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Associations of polymorphisms in the Mx1 gene with immunity traits in large White x Meishan [F.sub.2] offspring.

ABSTRACT: The mouse myxovirus resistance protein 1 (Mx1) is known to be sufficient to confer resistance to influenza viruses, and the gene encoding Mx1 is, therefore, an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; the full-length coding region of the pig Mx1 gene spans 2,545 bp (M65087) and is organized into 17 exons compared with the human ortholog mRNA. In this study, the exons 9, 10 and 11 and introns 6 and 9 of the porcine Mx1 gene were cloned and sequenced. Two SNPs were identified in exons 9, 10 and 11 but none of the SNPs led to an amino acid exchange, and the other eleven variants were detected in introns 6 and 9, respectively. Differences in allele frequency between Meishan and other pig breeds were observed within intron 6, of which an A [right arrow] G substitution at position 371 was detected as an SnaBI PCR-RFLP. The association analysis using the Large White x Meishan [F.sub.2] offspring suggested that the Mx1 genotype was associated with variation in several immunity traits that are of interest in pig breeding. However, further investigations in more populations are needed to confirm the above result. (Key Words: Pigs, Mx1 Gene, SnaBI Locus, Immunity Traits)

INTRODUCTION

The myxovirus resistance protein (Mx) is the key component of the antiviral state induced by type I (a/b) interferons (IFN) in many species (Haller et al., 1998), and the Mx1 gene plays an important role in immune response, induction of apoptosis, and signal transduction. They were discovered 20 years ago in an inbred mouse strain that showed an unusually high degree of resistance towards infection with influenza A virus (FLUAV) (Horisberger et al., 1983). Early experiments demonstrated that the mouse Mx1 protein had intrinsic antiviral activity and was the sole mediator of innate immunity against FLUAV in mice (Staeheli et al., 1986; Arnheiter et al., 1990). Later, Mx proteins were characterized as high-molecular-weight GTPases (Horisberger et al., 1990; Nakayama et al., 1991) with significant homology to mammalian dynamins and yeast VPS-1 (Obar et al., 1990; Rothman et al., 1990; Staeheli et al., 1993). In humans, two distinct Mx GTPases are encoded on chromosome 21, called MxA and MxB. Only MxA has detectable antiviral activity. Human MxA has a comparatively wide antiviral spectrum against different types of viruses, irrespective of their intracellular replication site. MxA-sensitive viruses include members of the bunyaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, togaviruses, picornaviruses, and hepatitis B virus, a DNA virus with a genomic RNA intermediate (Haller et al., 1998; Landis et al., 1998; Chieux et al., 2001; Gordien et al., 2001). In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is, therefore, an interesting candidate gene for disease resistance in farm animals (Morozumi et al., 2001). Immunity traits are as important as growth rate and body composition which are important characteristics in pig production. Efficient pig production systems not only demand lean individuals with high growth rate and a low conversion of feed to meat but also high resistance to disease (Wang et al., 2006). However, until now no evidence of association between polymorphisms and economic traits in the porcine Mx gene has been reported. The objectives of this research were to screen the gene for polymorphisms, thereby enabling further study of the functions of the Mx1 gene in immune competence, including considering it as candidate gene for immune capacity (disease-resistant) QTL.

[FIGURE 1 OMITTED]

MATERIALS AND METHODS

Pig populations and DNA preparation A three-generation resource family was investigated, and 109 [F.sub.2] offspring with immunity traits were used in this study. All the pigs were bred and raised at the genetic nucleus station of Huazhong Agricultural University, China.

Blood was collected in 50 mM EDTA at pH 8.0 to prevent coagulation, and genomic DNA was extracted from white blood corpuscles. DNA extraction was carried out according to the procedure described by Xiong and Deng (1999).

Traits

Immunity traits components: total erythrocytes (RBC), leukocyte counts (WBS), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red (cell) distribution width (RDW) were determined on blood corpuscle analyzing machine MEK-5216K (made in Japan). Albumin and total protein were tested on automatic biochemical analyzer. The effect of reducing reaction of nitroblue tetrazolium (NBT) on neutrophil was measured according to the procedure described by Shimizu et al. (1977). All the immunity traits were determined at the end of the 60th day.

Genomic DNA amplification and sequence analysis

PCR primers: intron 1, forward primer 1 F: 5'-GCATTTCTGCTGACGGG-3', reverse primer 1 R: 5'-AATAAACCATCTTCCTCCTT-3'; intron 6, forward primer 6 F: 5'-TAGGCAATCAGCCATACG-3', reverse primer 6 R: 5'-GGTCCTGTCTCCTTCGG-3'; intron 9, forward primer 9 F: 5'-CCAGAAAATAACAGAGGAGT-3', reverse primer 9 R: 5'-TCGCATCTTGGTAAACAG-3'. The polymerase chain (PCR) reactions were carried out in 25 [micro]l volumes containing the following reagents: 1.5 [micro]l of porcine genomic DNA (100-250 ng [micro]l-1), 0.5 [micro]l of dNTPs (10 mM each), 20 pmols of each primer, 2.5 [micro]l of 10x buffer, and 1 unit of Taq DNA polymerase. PCR was run in the GeneAmp PCR system 9600 (Perkin-Elmer Co., Norwalk, CT, USA) thermocycler as follows: initial denaturation at 94[degrees]C for 4 min, 35 cycles for 94[degrees]C for 45 s, optimal temperature for 45 s, 72[degrees]C for 1 min, and a final extension time of 10 min at 72[degrees]C.

[FIGURE 2 OMITTED]

The primer pairs 1, 6, and 9 produced 1,045 bp, 911 bp, and 589 bp fragments, respectively. The amplified fragment was cloned into the pGEM-T vector (TaKaRa, Dalian of China) and was sequenced using standard M13 primers. Sequence analysis revealed seven SNPs within intron 6, of which G371A can be detected as an SnaBI PCR-RFLP. For the PCR-RFLP assays, 7.5 [micro]l of PCR products were digested with 5 U SnaBI (TaKaRa) in 1x digestion buffer with 1x BSA added in a total volume of 10 [micro]l. Following digestion for 4 h at 37[degrees]C, the digested products were separated by electrophoresis on a 1.5% agarose gel in 1 x TAE buffer and stained with 0.5 [micro]g [ml.sup.-1] ethidium bromide. The 911 bp PCR product (Figure 1) was digested into two fragments of 371 bp and 540 bp. The result is shown in Figure 2.

Allele frequencies for the Mx1 SnaBI polymorphism (Table 1) are significantly different in native Chinese Meishan breed compared with western Large White and Landrace breeds. Allele B was found only in Meishan pigs, with frequency of 0.4444.

Association analysis

To study the association between carriers of different genotypes and the trait values, the SnaBI PCP-RFLP was tested in 109 [F.sub.2] pigs of a Large White x Meishan reference family (Zhang et al., 2005). The animals were born and raised in Huazhong Agricutural University Jingpin pig station. They were given twice daily diets formulated according to age under a standardized feeding regimen and free access to water (Zhang et al., 2006; Wang et al., 2007). The association between genotype and immunity traits was performed with the least squares method (GLM procedure, SAS version 8.0), and the model used to analyze the date was assumed to be:

[Y.sub.ij] = [mu]+[S.sub.i]+[G.sub.j]+[b.sub.ij][X.sub.ij]+[e.sub.ij]

where, [Y.sub.ij] is the observation of the trait; [micro] is the least square mean; [S.sub.i] is the effect of ith sex (i = 1 for male and 0 for female); Gj is the effect of jth genotype (j = AA, AB or BB); [b.sub.ij] is the regression coefficient of the carcass weight, and [e.sub.ij] is the random residual.

At this locus, the number of animals of genotypes AA, AB, and BB was 82, 15, and 12, respectively. According to the method of Liu (1998), both additive and dominance effects were also estimated using REG procedure of SAS version 8.0. where the additive effect was denoted as -1, 0, and 1 for AA, AB, and BB, respectively, and the dominance effect was denoted as 1, -1, and 1 for AA, AB, and BB, respectively.

RESULTS AND DISCUSSION

The results of tests for Mx1 genotypes and immunity traits are given in Table 2.

In Table 2, statistically significant associations with RBC, HCT, MCV, MCH, MCHC, and RDW were found, but no significant conclusion was made on other immunity traits. Results of the single marker analysis revealed pigs with the AB genotype had significantly higher RBC and HCT, and lower MCH and MCHC when compared with genotypes AA or BB (Table 2). Meanwhile, allele A seemed to be associated with increase in MCV and decrease in RDW. However, the present estimations are based on a weaker data structure, as BB genotype is carried only by the native Meishan pigs and the genotype AA mainly by western Large White and Landrace breeds. It can be assumed that the effects of genetic background influenced the results. Moreover, it is also likely that the intronic mutations are unlikely to be directly responsible for the effects on porcine immunity traits, therefore, the associations observed may simply reflect linkage disequilibrium between the Mx1 polymorphisms and casual genetic variation in some other gene distant from Mx1 or the chromosomal interval and gene interactions. According to the results obtained, analysis of more animals is necessary to confirm the association between the Mx1 genotype and immunity traits in [F.sub.2] intercross pedigree pigs.

ACKNOWLEDGEMENTS

We would like to thank the staff at Huazhong Agricultural University Jingpin Pig Station and teachers and graduate students at Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture for managing and slaughtering the research flocks. This study was financially supported by the National High Technology Development Project (863 Project) (2001-AA-243031).

Received March 5, 2007; Accepted June 10, 2007

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X. L. Li (1), W. L. He (1), C. Y. Deng (2), * and Y. Z. Xiong (2)

(1) College of Animal Science and Technology, HeNan University of Science and Technology, Luoyang, 471003, China

* Corresponding Author: C. Y. Deng. Tel: +86-379-64282574, Fax: +86-27-87394184,

E-mail: dengchy@mail.hzau.edu.cn

(2) Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Veterinary Medicine, Huazhong Agriculture University, Wuhan, 430070, P. R. China.
Table 1. Distribution of SnaBI-RFLP genotype and allele frequencies
among pig breeds

 Genotype Allele frequency
Breeds N
 AA AB BB A B

Large White 148 148 0 0 1 0
Landrace 20 20 0 0 1 0
Meishan 27 10 10 7 0.5556 0.4444

Table 2. Statistical analysis of Mx1 SnaBI-RFLP genotypes with
immunity traits

 Mx1 genotype ([bar.x] [+ or -] SE)
Traits
 AA

WBC (T/L) 26.6457 [+ or -] 2.1418
RBC (G/L) 7.9086 [+ or -] 0.1304 (Aa)
TP (g/L) 71.4448 [+ or -] 1.4731
ALB (g/L) 32.7331 [+ or -] 0.5286
NBT (%) 11.0146 [+ or -] 0.3736
HGB (mmol/L) 9.0038 [+ or -] 0.1009
 50.2767 [+ or -] 0.8753 (a)
MCV (fL) 63.5677 [+ or -] 0.4853 (a)
MCH (Pg) 1.1907 [+ or -] 0.0322 (a)
MCHC (mmol/L) 18.0389 [+ or -] 0.4795 (a)
RDW (% CV) 16.8931 [+ or -] 0.1216 (a)

 Mx1 genotype ([bar.x] [+ or -] SE)
Traits
 AB

WBC (T/L) 26.7110 [+ or -] 4.8948
RBC (G/L) 8.9171 [+ or -] 0.3000 (Bb)
TP (g/L) 74.8189 [+ or -] 3.3890
ALB (g/L) 33.9652 [+ or -] 1.2161
NBT (%) 11.3973 [+ or -] 0.8915
HGB (mmol/L) 8.8552 [+ or -] 0.2306
 55.7680 [+ or -] 2.0137 (b)
MCV (fL) 62.426 [+ or -] 1.1166 (ab)
MCH (Pg) 0.9644 [+ or -] 0.0822 (b)
MCHC (mmol/L) 14.3222 [+ or -] 1.2254 (b)
RDW (% CV) 17.357 [+ or -] 0.2798 (ab)

 Mx1 genotype ([bar.x] [+ or -] SE)
Traits
 BB

WBC (T/L) 22.0421 [+ or -] 5.2833
RBC (G/L) 8.6477 [+ or -] 0.324 (Abb)
TP (g/L) 70.8127 [+ or -] 3.6589
ALB (g/L) 32.6263 [+ or -] 1.3129
NBT (%) 10.6461 [+ or -] 0.9280
HGB (mmol/L) 9.0040 [+ or -] 0.2490
 52.7808 [+ or -] 2.1741 (ab)
MCV (fL) 60.8348 [+ or -] 1.2055 (b)
MCH (Pg) 1.1157 [+ or -] 0.0556 (ab)
MCHC (mmol/L) 19.3722 [+ or -] 0.8279 (a)
RDW (% CV) 17.5346 [+ or -] 0.3231 (b)

 Effect ([bar.x] [+ or -] SE)
Traits
 Additive

WBC (T/L) -2.3018 [+ or -] 2.8503
RBC (G/L) 0.3695 [+ or -] 0.1746 *
TP (g/L) -0.3160 [+ or -] 1.9721
ALB (g/L) -0.0534 [+ or -] 0.7076
NBT (%) -0.1843 [+ or -] 0.5002
HGB (mmol/L) 0.0001 [+ or -] 0.1343
 1.2521 [+ or -] 1.1718
MCV (fL) -1.3665 [+ or -] 0.6497 *
MCH (Pg) -0.0428 [+ or -] 0.0204 *
MCHC (mmol/L) -0.3040 [+ or -] 0.2824
RDW (% CV) 0.3208 [+ or -] 0.1628

 Effect ([bar.x] [+ or -] SE)
Traits
 Dominance

WBC (T/L) -1.1835 [+ or -] 2.8333
RBC (G/L) -0.3195 [+ or -] 0.1736
TP (g/L) -1.8451 [+ or -] 1.9613
ALB (g/L) -0.6428 [+ or -] 0.7038
NBT (%) -0.2835 [+ or -] 0.5112
HGB (mmol/L) 0.0743 [+ or -] 0.1335
 -2.1196 [+ or -] 1.1654
MCV (fL) -0.1125 [+ or -] 0.6462
MCH (Pg) 0.0343 [+ or -] 0.0198
MCHC (mmol/L) 0.5601 [+ or -] 0.2806 *
RDW (% CV) - 0.0716 [+ or -] 0.1620

All the data in the table are least square means [+ or -] standard
error. Values in each line with different lower-cased superscripts are
significantly different at p<0.05, with upper-cased superscripts
different at p<0.01. * p<0.05 and ** p<0.01.

WBC = White blood cell counts; RBC = Red blood cell counts; TP = Total
protein; ALB = Albumin; NBT = Nitrobiue tetrazolium; HGB = Hemoglobin;
HCT = Hematocrit; MCV = Mean corpuscular volume; MCHC = Mean
corpuscular hemoglobin concentration; MCH = Mean corpuscular
hemoglobin; RDW = Red (cell) distribution width.
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Author:Li, X.L.; He, W.L.; Deng, C.Y.; Xiong, Y.Z.
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9CHIN
Date:Nov 1, 2007
Words:2995
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