WO2022258078A1 - Primer set for identifying sex-linked dwarf gene and application thereof - Google Patents

Primer set for identifying sex-linked dwarf gene and application thereof Download PDF

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WO2022258078A1
WO2022258078A1 PCT/CN2022/105580 CN2022105580W WO2022258078A1 WO 2022258078 A1 WO2022258078 A1 WO 2022258078A1 CN 2022105580 W CN2022105580 W CN 2022105580W WO 2022258078 A1 WO2022258078 A1 WO 2022258078A1
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primer
chicken
primer pair
gene
sex
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PCT/CN2022/105580
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French (fr)
Chinese (zh)
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孙从佼
徐桂云
王喜琼
庄景杰
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中国农业大学
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Publication of WO2022258078A1 publication Critical patent/WO2022258078A1/en
Priority to ZA2022/13940A priority Critical patent/ZA202213940B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to the field of animal breeding, in particular to a primer set for identifying linked dwarf genes and its application.
  • the chicken sex-linked dwarf gene (dw gene) is an allele formed by a nearly 1.7 kb deletion mutation in the 3'-untranslated region (3'-UTR) of the growth hormone receptor (GHR) gene on the chicken sex chromosome.
  • GHR growth hormone receptor
  • Russian scholar Behtank discovered the dw gene.
  • the dw gene is located between the in site of the liver necrosis gene and the we site of the wingless gene on the chicken Z chromosome, close to the silver (S) and gold (s) feather genes and the slow feather (K) and fast feather (k) genes, dw
  • the exchange values of the gene with the silver gene and the slow feather gene were 7% and 6.6%, respectively.
  • the dw gene is one of the eight known dwarf genes, which is different from most of the dwarf genes that have obvious pathogenic or lethal effects. Many studies have shown that this gene is the only recessive sex-linked mutation gene that is harmless to the health of chickens and beneficial to humans. Compared with normal-sized chickens, the recessive homozygous dwarf chickens bred from it are only 2/3 of the weight of normal-sized chickens, and have low basal metabolic rate, low feed consumption, high stocking density, adaptability and Strong anti-stress and many other advantages.
  • the dw gene belongs to sex-linked inheritance, and the normal gene DW is dominant to dw, so only dwarf homozygous roosters and hens carrying the dwarf gene show dwarf traits.
  • the obvious external feature was about 20% shortening of the tibia, and the Z DW Z dw heterozygous cocks were phenotypically normal and could not be distinguished from the Z DW Z DW homozygous non-dwarf cocks. Therefore, the accurate identification of the genotype of sex-linked dwarf gene carrier chickens at the molecular level can speed up the breeding efficiency of dwarf chickens.
  • the present invention provides a primer set for identifying linked dwarf genes and its application.
  • the primer set provided by the invention can rapidly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken.
  • the present invention provides the following technical solutions:
  • the present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2;
  • the nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: 3;
  • the nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2; the nucleotide sequence of the reverse primer of the primer pair 2 is shown in SEQ ID NO: 3.
  • the present invention also provides the application of the primer set described in the above technical scheme in identifying linked dwarf gene-carrying chickens.
  • the present invention also provides a reagent or kit for identifying chickens carrying the linked dwarf gene, the reagent or kit includes the primer set described in the above technical scheme.
  • the present invention also provides a method for identifying sex-linked dwarf gene-carrying chickens, comprising the following steps:
  • Judgment of the amplified product includes: when the amplified product of 600bp-700bp is contained, the sample chicken is a sex-linked dwarf gene carrier chicken.
  • the PCR reaction program includes: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 5 min.
  • the reaction system of the PCR amplification is calculated in 20 ⁇ L, comprising: PCR SuperMix 10 ⁇ L, 0.4 ⁇ L each of the forward primer of primer pair 1 and the forward primer of primer pair 2 in the primer set, 0.2 ⁇ L each of the reverse primer of primer pair 1 and the reverse primer of primer pair 2 in the primer set ⁇ L, 2.5 ⁇ L of DNA template and remaining deionized water.
  • the The concentration of PCR SuperMix is 10 ⁇ moL/L;
  • the working concentration of the forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 2 and the reverse primer of primer pair 2 in the primer set is 10 ⁇ moL/L;
  • the concentration of the DNA template is 100 ng/ ⁇ L.
  • the judging method includes electrophoresis or sequencing.
  • the judgment of the amplified product also includes: when the amplified product of 600bp-700bp and the amplified product of greater than 700bp and less than or equal to 800bp are contained, the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene;
  • the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene
  • the sample chicken is a homozygous chicken that does not carry the sex-linked dwarf gene.
  • the present invention also provides the application of the above-mentioned primer set or the above-mentioned reagent or kit or the above-mentioned method in assisting in breeding new breeds of dwarf chickens.
  • the present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2; the nucleotide sequence of the forward primer of primer pair 1 is shown in SEQ ID NO: 1; the primer pair The nucleotide sequence of the reverse primer of 1 is shown in SEQ ID NO: 3; the nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2; the reverse primer pair 2 The nucleotide sequence of the primer is shown in SEQ ID NO:3.
  • the upstream primers for detecting the dw gene are designed inside the missing section of the GHR gene
  • the upstream primers for detecting the DW gene are designed upstream of the missing section of the GHR
  • the downstream primers are uniformly designed downstream of the missing section of the GHR.
  • the inside of the segment and both ends of the deleted segment are simultaneously amplified, so that Z DW Z DW and Z DW W can only detect the amplified product of the non-deleted segment, and Z dw W and Z dw Z dw can only detect the amplified product of the deleted segment.
  • Fragments, while heterozygous Z DW Z dw individuals can detect both fragments at the same time, and the genotype of chickens carrying the sex-linked dwarf gene can be quickly and accurately identified through a specific primer set.
  • Fig. 1 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder), 1,2,3,4,5,6,7,8,9 among the figure
  • Lane 10 is a Yangtai black chicken with a 713bp band and its genotype is Z DW Z DW
  • lane 11 is a Bailaihang rooster with a 714bp band and its genotype is Z DW Z DW
  • lane 12 is a Dwarf purebred rooster, with a 614bp band, genotype Z dw Z dw ;
  • Fig. 2 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9
  • Lane 10 is a Yangtai black chicken with two bands of 713bp and 614bp, and its genotype is Z DW Z dw
  • lane 11 is a rooster of Bailaihang, with a band of 714bp, its genotype is Z DW Z DW
  • Lane 12 is a dwarf purebred rooster with a 614bp band, and its genotype is Z dw Z dw
  • Lane 13 is a negative control deionized water
  • M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder)
  • Lane 10 is a Yangtai black chicken with two bands of 713bp and 614bp, and its genotype is Z DW Z dw
  • Fig. 3 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9
  • Lane 10 is Yangtai black chicken with a 713bp band and its genotype is Z DW W;
  • lane 11 is Bailai aircraft carrier chicken with a band of 714bp and its genotype is Z DW W;
  • lane 12 is dwarf Chicken pure line hen, there is a 614bp band, the genotype is Z dw W;
  • lane 13 is the negative control deionized water;
  • Fig. 4 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9
  • Lane 10 is Yangtai black chicken with a 614bp band and its genotype is Z dw W
  • lane 11 is a Bailai aircraft carrier chicken with a band of 714bp and its genotype is Z DW W
  • lane 12 is dwarf Chicken pure line hen, there is a 614bp band, the genotype is Z dw W
  • lane 13 is the negative control deionized water
  • M swimming lane is DNA molecular weight marker (100 ⁇ 1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9
  • Lane 10 is Yangtai black chicken with a 614bp band and its genotype is Z dw W
  • lane 11 is a Bailai aircraft carrier chicken with a band of 7
  • Figure 5 is the gel electrophoresis of chickens of different genotypes in Comparative Example 1, wherein the M swimming lane is the DNA molecular weight marker (100 ⁇ 1500bp Ladder), and the 1, 2, and 3 swimming lanes in the figure are the genotype Z DW Z dw Yang Tai Xiao Black chicken rooster; Lanes 4, 5, and 6 are Yangtai black chickens whose genotype is Z DW Z DW ; Lanes 7, 8, and 9 are Yangtai black chickens whose genotype is Z DW W; 10, 11, and 12 The lanes are hens whose genotype is Z dw W Yangtai black chicken.
  • the M swimming lane is the DNA molecular weight marker (100 ⁇ 1500bp Ladder)
  • the 1, 2, and 3 swimming lanes in the figure are the genotype Z DW Z dw Yang Tai Xiao Black chicken rooster
  • Lanes 4, 5, and 6 are Yangtai black chickens whose genotype is Z DW
  • the present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2;
  • the nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1: 5'-atgcctatttctgtgagg-3'; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: 3: 3'-atgtcctatctccttctactg-5';
  • the nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2: 5'-gggatgttcattgccttt-3'; the nucleotide sequence of the reverse primer of the primer pair 2 is as SEQ ID NO: 3: 3'-atgtcctatctccttctactg-5'.
  • the primer pair 1 of the present invention is designed according to the upstream and downstream of the GHR gene deletion in sex-linked dwarf chickens; wherein, the forward primer of the primer pair 1 is designed at 242 bp upstream of the GHR gene deletion, and the reverse primer is designed according to the GHR gene Design at the 372bp downstream of the missing segment; primer pair 1 is used to detect chicken non-dwarf allele DW gene; said primer pair 2 is designed according to the internal and downstream of the missing segment of the sex-linked dwarf chicken GHR gene; wherein, the primer The forward primer pair 2 was designed inside the GHR gene deletion segment, and the reverse primer was designed according to the downstream 372 bp of the GHR gene deletion segment; primer pair 2 was used to detect the chicken sex-linked dwarf mutant allele dw gene.
  • the specific primer set of the invention can quickly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken.
  • the method for synthesizing the primer pair is not particularly limited in the present invention, and it is preferably synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
  • the present invention also provides the application of the above-mentioned primer set in identifying linked dwarf gene-carrying chickens.
  • the specific primer set of the invention can quickly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken.
  • the present invention also provides a reagent or kit for identifying chickens carrying the linked dwarf gene, the reagent or kit includes the above primer set.
  • the present invention also provides a method for identifying sex-linked dwarf gene-carrying chickens, comprising the following steps:
  • Judgment of the amplified product includes: when the amplified product of 600bp-700bp is contained, the sample chicken is a sex-linked dwarf gene carrier chicken.
  • the DNA template is preferably the genome DNA of the chicken sample.
  • the present invention has no special requirements for the extraction method of the DNA template, and preferably adopts the blood/cell/tissue genomic DNA extraction kit (DP304) provided by Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA of the sample chicken.
  • DP304 blood/cell/tissue genomic DNA extraction kit
  • the PCR reaction program preferably includes: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, and 35 cycles; extension at 72°C for 5min;
  • the reaction system is calculated in 20 ⁇ L, preferably including: PCR SuperMix 10 ⁇ L, 0.4 ⁇ L each of the forward primer of primer pair 1 and the forward primer of primer pair 2 in the primer set, and 0.2 ⁇ L of each of the reverse primer of primer pair 1 and primer pair 2 in the primer set , 2.5 ⁇ L of DNA template and the remaining deionized water;
  • the concentration of PCR SuperMix is preferably 10 ⁇ moL/L; In the primer group, the working concentration of the forward primer to 1, the reverse primer to 1, the forward primer to 2 and the reverse primer to 2 are all It is preferably 10 ⁇ moL/L; the concentration of the DNA template is preferably 100 ng/ ⁇ L.
  • the reverse primer of primer pair 1 and primer pair 2 of the present invention is the same, and is a common reverse primer.
  • the forward primer of primer pair 1, the forward primer of primer pair 2 and the volume of the common reverse primer are preferably same.
  • the present invention has no special requirements on the source of each reagent of the reaction system of the PCR amplification, and the commercially available products well known to those skilled in the art can be used; PCR SuperMix is preferably purchased from Beijing Quanshijin Biotechnology Co., Ltd. PCR SuperMix(+dye), Cat. No. AS111-11.
  • the determination method preferably includes electrophoresis or sequencing, more preferably electrophoresis.
  • the judgment of the amplification product includes: when the amplification product contains 600bp ⁇ 700bp, the sample chicken is a sex-linked dwarf gene carrier chicken; the judgment of the amplification product preferably also includes: when the amplification product contains 600bp ⁇ 700bp When the amplification product and the amplification product greater than 700bp and less than or equal to 800bp, then the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene; the genotype of the heterozygous chicken carrying a sex-linked dwarf gene is preferably Z DW Z dw ;
  • the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene;
  • the genotype of the homozygous chicken carrying a sex-linked dwarf gene is preferably Z dw W or Z dw Z dw ; further typing is preferably distinguished according to the sex of the sample chicken;
  • the sample chicken is a homozygous chicken that does not carry a sex-linked dwarf gene; the genotype of the homozygous chicken that does not carry a sex-linked dwarf gene is preferably Z DW W or Z DW Z DW ; further typing is preferably distinguished according to the sex of the sample chicken.
  • the present invention also provides the application of the above-mentioned primer set or the above-mentioned reagent or kit or the above-mentioned method in assisting in breeding new breeds of dwarf chickens.
  • the invention can rapidly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken through the above primer set, and can assist in breeding new breeds of dwarf chickens.
  • a primer set for identifying linked dwarf genes consisting of a primer pair 1 for detecting chicken non-dwarf allele DW gene and a primer pair 2 for detecting chicken sex-linked dwarf allele dw gene; the primer set is produced by Sangon Bioengineering ( Shanghai) Co., Ltd. synthesis;
  • the nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1: 5'-atgcctatttctgtgagg-3'; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: Shown in 3: 3'-atgtcctatctccttctactg-5'; the size of the amplification product of the primer pair 1 is 713bp;
  • the nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2: 5'-gggatgttcattgccttt-3'; the nucleotide sequence of the reverse primer of the primer pair 2 is as SEQ ID NO: Shown in 3: 3'-atgtcctatctccttctactg-5'; the size of the amplified product of the primer pair 1 is 614bp.
  • the genotypes of sex-linked dwarf gene carrier chickens were identified using the primer set of Example 1.
  • Each chicken to be identified adopts the following PCR reaction system and the following PCR reaction procedure, uses the genomic DNA as a template, and utilizes the primer set in Example 1 to carry out PCR amplification.
  • the reaction system is a 20 ⁇ L system: PCR SuperMix (+dye) (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AS111-11) 10 ⁇ L (concentration: 10 ⁇ moL/L), the forward primer of primer pair 1 and the forward primer of primer pair 2 each 0.4 ⁇ L (both at a concentration of 10 ⁇ moL/L), 0.2 ⁇ L of each of the reverse primers of primer pair 1 and primer pair 2 (both at a concentration of 10 ⁇ moL/L), 2.5 ⁇ L of a DNA template (at a concentration of 100 ng/ ⁇ L), and 6.3 ⁇ L of ionized water.
  • the PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, renaturation at 58°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 5 min;
  • the PCR amplification products were stored at 4°C for future use.
  • Agarose gel electrophoresis detection Take 5 ⁇ L of PCR amplification product, perform 1.2% agarose gel electrophoresis (120V, 40min), use the above-mentioned amplification product to apply samples, and detect with a gel imager. The detection results are shown in Table 1 and Figure 1 ⁇ 4 ( Figures 1 to 4 are the electrophoresis diagrams of 10 Yangtai black chickens of different genotypes randomly selected, with Bailaihang and dwarf pure-line chickens as positive controls and water as negative control respectively).
  • the PCR amplification products of Yangtai black chicken with genotype Z dw W all showed a band of 600bp-700bp (Fig. 4).
  • Figure 4 except the first lane on the left is Marker, the other lanes are chickens to be identified.
  • the PCR products of each chicken to be identified were sequenced, and the results showed that the sequences of the PCR amplification products of the Yangtai black chicken hen whose genotype was Z DW W were all shown in SEQ ID NO: 5, and the sizes were both 614bp.
  • Figure 2 except the first lane on the left is Marker, the other lanes are chickens to be identified.
  • the identification primer set is as follows: the nucleotide sequence of the forward primer of primer pair 3 is shown in SEQ ID NO: 6: 5'-ggcagaaaccccaagtatg-3', reverse The nucleotide sequence of the forward primer is shown in SEQ ID NO: 7: 5'-tcgggacagatcaaagaca-3'; the nucleotide sequence of the primer pair 4 forward primer is shown in SEQ ID NO: 8: 5'-tgcagcaaaaattaaaaacag-3' , the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 9: it is 5'-cgtattcaattcctgtgtttc-3'.
  • the PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 5 min.
  • the reaction system for PCR amplification is a 20 ⁇ L system: PCR SuperMix (+dye) (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AS111-11) 10 ⁇ L (concentration: 10 ⁇ moL/L), the forward primer of primer pair 3 and the forward primer of primer pair 4 each 0.4 ⁇ L (both at a concentration of 10 ⁇ moL/L), 0.4 ⁇ L of each of the reverse primers of primer pair 3 and primer pair 4 (both at a concentration of 10 ⁇ moL/L), 2.5 ⁇ L of a DNA template (at a concentration of 100 ng/ ⁇ L), and 5.9 ⁇ L of ionized water.
  • PCR SuperMix (+dye) purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AS111-11
  • 10 ⁇ L concentration: 10 ⁇ moL/L
  • the forward primer of primer pair 3 and the forward primer of primer pair 4 each 0.4 ⁇ L (both at a
  • the judgment of the amplification product of the primer set is as follows: when the amplification product of 200bp ⁇ 300bp and the amplification product of greater than 500bp and less than 600bp are contained, the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene; The genotype of the heterozygous chicken with sex-linked dwarf gene is Z DW Z dw ;
  • the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene; the genotype of the homozygous chicken carrying a sex-linked dwarf gene is Z dw W or Z dw Z dw ; further classification is based on the sex of the sample chicken;
  • the sample chicken is a homozygous chicken that does not carry a sex-linked dwarf gene; the genotype of the homozygous chicken that does not carry a sex-linked dwarf gene is Z DW W Or Z DW Z DW ; further typing is distinguished according to the sex of the sample chicken.
  • the samples used are 3 blood DNA samples of Yangtai black chickens of the genotypes randomly selected in Application Example 1, and the measurement results are shown in Figure 5 (in which the M swimming lane is the DNA molecular weight marker (100-1500bp Ladder), 1 and 2 in the figure
  • Lanes 3 and 3 are Yangtai black chickens whose genotype is Z DW Z dw
  • lanes 4, 5 and 6 are Yangtai black chickens whose genotype is Z DW Z DW
  • lanes 7, 8 and 9 are Yangtai black chickens whose genotype is Z DW W Yangtai little black chicken hen
  • lanes 10, 11, and 12 are genotype Z dw W Yangtai little black chicken hen).
  • the present invention can quickly and accurately identify the genotype of sex-linked dwarf gene-carrying chickens through a specific primer set; in addition, the primers designed by the present invention have strong specificity, and the results are stable and reliable, and can be determined within 3 hours. Identify the genotype of the chicken, which can be used for early selection, according to the genotype to eliminate or eliminate the roosters or hens carrying the sex-linked dwarf gene in the normal population, reduce the cost of feeding, and improve the uniformity of the population.

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Abstract

Disclosed are a primer set for identifying a sex-linked dwarf gene and an application thereof. In the present invention, an upstream primer for detecting a dw gene is designed in a GHR gene deletion segment, an upstream primer for detecting a DW gene is designed upstream of the GHR deletion segment, and a downstream primer is uniformly designed downstream of the GHR deletion segment; amplification is simultaneously carried out on the interior of the deletion segment and the two ends of the deletion segment by means of multiple PCR reactions; in this way, ZDWZDW and ZDWW individuals can only detect an amplification product of a non-deleted segment, ZdwW and ZdwZdw individuals can only detect a deleted segment, and a heterozygote ZDWZdw individual can simultaneously detect such two segments. The genotype of a chicken individual at a sex-linked dwarf gene locus can be rapidly and accurately identified by means of the present primer set.

Description

一种鉴定性连锁矮小基因的引物组及其应用A kind of primer set and its application for identifying linked dwarf genes
本申请要求于2021年07月30日提交中国专利局、申请号为CN202110870820.6、发明名称为“一种鉴定性连锁矮小基因的引物组及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application submitted to the China Patent Office on July 30, 2021, the application number is CN202110870820.6, and the title of the invention is "a primer set for identifying linked dwarf genes and its application", all of which The contents are incorporated by reference in this application.
技术领域technical field
本发明涉及动物育种领域，特别是涉及一种鉴定性连锁矮小基因的引物组及其应用。The invention relates to the field of animal breeding, in particular to a primer set for identifying linked dwarf genes and its application.
背景技术Background technique
鸡性连锁矮小基因(dw基因)是鸡性染色体上的生长激素受体(GHR)基因3’-非翻译区(3’-UTR)存在近1.7kb的缺失突变形成的等位基因。1935年苏联学者Behtank就发现了dw基因。dw基因位于鸡Z染色体上肝脏坏死基因in位点与无翼基因we位点之间，靠近银色(S)及金色(s)羽基因和慢羽(K)、快羽(k)基因，dw基因与银色基因和慢羽基因的交换值分别为7％和6.6％。The chicken sex-linked dwarf gene (dw gene) is an allele formed by a nearly 1.7 kb deletion mutation in the 3'-untranslated region (3'-UTR) of the growth hormone receptor (GHR) gene on the chicken sex chromosome. In 1935, Soviet scholar Behtank discovered the dw gene. The dw gene is located between the in site of the liver necrosis gene and the we site of the wingless gene on the chicken Z chromosome, close to the silver (S) and gold (s) feather genes and the slow feather (K) and fast feather (k) genes, dw The exchange values of the gene with the silver gene and the slow feather gene were 7% and 6.6%, respectively.
dw基因是已知的8种矮小基因中的一种，有别于其他具有明显致病或致死效应的大多数矮小基因。诸多研究都表明，此基因是唯一对鸡本身健康无害，而对人类有利的隐形伴性突变基因。相比于正常体型鸡，由其培育而成的基因隐性纯合的矮小鸡仅有正常体型鸡体重的2/3，具有基础代谢率低、耗料量少、饲养密度大、适应性和抗应激强等诸多优势。The dw gene is one of the eight known dwarf genes, which is different from most of the dwarf genes that have obvious pathogenic or lethal effects. Many studies have shown that this gene is the only recessive sex-linked mutation gene that is harmless to the health of chickens and beneficial to humans. Compared with normal-sized chickens, the recessive homozygous dwarf chickens bred from it are only 2/3 of the weight of normal-sized chickens, and have low basal metabolic rate, low feed consumption, high stocking density, adaptability and Strong anti-stress and many other advantages.
而dw基因属于伴性遗传，正常基因DW对dw显性，因此只有矮小型纯合子的公鸡和携带矮小型基因的母鸡才表现矮小性状。明显的外部特征为胫骨缩短20％左右，而Z DWZ dw的杂合子公鸡表型正常，不能与Z DWZ DW的纯合非矮小公鸡区分。所以能从分子水平上准确鉴定性连锁矮小基因携带鸡的基因型可以加快矮小鸡育种效率。 The dw gene belongs to sex-linked inheritance, and the normal gene DW is dominant to dw, so only dwarf homozygous roosters and hens carrying the dwarf gene show dwarf traits. The obvious external feature was about 20% shortening of the tibia, and the Z DW Z dw heterozygous cocks were phenotypically normal and could not be distinguished from the Z DW Z DW homozygous non-dwarf cocks. Therefore, the accurate identification of the genotype of sex-linked dwarf gene carrier chickens at the molecular level can speed up the breeding efficiency of dwarf chickens.
发明内容Contents of the invention
为了解决上述问题，本发明提供了一种鉴定性连锁矮小基因的引物组及其应用。本发明提供的引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型。In order to solve the above problems, the present invention provides a primer set for identifying linked dwarf genes and its application. The primer set provided by the invention can rapidly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken.
为了实现上述目的，本发明提供如下技术方案：In order to achieve the above object, the present invention provides the following technical solutions:
本发明提供了一种鉴定性连锁矮小基因的引物组，包括引物对1和引物对2；The present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2;
所述引物对1的正向引物的核苷酸序列如SEQ ID NO：1所示；所述引物对1的反向引物的核苷酸序列如SEQ ID NO：3所示；The nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: 3;
所述引物对2的正向引物的核苷酸序列如SEQ ID NO：2所示；所述引物对2的反向引物的核苷酸序列如SEQ ID NO：3所示。The nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2; the nucleotide sequence of the reverse primer of the primer pair 2 is shown in SEQ ID NO: 3.
本发明还提供了上述技术方案所述引物组在鉴定性连锁矮小基因携带鸡中的应用。The present invention also provides the application of the primer set described in the above technical scheme in identifying linked dwarf gene-carrying chickens.
本发明还提供了一种鉴定性连锁矮小基因携带鸡的试剂或试剂盒，所述试剂或试剂盒包括上述技术方案所述引物组。The present invention also provides a reagent or kit for identifying chickens carrying the linked dwarf gene, the reagent or kit includes the primer set described in the above technical scheme.
本发明还提供了一种鉴定性连锁矮小基因携带鸡的方法，包括以下步骤：The present invention also provides a method for identifying sex-linked dwarf gene-carrying chickens, comprising the following steps:
以样本鸡的基因组DNA或总RNA反转录得到的cDNA为模板，利用上述引物组或者上述技术方案所述的试剂或试剂盒进行PCR扩增，得到扩增产物；Using the cDNA obtained by reverse transcription of the genomic DNA or total RNA of the sample chicken as a template, using the above primer set or the reagent or kit described in the above technical scheme to perform PCR amplification to obtain an amplification product;
扩增产物的判断包括：当含有600bp～700bp的扩增产物时，则样本鸡为性连锁矮小基因携带鸡。Judgment of the amplified product includes: when the amplified product of 600bp-700bp is contained, the sample chicken is a sex-linked dwarf gene carrier chicken.
优选的，所述PCR反应程序包括：94℃预变性5min；94℃变性30s，58℃复性30s，72℃延伸40s，循环35次；72℃延伸5min。Preferably, the PCR reaction program includes: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 5 min.
优选的，所述PCR扩增的反应体系以20μL计，包括：
Figure PCTCN2022105580-appb-000001
PCR SuperMix 10μL，所述引物组中引物对1的正向引物和引物对2的正向引物各0.4μL，所述引物组中引物对1的反向引物和引物对2的反向引物各0.2μL，DNA模板2.5μL和剩余去离子水。 Preferably, the reaction system of the PCR amplification is calculated in 20 μL, comprising:
Figure PCTCN2022105580-appb-000001
PCR SuperMix 10 μL, 0.4 μL each of the forward primer of primer pair 1 and the forward primer of primer pair 2 in the primer set, 0.2 μL each of the reverse primer of primer pair 1 and the reverse primer of primer pair 2 in the primer set μL, 2.5 μL of DNA template and remaining deionized water.
优选的，所述
Figure PCTCN2022105580-appb-000002
PCR SuperMix的浓度为10μmoL/L；所述引物组中引物对1的正向引物、引物对1的反向引物、引物对2的正向引物和引物对2的反向引物的工作浓度均为10μmoL/L；所述DNA模板的浓度为100ng/μL。 Preferably, the
Figure PCTCN2022105580-appb-000002
The concentration of PCR SuperMix is 10 μ moL/L; The working concentration of the forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 2 and the reverse primer of primer pair 2 in the primer set is 10 μmoL/L; the concentration of the DNA template is 100 ng/μL.
优选的，所述判断的方法包括电泳或测序。Preferably, the judging method includes electrophoresis or sequencing.
优选的，扩增产物的判断还包括：当含有600bp～700bp的扩增产物和大于700bp且小于等于800bp的扩增产物时，则样本鸡为携带有性连锁 矮小基因的杂合鸡；Preferably, the judgment of the amplified product also includes: when the amplified product of 600bp-700bp and the amplified product of greater than 700bp and less than or equal to 800bp are contained, the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene;
当仅含有600bp～700bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的纯合鸡；When only the amplification product of 600bp-700bp is contained, the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene;
当仅含有大于700bp且小于等于800bp的扩增产物时，则样本鸡为未携带有性连锁矮小基因的纯合鸡。When there are only amplified products greater than 700bp and less than or equal to 800bp, the sample chicken is a homozygous chicken that does not carry the sex-linked dwarf gene.
本发明还提供了上述引物组或上述试剂或试剂盒或上述方法在辅助选育矮小鸡群新品种中的应用。The present invention also provides the application of the above-mentioned primer set or the above-mentioned reagent or kit or the above-mentioned method in assisting in breeding new breeds of dwarf chickens.
本发明的有益效果：Beneficial effects of the present invention:
本发明提供一种鉴定性连锁矮小基因的引物组，包括引物对1和引物对2；所述引物对1的正向引物的核苷酸序列如SEQ ID NO：1所示；所述引物对1的反向引物的核苷酸序列如SEQ ID NO：3所示；所述引物对2的正向引物的核苷酸序列如SEQ ID NO：2所示；所述引物对2的反向引物的核苷酸序列如SEQ ID NO：3所示。本发明在GHR基因缺失段内部设计检测dw基因的上游引物，在GHR缺失段的上游设计检测DW基因的上游引物，并统一将下游引物设计在GHR缺失段的下游，通过多重PCR反应，利用缺失段内部和缺失区段两端同时进行扩增，这样Z DWZ DW和Z DWW只能检测到未缺失区段的扩增产物，Z dwW和Z dwZ dw只能检测到缺失后的片段，而杂合子Z DWZ dw个体则能同时检测到两种片段，通过特定的引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型。 The present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2; the nucleotide sequence of the forward primer of primer pair 1 is shown in SEQ ID NO: 1; the primer pair The nucleotide sequence of the reverse primer of 1 is shown in SEQ ID NO: 3; the nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2; the reverse primer pair 2 The nucleotide sequence of the primer is shown in SEQ ID NO:3. In the present invention, the upstream primers for detecting the dw gene are designed inside the missing section of the GHR gene, the upstream primers for detecting the DW gene are designed upstream of the missing section of the GHR, and the downstream primers are uniformly designed downstream of the missing section of the GHR. The inside of the segment and both ends of the deleted segment are simultaneously amplified, so that Z DW Z DW and Z DW W can only detect the amplified product of the non-deleted segment, and Z dw W and Z dw Z dw can only detect the amplified product of the deleted segment. Fragments, while heterozygous Z DW Z dw individuals can detect both fragments at the same time, and the genotype of chickens carrying the sex-linked dwarf gene can be quickly and accurately identified through a specific primer set.
附图说明Description of drawings
图1为应用例1中不同基因型鸡的凝胶电泳图，其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3、4、5、6、7、8、9、10泳道为扬泰小黑鸡，有一条713bp的条带，基因型为Z DWZ DW；11泳道为白来航公鸡，有一条714bp的条带，基因型为Z DWZ DW；12泳道为矮小鸡纯系公鸡，有一条614bp条带，基因型为Z dwZ dw； Fig. 1 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100～1500bp Ladder), 1,2,3,4,5,6,7,8,9 among the figure Lane 10 is a Yangtai black chicken with a 713bp band and its genotype is Z DW Z DW ; lane 11 is a Bailaihang rooster with a 714bp band and its genotype is Z DW Z DW ; lane 12 is a Dwarf purebred rooster, with a 614bp band, genotype Z dw Z dw ;
图2为应用例1中不同基因型鸡的凝胶电泳图，其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3、4、5、6、7、8、9、10泳道为扬泰小黑鸡，有两条713bp和614bp的条带，基因型为Z DWZ dw；11泳道为白来航公鸡，有一条714bp的条带，基因型为Z DWZ DW；12泳道为矮小鸡纯系公鸡，有一条614bp条带，基因型为Z dwZ dw；13泳道为阴性对照 去离子水； Fig. 2 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100～1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9 Lane 10 is a Yangtai black chicken with two bands of 713bp and 614bp, and its genotype is Z DW Z dw ; lane 11 is a rooster of Bailaihang, with a band of 714bp, its genotype is Z DW Z DW ; Lane 12 is a dwarf purebred rooster with a 614bp band, and its genotype is Z dw Z dw ; Lane 13 is a negative control deionized water;
图3为应用例1中不同基因型鸡的凝胶电泳图，其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3、4、5、6、7、8、9、10泳道为扬泰小黑鸡，有一条713bp的条带，基因型为Z DWW；11泳道为白来航母鸡，有一条714bp的条带，基因型为Z DWW；12泳道为矮小鸡纯系母鸡，有一条614bp条带，基因型为Z dwW；13泳道为阴性对照去离子水； Fig. 3 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100～1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9 Lane 10 is Yangtai black chicken with a 713bp band and its genotype is Z DW W; lane 11 is Bailai aircraft carrier chicken with a band of 714bp and its genotype is Z DW W; lane 12 is dwarf Chicken pure line hen, there is a 614bp band, the genotype is Z dw W; lane 13 is the negative control deionized water;
图4为应用例1中不同基因型鸡的凝胶电泳图，其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3、4、5、6、7、8、9、10泳道为扬泰小黑鸡，有一条614bp的条带，基因型为Z dwW，11泳道为白来航母鸡，有一条714bp的条带，基因型为Z DWW；12泳道为矮小鸡纯系母鸡，有一条614bp条带，基因型为Z dwW；13泳道为阴性对照去离子水； Fig. 4 is the gel electrophoresis figure of different genotype chickens in application example 1, and wherein M swimming lane is DNA molecular weight marker (100～1500bp Ladder), among the figure 1,2,3,4,5,6,7,8,9 Lane 10 is Yangtai black chicken with a 614bp band and its genotype is Z dw W; lane 11 is a Bailai aircraft carrier chicken with a band of 714bp and its genotype is Z DW W; lane 12 is dwarf Chicken pure line hen, there is a 614bp band, the genotype is Z dw W; lane 13 is the negative control deionized water;
图5为对比例1中不同基因型鸡的凝胶电泳图，其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3泳道为基因型为Z DWZ dw扬泰小黑鸡公鸡；4、5、6泳道为基因型为Z DWZ DW扬泰小黑鸡；7、8、9泳道为基因型为Z DWW扬泰小黑鸡母鸡；10、11、12泳道为基因型为Z dwW扬泰小黑鸡母鸡。 Figure 5 is the gel electrophoresis of chickens of different genotypes in Comparative Example 1, wherein the M swimming lane is the DNA molecular weight marker (100 ~ 1500bp Ladder), and the 1, 2, and 3 swimming lanes in the figure are the genotype Z DW Z dw Yang Tai Xiao Black chicken rooster; Lanes 4, 5, and 6 are Yangtai black chickens whose genotype is Z DW Z DW ; Lanes 7, 8, and 9 are Yangtai black chickens whose genotype is Z DW W; 10, 11, and 12 The lanes are hens whose genotype is Z dw W Yangtai black chicken.
具体实施方式Detailed ways
本发明提供了一种鉴定性连锁矮小基因的引物组，包括引物对1和引物对2；The present invention provides a primer set for identifying linked dwarf genes, including primer pair 1 and primer pair 2;
所述引物对1的正向引物的核苷酸序列如SEQ ID NO：1所示：5’-atgcctatttctgtgagg-3’；所述引物对1的反向引物的核苷酸序列如SEQ ID NO：3所示：3’-atgtcctatctccttctactg-5’；The nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1: 5'-atgcctatttctgtgagg-3'; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: 3: 3'-atgtcctatctccttctactg-5';
所述引物对2的正向引物的核苷酸序列如SEQ ID NO：2所示：5’-gggatgttcattgccttt-3’；所述引物对2的反向引物的核苷酸序列如SEQ ID NO：3所示：3’-atgtcctatctccttctactg-5’。The nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2: 5'-gggatgttcattgccttt-3'; the nucleotide sequence of the reverse primer of the primer pair 2 is as SEQ ID NO: 3: 3'-atgtcctatctccttctactg-5'.
本发明所述引物对1是根据伴性矮小型鸡GHR基因缺失段上游和下游设计；其中，引物对1的正向引物是在GHR基因的缺失段上游242bp处设计，反向引物根据GHR基因的缺失段的下游372bp处设计；引物对1用于检测鸡非矮小等位基因DW基因；所述引物对2是根据伴性矮小型 鸡GHR基因缺失段内部和缺失段下游设计；其中，引物对2的正向引物是在GHR基因缺失段内部设计，反向引物根据GHR基因的缺失段的下游372bp处设计；引物对2用于检测鸡性连锁矮小突变等位基因dw基因。本发明所述的特定引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型。本发明对所述引物对的合成方法并没有特殊限定，优选由生工生物工程(上海)股份有限公司合成。The primer pair 1 of the present invention is designed according to the upstream and downstream of the GHR gene deletion in sex-linked dwarf chickens; wherein, the forward primer of the primer pair 1 is designed at 242 bp upstream of the GHR gene deletion, and the reverse primer is designed according to the GHR gene Design at the 372bp downstream of the missing segment; primer pair 1 is used to detect chicken non-dwarf allele DW gene; said primer pair 2 is designed according to the internal and downstream of the missing segment of the sex-linked dwarf chicken GHR gene; wherein, the primer The forward primer pair 2 was designed inside the GHR gene deletion segment, and the reverse primer was designed according to the downstream 372 bp of the GHR gene deletion segment; primer pair 2 was used to detect the chicken sex-linked dwarf mutant allele dw gene. The specific primer set of the invention can quickly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken. The method for synthesizing the primer pair is not particularly limited in the present invention, and it is preferably synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
本发明还提供了上述引物组在鉴定性连锁矮小基因携带鸡中的应用。本发明所述的特定引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型。The present invention also provides the application of the above-mentioned primer set in identifying linked dwarf gene-carrying chickens. The specific primer set of the invention can quickly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken.
本发明还提供了一种鉴定性连锁矮小基因携带鸡的试剂或试剂盒，所述试剂或试剂盒包括上述的引物组。The present invention also provides a reagent or kit for identifying chickens carrying the linked dwarf gene, the reagent or kit includes the above primer set.
本发明还提供了一种鉴定性连锁矮小基因携带鸡的方法，包括以下步骤：The present invention also provides a method for identifying sex-linked dwarf gene-carrying chickens, comprising the following steps:
以样本鸡的基因组DNA或总RNA反转录得到的cDNA为模板，利用上述引物组进行PCR扩增，得到扩增产物；Using the cDNA obtained by reverse transcription of the genomic DNA or total RNA of the sample chicken as a template, using the above primer set to perform PCR amplification to obtain the amplification product;
扩增产物的判断包括：当含有600bp～700bp的扩增产物时，则样本鸡为性连锁矮小基因携带鸡。Judgment of the amplified product includes: when the amplified product of 600bp-700bp is contained, the sample chicken is a sex-linked dwarf gene carrier chicken.
在本发明中，所述DNA模板优选为样本鸡的基因组DNA。本发明对所述DNA模板的提取方法没有特殊要求，优选采用天根生化科技(北京)有限公司提供的血液/细胞/组织基因组DNA提取试剂盒(DP304)提取样本鸡的基因组DNA。In the present invention, the DNA template is preferably the genome DNA of the chicken sample. The present invention has no special requirements for the extraction method of the DNA template, and preferably adopts the blood/cell/tissue genomic DNA extraction kit (DP304) provided by Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA of the sample chicken.
在本发明中，所述PCR反应程序优选包括：94℃预变性5min；94℃变性30s，58℃复性30s，72℃延伸40s，循环35次；72℃延伸5min；所述PCR扩增的反应体系以20μL计，优选包括：
Figure PCTCN2022105580-appb-000003
PCR SuperMix10μL，所述引物组中引物对1的正向引物和引物对2的正向引物各0.4μL，所述引物组中引物对1的反向引物和引物对2的反向引物各0.2μL，DNA模板2.5μL和剩余去离子水；所述
Figure PCTCN2022105580-appb-000004
PCR SuperMix的浓度优选为10μmoL/L；所述引物组中引物对1的正向引物、引物对1的反向引物、引物对2的正向引物和引物对2的反向引物的工作浓度均优选为10μmoL/L；所述DNA模板的浓度优选为100ng/μL。本发明所述引物对1 和引物对2的反向引物相同，是公用反向引物，在反应体系中引物对1的正向引物、引物对2的正向引物和公用反向引物的体积优选相同。本发明对所述PCR扩增的反应体系的各试剂来源没有特殊要求，采用本领域技术人员所熟知的市售商品即可；本发明所述
Figure PCTCN2022105580-appb-000005
PCR SuperMix优选购自北京全式金生物技术有限公司的
Figure PCTCN2022105580-appb-000006
PCR SuperMix(+dye)，货号为AS111-11。 In the present invention, the PCR reaction program preferably includes: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, and 35 cycles; extension at 72°C for 5min; The reaction system is calculated in 20 μL, preferably including:
Figure PCTCN2022105580-appb-000003
PCR SuperMix 10 μL, 0.4 μL each of the forward primer of primer pair 1 and the forward primer of primer pair 2 in the primer set, and 0.2 μL of each of the reverse primer of primer pair 1 and primer pair 2 in the primer set , 2.5 μL of DNA template and the remaining deionized water;
Figure PCTCN2022105580-appb-000004
The concentration of PCR SuperMix is preferably 10 μ moL/L; In the primer group, the working concentration of the forward primer to 1, the reverse primer to 1, the forward primer to 2 and the reverse primer to 2 are all It is preferably 10 μmoL/L; the concentration of the DNA template is preferably 100 ng/μL. The reverse primer of primer pair 1 and primer pair 2 of the present invention is the same, and is a common reverse primer. In the reaction system, the forward primer of primer pair 1, the forward primer of primer pair 2 and the volume of the common reverse primer are preferably same. The present invention has no special requirements on the source of each reagent of the reaction system of the PCR amplification, and the commercially available products well known to those skilled in the art can be used;
Figure PCTCN2022105580-appb-000005
PCR SuperMix is preferably purchased from Beijing Quanshijin Biotechnology Co., Ltd.
Figure PCTCN2022105580-appb-000006
PCR SuperMix(+dye), Cat. No. AS111-11.
在本发明中，所述判断的方法优选包括电泳或测序，更优选为电泳。In the present invention, the determination method preferably includes electrophoresis or sequencing, more preferably electrophoresis.
在本发明中，扩增产物的判断包括：当含有600bp～700bp的扩增产物时，则样本鸡为性连锁矮小基因携带鸡；扩增产物的判断优选还包括：当含有600bp～700bp的扩增产物和大于700bp且小于等于800bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的杂合鸡；所述携带有性连锁矮小基因的杂合鸡的基因型优选为Z DWZ dw； In the present invention, the judgment of the amplification product includes: when the amplification product contains 600bp～700bp, the sample chicken is a sex-linked dwarf gene carrier chicken; the judgment of the amplification product preferably also includes: when the amplification product contains 600bp～700bp When the amplification product and the amplification product greater than 700bp and less than or equal to 800bp, then the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene; the genotype of the heterozygous chicken carrying a sex-linked dwarf gene is preferably Z DW Z dw ;
当仅含有600bp～700bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的纯合鸡；所述携带有性连锁矮小基因的纯合鸡的基因型优选为Z dwW或Z dwZ dw；进一步分型优选根据样本鸡的性别区分； When only containing the amplification product of 600bp～700bp, then the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene; the genotype of the homozygous chicken carrying a sex-linked dwarf gene is preferably Z dw W or Z dw Z dw ; further typing is preferably distinguished according to the sex of the sample chicken;
当仅含有大于700bp且小于等于800bp的扩增产物时，则样本鸡为未携带有性连锁矮小基因的纯合鸡；所述未携带有性连锁矮小基因的纯合鸡的基因型优选为Z DWW或Z DWZ DW；进一步分型优选根据样本鸡的性别区分。 When only containing amplification products greater than 700bp and less than or equal to 800bp, the sample chicken is a homozygous chicken that does not carry a sex-linked dwarf gene; the genotype of the homozygous chicken that does not carry a sex-linked dwarf gene is preferably Z DW W or Z DW Z DW ; further typing is preferably distinguished according to the sex of the sample chicken.
本发明还提供了上述引物组或上述试剂或试剂盒或上述方法在辅助选育矮小鸡群新品种中的应用。本发明通过上述引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型，可以辅助选育矮小鸡群新品种。The present invention also provides the application of the above-mentioned primer set or the above-mentioned reagent or kit or the above-mentioned method in assisting in breeding new breeds of dwarf chickens. The invention can rapidly and accurately identify the genotype of the sex-linked dwarf gene-carrying chicken through the above primer set, and can assist in breeding new breeds of dwarf chickens.
为了进一步说明本发明，下面结合实施例和附图对本发明提供的一种鉴定性连锁矮小基因的引物组及其应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。In order to further illustrate the present invention, a primer set and its application for identifying linked dwarf genes provided by the present invention will be described in detail below in conjunction with the examples and accompanying drawings, but they should not be construed as limiting the protection scope of the present invention.
实施例1Example 1
一种鉴定性连锁矮小基因的引物组，由检测鸡非矮小等位基因DW基因引物对1和检测鸡性连锁矮小等位基因dw基因引物对2组成；所述引物组由生工生物工程(上海)股份有限公司合成；A primer set for identifying linked dwarf genes, consisting of a primer pair 1 for detecting chicken non-dwarf allele DW gene and a primer pair 2 for detecting chicken sex-linked dwarf allele dw gene; the primer set is produced by Sangon Bioengineering ( Shanghai) Co., Ltd. synthesis;
所述引物对1的正向引物的核苷酸序列如SEQ ID NO：1所示： 5’-atgcctatttctgtgagg-3’；所述引物对1的反向引物的核苷酸序列如SEQ ID NO：3所示：3’-atgtcctatctccttctactg-5’；所述引物对1的扩增产物大小为713bp；The nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1: 5'-atgcctatttctgtgagg-3'; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: Shown in 3: 3'-atgtcctatctccttctactg-5'; the size of the amplification product of the primer pair 1 is 713bp;
所述引物对2的正向引物的核苷酸序列如SEQ ID NO：2所示：5’-gggatgttcattgccttt-3’；所述引物对2的反向引物的核苷酸序列如SEQ ID NO：3所示：3’-atgtcctatctccttctactg-5’；所述引物对1的扩增产物大小为614bp。The nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2: 5'-gggatgttcattgccttt-3'; the nucleotide sequence of the reverse primer of the primer pair 2 is as SEQ ID NO: Shown in 3: 3'-atgtcctatctccttctactg-5'; the size of the amplified product of the primer pair 1 is 614bp.
应用例1Application example 1
利用实施例1的引物组鉴定性连锁矮小基因携带鸡的基因型。The genotypes of sex-linked dwarf gene carrier chickens were identified using the primer set of Example 1.
1、待鉴定鸡1. Chicken to be identified
应用本发明的方法为江苏北农大农牧科技有限公司鉴定扬泰小黑鸡群体，选择父母代公鸡975只，母鸡58只，商品代母鸡76只，采集每只待鉴定鸡的血液，-20℃保存。Apply the method of the present invention to identify the Yangtai small black chicken population for Jiangsu Beinongda Agriculture and Animal Husbandry Technology Co., Ltd., select 975 parental cocks, 58 hens, and 76 commercial hens, and collect the blood of each chicken to be identified. Store at -20°C.
2、已知基因型鸡2. Known genotype chicken
1)5只基因型为Z DWZ DW的白来航公鸡，来自中国农业大学动物科学技术学院家禽遗传资源与育种试验基地。 1) Five Bailaihang roosters with genotype Z DW Z DW were from the Experimental Base of Poultry Genetic Resources and Breeding of the College of Animal Science and Technology, China Agricultural University.
2)5只基因型为Z DWW的白来航母鸡，来自中国农业大学动物科学技术学院家禽遗传资源与育种试验基地。 2) Five Bailai aircraft carrier chickens with genotype Z DW W were from the Experimental Base of Poultry Genetic Resources and Breeding of the College of Animal Science and Technology, China Agricultural University.
3)5只基因型为Z dwZ dw的矮小纯系公鸡，来自中国农业大学动物科学技术学院家禽遗传资源与育种试验基地。 3) Five dwarf purebred roosters with genotype Z dw Z dw were from the Experimental Base of Poultry Genetic Resources and Breeding of the College of Animal Science and Technology, China Agricultural University.
4)5只基因型为Z dwW的矮小纯系母鸡，来自中国农业大学动物科学技术学院家禽遗传资源与育种试验基地。 4) Five dwarf pure-line hens with genotype Z dw W were from the Poultry Genetic Resources and Breeding Experimental Base of the College of Animal Science and Technology, China Agricultural University.
3、鉴定基因型3. Identification of genotype
1)试样的制备和保存1) Preparation and storage of samples
每只待鉴定鸡和已知基因型鸡各取10μL含抗凝剂的鸡血，用天根生化科技(北京)有限公司提供的血液/细胞/组织基因组DNA提取试剂盒(DP304)提取基因组DNA，将DNA样品保存于-20℃冰箱中备用。Take 10 μL chicken blood containing anticoagulant from each chicken to be identified and chicken with known genotype, and use the blood/cell/tissue genomic DNA extraction kit (DP304) provided by Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract genomic DNA , DNA samples were stored in a -20°C refrigerator for later use.
2)PCR扩增2) PCR amplification
每只待鉴定鸡均采用如下PCR反应体系和如下PCR反应程序，以基因组DNA为模板，利用实施例1的引物组进行PCR扩增。反应体系为20μL体 系：
Figure PCTCN2022105580-appb-000007
PCR SuperMix(+dye)(购自北京全式金生物技术有限公司，货号为AS111-11)10μL(浓度为10μmoL/L)、引物对1的正向引物和引物对2的正向引物各0.4μL(浓度均为10μmoL/L)、引物对1的反向引物和引物对2的反向引物各0.2μL(浓度均为10μmoL/L)、DNA模板2.5μL(浓度为100ng/μL)、去离子水6.3μL。PCR反应程序为：94℃预变性5min；94℃变性30s，58℃复性30s，72℃延伸40s，循环35次；72℃延伸5min；循环反应结束后在72℃保持5min。PCR扩增产物于4℃下保存备用。 Each chicken to be identified adopts the following PCR reaction system and the following PCR reaction procedure, uses the genomic DNA as a template, and utilizes the primer set in Example 1 to carry out PCR amplification. The reaction system is a 20μL system:
Figure PCTCN2022105580-appb-000007
PCR SuperMix (+dye) (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AS111-11) 10 μL (concentration: 10 μmoL/L), the forward primer of primer pair 1 and the forward primer of primer pair 2 each 0.4 μL (both at a concentration of 10 μmoL/L), 0.2 μL of each of the reverse primers of primer pair 1 and primer pair 2 (both at a concentration of 10 μmoL/L), 2.5 μL of a DNA template (at a concentration of 100 ng/μL), and 6.3 μL of ionized water. The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, renaturation at 58°C for 30 s, extension at 72°C for 40 s, and 35 cycles; extension at 72°C for 5 min; The PCR amplification products were stored at 4°C for future use.
3)PCR扩增产物的检测及结果判定3) Detection of PCR amplification products and judgment of results
琼脂糖凝胶电泳检测：取5μL PCR扩增产物，1.2％的琼脂凝胶电泳(120V，40min)，用上述扩增产物点样，凝胶成像仪检测，检测结果见表1和图1～4(图1～4为随机挑选10只不同基因型的扬泰小黑鸡，分别以白来航和矮小纯系鸡作阳性对照，水作阴性对照的电泳图)。Agarose gel electrophoresis detection: Take 5 μL of PCR amplification product, perform 1.2% agarose gel electrophoresis (120V, 40min), use the above-mentioned amplification product to apply samples, and detect with a gel imager. The detection results are shown in Table 1 and Figure 1～ 4 (Figures 1 to 4 are the electrophoresis diagrams of 10 Yangtai black chickens of different genotypes randomly selected, with Bailaihang and dwarf pure-line chickens as positive controls and water as negative control respectively).
表1 不同检测鸡的检测结果Table 1 The detection results of different detection chickens
Figure PCTCN2022105580-appb-000008
Figure PCTCN2022105580-appb-000008
检测结果表明基因型为Z DWZ DW的扬泰小黑鸡公鸡和基因型为Z DWW的扬泰小黑鸡母鸡的PCR扩增产物均显示为700bp～800bp的一条带(图1和图3)。图1和图3中，除左边第一条泳道为Marker外，其他泳道为待鉴定鸡。对每一只待鉴定鸡的PCR产物进行测序，结果表明基因型为Z DWZ DW的扬泰小黑鸡公鸡和基因型为Z DWW的扬泰小黑鸡母鸡的PCR扩增产物的序列如SEQ ID NO：4所示：gggatgttcattgcctttctctgacgtgaacagaagtgcatgacgtgcagtgaatgcagatcttgctcatgatgtctcccactgaactcactgatgctctctttgtgaccaagggctacaggactgagcccaaacattgggtgggatttttacgttcagaacgtactctatggtaaatgtgcattaggtattctgctaatttgctgctatatttttctaacagctacattatttagttacatataaaatttcatagatgatagacactaatgcaagtaaagcctatgtttgtttggaaaaagtttcttactctgtttctattgccaaaaatagttaaatacctcaatcaaactcagaatgtcattttggtactttactggtcacacaagccattattcgccggtcagaagatgtgtctttcagtttctatttaactttccttatgtcagtgttttgtttgttttcctcgaagtttaataaatgtattgtctttgatctgtcccgacagaatgtgttttattgcagataattcagtgtttgtggaaaacagagattct gccaggagaaatcgagtatttcatagtgagtgaagaagaatacagctttcctttgaacacaagcaatatctgctggcattggtgttttcacttattgatatttatttagttaaaaatagaactacatacctgaatcagtagaaggagataggacat，大小均为713bp。 The test results showed that the PCR amplification products of the Yangtai black chicken rooster with the genotype Z DW Z DW and the Yangtai black chicken hen with the genotype Z DW W showed a band of 700bp to 800bp (Figure 1 and image 3). In Figure 1 and Figure 3, except the first lane on the left is Marker, the other lanes are chickens to be identified. The PCR products of each chicken to be identified were sequenced, and the results showed that the genotype of the Yangtai small black chicken rooster with the genotype Z DW Z DW and the genotype of the Yangtai small black chicken hen with the genotype Z DW W were relatively high.序列如SEQ ID NO：4所示：gggatgttcattgcctttctctgacgtgaacagaagtgcatgacgtgcagtgaatgcagatcttgctcatgatgtctcccactgaactcactgatgctctctttgtgaccaagggctacaggactgagcccaaacattgggtgggatttttacgttcagaacgtactctatggtaaatgtgcattaggtattctgctaatttgctgctatatttttctaacagctacattatttagttacatataaaatttcatagatgatagacactaatgcaagtaaagcctatgtttgtttggaaaaagtttcttactctgtttctattgccaaaaatagttaaatacctcaatcaaactcagaatgtcattttggtactttactggtcacacaagccattattcgccggtcagaagatgtgtctttcagtttctatttaactttccttatgtcagtgttttgtttgttttcctcgaagtttaataaatgtattgtctttgatctgtcccgacagaatgtgttttattgcagataattcagtgtttgtggaaaacagagattct gccaggagaaatcgagtatttcatagtgagtgaagaagaatacagctttcctttgaacacaagcaatatctgctggcattggtgttttcacttattgatatttatttagttaaaaatagaactacatacctgaatcagtagaaggagataggacat，大小均为713bp。
基因型为Z dwW的扬泰小黑鸡的PCR扩增产物均显示为600bp～700bp的一条带(图4)。图4中，除左边第一条泳道为Marker外，其他泳道为待鉴定鸡。对每一只待鉴定鸡的PCR产物进行测序，结果表明基因型为Z DWW的扬泰小黑鸡母鸡的PCR扩增产物的序列均如SEQ ID NO：5所示：atgcctatttctgtgaggcagatgtgaaaaaatgtattgctgtgatttcacaggaagaggatgagccgcgtgttcaggagcaaagctgtaacgaggacacttacttcaccacagaaagccttaccactaccggtatcaatcttggagcttcaatggcagaaaccccaagtatggaaatgcctgtcccagactacacttctattcatattgttcactctccacaaggccttgtgctcaatgcactcaatcaaactcagaatgtcattttggtactttactggtcacacaagccattattcgccggtcagaagatgtgtctttcagtttctatttaactttccttatgtcagtgttttgtttgttttcctcgaagtttaataaatgtattgtctttgatctgtcccgacagaatgtgttttattgcagataattcagtgtttgtggaaaacagagattctgccaggagaaatcgagtatttcatagtgagtgaagaagaatacagctttcctttgaacacaagcaatatctgctggcattggtgttttcacttattgatatttatttagttaaaaatagaactacatacctgaatcagtagaaggagataggacat，大小均为614bp。 The PCR amplification products of Yangtai black chicken with genotype Z dw W all showed a band of 600bp-700bp (Fig. 4). In Figure 4, except the first lane on the left is Marker, the other lanes are chickens to be identified. The PCR products of each chicken to be identified were sequenced, and the results showed that the sequences of the PCR amplification products of the Yangtai black chicken hen whose genotype was Z DW W were all shown in SEQ ID NO: 5, and the sizes were both 614bp.
基因型为Z DWZ dw的扬泰小黑鸡的PCR扩增产物均显示为600bp～700bp的一条带和700bp～800bp的一条带(图2)。图2中，除左边第一条泳道为Marker外，其他泳道为待鉴定鸡。对每一只待鉴定鸡的PCR产物进行测序，结果表明基因型为Z DWZ dw的扬泰小黑鸡公鸡的PCR扩增产物的序列均如SEQ ID NO：4和如SEQ ID NO：5所示，大小分别为713bp和614bp。 The PCR amplification products of Yangtai black chicken with genotype Z DW Z dw all showed a band of 600bp-700bp and a band of 700bp-800bp (Figure 2). In Figure 2, except the first lane on the left is Marker, the other lanes are chickens to be identified. The PCR products of each chicken to be identified were sequenced, and the results showed that the sequences of the PCR amplification products of the Yangtai black chicken rooster whose genotype was Z DW Z dw were all such as SEQ ID NO: 4 and SEQ ID NO: 5 As shown, the sizes are 713bp and 614bp, respectively.
对比例1Comparative example 1
一种与应用例1相似的鉴定方法，区别在于，鉴定引物组如下所示：引物对3的正向引物核苷酸序列如SEQ ID NO：6所示：5’-ggcagaaaccccaagtatg-3’，反向引物核苷酸序列如SEQ ID NO：7所示：5’-tcgggacagatcaaagaca-3’；引物对4正向引物核苷酸序列如SEQ ID NO：8所示：为5’-tgcagcaaaaattaaaaacag-3’，反向引物核苷酸序列如SEQ ID NO：9所示：为5’-cgtattcaattcctgtgtttc-3’。An identification method similar to Application Example 1, the difference is that the identification primer set is as follows: the nucleotide sequence of the forward primer of primer pair 3 is shown in SEQ ID NO: 6: 5'-ggcagaaaccccaagtatg-3', reverse The nucleotide sequence of the forward primer is shown in SEQ ID NO: 7: 5'-tcgggacagatcaaagaca-3'; the nucleotide sequence of the primer pair 4 forward primer is shown in SEQ ID NO: 8: 5'-tgcagcaaaaattaaaaacag-3' , the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 9: it is 5'-cgtattcaattcctgtgtttc-3'.
PCR反应程序为：94℃预变性5min；94℃变性30s，54℃复性30s， 72℃延伸30s，循环35次；72℃延伸5min。The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 5 min.
PCR扩增的反应体系为20μL体系：
Figure PCTCN2022105580-appb-000009
PCR SuperMix(+dye)(购自北京全式金生物技术有限公司，货号为AS111-11)10μL(浓度为10μmoL/L)、引物对3的正向引物和引物对4的正向引物各0.4μL(浓度均为10μmoL/L)、引物对3的反向引物和引物对4的反向引物各0.4μL(浓度均为10μmoL/L)、DNA模板2.5μL(浓度为100ng/μL)、去离子水5.9μL。 The reaction system for PCR amplification is a 20 μL system:
Figure PCTCN2022105580-appb-000009
PCR SuperMix (+dye) (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AS111-11) 10 μL (concentration: 10 μmoL/L), the forward primer of primer pair 3 and the forward primer of primer pair 4 each 0.4 μL (both at a concentration of 10 μmoL/L), 0.4 μL of each of the reverse primers of primer pair 3 and primer pair 4 (both at a concentration of 10 μmoL/L), 2.5 μL of a DNA template (at a concentration of 100 ng/μL), and 5.9 μL of ionized water.
该引物组的扩增产物的判断为：当含有200bp～300bp的扩增产物和大于500bp且小于600bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的杂合鸡；所述携带有性连锁矮小基因的杂合鸡的基因型为Z DWZ dw； The judgment of the amplification product of the primer set is as follows: when the amplification product of 200bp～300bp and the amplification product of greater than 500bp and less than 600bp are contained, the sample chicken is a heterozygous chicken carrying a sex-linked dwarf gene; The genotype of the heterozygous chicken with sex-linked dwarf gene is Z DW Z dw ;
当仅含有200bp～300bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的纯合鸡；所携带有性连锁矮小基因的述纯合鸡的基因型为Z dwW或Z dwZ dw；进一步分型根据样本鸡的性别区分； When only 200bp-300bp amplification products are contained, the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene; the genotype of the homozygous chicken carrying a sex-linked dwarf gene is Z dw W or Z dw Z dw ; further classification is based on the sex of the sample chicken;
当仅含有大于500bp且小于600bp的扩增产物时，则样本鸡为未携带有性连锁矮小基因的纯合鸡；所述未携带有性连锁矮小基因的纯合鸡的基因型为Z DWW或Z DWZ DW；进一步分型根据样本鸡的性别区分。 When there are only amplified products greater than 500bp and less than 600bp, the sample chicken is a homozygous chicken that does not carry a sex-linked dwarf gene; the genotype of the homozygous chicken that does not carry a sex-linked dwarf gene is Z DW W Or Z DW Z DW ; further typing is distinguished according to the sex of the sample chicken.
使用样本为应用例1中随机挑选的基因型的扬泰小黑鸡血液DNA样本各3个，测定结果见图5(其中M泳道为DNA分子量标记(100～1500bp Ladder)，图中1、2、3泳道为基因型为Z DWZ dw扬泰小黑鸡公鸡；4、5、6泳道为基因型为Z DWZ DW扬泰小黑鸡；7、8、9泳道为基因型为Z DWW扬泰小黑鸡母鸡；10、11、12泳道为基因型为Z dwW扬泰小黑鸡母鸡)。 The samples used are 3 blood DNA samples of Yangtai black chickens of the genotypes randomly selected in Application Example 1, and the measurement results are shown in Figure 5 (in which the M swimming lane is the DNA molecular weight marker (100-1500bp Ladder), 1 and 2 in the figure Lanes 3 and 3 are Yangtai black chickens whose genotype is Z DW Z dw ; lanes 4, 5 and 6 are Yangtai black chickens whose genotype is Z DW Z DW ; lanes 7, 8 and 9 are Yangtai black chickens whose genotype is Z DW W Yangtai little black chicken hen; lanes 10, 11, and 12 are genotype Z dw W Yangtai little black chicken hen).
由图5可知，对比例1中的引物组存在明显的引物二聚体，导致非矮小等位基因DW基因的区分效果不佳，无法区分基因型为Z DWZ dw的公鸡杂合子。 It can be seen from Figure 5 that there are obvious primer dimers in the primer set in Comparative Example 1, resulting in poor discrimination of the non-dwarf allele DW gene, and it is impossible to distinguish rooster heterozygotes with genotype Z DW Z dw .
综上所述，本发明通过特定的引物组能够快速、准确地鉴定性连锁矮小基因携带鸡的基因型；另外，本发明所设计引物特异性强，结果稳定可靠，3小时内即可确定待鉴定鸡的基因型，可用于早期选择，根据基因型剔除或淘汰正常群体中携带有性连锁矮小基因的公鸡或母鸡，减少饲养成本，提高群体均匀度。In summary, the present invention can quickly and accurately identify the genotype of sex-linked dwarf gene-carrying chickens through a specific primer set; in addition, the primers designed by the present invention have strong specificity, and the results are stable and reliable, and can be determined within 3 hours. Identify the genotype of the chicken, which can be used for early selection, according to the genotype to eliminate or eliminate the roosters or hens carrying the sex-linked dwarf gene in the normal population, reduce the cost of feeding, and improve the uniformity of the population.
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明， 任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可以做各种改动和修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

  1. 一种鉴定性连锁矮小基因的引物组，其特征在于，包括引物对1和引物对2；A primer set for identifying linked dwarf genes, characterized in that it includes primer pair 1 and primer pair 2;
    所述引物对1的正向引物的核苷酸序列如SEQ ID NO：1所示；所述引物对1的反向引物的核苷酸序列如SEQ ID NO：3所示；The nucleotide sequence of the forward primer of the primer pair 1 is shown in SEQ ID NO: 1; the nucleotide sequence of the reverse primer of the primer pair 1 is shown in SEQ ID NO: 3;
    所述引物对2的正向引物的核苷酸序列如SEQ ID NO：2所示；所述引物对2的反向引物的核苷酸序列如SEQ ID NO：3所示。The nucleotide sequence of the forward primer of the primer pair 2 is shown in SEQ ID NO: 2; the nucleotide sequence of the reverse primer of the primer pair 2 is shown in SEQ ID NO: 3.
  2. 权利要求1所述引物组在鉴定性连锁矮小基因携带鸡中的应用。The application of the primer set described in claim 1 in chickens carrying dwarf gene of identification linkage.
  3. 一种鉴定性连锁矮小基因携带鸡的试剂或试剂盒，其特征在于，所述试剂或试剂盒包括权利要求1所述的引物组。A reagent or kit for identifying linked dwarf gene-carrying chickens, characterized in that the reagent or kit includes the primer set according to claim 1.
  4. 一种鉴定性连锁矮小基因携带鸡的方法，其特征在于，包括以下步骤：A method for identifying sex-linked dwarf gene-carrying chickens, comprising the following steps:
    以样本鸡的基因组DNA或总RNA反转录得到的cDNA为模板，利用权利要求1所述引物组或者权利要求3所述的试剂或试剂盒进行PCR扩增，得到扩增产物；Using the cDNA obtained by reverse transcription of the genomic DNA or total RNA of the sample chicken as a template, utilize the primer set described in claim 1 or the reagent or kit described in claim 3 to carry out PCR amplification to obtain the amplified product;
    扩增产物的判断包括：当含有600bp～700bp的扩增产物时，则样本鸡为性连锁矮小基因的携带者。The judgment of the amplified product includes: when the amplified product of 600bp-700bp is contained, the sample chicken is a carrier of the sex-linked dwarf gene.
  5. 根据权利要求4所述的方法，其特征在于，所述PCR反应程序包括：94℃预变性5min；94℃变性30s，58℃复性30s，72℃延伸40s，循环35次；72℃延伸5min。The method according to claim 4, wherein the PCR reaction program comprises: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 58°C refolding for 30 seconds, 72°C extension for 40 seconds, and 35 cycles; 72°C extension for 5 minutes .
  6. 根据权利要求4所述的方法，其特征在于，所述PCR扩增的反应体系以20μL计，包括：2×Easy
    Figure PCTCN2022105580-appb-100001
    PCR SuperMix 10μL，所述引物组中引物对1的正向引物和引物对2的正向引物各0.4μL，所述引物组中引物对1的反向引物和引物对2的反向引物各0.2μL，DNA模板2.5μL和剩余去离子水。     The method according to claim 4, characterized in that, the reaction system of the PCR amplification is calculated in 20 μL, comprising: 2×Easy
    Figure PCTCN2022105580-appb-100001
    PCR SuperMix 10 μL, 0.4 μL each of the forward primer of primer pair 1 and the forward primer of primer pair 2 in the primer set, 0.2 μL each of the reverse primer of primer pair 1 and the reverse primer of primer pair 2 in the primer set μL, 2.5 μL of DNA template and remaining deionized water.
  7. 根据权利要求6所述的方法，其特征在于，所述2×Easy
    Figure PCTCN2022105580-appb-100002
    PCR SuperMix的浓度为10μmoL/L；所述引物组中引物对1的正向引物、引物对1的反向引物、引物对2的正向引物和引物对2的反向引物的工作浓度均为10μmoL/L；所述DNA模板的浓度为100ng/μL。     The method according to claim 6, wherein the 2×Easy
    Figure PCTCN2022105580-appb-100002
    The concentration of PCR SuperMix is 10 μ moL/L; The working concentration of the forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 2 and the reverse primer of primer pair 2 in the primer set is 10 μmoL/L; the concentration of the DNA template is 100 ng/μL.
  8. 根据权利要求4所述的方法，其特征在于，所述判断的方法包括电泳或测序。The method according to claim 4, characterized in that the judging method comprises electrophoresis or sequencing.
  9. 根据权利要求4所述的方法，其特征在于，扩增产物的判断还包括：当含有600bp～700bp的扩增产物和大于700bp且小于等于800bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的杂合鸡；The method according to claim 4, wherein the judgment of the amplified product further comprises: when the amplified product of 600bp～700bp and the amplified product of greater than 700bp and less than or equal to 800bp are contained, the sample chicken is carrier Heterozygous chickens linked to the dwarf gene;
    当仅含有600bp～700bp的扩增产物时，则样本鸡为携带有性连锁矮小基因的纯合鸡；When only the amplification product of 600bp-700bp is contained, the sample chicken is a homozygous chicken carrying a sex-linked dwarf gene;
    当仅含有大于700bp且小于等于800bp的扩增产物时，则样本鸡为未携带有性连锁矮小基因的纯合鸡。When there are only amplified products greater than 700bp and less than or equal to 800bp, the sample chicken is a homozygous chicken that does not carry the sex-linked dwarf gene.
  10. 权利要求1所述引物组或权利要求3所述试剂或试剂盒或权利要求4～9任一项所述方法在辅助选育矮小鸡群新品种中的应用。The application of the primer set according to claim 1 or the reagent or kit according to claim 3 or the method according to any one of claims 4 to 9 in assisted breeding of new breeds of dwarf chickens.
PCT/CN2022/105580 2021-07-30 2022-07-14 Primer set for identifying sex-linked dwarf gene and application thereof WO2022258078A1 (en)

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