was achieved by use of a pig-rodent somatic cell hybrid panel (Yerle et al., 1996).

 

Results

Leptin Receptor (LEPR)

Daily gain and backfat thickness are important traits for livestock producers to consider in order to produce efficient, fast growing and lean animals. The leptin receptor gene (LEPR), is a high affinity receptor (for review see Tartaglia, 1997) that mediates the regulation of the well known “obesity” gene, leptin (Zhang et al., 1997). Mutations in LEPR have been reported to be associated with obesity in humans and rodents (Reichart et al., 2000; Clement et al., 1998; Chen et al., 1996). Given the physiological role of LEPR, it is interesting as a candidate gene for backfat deposition and daily gain in the pig. Vincent et al. (1997) identified a HinfI polymorphism in porcine LEPR and mapped its location to pig chromosome 6 (SSC6). A few studies have reported on associations between leptin levels and production traits in pigs (Ramsay et al. 1998; Robert et al. 1998), but the effects of LEPR on pork quality traits of economic importance to the industry have not been investigated.

 

A leptin receptor (LEPR) MboI RFLP, developed by Vincent at Iowa State University (M.F. Rothschild, personal communication), was found to be polymorphic in all breeds analyzed; Berkshire (n=177), Duroc (n=76), Landrace (n=49), and Hampshire (n=149). Allele 2 was the most frequent (.91) in the total population and was found to be associated with leaner animals (Table 1).

 

Total population analysis revealed effects (P<.05) of LEPR on last lumbar backfat with the 11-animals having the fattest phenotype (Table 1). Although not significant (P>.05), last rib and 10th rib backfat (not shown) had similar numerical trends. Average daily gain was also different (P<.05) between the genotypes (Table 1). The results of the individual breed analyses (not shown) revealed differences (P<.05) between LEPR genotypes and off flavor score for Berkshire; average daily gain and Japanese color score for Duroc; glycolytic potential, loin glycogen and lactate concentration, intramuscular fat, and quality index (which includes Minolta, IMF, & pHu) for Hampshire; 10th rib backfat, average daily gain, loin pHu, Minolta and Hunter color values, and quality index for Landrace.

 

As previously reported in other mammalian species, LEPR appears to have the greatest effects on backfat and average daily gain in the pig, but may also be associated with correlated pork quality differences within genetic lines.

 

Table 1.  LS Means1 and Std. Errors for Total Population Analysis

LEPR2

n

11

n = 21

12

n = 41

22

n = 398

P value

Last Lumbar BF (cm)

438

2.56 + .15b

2.31 + .11ab

2.22 + .08a

*

Last Rib BF (cm)

440

2.52 + .14

2.50 + .11

2.39 + .07

.374

Av. Daily Gain (kg/d)

 445

.77 + .02a

.83 + .02b

.82 + .02b

*

MC4R3

n

11

n = 80

12

n = 202

22

n = 169

P value

Last Lumbar BF (cm)

443

2.06 + .09a

2.24 + .08b

2.42 + .08c

***

Last Rib BF (cm)

445

2.29 + .09a

2.41 + .08ab

2.51 + .08b

*

10th Rib BF (cm)

433

2.26 + .09

2.38 + .07

2.47 + .07

.085

Instron (kg)

449

5.37 + .13b

5.06 + .11a

5.06 + .11a

*

Loin Muscle Area (cm2)

445

38.03 + .83

36.79 + .72

36.58 + .74

.134

Av. Daily Gain (kg/d)

451

.81 + .02

.81 + .02

.83 + .02

.153

Color (1-5)

450

2.7 + .10

2.9 + .08

2.9 + .08

.079

MC5R4

n

11

n = 287

12

n = 49

22

n = 45

P value

10th Rib BF (cm)

365

2.49 + .10b

2.41 + .12b

2.15 + .14a

*

Last Rib BF (cm)

376

2.54 + .09

2.40 + .11

2.32 + .12

.139

Loin Muscle Area (cm2)

376

36.16 + .80

37.00 + .95

38.45 + 101

.097

PPARg5

 n

11

n = 287

12

n = 147

22

n = 12

P value

Off Flavor (1-10)

444

4.9 + .21b

4.4 + .25a

--

*

Av. Daily Gain (kg/d)

445

.81 + .01

.83 + .02

.85 + .03

.075

Tenderness (1-5)

423

6.8 + .20

7.2 + .25

--

.095

Juiciness (1-5)

423

5.3 + .17

5.1 + .20

--

.092

* P<.05, ** P<.01, *** P<.001, -- 22-PPARg excluded from analysis

1 LS Means with common subscripts are equal (P>.05)

2   LEPR MboI RLFP genotypes

3  MC4R TaqI RFLP genotypes

4 MC5R BsaHI RFLP genotypes

5  PPARg BsrI RFLP genotypes

 

Melanocortin-4 Receptor (MC4R)

Melanocortin-4 receptor (MC4R) has been found to play a significant role in regulating leptin’s effects on food intake and body weight (Seeley et al., 1997; Fan et al., 1997). Kim et al. (2000) demonstrated that a missense mutation in MC4R was associated with backfat thickness, growth, and feed intake in different genetic lines of pigs. No studies to date have reported associations between meat quality characteristics and MC4R genotypes.

 

Results with the TaqI MC4R RFLP, developed by Kim et al. (2000), show an allelic frequency of .60 for allele 2, which was associated with much fatter animals in the total population. Genotypic frequencies varied within the breeds, however the heterozygote 12-animals were the most frequent (.45) in the total population.

 

The effect of MC4R on last lumbar backfat was highly significant (P<.001) (Table 1). Approximately, 0.18 cm in last lumbar backfat is added with the inclusion of each 2 allele. MC4R genotype groups were also different (P<.05) for last rib backfat. and 10th rib backfat approached significance (P=.085) with the fatter 22-animals being in line with the last lumbar result. These backfat results are also in agreement with Kim et al. (2000) who demonstrated that 11-homozygote pigs had approximately 9 % less backfat than 22-pigs. Instron force was less (P<.05) for the 22-genotype (Table 1), indicating increased tenderness in the fatter animals. Interesting trends in loin muscle area, average daily gain, and color score were also noted. The results of the individual breed analysis (not shown) revealed differences (P<.05) between MC4R genotypes and soundness score, last lumbar backfat, last rib backfat, average backfat, average daily gain, and loin lactate concentration for Berkshire; and juiciness score, intramuscular fat, Minolta, Hunter color, and quality index for Landrace.

 

Our results suggest that MC4R has its greatest effects on backfat and Instron tenderness for the total population. However, differences between MC4R genotypes were also reported for average daily gain and meat quality traits within the breed populations. Further analysis in larger populations will be needed with this marker to fully characterize the effects of MC4R on meat quality.

 

Mealnocortin-5 Receptor (MC5R)

Melanocortin-5 receptor (MC5R) has been found to be associated with thermoregulation through gland secretion (van der Kraan et al., 1998; Chen et al., 1997) and serves a possible role in lipolysis of adipocytes (Boston, 1999). Kim et al. (1999) mapped porcine MC5R to SSC 6 and detected two single nucleotide polymorphisms within the porcine sequence. Previous analyses have not investigated the association between the porcine MC5R gene and fat deposition, growth or carcass quality traits in pigs.

 

Results indicate that the MC5R BsaHI RFLP, developed by Kim et al. (1999) was polymorphic in Berkshire, Duroc, Landrace and Hampshire populations. The frequency of alelle 1 was .82. Genotypic frequencies varied within the breeds, however, the homozygote 11-animals were the most frequent (.75) in the total population.

 

Total population analysis revealed effects (P<.05) of MC5R on 10th rib backfat (Table 1), with the 11 and 12-animals being fatter. Similar numerical differences were also noted for the last rib location (P=.14). Loin muscle area also approached significance for the total population analysis (P=.10). The results of the individual breed analysis (not shown) revealed differences (P<.05) between MC5R genotypes with color and Japanese subjective scores, Minolta and Hunter color values, as well as, Instron tenderness for Berkshire; quality index for Hampshires; all backfat measures and intramuscular fat % for Landrace.

 

This MC5R BsaHI RFLP appears to have its greatest effects on backfat in the total population. Further analysis is needed to fully characterize the differences in meat quality characteristics such as color and intramuscular fat % within the breed populations.

 

Peroxisome Proliferator Activated Receptor-g (PPARg)

Peroxisome Proliferator Activated Receptor-gamma (PPARg) is a member of the nuclear receptor superfamily (for review see Green, 1995) and regulates the expression of several genes encoding proteins involved in adipocyte differentiation (Rosen et al., 2000; Spiegelman et al., 1997) and fat deposition (for review see Schoonjans et al., 1996). Genetic mutations in PPARg have been found to be associated with extreme obesity in humans (Freake, 1999). In pigs, PPARg expression levels in adipose tissue vary among different breeds and ages (Grindflek et al., 1998). An association study (Grindflek, in manuscript), reported a difference in loin fatty acid composition in Norwegian pigs for a BsrI RFLP, but no significant differences were noted for backfat or intramuscular fat measurements.

 

Our results show that a BsrI PPARg RFLP (Grindflek, personal communication) was polymorphic in Berkshire, Duroc, Landrace, and Hampshire populations. The frequency of allele 1 was .81. Total population analysis revealed effects (P<.05) on off flavor score (Table 1), although interesting trends in average daily gain, tenderness and juiciness approached significance. The results of the individual breed analysis (not shown) revealed differences (P<.05) between PPARg genotypes and loin muscle area and marbling for Duroc, average daily gain for Hampshire; and last rib backfat, and Instron tenderness for Landrace.  This PPARg RFLP remains an interesting candidate gene for meat quality traits within specific lines of swine. These results, while promising, warrant larger scale investigation to determine the potential use of PPARg  in future selection programs.

 

Heart Fatty Acid Binding Protein (HFABP)

Intramuscular fat percentage (IMF) has been found to be positively associated with sensory attributes of pork (Fernandez et al., 1999; Touraille et al., 1989). Hovenier et al. (1992) reported that backfat reduction is not completely related with reductions in IMF. Therefore, it may be possible to treat the two traits separately in a breeding scheme with the proper selection tools.

 

Heart Fatty Acid Binding Protein-1 (HFABP) is a member of the fatty acid binding protein family (FABP), which is involved in fatty acid transport from the cell membrane to the intracellular sites of fatty acid utilization (Veerkamp and Maatman, 1995). Given this physiological role, HFABP has been considered to be an interesting candidate gene for IMF and backfat in pigs. Gerbens et al. (1997) mapped HFABP to pig chromosome 6. QTL studies have also identified IMF and BF loci in this region of chromosome 6 (Ovilo et al. 2000b; de Koning et al., 1999), further implicating HFABP as a strong candidate gene for IMF and backfat in the pig.

 

Gerbens et al. (1997) reported three polymorphic sites in the porcine HFABP gene (HaeIII, MspI, and HinfI) and conducted an association study to determine the genotype effects on traits in pigs (Gerbens et al., 1999). This study reported IMF and backfat differences between HFABP genotype groups and thus hypothesized that HFABP, or a closely linked marker, controlled IMF differences in pigs. Two of these markers were also informative in a Norwegian pig population (E. Grindflek, personal communication), and Ovilo et al. (2000a) also reported differences in genotype groups for the HFABP HaeIII RFLP.

 

Results from our population show that the HaeIII HFABP RFLP (originally reported by Gerbens et al., 1997) is polymorphic in Berkshire, Duroc, Landrace and Hampshire populations. The frequency of allele 1 was .63. Total population analysis reveals effects of HFABP on ultimate loin pH (P<.05) and quality index (P<.01) (Table 2), and intramuscular fat % and pork flavor also showed interesting trends. The results of the individual breed analysis (not shown) revealed differences between HFABP genotypes and loin muscle area, loin glycogen concentration, intramuscular fat, Instron tenderness, and quality index for Berkshire. The Berkshire analysis results are similar to those of Gerbens et al. (1999), which found an advantage of the HaeIII HFABP 12-heterozygote for intramuscular fat % and backfat.  Loin glycogen concentration was also significantly different (P<.05) in Duroc; last rib backfat, flavor score, and water holding capacity for Hampshire; and last rib backfat, and intramuscular fat for Landrace. HFABP remains an interesting candidate gene for meat quality traits of importance within specific breeds of swine.

 

Table 2.  LS Means1 and Std. Errors for Total Population Analysis

HFABP2

n

11

n = 176

12

n = 202

22

n = 59

P value

 Loin pHu

436

5.58 + .02b

5.54 + .02a

5.60 + .03b

*

Quality Index

(IMF, MIN & pHu)

435

50.1 + 2.1b

45.3 + 1.8a

49.5 + 2.5ab

**

Intramuscular Fat (%)

437

2.75 + .15

2.50 + .12

2.50 + .16

.058

Pork Flavor (1-10)

426

1.2 + .06

1.1 + .05

1.1 + .08

.090

CASTRsa3

n

11

n = 255

12

n = 154

22

n = 34

P value

Last Lumbar BF (cm)

436

2.30 + .08b

2.38 + .08b

2.00 + .12a

**

Last Rib BF (cm)

437

2.43 + .08

2.44 + .08

2.23 + .11

.098

10th Rib BF (cm)

425

2.38 + .09b

2.43 + .09b

2.15 + .12a

*

Loin Muscle Area (cm2)

437

36.46 + .72a

36.98 + .71a

39.78 + .99b

***

 Loin pHu

442

5.55 + .02

5.59 + .02

5.56 + .03

.097

Tenderness (1-10)

433

7.2 + .23

6.8 + .23

6.7 + .38

.102

CASTMsp4

n

11

n = 47

12

n = 152

22

n = 223

P value

Last Lumbar BF (cm)

415

2.07 + .11a

2.27 + .08b

2.30 + .09b

*

Last Rib BF (cm)

417

2.29 + .11

2.44 + .08

2.44 + .08

.242

10th Rib BF (cm)

405

2.18 + .12a