公牛精子耐冻性相关基因多态性位点的挖掘与功能验证

doi:10.11843/j.issn.0366-6964.2025.04.044

Abstract

Abstract:

The study aimed to identify key genes and molecular markers that affect the freezability of bull sperm and apply them to molecular breeding of dairy cattle. In this study, 11 candidate genes related to bull sperm freezability were identified based on the previous omics study, such as ATP5F1A, DNASE2, and LPO. Primers were designed for the exons of these genes to detect genetic variation sites via PCR pooled sequencing. Subsequently, the Time-of-Flight mass spectrometry was used to genotype important loci in a resource population of 130 healthy bulls and association analysis was performed. Among them, there were 55 bulls in the high sperm freezability group, with an average fresh sperm motility of 0.68 and an average post-freeze motility of 0.42, and 75 bulls in the low sperm freezability group, with an average fresh sperm motility of 0.65 and an average post-freeze motility of 0.32. Finally, 14 significant SNPs were identified, among which the rs110909003 SNP (C/T) located in the exon of ATP5F1A was significantly associated with the bull sperm freezability (P=0.009). Western blot analysis was performed to detect the expression of ATP5F1A in sperm with different genotypes at the rs110909003 locus. The results showed a significant increase in ATP5F1A protein expression in sperm from bulls with the CC genotype compared to those with the TT genotype. This study identified ATP5F1A significantly associated with bull sperm freezability, as well as an important molecular marker, the rs110909003 SNP, located within this gene. This SNP may influence sperm freezability by affecting the mitochondrial oxidative phosphorylation process. These findings provide valuable information for the molecular breeding of frozen semen quality traits in Holstein bulls.

Key words: Holstein cattle, sperm, freezability, functional genes, molecular markers

CLC Number:

S823.3

LI Xiaotong, WANG Pengyu, FANG Yingyan, YU Hongxi, ZHANG Yi, WANG Yachun, ZHANG Yuanpei, LI Yanqin, JIANG Li. Mining and Functional Verification of Gene Polymorphisms Loci Related to Bull Sperm Freezability[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1981-1988.

Figures/Tables 4

Table 1

Table 2

Table 3

Fig. 1

References 41

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增区域/bp Amplification region	退火温度/℃ Annealing temperature
ATP5F1A-E-1-F	GCCAGTTCCGCTATAACCAT	chr24：45850514~45851138	56
ATP5F1A-E-1-R	GCCATCTCAAGACACCACGAA
ATP5F1A-E-7-F	TGATGGATGGTGCTCATG	chr24：45842218~45842747	52
ATP5F1A-E-7-R	TTTCCATCGGTCCTATTT
DNASE2-E-4-F	CCCCTACAGACTGAGTGTTC	chr7：12677669~12678118	60
DNASE2-E-4-R	ATGGCCCTTGACTACCTC
DNASE2-E-2-F	TCCTTCCTGTCTTCCTCCAT	chr7：12677037~12677506	60
DNASE2-E-2-R	CAGGCAGGTCAGGAATCAG
LPO-E-5-F	AGCGGCAACCCTCTGATT	chr19：9227370~9227883	59
LPO-E-5-R	CTGACCTGACTGTCCTCCC
LPO-E-9-F	GTCAACCTTATTATCCCCATT	chr19：9234022~9234651	58
LPO-E-9-R	CTGACACCCCAAATCCAG
LPO-E-11-F	TGGGAAGGAGAATTGGAC	chr19：9242716~9243351	54
LPO-E-11-R	ACCACTGTAGGGCACTTT
CUTA-E-1-F	TGCAGATCCAGGTGTTGA	chr23：7593114~7593706	54
CUTA-E-1-R	GCATCCAAAACAGCGACA
ITGA6-E-1-F	GAACGGCAACCACAACTCAAGC	chr2：24178781~24179629	60
ITGA6-E-1-R	GGCTGAACTGGGGAAGGC
ITGA6-E-7-F	TTACTGTTGATGTTCTCCTTTG	chr2：24128249~24129073	54
ITGA6-E-7-R	AAAACAAGCAGCTCATTG
ACSL4-E-2-F	TTTGTTATGGTGATTGAA	chrX：58099374~58100231	57
ACSL4-E-2-R	TTTCTACTCTATTTGTCTG
ACSL4-E-9-F	ACCCCTCTACTTGCTCTT	chrX：58051804~58052313	54
ACSL4-E-9-R	GGTCCTCTGCTTGCTATA
ACSL4-E-17-F	ACATCGGAAACCATTTTA	chrX：58034060~58034806	59
ACSL4-E-17-R	GCACAAATATCAGTCCCT

基因名 Gene name	SNP ID SNP_ID	位置 Location	等位基因 Allele	突变类型 Mutation type
ATP5F1A	rs382264973	24:45850779	G/C	synonymous variant
	rs110909003	24:45842378	C/T	missense variant
DNASE2	rs42312321	7:12677843	T/C	missense variant
	rs380569688	7:12677265	T/G	splice donor 5th base variant
LPO	rs136605698	19:9227664	A/C	synonymous variant
	rs135860093	19:9234370	G/T	missense variant
	rs378135599	19:9242904	C/T	synonymous variant
CUTA	rs136744810	23:7593210	A/C	5′ UTR variant
ITGA6	rs451676306	2:24179226	G/T	5′ UTR variant
	rs209381216	2:24128761	T/C	missense variant
	rs133912789	2:24128525	A/C	synonymous variant
ACSL4	rs3423605874	X: 58099789	A/G
	rs384883555	X: 58052098	A/G	synonymous variant
	rs135718815	X: 58034271	A/G

SNP_ID	等位基因 Allele	最小等位基因频率 Minor allele frequency (MAF)		卡方值 χ²	P值 P value
SNP_ID	等位基因 Allele	LF	HF	卡方值 χ²	P值 P value
rs382264973	G/C	0.47(C)	0.49(G)	0.191	0.662
rs110909003	C/T	0.14(C)	0.31(C)	6.731	0.009^*
rs42312321	T/C	0.47(C)	0.40(C)	0.618	0.432
rs380569688	T/G	0.15(G)	0.13(G)	0.086	0.769
rs136605698	A/C	0.49(A)	0.47(C)	0.278	0.598
rs135860093	G/T	0.15(T)	0.18(T)	0.104	0.747
rs378135599	C/T	0.22(T)	0.23(T)	0.003	0.955
rs136744810	A/C	0.42(A)	0.33(A)	1.275	0.259
rs209381216	T/C	0.31(C)	0.30(C)	0.008	0.927
rs133912789	A/C	0.42(C)	0.36(C)	0.606	0.436
rs384883555	A/G	0.10(G)	0.15(G)	1.025	0.311
rs135718815	A/G	0.24(A)	0.28(A)	0.243	0.622

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Mining and Functional Verification of Gene Polymorphisms Loci Related to Bull Sperm Freezability

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