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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE
"ACTUAL PROBLEMS OF LIVESTOCK DEVELOPMENT, MODERN METHODS AND
DEVELOPMENT PROSPECTS" _SEPTEMBER 26-27, 2024_
ANALYSIS OF DGAT1 GENE POLYMORPHISM IN XINJIANG KAZAKH HORSES
1Xiaoyuan Sun, 2Yaru Wang, 3Gulibaheti Dawulietihan, 4Chi Tang, 5Na Yang, 6Li Xue, 7Khizat Serik, 8Iskhan Kairat, 9Kozhanov Zhassulan Ertaevich, 10Ruzikulov Nuriddin,
11Gemingguli Muhatai
1'2'4'5'11Tarim University, Alar, China, 1,2,4,5,11Key Laboratory of Tarim Basin Biological Resources Protection and Utilization, Alar, 1,2,5,11China, Key Laboratory of Tarim Livestock Science and Technology Corps, Alar , China, 3Agricultural (Animal Husbandry) Development Service Center of Tuerhong Township, Fuyun, China, 6Fuyun County Bureau of Agriculture and Rural Affairs, Fuyun,China, 7,8Kazakh National Agrarian Research University, Almaty,
Kazakhstan, 9Kazakh Research Institute of Livestock and Forage Production, Almaty,Kazakhstan, 10Samarkand state university of veterinary medicine, livestock and
biotechnologies,Samarkand,Uzbekistan) https://doi.org/10.5281/zenodo.13856353
Abstract. The study involved 60 Kazakh horses from Fuyun County, Altay Region, Xinjiang, China, serving as experimental subjects to investigate polymorphisms in the DGAT1 gene, specifically in Exon 1 and Exons 6-9 sequences. The results indicated significant polymorphisms within these sequences, identifying three mutation sites: g.3041T>C, g.7817A>G, and g.8036T>C. Among these mutations, g.3041T>C and g.8036T>C were classified as missense mutations. The g.3041T>C mutation resulted in a valine-to-alanine substitution and maintained Hardy-Weinberg equilibrium, whereas g.7817A>G was found to be in Hardy-Weinberg disequilibrium. The g.8036T>C mutation led to a methionine-to-threonine change and also exhibited Hardy-Weinberg disequilibrium. The distribution of haplotypes was relatively uniform, with comparable proportions of Kazakh horses represented in each haplotype across the three sampling points.
Keywords: Kazakh horses;DGAT1;Genepolymorphism.
1 Introduction
Previous studies have shown that DGAT(Diacylglycerol acyl-Coenzyme A acyltransferase) can play a role in adipogenesis, and its function is mainly to encode DGAT1[1]. Studies have shown that after knocking out the DGAT1 gene in mice, the mice show a complete inhibition of lactation, indicating that the DGAT1 coding gene may be associated with lactation traits[2].Studies have shown that there are polymorphisms in the DGAT1 gene in dairy animals such as cattle and sheep, and double base mutation was found in exon 8 of the bovine DGAT1 gene, which is significantly correlated with lactation performance[3,4]. However, in the lactation performance of horses, the study of the DGAT1 gene on lactation performance-related genes has not been involved. DGAT1 can be involved in the synthesis of triglycerides, which can affect the lactation performance of dairy animals such as cattle and sheep. Therefore, in this study, the equine DGAT1 gene was amplified to detect whether there were polymorphic sites, which provided a preliminary theoretical basis for molecular breeding of the Kazakh equine Altai type.
2 Methods
Sixty Kazakh horses from Dure Town(88.53°E, 46.51°N;n=20), Kalatongke Township (89.70°E,46.85°N;n=20), and Qakultu Town (89.54°E,46.33N;n=20) in Fuyun County were chosen as the subjects for this study. Their DNA was extracted after blood collection from the
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "ACTUAL PROBLEMS OF LIVESTOCK DEVELOPMENT, MODERN METHODS AND
DEVELOPMENT PROSPECTS" _SEPTEMBER 26-27, 2024_
jugular vein.This study aimed to identify if the same polymorphism is present in the horse DGAT1 gene and to explore potential polymorphisms in other exons. Primers were designed based on the sequences of Exon 1, Exon 6 to Exon 9 of the DGAT1 gene (accession number: NC_009152.3) available on NCBI.After the sequencing end, the polymorphisms were analyzedD 3 Results and discussion
(1) Following the identification of inaccurate fragments, a BLAST analysis was performed on the NCBI website to assess homology. The comparison results revealed that the amplified sequence exhibited over 95% similarity with the published sequence. Specifically, the similarity for Exon1 was 100%, while Exon6-Exon9 demonstrated a similarity of 99.78%, indicating that the amplified sequence corresponds to the Kazakhstan sequence of the equine DGAT1 genes Exon1 and Exon6-Exon9.The results indicated the presence of three SNPs in the target sequence region of the selected horses in this study(Table.1). The mutation information is presented in Table.1.In the DGAT1 gene, the base at the g.3041 site is mutated from T to C, resulting in a corresponding alteration of the encoded amino acid from valine to alanine. Additionally, the base A at the g.7817 site is mutated to G within the sixth exon, and the intronic region between exon 9 and exon 9 shows that the base T at the g.8036 position of the DGAT1 gene Exon6-Exon9 is mutated to C, leading to a change in the originally encoded methionine to threonine(Fig.1). As shown in Table.2, the allele frequencies at the g.3041 site of the DGAT1 gene sequence in the tested samples were 0.908 for T and 0.092 for C, with a polymorphic information content of 0.153 (PIC<0.25).The analysis reveals low polymorphism and an equilibrium state according to the Hardy-Weinberg test (P > 0.05). The allele frequencies of A and G at the g.7817 site of the DGAT1 gene sequence in the tested effective samples are 0.883 and 0.117, respectively. This yields a polymorphic information content (PIC) of 0.185 (PIC < 0.25), indicating low polymorphism and an imbalance according to Hardy-Weinberg detection (P < 0.05). At the g.8036 position of the DGAT1 gene sequence, the effective samples exhibit T and C alleles with frequencies of 0.842 and 0.158, respectively. The PIC for this site is 0.231 (PIC < 0.25), which also indicates low polymorphism and an imbalance according to the Hardy-Weinberg test (P > 0.05).
Table.1 Information about Exon1 and Exon6-Exon9 mutation sites of DGAT1 gene
Sequence Mutation site Mutation type Encoding the amino acid positions Mutant amino acids
Exonl g.3041T>C Missense mutation 23 Val>Ala
g.7817A>G Intron mutation
Exon6-Exon9
g.8036T>C Missense mutation 258 Met>Thr
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Fig.1 The Exonl and Exon 6-Exon9 mutation sites in the DGAT1 gene
Table.2 Information about Exonl and Exon6-Exon9 mutation sites of DGAT1 gene
Mutation site Gene type Quantit y Genotype frequency Allele frequency PIC Chi-square value
TT 51 0.850 T C
g.3041T>C TC CC 7 2 0.117 0.033 0.908 0.09 2 0.15 3 5.379
P
0.0 68
AA 50 0.833 g.7817A>G AG 6 0.100
GG 4 0.067
A
0.883
G
0.11 0.18 7 5
15.903
0.0 00
g.8036T>C TT 46 0.767
TC 9 0.150
CC 5 0.083
T
0.842
C
0.15 8
0.23 1
11.469
0.0 03
(2)A total of 13 haplotypes were identified from 60 sequences of the DGAT1 gene Exon 1(Table.4), exhibiting a gene diversity of 0.710, a nucleotide diversity of 0.00305, and an average number of nucleotide differences (k) of 1.274. The Tajima's D value and Fu and Li's D value were -1.89102 and -3.92000, respectively. Additionally, there were 404 conserved sites and 14 single mutation sites, including 4 sites with parsimonious information. From the 60 Exon 6-Exon 9 sequences, 4 haplotypes were identified(Table.4), with a gene diversity of 0.491, a nucleotide diversity of 0.00074, and an average number of nucleotide differences (k) of 0.677. The corresponding Tajima's D value and Fu and Li's D value were -1.89102 and -0.46069, respectively,
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along with 915 conserved sites and 4 single mutation sites, including 2 sites with parsimonious information. The negative values of Tajima's D and Fu and Li's D for both Exon 1 and Exon 6-Exon 9 sequences indicate a lack of significant differentiation in Exon 1(P>0.05), while Exon 6-Exon 9showed significant differentiation (P<0.05). This suggests that the Kazakh horse Altay type is generally in a balanced state, with no evidence of large-scale population expansion, although it may have experienced small-scale migration and expansion.
Table.4 Sequence polymorphisms of DGAT1 gene Exonl and Exon6-Exon9
Group S H Hd Hds n K Tajama's D Value P1 Fu and Li's P2 Value
1 0.71 0.04 0.003 1.27 <0.0 <0.
Exonl -1.89102 -3.92000
3 0 0 05 4 5 02
60
0.49 0.06 0.000 0.67 >0.
Exon6-Exon9 4 -0.46049 >0.1 -1.27574
1 4 74 7 1
Note: S is the total number of sequences, H is the number of haplotypes, Hd is the haplotype diversity, Hds is the standard deviation of haplotype diversity, n is the nucleotide diversity, K is the average nucleotide difference number, Pi is the significance of Tajama's D value, P2 is the significance of Fu and Li's value.
(3)As can be seen from Table.5, the molecular variance analysis showed that 88.99% of the variation in the Exoni sequence of DGAT1 gene occurred within the population, and 11.01% of the variation came from among the populations. In the sequence of Exon6-Exon9, 98.56% of the variation came from within populations, and 1.44% came from among populations. Combined with the analysis of the haplotype network and mismatch distribution map in the above study,itwas further suggested that the variation caused by the mutation of DGAT1 gene was caused by the adaptation of Kazakh horses to the local environment during their long-term life. During this period, this population did not experience a certain number expansion, which may be related to the fact that Kazak horses maintained this breed selection.
Table.5 Molecular variance analysis of genetic differences in DGAT1 gene Exon1 and Exon6-Exon9
Sequence Source of variation df Quadratic Variance component Percentag e of F P
sum s variation
Exon1 Among Population Within Population 2 57 4.083 33.500 0.07270Va 0.58722Vb 11.01 88.99 3.474 0.038
Total 59 37.583 0.66042
Exon6- Among Population Within Population 2 0.867 0.00491Va 1.44 1.22 0.302
Exon9 57 19.100 0.33509Vb 98.56
Total 59 19.967 0.34000
5 Conclusion
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "ACTUAL PROBLEMS OF LIVESTOCK DEVELOPMENT, MODERN METHODS AND
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In summary, the polymorphisms in the DGAT1 gene, specifically in Exon 1 and Exons 6-9, are notably high, with a total of three mutation sites identified: g.3041T>C, g.7817A>G, and g.8036T>C. Among these, g.3041T>C and g.8036T>C represent missense mutations. The distribution of haplotypes is relatively uniform, with the proportion of Kazakh horses being consistent across the three sampling points for each haplotype. Notably, the sixth haplotype in Exon 1 is shared by Kazakh horses at the D and K sampling sites, suggesting a historical period of genetic exchange. Molecular variance analysis indicates that the mutations at the three DGAT1 gene sites originated within the population, demonstrating that these mutations are associated with the adaptation to the environment and the long-term domestication of Kazakh horses.
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