A SNP in ADIPOQ Gene can be Used as Molecular Marker for Growth Traits in Goats
Qingming An1, Xiaoting Ding2, Dahui Wang1, Zijing Zhang3, Yu-Jia Sun4,
Xu Wang2, Peng Yang2, Xian Liu5, Baorui Ru5, Xianwei Wang5, Zejun Xu5,
Hua He6 and Yongzhen Huang2*
1College of Agriculture and Forestry Engineering, Tongren University, Tongren, Guizhou, 554300, People’s Republic of China
2College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi, 712100, People’s Republic of China
3Institute of Animal Husbandry and Veterinary Science, Henan Academy of Agricultural Sciences, Zhengzhou, Henan, 45002, People’s Republic of China
4Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education, Yangzhou University, Yangzhou, 225009, People’s Republic of China
5Henan Provincial Animal Husbandry General Station, Zhengzhou, Henan, 450008, People’s Republic of China
6College of Veterinary Medicine, Northwest AandF University, Yangling Shaanxi, 712100, People’s Republic of China
Qing-Ming An and Xiao-Ting Ding contributed equally as a first author.
ABSTRACT
This study investigated single nucleotide polymorphisms (SNPs) in intronic regions of the ADIPOQ gene and estimated their contributions to growth traits of goats. We first found a SNP site at the 14059th base position in the second introssn of the ADIPOQ gene (NC_030808.1: g. 14059 C>T). At the same time, we performed association analysis between this SNP and growth traits in goats. These results showed that the base (C or T) at the SNP was associated with changes in body length with CT and TT genotypes giving greater values than CC genotypes. This site therefore can be used as a molecular breeding marker for early selection of growth traits in goats.
Article Information
Received 26 January 2021
Revised 10 April 2021
Accepted 18 April 2021
Available online 11 February 2025
(early access)
Published 24 September 2025
Authors’ Contribution
QA, XD and YH conceived the study and designed the work. XD, DW, ZZ, YS, XW, PY and XL analyzed and interpreted the data. QA and XD wrote the manuscript. BR, XW, ZX and HH revised the manuscript critically. All authors read and approved the final manuscript.
Key words
Goat, ADIPOQ gene, SNP, Growth traits, Association analysis
DOI: https://dx.doi.org/10.17582/journal.pjz/20210126020127
* Corresponding author: [email protected]
0030-9923/2025/0006-2563 $ 9.00/00
Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Introduction
Molecular breeding technology has many advantages over conventional breeding, which can effectively avoid the problems of long generation interval, slow progress, low improvement efficiency, waste of excellent germplasm resources. The key to molecular breeding is molecular marker-assisted selection (MAS). Goat ADIPOQ gene is a candidate to target for goat growth traits. It was first found on human chromosome 3, with a total length of 17 kb, including three exons and two introns, encoding 247 amino acids (Wang et al., 2007; Pajvani et al., 2003). The ADIPOQ protein binds with AdipoR1 or AdipoR2 receptors and can activate adenylate activated protein kinase, p38 mitogen activated protein kinase, Jun amino terminal kinase, nuclear factor kB and other signal pathways and so play a role in the target tissue. Studies have shown that ADIPOQ can directly stimulate adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle and liver, lead to phosphorylation of acetyl CoA carboxylase, promote fatty acid oxidation, reduce lipid accumulation in skeletal muscle, improve insulin resistance in the liver and reduce the production of liver sugar and the synthesis of very low-density lipoprotein (Yamauchi et al., 2002). It was found that the binding of ADIPOQ to the AdipoR1/2 receptor increased the expression of the myhc1 and myhc2x genes, decreased the expression of the myh2b gene, and changed the composition of muscle fiber via AMPK and peroxisome proliferator-activated receptor alpha (PPARα) signal pathways (Zhang et al., 2011). In human, monkey and mouse, it was found that ADIPOQ gene is expressed in adipose and the level of ADIPOQ in blood reduced when there exists over-deposition of fat, while the expression of the ADIPOQ gene and the level of ADIPOQ in blood increased when weight decreased (Zhou et al., 2018). Therefore, the ADIPOQ gene is related to body weight. Adiponectine (ADIPOQ), the product of the ADIPOQ gene expression, which belongs to the family of adipocytokines, is a specific hormone secreted by adipocytes and it is mainly related to insulin tolerance regulation, obesity, cardiovascular disease, type II diabetes and other physiological diseases (Yang et al., 2006; Liu et al., 2018). Therefore, the ADIPOQ gene is also related to metabolism.
It was found that the variants of c.– 67G / A and c.– 892C/T in the promoter region of the ADIPOQ gene had an effect on the carcass traits and meat quality in pig (Cieslak et al., 2013). In the promoter region of the ADIPOQ gene in cattle, the variants of c. – 176A/G had an effect on carcass traits; the variants c.- 199 C/T and c.-34 G/A also had some effect on the area of the eye muscle and the thickness of the back fat (Shin and Chung, 2013; Morsei et al., 2006). Therefore, there may also be a relationship between the ADIPOQ gene and the growth traits in goats. However, molecular markers of goats are rarely reported. The purpose of this paper is to investigate the possibility of a molecular genetic marker related to the growth traits of goats.
Materials and Methods
Animals and data collection
In order to explore the genetic variation of the goat ADIPOQ gene, 339 individuals representing three goat breeds in China were sampled: Guizhou white goats (N=98), Guizhou black goats (N=155) and Henan hybrid goats (N=86). These breeds are important for goat meat production in China and are reared in the Provinces of Guizhou and Henan. The Guizhou white goats were from three farms (Dejiang County, Guizhou Province); the Guizhou black goats were from three farms (Bijie City, Guizhou Province, China); and the Henan hybrid goats were from four farms (Yongcheng City, Shangqiu City, Henan Province). On each farm, the goats were randomly selected, almost all of them were females, with the age generally between 2-2.5 years old. Basic data (withers height, body length, heart girth, circumference of cannon bone, body weight) for the corresponding individuals were also recorded. The DNA samples were anticoagulated with acid citrin dextrose (ACD) and quickly brought back to the laboratory in an ice box at - 80oC.
Genotyping
The genomic DNA of the samples was extracted using the phenol chloroform method.
The PCR amplification of second intron of the goat ADIPOQ gene was conducted using 2×Taq PCR Starmix, with a reaction volume of 10 μL, which contained 1μL of DNA pool, 5 μL of 2×Taq PCR Starmix, 0.5 μL of upstream primers, 0.5 μL of downstream primers and 3μL ddH2O. F: 5’-GGTTAAGCTTCTTTACCACAGAGTG-3’; R: 5’-CTTCCACACTGACCGAAGTC-3’. Thermal cycling conditions were as follows: pre denaturation at 95 oC for 300 s and one cycle; denaturation at 95 oC for 30 s; annealing at 54.1 oC for 30 s; extension at 72 oC for 30 s and 35 cycles; final extension at 72 oC for 600 s and one cycle; and preservation at 12 oC. The amplified products of PCR were analyzed by 1% agarose gel electrophoresis for 0.5 h. The amplified products were sequenced by Shanghai biotechnology Service Company.
To define genotypes of the variants, 10 μ L PCR products were digested with endonuclease AvaII (Takara company). The AvaII digestion system was conducted using 1× NE buffer, with a reaction volume of 10 μL, which contained 5μL of PCR product, 0.5μL of 1× NE buffer, 0.1 μL of AvaII and 4.4 μL of ddH2O; digestion took place in a water bath for 40-60 min.
Data analysis
The effective allele number (NE), heterozygosity (He), polymorphism information content (PIC), genotype frequency and allele frequency of goat ADPIOQ gene g. 14059 C > T were counted by Microsoft Excel software, and Hardy-Weinberg test was carried out (P<0.01, χ2-test).
Genotype frequencies were determined for each breed by direct counting using formula
PBB, NBB/N
where P BB is the genotype frequency given a certain nucleotide position, NBB is number of individuals with BB genotype in the population, and N is total number of individuals in the population
Gene frequency was determined using formula
PB=(2NBB+NBb1+NBb2+NBb3+NBb4+……+NBbn)/2N
where PB is frequency of allele B; NBB, Number of individuals with BB genotype in the population; NBbi is number of individuals with Bbi genotype in the population for i = 1,…, n; and b1~bn, n different alleles in a multi-allelic system with allele B.
Population genetic indexes, such as He, Ho, Ne, and PIC were calculated according to Nei and Roychoudhurg (1974), respectively. The formulas were as follows:
where Pi is the frequency of the i allele and n is the number of alleles.
General linear model (GLM) and one way ANOVA in SPSS 20.0 were used to analyze the correlation among genotypes, combination genotypes and body size traits of SNP loci. The model used in the analysis is Yij= μ+Gi+ εij, where Yij is the observed value of body size traits, μ is the observed mean value of traits, Gi is the genotype effect, and εij is the random error (Boldman et al., 1993; Zhao et al., 2004; Hendersor, 1986).
Results
Mutation in the intron 2 of ADIPOQ gene
Figure 1 shows A C/T mutation or natural cleavage site in the intron 2 region of the ADIPOQ gene in goat by comparing the sequence of the target fragment with the reference sequence (80105499th position of NC_030808.1 exists in the amplification region of the primer pair).
PCR–RFLP analysis of ADIPOQ gene
Figure 2 shows the product after endonuclease AvaII digestion. CC genotype shows two bands of 46bp and 272bp, the CT genotype shows three bands of 272bp, 46bp and 318bp, and the TT genotype shows one band of 318bp. However, the 46bp fragment is too small to be displayed.
Population genetic analysis
There are two alleles (C and T) and three genotypes (CC, CT and TT) at the 14059th position of the ADIPOQ gene (Table I). No matter which breed, C gene frequency is higher than T gene frequency, and CT genotype frequency is higher than CC and TT, and TT genotype frequency is the lowest in the three genotypes.
Table I. Population genetic analysis of ADIPOQ gene in goats.
|
Breed |
Sample size |
Genotype frequency |
Allele frequency |
|||||||
|
PCC |
PCT |
PTT |
C |
T |
He |
Ne |
PIC |
X2(1) |
||
|
Guizhou white goat |
98 |
0.204 (20) |
0.724 (71) |
0.072 (7) |
0.566 |
0.434 |
0.491 |
1.965 |
0.371 |
22.097 |
|
Guizhou black goat |
155 |
0.200 (31) |
0.645 (100) |
0.155 (24) |
0.523 |
0.477 |
0.499 |
1.996 |
0.374 |
13.206 |
|
Henan Hybrid goat |
86 |
0.128 (11) |
0.814 (70) |
0.058 (5) |
0.535 |
0.465 |
0.498 |
1.990 |
0.374 |
34.775 |
|
Total goat |
339 |
0.183 (62) |
0.711 (241) |
0.106 (36) |
0.538 |
0.462 |
0.497 |
1.988 |
0.374 |
62.738 |
Note: X20.05(1)= 6.635, X2(1)> X20.01(1), P<0.01. There was highly significant difference (P < 0.01).
Table II. Average growth trait values for three genotypes of goats defined from SNP analysis of the ADIPOQ gene.
|
Growth traits |
Genotype (Mean±Standard error) |
F-test P-value |
||
|
CC |
CT |
TT |
||
|
Withers height (cm) |
58.95±1.005 |
61.04±0.54 |
62.44±1.03 |
0.083 |
|
Body length (cm) |
60.47 b±1.11 |
64.31 a±0.73 |
65.035 a±1.50 |
0.035* |
|
Number |
42 |
169 |
29 |
|
|
Heart girth (cm) |
72.46±0.99 |
75.72±0.59 |
74.23±1.50 |
0.034 |
|
Number |
62 |
240 |
36 |
|
|
Circumference of cannon bone (cm) |
9.00±0.17 |
9.37±0.10 |
9.08±0.26 |
0.194 |
|
Number |
28 |
113 |
12 |
|
|
Body weight (kg) |
27.11±1.04 |
27.94±0.632 |
28.29±0.98 |
0.791 |
|
Number |
51 |
171 |
31 |
|
Note: For growth traits with F-test P-value less than 0.05, there is no significant difference (P>0.05, post hoc t-test) between means with the same superscript letters.
The genetic variation degree of the three populations is similar, and the heterozygosity of Guizhou black goat is highest. In terms of polymorphism information content (PIC), all the goat breeds tested were in moderate polymorphism (PIC > 0.5 was high polymorphism, 0.25 < PIC < 0.5 was moderate polymorphism, PIC < 0.25 was low polymorphism). Among them, Guizhou black goat and Henan hybrid goat gave the highest (0.374). The Chi-squared test for Hardy Weinberg equilibrium showed that the χ2 value of three goat populations reached a significant level, that is, the gene frequency and genotype frequency of these three goat populations were in the state of Hardy Weinberg disequilibrium (P < 0.01).
Association analysis
The results show that different genotypes, which the mutation at the 14059th position of the ADIPOQ gene in goats formed, were associated with the growth traits body length and heart girth (P < 0.05, F-test). Specifically, mutation of the C/T base at this locus was associated with body length, and mean values for the CT type and TT type were significantly (P < 0.05, post-hoc t-test) higher than for the CC type. It is suggested that the allele C of the ADIPOQ gene is closely related to the growth traits (body length) of goats (Table II). Therefore, CT and TT can be used as molecular breeding gene markers for early selection of growth traits in goats.
Discussion
It is predicted that the mutation in the ADIPOQ gene would affect the metabolism of sugar and lipid, and then affect the growth and development and meat quality in goats. The + 67bp G > C mutation in exon 2 of the ADIPOQ gene affects the live weight and carcass weight before slaughter in Tebetan sheep (Yang et al., 2014). In Qinchuan cattle, the missense mutation G to C at 64bp of exon 2 of the ADIPOQ gene had an effect on live weight, carcass weight and eye muscle area before slaughter; the missense mutation C to T at 50 bp of exon 3 not only affected live weight, carcass weight, leg hip circumference before slaughter, but also back fat thickness and tenderness (Yang, 2009). The ADIPOQ gene g.81966377 T > C locus in Hanwoo cattle has an influence on the eye muscle area and the marbling score, on the backfat thickness and carcass weight (MN et al., 2019); the dominant allele C of the ADIPOQ gene g.8196235 C > T locus is related to low marbling score; the dominant allele D of g.81966364 D > I locus also shows low additive effect on marbling score (Choi et al., 2015). Dai et al. (2006) showed that the g.81967079 G > A mutation in the ADIPOQ gene had an effect on backfat thickness and eye muscle area in pigs. There exists a single base mutation site in the ADIPOQ gene related to growth traits in each of these different livestock breeds.
It is very important to study the impact of mutations on the growth traits of livestock and poultry. This paper only studies one mutation site, and more genes and gene mutation sites which could be related to growth traits need further study.
Conclusion
In this study, we determined that the single nucleotide mutation in the ADIPOQ gene had an effect on growth traits. We conclude that this SNP could be used as a molecular marker in future breeding programs that aim to select for growth traits in goats.
Declarations
Acknowledgements
This study was supported by the Program of National Natural Science Foundation of China (31601926), Innovative Talents of Guizhou Province (2022-(2020)-037). Doctoral Talent Program of Tongren (Tongren Scientific Research 2023-4), Science and Technology Program of Guizhou Province (Science and technology cooperation support projects 2018-1161), Science and Technology Top Talent support Project of Guizhou Provincial Department of Education (KY2017-089), Science and Technology Program of Tongren (2020-75), Special Program for Self-Innovation of Henan Academy of Agricultural Sciences (2019ZC41), Funds for Science Research and Development of Henan Academy of Agricultural Science (2019CY08).
Funding
This study was supported by the Science and Technology Program of Guizhou Province (Science and technology cooperation support projects 2018-1161), Innovative Talents of Guizhou Province (2022-(2020)-037), Doctoral Talent Program of Tongren (Tongren Scientific Research 2023-4), Program of Special Fund for Henan Agriculture Research System (HARS-22-15-Z1).
Ethics approval and IRB approval
A series of animal experiments in our study were followed the relevant laws and policies about animal welfare. Furthermore, all operating procedures involved animals were approved by the Faculty Animal Policy and Welfare Committee of Northwest A and F University (FAPWC-NWAFU, protocol number NWAFAC1008). This article does not contain any studies with human participants or animals performed by any of the authors.
Statement of conflict of interest
The authors have declared no conflict of interest.
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