A molecular marker for identifying feed efficiency traits of ducks based on prkacb gene and an identification method and application thereof

Abstract

The present application relates to a kind of molecular marker based on PRKACB gene identification duck feed efficiency character and its identification method and application, belong to molecular marker technical field, the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1, wherein, the base at the 588th position of the nucleotide sequence is G or A.The present application is developed molecular marker based on the research on the relationship between PRKACB gene and duck feed efficiency character, by identifying the type of the molecular marker present in duck genome, the feed efficiency character of duck can be selected, a kind of poultry feed efficiency early selection breeding method is established, a new molecular marker assisted breeding method is provided for the growth character detection of duck, which is simple, fast, low cost, does not need special instrument, is suitable for the needs of molecular marker assisted breeding experiment.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a molecular marker for identifying duck feed efficiency traits based on the PRKACB gene, its identification method, and its application. Background Technology

[0002] In poultry production, feed contributes the most to livestock production costs. Improving feed efficiency is crucial for enhancing the economic value and sustainability of poultry production. Feed efficiency is measured by feed conversion ratio (FCR) or residual feed intake (RFI). FCR is defined as the ratio of feed intake to weight gain over a specific period; the lower the FCR, the better the feed efficiency. RFI is defined as the difference between an animal's actual feed intake and its expected feed intake. A negative RFI indicates a lower feed conversion ratio and better feed efficiency, while a positive RFI indicates a higher feed conversion ratio and worse feed efficiency. (Wen C, Yan W, Mai C, et al. Joint contributions of the gut microbiota andhost genetics to feed efficiency in chickens. Microbiome. 2021, 9(1):126; LiuJ, Stewart SN, Robinson K, et al. Linkage between the intestinal microbiota and residual feed intake in broiler chickens.[J] Anim Sci Biotechnol. 2021,12(1):22; Zhang D, Zhang X, Li F, et al. Polymorphisms in ovine ME1 and CA1genes and their association with feed efficiency in Hu sheep.[J] Anim BreedGenet. 2021, 138(5):589-599.).

[0003] PRKACB encodes different catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase (PKA). Under steady state, PKA exists in a tetrameric inactive state by binding two catalytic subunits and two regulatory subunits. When cAMP binds to the regulatory subunit, the catalytic and regulatory subunits detach, the catalytic subunit becomes activated and phosphorylates the substrate. PRKACB regulates the cAMP pathway, and cAMP activation inhibits lipogenesis, promotes lipolysis, reduces lipid deposition, and leads to higher feed efficiency (Itoh T, Omori Y, Seino M, et al. Gene Rearrangement and Expression of PRKACA and PRKACB GovernMorphobiology of Pancreatobiliary Oncocytic Neoplasms.[J] Mod Pathol. 2024,37(1):100358;Yi X, Yi S, Wang J, et al. Differential lipid metabolism in beefcattle: A comparative study of high and low residual feed intake bulls.[J]Anim Nutr. 2025, 22:214-229;Huang D, Wang Y, Qi P, et al. al. Transcriptomeanalysis of divergent residual feed intake phenotypes in the M[J].longissimus thoracis et lumborum of Wannan Yellow rabbits. Front Genet. 2023,14:1247048.) However, current research on the PRKACB gene mainly focuses on mice and humans, and the research content is mostly centered on the metabolic regulation of fatty acids and the lipid deposition mechanism in the body. However, in the poultry field, especially in research on ducks, the exploration of the PRKACB gene is still insufficient. Therefore, it is particularly important to explore in depth the specific impact of PRKACB gene variation and expression pattern on feed conversion ratio in broiler ducks and reveal the underlying molecular mechanism. This will not only help us to more comprehensively understand the key role of the PRKACB gene in the growth, development and feed utilization of broiler ducks, but also provide a strong theoretical basis for improving the feed efficiency of broiler ducks, which will help reduce the breeding cost of broiler ducks and promote the sustainable development of poultry farming. To this end, this invention proposes a molecular marker for identifying duck feed efficiency traits based on the PRKACB gene, as well as its identification method and application. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker for identifying duck feed efficiency traits based on the PRKACB gene, along with its identification method and application, in order to solve the above-mentioned problems. This invention targets SNP (single nucleotide polymorphism) molecular markers of candidate genes related to duck feed utilization traits, thereby addressing the challenge of slow progress in conventional phenotypic breeding and achieving early identification of feed utilization traits.

[0005] The present invention achieves the above objectives through the following technical solutions: A molecular marker for identifying duck feed efficiency traits based on the PRKACB gene, wherein the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and the 588th base of the nucleotide sequence is G or A.

[0006] Application of a molecular marker based on the PRKACB gene for identifying duck feed efficiency traits.

[0007] As a further optimization of the present invention, if the molecular marker type of the duck to be tested is AA, the duck has the best feed efficiency trait; if the molecular marker type of the duck to be tested is GA, the duck has a poor feed efficiency trait.

[0008] A method for identifying duck feed efficiency traits using molecular markers as described above includes the following steps: (1) Extract total DNA from the venous blood of the ducks to be tested; (2) Design specific amplification primers with the molecular marker site and the sequence composed of its upstream and downstream bases as the target sequence, use the total DNA as a template, and perform PCR amplification using the specific amplification primers to obtain the amplification product; (3) Genotyping and sequencing of the amplification products to obtain the molecular marker type of the duck to be tested; (4) Determine the duck feed efficiency trait based on the molecular marker type.

[0009] As a further optimization of the present invention, the sequence of the specific amplification primers is as follows: SEQ ID NO.2: Forward primer: GGGTTCATGGTGGGTCT; SEQ ID NO.3: Reverse primer: AGTCGGGTGATGGCTCTCTA.

[0010] As a further optimization of the present invention, the genotyping detection method involves obtaining enzyme digestion products by digesting the amplification products with restriction endonucleases, detecting the enzyme digestion products using agarose gel electrophoresis, and performing genotyping based on the images. If the enzyme digestion products: If it contains 1 band, it is type AA; if it contains 3 bands, it is type GA.

[0011] As a further optimization of the present invention, the amplification product is digested with BsrBI restriction endonuclease.

[0012] As a further optimization of the present invention, if the molecular marker type of the duck to be tested is AA, the duck has the best feed efficiency trait; if the molecular marker type of the duck to be tested is GA, the duck has a poor feed efficiency trait.

[0013] As a further optimization of the present invention, the enzyme digestion products are detected by agarose gel electrophoresis at a concentration of 1.5%-2.0%.

[0014] The present invention has the following beneficial effects: This invention develops a molecular marker based on research into the relationship between the PRKACB gene and duck feed efficiency traits. By identifying the types of these molecular markers present in the duck genome, selection of duck feed efficiency traits can be achieved, establishing a breeding method for early selection of poultry feed efficiency. This provides a new molecular marker-assisted breeding method for detecting duck growth traits. This method is simple, rapid, low-cost, and does not require special instruments, making it suitable for the needs of molecular marker-assisted breeding experiments. Attached Figure Description

[0015] Figure 1 Agarose gel electrophoresis images of PCR amplification products from a portion of the samples; Figure 2 Agarose gel electrophoresis image of the enzyme digestion products obtained by enzyme digestion of PCR amplification products from a portion of the samples; Figure 3 This is the genotype verification sequencing result for the G25090364A site (site 588 in SEQ ID NO.1) in the duck PRKACB gene. Detailed Implementation

[0016] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] 1. Materials Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0018] 2. Method 2.1 Primer Design The DNA sequence corresponding to the PRKACB gene (Gene ID: NC_092594.1) shown in SEQ ID NO.1 was found in the duck genome database. Using the partial DNA sequence of the PRKACB gene (the sequence consisting of the molecular marker site and its upstream and downstream bases) as a template, specific amplification primers were designed. The specific amplification primer sequences are shown below: SEQ ID NO.2: Forward primer: GGGTTCATGGTGGGTCT; SEQ ID NO.3: Reverse primer: AGTCGGGTGATGGCTCTCTA.

[0019] The amplifiable region of the primer is 600 bp in length, and the amplified sequence is shown in SEQ ID NO.4, which contains the molecular marker of the G / A mutation at the G25090364A site (the 588th site in SEQ ID NO.1).

[0020] 2.2 Extraction of total DNA from blood A total of 549 Qiangying ducks were selected, and blood was collected from the medial metatarsal vein. Total DNA was extracted from the blood. The total DNA was extracted from the blood samples from the medial metatarsal vein using a blood DNA extraction kit produced by Tiangen Biotech Co., Ltd. The extraction steps were performed according to the kit instructions.

[0021] 2.3 PCR Amplification Using Mix produced by Shanghai Yisheng Biotechnology Co., Ltd., PCR amplification of the target fragment of the PRKACB gene was performed using pre-synthesized sequencing-specific primers. The PCR amplification system is shown in Table 1. Table 1 PCR amplification system The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; first step denaturation at 95℃ for 45 s; second step annealing at 59.1℃ for 45 s (annealing temperature is set according to the primers); third step extension at 72℃ for 30 s, with the second and third steps repeated 34 times for a total of 35 cycles; and a final extension at 72℃ for 10 min.

[0022] 2.4 Detection and Sequencing of PCR Amplification Products PCR amplification products were detected using 2% agarose gel electrophoresis, such as... Figure 1 As shown, after imaging with a gel imaging system, a 600bp band was obtained, which was consistent with the predicted length, indicating that the target fragment was obtained. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. (Nanjing), and the sequence is shown in SEQ ID NO.4, which is consistent with the predicted result.

[0023] 2.5 Genotyping 2.5.1 Prepare the enzyme digestion system as shown in Table 2. The digestion conditions are 37℃, PCR overnight, and the enzyme digestion is performed using Beijing Bio-Labs Technology Co., Ltd. The PCR amplification product was digested with a restriction endonuclease [5'-CC^GCTC-3']. Table 2 Enzyme digestion system 2.5.2. Detection was performed using 1.5% mass ratio low-voltage agarose gel electrophoresis to obtain results such as... Figure 2 The results shown are partial; among them, if the enzyme digestion product contains 1 band, it is of type AA; if it contains 3 bands, it is of type GA.

[0024] 2.6. Enzyme digestion and sequencing verification Statistical analysis of the agarose gel electrophoresis images of gene restriction enzyme genotyping revealed two genotypes: AA and GA. One individual from each genotype was selected for sequencing comparison. The sequencing comparison images are shown below. Figure 3 As shown in the sequencing results, G mutated into A, and the arrows indicate the mutation locations, which is consistent with the enzyme digestion typing results.

[0025] 2.7 Effect Verification To determine the association between the G / A polymorphism at the G25090364A locus of the duck PRKACB gene and important phenotypic traits in ducks, 549 Qiangying ducks from step 2.2 above were used as experimental materials. Feed intake (ADFI), average daily gain (ADG), and metabolite gain (AGR) were recorded from 21 to 38 days of age. The feed conversion ratio (FCR) and residual feed intake (RFI) were used to genotype 549 Qiangying ducks using the genotyping method described in step 2.5 above. The results are shown in Table 3. Table 3. Genotype detection results for individuals with different phenotypes Experimental conclusion: The chi-square test results showed that the genotypes of the experimental duck population were in Hardy-Weinberg equilibrium (P>0.05).

[0026] 2.8 Statistical Analysis The association between the three genotypes and duck feed efficiency traits was analyzed using analysis of variance in SPSS software. The association results between different genotypes and each trait are shown in Table 4. Table 4. Association analysis between duck PRKACB genotype and duck feed efficiency traits. Note: Different lowercase letters in the same line indicate significant differences (P<0.05), and different uppercase letters in the same line indicate extremely significant differences (P<0.01).

[0027] Experimental Conclusions: As shown in Table 4, for the PRKACB gene G25090364A locus, the AA genotype individuals had significantly lower daily feed intake (ADFI), feed conversion ratio (FCR), and residual feed intake (RFI) traits than the GA genotype individuals (P<0.01); the two genotypes also showed significant differences in average daily gain (ADG) and metabolite gain (…). There was no significant difference in feed efficiency (P>0.05) in the AA genotype, which indicates that individuals with the AA genotype had the best feed efficiency trait, while individuals with the GA genotype had the worst feed efficiency trait.

[0028] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A molecular marker for identifying duck feed efficiency traits based on the PRKACB gene, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 588th base of the nucleotide sequence is G or A.

2. The application of the molecular marker as described in claim 1 in identifying duck feed efficiency traits.

3. The application according to claim 2, characterized in that, If the molecular marker type of the duck to be tested is AA, the duck has the best feed efficiency trait; if the molecular marker type of the duck to be tested is GA, the duck has a poor feed efficiency trait.

4. A method for identifying duck feed efficiency traits using the molecular markers as described in claim 1, characterized in that, Includes the following steps: (1) Extract total DNA from the venous blood of the ducks to be tested; (2) Design specific amplification primers with the molecular marker site and the sequence composed of its upstream and downstream bases as the target sequence, use the total DNA as a template, and perform PCR amplification using the specific amplification primers to obtain the amplification product; (3) Genotyping and sequencing of the amplification products to obtain the molecular marker type of the duck to be tested; (4) Determine the duck feed efficiency trait based on the molecular marker type.

5. The method for identifying duck feed efficiency traits using molecular markers according to claim 4, characterized in that, The sequence of the specific amplification primers is as follows: SEQ ID NO.2: Forward primer: GGGTTCATGGTGGGTCT; SEQ ID NO.3: Reverse primer: AGTCGGGTGATGGCTCTCTA.

6. The method for identifying duck feed efficiency traits using molecular markers according to claim 4, characterized in that, The genotyping detection method involves obtaining restriction endonuclease digestion of the amplification products, detecting the digested products using agarose gel electrophoresis, and performing genotyping based on the images. If the digested products: If it contains 1 band, it is type AA; if it contains 3 bands, it is type GA.

7. The method for identifying duck feed efficiency traits using molecular markers according to claim 6, characterized in that, The amplified products were digested with BsrBI restriction endonuclease.

8. The method for identifying duck feed efficiency traits using molecular markers according to claim 6, characterized in that, If the molecular marker type of the duck to be tested is AA, the duck has the best feed efficiency trait; if the molecular marker type of the duck to be tested is GA, the duck has a poor feed efficiency trait.

9. A method for identifying duck feed efficiency traits using molecular markers according to claim 6, characterized in that, The enzyme digestion products were detected by agarose gel electrophoresis at a concentration of 1.5%–2.0%.

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