A molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, its identification method and application

Abstract

This invention relates to a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, its identification method, and its application, belonging to the field of molecular marker technology. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.1, wherein the 193rd base of the nucleotide sequence is G or A. This invention provides a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, its identification method, and its application. Based on research on the relationship between the PLA2G1B gene and duck feed utilization traits, a molecular marker was developed. By identifying the type of this molecular marker present in the duck genome, selection of duck feed utilization traits can be performed, establishing a breeding method for early selection of poultry feed utilization. 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.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, its identification method, and its application. Background Technology

[0002] Residual Feed Intake (RFI) is defined as the difference between the actual feed intake and the expected feed intake of an animal given a certain body weight and weight gain. Therefore, the larger the RFI value, the lower the relative feed efficiency. In recent years, more and more breeders have begun to use RFI as the main indicator for measuring feed efficiency, and this trend has become more and more obvious with the development of the times. This is because using RFI for breeding can not only take into account daily weight gain and metabolic weight, but also has a relatively weak impact on important economic traits such as market weight. Moreover, the heritability of RFI is between 0.2 and 0.4 (Chen C, Su Z, Li Y, et al. Estimation of the genetic parameters of traits relevant to feed efficiency: result from broiler lines divergent for high or low abdominal fat content[J]. Poult Sci, 2021, 100(2): 461-466; Zhang Y, Guo ZB, Xie M, et al. Genetic parameters for residual feed intake in a random population of Pekin duck[J].Asian-Australas J Anim Sci, 2017, 30(2): 167-170.), belongs to the trait of medium heritability, so RFI can be used as a measure of feed efficiency in breeding.

[0003] PLA2G1 is a class of enzymes that catalyze the hydrolysis and removal of the sn-2 fatty acid acyl group in phospholipids, including cell-secreted enzymes and intracellular phospholipases. Evidence from in vitro cell culture experiments suggests that PLA2G1B in pancreatic extract is responsible for the digestion of phospholipids and the absorption of lipids by intestinal epithelial cells (HOMAN R, HAMELEHLE K L. Phospholipase A2 relieves phosphatidylcholine inhibition of micellar cholesterol absorption and transport by human intestinal cell line Caco-2[J]. J Lipid Res, 1998, 39(6): 1197-209; MACKAY K, STARR JR, LAWN RM, et al. Phosphatidylcholine hydrolysis is required for pancreatic cholesterol esterase-and phospholipase A2-facilitated cholesterol uptake into intestinal Caco-2 cells[J]. J Biol Chem, 1997, 272(20): 13380-9.). Studies using PLA2G1B knockout mice have shown that when animals are fed a normal low-fat diet, there is no difference in fat and cholesterol absorption between PLA2G1B knockout mice and PLA2G1B wild-type mice. Although a delay in fat and cholesterol absorption was observed when PLA2G1B knockout mice were fed a long-term high-fat / high-cholesterol diet, the total amount of fat and cholesterol absorbed by PLA2G1B knockout mice was similar to that of PLA2G1B mice fed a high-fat / high-cholesterol diet (HUGGINS KW, BOILEAU AC, HUID Y. Protection against diet-induced obesity and obesity-related insulin resistance in Group 1B PLA2-deficient mice[J]. Am J Physiol Endocrinol Metab, 2002, 283(5): E994-E1001.).

[0004] Current research on the PLA2G1B gene primarily focuses on mice, fish, and humans, with studies largely centered on fatty acid metabolism regulation and lipid deposition mechanisms. However, research on the PLA2G1B gene, particularly in ducks, is still insufficient. Nevertheless, based on existing findings, it can be inferred that the PLA2G1B gene may be an important candidate gene for regulating duck feed efficiency. Therefore, in-depth exploration of the variations and expression patterns of the PLA2G1B gene on the feed conversion ratio of broiler ducks, and revealing the underlying molecular mechanisms, is crucial. This will not only help us to more comprehensively understand the key role of the PLA2G1B gene in the growth, development, and feed utilization of broiler ducks, but also provide a strong theoretical basis for improving feed efficiency in broiler ducks, helping to reduce the cost of broiler duck farming and promoting the sustainable development of the poultry industry. To this end, this invention proposes a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, along with its identification method and application. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, as well as its identification method and application, in order to solve the above-mentioned problems.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] This invention provides a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 193rd base of the nucleotide sequence is G or A.

[0008] The present invention also provides an application of the above-mentioned molecular marker in identifying duck feed utilization traits.

[0009] 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 utilization trait; if the molecular marker type of the duck to be tested is GA, the duck has a moderate feed utilization trait; and if the molecular marker type of the duck to be tested is GG, the duck has a poor feed utilization trait.

[0010] The present invention also provides a method for identifying duck feed utilization traits using the above-mentioned molecular markers, comprising the following steps:

[0011] (1) Extract total DNA from the blood of the medial metatarsal vein of the duck to be tested;

[0012] (2) Design specific amplification primers with molecular marker nucleotide sequences as target sequences, use total DNA as template, and perform PCR amplification using specific amplification primers to obtain amplification products;

[0013] (3) Sequencing and genotyping of the amplification products to obtain the molecular marker type of the duck to be tested;

[0014] (4) Determine the duck feed utilization trait based on the molecular marker type.

[0015] As a further optimization of the present invention, the sequence of the specific amplification primers is as follows:

[0016] SEQ ID NO.2: Forward primer: ACGCCATCTCCACTAACAT;

[0017] SEQ ID NO.3: Reverse primer: GTTTTCCAGAGAATCCCAAG.

[0018] As a further optimization of the present invention, the method for genotyping detection is to obtain enzyme digestion products by digesting the amplification products, detect the enzyme digestion products by agarose gel electrophoresis, and perform genotyping based on the images. If the enzyme digestion product contains 1 band, it is AA type; if it contains 2 bands, it is GG type; and if it contains 3 bands, it is GA type.

[0019] As a further optimization of the present invention, the amplification product is digested with PspXⅠ restriction endonuclease.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention, based on research into the relationship between the PLA2G1B gene and duck feed utilization traits, developed a molecular marker. By identifying the types of this molecular marker present in the duck genome, selection of ducks for feed utilization traits can be achieved, establishing a breeding method for early selection of poultry feed utilization. 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

[0022] Figure 1 Agarose gel electrophoresis images of PCR amplification products from a portion of the samples;

[0023] 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;

[0024] Figure 3 This is the genotype verification sequencing result for the G7980795A site (site 193 in SEQ ID NO.1) in the duck PLA2G1B gene. Detailed Implementation

[0025] 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.

[0026] 1. Materials

[0027] 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.

[0028] 2. Method

[0029] 2.1 Primer Design

[0030] The DNA sequence corresponding to the PLA2G1B gene (Gene ID: NC_051787.1) shown in SEQ ID NO.1 was found in the duck genome database. Using the partial DNA sequence of the PLA2G1B 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:

[0031] SEQ ID NO.2: Forward primer: ACGCCATCTCCACTAACAT;

[0032] SEQ ID NO.3: Reverse primer: GTTTTCCAGAGAATCCCAAG.

[0033] The amplifiable region of the primer is 731 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 G7980795A site (the 193rd site in SEQ ID NO.1).

[0034] 2.2 Extraction of total DNA from blood

[0035] Forty-five 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.

[0036] 2.3 PCR amplification

[0037] Using Mix produced by Shanghai Yisheng Biotechnology Co., Ltd., PCR amplification of the target fragment of the PLA2G1B gene was performed using pre-synthesized sequencing-specific primers. The PCR amplification system is shown in Table 1.

[0038] Table 1 PCR amplification system

[0039] Components Dosage DNA template 1μL Forward primer 1μL Reveres primer 1μL Mix 10μL <![CDATA[ddH2O]]> 7μL total 20μL

[0040] 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 64.8℃ 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 31 times for a total of 32 cycles; and a final extension at 72℃ for 10 min.

[0041] 2.4 Detection and sequencing of PCR amplification products

[0042] PCR amplification products were detected using 2% agarose gel electrophoresis, such as... Figure 1 As shown, after imaging with a gel imaging system, a band of approximately 731 bp in length was obtained, which is 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.

[0043] 2.5 Genotyping

[0044] 2.5.1 Prepare the enzyme digestion system as shown in Table 2. The enzyme digestion conditions are 37℃ water bath for 1 hour. Use Psp XⅠ restriction endonuclease from Beijing Biolabs Technology Co., Ltd. to digest the PCR amplification product.

[0045] Table 2 Enzyme digestion system

[0046] Components Dosage PCR amplification products 0.4μL PspXⅠ 0.4 μL (5 U / μL) buffer 2μL <![CDATA[ddH2O]]> 7.2μL total 10μL

[0047] 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; contains 2 bands, it is of type GG; and contains 3 bands, it is of type GA.

[0048] 2.6 Enzyme digestion and sequencing verification

[0049] Statistical analysis of the gene genotyping agarose gel electrophoresis images yielded three genotypes: AA, GA, and GG. One individual was selected from each of these three genotypes for sequencing alignment. The sequencing alignment 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.

[0050] 2.7 Effect Verification

[0051] To determine the association between the G / A polymorphism at the G7980795A locus of the PLA2G1B gene in ducks and important phenotypic traits, 475 Qiangying ducks from section 2.2 were used as experimental materials. Feed intake (ADFI), average daily gain (ADG), and metabolite weight gain (MBW) were recorded from 21 to 42 days of age. 0.75 ), feed conversion ratio (FCR), and residual feed intake (RFI) were determined. The 2.5 genotyping method was used to genotype 475 Qiangying ducks, and the results are shown in Table 3.

[0052] Table 3. Genotype detection results for individuals with different phenotypes

[0053]

[0054] Experimental conclusion: The chi-square test results showed that the genotypes of the experimental duck population were in Hardy-Weinberg equilibrium (P>0.05).

[0055] 2.8 Statistical Analysis

[0056] The association between the three genotypes and the duck feed utilization trait was analyzed using the least squares method in SAS 9.4 software. The association analysis results between different genotypes and each trait are shown in Table 4.

[0057] Table 4. Association analysis between duck PLA2G1B genotype and duck feed utilization rate trait.

[0058]

[0059] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05), and different uppercase letters in the same row indicate extremely significant differences (P < 0.01).

[0060] Experimental conclusions: As shown in Table 4, for the PLA2G1B gene G7980795A locus, the feed conversion ratio (FCR) of GG genotype individuals was significantly higher than that of AA genotype individuals. The FCR, age-dependent feed intake (ADFI), and residual feed intake (RFI) of GG genotype individuals were extremely significantly higher than those of AA genotype individuals. There were no significant differences among the three genotypes in terms of average daily gain (ADG) and metabolite weight gain (MBW 0.75). Therefore, it can be concluded that AA genotype individuals have the best feed utilization trait, GA genotype individuals have a moderate feed utilization trait, and GG genotype individuals have a poor feed utilization trait.

[0061] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, 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 modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method based on PLA2G1B The application of molecular markers of genes in identifying duck feed utilization traits is characterized by, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 193rd base of the nucleotide sequence is G or A; If the molecular marker type of the Qiangying duck to be tested is AA, the feed utilization trait of the Qiangying duck is the best; if the molecular marker type of the Qiangying duck to be tested is GA, the feed utilization trait of the Qiangying duck is moderate; if the molecular marker type of the Qiangying duck to be tested is GG, the feed utilization trait of the Qiangying duck is poor.

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