CN114703299B - Molecular marker for identifying duck feed utilization rate character based on neuropeptide Y5 receptor NPY5R gene, and method and application thereof - Google Patents
Molecular marker for identifying duck feed utilization rate character based on neuropeptide Y5 receptor NPY5R gene, and method and application thereof Download PDFInfo
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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
Technical Field
The invention relates to the technical field of molecular markers, in particular to a molecular marker for identifying duck feed utilization rate characters based on a neuropeptide Y5 receptor NPY5R gene, and a method and application thereof.
Background
The feed efficiency trait is one of important economic traits in livestock and poultry production, wherein Feed Conversion Rate (FCR) and Residual Feed Intake (RFI) are one of indexes 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 reverse feeding along with the feedback conversion ratio in Pekin products [ J ]. Anim Biotechnol, 2021. RFI and FCR can be used to assess poultry feed efficiency utilization, however, there are many factors that affect feed efficiency, including feed intake and genetic factors (panyun-induced "pig feed efficiency differentiation and genome wide association analysis" [ D ]. Ghlin university, 2016; zhuyuan "lamb productivity and ruminal microflora and liver transcriptome studies with different residual feed intake" [ D ]. Langu university, 2019.).
Neuropeptide Y, a type 5 receptor NP5YR, has been found to mediate animal feeding behavior while also being involved in energy metabolism and belongs to one of the members of the G protein-coupled receptor superfamily (Michel M C, beck-Sickinger A, cox H, et al. XVI. International Union of pharmacological receptors for the nomenclature of neuropeptide Y, peptide YY, and cosmetic polypeptide receptors [ J ]. Pharmacol Rev,1998,50 (1): 143-150.). Tang-Christensen et al used Oligonucleotides (ODN) antisense to NPY5R to study the effect of NPY5R and NPY on food intake, and found that ODN significantly reduced spontaneous food intake by repeated central administration, resulting in significant weight loss (Tang-Christensen M, kristensen P, stidsen C E, et al. Central administration of Y5 receptor inhibitors specific genes food intake and ingredients feeding response to exogenous neuropeptide Y [ J ]. J Endocrinol,1998,159 (2): 307-12). Through duck studies on individuals with different Residual Food Intake (RFI), researchers have detected high mRNA expression levels of NPY and NPY5R in the hypothalamus of high RF I individuals (Zeng T, chen L, du X, et al. Therefore, the NPY5R is presumed to participate in the regulation of the feed efficiency of ducks, and an guess about the correlation of the gene polymorphism and the poultry feed efficiency character is provided for researching the feed efficiency of the ducks.
In addition, chinese patent document CN111676295A discloses a research method of genes related to feed intake regulation, and specifically discloses that polymorphism detection primers are designed according to the known CCK, CCKAR, NPY and NPY5R genes of ducks in NCBI GenBank for developing SNPs sites related to feed intake of ducks based on the above genes, and as a result, it is found that CCKAR genes have SNPs sites related to feed intake of ducks, and there is no report on SNPs sites of CCK, NPY and NPY5R genes. At present, no other documents relate to the development of SNPs sites of NPY5R related to the feed intake of ducks.
The research aims to improve the efficiency of duck feed, fundamentally reduce the cost expenditure and increase the income of duck breeding industry, early researchers often use conventional breeding but the efficiency is not high, and with the technical development, molecular markers assist in breeding and reflect on eye curtains, characters can be fundamentally improved from heredity, and therefore the genetic progress is accelerated. Based on the content, the molecular marker for identifying the utilization rate character of the duck feed based on the neuropeptide Y5 receptor NPY5R gene, and the method and the application thereof are provided.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a molecular marker for identifying duck feed utilization rate traits based on neuropeptide Y5 receptor NPY5R genes, and a method and application thereof, and aims at the SNP (single nucleotide polymorphism) molecular marker of candidate genes related to the duck feed utilization rate traits to solve the problems of slow development of conventional phenotypic breeding and realization of early identification of the feed utilization rate traits.
The invention realizes the purpose through the following technical scheme:
the invention provides a molecular marker based on neuropeptide Y5 receptor NPY5R gene duck feed utilization rate character, wherein an 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 site of the nucleotide sequence.
The invention also provides application of the molecular marker based on the utilization rate character of the neuropeptide Y5 receptor NPY5R gene duck feed in identification of the utilization rate character of the duck feed.
The invention also provides a method for identifying the character of duck feed utilization rate by using the molecular marker, which comprises the following steps:
(1) Extracting the total DNA of the duck wing venous blood;
(2) Designing a specific amplification primer by taking the site where the molecular marker is located and a sequence consisting of upstream and downstream bases as a target sequence, and carrying out PCR amplification by using the specific amplification primer by taking the total DNA as a template to obtain an amplification product;
(3) Carrying out genotyping detection and sequencing on the amplification product to obtain the molecular marker type of the duck to be detected;
(4) And judging the character of the utilization rate of the duck feed according to the type of the molecular marker.
The further improvement is that the length of the amplified product at the upstream and downstream of the site where the molecular marker is located is between 200 and 250 bp.
In a further improvement, the specific amplification primer sequence is:
SEQ ID NO.2:Forward primer：ATTCTTCTTT GAGTTAGGCA；
SEQ ID NO.3:Reverse primer：GCAGACAGAC AGGGTCCGAG。
the further improvement is that the genotyping detection method comprises the steps of carrying out non-denaturing polyacrylamide gel electrophoresis and silver staining on a PCR amplification product to obtain an image, and carrying out genotyping according to the image:
(1) The strip comprising 2 strips with closer distance is CC type;
(2) The type TT is formed by 2 strips with longer strip spacing;
(3) Containing 4 bands, it is type TC.
The further improvement is that the specific steps for judging the meat duck feed utilization rate character according to the molecular marker type in the step (4) are as follows:
(1) If the type of the molecular marker of the duck to be detected is CC type, the utilization rate character of the duck feed is extremely high;
(2) If the molecular marker type of the duck to be detected is TT type, the utilization rate of the duck feed is poor;
(3) If the molecular marker type of the duck to be detected is TC type, the utilization rate of the duck feed is moderate.
The invention has the beneficial effects that: the invention provides a molecular marker for identifying duck feed utilization rate character based on neuropeptide Y5 receptor NPY5R gene, a method and application thereof, wherein the mutation of the NPY5R gene is detected by using a PCR-SSCP method, the feed utilization rate character of ducks is selected according to genotypes, and a breeding method for early selection of poultry feed utilization rate is established.
Drawings
FIG. 1 is an agarose gel electrophoresis of a portion of sample DNA;
FIG. 2 is an agarose gel electrophoresis of a PCR amplification product from a portion of the sample;
FIG. 3 is a polyacrylamide gel electrophoresis diagram of a PCR amplification product of a part of samples;
FIG. 4 is a sequence diagram of different genotype individuals at position 782 of the duck NPY5R genome.
Detailed Description
The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.
1. Material
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 in this example are commercially available products unless otherwise specified.
2. Method of producing a composite material
2.1 obtaining polymorphic sites of duck NPY5R gene
2.1.1 genomic DNA extraction and detection
388 white feather meat ducks are selected, blood is collected from the vein of the wings, and the total DNA in the blood sample of the vein of the duck wings is extracted by using a blood DNA extraction kit produced by Dalibao biology company, which is specifically carried out according to the kit use instruction.
The DNA concentration and OD value were measured using a NanoDrop 2000. The DNA was detected by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1, the extracted genomic DNA was of good quality, single and clear main band.
2.1.2 primer design
Finding out a DNA sequence corresponding to a gene NPY5R shown by SEQ ID NO.1 from a duck genome database, taking a partial DNA sequence of the gene NPY5R shown by SEQ ID NO.1 as a template, and paying attention to the fact that an SNP locus is arranged at a middle position as much as possible in the process of primer design, so that the conditions of hairpin structure, primer dimer, mismatch and the like are avoided, the primer sequence is optimized, and the primer sequence is shown 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, the sequence is shown in SEQ ID NO.4, and the primer comprises a T/C mutant molecular marker locus.
2.1.3 PCR amplification
Carrying out PCR amplification reaction on a target fragment of the NPY5R gene by using Mix produced by Shanghai biological engineering Limited company through a synthesized sequencing specific primer, wherein the PCR amplification system is as follows:
the PCR amplification conditions were: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 34 cycles; then extending for 10min at 72 ℃; finally, storing at 4 ℃.
2.1.4 PCR amplification product detection
Detecting the PCR amplification product by agarose gel electrophoresis of 1 percent of mass ratio, obtaining a strip with the length of 200-250 bp after imaging by a gel imager, and indicating that the target fragment is obtained. And (3) sending the PCR product to Shanghai biological engineering for sequencing, wherein the sequence is shown as SEQ ID NO.4 and is consistent with the prediction result.
2.1.5 PCR product denaturation and SSCP detection
Firstly, denaturing a product after PCR amplification, then carrying out polyacrylamide gel detection, and finally judging mutation according to different banding patterns, wherein the specific steps are as follows:
(1) According to the specification, a non-denatured polyacrylamide gel is configured, and a non-denatured acrylamide gel system is as follows:
(2) And adding TEMED finally, pouring into a mold immediately after adding (the mold is required to be closed before pouring, glue leakage during glue pouring is prevented, and the sizes of a glue strip and a hole comb are required to be consistent), inclining for about 45 degrees, slowly pouring glue solution from the middle of a vertical plate (the generation of bubbles can be effectively avoided), stopping glue pouring when pouring is carried out until the glue solution is about to be away from the upper edge of the mold, inserting a pre-prepared comb, polymerizing for 40 minutes at room temperature, storing redundant acrylamide at 4 ℃, observing the gel polymerization condition of the glass plate at any time, and adding a proper amount of non-denatured acrylamide gel system mixed solution.
(3) Preparing an electrophoresis clamping groove in the gel polymerization waiting process, installing a glass plate after polymerization is completed, adding 1 multiplied by TBE into the electrophoresis clamping groove, leading TBE solution to exceed a sample adding hole by about 3cm, and removing bubbles in the TBE solution.
(4) Putting 3 mu L of PCR amplification product into a PCR tube, adding 7 mu L of denaturation reagent, centrifuging for a short time, mixing uniformly, denaturing at 98 ℃ for 10min, taking out quickly, putting into an ice box at-20 ℃ for 10min, and spotting with a 10 mu L pipette.
(5) The power is turned on, 220V electrophoresis is carried out for 10min, then the voltage is modulated to 120V, and electrophoresis is carried out for 21h.
(6) After the electrophoresis is finished, the electrophoresis apparatus is closed, the glass plate is taken out, the gel is carefully taken out, and the gel is put into a white porcelain plate filled with clear water to be washed for 1 to 2 times.
(7) Put the gel into staining solution and shake gently for 15min in the dark. Dyeing liquid: consists of silver nitrate and pure water, wherein the concentration of the silver nitrate is 0.2 percent.
(8) And after dyeing is finished, recovering AgNO3, washing for 1-3 times by deionized water for 2min each time, and washing off redundant dyeing liquid.
(9) The developing liquid develops color to make the strip clear, the background is light yellow, the dyeing time is long enough to make the strip clear and visible, and the developing liquid is poured out immediately after developing color. Color development liquid: 500mL, where 2% NaOH +0.04% Na 2 CO 3 + 420. Mu.L of formaldehyde.
(10) And taking a picture and storing.
2.1.6 genotyping
The PCR-SSCP pictures are shown in FIG. 2. Different band types represent different genotypes, and it can be seen from fig. 2 that there are three genotypes at position 782 of the NPY5R gene, i.e., TT, TC, and CC, wherein the CC genotype is 2 bands at closer intervals, the TT genotype has 2 bands at farther intervals, and the TC genotype has 4 bands.
2.1.7 DNA verification sequencing
Counting the developed genotyping glue map to obtain three types of TT, TC and CC, respectively selecting an individual for sequencing comparison, wherein a sequencing comparison map is shown in figure 3; the sequencing results show that T is mutated to C, and the arrow marks the mutation position, which is consistent with the PCR-SSCP results.
2.2 Correlation analysis of NPY5R gene T782C mutant site and duck feed utilization rate character
2.2.1 genotyping
In order to determine the relevance of the T/C polymorphism at 782 th site of NPY5R gene and important characters of ducks, 388 white feather ducks are used as test materials, and are recorded from hatching, worn with wing numbers and normally bred. And (3) counting RFI (residual feed intake), FI (individual daily feed intake), ADG (daily gain), FCR (feed-weight ratio) and MBW (metabolite gain) corresponding to the age of 388 white feather meat ducks in 21-42 days. 388 ducks were genotyped by the genotyping method of 2.1.6, and the results are shown in table 1.
TABLE 1 results of genotype measurements for individuals with different phenotypes
Chi-square test results show that the genotype of the experimental duck population is in Hardy-Weinberg balance.
2.2.2 statistical analysis
The number of the TT, TC and CC genotypes is counted by a gel chart, the difference between the three genotypes and slaughter performance is analyzed by adopting One-way ANOVA in SPSS20.0, and the correlation analysis results between different genotypes and various characters are shown in a table 2:
TABLE 2 correlation analysis of duck NPY5R genotype and duck feed utilization rate traits
Note: different lower case letters in the same row indicate significant difference (P < 0.05), and no letter indicates insignificant difference (P > 0.05).
As can be seen from the table, CC individuals are significantly lower than TT individuals (P < 0.05) in terms of RFI (residual feed intake) by comparing the feed efficiency traits of different genotypes of individuals; there were no significant differences in the three genotypes on FI, BW42, BWG and FCR. As RFI is a negative selection character, the conclusion is drawn that the feed conversion character of CC genotype individuals is extremely high, the feed conversion character of TC genotype individuals is moderate, and the feed conversion character of TT genotype individuals is poor.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> agriculture university of Anhui
<120> molecular marker for identifying duck feed utilization rate character based on neuropeptide Y5 receptor NPY5R gene, and method and application thereof
<141> 2022-05-09
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ggctccagac cccgcacgga gcgctctccc gaaatgcacc cgcgcggcgg ccggctgagc 180
acccagcacc gagcacccag caccgcagca gcagcagctg ccgggtgcct ggccctggcg 240
cctccgcagc acccgcataa gatgcgctgc cgggcatcct cgccctgctc ccgcagagcc 300
gtgccggggc tatgagcggt gcctgggggc tgcgcactcg cggccccttc tgcagcgggc 360
tccccgtggc tgcaggtgag cagggggcag cggggtgggc agggggcaga ggtgtaaatt 420
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Claims (3)
1. Receptor based on neuropeptide Y5NPY5RThe molecular marker for genetic identification of the duck feed utilization rate traits is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, wherein the 782 th base of the nucleotide sequence is T or C.
2. Use of the molecular marker of claim 1 for identifying a duck feed utilization trait.
3. A method for identifying a duck feed utilization rate trait using the molecular marker of claim 1, comprising the steps of:
(1) Extracting the total DNA of the duck wing venous blood;
(2) Designing a specific amplification primer by taking the site where the molecular marker is located and a sequence consisting of upstream and downstream bases as a target sequence, and performing PCR amplification by using the specific amplification primer by taking the total DNA as a template to obtain an amplification product;
wherein the length of an upstream and downstream amplification product of a site where the molecular marker is located is between 200 and 250 bp;
the specific amplification primer sequence is as follows:
SEQ ID NO.2: Forward primer：ATTCTTCTTT GAGTTAGGCA；
SEQ ID NO.3: Reverse primer：GCAGACAGAC AGGGTCCGAG；
(3) Carrying out genotyping detection and sequencing on the amplification product to obtain the molecular marker type of the duck to be detected;
(4) Judging the character of the utilization rate of the duck feed according to the type of the molecular marker, and specifically comprising the following steps: if the type of the molecular marker of the duck to be detected is CC type, the utilization rate character of the duck feed is extremely high; if the type of the molecular marker of the duck to be detected is TT type, the utilization rate character of the duck feed is poor; if the molecular marker type of the duck to be detected is TC type, the utilization rate of the duck feed is moderate.
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| NL2032971A NL2032971B1 (en) | 2022-05-09 | 2022-09-07 | Molecular marker for identifying trait of efficiency of duck feed utilization based on neuropeptide y5 receptor npy5r gene, method and use thereof |
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| CN114703298B (en) * | 2022-05-09 | 2022-10-25 | 安徽农业大学 | Molecular marker for identifying duck feed utilization rate character based on neuropeptide gene NPY and method and application thereof |
| CN114703299B (en) * | 2022-05-09 | 2022-12-23 | 安徽农业大学 | Molecular marker for identifying duck feed utilization rate character based on neuropeptide Y5 receptor NPY5R gene, and method and application thereof |
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| CN111676295A (en) * | 2020-05-28 | 2020-09-18 | 浙江省农业科学院 | Research method of gene related to feed intake regulation |
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