Application of NDUFS1 gene molecular marker influencing death rate of duck sperms in duck genetic breeding

Abstract

The invention discloses application of an NDUFS1 gene molecular marker influencing the death rate of duck sperms in duck genetic breeding, and belongs to the technical field of biology. According to the method, SNP genotyping is performed by using a sequencing technology, the NDUFS1 gene molecular marker remarkably related to the death rate of the sperms is obtained through whole genome association analysis and screening, the death rate of the sperms of the ducks can be efficiently and rapidly identified through the method, and a scientific basis is provided for early breeding of high-quality ducks.

Description

Technical Field

[0001] This invention relates to the application of the NDUFS1 gene molecular marker affecting duck sperm mortality rate in duck genetic breeding, and belongs to the field of biotechnology. Background Technology

[0002] Sperm mortality rate is a core parameter for measuring the quality of male duck sperm, directly reflecting sperm viability and resistance to environmental stress. This indicator plays a decisive role in maintaining sperm motility and completing the fertilization process during artificial insemination. The sperm mortality rate not only directly affects the fertilization and hatching rates of eggs, but is also a key factor in determining whether the genetic resources of superior male ducks can be efficiently transferred, reducing ineffective feeding costs, and improving overall economic benefits.

[0003] In terms of genetic analysis, although existing studies have used genome-wide association studies (GWAS) or candidate gene strategies to screen for gene markers related to sperm viability in other livestock and poultry, relevant research in waterfowl such as ducks remains insufficient. In particular, the direct genetic association mechanism between specific variations in the NDUFS1 gene (NADH: ubiquinone oxidoreductase core subunit S1) and the key economic trait of sperm mortality remains unclear. Current technologies severely lack research on SNP molecular markers targeting specific regions of the duck NDUFS1 gene, as well as validation of the association between these markers and sperm mortality, and even more so, their application in early detection and genetic selection of sperm mortality in breeding ducks. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of the NDUFS1 gene molecular marker, which affects the sperm mortality rate in ducks, in duck genetic breeding, thereby improving the reproductive performance of breeding ducks, optimizing artificial insemination efficiency, and accelerating breeding progress.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] Firstly, the application of the NDUFS1 gene molecular marker affecting duck sperm mortality rate in duck genetic breeding is provided. The NDUFS1 gene molecular marker is located at position 26651389 on chromosome 7 of the duck reference genome GCF_015476345.1_ZJU1.0 version, where the base is T or C.

[0007] Secondly, a method for detecting duck sperm mortality rate is provided, the method comprising detecting the duck sperm mortality rate trait based on the NDUFS1 gene molecular marker, wherein the NDUFS1 gene molecular marker is located at position 26651389 of chromosome 7 in duck reference genome version GCF_015476345.1_ZJU1.0, and the base here is T or C.

[0008] Furthermore, the method specifically includes:

[0009] Step S1: The duck DNA sample to be tested is subjected to PCR amplification using duck DNA-specific primer pairs to obtain amplification products; the duck DNA-specific primer pairs include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2;

[0010] Step S2: Perform Sanger sequencing on the amplified products;

[0011] Step S3: Determine the molecular marker genotype of the target site based on the sequencing results of step S2.

[0012] Furthermore, in step S1, the final concentration of the PCR amplification reaction system is 25 μl, specifically:

[0013]

[0014]

[0015] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 60 sec, for a total of 30 cycles; 72℃ extension for 2 min; and storage at 20℃.

[0016] Furthermore, the amplification product is 149 bp in length and contains the 26651389th base on chromosome 7 of the duck reference genome GCF_015476345.1_ZJU1.0 version.

[0017] Furthermore, in step S3, the molecular marker genotype of the target site is determined as follows: the sperm mortality rate of T / T genotype ducks is lower than that of T / C and C / C genotype ducks, and the sperm mortality rate of T / C genotype ducks is lower than that of C / C genotype ducks.

[0018] Thirdly, this invention provides the application of a reagent for detecting the NDUFS1 gene molecular marker in detecting sperm mortality in ducks. The NDUFS1 gene molecular marker is located at position 26651389 of chromosome 7 in the duck reference genome GCF_015476345.1_ZJU1.0 version, where the base is T or C. The reagent includes duck DNA-specific primers, and the duck DNA-specific primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2.

[0019] This invention uses the NDUFS1 gene molecular marker to detect the genotype of duck sperm mortality. It was found that at 43 weeks of age, the sperm mortality rate of T / T genotype individuals was lower than that of T / C and C / C genotype individuals, and the sperm mortality rate of T / C genotype individuals was lower than that of C / C genotype individuals. Using the genomic DNA of the ducks to be tested as a template, PCR amplification was performed using specific primer pairs. The PCR amplification products were then subjected to Sanger sequencing and SNP molecular marker genotyping. Based on the genotype of these SNP molecular markers, selection based on the sperm mortality rate of ducks can be achieved. In breeding, according to the breeding objectives, T / C and C / C genotype individuals are culled, while T / T genotype individuals are retained.

[0020] The present invention has the following beneficial effects: the method of the present invention can efficiently and rapidly identify the sperm mortality rate in ducks, providing a scientific basis for the early selection and breeding of high-quality ducks. Furthermore, the detection method disclosed in this invention is simple and easy to operate, can be carried out in a laboratory, and can also be applied to genomic breeding technology.

[0021] First, it solves the problem that traditional detection methods cannot accurately analyze duck sperm mortality at the gene level, making it difficult to meet the needs of modern breeding for genetic improvement. Second, it fills the gap in the application of existing gene marker technology in duck sperm mortality, providing effective molecular markers for rapid screening and breeding. Finally, it improves detection efficiency, solves the limitations in cost control and large-scale application, and achieves efficient and low-cost breeding of ducks. Attached Figure Description

[0022] Figure 1 This is a Manhattan plot of GWAS analysis of sperm mortality in ducks at 43 weeks of age.

[0023] Figure 2 These are Sanger sequencing results of PCR amplification products from three genotypes.

[0024] Figure 3 This is a phenotypic distribution map of individuals with three genotypes of the chr7:26651389 molecular marker.

[0025] Upstream primer (SEQ ID NO: 1): 5'-TTGTTCAGCCTGTTGCC-3'

[0026] Downstream primer (SEQ ID NO: 2): 5'-ATGCCTGCCTCTGGTCT-3'

[0027] SEQ ID NO: 3

[0028] TTTGTTCAGCCTGTTGCCTTTTATATTCCTCAAATCTCCCACTTTGAAATATTTTTCCTTCTTTTTTTTTTTCTTTTTTATTTTGATTCCATTCTGTGCATTTCCCCCCTTTCTGATGGCAGTCAGACAGCCAGACCAGAGGCAGGCAT

[0029] SEQ ID NO: 4

[0030] TTTGTTCAGCCTGTTGCCTTTTATATTCCTCAAATCTCCCACTTTGCAAATATTTTTCCTTCTTTTTTTTTTTTTTTTATTTTGATTCCATTCTGTGCATTTCCCCCCTTTCTGATGGCAGTCAGACAGCCAGACCAGAGGCAGGCAT Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0032] Example 1

[0033] This embodiment measured the sperm mortality rate of male mountain ducks at 43 weeks of age. SNP genotyping was performed using sequencing technology, and the NDUFS1 gene molecular marker, significantly associated with sperm mortality, was identified through genome-wide association analysis. The results are as follows: Figure 1 As shown.

[0034] This embodiment uses the following experiments to identify and apply the NDUFS1 gene molecular marker related to duck sperm mortality.

[0035] 1. Phenotyping and Genotyping

[0036] (1) Experimental materials and sperm mortality phenotype determination

[0037] Forty-three male mountain ducks were selected as experimental animals and raised under identical conditions, with free access to food and water throughout the process. Semen was collected at 43 weeks of age, and the sperm mortality rate of each duck was measured using a fully automated sperm analyzer to obtain phenotypic data on the sperm mortality rate.

[0038] (2) Extraction of genomic DNA

[0039] Blood was collected from the subwing vein of the individuals to be tested, and after anticoagulation, the blood was lysed, digested with proteinase K, extracted using the saturated sodium chloride method, dissolved in TE, and stored at -20°C.

[0040] (3) PCR amplification

[0041] Using the extracted genomic DNA as a template, the fragment containing the SNP molecular marker at position 26651389 of duck chromosome 7 was amplified.

[0042] Upstream primer: 5'-TTGTTCAGCCTGTTGCC-3' (SEQ ID NO:1)

[0043] Downstream primer: 5'-ATGCCTGCCTCTGGTCT-3' (SEQ ID NO:2)

[0044] The final concentration (25 μl) of the reaction system is:

[0045]

[0046] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 60 sec, for a total of 30 cycles; 72℃ extension for 2 min; storage at 20℃; 10 μl was used for agarose gel assay, and an amplification product with a single target band length of 149 bp was obtained, containing base position 26651389 of duck chromosome 7. The amplification product is shown in sequence SEQ ID NO:3 or SEQ ID NO:4, with a mutation at position 73 to C or T.

[0047] (4) Sequencing verification and genotyping

[0048] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram is shown below. Figure 2 As shown.

[0049] (5) Phenotypic determination of fertilization rate and hatching rate of fertilized eggs in individuals with different genotypes

[0050] From 424 genotyped male Shanma ducks, 6 individuals each of the T / T, T / C, and C / C genotypes (a total of 18 individuals) were randomly selected, requiring that the individuals be healthy and of similar weight (±5%). 180 300-day-old Shanma female ducks with an egg production rate of 90% or higher for three consecutive weeks were selected and randomly divided into three groups of 60 ducks each, paired with male ducks of the T / T, T / C, and C / C genotypes respectively. All female ducks were raised separately in the same environment, fed a uniform complete feed, and had free access to water and food, for two weeks to acclimatize to the environment. Semen was collected from the male ducks and mixed with semen from 6 male ducks in the same genotype group for artificial insemination of the female ducks. The insemination volume was 0.05 mL / duck (containing a sperm count ≥3×10^7). Eggs were collected after 5 consecutive days of insemination, and this process was repeated for 3 weeks. Fertilization rate = (number of fertilized eggs / total number of eggs put into incubation) × 100%; Hatching rate of fertilized eggs = (number of chicks hatched / number of fertilized eggs) × 100%.

[0051] 2. Results Analysis

[0052] Correlation analysis was performed on 424 male Shanma ducks aged 43 weeks with clearly recorded sperm mortality rates. Statistical tests were conducted using the t.test function in R 4.0 software. Pairwise comparisons of means were used to statistically analyze the genotype and sperm mortality rates of the experimental duck groups. P < 0.05 indicated significant differences, and P < 0.01 indicated highly significant differences. The results are shown in Table 1 and... Figure 3 As shown, among the tested individuals, there were 133 T / T genotypes, 146 T / C genotypes, and 145 C / C genotypes. The sperm mortality rates at 43 weeks of age differed significantly among the three genotypes (P<0.01). At 43 weeks of age, the average sperm mortality rate of T / T genotype individuals was 0.858%, significantly lower than that of T / C and C / C genotype individuals (P<0.01), specifically 1.476% lower than T / C and 4.446% lower than C / C, respectively. The average sperm mortality rate of T / C genotype individuals was 2.334%, significantly lower than that of C / C genotype individuals (P<0.01), specifically 2.97% lower than C / C, respectively. The average fertilization rate of T / T genotype individuals was 93.1%, and the average hatching rate of fertilized eggs was 91.6%, significantly higher than that of T / C and C / C genotype individuals (P<0.05). The results showed that the duck NDUFS1 gene molecular marker was significantly associated with duck sperm mortality and affected fertilization rate and hatchability of fertilized eggs. According to actual breeding goals, T / T genotype individuals can be selected to reduce duck sperm mortality and improve fertilization rate and hatchability of fertilized eggs.

[0053] Table 1. Association analysis of molecular marker at position 26651389 on chromosome 7 with sperm mortality rate.

[0054] genotype Quantity / piece Sperm mortality rate at 43 weeks of age Standard deviation CV T / T 133 <![CDATA[0.858% a ]]> 0.233 27.16％ T / C 146 <![CDATA[2.334% b ]]> 0.378 16.2％ C / C 145 <![CDATA[5.304% c ]]> 0.873 16.46％

[0055] Table 2. Association analysis between molecular marker at position 26651389 on chromosome 7 and fertilization rate.

[0056] genotype fertilization rate Standard deviation CV T / T <![CDATA[93.1% a ]]> 0.7 0.75％ T / C <![CDATA[92.4% b ]]> 0.8 0.87％ C / C <![CDATA[90.9% c ]]> 0.7 0.77％

[0057] Table 3. Association analysis between molecular marker at position 26651389 on chromosome 7 and hatchability of fertilized eggs.

[0058] genotype Hatching rate of fertilized eggs Standard deviation CV T / T <![CDATA[91.6% a ]]> 0.6 0.65％ T / C <![CDATA[90.9% b ]]> 0.7 0.77％ C / C <![CDATA[88.8% c ]]> 0.7 0.79％

[0059] Note: Data with the same subtitle in the same column indicate no significant difference, while data with different subtitles indicate significant difference (P<0.05).

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. The application of the NDUFS1 gene molecular marker affecting duck sperm mortality rate in duck genetic breeding, characterized by: The NDUFS1 gene molecular marker is located at position 26651389 on chromosome 7 of the duck reference genome GCF_015476345.1_ZJU1.0 version, where the base is either T or C.

2. A method for detecting the mortality rate of duck sperm, characterized in that, The method includes detecting the sperm mortality trait in ducks based on the NDUFS1 gene molecular marker, which is located at position 26651389 on chromosome 7 of the duck reference genome GCF_015476345.1_ZJU1.0 version, where the base is T or C.

3. The method according to claim 1, characterized in that, The method specifically includes: Step S1: The duck DNA sample to be tested is subjected to PCR amplification using duck DNA-specific primer pairs to obtain amplification products; the duck DNA-specific primer pairs include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2; Step S2: Perform Sanger sequencing on the amplified products; Step S3: Determine the molecular marker genotype of the target site based on the sequencing results of step S2.

4. The method according to claim 3, characterized in that, In step S1, the final concentration of the PCR amplification reaction system is 25 μl, specifically: The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 60 sec, for a total of 30 cycles; 72℃ extension for 2 min; and storage at 20℃.

5. The application according to claim 3, characterized in that, The amplification product is 149 bp in length and contains the 26651389th base on chromosome 7 of the duck reference genome GCF_015476345.1_ZJU1.0 version.

6. The application according to claim 3, characterized in that, In step S3, the molecular marker genotype of the target site is determined as follows: the sperm mortality rate of T / T genotype ducks is lower than that of T / C and C / C genotype ducks, and the sperm mortality rate of T / C genotype ducks is lower than that of C / C genotype ducks.

7. The application of a reagent for detecting the NDUFS1 gene molecular marker in the detection of duck sperm mortality, characterized in that, The NDUFS1 gene molecular marker is located at position 26651389 of chromosome 7 in the duck reference genome GCF_015476345.1_ZJU1.0 version, where the base is T or C. The reagents include duck DNA-specific primers, and the duck DNA-specific primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2.

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