Molecular marker for identifying duck feed utilization rate character based on PLA2G1B gene as well as identification method and application thereof

Abstract

The invention relates to a molecular marker for identifying the duck feed utilization rate character based on a PLA2G1B gene and an identification method and application of the molecular marker, and belongs to the technical field of molecular markers, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the 193rd base of the nucleotide sequence is G or A. The molecular marker has the advantages that the molecular marker can be used for identifying the duck feed utilization rate character based on the PLA2G1B gene; according to the molecular marker for identifying the duck feed utilization rate character based on the PLA2G1B gene and the identification method and application of the molecular marker, the molecular marker is developed on the basis of research on the relationship between the PLA2G1B gene and the duck feed utilization rate character, and the duck feed utilization rate character can be identified by identifying the type of the molecular marker in a duck genome. According to the method, the feed utilization rate characters of the ducks can be selected, a breeding method for early selection of the feed utilization rate of poultry is established, a novel molecular marker assisted breeding method is provided for detection of the growth characters of the ducks, and the method is simple, rapid, low in cost, free of special instruments and suitable for the requirements of molecular marker assisted breeding experiments.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a molecular marker for identifying duck feed utilization traits based on a PLA2G1B gene, and an identification method and application thereof. Background Art

[0002] Residual Feed Intake (RFI) is defined as the difference between the actual feed intake and the expected feed intake of an animal given its body weight and body 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 to measure feed efficiency, and with the development of the times, this trend has become more and more obvious. This is because the use of RFI for breeding can not only take into account daily weight gain and metabolic weight, but also has a relatively weak effect on important economic traits such as market weight, and 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.), which is a medium heritability trait, so RFI can be used as a measure of feed efficiency in breeding.

[0003] PLA2G1 is a class of enzymes that catalyze the hydrolytic removal of sn-2 fatty acid acyl groups from phospholipids, including enzymes secreted by cells as well as intracellular phospholipases. Evidence from in vitro cell culture experiments shows that PLA2G1B in pancreatic extracts is responsible for the digestion of phospholipids and lipid absorption 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 the animals were fed a normal low-fat diet, there was 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] However, the current research on the PLA2G1B gene mainly focuses on mice, fish and humans, and most of the research content revolves around the metabolic regulation of fatty acids and the deposition mechanism of lipids in the body. However, in the field of poultry, especially in the research on ducks, the exploration of the PLA2G1B gene is still insufficient. However, based on the existing findings, it can be inferred that the PLA2G1B gene may be an important candidate gene for regulating duck feed efficiency. Therefore, it is particularly important to deeply explore the variation of the PLA2G1B gene and its expression pattern on the specific effects of the feed conversion rate of meat ducks and reveal the molecular mechanism behind it. This not only helps us to more comprehensively understand the key role of the PLA2G1B gene in the growth and development of meat ducks and feed utilization, but also can provide a strong theoretical basis for improving the feed efficiency of meat ducks, help reduce the breeding cost of meat ducks, and promote the sustainable development of poultry farming. To this end, the present invention proposes a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene and its identification method and application. Summary of the invention

[0005] The purpose of the present invention is to provide a molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene and an identification method and application thereof in order to solve the above problems.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The 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 molecular marker in identifying the trait of duck feed utilization rate.

[0009] As a further optimization scheme of the present invention, if the duck molecular marker type to be tested is AA type, the duck feed utilization trait is the best; if the duck molecular marker type to be tested is GA type, the duck feed utilization trait is medium; if the duck molecular marker type to be tested is GG type, the duck feed utilization trait is poor

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

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

[0012] (2) Designing specific amplification primers using the nucleotide sequence of the molecular marker as the target sequence, using the total DNA as a template, and performing PCR amplification using the specific amplification primers to obtain an amplified product;

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

[0014] (4) Determine the feed utilization traits of ducks based on the molecular marker type.

[0015] As a further optimization scheme of the present invention, the sequence of the specific amplification primer is:

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

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

[0018] As a further optimization scheme of the present invention, the method of genotyping detection is to obtain an enzyme digestion product by enzymatic digestion of the amplified product, detect the enzyme digestion product by agarose gel electrophoresis, and perform genotyping based on the image. If the enzyme digestion product: contains 1 band, it is AA type; contains 2 bands, it is GG type; contains 3 bands, it is GA type.

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

[0020] The beneficial effects of the present invention are:

[0021] The present invention is based on the study of the relationship between the PLA2G1B gene and the feed utilization trait of ducks, develops a molecular marker, and by identifying the type of the molecular marker present in the duck genome, the feed utilization trait of ducks can be selected, and a breeding method for early selection of feed utilization of poultry is established, and a new molecular marker-assisted breeding method is provided for the detection of growth traits of ducks. The method is simple, rapid, low-cost, does not require special instruments, and is suitable for the needs of molecular marker-assisted breeding experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Agarose gel electrophoresis of PCR amplification products of some samples;

[0023] Figure 2 It is the agarose gel electrophoresis diagram of the digestion products obtained by digestion of PCR amplification products of some samples;

[0024] Figure 3 The sequencing results were verified for the genotype of the G7980795A site in the duck PLA2G1B gene (the 193rd site in SEQ ID NO.1). DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0026] 1. Materials

[0027] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0028] 2. Methods

[0029] 2.1 Primer design

[0030] The DNA sequence corresponding to the PLA2G1B gene (Gene ID: NC_051787.1) as shown in SEQ ID NO.1 was found from the duck genome database, and the specific amplification primers were designed using the partial DNA sequence of the PLA2G1B gene (the sequence of the site where the molecular marker is located and its upstream and downstream bases) as a template. The specific amplification primer sequences are as follows:

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

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

[0033] The length of the amplifiable region of the primer is 731 bp, and the amplified sequence is shown in SEQ ID NO.4, which contains a molecular marker of a G / A mutation at the G7980795A site (the 193rd site in SEQ ID NO.1).

[0034] 2.2 Extraction of total blood DNA

[0035] 475 Qiangying ducks were selected, blood was collected from the medial metatarsal vein, and total blood DNA was extracted. The blood DNA extraction kit produced by Tiangen Biotechnology Co., Ltd. was used to extract total DNA from the medial metatarsal vein blood samples of ducks. The extraction steps were carried out according to the instructions of the kit.

[0036] 2.3 PCR amplification

[0037] Mix produced by Shanghai Yisheng Biotechnology Co., Ltd. was used to perform PCR amplification reaction on the target fragment of PLA2G1B gene through the 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] PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; first step denaturation at 95°C for 45 s; second step annealing at 64.8°C for 45 s (annealing temperature was set according to primers); third step extension at 72°C for 30 s, wherein the second to third steps were cycled 31 times, for a total of 32 cycles; and extension at 72°C for 10 min.

[0041] 2.4 PCR amplification product detection and sequencing

[0042] The PCR amplification products were detected by 2% agarose gel electrophoresis. Figure 1 As shown, after imaging with a gel imager, a band of approximately 731 bp in length 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 was shown in SEQ ID NO.4, which was consistent with the predicted result.

[0043] 2.5 Genotyping

[0044] 2.5.1 Prepare the enzyme digestion system as shown in Table 2. The digestion condition is 37°C water bath for 1 hour. Use PspⅩⅠ restriction endonuclease from Beijing Biolab Technology Co., Ltd. to digest the PCR amplification product.

[0045] Table 2 Enzyme digestion system

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

[0047] 2.5.2 Using 1.5% mass ratio low voltage agarose gel electrophoresis, the following Figure 2 The results shown (partial results); among them, if the enzyme digestion product: contains 1 band, it is AA type; contains 2 bands, it is GG type; contains 3 bands, it is GA type.

[0048] 2.6 Enzyme Digestion and Sequencing Verification

[0049] The agarose gel electrophoresis diagram of gene restriction typing was statistically analyzed to obtain three typings: AA, GA, and GG. One individual was selected for each of the three typings for sequencing comparison. The sequencing comparison diagram is shown in FIG. Figure 3 As shown, in the sequencing result, G mutated into A, and the arrow marked the mutation position, which was consistent with the enzyme digestion typing result.

[0050] 2.7 Effect Verification

[0051] To determine the association between the G / A polymorphism of the G7980795A locus of duck PLA2G1B gene and important phenotypic traits of ducks, 475 Qiangying ducks in 2.2 were used as experimental materials, and the feed intake (ADFI), average daily gain (ADG), metabolic body weight gain (MBW) at 21 to 42 days of age were statistically analyzed. 0.75 ), feed-to-weight ratio (FCR), residual feed intake (RFI), 475 Qiangying ducks were genotyped using the 2.5 genotyping method, and the results are shown in Table 3:

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

[0053]

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

[0055] 2.8 Statistical analysis

[0056] The association between the three genotypes and the duck feed utilization traits was analyzed using the least squares analysis method in SAS9.4 software. The results of the association analysis between different genotypes and each trait are shown in Table 4:

[0057] Table 4 Association analysis between duck PLA2G1B genotype and duck feed utilization traits

[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 conclusion: As can be seen from Table 4, for the G7980795A site of the PLA2G1B gene, the feed-to-weight ratio (FCR) of GG type individuals was significantly higher than that of AA type individuals, and the feed-to-weight ratio (FCR), age-dependent feed intake (ADFI) and residual feed intake (RFI) of GG type individuals were extremely significantly higher than those of AA type individuals. There were no significant differences in average daily weight gain (ADG) and metabolic body weight gain (MBW0.75) among the three genotypes. It can be concluded that the feed utilization trait of AA genotype individuals was the best, the feed utilization trait of GA genotype individuals was medium, and the feed utilization trait of GG genotype individuals was poor.

[0061] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A molecular marker for identifying duck feed utilization traits based on the PLA2G1B gene, characterized in that: 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.

2. Use of the molecular marker as claimed in claim 1 in identifying the feed utilization trait of ducks.

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

4. A method for identifying the trait of feed utilization rate of ducks using the molecular marker according to claim 1, characterized in that: The following steps are involved: (1) Extracting total DNA from the medial metatarsal vein blood of the duck to be tested; (2) designing specific amplification primers using the nucleotide sequence of the molecular marker as the target sequence, using the total DNA as a template, and performing PCR amplification using the specific amplification primers to obtain an amplified product; (3) sequencing and genotyping the amplified products to obtain the molecular marker type of the duck to be tested; (4) Determine the feed utilization traits of ducks based on the molecular marker type.

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

6. The method for identifying duck feed utilization traits using molecular markers according to claim 4, characterized in that: The method of genotyping detection is to obtain a digestion product by digesting the amplified product, detect the digestion product by agarose gel electrophoresis, and perform genotyping according to the image. If the digestion product: If it contains 1 band, it is type AA; If it contains 2 bands, it is GG type; If it contains 3 bands, it is GA type.

7. The method for identifying duck feed utilization traits using molecular markers according to claim 6, characterized in that: The amplified product was digested with PspⅩⅠ restriction endonuclease.

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