A molecular marker for cat red hair trait locus and its application

Abstract

The present invention relates to a molecular marker of a cat's red hair trait locus and its application. The molecular marker is a point mutation C>T at 110431605bp (reference genome Felis_catus_9.0) on the cat's X chromosome, which is an X-linked dominant inheritance. After the mutation of this site, the cat's hair shows orange color. The present invention clarifies the control gene of the red gene and the corresponding genotyping method. The technology of the present invention can detect the site to determine the genotype of the red gene, thereby more accurately judging the cat's hair color, and at the same time helping breeders to more efficiently and accurately breed the hair color of concern in cat breeding, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a molecular marker of a cat red hair trait site and an application thereof. Background Art

[0002] Red (orange) is one of the eight basic colors of cats and is controlled by the red gene. Common orange cats, tortoise shells and calico cats are all related to the red gene. Previous scientific studies have found that the red gene is related to gender, and inferred that the red gene is located on the X chromosome and is an X-linked dominant inheritance, but which gene specifically controls the red gene has not been discovered. Studies have been conducted to find the specific location of the red gene and locate it in a region of the cat's X chromosome. The red gene will affect the brown gene that also controls the basic color of the cat's hair, causing the cat to not show the hair color corresponding to different genotypes of the brown gene. Therefore, the positioning and detection of the red gene is crucial to the accurate judgment of the eight basic colors of cats. At the same time, it can help breeders to more efficiently breed the hair color of concern in cat breeding. The positioning of the red gene and the establishment of genotyping detection methods are very important.

[0003] Single nucleotide polymorphism (SNP) refers to the change of a single base in the DNA sequence of a population genome, including the insertion, deletion and substitution of a single base. SNP variant sites have the following advantages: large number, wide distribution; rich representativeness, and can be converted into molecular markers through technologies such as CAPs (restriction site polymorphism markers), dCAPs (interventional restriction site polymorphism markers), KASP (competitive allele-specific PCR) and AS-PCR (allele-specific PCR technology), which is convenient for molecular marker detection. Molecular markers based on SNPs will not separate the markers and target genes, and can greatly improve the accuracy and stability of the markers. However, there is currently no report on SNP molecular markers related to cat red hair trait sites. Summary of the invention

[0004] The present invention aims at the technical problems existing in the prior art, and provides a molecular marker for a cat red hair trait locus and its application. The genotyping detection method developed by the molecular marker can accurately judge the three phenotypes of cats, namely orange, non-orange and tortoise shell, and has very important value for accurate judgment of cat fur color and precise breeding.

[0005] One of the purposes of the present invention is to provide a molecular marker associated with cat coat color traits, characterized in that the sequence of the molecular marker is as shown in SEQ ID NO.1, and there is a C>T mutation at the 11th position of SEQ ID NO.1.

[0006] Preferably, the molecular marker is located at position 110431605bp on the cat's X chromosome, which is X-linked dominant inheritance. When the site mutates from C to T, the cat's hair shows orange color.

[0007] Preferably, the reference genome of cat is Felis_catus_9.0.

[0008] Another object of the present invention is to provide an identification reagent for molecular markers associated with cat fur color traits, characterized in that the reagent detects the target site by one or more of the following methods: amplification sequencing (based on a first-generation sequencing platform), restriction fragment length polymorphism (RFLP), random amplification polymorphism (RAPD), primer invasion analysis (invader assay), single-strand conformation polymorphism (SSCP), UCAN (RNase H), denaturing gradient gel electrophoresis (DGGE), allele-specific PCR (ASPCR), ligase detection reaction (LDR), allele-specific PCR (ARMS-PCR), dynamic allele-specific hybridization (DASH), TaqMan probe method, KASP technology, denaturing high performance liquid chromatography (DHPLC), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD I-TOF-MS), mass spectrometry (MassArray), HRM, liquid phase chip multi-gene typing technology, solid phase chip multi-gene typing technology, SNaPshot method, exon sequencing / whole genome sequencing (based on second-generation sequencing technology and third-generation sequencing technology).

[0009] Preferably, the reagents include a primer and a probe combination product.

[0010] Preferably, the primer combination is SEQ ID NO.2 and SEQ ID NO.3:

[0011] Preferably, the reagents also include DNA extraction reagent, amplification buffer, dNTP, Mg 2+ , DNA polymerase, a positive control, a negative control, water, and at least one of a sulfite sequencing reagent.

[0012] Preferably, the reagent is used to detect the genotyping result of at least one target molecular marker.

[0013] Another object of the present invention is to provide an identification kit for molecular markers associated with cat fur color traits, characterized in that the kit includes the above-mentioned identification reagent.

[0014] Preferably, the test samples of the kit include cat oral swab cell preservation fluid, blood samples or umbilical cord samples, etc.

[0015] Another object of the present invention is to provide the use of the molecular marker or the identification reagent or the identification kit in identifying cat coat color traits or in coat color selection in cat breeding.

[0016] Preferably, the judgment criteria include that for female cats, when the genotype is CC, the color is non-orange, when the genotype is CT, the color is tortoiseshell, and when the genotype is TT, the color is orange; for male cats, when the genotype is CC, the color is non-orange, and when the genotype is TT, the color is orange.

[0017] The beneficial effects of the present invention are as follows: the present invention discloses the molecular marker g.110431605C>T for controlling orange hair color of cats for the first time in the world, and develops a genotyping detection method based on the molecular marker. The method proposed by the present invention can accurately judge the three phenotypes of cats, namely orange, non-orange and tortoise shell, and has very important value for accurate judgment of cat hair color and accurate breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a CC typing result diagram in the molecular marker detection result of the present invention;

[0019] Figure 2 It is a CT typing result diagram in the molecular marker detection result of the present invention;

[0020] Figure 3 This is a diagram of the TT typing results in the molecular marker detection results of the present invention. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein belong to the meanings commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] Example 1

[0023] A method for screening SNP molecular markers for identifying orange hair in cats, comprising the following steps:

[0024] 1) Genomic DNA extraction: Oral swab samples were collected from 24 cats with orange-related fur colors. Genomic DNA was extracted according to the operating procedures of the Tiangen Automated Oral Swab DNA Extraction Kit. An appropriate amount of TE buffer was added to dissolve the precipitate and stored at 4°C or -20°C for later use. There were 8 orange, tortoiseshell or non-orange cats, and the male-female ratio of each fur color was 1:1.

[0025] 2) High-throughput sequencing analysis: The genome of 24 cats was resequenced using the MGI DNBSEQ-T7 sequencer platform, and the sequencing depth of each sample was 15X. After filtering the data (software bcftool s1.15, mis 0.1, maf0.05), the data was aligned to the reference genome Feli s_catus_9.0 using the software bwa-0.7.17 (default parameters), and variants were detected using GATK4.4.0.0 software (default parameters). The variants within the range locked by previous scientific studies (chrX: 101,601,853-111,310,076, the reference genome is Feli s_catus_9.0) were sorted (extracted using vcftool ls0.1.16).

[0026] 3) Preliminary screening of SNP variant sites: The variants that completely matched the genotypes of the 24 samples (including . / .) were screened out among the sorted variants, and finally two SNPs were screened out, namely g.110135598T>A and g.110431605C>T located on chromosome X.

[0027] 4) Preliminary verification of SNP variant sites: 31 samples of known gender and coat color (orange, hawksbill or non-orange) were collected for first-generation sequencing verification of the two selected SNP sites. The results showed that the high-throughput sequencing typing results of the g.110431605C>T site in 31 samples were consistent with the coat color phenotype, while the high-throughput sequencing typing results of g.110135598T>A in two samples were inconsistent with the coat color phenotype. Further screening of g.110431605C>T was more suitable as a molecular marker for the red gene.

[0028] 5) SNP variant site re-verification: Another 75 samples of known gender and coat color (orange, tortoise or non-orange) were collected to verify the g.110431605C>T site using high-throughput sequencing. The results are shown in Table 1. There is a one-to-one correspondence between the variant type and phenotype of this site, further confirming that g.110431605C>T can be used as a molecular marker for the red gene. Figure 1-3 As shown, the CC typing result diagram, CT typing result diagram and TT typing result diagram in the SNP molecular marker detection result of the present invention are respectively as shown in Figure 1-3 shown.

[0029] Table 1 Statistics of first-generation sequencing verification results

[0030]

[0031] Example 2

[0032] A method for identifying orange hair of a cat comprises the following steps:

[0033] 1) Extract DNA from the sample to be tested: Use Tiangen automated oral swab DNA extraction kit to process cat oral swab samples, and use magnetic bead method to prepare DNA;

[0034] 2) Prepare the PCR reaction amplification system: 1 μL DNA template, 0.5 μL 10 mmol / L forward and reverse primers, 10 μL 2×Taq PCR Mastermix, and add ddH2O to a total reaction volume of 20 uL; wherein the forward primer sequence is 5'-GCGAGATCATGACCTGAGCTG-3' (SEQ ID NO.2), and the reverse primer sequence is 5'-ACTGGGTGACGGCTAGCATAG-3' (SEQ ID NO.3);

[0035] 3) PCR reaction conditions were set as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 65°C annealing for 30 sec, 72°C extension for 30 sec, for a total of 10 cycles; 95°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 30 sec, for a total of 30 cycles; 72°C final extension for 10 min, and storage at 4°C;

[0036] 4) PCR product recovery: prepare agarose gel with a concentration of 2% (w / v), take 5 μL of sample and add it to the spot well, and take 5 μL of DNA Marker and add it to the adjacent well, turn on the power and run electrophoresis at 80V for 40 min. After the electrophoresis is completed, place the gel block in a gel imager and take a picture, observe the size of the target amplified fragment in the gel image, and use a PCR product purification kit (Qiagen, Cat. No. 28004) to recover the PCR amplified product;

[0037] 5) Sequencing analysis: The PCR amplification products were sequenced and analyzed using an AB I 3730 sequencer to obtain the PCR product sequence and peak graph file; the peak graph was viewed using Chromas software for genotyping;

[0038] 6) Genotyping result judgment criteria: In female cats, "CC" corresponds to not carrying the red gene, showing non-orange color; "CT" corresponds to carrying one copy of the red gene, showing tortoise shell color; "TT" corresponds to carrying two copies of the red gene, showing orange color; in male cats, "CC" corresponds to not carrying the red gene, showing non-orange color; "TT" corresponds to carrying one copy of the red gene, showing orange color. The "CT" typing in male cats is extremely rare and occurs when the genome mutates.

[0039] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A molecular marker associated with a cat coat color trait, characterized in that: The sequence of the molecular marker is shown in SEQ ID NO.1, and there is a C>T mutation at the 11th position of SEQ ID NO.

1.

2. A reagent for identifying the molecular marker associated with the cat fur color trait according to claim 1, characterized in that: The reagent is realized by one or more of the following methods: restriction fragment length polymorphism, random amplification polymorphism, primer invasion analysis technology, single-strand conformation polymorphism, UCAN based on RNase H, denaturing gradient gel electrophoresis, allele-specific PCR, ligase detection reaction, dynamic allele-specific hybridization, TaqMan probe method, denaturing high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, HRM method, liquid chip multi-gene typing technology, solid chip multi-gene typing technology, SNaPshot method, exon sequencing, whole genome sequencing.

3. The use according to claim 2, characterized in that The reagents include primer and probe combination products.

4. The use according to claim 3, characterized in that The primer combination is SEQ ID NO.2 and SEQ ID NO.

3.

5. The use according to claim 2, characterized in that The reagents also include DNA extraction reagents, amplification buffer, dNTP, Mg 2+ , DNA polymerase, a positive control, a negative control, water, and at least one of a sulfite sequencing reagent.

6. The use according to claim 2, characterized in that The reagent is used to detect the genotyping result of at least one target molecular marker.

7. A kit for identifying the molecular marker associated with cat fur color traits according to claim 1, characterized in that: The kit comprises the reagent according to any one of claims 2 to 6.

8. The use of the molecular marker according to claim 1 or any one of claims 2 to 7 in identifying cat coat color traits or in coat color selection in cat breeding, characterized in that: The judgment criteria include that for female cats, when the genotype is CC, the color is non-orange, when the genotype is CT, the color is tortoiseshell, and when the genotype is TT, the color is orange; for male cats, when the genotype is CC, the color is non-orange, and when the genotype is TT, the color is orange.

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