CN106244713B - A method and application for detecting the five-toed character of Beijing You Chicken - Google Patents

Abstract

The invention discloses a method for detecting the five-toe character of Beijing fatty chicken. The method comprises the step of detecting the genotype of the rs80659072 mutation site in the ZRS region in the 5 th intron of the LMBR1 gene by a real-time fluorescent quantitative PCR technology. The real-time fluorescent quantitative TaqMan SNP typing technology adopted by the invention has the advantages of high detection speed, high sensitivity and high automation degree, the typing can be judged through one PCR reaction, the operation is simple and convenient, the whole reaction process is carried out in a closed tube, and the cross contamination is reduced. On the basis of the invention, molecular marker-assisted breeding is adopted, and the five-toe strain pure line of the Beijing fatty chicken can be quickly constructed.

Description

A method and application for detecting the five-toed character of Beijing You Chicken

technical field

The invention relates to the field of biological detection, in particular, to a method and application for detecting the five-toed character of Beijing You Chicken.

Background technique

Beijing oil chicken is a famous local breed in my country. It is native to Beijing area. This chicken breed has high quality meat and eggs and is very popular among farmers and consumers. It has been included in the national key protection list of livestock and poultry breed resources by the Ministry of Agriculture. In recent years, it has been promoted in Beijing and most provinces and cities in the country, and the breeding volume and breeding scale have increased year by year. The chicken has a unique appearance with five toes and three hairs (crested head, beard, hairy legs).

Among them, the five-toed is the most obvious recognizable character of Beijing oil chicken after slaughtering and listing. It can be used as an important breed identification of Beijing oil chicken to help consumers make consumption choices. Therefore, it is of great significance to strengthen the selection and breeding of the five-toed traits of Beijing You Chicken, both from the perspective of promoting the development of the breed itself, as well as from the perspective of farmers and consumers.

However, the five toes of chickens is a relatively complex quality trait with incomplete dominant inheritance. The progress of selection by phenotype of toe number is very slow, and it is difficult to obtain significant effects.

In 2010, Dorshorst et al. detected the mutation rs80659072 in the ZRS region in the fifth intron of the LMBR1 gene in Siklie and WhiteSultan chicken breeds, which was associated with five toes, of which the GG type showed four toes, and the GT and TT types showed five toes or more. Polydactyly. In 2016, Zhang et al. also indicated that this locus is completely related to the polydactyly of Beijing oil chicken, and established a PCR-RFLP method based on enzyme digestion to detect this locus (Patent Application No. 201510044061.2).

However, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method has complicated detection steps, including: 1. Design primers according to the upstream and downstream sequences of the restriction sites, and use PCR to amplify the target fragment; ②Agarose gel detection of the amplified product; ③Restriction endonuclease digestion of the amplified product; ④Agarose gel electrophoresis detection of the digestion product. Not only are the steps cumbersome and time-consuming, but also need to transfer tubes and add samples many times, and the DNA concentration, DNA quality, amplification conditions, endonuclease digestion conditions, and agarose gel quality will affect the validity and quality of judging. accuracy.

In the actual breeding process of the five-toed trait of Beijing You Chicken, it is required to be able to detect the genotype quickly, and the result must be accurate, because once the judgment is wrong, it will bring great losses to the breeding. Therefore, it is particularly necessary to develop a method that can quickly and accurately detect the five-toed traits of Beijing oil chickens, so as to provide strong technical support for the selection and breeding of Beijing oil chickens.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method that can accurately and efficiently detect the defects of the existing five-toed traits of Beijing oil chicken by using the PCR-RFLP method, which are cumbersome, time-consuming, low in sensitivity, and easily affected by experimental conditions. Quantitative real-time PCR method for the detection of five-toed traits in Beijing You Chicken.

To achieve the above object, the present invention adopts the following technical solutions:

First of all, the present invention provides a method for detecting the five-toed traits of Beijing You Chicken, which includes the step of detecting the genotype of the rs80659072 mutation site in the ZRS region in the fifth intron of the LMBR1 gene by real-time fluorescence quantitative PCR technology.

Preferably, the above method includes designing a pair of fluorescent quantitative PCR primers and a TaqMan probe used in conjunction with the primers according to the sequence of the ZRS region in the fifth intron of the LMBR1 gene, using these primers and probes, using real-time fluorescent quantitative PCR technology Steps for detecting the genotype of the rs80659072 mutation site;

Wherein, the nucleotide sequence of the region where the ZRS is located in the fifth intron of the LMBR1 gene is shown in SEQ ID NO: 5.

In a preferred aspect of the present invention, the primers for fluorescent quantitative PCR are: the nucleotide sequences of the fluorescent quantitative PCR primer pairs used to amplify the ZRS region are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and

The nucleotide sequences of the TaqMan fluorescent probes used in conjunction with the above PCR primers are: the nucleotide sequence of the wild-type allele probe is shown in SEQ ID NO: 3, and the nucleotide sequence of the mutant-type allele probe is shown in SEQ ID NO: 3. As shown in SEQ ID NO:4.

Preferably, the reaction system of the above fluorescent quantitative PCR is: 1 μL of genomic DNA, 0.25 μL of 40-fold concentrated probe and primer mixture, 5 μL of general PCR mixture, and make up to 10 μL with water.

Preferably, the reaction conditions of the above-mentioned fluorescence quantitative PCR are: 95°C, pre-denaturation for 10 min; denaturation at 95°C for 15s, annealing at 60°C for 1 min, for a total of 40 cycles.

The present invention provides a primer for identifying traits of five-toed chickens in Beijing You Chicken, which is a primer pair for amplifying the ZRS region, and its nucleotide sequences are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2 .

Further, the present invention also provides a TaqMan fluorescent probe used in conjunction with the above-mentioned PCR primers, which includes the nucleotide sequence of the wild-type allele probe as shown in SEQ ID NO: 3; the mutant allele The nucleotide sequence of the gene probe is shown in SEQ ID NO:4.

Further, the present invention also provides a kit, which comprises a combination of the above-mentioned primers and the above-mentioned fluorescent probes.

Preferably, the kit of the present invention is used in the detection of the five-toed traits of Beijing You Chicken.

Further, the present invention also provides the application of the above-mentioned method for detecting the five-toed character of the Beijing You Chicken to the breeding of the five-toed trait of the Beijing You Chicken.

The invention adopts the real-time fluorescent quantitative TaqMan SNP typing technology to detect the genotype of the target sequence by designing specific primer pairs and probes used in conjunction with the primer pairs. Real-time fluorescence quantitative PCR technology can quickly and sensitively detect the genotype of the mutation site in the target region of the chicken to be tested, and on this basis, molecular marker-assisted breeding can quickly construct a pure line of the Beijing You Chicken five-toed line. In addition, the real-time fluorescent quantitative TaqMan SNP typing technology used in the present invention has fast detection speed, high sensitivity, and high degree of automation. Types can be determined after one PCR reaction, and the operation is simple and convenient, and the whole reaction process is carried out in a closed tube, which reduces crossover. Pollution. It can meet the accuracy and timeliness requirements of breeding work.

In addition, the probe used in combination with the specific primer pair used in the present invention is a TaqMan MGB probe, and the quenching group at the 3' end of the probe is a non-luminescent quenching group (Non-Fluorescent Quencher, NFQ). , so when the quencher group absorbs the energy of the reporter group, it does not emit light, which greatly reduces the interference of the background signal; the TaqMan MGB probe also has stronger sequence specificity, and the experimental results are more accurate and reliable.

Description of drawings

Figure 1 is an agarose gel electrophoresis image of the PCR amplification products of some individuals, in the figure, lanes 1-10 are different individuals, and lane M is a 100bp Marker;

Figure 2 shows the genotype of individuals detected by fluorescence quantitative PCR;

Figure 3 shows the sequencing results of individuals with different genotypes.

Detailed ways

The current method for detecting polydactyly in Beijing oil chicken is to detect the genotype of the locus related to the five toes of Beijing oil chicken based on the enzyme digestion PCR-RFLP method. In order to overcome the above-mentioned defects, the present invention provides a method for efficiently detecting the traits of the five-toed Beijing oil chicken by using real-time fluorescent PCR technology.

The present invention will be described below in conjunction with specific embodiments, but should not be construed as a limitation of the present invention. Under the premise that does not deviate from the spirit and essence of the present invention, modifications or replacements made to the present invention all belong to the scope of the present invention:

Example 1 Detection of mutation site rs80659072

With reference to the mutation site rs80659072 (NCBI accession number) and its surrounding sequence (nucleotide sequence shown in SEQ ID NO: 5), design primers, upstream primer: 5'-ACATACCAAGAATGTGCATGTGC-3', downstream primer: 5'-TTTGAGGTAACTTCCTTGCTTAA -3', the length of the amplified product is 448bp.

A total of 155 individuals of various toe types were randomly selected from the large flock of Beijing oil chickens. Anticoagulant was collected, and genomic DNA was extracted using a DNA extraction kit (Tiangen Biochemical, Beijing), and the above-mentioned primers and surrounding sequences were used for PCR amplification. After the amplified product was detected by 2% agarose gel, sequencing was completed by Beijing Qingke Jiamei Biotechnology Co., Ltd. and reverse sequencing was performed.

Figure 1 shows the agarose gel electrophoresis detection results after PCR amplification of some of the above-mentioned individuals, wherein the products corresponding to the individuals in lanes 8 and 10 are very weak and cannot be used for subsequent sequencing experiments. Using NanoDrop2000 UV spectrophotometer to measure the genome concentration, it was found that the DNA concentration of these four individuals was low, only 10-15ng/μL. The amount of DNA template added was increased to 5 μL, and the PCR amplification was repeated, but the ideal PCR product amplification concentration was still not reached. Therefore, after re-extracting genomic DNA from these 4 individuals, and re-PCR amplification, satisfactory results were obtained.

The sequencing results of all 155 chickens are shown in Table 1: from Table 1, it can be seen that except for the oil chicken with double four-toed (4/4), no matter which phenotype, left five-right four (5/4), left Four-right five (4/5), double five toes (5/5), six toes (including double six toes and single six toes, 6/-), there are only two genotypes of TT and GT. The results showed that the T locus is a necessary but not sufficient condition for the five-toed/six-toed phenotype of Beijing You Chicken.

Table 1 Genotype distribution of rs80659072 mutation sites in oil chickens with different phenotypes

Example 2 Establishment of Fluorescence Quantitative PCR Detection Method

After repeated comparison screening and verification in the present invention, according to the ZRS region in the 5th intron of the LMBR1 gene and its adjacent sequences (the nucleotide sequence is shown in SEQ ID NO: 5), a pair of pairs for detecting chicken rs80659072 mutation is obtained Real-time PCR primers for the locus and TaqMan probes used in conjunction with the primers.

Upstream primer F: 5'-TCAGTGGCAAAAAACGAGCAAAAAT-3' (SEQ ID NO.1)

Downstream primer R: 5'-CACACAGAAATGAGTAGGAAGTCCAA-3' (SEQ ID NO. 2).

The upstream primer consists of 25 bases, corresponding to 8467207-8467231 bases on chicken chromosome 2 (www.ensembl.org, galgal4), and the downstream primer consists of 26 bases, corresponding to 8467272-8467299 bases on chicken chromosome 2 (www.ensembl.org, galgal4), the amplified fragment length of this primer pair is 93 bp.

The nucleotide sequence of the TaqMan fluorescent probe used in conjunction with the above-mentioned specific primers is:

Wild-type allele probe: 5'-ATGCAATGAAAGCTC-3' (SEQ IN NO.3);

Mutant allele probe: 5'-CATGCAATTAAAGCTC-3' (SEQ IN NO. 4).

The wild-type probe consists of 15 bases, and the mutant probe consists of 16 bases, corresponding to the 8467241-8467255 and 8467240-8467255 base sequences of chromosome 2. Among them, the probe of the wild-type allele is directed to the wild-type base G, and the fluorophore VIC is connected to the 5' end of the probe. The probe of the mutant allele is directed against the mutant base T, and the 5' end of the probe is connected with a fluorophore FAM; the 3' end of the two probes are labeled with a quenching group, which is non-luminescent Quencher group (Non-Fluorescent Quencher, NFQ).

TaqMan probe real-time fluorescent quantitative PCR test was performed using Bio-Rad iQ5 real-time quantitative PCR instrument.

Using the genomic DNAs of 155 individuals with known genotypes in the above Example 1 as templates, a TaqMan fluorescence quantitative PCR detection method was established. The reaction system is as follows:

Genotyping Master Mix由ABI(美国)合成，购自英潍捷基(上海)贸易有限公司，ddH2O为灭菌去离子水。Wherein, the genomic DNA is the genomic DNA obtained in Example 1, and 40×SNP Genotyping AssayMix (upstream and downstream primer concentrations of 36 μM and probe concentration of 8 μM each) were synthesized by ABI (United States) and purchased from Yingweijieji (Shanghai) trade co., LTD),

Genotyping Master Mix was synthesized by ABI (USA), purchased from Yingweijieji (Shanghai) Trading Co., Ltd., and ddH2O was sterile deionized water.

PCR reaction conditions: 95 °C, pre-denaturation for 10 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, a total of 40 cycles, and real-time detection of the signal intensities of the two fluorescent dyes throughout the amplification.

During the detection process, two blank controls without template DNA were set for each reaction system to facilitate the software to correct the background color.

After the reaction, you can also use the instrument's own software, the Allelic Disc function interface in the data analysis module to directly judge the type, and the type judgment result is shown in Figure 2:

From Figure 2, it can be seen that individuals of the same genotype are clustered together, where the squares represent TT homozygotes; the triangles represent GT heterozygotes; the circles represent GG homozygotes.

The reverse sequencing results of individuals with different genotypes are shown in Figure 3, a) There is only one peak at the mutation site indicated by the arrow, corresponding to the A base. Because it is reverse sequencing, the genotype of the corresponding individual is TT mutant, b ) There are 2 peaks at the mutation site indicated by the arrow, corresponding to two bases A and C. Because of reverse sequencing, the corresponding individual genotype is GT type, which is a heterozygote; c) At the mutation site indicated by the arrow There is a peak at the point, and the corresponding base is C. Because of reverse sequencing, the genotype of the corresponding individual is GG and wild type.

To sum up, the results showed that the Taqman probe detection results for 155 individuals were completely consistent with the known PCR product sequencing results, indicating that the TaqMan probe real-time fluorescence quantitative PCR method was accurate and reliable.

When the method provided by the present invention detects 155 individuals, a 96-well plate is used, and only two PCR plates are used to complete the judgment of the results. It only takes 4-5 hours. And including the 4 ones that cannot be amplified in Example 1, this is mainly because the TaqMan SNP typing method has relatively low requirements on the genome, ranging from 10-100ng can meet the needs, even if the genome concentration is very low or the extraction quality is very low. Slightly worse, there is no need to re-extract the genome, and it will not affect the judgment of the detection and typing results.

However, if the method of direct sequencing of PCR products in Example 1 or the method of PCR-RFLP is used, both have high requirements on the genome, otherwise the PCR amplification will be unstable, or the amount of the product will be too small, which will affect the subsequent enzyme digestion and sequencing work. . In addition, in addition to the PCR amplification step, the method of direct sequencing of PCR products or the method of PCR-RFLP also needs to increase the step of sequencing or enzyme digestion, which takes at least 10 hours. Therefore, the TaqMan SNP typing method of the present invention has very obvious advantages.

Example 3 Study on the genetic law of chickens with different toe number phenotypes and genotypes

The roosters and hens with different toe number phenotypes and combinations of genotypes were selected for mating, and the inheritance laws of different genotypes were studied. The specific experimental operations are as follows:

(1) Double four-toed phenotype: 7 GG roosters, mated with 20 GG hens; 3 TT roosters, mated with 7 TT hens and 7 GT hens; (2) Double five-toed phenotype : 8 TT roosters with 20TT hens and 6 GT hens; two GT roosters and 2 GT hens.

The offspring of the above-mentioned mating types were separately registered for the toe pattern. The results are shown in Table 2:

Table 2 Results of toe type in the offspring of chickens with different toe number phenotypes and genotypes

It can be seen from Table 2 that 100% of the offspring of CC type and CC type are double four-toed individuals. The test results fully demonstrate that if GG genotype roosters and hens are mated, the offspring are completely double four-toed individuals.

Most of the offspring of AA and AA or AC types show double five toes. The specific analysis is as follows:

The TT type with double four-toed phenotype is mated with TT type or GT type chickens, and the offspring will still have a higher proportion of five-toed chickens. , there are still about 18% double-four-toed individuals in the population.

However, even when the double-five-toed TT type is mated with TT or GT chickens, the offspring will still show a low proportion of the double-four-toed phenotype, about 4%.

In other words, there may be some inhibitory factors that inhibit its expression, but it does not affect the inheritance of the five toes. The mutation site rs80659072 can be used as a candidate marker for the five-toed trait of Beijing oil chicken in breeding. Although the offspring may not all show five-toed, it will greatly increase the proportion of the five-toed phenotype in the offspring of the population.

Claims (6)

1. a method for detecting the five-toed character of Beijing oil chicken, is characterized in that, comprises the step of detecting the genotype of the rs80659072 mutation site in the ZRS region in the 5th intron of LMBR1 gene by real-time fluorescence quantitative PCR technology; According to the sequence of the ZRS region in the fifth intron of the LMBR1 gene, a pair of fluorescent quantitative PCR primers and TaqMan probes used in conjunction with the primers were designed. Using these primers and probes, real-time fluorescent quantitative PCR technology was used to detect the mutation site of rs80659072 genotype steps; Wherein, the nucleotide sequence of the region where the ZRS is located in the fifth intron of the LMBR1 gene is shown in SEQ ID NO: 5; The primers of the fluorescent quantitative PCR are: the nucleotide sequences of the fluorescent quantitative PCR primer pairs used to amplify the sequence of the ZRS region are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; and The nucleotide sequences of the TaqMan fluorescent probes used in conjunction with the above PCR primers are: the nucleotide sequence of the wild-type allele probe is shown in SEQ ID NO: 3, and the nucleotide sequence of the mutant-type allele probe is shown in SEQ ID NO: 3. As shown in SEQ ID NO: 4; The reaction conditions of the fluorescent quantitative PCR were: 95°C, pre-denaturation for 10 min; denaturation at 95°C for 15s, annealing at 60°C for 1 min, a total of 40 cycles. 2. The method according to claim 1, wherein the reaction system of the fluorescent quantitative PCR is: 1 μL of genomic DNA, 0.25 μL of a 40-fold concentrated probe and primer mixture, 5 μL of a general PCR mixture, replenished with water Make up to 10 μL. 3. Primers and probes for identifying the five-toed traits of Beijing You Chicken, which are primer pairs for amplifying the ZRS region, the nucleotide sequences of which are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2; The probes include the nucleotide sequence of the wild-type allele probe as set forth in SEQ ID NO:3; and the nucleotide sequence of the mutant allele probe as set forth in SEQ ID NO:4. 4. A kit, characterized in that, comprising the primers and probes of claim 3. 5. the application of the method described in claim 1 or 2 in breeding Beijing oil chicken five-toed character, it is characterized in that, with non-GG type oil chicken rooster and hen mating, select the offspring with five-toed character. 6. The application of the test kit of claim 4 in detecting the five-toed character of Beijing You Chicken.

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