CN114134238B - RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks and application thereof - Google Patents
RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks and application thereof Download PDFInfo
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Abstract
The invention discloses a RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks and application thereof, belonging to the technical field of biology and comprising the following steps: SNP1, SNP2, and/or SNP 8; wherein, include: SNP1, SNP2, and/or SNP 8; wherein, the SNP1 is located at the position 35658560 of an intron region of a RALGAPA1 gene of chromosome 5, and the allele is T & gtG; the SNP2 is located at the 35658263 locus of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G & gtT; the SNP8 is located at the position 35656935 of an intron region of a RALGPA 1 gene of a chromosome 5, and the allele is G > A; the method can accurately identify the egg laying characteristics of Muscovy ducks of different ages of weeks, and provides a new molecular marker for molecular marker-assisted selective breeding.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks and application thereof.
Background
The Muscovy duck is strong in adaptability, strong in fertility and high in meat yield, is native to tropical regions in Central and south America, and is suitable for breeding in southern China. The Muscovy duck is suitable for being prepared by a plurality of Chinese-style methods such as taro braised duck, braised Muscovy duck, dried bamboo shoot braised Muscovy duck, four-material Muscovy duck soup, salt baked Muscovy duck, agrocybe cylindracea Muscovy duck soup, ginger duck and the like, and has a good market prospect in south China. Although the large-scale breeding of Muscovy ducks is developed in China, the breeding progress of Muscovy ducks is laggard, and particularly the reproductive performance seriously restricts the improvement of productivity. Reproductive trait heritability is low, and selection breeding based on traits is difficult to make great progress.
The ovary is a female reproductive organ and is closely related to egg laying characteristics, and the ovarian function of poultry directly influences the egg laying amount. The follicular development process is complex and is associated with a variety of endocrine, autocrine and paracrine factors. The number of primary follicles in the ovary is fixed, and the quiescence, survival and activation of the follicles depend on the process of homeostasis. TGF-. beta.s, FGFs, IGFs, DRD2, FSHR, GnRH, STAR and PRLR were shown to be involved in the ovarian function of poultry, affecting egg production.
The ralga 1 gene is not only involved in immunity, but its expression level is also involved in the duration of fertility. The molecular marker assisted selection can accelerate the breeding progress, and the commonly used molecular markers include RFLP, RAPD, SSR, AFLP and the like, but the molecular markers need gel electrophoresis detection when being used, so the speed is low, and pollution is also caused. An SNP is a single nucleotide polymorphism, including insertions, deletions, mutations, etc. of genomic bases, which can be detected precisely using Sanger sequencing. Sanger sequencing is low in price and high in accuracy, and large-scale detection can be realized by means of modern equipment. Thus, Sanger sequencing was used to detect SNPs of ralga 1 gene, and these were correlated with egg laying traits and used as molecular markers for molecular marker-assisted selective breeding.
Disclosure of Invention
The invention aims to provide a RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks and application thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a RALGAPA1 gene SNP molecular marker related to egg laying traits of Muscovy ducks, which comprises the following components: SNP1, SNP2, and/or SNP 8; wherein, the SNP1 is located at the 35658560 site of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is T > G; the SNP2 is located at the 35658263 locus of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G & gtT; the SNP8 is located at the position 35656935 of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G > A.
The invention also provides an application of the detection reagent of the RALGAPA1 gene SNP molecular marker related to the egg laying traits of the Muscovy ducks in the detection of the egg laying traits of the Muscovy ducks.
The invention also provides a primer for amplifying the RALGAPA1 gene SNP molecular marker related to the egg laying traits of the Muscovy ducks, which comprises a primer with a sequence shown as SEQ ID No. 3-6.
The invention also provides application of the primer in identifying the egg laying traits of Muscovy ducks or preparing a kit for identifying the egg laying traits of Muscovy ducks.
The invention also provides a kit for identifying the egg laying traits of Muscovy ducks, which comprises the primer.
The invention also provides a method for identifying the egg laying traits of the Muscovy ducks, which identifies the genotypes of the molecular markers in the Muscovy ducks to be detected.
Further, the method comprises the steps of:
(1) extracting the genome DNA of the Muscovy duck to be detected;
(2) carrying out PCR amplification by using the primers to obtain amplification primers;
(3) sequencing the amplification product to obtain the genotype of the molecular marker;
(4) and judging the egg laying character of the muscovy duck of the sample to be detected according to the genotyping result.
Further, in step (2), the amplification system for PCR amplification comprises: 2xTaq MasterMix 15 muL, mixed primers of 1.2 muL, ultrapure water of 10.6 muL and DNA of 2 muL;
the reaction procedure of the PCR amplification is as follows: 94 ℃ for 2min, 94 ℃ for 30s, 53 ℃ for 30s, 72 ℃ for 30s, 30cycles, 72 ℃ for 2 min.
The invention also provides the RALGAPA1 gene SNP molecular marker related to the egg laying traits of the Muscovy ducks, the primer and the application of the kit in Muscovy duck breeding.
The invention also provides a haplotype block set related to the egg laying traits of Muscovy ducks, which comprises a haplotype block 1 and/or a haplotype block 2;
wherein, the haplotype block 1 consists of SNP1(T35658560G), SNP2(G35658263A) and SNP3(T35658203A), and comprises 5 haplotype block combinations of H11(TGT), H12(AGT) and H13(ATG) with haplotype frequency more than 0.01, H11H11, H11H12, H11H13, H12H12 and H12H 13;
the haplotype block 2 consists of 6 haplotype block combinations of SNP3(T35658203A), SNP4(T35658202C), SNP5(A35658060T), SNP6(C35657186T), SNP7(G35657058C), SNP8(G35656935A), SNP9(A35656927G), SNP10(C35656736T), SNP11(A35656608G) and SNP12(A35656564C), wherein the haplotype frequency is more than 0.01, H21(CGCAGGTATT), H22(CGCGGGTTTA), H23(AATGGCCTCA) and H24(AATGACCTCA), and H21H21, H21H22, H21H23, H21H24, H22H23 and H23H 24.
The invention discloses the following technical effects:
according to the invention, by analyzing the RALGAPA1 gene, a plurality of SNP sites which are obviously related to the egg laying traits of Muscovy ducks exist in the gene, a new SNP molecular marker is provided for MAS, and the SNP1, the SNP2 and the SNP8 are related to the egg laying traits through experimental verification. The SNP1 has obvious correlation with the egg yield of the Muscovy ducks of the population at 48-57 weeks, the total egg yield and the qualified egg yield (P is less than 0.05), and compared with GG and TG genotypes, the egg yield of the TT genotype at the position is higher. SNP2 is obviously related to egg yield of 43-57 weeks, total egg yield, qualified egg yield and 300 days (P is less than 0.05), and the GG genotype of the locus has obvious egg laying advantages. SNP8 was significantly associated with egg production at 29-40 weeks of age and day of onset (P <0.05), GA heterozygous genotypes had egg production advantages. The association of other SNP loci with egg laying traits does not reach a significant level (P > 0.05). Also, SNP1, SNP2, and SNP3 can be combined, and SNP3-SNP12 can be combined. SNP1, SNP5, SNP7, and SNP8 can also be used as optimized Tagger for haplotype block 1 and haplotype block 2 for selection of haplotype blocks. Therefore, the molecular marker provided by the invention can accurately identify the egg laying performance of the Muscovy ducks of different ages in week, so as to provide scientific data for the breeding of the Muscovy ducks.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram of sequencing peaks of different genotypes of SNP1-SNP 12;
FIG. 2 shows SNP combination haplotype blocks, A. haplotype block 1 and B. haplotype block 2.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Examples
1 materials and methods
1.1 animal samples
382 female white muscovy ducks (Renma duck farm, south poultry Breeding Co., Ltd., Guangdong Wen) with the age of 60 weeks were selected. 2mL of subcutaneous venous blood was collected and stored at-80 ℃ and used as a DNA extraction sample. And recording the family information of the selected population and reproductive traits such as the age of the laying date, the daily egg yield, the total egg yield, the egg yield qualification number and the like.
1.2 Main Instrument
BIO-RAD T100 PCR instrument (brand: BIO-RAD; cat # 1861096; Berle, USA) and Hexagon DYCP-31DN nucleic acid electrophoresis instrument (brand: Hexagon, cat # 413: 1402; Hexagon Biotech Co., Ltd.).
1.3 Primary reagents
Animal genome DNA extraction kit (brand: Ongke; Cat number: TSP 201-50; Beijing Ongke Biotechnology Co., Ltd.), 2xTaq MasterMix (containing dye) (brand: Kangshi; Cat number: CW 0682; Jiangsu Kangshi Biotechnology Co., Ltd.), DNA marker (brand: Novozan; Cat number: MD 101; Jiangsu Novozan Biotechnology Co., Ltd.), high purity low agarose electroosmosis (brand: Ongke; Cat number: TSJ 001; Beijing Ongke Biotechnology Co., Ltd.).
2 method of experiment
2.1 primer design
Primers were designed using the Primer-BLAST tool of NCBI (national Center for Biotechnology Information Search database) according to the Muscovy Duck (domesticated) genome (GCA _009194515.1) RALGAPA1 gene (ENSCMG 00000005936) published by Ensembl, and the Primer synthesis service was provided by Shanghai Biotechnology engineering, Inc. The information on the primer sequences is shown in Table 1.
TABLE 1 PCR amplification primer sequences
2.2 extraction of blood genomic DNA
Extracting the blood genome DNA by referring to an animal genome DNA extraction kit operation manual.
2.3 PCR amplification of prolactin Gene exon sequences
A PCR amplification reaction system (Table 2) was prepared using the blood genome DNA of the 382 Muscovy duck half-sib individuals as templates. The reaction sequence is shown in table 3. After the reaction was complete, Sanger sequencing service was provided by Tianyihui Gene technology, Inc., Guangzhou.
TABLE 2 reaction System
TABLE 2 reaction procedure
2.4SNPs determination and genotyping
Genomic DNA of 30 Muscovy ducks was randomly extracted from 382 Muscovy ducks as a template for detecting SNP positions. The Sanger sequencing results of the PCR amplification products were compared using SnapGene 4.3.6 software and genotyped by base peak mapping.
2.5 correlation analysis of genotype and reproductive traits
The association analysis was performed using the GLM program of the SAS 9.4 software. The model is as follows:
Yijlkhno＝μ+Gi+Mj+Bl+GMk+GBh+MBn+GMBO+eijlkhno
Yijlkhnodenotes the phenotypic value,. mu.denotes the mean value, GiIndicating genotype effects, MjIndicates the maternal effect, GMkIndicating the interaction between genotype and mother character, GBhShowing the interaction effects of genotype and maternity, MBnIndicating the interaction between genotype and mother character, GMBoIndicating the interaction between genotype and maternity, eijlkhnoRepresenting the residual error.
Hardy Winberg Equilibrium (HWE) computational analysis was performed using Plink 1.9 software, and haploinsure analysis was performed using Haploview 4.6 and Phase 2.1 software.
3 results
3.1 amplification of RALGAPA1 Gene and SNP screening
PCR amplification was performed using the primers in Table 1 to obtain PCR products of 798bp (SEQ ID No.1) and 917bp (SEQ ID No.2), respectively, located in the exon 14, intron 14, exon 15, intron 15 and exon 16 regions of the RALGAPA1 gene. A total of 12 SNP sites were obtained by software analysis: SNP1(T35658560G), SNP2(G35658263A), SNP3(T35658203A), SNP4(T35658202C), SNP5(a35658060T), SNP6(C35657186T), SNP7(G35657058C), SNP8(G35656935A), SNP9(a35656927G), SNP10(C35656736T), SNP11(a35656608G), SNP12(a 35656564C). These SNPs were all located in the intron region of RALGAPA1 gene on chromosome 5 (FIG. 1, Table 4), and were in accordance with Hardy-Weinberg equilibrium (Table 5).
SEQ ID No.1(RALGAPA1-SNP-F1/R1 product sequence):
SEQ ID No.1(RALGAPA1-SNP-F2/R2 product sequence):
TABLE 4SNP essential information
TABLE 5 Hardy Weinberg equilibrium
3.2 Association of SNP sites with reproductive traits
By GLM linear analysis, SNP1, SNP2, and SNP8 were associated with egg laying traits (tables 6-8). The SNP1 has obvious correlation with the egg yield of the Muscovy ducks of the population at 48-57 weeks, the total egg yield and the qualified egg yield (P is less than 0.05), and compared with GG and TG genotypes, the egg yield of the TT genotype at the position is higher. SNP2 is obviously related to egg yield of 43-57 weeks, total egg yield, qualified egg yield and 300 days (P is less than 0.05), and the GG genotype of the locus has obvious egg laying advantages. SNP8 was significantly associated with egg production at 29-40 weeks of age and day of onset (P <0.05), GA heterozygous genotypes had egg production advantages.
TABLE 3 SNP1 Association information
Note: and +/-represents that the degree of freedom is 0 without standard error value.
TABLE 4SNP 2 Association information
TABLE 5 SNP8 Association information
3.3 haplotype Association with egg laying traits
SNPs 1-SNP3 make up a haplotype block, referred to as haplotype block 1, and SNP3-SNP12 make up a haplotype block, referred to as haplotype block 2 (Table 9). Haplotype block 1 has a haplotype frequency of more than 0.01 and comprises 5 haplotype block combinations of H11(TGT), H12(AGT) and H13(ATG), and H11H11, H11H12, H11H13, H12H12 and H12H 13. Haplotype block 2 with haplotype frequency greater than 0.01 has 6 haplotype block combinations of H21(CGCAGGTATT), H22(CGCGGGTTTA), H23(AATGGCCTCA) and H24(AATGACCTCA), and H21H21, H21H22, H21H23, H21H24, H22H23 and H23H24 (FIG. 2). Haplotype block 1 was significantly associated with 49 weeks old egg production (P <0.05) and haplotype block 2 was significantly associated with 48, 49, 53, 54 weeks old egg production and total egg production (P <0.05) by association analysis with reproductive traits (table 10). Haplotype block 1 and haplotype block 2 gradually increase with the increase of the day-old muscovy duck, and the correlation between the two types of muscovy duck and the egg yield is gradually increased.
TABLE 6 haplotype distribution and frequency
TABLE 7 haplotype Block Association
Discussion 4
The SNP1, the SNP2 and the SNP8 are all obviously related to reproductive traits and are used for breeding the reproductive traits of Muscovy ducks, the SNP1, the SNP2 and the SNP3 can be used as a combination, and the SNP3-SNP12 can be used as a combination. SNP1, SNP5, SNP7, and SNP8 are also useful as optimized Tagger (Table 11) for haplotype block 1 and haplotype block 2 for selection of haplotype blocks.
TABLE 8 haplotype Block Tagger
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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Claims (6)
1. The application of a detection reagent of RALGAPA1 gene SNP molecular marker related to the laying traits of Muscovy ducks in detecting the laying traits of Muscovy ducks, wherein the RALGAPA1 gene SNP molecular marker related to the laying traits of Muscovy ducks comprises: SNP1, SNP2, and SNP 8; wherein, the SNP1 is located at the 35658560 site of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is T > G; the SNP2 is located at the 35658263 locus of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G & gtT; the SNP8 is located at the position 35656935 of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G > A.
2. The application of a primer in identifying the egg laying traits of Muscovy ducks or preparing a kit for identifying the egg laying traits of Muscovy ducks, wherein the nucleotide sequence of the primer is shown in SEQ ID Nos. 3-6, and the primer is used for amplifying the RALGAPA1 gene SNP molecular marker related to the egg laying traits of Muscovy ducks in claim 1.
3. A method for identifying the egg-laying traits of Muscovy ducks, which is characterized by identifying the genotypes of the molecular markers as claimed in claim 1 in a sample Muscovy duck to be detected.
4. A method according to claim 3, characterized by the steps of:
(1) extracting the genome DNA of the Muscovy duck to be detected;
(2) performing PCR amplification by using the primer of claim 2 to obtain an amplification primer;
(3) sequencing the amplification product to obtain the genotype of the molecular marker;
(4) and judging the egg laying character of the muscovy duck of the sample to be detected according to the genotyping result.
5. The method according to claim 4, wherein in the step (2), the PCR-amplified amplification system comprises: 2xTaq MasterMix 15. mu.L, mixed primers each 1.2. mu.L, ultrapure water 10.6. mu.L and DNA 2. mu.L;
the reaction procedure of the PCR amplification is as follows: 94 ℃ for 2min, 94 ℃ for 30s, 53 ℃ for 30s, 72 ℃ for 30s, 30cycles, 72 ℃ for 2 min.
6. The application of RALGAPA1 gene SNP molecular marker and primers related to the egg laying traits of Muscovy ducks in Muscovy duck breeding, wherein the RALGAPA1 gene SNP molecular marker related to the egg laying traits of Muscovy ducks comprises: SNP1, SNP2, and SNP 8; wherein, the SNP1 is located at the 35658560 site of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is T > G; the SNP2 is located at the 35658263 locus of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G & gtT; the SNP8 is located at the 35656935 locus of the intron region of the RALGAPA1 gene of chromosome 5, and the allele is G > A;
the nucleotide sequence of the primer is shown as SEQ ID No.3-6, and the primer is used for amplifying the RALGAPA1 gene SNP molecular marker related to the egg laying traits of the Muscovy ducks.
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| CN111440878A (en) * | 2020-03-08 | 2020-07-24 | 湖北省农业科学院畜牧兽医研究所 | Haplotype marker related to quality traits of egg shells in CDH17 genes and application |
| AU2020102716A4 (en) * | 2020-03-08 | 2020-12-03 | Institute Of Animal Science And Veterinary Medicine, Hubei Academy Of Agricultural Sciences | Haplotype markers of KRT14 gene associated with eggshell quality traits and its application |
| CN112852976A (en) * | 2021-03-17 | 2021-05-28 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof |
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| CN111440878A (en) * | 2020-03-08 | 2020-07-24 | 湖北省农业科学院畜牧兽医研究所 | Haplotype marker related to quality traits of egg shells in CDH17 genes and application |
| AU2020102716A4 (en) * | 2020-03-08 | 2020-12-03 | Institute Of Animal Science And Veterinary Medicine, Hubei Academy Of Agricultural Sciences | Haplotype markers of KRT14 gene associated with eggshell quality traits and its application |
| CN112852976A (en) * | 2021-03-17 | 2021-05-28 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof |
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| 利用DNA池和测序技术快速筛查候选效应单倍型;杜红丽等;《农业生物技术学报》;20080415(第02期);281-285 * |
| 家养动物选择信号研究进展;潘章源等;《遗传》;20161231(第12期);1069-1080 * |
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