CN110904244B - Method for rapidly identifying gray feather heterozygote of Chinese goose species - Google Patents
Method for rapidly identifying gray feather heterozygote of Chinese goose species Download PDFInfo
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Abstract
The invention discloses a method for rapidly identifying a gray feather heterozygote of a Chinese goose species in the field of goose species detection, which comprises the following steps of S1: collecting biological detection materials of individuals with gray feather to be detected; s2: extracting genome DNA in a biological test material of an individual to be tested; s3: taking the extracted genome DNA as a template, and taking a designed and synthesized PCR primer combination as a primer to carry out conventional PCR reaction; s4: separating PCR products by using agarose gel electrophoresis with the concentration of 1%, taking DL2000 as DNAmarker and taking Goldview I as DNA stain; s5: observing the amplification product under an ultraviolet lamp or a gel imaging system, judging that the detected individual is a heterozygous individual if an amplification band exists at about 300bp, and on the contrary, judging that the detected individual is a homozygous gray feather individual if no amplification band exists at about 300 bp. The method has the advantages that the gray feather heterozygous individuals are quickly and accurately identified, so that the goose population reserved for breeding is removed, the breeding population can be homozygous on the gray feather phenotype only by detection of one generation, the breeding efficiency is improved, and the method is provided for identifying the purity of the population.
Description
Technical Field
The invention relates to the technical field of goose species detection, in particular to a method for rapidly identifying a gray feather heterozygote of a Chinese goose species.
Background
The goose yield of China accounts for about 94% of the total world, the goose industry has obvious advantages and characteristics, rapid development is achieved in recent years, and pure breeding and hybrid production of geese are also continuously developed. Chinese goose species all originate from swan geese except Xinjiang Yili geese. From the feather color, the goose species are classified into gray goose species and white goose species. In the 2011 published "national livestock and poultry genetic resource will, poultry will", the national livestock and poultry genetic resource committee recorded 17 varieties of Chinese gray-feather gooses and 12 varieties of white-feather gooses originating from swan gooses.
Researches show that the gray feather in Chinese goose species is dominant relative to the white feather, that is, the goose with the gray feather as the phenotype is a homozygote or a heterozygote. In the breeding process of gray-feather goose species, breeders all want the selected individuals to be homozygous gray-feather individuals, but the individuals are difficult to distinguish from the phenotypes, and the genotypes of the individuals can only be presumed by observing the phenotypes of offspring and utilizing the Mendelian genetic law. However, in goose breeding, accurate pedigree records are generally lacking, and even if pedigree records exist, the genotype of a parent can only be deduced according to the phenotype of a progeny, so that the time consumption is long and the cost is high. Therefore, heterozygous individuals cannot be really detected and rejected in the breeding population of the gray-feather geese.
In addition, in southern areas of China, people have consumption preference for the gray feather varieties, and the gray feather goose varieties are considered to be local varieties and have good meat quality and flavor. However, the gray-feather goose species generally have the defect of low egg yield, and the breeding geese are usually hybridized with the white-feather female geese with relatively high reproduction rate to produce the gray-feather commercial geese. The gray-feather gooses on the market are pure local varieties or filial generations of the gray gooses and the white gooses, and no rapid and accurate identification method exists so far.
Based on the above, the invention designs a method for rapidly identifying the gray feather heterozygote of the Chinese goose species, so as to solve the above-mentioned problems.
Disclosure of Invention
The invention aims to provide a method for rapidly identifying a gray feather heterozygote of a Chinese goose, which designs a PCR primer combination for identifying the genotype of the gray feather heterozygote of the Chinese goose, and judges whether a gray feather individual is a heterozygote individual or not according to the existence and the nonexistence of a PCR product strip by utilizing a conventional PCR technology and an electrophoresis technology, thereby providing a simple, rapid and accurate method for breeding a gray feather homozygous strain and identifying whether the gray feather individual is a homozygous local variety or a filial generation of the gray goose and the white goose.
In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly identifying a gray feather heterozygote of a Chinese goose species is a PCR primer combination for identifying the genotype of the gray feather heterozygote of the Chinese goose species, and comprises the following steps: upstream primers (5 '-3'): CTCAGCACAGGTGAGCT, respectively; and downstream primers (5 '-3'): CCACCTCCTGGTGGGAG, comprising the following steps:
s1: collecting biological detection materials of individuals with gray feather to be detected;
s2: extracting genome DNA in a biological test material of an individual to be tested;
s3: taking the extracted genome DNA as a template, and taking a designed and synthesized PCR primer combination as a primer to carry out conventional PCR reaction;
s4: separating PCR products by using agarose gel electrophoresis with the concentration of 1%, taking DL2000 as a DNA marker and taking Goldview I as a DNA stain;
s5: observing the amplification product under an ultraviolet lamp or a gel imaging system, judging that the detected individual is a heterozygous individual if an amplification band exists at the position 317bp, and on the contrary, judging that the detected individual is a homozygous gray feather individual if no amplification band exists at the position 317 bp.
Preferably, the biological sample includes, but is not limited to, blood sample, skin sample, hair follicle sample.
Preferably, the reaction system of step S3 is 20uL, which includes 0.5uL of each of the upstream and downstream primers, 2uL of template DNA, 1.5uL of dNTP, 1uL of taqDNA polymerase, 12.5uL of sterilized deionized water, and 2uL of 10 × solution buffer.
Preferably, the reaction procedure of step S3 is: the above reaction procedure was cycled 30 times at 95 ℃ for denaturation 30s, 55 ℃ for annealing 30s, and 72 ℃ for extension 30 s.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the breeding process of a new variety or a new strain of the gray-feather goose, the method can be used for quickly and accurately identifying the gray-feather heterozygous individuals, so that the individuals are removed from the reserved goose group, the breeding group can be homozygous on the gray-feather phenotype only by detection of one generation, and the breeding efficiency is improved.
(2) By using the method, whether the gray feather individual is the filial generation of the gray goose and the white goose can be rapidly identified, and a method is provided for identifying the purity of the population.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced 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 that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of an amplification product observed under a gel imaging system according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, Marker is DL2000 mark; A. b is an electrophoresis strip of the gray feather heterozygous individual, namely an amplification strip is arranged at the position of about 300 bp; C. d is an electrophoresis band of individuals homozygous for gray feather, namely no band.
The invention provides a technical scheme that: a method for rapidly identifying a gray feather heterozygote of a Chinese goose species is used for identifying a PCR primer combination of the gray feather heterozygote genotype of the Chinese goose species, and comprises the following steps: upstream primers (5 '-3'): CTCAGCACAGGTGAGCT, respectively; and downstream primers (5 '-3'): CCACCTCCTGGTGGGAG, comprising the following steps:
s1: collecting biological detection materials of individuals to be detected with the gray feather, including but not limited to blood samples, skin samples and hair follicle samples;
s2: extracting genome DNA in a biological test material of an individual to be tested;
s3: taking the extracted genome DNA as a template, and a designed and synthesized PCR primer combination as a primer, carrying out conventional PCR reaction, wherein the reaction system is 20uL, the reaction system comprises 0.5uL of each of upstream and downstream primers, 2uL of template DNA, 1.5uL of dNTP, 1uL of taqDNA polymerase, 12.5uL of sterilized deionized water and 10 Xsolutionbuffer 2uL, and the reaction program is as follows: performing denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, and circulating the reaction program for 30 times;
s4: separating PCR products by using agarose gel electrophoresis with the concentration of 1%, taking DL2000 as a DNA marker and taking Goldview I as a DNA stain;
s5: the amplification products were observed under uv lamp or gel imaging system, as shown in fig. 1, from left to right: a is DNAmarker (DL 2000); B. c is a gray feather heterozygote; D. e is an individual with homozygous gray feather,
if the amplified band exists at about 300bp, the detected individual is judged to be a heterozygous individual, according to the design result of the PCR primer, if the amplified product exists, the product length is 317bp, otherwise, if the amplified band does not exist at about 300bp, the detected individual is judged to be a homozygous gray feather individual.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (4)
1. A method for rapidly identifying a gray feather heterozygote of a Chinese goose species is used for identifying a PCR primer combination of the gray feather heterozygote genotype of the Chinese goose species, and comprises the following steps: upstream primers (5 '-3'): CTCAGCACAGGTGAGCT; and downstream primers (5 '-3'): CCACCTCCTGGTGGGAG, characterized by: the method comprises the following steps:
s1: collecting biological detection materials of individuals with gray feather to be detected;
s2: extracting genome DNA in a biological test material of an individual to be tested;
s3: taking the extracted genome DNA as a template, and taking a designed and synthesized PCR primer combination as a primer to carry out conventional PCR reaction;
s4: separating PCR products by using agarose gel electrophoresis with the concentration of 1%, taking DL2000 as a DNA marker and taking GoldviewI as a DNA stain;
s5: observing the amplification product under an ultraviolet lamp or a gel imaging system, judging that the detected individual is a heterozygous individual if an amplification band exists at the position 317bp, and on the contrary, judging that the detected individual is a homozygous gray feather individual if no amplification band exists at the position 317 bp.
2. The method for rapidly identifying the gray feather heterozygote of Chinese goose species according to claim 1, which is characterized in that: the biological sample includes, but is not limited to, blood sample, skin sample, hair follicle sample.
3. The method of claim 1, wherein the method comprises the steps of: the reaction system of step S3 is 20uL, including upstream and downstream primers each 0.5uL, template DNA 2uL, dNTP 1.5uL, taqDNA polymerase 1uL, sterile deionized water 12.5uL, 10 Xsolutionbuffer 2 uL.
4. The method of claim 3, wherein the method comprises the steps of: the step S3 reaction procedure is: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and the above reaction procedure was repeated 30 times.
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