CN114703299B - A molecular marker for identifying duck feed utilization traits based on the neuropeptide Y5 receptor NPY5R gene and its method and application - Google Patents

Abstract

The invention discloses a molecular marker for identifying duck feed utilization rate traits based on a neuropeptide Y5 receptor NPY5R gene, a method and application thereof, wherein the NPY5R gene has a nucleotide sequence shown as SEQ ID NO.1, the molecular marker is T or C, and the molecular marker is positioned at the 782 th position of the nucleotide sequence. The invention utilizes a PCR-SSCP method to detect the mutation of the NPY5R gene, selects the feed utilization rate character of ducks according to the genotype, establishes a breeding method for early selection of poultry feed utilization rate, has the advantages of simplicity, rapidness, low cost, no need of special instruments and suitability for the needs of experiments.

Description

A method for identifying duck feed utilization traits based on the neuropeptide Y5 receptor NPY5R gene Molecular markers and their methods and applications

technical field

The invention relates to the technical field of molecular markers, in particular to a molecular marker for identifying duck feed utilization traits based on the neuropeptide Y5 receptor NPY5R gene, and a method and application thereof.

Background technique

Feed efficiency traits are one of the important economic traits in livestock and poultry production, among which feed conversion ratio (FCR) and residual feed intake (RFI) are one of the indicators for evaluating feed efficiency. Feed conversion efficiency (FCR) refers to how much product is obtained by feeding a unit of feed (Liang S, Guo Z, Tang J, et al. Genomic divergence during artificial selection by feed conversion ratio in Pekin ducks[J]. Anim Biotechnol, 2021:1 -9), with the in-depth exploration of feed efficiency, researchers define the difference between the amount of feed actually consumed by livestock and poultry in a certain period of time minus the amount of feed required to maintain growth as residual feed intake (RFI) (Koch R M, Swiger L A, Chambers D, et al. Efficiency of feed use in beef cattle [J]. Journal of Animal Science, 1963, 22 (2): 486-494.). RFI and FCR can be used to evaluate the utilization of feed efficiency in poultry, but there are many factors that affect feed efficiency, including feed intake and genetic factors (Pan Yunzhi "Pig Feed Efficiency Differentiation Selection and Genome-wide Association Analysis" [D]. Jilin University, 2016; Zhang Xiaoxue, "Study on Lamb Production Performance, Rumen Microflora and Liver Transcriptome with Different Residual Feed Intake" [D]. Lanzhou University, 2019.).

Studies have found that neuropeptide Y and its type 5 receptor NP5YR mediate animal feeding behavior and are also related to energy metabolism, which belongs to a member of the G protein-coupled receptor superfamily (Michel M C, Beck-Sickinger A, Cox H, et al. XVI. International Union of Pharmacology recommendations for thenomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors [J]. Pharmacol Rev, 1998, 50(1): 143-150.). Tang-Christensen et al. used oligonucleotides (ODN) antisense to NPY5R to study the effects of NPY5R and NPY on food intake. Through repeated central administration, they found that ODN significantly reduced spontaneous food intake, resulting in significant weight loss (Tang-Christensen et al. Christensen M, Kristensen P, Stidsen CE, et al.Central administration of Y5 receptor antisense decreases spontaneous food intake and attenuates feeding in response to exogenous neuropeptide Y[J].J Endocrinol,1998,159(2):307-12.). By studying ducks with different residual feed intake (RFI) individuals, researchers detected higher mRNA expression levels of NPY and NPY5R in the hypothalamus of individuals with high RF I (Zeng T, Chen L, Du X, et al. Association analysis between feed efficiency studies and expression of hypothal amic neuropeptide genes in laying ducks[J].Anim Genet,2016,47(5):606-609.). Therefore, we presume that NPY5R is involved in the regulation of duck feed efficiency, which provides a conjecture about the relationship between the polymorphism of this gene and poultry feed efficiency traits for the study of duck feed efficiency.

In addition, the Chinese patent document CN111676295A discloses a research method for genes related to feed intake regulation, and specifically discloses the design of polymorphism detection primers based on the known duck CCK, CCKAR, NPY and NPY5R genes in NCBI GenBank for use in The above genes developed SNPs loci related to duck feed intake, and it was found that CCKAR gene had SNPs loci related to duck feed intake, but there were no reports on the SNPs loci of CCK, NPY and NPY5R genes. At present, there is no other literature about the SNPs loci related to NPY5R and duck feed intake have been developed.

In order to improve the feed efficiency of ducks, this research fundamentally reduces the cost of expenditure and increases the income of the duck industry. Early researchers often used conventional breeding but the efficiency was not high. With the development of technology, molecular marker-assisted breeding has come into play. Eyelids, which can fundamentally improve traits genetically, thereby accelerating genetic progress. Based on the above content, a molecular marker and its method and application for identifying duck feed utilization traits based on the neuropeptide Y5 receptor NPY5R gene were proposed.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a molecular marker based on the neuropeptide Y5 receptor NPY5R gene identification of duck feed utilization traits and its method and application, aiming at candidates related to the feed utilization traits of ducks Gene SNP (single nucleotide polymorphism) molecular markers to solve the problem of slow progress in conventional phenotypic breeding and to achieve early identification of feed utilization traits.

The present invention achieves the above object through the following technical solutions:

The present invention provides a molecular marker based on neuropeptide Y5 receptor NPY5R gene duck feed utilization traits, the NPY5R gene has a nucleotide sequence as shown in SEQ ID NO.1, and the molecular marker is T or C , the molecular marker is located at the 782nd position of the nucleotide sequence.

The present invention also provides an application of the above-mentioned molecular marker based on the neuropeptide Y5 receptor NPY5R gene duck feed utilization traits in identifying the duck feed utilization traits.

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

(1) extract the total DNA of duck wing vein blood;

(2) Design specific amplification primers with the target sequence consisting of the site where the molecular marker is located and its upstream and downstream bases, and use the total DNA as a template to perform PCR amplification using the specific amplification primers to obtain amplified product;

(3) Carry out genotyping detection and sequencing to the amplified product, and obtain the molecular marker type of the duck to be tested;

(4) Judging the traits of duck feed utilization according to the type of molecular markers.

A further improvement is that the length of the amplification products upstream and downstream of the site where the molecular marker is located is between 200-250 bp.

A further improvement is that the sequence of the specific amplification primer is:

SEQ ID NO.2: Forward primer: ATTCTTCTTT GAGTTAGGCA;

SEQ ID NO. 3: Reverse primer: GCAGACAGAC AGGGTCCGAG.

A further improvement is that the genotyping detection method is to perform non-denaturing polyacrylamide gel electrophoresis and silver staining on the PCR amplification product to obtain an image, and perform genotyping according to the image:

(1) Containing 2 strips with close spacing, it is CC type;

(2) Containing 2 strips with a long distance between strips, it is TT type;

(3) If it contains 4 bands, it is TC type.

A further improvement is that in the step (4), the specific steps for judging the feed utilization rate traits of meat ducks according to the type of molecular markers are:

(1) If the molecular marker type of the duck to be tested is CC type, the feed utilization rate of the duck is extremely high;

(2) If the molecular marker type of the duck to be tested is TT type, the feed utilization traits of the duck are poor;

(3) If the molecular marker type of the duck to be tested is TC type, the feed utilization traits of the duck are moderate.

The beneficial effect of the present invention is: the present invention provides a kind of molecular marker and its method and application based on neuropeptide Y5 receptor NPY5R gene identification duck feed utilization traits, utilizes PCR-SSCP method to detect the mutation of NPY5R gene, according to The genotype selects the traits of feed utilization efficiency of ducks, and establishes a breeding method for early selection of poultry feed utilization efficiency. This method is simple, fast, low-cost, does not require special instruments, and is suitable for the needs of experiments.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of part sample DNA;

Fig. 2 is the agarose gel electrophoresis figure of partial sample PCR amplification product;

Fig. 3 is the polyacrylamide gel electrophoresis figure of partial sample PCR amplification product;

Fig. 4 is the sequencing map of the 782th individual of different genotypes in the duck NPY5R genome.

detailed description

The application will be described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific embodiments are only used to further illustrate the application, and cannot be interpreted as limiting the protection scope of the application. The above application content makes some non-essential improvements and adjustments to this application.

1. Materials

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 all commercially available products unless otherwise specified.

2. Method

2.1 Obtaining polymorphic sites of duck NPY5R gene

2.1.1 Genomic DNA extraction and detection

388 white-feathered meat ducks were selected, and blood was collected from the wing veins. The blood DNA extraction kit produced by Dalian Bao Biology Co., Ltd. was used to extract the total DNA in the duck wing vein blood samples, and the details were carried out according to the kit instruction manual.

DNA concentration and OD value were measured with NanoDrop2000. The DNA was detected by 1.5% agarose gel electrophoresis, and the results are shown in Figure 1. The quality of the extracted genomic DNA was good, and the main band was single and clear.

2.1.2 Primer design

Find the DNA sequence corresponding to the gene NPY5R shown in SEQ ID NO.1 from the duck genome database, use the partial DNA sequence of the gene NPY5R shown in SEQ ID NO.1 as a template, and set the SNP site as much as possible in the primer design process. In the middle position, avoid the occurrence of hairpin structure, primer dimer and mismatching, etc., and optimize the primer sequence. The primer sequence is as follows:

SEQ ID NO.2: Forward primer: ATTCTTCTTT GAGTTAGGCA;

SEQ ID NO. 3: Reverse primer: GCAGACAGAC AGGGTCCGAG.

The length of the amplified fragment of the primer is 237bp, and the sequence is shown in SEQ ID NO.4, which contains the molecular marker site for T/C mutation.

2.1.3 PCR amplification

Use the Mix produced by Shanghai Sangon Bioengineering Co., Ltd. to carry out PCR amplification reaction on the target fragment of NPY5R gene through the synthesized sequencing-specific primers. The PCR amplification system is:

PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, a total of 34 cycles; extension at 72°C for 10 min; and finally storage at 4°C.

2.1.4 PCR amplification product detection

The PCR amplification product was detected by 1% agarose gel electrophoresis by mass ratio, and a band with a length of 200-250 bp was obtained after imaging with a gel imager, which was consistent with the predicted length, indicating that the target fragment was obtained. The PCR product was sent to Shanghai Sangon Bioengineering for sequencing. The sequence is shown in SEQ ID NO.4, which is consistent with the predicted result.

2.1.5 PCR product denaturation and SSCP detection

The PCR-amplified product is first denatured, then detected by polyacrylamide gel, and finally its mutation is determined according to the results of different band patterns. The specific steps are as follows:

(1) Configure non-denatured polyacrylamide gel according to the instructions. The non-denatured acrylamide gel system is as follows:

(2) TEMED is added last, and poured into the mold immediately after adding (make sure the mold is closed before pouring to prevent glue leakage during glue filling, and the size of the glue strip and the hole comb should be consistent), tilt at an angle of about 45°, and the glue liquid from Pour slowly into the middle of the vertical plate (which can effectively avoid the generation of air bubbles), stop pouring glue when pouring is about from the top edge of the mold, insert the comb prepared in advance, polymerize at room temperature for 40 minutes, store excess acrylamide at 4°C, and observe the glass at any time Check the polymerization of the slab gel, and add an appropriate amount of non-denaturing acrylamide gel system mixture.

(3) Prepare the electrophoresis card tank while the gel polymerization is waiting. After the polymerization is completed, install the glass plate and add 1 × TBE to the electrophoresis card tank, so that the TBE solution exceeds the sample hole by about 3 cm, and drive away the air bubbles.

(4) Take 3 μL of the PCR amplification product and place it in a PCR tube, add 7 μL of denaturing reagent, briefly centrifuge and mix, denature at 98°C for 10 min, quickly take it out and place it in a -20°C ice box for 10 min, and apply the sample with a 10 μL pipette.

(5) Turn on the power, run electrophoresis at 220 volts for 10 minutes, then adjust the voltage to 120 volts, and perform electrophoresis for 21 hours.

(6) After the electrophoresis is over, close the electrophoresis apparatus, take out the glass plate, carefully take out the gel, put it into a white porcelain dish filled with clean water and wash it 1-2 times.

(7) Put the gel into the staining solution and shake gently for 15 minutes in the dark. Staining solution: composed of silver nitrate and pure water, the concentration of silver nitrate is 0.2%.

(8) After dyeing, recover AgNO3, wash with deionized water 1-3 times, each time for 2 minutes, and wash away excess dyeing solution.

(9) The chromogenic solution develops the color, so that the bands are clear and the background is light yellow. The staining time is until the bands are clearly visible, and the chromogenic solution is poured out immediately after the color development. Chromogenic solution: 500 mL, including 2% NaOH + 0.04% Na ₂ CO ₃ + 420 μL formaldehyde.

(10) Take pictures and save them.

2.1.6 Genotyping

The PCR-SSCP picture is shown in Figure 2. Different band types represent different genotypes. It can be seen from Figure 2 that there are three genotypes at the 782nd position of the NPY5R gene, namely TT, TC, and CC. Among them, the CC genotype is two closely spaced bands, and the TT The genotype has 2 widely spaced bands and the TC genotype has 4 bands.

2.1.7 DNA verification sequencing

The color-developed genotyping gel map was counted to obtain three types of TT, TC, and CC, and one individual was selected for sequencing comparison of these three types. The sequence comparison diagram is shown in Figure 3; the sequencing results T in the middle is mutated into C, and the arrow marks the mutation position, which is consistent with the results of PCR-SSCP.

2.2 Association analysis of NPY5R gene T782C mutation site and duck feed utilization traits

2.2.1 Genotyping

In order to determine the relationship between the T/C polymorphism at position 782 of NPY5R gene and the important traits of ducks, 388 white-feathered meat ducks were used as experimental materials, recorded from hatching, wearing wings, and reared normally. RFI (residual feed intake), FI (individual daily feed intake), ADG (daily gain), FCR (feed-to-weight ratio), MBW (metabolized body weight ). Using the genotyping method in 2.1.6, 388 ducks were genotyped, and the results are shown in Table 1.

Table 1 The genotype detection results of individuals with different phenotypes

Chi-square test results showed that the genotypes of the experimental duck population were in Hardy-Weinberg equilibrium.

2.2.2 Statistical analysis

The number of TT, TC, and CC genotypes was counted through the gel map, and the difference between the three genotypes and the slaughter performance was analyzed by One-way ANOVA in SPSS20.0. The results of the correlation analysis between different genotypes and various traits are shown in Table 2 Shown:

Table 2 Correlation analysis between duck NPY5R genotype and duck feed utilization traits

Note: Different lowercase letters in the same row indicate significant differences (P<0.05), and no letters indicate no significant differences (P>0.05).

It can be seen from the table that by comparing the feed efficiency traits of individuals of different genotypes, CC individuals were significantly lower than TT individuals in terms of RFI (residual feed intake) (P<0.05); while in FI, BW42, BWG and FCR There were no significant differences among the above three genotypes. Since RFI is a negative selective trait, it can be concluded that CC genotype individuals have extremely high feed conversion traits, TC genotype individuals have moderate feed conversion traits, and TT genotype individuals have poor feed conversion traits.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

sequence listing

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Claims (3)

1. A molecular marker based on neuropeptide Y5 receptor NPY5R gene identification duck feed utilization traits, characterized in that the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1, wherein the nucleoside The 782nd base of the acid sequence is T or C. 2. The application of a molecular marker as claimed in claim 1 in identifying duck feed utilization traits. 3. a method utilizing molecular markers as claimed in claim 1 to identify duck feed utilization proterties, is characterized in that, comprises the following steps: (1) Extract the total DNA from duck wing vein blood; (2) Design specific amplification primers with the target sequence consisting of the site where the molecular marker is located and its upstream and downstream bases, and use the total DNA as a template to perform PCR amplification using the specific amplification primers to obtain amplified product; Wherein, the length of the upstream and downstream amplification products at the site where the molecular marker is located is between 200 and 250 bp; The specific amplification primer sequence is: SEQ ID NO.2: Forward primer: ATTCTTCTTT GAGTTAGGCA; SEQ ID NO.3: Reverse primer: GCAGACAGAC AGGGTCCGAG; (3) Perform genotyping detection and sequencing on the amplified product to obtain the molecular marker type of the duck to be tested; (4) Judging the feed utilization traits of ducks according to the type of molecular markers, specifically: if the molecular marker type of the duck to be tested is CC type, the duck feed utilization rate traits are extremely high; if the duck molecular marker type to be tested is TT type, the duck The traits of feed utilization rate are poor; if the type of molecular marker of the duck to be tested is TC type, the traits of feed utilization rate of the duck are moderate.

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