In ternationa l Scholars Journa ls African Journal of Pig Farming ISSN 2375-0731 Vol. 5 (4), pp. 001-006, April, 2017. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Polymorphism and association of microsatellite SJ01 with birth weight and early growth traits in pigs Y. J. Zhang1,2, G. C. Li1 and Y. L. Jiang1* 1 Laboratory of Animal Molecular Genetics, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271018, China. 2 College of Life Science, Linyi Normal University, Linyi 276005, China. Accepted 22 November, 2016 Myostatin is a negative regulator of animal skeletal muscle development and SJ01 is a microsatellite locus flanking porcine myostatin gene. In the present study, the polymorphism of microsatellite SJ01 in three pig populations, that is, Duroc (n = 420), Yorkshire (n = 414) and Landrace (n = 119), were investigated, and its associations with birth weight and early growth traits were analyzed. The results indicated that genotype BB was predominant in Yorkshire pigs and allele B was predominant in both Yorkshire and Landrace pigs, whereas allele A was more prevalent in Duroc pigs. Among the three pig breeds, Duroc population had the highest heterozygosity and deviated from Hardy-Weinberg equilibrium (P < 0.05). The average daily gain from 28 d to 70 d in Yorkshire pigs and the body weight at 70 d in Landrace pigs were significantly different between SJ01 genotypes (P < 0.05). These results suggest that microsatellite SJ01 is a potential DNA marker for early growth trait selection in Yorkshire and Landrace pigs as well as for refining QTL for early growth traits on SSC15. Key words: Pig, myostatin, microsatellite SJ01, polymorphism, early growth traits. INTRODUCTION Myostatin is a negative regulator of skeletal muscle mass in mammals (McPherron et al., 1997). Natural mutations, such as deletions, point mutations in myostatin coding re- gion, intronic region or 3′-untranslated region, bring about double-muscling traits in beef cattle (McPherron and Lee, 1997), sheep (Clop et al., 2006; Boman et al., 2009), human (Schuelke et al., 2004) and dogs (Mosher et al., 2007). In pigs differing in muscular traits, such as wild boar, Pietrain and Laiwu pigs, however, no mutation with similar effect in myostatin gene was reported (Jiang et al., 2002a; Stinckens et al., 2008). Recent studies reported associations of polymorphisms in the promoter region of porcine myostatin gene with production traits (Jiang et al., 2002b), meat quality traits (Guimaraes et al., 2007) and mRNA level (Stinckens et al., 2008), suggesting that, in pigs, myostatin might also play important roles in the development of skeletal muscle and its related traits. Microsatellite loci are frequently used as DNA markers to locate quantitative trait loci (QTL), but usually to a *Corresponding author. E-mail: yunliangjiang@gmail.com. relatively wide range of chromosomal fragment, in which many genes reside. To find the functional gene(s) or quantitative trait nucleotide (QTN) fine mapping with more markers is required. Microsatellite SJ01, which was iden- tified by our previous study, is a locus located 42 kb downstream of porcine myostatin gene (Jiang et al., 2004) whether it is associated with production traits and can be used as DNA marker remain unknown. Birth weight and early growth traits were two econo- mically important traits in that, on one hand, they directly affect the mortality rate of weanling pigs and on another hand, are associated with growth rate and carcass traits on test. A QTL mapping study based on a F2 pedigree constructed with wild boar ×Yorkshire suggested one QTL for average daily gain from birth to 70 d on SSC15 (50 cM, male; 78 cM, female) (Knott et al., 1998), the map- ping interval was partially overlapped with myostatin gene and SJ01 locus. The production traits associated polymorphisms involve three sites in the promoter region of porcine myostatin gene (Jiang et al., 2002a; Guimaraes et al., 2007; Yu et al., 2007; Stinckens et al., 2008), the detection of which is not practical for use in molecular pig breeding. Therefore, Table 1. Means and s.d. of birth weight and early growth traits. Trait Breed Duroc Yorkshire Landrace BW0 (kg) 1.44 ± 0.31 1.49 ± 0.31 1.46 ± 0.25 BW28 (kg) 7.12 ± 1.52 7.53 ± 2.05 8.53 ± 2.15 BW70 (kg) 21.13 ± 5.27 21.78 ± 5.53 24.77 ± 5.65 ADG1 (kg) 0.20 ± 0.05 0.20 ± 0.06 0.25 ± 0.08 ADG2 (kg) 0.36 ± 0.10 0.50 ± 0.13 0.35 ± 0.07 BW0, BW28 and BW70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. ADG1 and ADG2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. It is necessary to find another closely linked marker. Given the important role of myostatin in meat traits and the short physical distance between myostatin and SJ01, we speculate that SJ01 is likely associated with some production traits in pigs and therefore is a candidate marker. In this study, the polymorphism at SJ01 locus and its associations with porcine body weight and early growth traits were analyzed in three pig breeds (Duroc, Yorkshire and Landrace) which are currently used for worldwide pork production. MATERIALS AND METHODS Population and traits Three western pig breeds, including 420 Duroc, 414 Yorkshire and 119 Landrace individuals were randomly sampled from Pig Breed- ing Center of Shandong Academy of Agricultural Sciences. Animals were reared under the same environment and had free access to feed and water. Data of the traits including birth weight (BW0), body weight on day 28 (BW28) and body weight on day 70 (BW70) of each individual were collected on farm. Sampling was performed by taking a notch from the ear of pigs and put into a 1.5-mL Eppendorf tube containing 70% ethanol and stored at −20ºC. The total number of production records was 2757 and the mean and standard deviation (s.d.) of the five traits are presented in Table 1. Isolation of genomic DNA Genomic DNA from ear notch was isolated with phenol/chloroform method (Sambrook and Russell, 2001), dissolved in Tris-EDTA buf- fer (pH 8.0) and stored at −20ºC. The concentration was checked by running 0.7% agrose electrophoresis and using UV spectro- photometer. Genotyping Primers for SJ01 were from STS database of National Centre for Biological Information (UniSTS accession number: 81993): Forward 5′-CAGAACATAAATGCCAAGAG-3′ and Reverse 5′-AGTATTTA GTGAACACCTCG-3′. PCR was performed by mixing 0.3 µL (50 - 100 ng) of genomic DNA, 1.6 µL of dNTPs (2.5 mM each), 1.2 µL of MgCl2 (25 mM), 0.5 µL each of primers (10 µM), 0.1 µL of rTaq polymerase (5 U/µL, TaKaRa, Dalian China) and 2.0 µL of 1×rTaq buffer in a 20 µL volume and running on a Mastercycler gradient (Eppendorf, Germany) according to the following program: 95ºC for 3 min, 32 cycles of 95ºC for 30 s, 55ºC for 30 s and 72ºC for 30 s and final extension at 72ºC for 5 min. The PCR products were electrophoresed on 1.5% agarose gel to check amplification efficiency. Genotyping of SJ01 was carried out by running 12% polyacryl- amide gels (arc：bis = 19:1) with 1×TBE as electrophoresis buffer at 180 V for 10 h. After electrophoresis, the gels were silver-stained and the genotype was determined according to the electrophoresis band patterns. Sequencing The PCR products were purified with Gel Extraction System B (BioDev, China), inserted into pMD-18T vector and was used to transform E. coli DH5 α competent cells. At least four clones were sequenced using the BigDye v3.1 kit (Applied Biosystem) by Shanghai Songon Co. Ltd (Shanghai, China). Statistics The genotype frequency, allele frequency, observed heterozygosity (Ho), expected heterozygosity (He), average heterozygosity (Ha), number of available alleles (na), number of effective alleles (ne) and polymorphic information content (PIC) were obtained and the deviation from Hardy-Weinberg equlibrium was tested with Popgen 32 software. Associations of SJ01 genotypes with BW0, BW28, BW70, average daily gain from birth to day 28 (ADG1) and average daily gain from day 28 to day 70 (ADG2) were performed separately in Duroc,Yorkshire and Lanrace pigs with the PROC GLM procedure of SAS 8.2 software (SAS Institute Inc, 1998). In the statistics model, genotype, farm-year-season and sex were treated as fixed effects, litter size as co-variable to eliminate the influences of litter size on birth weight (Model 1), birth weight as co-variable to eliminate the influences of birth weight on body weight (Model 2) and start body weight as co-variable to eliminate the influences of start body weight on average daily gain (Model 3). Model 1: Yijklm=µ+Di+Cj+Sk+Zl+eijklm; Model 2: Yijklm=µ+Di+Cj+Sk+Bl+eijklm; Model 3: Yijklm=µ+Di+Cj+Sk+Pl+eijkml; where Yijklm is the birth weight (BW0), body weight (BW28, BW70) Figure 1. Genotyping and identification by sequencing of microsatellite SJ01 locus. a. PCR amplification; b. genotyping. Lanes 1-11 represent genotypes AA, BD, AB, AC, BC, BE, DD, DD, CD, BB, AD and CC, respectively. c. Sequencing result, indicating dinucleotide (TG) repeats of 16, 17 and 22, respectively. and average daily gain (ADG1, ADG2) of each individual for models 1-3 respectively; µ is the overall mean of population; Di is the mean effect of genotype i; Cj is the farm-year-season effect; Sk is the effect of sex; Zl is the effect of litter size (Model 1); Bl is the effect of birth weight (Model 2); Pl is the effect of start body weight (Model 3); eijklm is the residual effect. The difference between genotypes was considered as significant when P < 0.05. AC, BC, BE, DD, CD, BB, AD and CC is shown in Figure 1b. Among the five alleles, alleles A and B were reported by our previous study, which were composed of 13 and 19 repeats of dinucleotide (TG), respectively (Jiang et al., 2004); alleles C, D and E were composed of 16, 17 and 22 repeats of dinucleotide (TG), respectively (Figure 1c) and were reported for the first time by present study. RESULTS Genotypes and alleles Analysis of SJ01 polymorphism revealed 13 genotypes (AA, AB, BB, BC, BD, BE, AC, AD, AE, CC, CD, DD and EE) and five alleles (A, B, C, D and E) in Duroc (n = 420), Yorkshire (n = 414) and Landrace (n = 119) pig popula- tions. The electriphoresis result of genotypes AA, BD, AB, Population genetic parameters at SJ01 locus Differences in genotype frequency and allele frequency were found between three pig populations of Duroc, Yorkshire and Landrace (Table 2). For genotypes with frequency exceeding 0.1, genotypes AB, AA and AD in Duroc, AB, BB and BE in Yorkshire and BB and BD in Landrace were identified. Genotypes BC, AE, CC, CD and EE occurred at low frequencies in all of the three pig Table 2. Genotype frequency of microsatellite SJ01 in Duroc, Yorkshire and Landrace populations. Breed (n) AA AB BB BC BD BE AC AD AE CC CD DD EE Duroc (420) 0.2167(91) 0.2905(122) 0.0762(32) 0.0048(2) 0.081(34) 0.0024(1) 0.0357(15) 0.2500(105) 0.0000(0) 0.0024(1) 0.0143(6) 0.0262(11) 0.0000(0) Yorkshire(414) 0.0024 (1) 0.1232(51) 0.6594(273) 0.0000(0) 0.0773(32) 0.1232(51) 0.0000 (0) 0.0024 (1) 0.0048(2) 0.0000(0) 0.0000(0) 0.0024 (1) 0.0048(2) Landrace(119) 0.0000 (0) 0.0000(0) 0.5210(620) 0.0000(0) 0.4034(48) 0.0084 (1) 0.0000 (0) 0.0000 (0) 0.0000(0) 0.0000(0) 0.0000(0) 0.0672 (8) 0.0000(0) Table 3. Allele frequency and population genetic parameters of microsatellite SJ01 in Duroc, Yorkshire and Landrace populations. Breed A B C D E H-W test Ho He Ha na ne PIC Duroc 0.5048 0.2655 0.0298 0.1988 0.0012 0.0252 0.6786 0.6351 0.6343 5.0000 2.7347 1.1309 Yorkshire 0.0676 0.8213 - 0.0423 0.0688 0.4226 0.3309 0.3148 0.3144 4.0000 1.4587 0.6618 Landrace - 0.7269 - 0.2689 0.0042 0.9308 0.4118 0.4010 0.3993 3.0000 1.6647 0.6080 Table 4. Least squares means for performances among genotypes of microsatellite SJ01 in Duroc populations. Performance AA AB AC AD BD P-value BW0 1.41 ± 0.03 (n = 88) 1.47 ± 0.03 (n = 112) 1.41 ± 0.09 (n=13) 1.47 ± 0.03 (n = 101) 1.49 ± 0.05 (n = 34) 0.7686 BW28 7.18 ± 0.17 (n = 67) 7.28 ± 0.16 (n = 72) 7.12 ± 0.50 (n=8) 7.10 ± 0.18 (n = 57) 7.06 ± 0.26 (n = 27) 0.8664 BW70 21.31 ± 0.72 (n = 57) 21.51 ± 0.64 (n = 71) 19.39 ± 2.01 (n=7) 20.00 ± 0.73 (n = 56) 20.76 ± 1.13 (n = 22) 0.3297 ADG1 0.20 ± 0.01 (n = 65) 0.21 ± 0.01 (n = 66) 0.20 ± 0.02 (n=7) 0.20 ± 0.01 (n = 55) 0.20 ± 0.01 (n = 27) 0.8664 ADG2 0.36 ± 0.01 (n = 43) 0.36 ± 0.01 (n = 43) 0.36 ± 0.07 (n=2) 0.35 ± 0.02 (n = 29) 0.36 ± 0.02 (n = 17) 0.9808 BW0, BW28 and BW70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. ADG1 and ADG2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. populations. In Yorkshire pigs, genotype BB was predominant, while in Duroc and Landrace pigs, no predominant genotype was found. As for allele frequencies, five, four and three alleles were detected in Duroc, Yorkshire and Landrace pig populations, respectively and allele B was predo- minant in Yorkshire and Landrace, whereas allele A was more prevalent in Duroc populations (Table 3). At SJ01 locus, Yorkshire and Landrace, but not Duroc, were in a state of Hardy-Weinberg equili- brium (P > 0.05). Information obtained from other genetic parameters of Ho, He and PIC (Table 3) indicated that, at this locus, Duroc is genetically more diverse compared to Yorkshire and Land- race pig populations. Associations of SJ01 genotype with birth weight and early growth traits The relationship of SJ01 genotype with birth weight and some early growth traits, that is, body weight at 28 d and 70 d and average daily gain from birth to 28 d and from 28 to 70 d, were analy- zed in present study. The results showed that, in Duroc pig populations, differences in birth weight, body weight at 28 and 70 d and average daily gain traits were not significant between genotypes of SJ01 locus (P > 0.1) (Table 4); whereas in York- shire pig populations, the least squares means for average daily gain from 28 to 70 d were signifi- cantly different among genotypes of SJ01 locus (P < 0.05) (Table 5) and in Landrace pigs, the least squares means for body weight at 70 d were significantly different among genotypes of SJ01 locus (P < 0.05) (Table 6). The effect of SJ01 on Table 5. Least squares means for performances among genotypes of microsatellite SJ01 in Yorkshire populations. Performance AB BB BD BE P-value BW0 1.44 ± 0.06 (n =51) 1.48 ± 0.02 (n = 272) 1.57±0.07 (n = 32) 1.56±0.05 (n = 51) 0.2361 BW28 7.54 ± 0.34 (n =34) 7.63 ± 0.17 (n = 161) 7.01±0.49 (n = 16) 6.80±0.37 (n = 29) 0.1581 BW70 20.45 ± 1.01 (n = 28) 22.05 ± 0.40 (n = 176) 22.48±1.21 (n = 19) 19.87±0.84 (n = 40) 0.0631 ADG1 0.21 ± 0.01 (n = 30) 0.21 ± 0.00 (n = 149) 0.17±0.02 (n = 12) 0.19±0.01 (n = 28) 0.0910 ADG2 0.42 ± 0.03 (n = 18) 0.49 ± 0.01 (n = 104) 0.53±0.04 (n = 9) 0.47±0.02 (n = 23) 0.0464 BW0, BW28 and BW70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. ADG1 and ADG2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. Table 6. Least squares means for performances among genotypes of microsatellite SJ01 in Landrace populations. Performance BB BD DD P-value BW0 1.46 ± 0.03 (n = 61) 1.44 ± 0.04 (n = 48) 1.47 ± 0.10 (n = 8) 0.8384 BW28 8.35 ± 0.25 (n = 49) 8.51 ± 0.36 (n = 25) 7.14 ± 1.22 (n = 2) 0.5400 BW70 24.53 ± 0.83 (n = 27) 26.60 ±1.33 (n = 9) 33.02 ± 2.83 (n = 2) 0.0268 ADG1 0.25 ± 0.01 (n = 48) 0.25 ± 0.01 (n = 25) 0.20 ± 0.04 (n = 2) 0.5400 ADG2 0.38 ± 0.03 (n = 20) 0.35 ± 0.04 (n = 3) - 0.6554 BW0, BW28 and BW70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. ADG1 and ADG2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. other production traits of Yorkshire and Landrace pigs was not significantly different (P > 0.5). DISCUSSION Myostatin is an important growth/differentiation factor regulating skeletal muscle development, therefore, is also associated with meat production and quality traits as well as with other related traits. Porcine myostatin gene is mapped in a SW1065-(53.1)-SW1263- (55.0)-myostatin- (55.0)-SWR1533 -(57.9) linkage group on SSC15q2.3 (Sonstegard et al., 1998), the flanking microsatellite loci SW1263 and SWR1553 were located at 55.0 and 57.9 cM, respectively. The microsatellite locus SJ01 is located 42 kb downstream of myostatin gene, more close to myo- statin gene than SWR1553. Compared with other microsatellite loci located in nonstructural gene region, the number of genotypes and alleles were rather few (Jiang et al., 2004 and present study) and the hetero- zygosity and diversity were rather low for SJ01, sug- gesting relatively higher selection force and more conser- vation in this region than microsatellite loci located in nonstructural gene regions. According to the results of SJ01 polymorphism, of the three pig breeds used in this study, Duroc pigs were more diverse and its genetic component was more complex. A QTL for average daily gain from birth to 70 d was mapped on SSC15 (50 cM, male; 78 cM, female) (Knott et al., 1998), where myostatin gene and SJ01 locus also reside. In this study, SJ01 locus is found to be associated with average daily gain from 28 to 70 d in Yorkshire pigs and with body weight at 70 d in Landrace pigs. Besides, a nearly significant association was also noted for SJ01 with body weight at 70 d and average daily gain from birth to 28 d in Yorkshire pigs. These data suggest that this region likely harbors a QTL affecting early growth and SJ01 is a useful marker for refining this QTL. Growth traits, such as body weight and average daily gain, are controlled by multiple genes. In addition to the aforementioned QTL for average daily gain from birth to 70 d on SSC15 (Knott et al., 1998), other QTLs for body weight of 10 weeks were found on SSC1, 4, 6, 7 and 9 and for average daily gain from 4 weeks to 13 weeks on SSC9 and SSC10, from 3 weeks to 10 weeks on SSC 1, 2, 4 and 7, respectively (http://www. animalgenome.org/ QTLdb /pig.html). The effect of each gene or QTL on early growth traits is somewhat dependent on genetic background and environmental effect, giving rise to the differences between breeds concerning the relationship of SJ01 genotype with these traits, as shown in this study. Although three relatively large populations of Duroc, Yorkshire and Landrace were used for association study, some genotypes were present at low frequency, that is, genotype DD in Landrace population. 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