SNP markers associated with chest girth in Tianhua mutton sheep and their application

Abstract

The present invention discloses a SNP marker associated with the chest girth of Tianhua mutton sheep and its application, belonging to the fields of molecular biology and genetic breeding technology. The SNP marker is located at bp 110958643 on chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the base mutation is T>G (rs399622340). Whole-genome resequencing and chest girth measurement data show that the SNP marker has a significant effect on the chest girth of Tianhua mutton sheep, and the G allele has a better chest girth. Therefore, rs399622340 can be used in molecular marker selection breeding for the chest girth trait of Tianhua mutton sheep, which can accelerate the breeding process of Tianhua mutton sheep for body shape traits.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to SNP markers related to the chest circumference of Tianhua mutton sheep and applications thereof. Background Art

[0002] The Tianhua mutton breed is a new fine-wool mutton breed adapted to high-altitude, cold, and arid environments, suitable for grazing, semi-grazing, and confinement, and confinement. The Tianhua mutton breed has undergone four generations of continuous breeding. Its sire is a South African mutton Merino (62.5% pedigree) and its dam is a Gansu Alpine Fine-wool sheep (37.5% pedigree). The Tianhua mutton breed is characterized by large size, rapid growth, and high fertility. It combines the dual-purpose meat and wool characteristics of the South African mutton Merino with the adaptability and wool properties of the Gansu Alpine Fine-wool sheep. Body conformation in sheep is a complex trait. Chest girth is a key indicator of animal size, and since body size is correlated with economic profitability, improving chest girth can be a breeding goal. Molecular marker-assisted selection (MAS) is a method of selecting animals based on molecular markers that influence traits. Compared to traditional phenotypic selection, MAS can accelerate breeding processes and improve efficiency.

[0003] Single nucleotide polymorphisms (SNPs) are variations caused by changes in a single nucleotide sequence in the genome. SNPs are also the most common type of variation in the genome. Genome-wide association studies (GWAS) are based on whole-genome resequencing to screen molecular markers that associate traits with variant sites. They are an ideal method for identifying variant sites associated with complex traits.

[0004] The body size of sheep is related to the growth and development of muscles and bones, as well as the digestive and metabolic efficiency of the animal's body. IGSF8 Located in chromosome 1 of Tianhua sheep, it is reported to be involved in the animal body bone mass and bone homeostasis regulation network: for example, in mice, IGSF8 The mutant mice exhibited shorter femur length. DCAF8 It is also located on chromosome 1 of Tianhua sheep. It encodes cullin-associated factor 8, which can bind to muscle-specific ring finger protein and target MyHC, causing muscle atrophy. PIGM and CASQ1 In pigs, an association with muscle development has been reported. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide SNP markers related to the chest circumference of Tianhua mutton sheep and their applications, so as to accelerate the breeding process of Tianhua mutton sheep and improve the breeding efficiency.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a SNP marker associated with the chest circumference of Tianhua mutton is disclosed. The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1. The 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site. The SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the SNP site is T or G.

[0008] Preferably, the chest circumference of Tianhua mutton individuals with GG and TG genotypes of the SNP markers associated with the chest circumference of Tianhua mutton is significantly higher than that of Tianhua mutton individuals with TT genotype.

[0009] The second aspect of the present invention discloses the use of the above-mentioned SNP markers associated with the chest circumference of Tianhua mutton sheep in the breeding of Tianhua mutton sheep.

[0010] The third aspect of the present invention discloses the use of the above-mentioned SNP markers associated with chest girth of Tianhua mutton sheep in the prediction of chest girth traits of Tianhua mutton sheep.

[0011] The fourth aspect of the present invention discloses the use of the above-mentioned SNP markers associated with the chest girth of Tianhua mutton sheep in the preparation of a reagent for predicting the chest girth trait of Tianhua mutton sheep.

[0012] The fifth aspect of the present invention discloses the use of the above-mentioned SNP markers associated with the chest girth of Tianhua mutton sheep in the preparation of a kit for screening and identifying Tianhua mutton sheep strains with wide chest girth.

[0013] The sixth aspect of the present invention discloses a method for detecting the chest girth trait of Tianhua mutton sheep, by detecting the above-mentioned SNP markers related to the chest girth of Tianhua mutton sheep to be tested, and predicting the chest girth trait of the Tianhua mutton sheep to be tested.

[0014] Preferably, the genomic DNA of the Tianhua mutton to be tested is extracted and PCR amplified, and then the PCR amplification product is sequenced by HISEQ2000, SOLiD, 454 or single-molecule sequencing method, and the genotype of the SNP marker of the Tianhua mutton to be tested is determined according to the sequencing results to predict the chest girth trait of the Tianhua mutton.

[0015] Preferably, the chest circumference of Tianhua mutton sheep individuals with GG and TG genotypes at the SNP marker site is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

[0016] A seventh aspect of the present invention discloses a method for screening Tianhua mutton sheep having a wide chest girth trait, comprising detecting SNP markers associated with the chest girth of Tianhua mutton sheep, selecting Tianhua mutton sheep with a genotype of GG or TG as breeding stock for the wide chest girth Tianhua mutton sheep line, and eliminating Tianhua mutton sheep with a TT genotype;

[0017] The sequence of the SNP marker is shown in SEQ ID NO. 1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO. 1 is T or G.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a SNP marker associated with the chest girth of Tianhua mutton. Based on the whole genome resequencing of Tianhua mutton, the SNP molecular marker rs399622340 significantly associated with the chest girth of Tianhua mutton was screened. The chest girth of Tianhua mutton with different SNP genotypes was significantly different. The average chest girth of the genotype of the SNP molecular marker TT was 110.1 cm, the average chest girth of the genotype of TG was 114.4 cm, and the average chest girth of the genotype of GG was 117.0 cm, indicating that the chest girth of Tianhua mutton GG type was significantly higher than that of TG type, and that the chest girth of TG type was significantly higher than that of TT type ( P <0.01) The G allele has a positive effect on chest girth in Tianhua mutton sheep. Therefore, the SNP (rs399622340) molecular marker was applied in breeding practice to select Tianhua mutton sheep with GG or TG morphs as large-bodied breeding stock. This would accelerate the selection process for body conformation traits in Tianhua mutton sheep and provide a basis and material for molecular marker-assisted selection for chest girth, thereby improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The Manhattan plot of the SNP effect distribution of the whole genome and the SNP site quantile plot of the chest girth of Tianhua mutton sheep in Example 1 of the present invention are shown;

[0021] Figure 2 This is a genome-wide SNP quantile map of chest circumference of Tianhua mutton sheep in Example 1 of the present invention;

[0022] Figure 3 This is a statistical analysis result of the chest circumference of Tianhua mutton sheep in each genotype group of the SNP in Example 2 of the present invention; wherein, *** represents P <0.01. DETAILED DESCRIPTION

[0023] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0024] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0025] All features, such as values, amounts, contents, and concentrations, described herein as numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0026] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0027] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0028] It's important to note that SNP (single nucleotide polymorphism) is a type of molecular genetic marker proposed in 1996 by Lander, a researcher at the Human Genome Research Center at the Massachusetts Institute of Technology. It primarily refers to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. SNPs represent polymorphisms involving variations in only a single base, manifesting as transitions, transversions, insertions, and deletions.

[0029] The present invention provides a SNP marker associated with the chest girth of Tianhua mutton. The nucleotide sequence is shown in SEQ ID NO.1 in Table 1. The polymorphic site of the SNP marker associated with the chest girth of Tianhua mutton is located at position 151 from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is T / G. That is, the SNP marker associated with the chest girth of Tianhua mutton is located at position 110958643 of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, located at IGSF9Genetically, the rs399622340 molecular marker (ARS-Ul Ramb v2.0 / XR_006061305.2 / Chr1: 110958493- 110958793 / "T":rs399622340) is significantly associated with chest circumference in Tianhua mutton sheep and can be used for early prediction of chest circumference and marker-assisted selection breeding in Tianhua mutton sheep.

[0030] Table 1 Sequence Listing

[0031]

[0032] The present invention also provides a method for selecting and breeding the chest circumference of Tianhua mutton sheep, detecting the above-mentioned SNP in the genome of Tianhua mutton sheep, selecting individuals with genotypes of GG or TG as breeding sheep for the large-bodied Tianhua mutton sheep breed, and eliminating individuals with TT genotypes.

[0033] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0034] The following examples utilize conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art.

[0035] In the following embodiments, the method for SNP marker detection in the Tianhua mutton to be tested is not particularly limited. Sequencing, single strand conformation polymorphism polymerase chain reaction (PCR single strand conformation polymorphism, PCR-SSCP), restriction fragment length polymorphism polymerase chain reaction (PCR-restriction fragment length polymorphism, PCR-RFLP) and time-of-flight mass spectrometry and other technologies can all achieve the detection of SNP. Among them, sequencing is a detection technology with the highest accuracy, strong flexibility, large throughput and short detection cycle. It is only necessary to design a pair of primers upstream and downstream of the SNP site to amplify the product, and then the genotype of the SNP site can be directly detected by sequencing. Therefore, the present invention adopts the method of sequencing to carry out SNP marker detection. According to some specific examples of the present invention, the Tianhua mutton to be tested is subjected to the detection of the SNP markers described above, and the Tianhua mutton with the wide chest girth trait is screened.

[0036] In the following examples, the method for sequencing the PCR amplification products is not particularly limited, so long as the sequence of the PCR amplification product, i.e., the fragment containing the SNP marker associated with chest circumference of Tianhua mutton, can be effectively obtained. According to some specific examples of the present invention, the PCR amplification products can be sequenced using at least one method selected from HISEQ2000, SOLiD, 454, and single-molecule sequencing. This allows for high-throughput, rapid, efficient, and accurate sequencing results.

[0037] Example 1 Obtaining SNP markers associated with chest circumference of Tianhua sheep

[0038] 1. A total of 540 Tianhua mutton sheep were selected from two large-scale breeding farms for the experimental group. Chest circumference of the experimental group was measured according to the Chinese agricultural industry standard "Technical Specifications for Determination of Breeding Sheep Production Performance" (NY / T 1236-2023): With the sheep standing upright on a firm, flat surface, a tape measure was used to measure the length of the thorax from the rear end of the shoulder blade to the chest in centimeters (cm), rounded to one decimal place. The age, breeding, and sex of the experimental group were recorded. Chest circumference data were summarized using Excel. After excluding extreme values ​​outside the range of "mean ± 3 standard deviations," chest circumference data for the remaining 533 Tianhua mutton sheep were used for subsequent analysis (Table 2).

[0039] Table 2 Descriptive statistics of chest circumference of Tianhua sheep

[0040]

[0041] 2. 4-5 mL of whole blood was collected from 533 individuals using the jugular vein blood collection method in EDTA anticoagulant tubes, transported at low temperature and stored at -20°C in the laboratory. ® DNA Library Prep Kit for ILM was used to construct resequencing libraries for DNA that passed the quality inspection. 200 ng of DNA that passed the quality inspection was placed in a 0.2 mL PCR tube and 4 μL of GenoBaits was added to the tube. ® End Repair Buffer and 2.6 μL GenoBaits ® End Repair Enzyme, add water to 20 μL, place in ABI 9700 PCR instrument, incubate at 37℃ for 20 min, denature at 72℃ for 20 min to complete DNA fragmentation, end repair and A-tailing process. Add 2 μL GenoBaits ® Ultra DNA Ligase, 8 μL GenoBaits ®Ultra DNA Ligase Buffer and 4 μL GenoBaits® Adapter for ILM were added to 40 μL of water and placed on an ABI 9700 PCR instrument at 22°C for 60 min to complete the ligation of sequencing adapters. 48 μL of GenoPrep DNA Clean Beads were then added to the ligation product to purify the ligation product. After purification, fragments were screened using 0.68 + 0.2x magnetic beads, and ligation products with inserts between 300 and 350 bp were retained. 10 μL of sequencing adapters with barcode sequences and 10 μL of GenoBaits® Adapter for ILM were added to the PCR tube from the previous step. ® PCR Master Mix was added and the volume was filled to 20 μL. Amplification was performed using an ABI 9700 PCR instrument with the following program: 98°C pre-denaturation for 2 min, 98°C denaturation for 30 s, 65°C annealing for 30 s, and 72°C extension for 40 s for five cycles, followed by 72°C extension for 4 min. 20 μL of GenoPrep DNA Clean Beads were added to the second-round PCR product and placed on a magnetic rack until the solution clarified. The supernatant was discarded, and the beads were washed with 100 μL of 80% ethanol. 35 μL of 10 mM Tris-HCl was added to obtain the purified DNA library. After library construction, preliminary quantification was performed using Qubit 2.0, and the effective concentration of the library was accurately quantified by qPCR to ensure library quality. After passing the quality test, the library was sequenced using the BGI-2000 / MGI-T7 sequencing platform in PE150 mode.

[0042] 3. Clean reads were filtered from raw reads using fastp (version 0.20.0). Paired reads containing more than 10 N bases or containing more than 40% of low-quality (Q ≤ 20) bases were removed. After filtering, qualified sequences were aligned to the NCBI sheep reference genome ARS-Ul Ramb v2.0 (GCF 016772045.1) using BWA-MEM (0.7.13-r1126). The aligned bam files were sorted using Picard v2.18.2 (http: / / broadinstitute.github.io / picard / ) to remove duplicate sequences. Qualimap (v2.2) was used to calculate the alignment rate and average depth for all samples. Finally, the HaplotypeCaller, CombineGVCFs, GenotypeGVCFs, and VariantFiltratio tools in the Genome Analysis Toolkit (GATK, version 3.6-0-g89b7209) software package were used to identify SNP loci. Multiple GVCF files were merged into a single GVCF file using the CombineGVCFs module, and the merged GVCF file was processed using the GenotypeGVCFs module. The final VCF file was output. PLINK software was used to remove SNPs with a missingness rate >2% and a minor allele frequency <5% in 533 samples, as well as SNPs located on sex chromosomes. This resulted in a 75.41 Gb VCF file containing 25,544,659 SNPs. PLINK software was also used to generate a principal component file for this VCF file.

[0043] 4. The rMVP software package was used to perform an association analysis between the detected SNPs and chest circumference. The first three principal components were used, and field, age, sex, and pseudo-quantitative trait nucleotides (QTNs) were added as covariates. The association model was the FarmCPU model:

[0044] y = Xb i + G j b j + Z k D k + e

[0045] in,y is the chest phenotype value vector, X is a fixed effect (including session, age, gender and the first three principal components), bi Represents the corresponding vector effect, G j As a fixed effect j QTNs genotype matrix, b j For SNP effect carriers, Z k is the matrix of random effects, D k is a random individual being predicted, e are random residuals.

[0046] 5. Use -log 10 P = 6 was used as the significant threshold at the genomic level to screen significant association sites, and bedtools software was used to align the significant SNPs to the sheep reference genome (ARS-Ul Ramb v2.0) for gene annotation.

[0047] 6. GWAS results showed that 59 SNPs from 110849007 bp to 113750956 bp on chromosome 1 of Tianhua sheep were significantly associated with chest circumference ( Figure 1 and Figure 2 ), which contains IGSF8 、 IGSF9 、 PIGM 、 CASQ1 、 USF1 、 NDUS2 and DCAF8 , these genes are all related to bone development or muscle development. Among them, SNP (rs399622340) is located IGSF9 Genetically. Detailed information is as follows (Table 3):

[0048] Table 3 SNP information

[0049]

[0050] Example 2 Detection of the effect of SNP markers

[0051] The typing results of SNP (rs399622340) in 533 individuals were extracted using VCFtools software, and the association between different genotypes of this SNP and chest circumference was analyzed using the one-way analysis of variance multiple comparison model in Graphpad Prism software (Table 4).

[0052] Table 4 Association between SNPs and chest circumference

[0053]

[0054] The above results showed that SNP (rs399622340) significantly affected the chest circumference of Tianhua sheep, among which the chest circumference of GG type was significantly higher than that of TG type, and that of TG type was significantly higher than that of TT type ( P <0.01) ( Figure 3 The G allele has a positive effect on the chest girth of Tianhua sheep. In breeding practice, GG or TG type individuals can be selected as large-bodied breed sheep.

[0055] Example 3: Breeding Tianhua Sheep with Wide Chest Girth Trait Using SNP Markers Related to Chest Girth of Tianhua Sheep

[0056] A pair of primers were designed upstream and downstream of the SNP (rs399622340) molecular marker to amplify the product, and then the PCR amplification product was sequenced by HISEQ2000, SOLiD, 454 or single-molecule sequencing methods. The genotype of the SNP (rs399622340) molecular marker of the Tianhua mutton sheep to be tested was determined according to the sequencing results. Tianhua mutton sheep individuals with a genotype of GG or TG were selected as breeding sheep for the large-bodied Tianhua mutton sheep breed, and individuals with the TT genotype were eliminated to achieve the selection and breeding of Tianhua mutton sheep for the chest girth trait.

[0057] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of SNP markers associated with chest girth of Tianhua mutton sheep in Tianhua mutton sheep breeding, characterized in that: The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site. The SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the SNP site is T or G; the chest circumference of Tianhua mutton sheep individuals with GG and TG genotypes of the SNP marker associated with the chest circumference of Tianhua mutton is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

2. Application of SNP markers associated with chest girth of Tianhua mutton sheep in predicting chest girth traits of Tianhua mutton sheep, characterized in that: The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site. The SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the SNP site is T or G; the chest circumference of Tianhua mutton sheep individuals with GG and TG genotypes of the SNP marker associated with the chest circumference of Tianhua mutton is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

3. Use of a SNP marker associated with chest girth of Tianhua mutton sheep in the preparation of a reagent for predicting chest girth traits of Tianhua mutton sheep, characterized in that: The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site. The SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the SNP site is T or G; the chest circumference of Tianhua mutton sheep individuals with GG and TG genotypes of the SNP marker associated with the chest circumference of Tianhua mutton is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

4. Use of a SNP marker associated with chest girth of Tianhua mutton sheep in the preparation of a kit for screening or identifying a wide chest girth Tianhua mutton sheep strain, characterized in that: The nucleotide sequence of the SNP marker is shown in SEQ ID NO.1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site. The SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Rambv2.0, and the SNP site is T or G; the chest circumference of Tianhua mutton sheep individuals with GG and TG genotypes of the SNP marker associated with the chest circumference of Tianhua mutton is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

5. A method for detecting chest girth traits of Tianhua mutton sheep, characterized in that: The chest girth trait of the Tianhua mutton sheep to be tested is predicted by detecting SNP markers associated with the chest girth of the Tianhua mutton sheep to be tested; the nucleotide sequence of the SNP marker is shown in SEQ ID NO.1, the 151st base from the 5' end of the sequence shown in SEQ ID NO.1 is a SNP site, the SNP site is located at the 110958643 bp nucleotide of chromosome 1 of the sheep reference genome ARS-Ul Ramb v2.0, and the SNP site is T or G; the chest girth of Tianhua mutton sheep individuals with GG and TG genotypes of the SNP marker associated with the chest girth of Tianhua mutton sheep is significantly higher than that of Tianhua mutton sheep individuals with TT genotype.

6. The method for detecting chest girth traits of Tianhua mutton according to claim 5, characterized in that: The genomic DNA of the Tianhua mutton to be tested is extracted and PCR amplified, and the PCR amplification product is sequenced by HISEQ2000, SOLiD, 454 or single-molecule sequencing method. The genotype of the SNP marker of the Tianhua mutton to be tested is determined according to the sequencing results to predict the chest girth trait of the Tianhua mutton.

7. The method for detecting chest girth traits of Tianhua mutton according to claim 5, characterized in that: The chest circumference of Tianhua mutton sheep with GG and TG genotypes at the SNP marker site was significantly higher than that of Tianhua mutton sheep with TT genotype.

8. A method for screening Tianhua sheep with a wide chest girth trait, characterized in that: Detect SNP markers associated with chest girth in Tianhua mutton sheep, select Tianhua mutton sheep with genotype GG or TG as breeding sheep for wide chest girth Tianhua mutton sheep, and eliminate Tianhua mutton sheep with TT genotype; The sequence of the SNP marker is shown in SEQ ID NO. 1, and the 151st base from the 5' end of the sequence shown in SEQ ID NO. 1 is T or G.

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