Breeding of Duroc Breeds Homozygous for MUC13 Diarrhea-resistant Allele

Guorong RUAN,Xiaofang HE,Jing HUANG,Shijun XIAO,Jinxiong CHEN,Junshan LI,Yaxuan LIU,Yaohua SUN,Fuzhen CHEN,Guozhong LIN

1.Fujian Vocational College of Agriculture,Fuzhou 350119,China;

2.Institute of Animal Husbandry and Veterinary Science,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;

3.Candidate of National Key Laboratory for Animal Biotechnology,Jiangxi Agricultural University,Nanchang 330045,China;

4.Xiamen Guoshou Breeding Pig Development Co.,Ltd.,Xiamen 350021,China;

5.Fujian Guanghua Best Eco-agriculture Development Co.,Ltd.,Youxi 365100,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

Diarrhea is a major cause of death in piglets.According to the survey,the piglets that die of diarrhea account for 38.9% of the total dead piglets[1].To control diarrhea,the currently commonly-used method is to add large-dosage of antibiotics in feed or to inoculate with vaccines.However,the control effects are dissatisfactory.Even worse,drug resistance may occur in piglets,thereby affecting later growth of piglets and resulting in the excessive antibiotic residues in piglets.Therefore,the breeding of disease-resistant pigs has attracted more and more attention.The breeding will change the disease resistance of pigs at the gene level.Thus new pig breeds that are congenitally resistant to diarrhea will be selected.

Studies have shown that ETEC F4 is the most important kind of pathogenic bacteria that leads to the occurrence of diarrhea in newborn and sulking piglets[2-4].ETEC F4 has three serotypes,F4ab,F4ac and F4ad.Its main serotype is F4ac[5].The pathogenicity of ETEC F4 depends on whether it can specifically combine with receptor on the surface of epithelial cells of swine intestinal brush border.The receptor-free pigs are resistant to diarrhea; if not,the pigs are susceptible to diarrhea.Porcine ETEC F4 receptor is controlled by a single gene,and it is a recessive inheritance.The pigs that carry the dominant allele are vulnerable to infection of ETEC F4,but the pigs that are homozygous for recessive allele are resistant[6-7].Therefore,the positioning and identification of ETEC F4 receptor locus is the key for diarrhea-resistant breeding.Considering the refined localization of ETEC F4 receptor locus and identification of major gene,Joller et al.[8]fund the ETEC F4 receptor locus is located between SW207 and MUC4.Huang et al.[8-12]finally identified the target gene,MUC13,which encodes the ETEC F4 receptor using a range of modern genetic analysis.They found the molecular marker that can accurately identify the F4ac resistant and susceptible individuals (accuracy ＞97%).Performance measurement and BLUP genetic evaluation haven been considered to be the most effective methods to improve pig performance at home and abroad in recent 20 years.Many domestic and foreign enterprises have applied the methods above into pig breeding,and remarkable results have been achieved[13-16].So the combination of anti-diarrhea molecular markerassisted breeding technology,performance measurement and BLUP genetic evaluation and breeding technology will undoubtedly contribute to the breeding of new diarrhea-resistant pig lines with superior performance.

Among the introduced lean-type pig breeds,the frequencies of favorable resistant genes are relatively high in Duroc pigs[17-18].The Duroc pigs can probably be bred into resistant pig breed through generations of selection.Moreover,in the production of commercial pigs,Duroc pig is often used as a terminal male parent.So Duroc pigs are also characterized by wide range of impact.In this study,two national nuclear breeding farms that are qualified and skilled to carry out performance measurement were selected.Then a comprehensive breeding study on homozygosis for MUC13 resistant allele and performance measurement was carried out in Duroc pig population so as to breed F4ac diarrhea-specifically resistant new lines with superior performance.

Material and Methods

Material

According to blood relationship,production performance and body conformation,two nuclear breeding groups of Duroc pigs were organized.A total of 275 (25 ♂,250 ♀) pigs were selected,including220 pigs(20♂,200♀)from Xiamen Guoshou Breeding Pig Development Co.,Ltd.and 55 pigs(5♂,50♀)from Fujian Guanghua Best Eco-agriculture Development Co.,Ltd.The genotype detection,performance measurement and breeding were synchronized between the two groups.In addition,the feeding,management and nutritional level were also consistent between the two groups.During the test,a total of 6 boars were selected for blood exchange.

Breeding schemes

Based on the original breeding populations in two farms,two nuclear breeding populations were organized(25 ♂,250 ♀).The information on mating,conception,farrowing and growth of individuals were detailedly recorded.According to the performance of litters and individuals,the weaned piglets from the nuclear breeding populations were selected primarily.The litters in which genetic disorders occurred were excluded.Among the remaining litters,1 -2 ♂and 2-3♀were selected from each of the litters,respectively.

For the selected piglets,the performance measurement and genotype detection were completed.Based on the performance measurement results,the breeding values of individuals were calculated by using BLUP method.Then for the piglets with high comprehensive index and favorable resistant genotype,the body conformation identification was carried out.Only the excellent piglets that had higher comprehensive index and qualified body conformation were selected into the next-generation nuclear breeding population.The partially generation overlapping was allowed.

The three-consecutive-year repeated detection and selection were conducted.Moreover,the changes in production performance indicators were explored.On the other hand,the occurrence of diarrhea in piglets was compared between the nuclear breeding population and original propagating population.

Detection of MUC13 gene

Collection and preservation of samplesAccording to the requirement,the ear samples of the selected piglets from the nuclear breeding population of each generation were collected to identify their MUC13 genotypes.A piece of ear tissue was collected from each of the piglets with ear clamp.The ear samples were timely placed in sample collection tubes containing 75%of alcohol.Then the lids of tubes were tightened.The ear marks of sampled piglets were recorded on both tube lids and bodied with a marker pen.The collected ear samples were preserved at-20 ℃.

Extraction of DNAThe DNA of samples was extracted with phenolchloroform.The concentrations and quality of extracted DNA were examined with UV/VIS scanning spectrophotometer (Nanodrop ND-1000,thermal,USA).Only the qualified extracted DNA was preserved at-20 ℃.

Gene identificationAccording to the patented technology,developed by Jiangxi Agricultural University for identifying genotyping patterns of MUC13 locus in susceptible/resistant individuals,the PCR-snapshot primers were designed by the Sangon Biotech(Shanghai)Co.,Ltd.The sequences of PCR primers were as follows (5’-3’):Fp,GGAGAGACCAAACCCACAGA;Rp,CTCCTCACCAGCTCCTTAGC.The length of target fragment was 280 bp.The PCR reaction system was as follows (20 μl):DNA template 40 ng,10×buffer 2.0 μl,25 mmol/L MgCl21.2 μl,10 mmol/L dNTP 0.3 μl,Fp 0.4 μl,Rp 0.4 μl,Taq polymerase 0.5 μl,ultrapure water 13.2 μl.The PCR reaction conditions were as follows:predenaturation at 94 ℃for 3 min,denaturation at 94 ℃for 30 s,annealing at 61 ℃for 30 s,extension at 72 ℃for 45 s,36 cycles.

The MUC13 genotype was identified with PCR-SNaPshot.The SNaPShot reaction system was as follows:purified PCR product 1.5 μl,SNaPshot multiplex mix (containing Taq polymerase and fluorescently labeled dd-NTPs) 2.0 μl,deionized water 1.2 μl,SNaPshot primer 0.3 μl (sequence,TTTTTTTTTTTTTTTCCATGTACATTTCAGAGTCTGAGGGAT).The SNaPShot reaction conditions were as follows:96 ℃10 s,50 ℃5 s,60 ℃30 s,25 cycles.After the reaction,0.57 μl of 1 ×NEB buffer and 0.1 μl of CIP(NEB,USA) were added to the reaction product to purify the reaction product(37 ℃60 min,75 ℃15 min).Thus the fluorescently labeled dd-NTPs would be removed and the purification enzyme would be inactivated.At final,the genotyping patterns were examined with capillary electrophoresis.The mixture of SNaPshot reaction product(1 μl) and sample denaturant (8 μl)were denaturized at 95 ℃for 5 min.The sample denaturant was prepared by mixing Hi-Di formamide and GeneScan 120 LIZsize standard (v:v,20:1).Then the mixture was rapidly transferred to ice bath for 2 min.After cooled,the samples were centrifuged.The supernatants were used for gel electrophoresis (3130XL Genetic Analyzer,ABI,USA).The electrophoresis results were analyzed with GeneMapper 4.0 (ABI,USA).The identification standards were shown in Fig.1.

Observation of diarrhea in piglets

From each of the breeding population and propagating population,10 pairs of Duroc sows at the same litter were selected,respectively.They were arranged in the same piggery.For the equivalently-aged (difference less than one week) piglets,their mothers’ear markers,farrowing times and piglet numbers per litter were recorded.In addition,the ear markers,ages,durations and treating times of piglets that were infected with diarrhea were also recorded.The incidences of diarrhea and survival rates were calculated.

Determination of growth performance

According to the requirements by"Swine Performance Measurement Technical Specification" (NY/T 822-2004),the entire litter in which there was one or more piglets having low reproductive performance or infected with genetic disorders was excluded.The selected weaned piglets from the remaining litters of each generation(1-2♂and 2-3♀/litter) were concentrated in measurement house.They were bred under conventional conditions.When their body weights grew to 85 -105 kg,performance measurement was completed.In addition,the live backfat thickness was determined by using B-device (ALOKA 500).All the measured data was corrected to 100 kg of body weight using GBS.

Comparison of growth performance among different genotypes

Based on the results of performance measurement and MUC13 genotype detection,the effects of 3 kinds of MUC13 genotypes on growth performance of 525 piglets from three batches of the two farms were investigated.

Data analysis

The incidences of diarrhea and survival rates of piglets were analyzed using the Chi-square test module of SPSS 13.0,and the data of growth performance indexes was analyzed using the One-Way ANOVA and Post Hoc modules.

Results and Analysis

Detection of MUC13 gene

As shown in Table1,through a three-generation genetic identification and screening,the frequency of favorable diarrhea-resistant allele G was significantly increased,but the frequency of unfavorable allele A in nuclear breeding population was decreased generation by generation,until completely removed.

Occurrence of diarrhea in piglets and survival rate of piglets during lactation

Table1 Genotyping patterns at the MUC13 locus of the three-generation pigs

Table2 Occurrence of diarrhea in piglets from nuclear breeding population and ordinary propagating population

Table3 Growth performance of breeding pigs of three generations

Table4 Growth performance of breeding pigs of different genotypes

Table2 showed that under conditions of same litter,same feeding and same management,the incidences of diarrhea in piglets from nuclear population and propagating population were 8.7% and 18.4% respectively.There was a significant difference in incidence of diarrhea in piglets between the two populations (P = 0.006).The diarrhea-infected piglets from the nuclear population were cured in a relatively short time,but the treatment of diarrhea-infected piglets from the propagating population required 3-4 d more under the same treating conditions.The mortalities of diarrhea in piglets from the nuclear population and the propagating population were 12.5% and 16.7%,respectively.However,the difference in mortality of diarrhea was not significant between the two populations (P＞0.05).There was also no significant difference in survival rate of piglets between the two populations at the lactation (94.57%vs.92.23%,P＞0.05).

Performance determination and breeding results

Through three-consecutive-year performance measurement and BLUP-index selection,the growth rate and live backfat thickness were all improved to some extent; the days required by piglets to grow to 100 kg of body weight were reduced from 181.34 to 177.48 d.However,there were no significant differences in days required by piglets to grow to 100 kg among three years (P＞0.05).The live backfat thickness was reduced from 12.23 to 10.77 mm.The multi-comparison analysis showed the live backfat thickness in 2010 was significantly higher than that in 2012(P ＜0.05).

Correlation between MUC13 genotype and growth performance

Table4 showed the days required by 100 kg AA genotype pigs that were susceptible to diarrhea was increased only by 1.56 d compared to that required by 100 kg GG genotype pigs that were resistant to diarrhea.The backfat thickness was increased by 0.36 mm in average.The days required by 100 kg GA genotype pigs that were susceptible to diarrhea was increased by 1.34 d compared to that required by GG genotype pigs.The backfat thickness was increased by 0.27 mm.However,no significant differences were shown in days required by 100 kg body weight or backfat thickness of 100 kg body weight among the three genotypes(P ＞0.05).It is indicated there are no significant differences in growth rate or live backfat thickness among different-MUC13 genotype pigs.

Conclusions and Discussion

Breeding of pigs homozygous for MUC13 resistant allele

Through three-consecutive-year MUC13 genotype identification and screening and performance measurement-based BLIP index selection,the frequency of MUC13 diarrhea-resistant allele G was increased from 0.86 to 1.00 and the frequency of GG genotype was increased from 0.75 to 1.00,realizing the goal of homozygosis for diarrhea-resistant favorable allele.At the same time,the growth rate and live backfat thickness were also improved to some extent.The days required by 100 kg body weight was reduced from 181.34 d to 177.48 d(P ＞0.05),and the live backfat thickness was reduced from 12.23 mm to 10.77 mm(P ＜0.05).However,due to the restriction by actual production time,the detection and measurement inside farms were lasted as long as six months.There were also great differences among individuals.Although the BLUP model could correct the error caused by different farms,different years,different seasons,etc.,certain error still existed in the measured data during the SPSS analysis.Therefore,in the future measurement and breeding,the mating of sows in nuclear population must be arranged in a small time range so as to reduce the adverse effect of long time pan.In addition,the nuclear breeding population should be remained homozygous for diarrhea-resistant allele.The sow candidates should be persistently selected from the original breeding population.If exotic genealogy is needed to be introduced,the MUC13 genotype of introduced individuals must be detected,thereby preventing the introduction of susceptible alleles.

Relationship between occurrence of diarrhea in sucking piglets and homozygosis for MUC13 resistant allele

There are many factors leading to the occurrence of diarrhea in sulking piglets.But enterotoxigenic E.coli is the major factor causing the diarrhea.It has been researched and validated that the resistance/susceptibility to diarrhea caused by E.coli in piglets can be deducted from the MUC13 genotype because the MUC13 genotype determines the encoding (or not) of enterotoxigenic E.coli receptor[9-12,19-20](patent number:200810136425).According to census,under conditions of natural selection,the degree of homozygosis for MUC13-resistant allele in Duroc pigs has reached 70%-80%.In this study,the occurrence of diarrhea in piglets from nuclear breeding population and original propagating population was investigated under conditions of same litter,same feeding and same management.The results showed the incidences of diarrhea in pre-weaned piglets from the breeding population and propagating population were 8.7% and 18.4%,respectively.The difference analysis showed the difference in incidence of diarrhea was significant(P ＜0.01).The survival rate of weaned piglets from the breeding population and propagating population were 94.6% and 92.3% (P ＞0.05).A total of 20 litters of piglets were selected for each of breeding population and propagating population,so the obtained results have certain reference value.But a larger range of study is needed to be conducted so as to validate the relationship between the homozygosis for MUC13-resistant allele and occurrence of diarrhea in preweaned piglets.

Relationship between homozygosis for MUC13 resistant allele and growth performance of pigs

The homozygosis for MUC13-resistant allele can reduce the incidence of diarrhea in piglets.But there are rare reports on the adverse effects of homozygosis for MUC13-resistant allele on other production performance indexes of piglets.In this study,the growth performance of different-genotype individuals was measured.The results showed the days by 100 kg GG,GA and AA genotype pigs were 181.25,182.59 and 182.81 d,respectively; and the backfat thicknesses of 100 kg GG,GA and AA genotype pigs were 11.50,11.77 and 11.86 mm.The difference analysis showed there were no significant differences in days required by 100 kg body weight and live backfat thickness among differentgenotype pigs(P ＞0.05).It is suggested the homozygosis for MUC13 resistant allele has no significant effects on growth rate and backfat thickness of pigs.

The three-consecutive-year comprehensive breeding that combined the molecular detection and performance measurement validated that the homozygosis for MUC13 resistant allele contributes to reducing the incidence of diarrhea in pre-weaned piglets; the growth performance measurement and BLUP evaluation can effectively reduce live backfat thickness;the homozygosis for MUC13 resistant allele has no significant effect on growth and development of pigs.

Acknowledgements

We would express our gratitude to researcher Ren Jun for his guidance on genetic tests and modifications to this paper.

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