CN106244713B - Method for detecting five-toe characters of Beijing fatty chicken and application thereof - Google Patents
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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
Technical Field
The invention relates to the field of biological detection, in particular to a method for detecting the five-toe character of Beijing fatty chicken and application thereof.
Background
The Beijing fatty chicken is a famous local variety in China, is native to Beijing area, has excellent meat and egg quality, is popular with farmers and consumers, and is listed in the national key protection list of livestock and poultry variety resources by Ministry of agriculture. In recent years, the method is popularized in Beijing city and most provinces and cities in China, and the culture quantity and the culture scale are increased year by year. The chicken has unique appearance, and has characteristics of five toes and three hair (chicken head, beard, hair and leg).
The five toes are the most obvious recognition traits of the Beijing fatty chickens slaughtered to market, and can be used as important variety identifications of the Beijing fatty chickens to help consumers to make consumption choices. Therefore, the method for enhancing the breeding of the five-toe character of the Beijing fatty chicken has important significance in promoting the development of the variety and in the aspects of breeders and consumers.
However, the five toes of the chicken are relatively complex quality traits and are incompletely inherited dominantly, and selection by the phenotype of the number of toes progresses very slowly, so that a remarkable effect is difficult to obtain.
In 2010, Dorshorst et al detected mutations rs80659072 in the ZRS region within the 5 th intron of the LMBR1 gene in Siklie and Whitestutan chicken species in association with five toes, with GG appearing as four toes and GT and TT appearing as five or more toes. In 2016, Zhang et al also showed that the site is completely related to the polydactylus of Beijing oil chicken, and the enzyme digestion-based PCR-RFLP method was established to detect the site (patent application No. 201510044061.2).
However, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method has complex detection steps, including ① designing primers according to sequences on the upstream and downstream of the restriction enzyme cutting site, amplifying a target fragment by using polymerase chain reaction, ② agarose gel detection of an amplification product, ③ restriction enzyme digestion of the amplification product, and ④ agarose gel electrophoresis detection of the restriction enzyme cutting product, has the disadvantages of complicated steps, long time consumption, multiple tube transfer and sample loading, and the effectiveness and accuracy of type judgment can be influenced by DNA concentration, DNA quality, amplification conditions, endonuclease digestion conditions, agarose gel quality and the like.
In the actual breeding process of the five-toe character of the Beijing fatty chicken, the genotype can be quickly detected, and the result must ensure the accuracy, because once the judgment is wrong, the great loss is brought to breeding. Therefore, a method capable of rapidly and accurately detecting the five-toe character of the Beijing fatty chicken is developed, and is particularly necessary for providing powerful technical support for the breeding of the Beijing fatty chicken.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative PCR method capable of accurately and efficiently detecting the five-toe character of Beijing fatty chicken, aiming at the defects that the existing PCR-RFLP method for detecting the five-toe character of the Beijing fatty chicken has complicated steps, long time consumption, low sensitivity and easily influenced detection results by experimental conditions.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for detecting the five-toe character of Beijing fatty chicken, which comprises the step of detecting the genotype of rs80659072 mutation site in ZRS region in the 5 th intron of LMBR1 gene by real-time fluorescent quantitative PCR technology.
Preferably, the method comprises the steps of designing a pair of fluorescent quantitative PCR primers and a TaqMan probe matched with the primers according to the sequence of a ZRS region in a 5 th intron of the LMBR1 gene, and detecting the genotype of the rs80659072 mutation site by using the primers and the probe and adopting a real-time fluorescent quantitative PCR technology;
wherein, the nucleotide sequence of the region of ZRS in the 5 th intron of the LMBR1 gene is shown as SEQ ID NO: 5, respectively.
In a preferred aspect of the present invention, the primers of the fluorescent quantitative PCR are: the nucleotide sequences of the fluorescent quantitative PCR primer pair for amplifying the ZRS region are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; and
the nucleotide sequence of the TaqMan fluorescent probe matched with the PCR primer is as follows: the nucleotide sequence of the wild type allele probe is shown as SEQ ID NO: 3, the nucleotide sequence of the mutant allele probe is shown as SEQ ID NO: 4, respectively.
Preferably, the reaction system of the fluorescent quantitative PCR is: genomic DNA 1. mu.L, 40-fold concentrated probe and primer mix 0.25. mu.L, universal PCR mix 5. mu.L, made up to 10. mu.L with water.
Preferably, the reaction conditions of the fluorescent quantitative PCR are: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 1min, for 40 cycles.
The invention provides a primer for identifying the five-toe character of Beijing fatty chicken, which is a primer pair for amplifying ZRS region, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Further, the invention also provides a TaqMan fluorescent probe matched with the PCR primer for use, which comprises a nucleotide sequence of a wild type allele probe shown as SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the mutant allele probe is shown as SEQ ID NO: 4, respectively.
Further, the invention also provides a kit, which comprises the combination of the primer and the fluorescent probe.
Preferably, the kit provided by the invention is applied to detecting the five-toe character of the Beijing fatty chicken.
Furthermore, the invention also provides application of the method for detecting the five-toe character of the Beijing fatty chicken in breeding the five-toe character of the Beijing fatty chicken.
The invention adopts real-time fluorescence quantitative TaqMan SNP typing technology to detect the genotype of a target sequence by designing a specific primer pair and a probe matched with the primer pair for use. The real-time fluorescent quantitative PCR technology is adopted, the genotype of the mutation site of the target region of the chicken to be detected can be rapidly and sensitively detected, and on the basis, the molecular marker is used for assisting in breeding, so that the pure line of the five-toe strain of the Beijing oil chicken can be rapidly constructed. In addition, the real-time fluorescent quantitative TaqMan SNP typing technology used 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. Can meet the requirements of breeding work on accuracy and timeliness.
In addition, the probe used in cooperation with the specific primer pair is a TaqMan MGB probe, and the quenching group at the 3' end of the probe is a Non-luminescent quenching group (NFQ), so that the quenching group does not emit light after absorbing the energy of the reporter group, and the interference of background signals is greatly reduced; the TaqMan MGB probe also has stronger sequence specificity, and the experimental result is more accurate and reliable.
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FIG. 1 is an agarose gel electrophoresis of a PCR amplification product of a part of individuals, wherein lanes 1-10 represent different individuals, and lane M represents a 100bp Marker;
FIG. 2 is a diagram showing the genotype of an individual detected by a fluorescent quantitative PCR method;
FIG. 3 shows the sequencing results of individuals with different genotypes.
Detailed Description
The method for detecting the multi-toe of the Beijing fatty chicken at present is to detect the genotype of a locus related to the five toes of the Beijing fatty chicken by a PCR-RFLP method based on enzyme digestion, has complex steps and long consumed time, and can not meet the requirement of later-stage construction of a pure line of the five-toe strain of the Beijing fatty chicken.
The invention is illustrated below with reference to specific examples, which should not be construed as limiting the invention. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention as set forth in the appended claims:
example 1 detection of mutation site rs80659072
With reference to mutation site rs80659072(NCBI accession number) and its surrounding sequence (nucleotide sequence shown in SEQ ID NO: 5), a primer, an upstream primer: 5'-ACATACCAAGAATGTGCATGTGC-3', downstream primer: 5'-TTTGAGGTAACTTCCTTGCTTAA-3', the length of the amplified product is 448 bp.
Individuals of various toe types were randomly selected from the Beijing fatty chicken flock, and a total of 155 individuals were selected. Collecting anticoagulation, extracting genome DNA by using a DNA extraction kit (Tiangen Biochemical, Beijing), and performing PCR amplification by using the site and the surrounding sequence of the primer. After the amplification product was detected by 2% agarose gel, sequencing was completed by camei biotechnology limited of kyoto, and reverse sequencing was performed.
FIG. 1 shows the result of agarose gel electrophoresis detection after PCR amplification of a part of the above individuals, wherein the products of lanes 8 and 10 corresponding to the individuals are very weak and cannot be used in the subsequent sequencing test. The genome concentration was measured using a NanoDrop2000 UV spectrophotometer and the 4 individuals were found to have a low DNA concentration of only 10-15 ng/. mu.L. The amount of DNA template added was increased to 5. mu.L and the PCR amplification was resumed, but the desired concentration of PCR product amplification was still not achieved. Therefore, satisfactory results were obtained only after re-extracting genomic DNA from these 4 individuals and re-PCR amplification.
The sequencing results for all 155 chickens are shown in table 1: it can be seen from Table 1 that, in addition to the double-four-toe (4/4) broiler, the remaining phenotypes, i.e., left five-right four (5/4), left four-right five (4/5), double five-toe (5/5), and six-toe (including double six-toe and single six-toe, 6 /), are the only genotypes of TT and GT. The results indicate that the T site is a necessary condition, but not a sufficient condition, for the five/six-toe phenotype of the Beijing fatty chickens.
TABLE 1 distribution of rs80659072 mutation site genotypes of different phenotype oil chickens
Example 2 establishment of fluorescent quantitative PCR detection method
Through repeated comparison, screening and verification, a pair of fluorescent quantitative PCR primers for detecting rs80659072 mutation sites of the chicken and a TaqMan probe matched with the primers are obtained according to a ZRS region in a 5 th intron of an LMBR1 gene and a sequence (the nucleotide sequence is shown as SEQ ID NO: 5) nearby the region.
An upstream primer F: 5'-TCAGTGGCAAAAAACGAGCAAAAAT-3' (SEQ ID NO.1)
A downstream primer R: 5'-CACACAGAAATGAGTAGGAAGTCCAA-3' (SEQ ID NO. 2).
The upstream primer consists of 25 bases and corresponds to 8467207-8467231 base positions (www.ensembl.org, galgal4) of the chicken chromosome 2, the downstream primer consists of 26 bases and corresponds to 8467272-8467299 base positions (www.ensembl.org, galgal4) of the chicken chromosome 2, and the length of an amplified fragment of the primer pair is 93 bp.
The nucleotide sequence of the TaqMan fluorescent probe matched with the specific primer is as follows:
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 type probe consists of 16 bases, and corresponds to 8467241-8467255 and 8467240-8467255 base sequences of No.2 chromosome. Wherein, the probe of the wild allele aims at the wild base G, and the 5' end of the probe is connected with a fluorescent group VIC. The probe of the mutant allele aims at the mutant base T, and the 5' end of the probe is connected with a fluorescent group FAM; the 3' end labels of both probes are labeled with quenching groups, which are Non-luminescent quenching groups (Non-Fluorescent Quencher, NFQ).
The TaqMan probe real-time fluorescent quantitative PCR assay was performed using a Bio-Rad iQ5 model real-time fluorescent quantitative PCR instrument.
The TaqMan fluorescent quantitative PCR detection method was established using the genomic DNA of 155 individuals whose genotypes were known in example 1 as a template. The reaction system is as follows:
wherein the genomic DNA was the genomic DNA obtained in example 1, 40 XSNP Genotyping AssayMix (36. mu.M each of upstream and downstream primer concentrations and 8. mu.M each of probe concentrations) was synthesized by ABI (USA) and purchased from Yinfei Jie Co., Ltd.),
genotyping Master Mix was synthesized by ABI (USA) from Yiwei Jie traded, Inc., and ddH2O was sterilized deionized water.
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 1min, and detecting the signal intensity of the two fluorescent dyes in the whole amplification in real time for 40 cycles.
In the detection process, 2 blank controls without template DNA are arranged in each reaction system, so that software can correct the background color conveniently.
After the reaction is finished, software of the instrument can be used, the Allelic Disc function interface in the data analysis module can be used for directly judging the type, and the type judging result is shown in figure 2:
from FIG. 2, it can be seen that individuals of the same genotype cluster together, where squares represent TT homozygotes; triangles represent GT heterozygotes; the circles represent the GG homozygotes.
The reverse sequencing results of individuals with different genotypes are shown in figure 3, a) only one peak at the mutation site indicated by the arrow corresponds to A base, and because the reverse sequencing is carried out, the genotype of the corresponding individual is TT mutant, b) 2 peaks at the mutation site indicated by the arrow correspond to A, C two bases, and because the reverse sequencing is carried out, the genotype of the corresponding individual is GT type and is heterozygote; c) there is a peak at the mutation site indicated by the arrow, the corresponding base is C, and the genotype of the corresponding individual is GG and the wild type because of reverse sequencing.
The results show that the TaqMan probe detection result for 155 individuals is completely consistent with the sequencing result of the known PCR product by comparison, which indicates that the TaqMan probe real-time fluorescence quantitative PCR method is accurate and reliable.
When the method provided by the invention is used for detecting 155 individuals, a 96-well plate is used, and the result is judged only by using two PCR plates. It took only 4-5 hours. And 4 of the samples which appear in example 1 can not be amplified, which is mainly because the TaqMan SNP typing method has low requirements on genome, can meet the requirements from 10 ng to 100ng, does not need to re-extract genome even if the genome concentration is very low or the extraction quality is slightly poor, and does not influence the judgment of detection typing results.
However, if the method of direct sequencing of PCR products or the method of PCR-RFLP in example 1 is adopted, the genome is highly required, otherwise, PCR amplification is unstable, or the amount of products is too small, which affects subsequent enzyme digestion and sequencing. In addition, in addition to the PCR amplification step, the method of directly sequencing the PCR product or the method of PCR-RFLP requires an additional sequencing or enzymatic cleavage step, which takes at least 10 hours more. Therefore, the TaqMan SNP typing method has very obvious advantages.
Example 3 genetic Studies of chickens with different toe phenotypes and genotypes
And selecting the cocks and the hens with different toe number phenotypes and different genotype combinations respectively for mating, and researching the genetic rules of different genotypes. The specific experimental operations were as follows:
(1) double four-toe phenotype: 7 GG-type cocks are mated with 20 GG-type hens; 3 TT roosters, and 7 TT hens, 7 GT hens; (2) double five-toe phenotype: 8 TT-roosters, with 20 TT-roosters and 6 GT-roosters; two GT cocks and 2 GT hens.
The offspring of the above mating types were observed and registered for toe type. The results are shown in Table 2:
TABLE 2 toe type results of offspring from mating of chickens of different toe number phenotype and genotype
As can be seen from Table 2, 100% of the CC-type and CC-type offspring were double four-toe, and the test results fully indicate that if the cock and hen of GG genotype were mated, the offspring were completely double four-toe individuals.
Most of the offspring of AA and AA or AC types are expressed as double five toes, and the specific analysis is as follows:
the TT type with the toe number phenotype of double four toes is mated with the TT type or GT type chicken, the offspring still has a high proportion of five-toe chicken, the proportion of double five toes is about 63 percent, the proportion of single five toes is about 19 percent, and of course, the proportion of double four-toe individuals in the population is still about 18 percent.
However, even if double five-toe TT-type and TT-or GT-type chickens mate, the offspring will still develop a low proportion of the double four-toe phenotype, about 4%.
That is, there may be some suppressors that inhibit its expression, but do not affect the inheritance of the five toes. The mutation site rs80659072 can be applied to breeding as a candidate marker for the five-toe character of Beijing fatty chickens, and although the offspring does not necessarily show all five toes, the proportion of the five-toe phenotype of the offspring of the group can be greatly improved.
Claims (6)
1. A method for detecting the five-toe character of Beijing fatty chicken is characterized by comprising the step of detecting the genotype of rs80659072 mutation site in ZRS region in the 5 th intron of LMBR1 gene by a real-time fluorescent quantitative PCR technology;
designing a pair of fluorescent quantitative PCR primers and a TaqMan probe matched with the primers according to a sequence of a ZRS region in a 5 th intron of an LMBR1 gene, and detecting the genotype of an rs80659072 mutation site by using the primers and the probe and adopting a real-time fluorescent quantitative PCR technology;
wherein, the nucleotide sequence of the region of ZRS in the 5 th intron of the LMBR1 gene is shown as SEQ ID NO: 5 is shown in the specification;
the primer of the fluorescent quantitative PCR is as follows: the nucleotide sequences of the fluorescent quantitative PCR primer pair for amplifying the sequence of the ZRS region are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; and
the nucleotide sequence of the TaqMan fluorescent probe matched with the PCR primer is as follows: the nucleotide sequence of the wild type allele probe is shown as SEQ ID NO: 3, the nucleotide sequence of the mutant allele probe is shown as SEQ ID NO: 4 is shown in the specification;
the reaction conditions of the fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 1min, for 40 cycles.
2. The method of claim 1, wherein the reaction system of the fluorescence quantitative PCR is: genomic DNA 1. mu.L, 40-fold concentrated probe and primer mix 0.25. mu.L, universal PCR mix 5. mu.L, made up to 10. mu.L with water.
3. The primer and the probe for identifying the five-toe character of the Beijing fatty chicken are primer pairs for amplifying ZRS regions, and the nucleotide sequences of the primer pairs are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the probe comprises a nucleotide sequence of a wild type allele probe shown as SEQ ID NO: 3 is shown in the specification; and the nucleotide sequence of the mutant allele probe is shown as SEQ ID NO: 4, respectively.
4. A kit comprising the primer of claim 3 and a probe.
5. Use of the method of claim 1 or 2 for breeding Beijing fatty chicken with five-toe traits, wherein non-GG-type fatty chicken cock and hen are mated and offspring with five-toe traits are selected.
6. The application of the kit of claim 4 in detecting the five-toe trait of Beijing fatty chicken.
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| CN102994643A (en) * | 2012-12-27 | 2013-03-27 | 北京市农林科学院 | Primer and probe for detecting FMO3 gene A1034T mutation of chicken |
| CN103374629A (en) * | 2012-04-28 | 2013-10-30 | 中国农业科学院北京畜牧兽医研究所 | Method for authenticating Beijing fatty chickens by microsatellite markers |
| CN104694538A (en) * | 2015-01-28 | 2015-06-10 | 中国农业大学 | SNP molecular marker related to chicken polydactyly character and application thereof |
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| CN103374629A (en) * | 2012-04-28 | 2013-10-30 | 中国农业科学院北京畜牧兽医研究所 | Method for authenticating Beijing fatty chickens by microsatellite markers |
| CN102994643A (en) * | 2012-12-27 | 2013-03-27 | 北京市农林科学院 | Primer and probe for detecting FMO3 gene A1034T mutation of chicken |
| CN104694538A (en) * | 2015-01-28 | 2015-06-10 | 中国农业大学 | SNP molecular marker related to chicken polydactyly character and application thereof |
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| Genomic Regions Associated with Dermal Hyperpigmentation, Polydactyly and Other Morphological Traits in the Silkie Chicken;BEN DORSHORST et al;《Journal of Heredity》;20100111;摘要，材料与方法，表1，第344页多趾部分，表2，图4 * |
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