M. Zappaterra, M. Mazzoni, P. Zambonelli, R. Davoli

Investigation of the Perilipin 5 gene expression and association study of its sequence polymorphism with meat and carcass quality traits in different pig breeds

Table S1 Primers for the sequencing, the genotyping and the gene expression study.

Primer name (sequence ID) / Primer sequence (5’-3’) / TM (°C) / Size (bp)
Sequencing
(ENSSSCG00000013513.2)
PLIN5 -FOR
PLIN5 -REV / 5’- TTGGGGCTCTGAAAAGTGAG -3’
5’- GCAGTTGTGGTTCAGATCCTG -3’ / 64°C / 600
Genotyping (ENSSSCG00000013513.2)
PLIN5 -FOR / 5’- CCAGGCTAGGGGTGGAATC -3’ / 63°C / 119
PLIN5 -REV / 5’- AAGAATCCAGCATCGCCATG -3’
PLIN5 (ENSSSCG00000013513.2)
PLIN5 -E-FOR
PLIN5 -E-REV / 5’- GCGGTCTCCGATGCTTATAG -3’
5’- CCCTGTTGTCTCCTCTGCTC -3’ / 64°C / 476
PLIN5 -I-FOR
PLIN5 -I-REV / 5’- GTGGAGCTCAAACGATCCAT -3’
5’- TCAGTCATGGGCAGGAAGT -3’ / 67°C / 89
LIPE (AY686758.1)
LIPE -E-FOR
LIPE -E-REV / 5’- CCGAGACGAGATTAGCACCA -3’
5’- CCTAGCGAACATGACCGAGT -3’ / 66°C / 247
LIPE -I-FOR
LIPE -I-REV / 5’- AAGTCTACAGTGTGAGGGCC -3’
5’- CGATGGGAGCTGAGTAGAGG -3’ / 70°C / 96
Normalising genes
HPRT1 (AK346023.1)
HPRT1-E-FOR
HPRT1-E-REV / 5’- GCCCCAGCGTCGTGATTA -3’
5’- AGAGGGCTACGATGTGATGG -3’ / 64°C / 183
HPRT1-I-FOR
HPRT1-I-REV / 5’- CCCAGCGTCGTGATTAGTGA -3’
5’- CCTTTTCCAAATCCTCGGCA -3’ / 66°C / 88
YWHAZ (AK344707.1)
YWHAZ-E-FOR
YWHAZ -E-REV / 5’- TGGAGCACTTACAAGGCGTA -3’
5’- ACCGTTTCTGCCCTTATCCA -3’ / 64°C / 168
YWHAZ -I-FOR
YWHAZ -I-REV / 5’- AAGGCGTAGTGGAAGTGGAT -3’
5’- GCTGTAGTCAAAGGTGTGCA -3’ / 66°C / 98
B2M (AK239552.1)
B2M-E-FOR
B2M-E-REV / 5’- AAACGGGGAGAAGATGAACG -3’
5’- ACATCTACCTGCTCAGACAGT -3’ / 63°C / 377
B2M-I-FOR
B2M-I-REV / 5’- CCTTCTGGTCCACACTGAGT -3’
5’- TCCCACTTAACTATCTTGGGCT -3’ / 66°C / 99

For all the considered genes in the table are reported the used primer, the annealing temperature expressed in °C and the size in base pairs (bp).

Each primer name is composed by the gene name, “I” for internal primer or “E” for external primer, “FOR” for the left primer and “REV” for the right primer.

1TM (°C) Annealing temperature.

Table S2 Immunostaining results for Perilipin 5 (PLIN5) protein and average fiber size in pig Semimembranosus muscle in Italian Large White and Italian Duroc samples. Samples belonging to the two breeds have been compared using Wilcoxon test, and the relative P-values were reported.

Immunostaining results
ILW1 / IDU2
Mean / SD / Mean / SD / P-value
PLIN5 staining total CSA3 (μm2) / 715 261.66 / 100 166.11 / 708 011.97 / 84 280.84 / 0.931
PLIN5 staining CSA percentage (%) / 92.94 / 5.13 / 94.96 / 1.92 / 0.931
Average PLIN5-positive myofiber size (μm2) / 7 673.47 / 776.34 / 7 459.79 / 909.47 / 0.931
Average PLIN5-negative myofiber size (μm2) / 6 677.67 / 2 967.15 / 6 156.80 / 2 607.23 / 0.792

1 Italian Large White pigs.

2 Italian Duroc pigs.

3 CSA stands for cross-sectional area.

Table S3 Additive and dominant effects of rs327694326 SNP on backfat fatty acid composition, with the means and differences of the estimated marginal posterior distribution for the genotypes in Italian Large White pigs.

Additive (a) and dominant (d) effects / Estimated means / Estimated differences between genotypes
Backfat fatty acids1 / TT-CT / CT-CC / TT-CC
a / P(>0) / d / P(>0) / TT / CT / CC / Mean / P(>0) / Mean / P(>0) / Mean / P(>0)
C18:1, cis-92 / 0.001 / 0.64 / 0.002 / 0.85 / 38.41 / 38.66 / 38.39 / -0.25 / 0.07 / 0.27 / 0.79 / 0.02 / 0.52
C18:1, cis-113 / -0.00002 / 0.48 / 0.0003 / 0.83 / 2.18 / 2.22 / 2.20 / -0.03 / 0.10 / 0.03 / 0.69 / -0.008 / 0.44
C18:2, trans-9, cis-124 / -0.000004 / 0.30 / -0.00002 / 0.05 / 0.021 / 0.019 / 0.022 / 0.002 / 0.98 / -0.002 / 0.09 / -0.0004 / 0.38
C18:3, Ω35 / -0.00014 / 0.20 / -0.00029 / 0.12 / 0.77 / 0.76 / 0.80 / 0.012 / 0.74 / -0.04 / 0.15 / -0.028 / 0.20
C18:3/C18:26 / -0.00001 / 0.14 / -0.00002 / 0.10 / 0.049 / 0.048 / 0.051 / 0.0006 / 0.67 / -0.003 / 0.10 / -0.002 / 0.14
C20:1, cis-117 / -0.00002 / 0.47 / 0.00036 / 0.90 / 0.80 / 0.83 / 0.81 / -0.040 / 0.04 / 0.028 / 0.73 / -0.012 / 0.38
C20:2, Ω68 / 0.0001 / 0.71 / 0.00015 / 0.88 / 0.77 / 0.79 / 0.77 / -0.014 / 0.08 / 0.013 / 0.75 / -0.001 / 0.48
C22:4, Ω69 / 0.00002 / 0.95 / 0.00002 / 0.85 / 0.093 / 0.092 / 0.088 / 0.0004 / 0.58 / 0.0037 / 0.87 / 0.0041 / 0.91
C22:6, Ω310 / -0.00001 / 0.30 / -0.00001 / 0.15 / 0.013 / 0.012 / 0.011 / 0.0007 / 0.80 / 0.0012 / 0.77 / 0.0019 / 0.90
LCFA11 / 0.0010 / 0.80 / 0.0018 / 0.86 / 73.26 / 73.41 / 73.15 / -0.15 / 0.13 / 0.26 / 0.84 / 0.11 / 0.68
MUFA12 / 0.0005 / 0.61 / 0.003 / 0.90 / 43.61 / 43.94 / 43.61 / -0.33 / 0.05 / 0.33 / 0.81 / -0.001 / 0.50
PUFA Ω313 / -0.00014 / 0.22 / -0.00031 / 0.11 / 0.84 / 0.82 / 0.87 / 0.014 / 0.77 / -0.043 / 0.14 / -0.028 / 0.20

1 All the fatty acids or fatty acid categories are expressed as percentage on the total fatty acids amount.

2 Oleic acid; 3 Cis-vaccenic acid; 4 Trans-9, cis-12 octadecenoic acid; 5 Linolenic acid; 6 the ratio between C18 tri-unsaturated and C18 di-unsaturated fatty acids; 7 Eicosenoic acid; 8 Eicosadienoic acid; 9 Docosatetraenoic acid; 10 Docosahexaenoic acid; 11 Long chain fatty acids; 12 Monounsaturated fatty acids; 13 Polyunsaturated fatty acids omega3.

P (>0): Posterior probability of a value being positive. In bold, probabilities above (or equal to) 0.90 or below 0.10. Oleic, cis-vaccenic, linolenic, eicosadienoic, docosahexaenoic, monounsaturated fatty acids, long chain and omega 3 fatty acids levels were reported in Table 4 as the observed P(>0) were close to the considered thresholds of 0.90 and 0.10.

Table S4 Additive and dominant effects of rs327694326 SNP on FCR and carcass phenotypes, with the means and differences of the estimated marginal posterior distribution for the genotypes in Italian Large White pigs.

Additive (a) and dominant (d) effects / Estimated means / Estimated differences between genotypes
Traits / TT-CT / CT-CC / TT-CC
a / P(>0) / d / P(>0) / TT / CT / CC / Mean / P(>0) / Mean / P(>0) / Mean / P(>0)
Lean1 (%) / -0.002 / 0.20 / -0.005 / 0.07 / 48.50 / 48.09 / 48.97 / 0.41 / 0.91 / -0.89 / 0.06 / -0.48 / 0.18
Backfat thickness2 / 0.003 / 0.71 / 0.009 / 0.90 / 28.29 / 29.02 / 27.67 / -0.73 / 0.10 / 1.35 / 0.90 / 0.62 / 0.74
Drip loss3 / 0.008 / 0.81 / 0.04 / >0.99 / 52.59 / 56.14 / 51.04 / -3.55 / <0.01 / 5.09 / 0.99 / 1.54 / 0.79
pH244 / 0.0002 / 0.77 / 0.001 / >0.99 / 5.96 / 6.05 / 5.92 / -0.09 / 0.01 / 0.14 / 0.98 / 0.04 / 0.77
CIE L* / -0.005 / 0.11 / -0.009 / 0.07 / 33.18 / 32.86 / 34.27 / 0.31 / 0.73 / -1.41 / 0.07 / -1.09 / 0.10
CIE a* / 0.003 / 0.91 / 0.003 / 0.86 / 9.17 / 9.22 / 8.69 / -0.05 / 0.42 / 0.53 / 0.88 / 0.48 / 0.88
CIE b* / -0.002 / 0.09 / -0.004 / 0.03 / 3.64 / 3.45 / 4.01 / 0.19 / 0.88 / -0.56 / 0.03 / -0.37 / 0.08

1 The percentage of lean mass measured with Fat-O-Meter.

2 Backfat thickness measured with Fat-O-Meter at 8 cm off the midline of the carcass between the third and fourth last ribs.

3 Drip loss measured with Filter Paper Press method

4 pH measured at 24 hours post-mortem.

CIE stands for International Commission on Illumination.

P (>0): Posterior probability of a value being positive. In bold, probabilities above (or equal to) 0.90 or below 0.10.

Table S5 Number of samples per each extreme group, genotypic and allelic frequencies inside the two tails of extreme and divergent samples, and the results of the two tailed Fisher’s exact test and Cochran-Armitage trend tests performed on the genotypic frequencies of rs327694326 SNP in Italian Large White pigs.

Groups / TT / CT / CC / Allele frequencies (%) / Fisher's exact
P-value / Χ2 / Cochran-Armitage
P-value
Traits / Type / N / N / % / N / % / N / % / T / C
Backfat fatty acid composition (%)
C18:1, cis-91 / low / 29 / 28 / 0.965 / 0 / 0.000 / 1 / 0.035 / 0.965 / 0.035 / 0.007 / 4.984 / 0.026
high / 35 / 24 / 0.686 / 7 / 0.200 / 4 / 0.114 / 0.786 / 0.214
C18:1, cis-112 / low / 29 / 28 / 0.965 / 0 / 0.000 / 1 / 0.035 / 0.965 / 0.035 / 0.007 / 4.984 / 0.026
high / 35 / 24 / 0.686 / 7 / 0.200 / 4 / 0.114 / 0.786 / 0.214
C18:2, trans-9, cis-123 / low / 28 / 20 / 0.714 / 7 / 0.250 / 1 / 0.036 / 0.839 / 0.161 / 0.597 / 0.172 / 0.679
high / 39 / 28 / 0.718 / 7 / 0.179 / 4 / 0.103 / 0.808 / 0.192
C18:3, Ω34 / low / 28 / 22 / 0.786 / 5 / 0.179 / 1 / 0.036 / 0.875 / 0.125 / 0.515 / 1.508 / 0.219
high / 37 / 24 / 0.649 / 10 / 0.270 / 3 / 0.081 / 0.784 / 0.216
C20:1, cis-115 / low / 37 / 28 / 0.757 / 8 / 0.216 / 1 / 0.027 / 0.865 / 0.135 / 0.788 / 0.332 / 0.564
high / 29 / 20 / 0.690 / 8 / 0.276 / 1 / 0.034 / 0.828 / 0.172
C20:2, Ω66 / low / 29 / 20 / 0.690 / 7 / 0.241 / 2 / 0.069 / 0.810 / 0.190 / 0.857 / 0.223 / 0.637
high / 35 / 23 / 0.657 / 8 / 0.229 / 4 / 0.114 / 0.771 / 0.229
C22:4, Ω67 / low / 34 / 26 / 0.765 / 7 / 0.206 / 1 / 0.029 / 0.868 / 0.132 / 1.000 / 0.038 / 0.846
high / 33 / 25 / 0.758 / 8 / 0.242 / 0 / 0.000 / 0.879 / 0.121
C22:6, Ω38 / low / 29 / 21 / 0.724 / 3 / 0.103 / 5 / 0.172 / 0.776 / 0.224 / 0.161 / 1.416 / 0.234
high / 32 / 25 / 0.781 / 6 / 0.188 / 1 / 0.031 / 0.875 / 0.125
LCFA9 / low / 35 / 27 / 0.771 / 5 / 0.143 / 3 / 0.086 / 0.843 / 0.157 / 0.387 / 0.474 / 0.491
high / 31 / 20 / 0.645 / 9 / 0.290 / 2 / 0.065 / 0.790 / 0.210
MUFA10 / low / 31 / 29 / 0.935 / 1 / 0.032 / 1 / 0.032 / 0.952 / 0.048 / 0.108 / 3.494 / 0.062
high / 38 / 28 / 0.737 / 7 / 0.184 / 3 / 0.079 / 0.829 / 0.171
PUFA Ω311 / low / 29 / 24 / 0.828 / 4 / 0.138 / 1 / 0.034 / 0.897 / 0.103 / 0.427 / 1.882 / 0.170
high / 40 / 27 / 0.675 / 10 / 0.250 / 3 / 0.075 / 0.800 / 0.200
C18:3/C18:212 / low / 28 / 23 / 0.821 / 4 / 0.143 / 1 / 0.036 / 0.893 / 0.107 / 0.777 / 0.974 / 0.324
high / 54 / 40 / 0.741 / 9 / 0.167 / 5 / 0.093 / 0.824 / 0.176
Carcass traits
CIE a* / low / 25 / 17 / 0.680 / 4 / 0.160 / 4 / 0.160 / 0.760 / 0.240 / 0.036 / 5.650 / 0.017
high / 38 / 33 / 0.868 / 5 / 0.132 / 0 / 0.000 / 0.934 / 0.066
CIE b* / low / 32 / 27 / 0.844 / 4 / 0.125 / 1 / 0.031 / 0.906 / 0.094 / 0.625 / 0.889 / 0.346
high / 32 / 24 / 0.750 / 6 / 0.188 / 2 / 0.063 / 0.844 / 0.156
CIE L* / low / 24 / 21 / 0.875 / 2 / 0.083 / 1 / 0.042 / 0.917 / 0.083 / 0.761 / 0.504 / 0.478
high / 38 / 30 / 0.789 / 6 / 0.158 / 2 / 0.053 / 0.868 / 0.132
Drip loss13 / low / 29 / 23 / 0.793 / 5 / 0.172 / 1 / 0.034 / 0.879 / 0.121 / 0.723 / 0.738 / 0.390
high / 30 / 21 / 0.700 / 7 / 0.233 / 2 / 0.067 / 0.817 / 0.183
Backfat thickness14 / low / 25 / 15 / 0.600 / 8 / 0.320 / 2 / 0.080 / 0.760 / 0.240 / 0.623 / 0.684 / 0.408
high / 32 / 23 / 0.719 / 7 / 0.219 / 2 / 0.063 / 0.828 / 0.172
Lean15 (%) / low / 33 / 22 / 0.667 / 9 / 0.273 / 2 / 0.061 / 0.803 / 0.197 / 0.828 / 0.306 / 0.580
high / 32 / 24 / 0.750 / 6 / 0.188 / 2 / 0.063 / 0.844 / 0.156
pH2416 / low / 36 / 30 / 0.833 / 5 / 0.139 / 1 / 0.028 / 0.903 / 0.097 / 0.682 / 0.802 / 0.370
high / 37 / 27 / 0.730 / 9 / 0.243 / 1 / 0.027 / 0.851 / 0.149

1Oleic acid; 2Cis-vaccenic acid; 3Trans-9, cis-12 octadecenoic acid; 4Linolenic acid; 5 Eicosenoic acid; 6Eicosadienoic acid; 7 Docosatetraenoic acid; 8 Docosahexaenoic acid; 9 Long chain fatty acids; 10Monounsaturated fatty acids; 11Polyunsaturated fatty acids omega3; 12 the ratio between C18 tri-unsaturated and C18 di-unsaturated fatty acids. All the fatty acids or fatty acid categories are expressed as percentage on the total fatty acids amount.

CIE stands for International Commission on Illumination.

13 Drip loss measured with Filter Paper Press method

14 Backfat thickness measured with Fat-O-Meter at 8 cm off the midline of the carcass between the third and fourth last ribs.

15 The percentage of lean mass measured with Fat-O-Meter.

16 pH measured at 24 hours post-mortem.

Figure S1 The full image of the Western-blot membrane. Beta actin was used as standard control (on the left part of the membrane) to assess the molecular weight of PLIN5 band (on the right part of the image). The top of the membrane was cut with the aim of identifying after the development with diaminobenzidin (DAB) which part of the membrane was incubated with Rabbit anti-PLIN5 antibody and which part was incubated with Rabbit anti-beta actin antibody. Three samples were randomly chosen and tested for both PLIN5 and beta actin.