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African Journal of Pig Farming ISSN 2375-0731 Vol. 7 (3), pp. 001-006, March, 2019. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Sequences polymorphism and variation of major 
histocompatibility complex DRB exon 2 of black 

Dahe pig 
 

Lizhou Tang1, Long Yu1, Jiangang Chen1, Junjie Wang1, Mei Ma1, 
Weidong Lu1* and Tongzuo Zhang2*

 
 

1
Yunnan-Guizhou Plateau Institute of Biodiversity, College of Biologic Resource and Environment Science, Qujing 

Normal University, Qujing, Yunnan 655011, China. 
2
Key Laboratory of the Qinghai-Tibetan Plateau Ecosystem and Biological Evolution and Adaptation, Northwest Institute 

of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai 810001, China. 
 

Accepted 22 November, 2018 
 
DNA sequences of swine leukocyte antigens (SLA) DRB exon 2 from five populations were used to investigate 
genetic polymorphism of black Dahe pig in Yunnan province of China. Our results showed that the SLA-DRB 
exon 2 of black Dahe pig obtained high variable rate of 23.50%, which was higher than the values analyzed for 
cattles or other livestocks. The average G + C content at the third synonymous variable coding positions (GC3) 
was closed to 0.78. The amino acids composition rate and majority of relative synonymous codon usage were 
near the ranges of 0.00 to 11.27 and 1.00 to 2.00, respectively. These significant polymorphism, high GC3 
content and differentiation of preferred codon usage could be resulted from over dominance selection, base 
compositional bias and the diversity of gene expression level. Aforementioned results adequately testified the 
SLA-DRB exon 2 may be an important genetic marker for studies in genetic diversity, population genetics, 
breeding reproduction investigation and exploring new functional genes of black Dahe pig. 

 
Key words: Black Dahe pig, DRB exon 2, genetic polymorphism, major histocompatibility complex, swine lymphocyte 
alloantigen, Yunnan province of China. 
 
 
INTRODUCTION 

 
Major histocompatibility complex (MHC) gene is a 
member of the multigene families, which include three 
gene regions (Classes I, II and III) and correlate with 
immunity function in vertebrate (Benacerraf, 1981; Gu 
and Nei, 1999). Recent studies showed that the compact 
chain, differ markedly in degree of expression and high 
polymorphism were the characters for some MHC genes, 
such as exon 2 of MHC-DRB and MHC-DQB (Kloch et 
al., 2010). Previous researches of MHC gene mostly 
concentrated their attention on rats, and found that this 
gene was also discovered in different chromosomes of 
more vertebrates, such as sheeps and cattles (Gorer,  
 

 
*Corresponding  author.  E-mail:  luweidonghxx@yahoo.com.cn,  
zhangtz@nwipb.ac.cn. Tel: +86-874-8998627. Fax:: +86-874-
8998627. 

 
 
 
 

 
1937). Some studies indicated MHC gene plays important 
role in the regulation of immune response, also could be 
related to the characteristics of disease resistance, 
reproductive capability, growth capability and fleshy 
quality in livestock and poultry (Benacerraf, 1981). 
Therefore, as an important candidate gene for 
investigation about disease resistance and susceptibility, 
the MHC gene has become a hotspot in the research 
area of genetic breeding, and has attracted the attention 
of some scientists, such as ecologists, geneticists and 
animal breeding scientists.  

The MHC gene of pigs, named swine lymphocyte 
alloantigen (SLA), have been originally pitched on 
chromosome number 7 and have been finally located on 
the centromere of chromosome number 7 (Rabin et al., 
1985). Other studies mostly investigated the relationship 
between SLA genotypes and immunoreactions or disease 



 
 
 

 
Table 1. The locations of sampled populations of black Dahe pig.  

 

Population Site Latitude (N) Longitude (E) Sample size 
     

1 Dahe town, Fuyuan county, Yunnan province 25°32'51.79" 104°18'10.16" 10 

2 Yingshang town, Fuyuan county, Yunnan province 25°28'37.52" 104°19'33.40" 10 

3 Zhongan town, Fuyuan county, Yunnan province 25°41'29.73" 104°14'52.72" 10 

4 Fucun town, Fuyuan county, Yunnan province 25°22'23.36" 104°26'29.68" 10 

5 Laochang town, Fuyuan county, Yunnan province 25°13'34.50" 104°31'15.63" 8 
     

 

 

resistance (Benacerraf, 1981; Shia et al., 1995). More 
scientists subsequently studied differentiation of parasites 
resistance of SLA genotypes in different pigs or tissues, 
and analyzed relationship between some economic 
characters and gene haplotypes based on molecular 
biologic techniques (Lee et al., 2005). As a crossbreed 
hybridized by Chinese Dahe pig and England Yorkshire 
pig, black Dahe pig has been formally approved as an 
endemic new species by the National Agriculture Ministry 
of China, and distributed on districts of Dahe-Yingshang 
towns in Qujing city of Yunnan province. This species has 
become the material source for Chinese famous ham 
brand (Xuanwei ham) because of its excellent characters, 
such as better meat, high intramuscular fat, quick growth 
rate, strong reproductive ability, resistance of crude feed 
and disease resistance (Huo et al., 2009). However, the 
piglets of black Dahe pig were also killed by these familiar 
epidemic diseases, especially the yellow and white scour 
of piglets (Huo et al., 2009). The clinic diseases could 
badly affect the pure reproduction, piglet supply and 
piglet production with high quality, and bring huge 
economic loses to the farmer. The medication therapy or 
vaccine inoculability could not settle the difficult problem 
of repetitive disease infection ultimately. But pig 
producing via the method of gene modification must 
satisfy modern people’s demands for food safety.  

Therefore, we analyzed the sequence, amino acid 
substitution and polymorphism of exon 2 of SLA-DRB 
gene for endemic black Dahe pig from Yunnan province 
of China. The aforementioned acquired information can 
facilitate us to develop subsequent studies of disease 
resistance and breeding, and also to investigate the 
relationship between other economic characters and SLA 
genotypes of this endemic breed. 
 

 
MATERIALS AND METHODS 
 
Population samples 

 
Samples for genetic polymorphism analysis totaled 48 individuals 
from 5 populations of black Dahe pig were collected and sequenced 

 

 
for this study (Table 1). The sampled muscle tissues for each pig 
were immediately preserved in 95% ethanol and transferred to 
Qujing Normal University, for storage at -20°C. 

 

DNA extraction, PCR amplification and sequencing 

 
Total DNA was isolated from ethanol-fixed muscle tissue after 
proteinase K digestion, followed by standard animal genome DNA 
extraction (Dingguo, Beijing, China) and ethanol precipitation. The 
sequences of exon 2 of MHC-DRB gene were amplified using 
primer pair DRB1 (5’-TAGGATCCCTCACAGCGCATTTCTT-3’) and 
DRB2 (5’-GTGTCTGCAGTACGTGTCCA-3’). PCR amplifications 
were performed in total reaction volumes of 15 µl, containing 1.5 µl 

of 10 × Buffer (include Mg
2+

), 1 µl of mixed dNTP, 1.2 µl of each 
primer (synthesized by Sangon, Shanghai, China), 1.5 µl (100 
ng/µl) of template DNA, and 0.12 µl Taq DNA polymerase (5 U/µl) 
(Sangon, Shanghai, China). The reaction mixtures were denatured 
at 94°C for 5 min and subjected to 30 cycles of 30 s at 94°C, 45 s at 
60°C, 45 s at 72°C, and a final extension step of 8 min at 72°C. 
PCR products were purified using a Geneclean Purification Kit, 
cloned and sequenced (Xu et al., 2005; Wu et al., 2007). 
 

 
Data analysis 

 
All obtained sequences for black Dahe pig were compared with the 
submitted sequences of Sus scrofa in GenBank (accession 
numbers GU263819, U52528). All sequences were aligned using 
Clustal X (Thompson et al., 1997) with the default settings and 
refined manually. The total number of mutations, sites with 
alignment gaps, polymorphic sites, singleton variable sites and 
parsimony informative sites were determined using DnaSP (Rozas  
et al., 2003). Maximum likelihood estimates of 
transition/transversion bias were conducted in MEGA5 (Tamura et 
al., 2011) based on using a user-specified topology and eliminating 
all missing positions. The nucleotide frequency, G + C content, 
relative synonymous codon usage, average nucleotide composition, 
amino acid composition and average nucleotide pair frequency 
were estimated in MEGA5 (Tamura et al., 2011). 
 
 
RESULTS 

 

Homologous comparison and clone sequencing 

 

As compared with exon 2 of SLA-DRB DNA sequences of 



  
 
 

 
Table 2. The maximum composite likelihood estimate of the pattern of nucleotide substitution.  

 
Nucleotide A T C G 

A - 5.04 5.71 12.94 

T 5.48 - 15.86 7.86 

C 5.48 14 - 7.86 

G 9.01 5.04 5.71 - 
 

Rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics. The 
nucleotide frequencies are 22.73% (A), 20.93% (T/U), 32.64% (C), and 23.71% (G). The transition/transversion rate ratios are 

k1 = 1.645 (purines) and k2 = 2.778 (pyrimidines). The overall transition/transversion bias is R = 1.052, where R = [A*G*k1 + 

T*C*k2]/[(A+G)*(T+C)]. 
 

 

Sus scrofa, the homologous percentage for all obtained 
black Dahe pig sequences were close to 90 to 100%. A 
total of 29 haplotypes were identified in 48 pig individuals. 
There are 200 sites determined using clone sequencing. 
 

 

Sequence polymorphism and variation 

 

Of the 200 sites aligned in all individuals, 5 missing sites, 
148 invariable sites and 47 polymorphic sites were 
observed respectively. These 47 polymorphic sites were 
constituted by two parts of 9 singleton variable sites (two 
variants) and 38 parsimony informative sites covered 29 
two variants, 8 three variants and 1 four variants. The 
estimation of overall transition/transversion bias (R) was 
1.052 when frequencies were observed for four 
nucleotides (A, 23.30%; T, 21.00%; C, 23.20%; G, 
32.50%) (Table 2). The molecular genetic indices 
analyses showed that 29 transitional sites and 30 
transversional sites were estimated for all 48 pigs. Among 
these substitutions, the rates of different transitions 
ranged from 9.01 to 15.86 with the highest T-C transition, 
while the transversional rates ranged from 5.04 to 7.86 
with the highest of T-G and C-G transversion (Table 2). 
 
 

 

Codon usage in black Dahe pig 

 

The overall codon usage analysis in coding sequences of 
48 black Dahe pig showed that the average G + C 
content at the third synonymous variable coding positions 
was 0.78. The amino acid composition, corresponding 
codon and their frequency and the relative synonymous 
codon usage were calculated for all pig individuals (Table 
3). The analysis of amino acids composition and their 
frequency indicated that amino acid compositional rate 
ranged from 0.00 to 11.27, and Phe obtained the highest 
values followed by lesser adjacent rate of Leu, Glu, Arg 
and Gly respectively (Table 3). There was clear 
heterogeneity of codon usage among different amino 
acid. Firstly, estimated codon compositional frequencies 
obtained a range from 0.00 to 6.30, while the highest rate 
in codon UUC and the lowest on CUU, GUU, GCA, UAA, 

 
 

 

UAG and GAU. Secondly, codon UUC was about six fold 
as frequent as UUU in the preferred Phe amino acid, 
while the codon CCA and UGU obtained a compositional 
rate of 1.00 in the secondly non-preferred amino acid of 
Pro and Cys. The trend of codon usage pattern indicated 
a significant biased codon usage because of majority of 
relative synonymous codon usage (RSCU) values near 
the range of 1.00 to 2.00, but obvious exceptions were 
observed for four comparative large RSCU in codons of 
GUG, CCA, CGG and AGC (Table 3). Correlative 
analyses showed that there was a significant positive 
correlation between the codon frequency and RSCU 

values (R
2
 = 0.442) (Figure 1). 

 

 

DISCUSSION 

 

We have presented evidence that the exon 2 of SLA-DRB 
of black Dahe pig obtained the variable rate of 23.50%, 
which was higher than the values of 13.70% analyzed for 
cattles. The results also showed that the parsimony 
informative sites not only included two variants, three 
variants and four variants, but even accounted for nearly 
80.85% of the 47 polymorphic sites. All the 
aforementioned results together indicated that there was 
higher polymorphism in the exon 2 of SLA-DRB gene of 
black Dahe pig than in other class II genes, which could 
be consistent with the supported viewpoint of previous 
studies (Hughes and Nei, 1989; Kloch et al., 2010; 
Spurgin and Richardson, 2011). In this study, a better 
explanation for this high polymorphism could be the over 
dominance selection that emphasized enhancing the rate 
of amino acid substitution and increasing the 
heterozygosity and persistence time of polymorphic 
alleles as compared with those of neutral alleles (Hughes 
and Nei, 1989). The transitions of T-C and C-T pyrimidine 
achieved the highest values of 15.86 and 14.00 
respectively among all transitional substitutions in this 
study (Table 2). These may induce enhancing of amino 
acid substitution rate (Table 3) instead of decreasing of 
disadvantageous selection, because the selection from 
advantageous mutations could reduce the level of 
nucleotide polymorphism (Akashi, 1994; Hughes and Nei, 
1989). This important genetic information of high 



 
 
 

 
Table 3. The amino acid composition and relative synonymous codon usage.  

 
Amino acid AF Codon F RSCU Amino acid AF Codon F RSCU 

Phe 11.27 UUU 1.00 0.27 Ser 4.74 UCU 0 0 

  UUC 6.30 1.73   UCC 1.10 2.21 

       UCA 0 0 

Leu 10.87 UUA 1.40 1.21   UCG 0 0 

  UUG 1.80 1.57      

     Pro 1.59 CCU 0 0 

Leu  CUU 0.10 0.11   CCC 0 0.13 

  CUC 1.20 1.05   CCA 1.00 3.88 

  CUA 0 0   CCG 0 0 

  CUG 2.40 2.06      

     Thr 4.63 ACU 0 0 

IIe 0.00 AUU 0 0   ACC 1.40 1.89 

  AUC 0 0   ACA 1.00 1.38 

  AUA 0 0   ACG 0.50 0.73 

Met 0.05 AUG 0 1.00 Ala 3.89 GCU 0 0.05 

Val 7.03 GUU 0.10 0.06   GCC 1.00 1.59 

  GUC 0 0.03   GCA 0.10 0.1 

  GUA 0 0   GCG 1.40 2.26 

  GUG 4.50 3.91      

     Cys 1.59 UGU 1.00 2.00 

Tyr 5.08 UAU 1.20 0.71   UGC 0 0 

  UAC 2.10 1.29      

Ter  UAA 0.10 1.50 ter  UGA 0 0 

Ter  UAG 0.10 1.50 Trp 0.00 UGG 0 0 

His 3.63 CAU 1.70 1.42 Arg 9.32 CGU 0.30 0.32 

  CAC 0.70 0.58   CGC 1.00 1.03 

       CGA 0 0 

Gb  CAA 0.30 0.41   CGG 2.90 2.86 

  CAG 1.30 1.59      

     Ser 4.74 AGU 0 0 

Asn 4.74 AAU 0.30 0.21   AGC 1.90 3.79 

  AAC 2.70 1.79      

     Arg 9.32 AGA 0.60 0.58 

Lys 4.59 AAA 0.80 0.54   AGG 1.20 1.22 

  AAG 2.20 1.46      

     Gly 8.28 GGU 0 0 

Asp 6.03 GAU 0.10 0.03   GGC 1.00 0.72 

  GAC 3.80 1.97   GGA 2.00 1.52 

       GGG 2.40 1.76 

Glu 10.22 GAA 0.90 0.27      

  GAG 5.70 1.73      
 

AF, Average amino acid frequency; F, codon frequency; RSCU, relative synonymous codon usage. 
 

 

polymorphism in exon 2 of SLA-DRB of black Dahe pig 
could provide us a good molecular marker for studying 
disease resistance breeding and correlative analyses of 
economic characters.  

The results also supported that there was a significant 
nucleotide base bias in the exon 2 of SLA-DRB gene of 

 
 

 

black Dahe pig. The content of G + C in the 200 sites was 
nearly 55.70%, and increased to 78% at the third 
synonymous variable coding positions (Table 2). This 
phenomenon of nucleotide bias could still influence codon 
usage and frequency of acid amino substitution, such as 
majority of RSCU values near the range of 1.00 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. The correlation between codon frequency and relative synonymous codon usage. The above and below lines 
indicate the 95% confidence interval. 

 

 

to 2.00, and the amino acid compositional rate ranged 
from 0.00 to 11.27 (Table 3). This significant bias of 
RSCU could be dominated by base compositional bias 
and also be associated with gene expression level (Gupta 
and Ghosh, 2001). Such codon usage biases may be 
result from natural selection acting on silent changes in 
DNA, or both mutational biases and natural selection 
(Bulmer, 1991; Akashi, 1994; Duret and Mouchiroud, 
1999; Grocock and Sharp, 2002). The selective 
advantage may depend on increasing the translation 
efficiency, especially during periods of competitive 
exponential growth (Duret and Mouchiroud, 1999; 
Grocock and Sharp, 2002).  

In conclusion, the significant polymorphism, high GC3 
content and preferred codon usage could indicate that the 
exon 2 of SLA-DRB gene of black Dahe pig must be an 
important genetic marker of chromosome for studies in 
genetic diversity, population genetics, breeding 
reproduction investigation and exploring new functional 
genes for disease resistance. The acquired sequences 

 
 

 

information of SLA-DRB could be useful for the 
researches on the relationship between the SLA 
haplotypes and the economic characters of black Dahe 
pigs, despite fewer samples of pigs used in this study. 
 

 

ACKNOWLEDGEMENTS 

 

This work was supported by the NSFC (30970366 to T-Z 
Zhang), the Applied Fundamental Research Project of 
Yunnan Provincial Science and Technology Department 
(2010ZC149 to L-Z Tang), and the Key Project of Qujing 
Normal University Scientific Research Fund (2009ZD004 
to L-Z Tang). 
 

 
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