CN110904244B - Method for rapidly identifying gray feather heterozygote of Chinese goose species - Google Patents

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

A method for rapid identification of Chinese geese hybrids with gray feathers

technical field

The invention relates to the technical field of goose species detection, in particular to a method for rapidly identifying Chinese goose species ash-feather hybrids.

Background technique

my country's meat goose production accounts for about 94% of the world's total. The goose industry has obvious advantages and characteristics. It has developed rapidly in recent years. The purebred breeding and hybrid production of goose are also developing continuously. Except for Xinjiang Yili goose, all Chinese goose species originated from Hongyan. From the perspective of feather color, it is divided into two types: gray-feathered goose species and white-feathered goose species. In 2011, the National Livestock and Poultry Genetic Resources Commission compiled and published "China Livestock and Poultry Genetic Resources: Poultry", which recorded 17 grey-feathered geese and 12 white-feathered geese originating from swan geese.

Previous studies have found that gray feathers are dominant traits relative to white feathers in Chinese goose species, that is to say, geese with gray feather phenotype may be homozygous or heterozygous. In the breeding process of grey feather goose species, breeders all hope that the selected individuals are homozygous grey feather individuals, but it is difficult to distinguish from their phenotype. Del's law of inheritance for genotype inference. However, in goose breeding, accurate pedigree records are usually lacking, and even if there are pedigree records, the genotypes of the parents can only be inferred from the offspring phenotype, which is time-consuming and costly. Therefore, it is often impossible to truly detect and eliminate heterozygous individuals in breeding populations of grey-feathered geese.

In addition, in southern China, people have a preference for gray feather varieties, and they believe that gray feather goose species are local local varieties with good meat and flavor. However, grey-feathered goose species generally have the disadvantage of low egg production. In breeding goose production, grey-feathered male geese and white-feathered female geese with relatively high reproductive rate are often crossed to produce grey-feathered commercial geese. Whether the gray-feathered geese on the market are homozygous local breeds or the hybrid offspring of gray geese and white geese, so far, there is no quick and accurate identification method.

Based on this, the present invention devises a method for rapid identification of Chinese goose species ash-feather hybrids, in order to solve the above-mentioned problems.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of method for quickly identifying Chinese goose species ash-feather hybrids, design the PCR primer combination for identifying Chinese goose species ash-feather hybrid genotypes, utilize conventional PCR technology and electrophoresis technology, according to PCR product strips The "yes" and "absence" of the band determine whether the gray feather individual is a heterozygous individual, so as to provide information for the selection and breeding of the gray feather homozygous line and the identification of whether the gray feather individual is a homozygous local breed or the hybrid progeny of gray goose and white goose. A simple, fast and accurate method.

In order to achieve the above object, the invention provides the following technical solutions: a method for rapidly identifying Chinese goose species ash-feather hybrids, for identifying the PCR primer combination of Chinese goose species ash-feather hybrid genotypes, comprising: upstream primers (5' -3'): CTCAGCACAGGTGAGCT; and the downstream primer (5'-3'): CCACCTCCTGGTGGGAG, including the following steps:

S1: Collect biological samples of gray feather individuals to be detected;

S2: Extracting genomic DNA from the biological sample of the individual to be tested;

S3: using the extracted genomic DNA as a template, the designed and synthesized PCR primer combinations are used as primers, and a conventional PCR reaction is carried out;

S4: Use 1% concentration agarose gel electrophoresis to separate PCR products, use DL2000 as DNA marker, and Goldview I as DNA stain;

S5: Observe the amplified product under UV lamp or gel imaging system. If there is an amplified band at the 317bp position, it is judged that the detected individual is a heterozygous individual. On the contrary, if there is no amplified band at the 317bp position, it is judged that the detected individual is a heterozygous individual. The tested individuals are homozygous gray-feathered individuals.

Preferably, the biological samples include but are not limited to blood samples, skin samples, and hair follicle samples.

Preferably, the step S3 reaction system is 20 uL, including 0.5 uL of upstream and downstream primers, 2 uL of template DNA, 1.5 uL of dNTP, 1 uL of taqDNA polymerase, 12.5 uL of sterile deionized water, and 2 uL of 10×solution buffer.

Preferably, the reaction procedure of step S3 is as follows: denaturation at a temperature of 95° C. for 30 s, annealing at 55° C. for 30 s, and extension at 72° C. for 30 s, and the above reaction procedure is cycled 30 times.

Compared with the prior art, the beneficial effects of the present invention are:

(1) in the breeding process of the new breed or new line of grey feather goose, using the method of the present invention, the grey feather heterozygous individual can be quickly and accurately identified, so as to be eliminated in the breed-keeping goose group, only one generation of detection is required , the breeding population can be homozygous in the gray feather phenotype, and the breeding efficiency can be improved.

(2) By using the method of the present invention, whether the gray feather individual is the hybrid offspring of the gray goose and the white goose can be quickly identified, which provides a method for the identification of the purity of the population.

Description of drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

FIG. 1 is a schematic diagram of the amplification product observed under the gel imaging system of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Please refer to Figure 1, Marker is the DL2000 marker; A and B are the electrophoresis bands of the gray feather heterozygous individuals, that is, there is an amplification band at about 300bp; C and D are the electrophoresis bands of the gray feather homozygous individuals, i.e. no banding.

The invention provides a technical scheme: a method for rapidly identifying Chinese goose species gray feather hybrids, and a PCR primer combination for identifying the Chinese goose species gray feather hybrid genotypes, including: upstream primers (5'-3') : CTCAGCACAGGTGAGCT; and the downstream primer (5'-3'): CCACCTCCTGGTGGGAG, including the following steps:

S1: Collect biological samples of gray feather individuals to be tested, including but not limited to blood samples, skin samples, and hair follicle samples;

S2: Extracting genomic DNA from the biological sample of the individual to be tested;

S3: Take the extracted genomic DNA as a template, design and synthesize the PCR primer combination as primers, and carry out a conventional PCR reaction. The reaction system is 20uL, including 0.5uL of upstream and downstream primers, 2 uL of template DNA, 1.5 uL of dNTP, and taqDNA polymerase. 1uL, sterilized deionized water 12.5uL, 10×solutionbuffer 2uL, the reaction procedure is: denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, and cycle the above reaction procedure 30 times;

S4: Use 1% concentration agarose gel electrophoresis to separate PCR products, use DL2000 as DNA marker, and Goldview I as DNA stain;

S5: Observe the amplified product under UV lamp or gel imaging system, as shown in Figure 1, from left to right: A is DNA marker (DL2000); B and C are gray feather hybrids; D and E are gray feather pure combined individual,

If there is an amplification band around 300bp, the detected individual is judged to be a heterozygous individual. According to the design results of the PCR primers, if there is an amplification product, the length of the product is 317bp. On the contrary, if there is no amplification band around 300bp band, it is judged that the detected individual is a homozygous gray feather individual.

In the description of this specification, description with reference to the terms "one embodiment," "example," "specific example," etc. means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one aspect of the present invention. in one embodiment or example. In this specification, schematic representations of the above terms 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 above-disclosed preferred embodiments of the present invention are provided only to help illustrate the present invention. The preferred embodiments do not exhaust all the details, nor do they limit the invention to only the described embodiments. Obviously, many modifications and variations are possible in light of the content of this specification. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is to be limited only by the claims and their full scope and equivalents.

Claims (4)

1. A method for rapidly identifying Chinese goose species ash-feather hybrids, the combination of PCR primers for identifying the genotypes of Chinese goose species ash-feather hybrids, comprising: upstream primers (5'-3'): CTCAGCACAGGTGAGCT; and downstream primers (5 '-3 '): CCACCTCCTGGTGGGAG, it is characterized in that: comprise concrete following steps: S1: Collect biological samples of gray feather individuals to be detected; S2: Extracting genomic DNA from the biological sample of the individual to be tested; S3: using the extracted genomic DNA as a template, the designed and synthesized PCR primer combinations are used as primers, and a conventional PCR reaction is carried out; S4: Use 1% agarose gel electrophoresis to separate PCR products, use DL2000 as DNA marker, and GoldviewI as DNA stain; S5: Observe the amplified product under UV lamp or gel imaging system. If there is an amplified band at the 317bp position, it is judged that the detected individual is a heterozygous individual. On the contrary, if there is no amplified band at the 317bp position, it is judged that the detected individual is a heterozygous individual. The tested individuals are homozygous gray-feathered individuals. 2 . The method for rapidly identifying Chinese goose species ash-feather hybrids according to claim 1 , wherein the biological test materials include but are not limited to blood samples, skin samples, and hair follicle samples. 3 . 3. a kind of method for quickly identifying Chinese goose species ash-feather hybrid according to claim 1, is characterized in that: described step S3 reaction system is 20uL, comprises each 0.5uL of upstream and downstream primers, template DNA 2uL, dNTP 1.5 uL, 1uL of taqDNA polymerase, 12.5uL of sterile deionized water, 2uL of 10×solutionbuffer. 4. A method for rapidly identifying Chinese goose species ash-feather hybrids according to claim 3, wherein the step S3 reaction program is: denaturation at a temperature of 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C For 30 s, the above reaction procedure was cycled 30 times.

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