Sheep SNP molecular markers and their application in detecting polyhedral traits

Abstract

The present invention relates to the field of molecular biology technology, and in particular to sheep SNP molecular markers and their application in polyhorn trait detection. The SNP molecular marker is a G / C mutation at 132749153bp of sheep chromosome 2; the sheep genome version number where the SNP molecular marker is located is Oar_v4.0. The molecular genetic marker provided by the present invention is not affected by the gender and age of the sheep, and can be used for early screening of the sheep to be tested, significantly shortening the selection cycle of excellent polyhorn breeding sheep, reducing breeding costs, and improving economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to sheep SNP molecular markers and applications thereof in polyhedral trait detection. Background Art

[0002] Molecular breeding, also known as molecular marker-assisted selection breeding, refers to the use of DNA molecular markers to select breeding materials and comprehensively improve important economic traits of livestock and poultry. It is a breeding method that organically combines traditional genetic breeding with modern molecular biology. With the rapid development of molecular biology, the application of molecular markers in livestock breeding is becoming more and more extensive, and its importance is becoming increasingly prominent.

[0003] Homeobox D1 HOXD1 ) is a protein-coding gene. This gene is a member of the Antp homeobox family and encodes a protein with a homeobox DNA binding domain. This nuclear protein functions as a sequence-specific transcription factor and is involved in differentiation and limb development. HOXD The gene cluster is part of the HOX gene family and is located on chromosome 2. HOXD1 , HOXD3 , HOXD4 , HOXD8 , HOXD9 , HOXD10 , HOXD11 , HOXD12 and HOXD13 The transcription factor composition of the gene is crucial for the occurrence of common diseases such as limb and bone developmental defects, neurological dysfunction, urogenital system and cardiovascular system, and the development of tissues and organs. HOXD1 Related disorders include congenital disorders of glycosylation and 3M syndrome type 1. HOXD1 The gene has been less studied in sheep and has not yet been identified HOXD1 Genes associated with the polyhorn trait in sheep.

[0004] Horns are the iconic features of Ceratidae (cattle, sheep, goats, etc.) and are one of their most successful organs in evolution. Polyhorn is an ancient and rare phenotype found in domestic sheep breeds. Polyhorned sheep are sheep with more than two horns on their heads. my country's polyhorned sheep breeds mainly include: Altay sheep, Mongolian sheep and Tibetan sheep, which are precious sheep genetic resources. Due to its high ornamental value and scientific research value, the breeding of polyhorned sheep breeds has received more and more attention in recent years. The phenotype of sheep horns will not be determined until they grow to half a year to one year old. Therefore, the development and application of SNP molecular markers related to the polyhorn trait of sheep has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] Based on this, the present invention has developed a SNP molecular marker associated with the polygonal trait of sheep, which is a G / C mutation at the 132749153bp of chromosome 2 of sheep; the sheep genome version number where the SNP molecular marker is located is Oar_v4.0.

[0006] Preferably, the sheep is Chinese polyhorn sheep.

[0007] More preferably, the sheep are Altai sheep, Mongolian sheep or Tibetan sheep.

[0008] Preferably, the SNP molecular marker is obtained by amplification using primers shown in SEQ ID NO. 1 and SEQ ID NO. 2. The SNP molecular marker is located at the 200 bp position of the amplified product fragment.

[0009] SEQ ID NO.1: 5'-ACGTTGGATGTGACGCTCGGATTCCCAAAG-3';

[0010] SEQ ID NO. 2: 5'-ACGTTGGATCTGGCGGTAACCTTGGAGT-3'.

[0011] Furthermore, the present invention provides primers for amplifying the SNP molecular marker.

[0012] Preferably, the primers are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0013] Furthermore, the present invention provides a reagent or a kit containing the primer.

[0014] Preferably, the reagent or kit also includes dNTPs, Taq DNA polymerase, Mg 2+ , PCR reaction buffer, and at least one of standard positive templates.

[0015] Furthermore, the present invention provides the use of the SNP molecular marker, the primer, or the reagent or kit in at least one of the following aspects:

[0016] (1) Detection of polyhorn phenotype in individual sheep;

[0017] (2) Early prediction of polyhorn phenotype in sheep;

[0018] (3) Molecular marker-assisted breeding of polyhorned sheep;

[0019] (4) Preparation of products; the products are reagents, kits or gene chips.

[0020] In the specific implementation process, the primers can also be combined with other specific primers for sheep phenotype detection for sheep classification and breeding research, which also falls within the protection scope of the present invention.

[0021] When the genotype of the polymorphic site of the SNP molecular marker is CC or CG, the sheep horn type is polyhorn; when the genotype is GG, the sheep horn type is bihorn.

[0022] Furthermore, the present invention provides a method for detecting sheep horn type, comprising: using the genomic DNA of the sheep to be tested as a template and using the primers to perform PCR amplification.

[0023] Preferably, the marker at the 200th bp of the PCR amplification product fragment is detected. When the genotype at the marker is CC or CG, the sheep horn type is multiple-horned; when the genotype is GG, the sheep horn type is two-horned.

[0024] In the specific implementation process, there is no particular limitation on the method for detecting PCR amplification product fragments, and it can be carried out using conventional detection methods in the art, including but not limited to detection by gene sequencing methods.

[0025] Preferably, the reaction conditions for PCR amplification are: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 20 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds, for a total of 45 cycles; and extension for 3 minutes after the last cycle.

[0026] Preferably, the reaction system for PCR amplification is 5 μL, including: 1.75 μL of water, 0.625 μL of 10×PCR Buffer, 0.325 μL of MgCl2 (25 mM), 0.1 μL of dNTP mix (25 mM), 0.2 μL of HotStar Taq (5 U / μL), 1 μL of primer (0.5 μM), and 1 μL of template DNA.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The SNP molecular marker of the present invention can be used for genotyping of sheep and the SNP molecular marker can be associated with the sheep polyhorn phenotype. The detection of this site not only provides new material for molecular breeding, but also provides a scientific basis for marker-assisted selection of sheep polyhorn traits.

[0029] The molecular genetic markers provided by the present invention are not limited by the age, gender, etc. of the sheep, and can be used for early screening of the sheep to be tested, effectively alleviating the problem of long selection time for excellent polyhorn breeding sheep in actual production, reducing breeding costs, and improving economic benefits, which will play a huge role in polyhorn sheep breeding.

[0030] The identification method of the present invention is simple to operate, inexpensive, highly accurate, and can realize automated detection, thus having high breeding practice application value. The present invention can make the accuracy of identification in horned sheep individuals reach 98.9%, thereby obtaining considerable economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the MassArray SNP typing results of the three types of sheep provided by the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0034] Example 1

[0035] This example provides a method for amplifying and genotyping the G / C mutation site at 132749153 bp of sheep chromosome 2 (sheep genome version number is Oar_v4.0), which specifically includes the following process:

[0036] 1. Primer design

[0037] According to the information of sheep genomic DNA sequence, a pair of primers were designed as follows: forward primer F 5'-ACGTTGGATGTGACGCTCGGATTCCCAAAG-3' and reverse primer R 5'-ACGTTGGATGCTGGCGGTAACCTTGGAGT-3', which were used to amplify the nucleotide fragment where the SNP to be tested was located.

[0038] 2. Genotyping test

[0039] First, prepare 4μL PCR master mix, including 1.75μL water, 0.625μL 10×PCR Buffer, 0.325μL MgCl2 (25mM), 0.1μL dNTP mix (25mM), 0.2μL HotStar Taq (5U / μL), 0.5μL forward primer and reverse primer (0.5μM) respectively. Add 4μL PCR master mix to each well of 384-well plate, then add 1μL template DNA, press and mix with 384-well sealing film, and centrifuge at 1200rpm for 1min.

[0040] The reaction plate was placed on a PCR instrument and the reaction was carried out according to the following PCR amplification reaction program: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 20 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds, for a total of 45 cycles; extension for 3 minutes after the last cycle; and storage at 4°C.

[0041] After the PCR reaction is completed, the PCR product is subjected to alkaline phosphatase (SAP) reaction to remove free dNTPS in the system. Prepare alkaline phosphatase treatment reaction solution (SAP mix) in a 1.5mL EP tube, including: 1.53μL water, 0.17μL SAPBuffer, 0.30pL SAP Enzyme. Add SAP mix to a 384-well PCR reaction plate, with 5μL PCR product and 2μL SAP mix in each well, seal the film and centrifuge. Set the SAP reaction program: 37℃ 40min; 85℃ 5min; store at 4℃.

[0042] Then, a single-base extension reaction was carried out in the ddNTP system to form a single-base extension product complementary to the SNP genotype to be detected. After resin purification and sample loading, the product was analyzed and data was collected using a mass spectrometer. The original data and genotyping diagram of the test results were obtained using TYPER4.0 software.

[0043] Example 2

[0044] This example uses the genotyping detection method in Example 1 to perform PCR amplification and sequencing on the genomic DNA of three sheep breeds. A total of 275 individuals of Altay sheep, Mongolian sheep and Tibetan sheep were tested, and the number of samples and horn phenotypes of each breed are shown in Table 1.

[0045] Table 1 Sample numbers and horn phenotypes of three sheep breeds

[0046]

[0047] The genotype and phenotype analysis results of the PCR amplification product at 200 bp are shown in Table 2 , and the schematic diagram of the genotyping results is shown inFigure 1 As shown, the inverted triangle close to the vertical axis represents the GG genotype, the middle square represents the CG genotype, and the upright triangle close to the horizontal axis represents the CC genotype.

[0048] Table 2 Distribution of SNP genotypes in 275 sheep individuals

[0049]

[0050] The genotyping results showed that among all polyhorned sheep individuals, 98.9% of the individuals were of CC or CG genotype, that is, the accuracy of using this SNP molecular marker to judge the polyhorned phenotype in the sheep population reached 98.9%; among all two-horned sheep individuals, 70.0% of the individuals were of GG phenotype, that is, the accuracy of using this SNP molecular marker to judge the two-horned phenotype reached 70.0%.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a SNP molecular marker, a primer for amplifying the SNP molecular marker, or a reagent or kit containing the primer in at least one of the following aspects: (1) Detection of polyhorn phenotype in individual sheep; (2) Early prediction of polyhorn phenotype in sheep; The SNP molecular marker is a G / C mutation at 132749153bp of sheep chromosome 2; the sheep genome version number where the SNP molecular marker is located is Oar_v4.0; The primers are shown in SEQ ID NO.1 and SEQ ID NO.2; When the genotype of the polymorphic site of the SNP molecular marker is CC or CG, the sheep horn type is polyhorn; when the genotype is GG, the sheep horn type is bihorn.

2. The use according to claim 1, characterized in that: The SNP molecular marker is obtained by amplification of the primers shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. A method for detecting sheep horn type, characterized in that: include: Using the genomic DNA of the sheep to be tested as a template, PCR amplification was performed using the primers shown in SEQ ID NO.1 and SEQ ID NO.2; The marker at the 200 bp position of the PCR amplification product fragment is detected. When the genotype at the marker is CC or CG, the sheep horn type is multiple-horned; when the genotype is GG, the sheep horn type is two-horned.

4. The detection method according to claim 3, characterized in that: The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 20 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds, for a total of 45 cycles; and extension for 3 minutes after the last cycle.

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