Beneficial Genotype of Swine FUT1 Gene Governing Resistance to E. Coli F18 Is Associated with Important Economic Traits

c Indian Academy of Sciences

RESEARCH NOTE

Beneﬁcial genotype of swine FUT1 gene governing resistance to E. coli F18 is associated with important economic traits

WEN-BIN BAO1,4, LAN YE1, ZHANG-YUAN PAN1,JINZHU1, GUO-QIANG ZHU2,4, XUE-GEN HUANG3,4 and SHENG-LONG WU1,4∗

1Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, and 2College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China 3Suzhou Taihu Pig Breeding Centre, Jiangsu Province, Suzhou, Jiangsu 215000, People’s Republic of China 4Jiangsu Engineering Research Centre for Molecular Breeding of Pig, Changzhou, Jiangsu 213149, People’s Republic of China

[Bao W.-B., Ye L., Pan Z.-Y., Zhu J., Zhu G.-Q., Huang X.-G. and Wu S.-L. 2011 Beneﬁcial genotype of swine FUT1 gene governing resistance to E. coli F18 is associated with important economic traits. J. Genet. 90, 315–318]

Introduction breeds (Shi et al. 2003;Yanet al. 2004;Wuet al. 2006;Bao et al. 2008). The above results suggested that it is not fea- Porcine post-weaning diarrhoea (PWD) and porcine edema sible to select AA resistant genotype in FUT1 directly from disease (ED) are bacterial diseases caused by enterotoxigenic Chinese native pig breeds. In addition, we should target the Escherichia coli F18 (ETEC F18). These diseases cause breeding of ETEC F18 resistant breeds in imported breeds economic loss to the swine industry. The pathogenicity of only. ECF18 bacteria depends on the existence of ECF18 receptor Sutai pig is the hybridization product of Duroc and Taihu in the brush border membranes of the small intestinal mucosa pigs after 15 years of practices. In 1999, it was approved by in piglets (Nagy and Fekete 1999). Using the candidate gene National Committee of Livestock and Poultry Species as a approach and linkage analysis, Vogeli et al. (1997) demon- α new breed. Using a small number of AG type (9.2%) indi- strated that (1, 2)-fucosyltransferase gene (FUT1) in chro- viduals in FUT1 gene detected from Sutai pigs, our group mosome 6qll is the candidate gene controlling the adhesion conducted proper selection and assortative mating. While to F18 receptor. Research by Meijerink et al. (1997) showed we were working on the selective breeding for resistance to the existence of a G/A mutation at nucleotide 307 of FUT1 ETEC F18 in Sutai pig, we paid careful attention not only open reading frame (ORF); and that pigs with genotype AA to the resistant varieties of ETEC F18 but also to the stabil- are resistant to ETEC F18 and pigs with genotype GG or the ity and improvement of some important economic traits and heterozygote AG are sensitive to ETEC F18. Breeding for general disease resistance ability. The effects on these addi- disease resistance could be implemented by marker-assisted tional traits directly determine the feasibility of further breed- selection using FUT1 gene as the ETEC F18R candidate ing for diseases resistance. Based on the polymorphic detec- gene (Meijerink et al. 1997). In another study, we adopted in tion of FUT1 gene in the breeding base population of Sutai vivo adherent experiment of pig intestinal epithelial cells and pigs resistant to ETEC F18, we analysed the relationship of F18 E. coli and showed that the intestinal epithelial cells of these genotypes to the growth and development, and repro- piglets with GG and AG genotypes in FUT1 gene can adhere ductive ability. Therefore, we could further study the genetic to the wild E. coli and can also adhere to the recombined effect of FUT1 gene. This is necessary for designing the new E. coli which is expressing F18 pilus and F18 subunit F resistance varieties system of Sutai pigs in future. in vitro (Wu et al. 2006). While studying the polymorphism at nucleotide 307 of FUT1 ORF in Chinese native pig breeds and imported breeds, some Chinese researchers found that the AA resistant Materials and methods genotype polymorphism at M307 was detected in imported Experimental materials breeds, the AA genotype was not found in any Chinese native Small amount of AG type (9.2%) individuals in FUT1 gene ∗ For correspondence. E-mail: [email protected]. detected from Sutai pigs were used for proper selection Keywords. pig; FUT1 gene; growth and development; reproductive performance.

Journal of Genetics, Vol. 90, No. 2, August 2011 315 Wen-Bin Bao et al. and assortative mating to establish an ETEC F18 resistant B-ultrasound scanner: scapuloposterior position around the resource population of 576 Sutai pigs. About 1.0 g of ear 4th or 5th rib, last rib, and lumbosacral junction at 4 cm tissue was collected from every individual and placed into to the dorsal line) of 60-kg pigs (gilt pigs) were measured. a 1.5 mL Eppendoff tube in ice boxes before taking to the After reaching reproductive ability stable for the 3rd and lab. A routine phenol–chloroform method was used to extract 4th parity, the total number born (TNB), number born alive DNA from each sample. (NBA), body weight of litter (BWL), number of alive weaning litter (NW) and weaning weight of litter (WWL) were PCR-RFLP analysis measured. Using the general linear model (GLM) in SPSS ver- Primers: 5 -CCA ACG CCT CCG ATT CCT GT-3 and sion 11.5 software (SPSS, Chicago, USA), several charac- 5 -GTG CAT GGC AGG CTG GAT GA-3 were designed teristic values and relationship between different genotypes according to FUT1 gene sequence in GenBank (accession after endonuclease digestion were predicted by least square no. L50534). Exactly 25 μL of PCR product included 100 ng procedure. of template DNA, 5 pmol of each primer, 10× PCR Buffer, 2 mM dNTP mixture and 1 U of Taq DNA polymerase (TakaRa, Dalian, China) was used. PCR conditions carried out by thermal cycle, were 95◦C for 5 min, and then 94◦Cfor 40 s, 60◦C for 40 s, 72◦C for 45 s, 30 cycles and an exten- sion at 72◦C for 10 min. PCR products were checked in 1% Results and discussion agarose gels stained with ethidium bromide. PCR product, PCR-RFLP analysis for FUT1 gene 10 μL each, was digested overnight at 37◦Cby2UofHin6I (Sangon, Shanghai, China) restriction enzyme. The digested PCR products were detected with 1% agarose electrophore- fragments were electrophoresed in 10% polyacrylamide gels sis. A clear specific DNA band was shown at position 161 bp in 1× TBE at a constant voltage of 120 V, stained by silver which agreed with the predicted amplified fragment size and visualized under ultraviolet light using transilluminator. There is a restriction recognition site for Hin6I at position M307 in FUT1 gene that can be completely digested by Hin6I to produce GG phenotype (117/44 bp). If this posi- Performance measurement and statistic analysis tion has a G→A mutation, Hin6I enzyme would not be Individual body weight of breeding base population of Sutai able to digest the fragment resulting in AA type (161 bp). pigs was measured for newborn litter and 35-day-weaning When both alleles are existed together they produced AG litter. The body length, height, back width and back fat thick- type (161/117/44 bp). The restriction map is shown in ness (three points averages were detected for live body by figure 1.

Figure 1. The genotyping results of FUTI gene by PCR-RFLP. Lanes 2, 4 were GG genotype; lanes 6, 7, 8 were AA genotype; lanes 1, 3 and 5 were AG genotype. M was pUC19 DNA/Msp I marker (Sangon, Shanghai, China).

316 Journal of Genetics, Vol. 90, No. 2, August 2011 Growth correlates of porcine FUT1

Polymorphism of FUT1 gene in Sutai pigs table 1. The average growth and development of AA type was better than those of AG and GG types. The weaning weight Among the 576 samples, the number of AA, AG and GG of litter at 35th day of AA and AG types were significantly genotypes in FUT1 gene were 135, 351 and 90, respectively. higher than that of GG type (P < 0.05). The body length Their corresponding genotype frequencies were 0.235, 0.609 of AA type was significantly higher than that of AG and GG and 0.156. The gene frequency for disease resistant gene A types (P < 0.05), with no significant difference between AG was 0.540, while the frequency for sensitive gene G was and GG types (P > 0.05). The body height and back width of 0.460. Before the selective breeding, no AA genotype was AA type were both significantly higher than those of AG type detected in Sutai pigs. The frequencies for AG and GG geno- (P < 0.05), with no significant difference from those of GG types were 0.092 and 0.908, respectively. The gene fre- type (P > 0.05). In terms of back fat thickness, AA type was quency for disease resistant gene A was 0.046, while the lower than AG and GG types with no significant difference frequency for sensitive gene G was 0.954 (Bao et al. 2008). among genotypes. This suggests that the artificial selection (selectively main- Jiang et al. (2005) proposed that AA genotype is a benefi- taining AG and AA types) substantially affected the FUT1 cial genotype for meat quality and carcass traits. Increasing gene distribution in the breeding base population of Sutai the frequency of the A allele not only enhances the groups’ pigs resistant to ETEC F18. anti-disease ability, but also improves the meat quality and carcass traits. He et al. (2006) found the fat content of pig with AA type in FUT1 gene was lower than that of AG and Relationship between FUT1 gene polymorphism, early growth GG types, with larger amount of total meat than AG and and development GG types and better meat quality than GG type. The results The result of least square method for three genotypes of of our study also indicate that AA genotype is a beneficial FUT1 gene and early growth and development are shown in genotype for growth and development performance. When

Table 1. The correlation between FUT1 gene and early growth and development in Sutai pigs.

Genotype Litter weight Weaning weight Body length Body height Back width Back fat (kg) of litter (kg) (cm) (cm) (cm) thickness (cm)

AA 1.26 ± 0.05 7.98 ± 0.280a 101.17 ± 0.89a 57.52 ± 0.62a 26.26 ± 0.37a 13.52 ± 0.59 AG 1.24 ± 0.08 7.69 ± 0.21a 98.36 ± 0.682b 56.00 ± 0.473b 25.35 ± 0.28b 14.12 ± 0.45 GG 1.17 ± 0.04 6.71 ± 0.403b 97.10 ± 1.35b 56.20 ± 0.93ab 25.85 ± 0.56ab 14.20 ± 0.90

Note: different superscript letters in the same column means signiﬁcant difference (P < 0.05); same letter label or no letter means insigniﬁcant difference (P > 0.05).

Table 2. The correlation between FUT1 gene resistant to F18 and the 3rd parity reproductive ability in Sutai pigs.

Genotype Number Total number Number born Body weight Number of alive Weaning weight born alive of litter (kg) weaning litter of litter (kg)

AA 135 15.81 ± 3.20 13.66 ± 1.49 15.66 ± 1.99 12.88 ± 1.86a 96.96 ± 6.97a AG 351 14.41 ± 2.86 12.64 ± 2.30 13.95 ± 2.80 10.99 ± 1.28b 78.90 ± 7.20b GG 90 14.23 ± 3.14 12.52 ± 2.35 13.77 ± 2.11 10.93 ± 1.29b 76.69 ± 8.26b

Note: same letter labels means insigniﬁcant difference; different letter labels means signiﬁcant difference.

Table 3. The correlation between FUT1 gene resistant to F18 and the 4th parity reproductive ability in Sutai pigs.

Genotype Number Total number Number born Body weight of Number of alive Weaning weight born alive litter (kg) weaning litter of litter (kg)

AA 135 15.92 ± 2.96 13.63 ± 2.12 15.10 ± 2.09 12.93 ± 1.80a 96.06 ± 9.26a AG 351 15.02 ± 2.77 12.74 ± 2.46 13.89 ± 2.72 11.05 ± 1.23b 79.90 ± 8.20b GG 90 14.93 ± 4.14 12.66 ± 2.86 13.96 ± 2.08 10.99 ± 1.19b 79.69 ± 9.05b

Note: same letter labels means insigniﬁcant difference; different letter labels means signiﬁcant difference.

Journal of Genetics, Vol. 90, No. 2, August 2011 317 Wen-Bin Bao et al. practising selective breeding for disease resistance, in addi- F18. The incidence and mortality rate of these diseases are tion to raise a population with resistance to infectious dis- relatively high, with low cure rate. Our data showed that the eases, we also expect to breed a population with stable or NW and WWL of the 3rd and the 4th parities of sows with even better production performances. Our experimental data AA genotype were significantly higher than those of AG and showed that selective breeding in Sutai pigs resistant to GG types (P < 0.05). The reason might be that pigs with ETEC F18 not only enhanced the frequency of the A allele AA genotype are resistant to ETEC F18 resulting in relatively but was also beneficial for better growth and development high survival rates before and after weaning and WWL. for production performance. The resistant Sutai pigs with AA genotype at position 307 in FUT1 were not only resistant to ED and PWD in piglets, but also had better early growth and Acknowledgments development abilities. This positive correlation is valuable and can be applied for future breeding studies. This research was supported by Genetically Modified Organ- isms Technology Major Project of Peoples Republic of China (2009ZX08006-004B), Jiangsu Province Science and Technol- Relationship between FUT1 gene polymorphism and ogy Supporting Project (agriculture) (BG2006302, BE2008364, reproductive ability BE2009330-2, BE2010371) and Jiangsu Academic Nature Science Fundamental Research Project (08KJB230004). The relationship between FUT1 gene in the breeding base population of Sutai pigs resistant to ETEC F18 and reproductive ability of the 3rd and 4th parities is shown in tables 2 and 3, respectively. The NW and WWL of AA type in the 3rd and References 4th parities were significantly higher than those of AG and GG types (P < 0.05). The TNB, NBA and BWL of AA type Bao W. B., Wu S. L., Musa H. H., Zhu G. Q. and Chen G. H. 2008 Genetic variation at the alpha (1) fucosyltransferase (FUT1) were also higher than those of AG and GG types, with no sig- gene in Asian wild boar and Chinese and Western commercial nificant difference (P > 0.05). Regarding the reproductive pig breeds. J. Anim. Breed. Genet. 125, 427–430. ability, AA type is a beneficial genotype. Bao W. B., Wu S. L., Cao J. J., Huang X. G. and Chen G. H. 2009 Research of Liu (2001, Study on the effects of halothane Polymorphism of FUT1 gene and its relationship with litter size gene and FUT1 gene on reproduction performance of pigs. in Duroc pigs. Chin.J.Anim.Sci.3, 5–7. He X. P., Peng Y. B., Fan B., Li K., Zhu M. J., Peng Z. Z. et al. Master Thesis, Zhejiang University, Hangzhou, China) found 2006 Study on the detection of swine α-1 fucosytransferase poly- that the effects of three genotypes of FUT1 on TNB and morphism and its correlation with traits. Swine Hlth. Prod. 3, NBA did not show significant difference. The dose effect 26–28. of A gene is outstanding. The foetal survival in pigs with Jiang X. P., Liu Y. G., Xiong Y. Z. and Deng C. Y. 2005 Effects of recessive AA homozygote was better than that in pigs with FUT1 gene on meat quality and carcass traits in swine. Hereditas (Beijing) 27, 566–570. dominant GG homozygote. Zhang (2004, Analysis of 1,2- Meijerink E., Fries R., Vogeli P., Masabanda J., Wiager G., Stricker fucosyltransferase (FUT1) polymorphism and its correlation C. et al. 1997 Two α (1,2) fucosyltransferase genes on porcine with piglet traits. Master Thesis, Shanxi Agricultural Uni- chromosome 6q11 are closely linked to the blood group inhibitor versity, China) stated that the polymorphism at M307 in (S) and Escherichia coli F18 receptor (ECF18R) Loci. Mamm. FUT1 gene significantly affects the TNB, with little effect on Genome 8, 736–741. Nagy B. and Fekete P. Z. 1999 Enterotoxigenic Escherichia coli the NBA. The order of average TNB for the three genotypes (ETEC) in farm animal. Vet. Res. 30, 259–284. was: AA > AG > GG.Baoet al. (2009) found that TNB in Shi Q. S., Huang S. Q., Liu X. C., He C. Q. and Jiang J. 2003 Poly- every parity of AA genotype in Duroc was higher than that morphism of E. coli F18 receptor gene in different pig breeds. of AG and GG types. In addition, the TNB in the 1st and the Acta Genet. Sin. 30, 221–224. 4th parities of AA type were significantly higher than those in Vogeli P., Meijerink E., Fries R., Stricker C. and Bertschinger H. U. 1997 A molecular test for the detection of E. coli F18 receptors: other two genotypes. This indicates that the polymorphism at a breakthrough in the struggle against edema and post-weaning M307 in FUT1 significantly affects the TNB in Duroc. The diarrhoea in swine. Schweiz. Arch. Tierheilk. 139, 479–484. reproductive ability of pigs is relatively stable in the 3rd and Wu S. L., Yuan Z. W., Ju H. P., Zhu G. Q., Wang J. Y., Song C. Y. the 4th parities. Therefore, the reproductive ability of pigs et al. 2006 Study on genetically susceptible piglets of small at the 3rd and the 4th parities is one of the most important intestinal epithelium receptors to pathogenic F18 fimbrial Escherichia coli adhesion in vitro. Chin.J.Vet.Sci.26, 622–625. indexes for selective breeding. In many swine diseases, PWD Yan X. M., Guo Y. M., Ding N. S., Ren J. and Huang L. 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Received 21 October 2010, in ﬁnal revised form 6 December 2010; accepted 4 January 2011 Published on the Web: 19 August 2011

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