A method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool

Abstract

The present invention discloses a whole genome SNPs screening method related to wool traits of dry Chinese meat Merino, which belongs to the field of genetic marker detection, and includes the following steps: S1, blood sample collection; S2, extraction of DNA from frozen anticoagulated blood to obtain a solution containing free DNA; S3, DNA quality control detection; S4, complete enzyme digestion of whole genome DNA, recovery of enzyme-cut fragments with set length of insertion, library construction, and genome database; S5, double-end sequencing of the genome database to obtain whole genome SNPs; S6, analysis of GWAS between wool traits and whole genome SNPs. The present invention adopts the above-mentioned whole genome SNPs screening method related to wool traits of dry Chinese meat Merino, and can reveal the genetic basis of high-yield and high-efficiency wool traits of sheep by performing association analysis between genotypes and wool traits of sheep, thereby accelerating the breeding process of ultra-fine wool strains and improving breeding stability.

Description

Technical Field

[0001] The invention relates to the field of genetic marker detection, and in particular to a method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool. Background Art

[0002] Since the 1990s, due to the large-scale import of Australian wool, the domestic wool price has fallen, the benefits of fine-wool sheep farming have declined, and the meat sheep market has gradually matured, which has had an impact on the wool sheep industry. The focus of the sheep industry has gradually shifted from wool production to meat production, and the benefits of meat production have further increased the impact on the wool industry, causing farmers to choose to raise local sheep with poor wool quality and a large number of lambs in pursuit of meat production. How to deal with this market impact and select new varieties that can produce both wool and meat through breeding methods to achieve the effects of "maintaining wool and increasing meat" and "harvesting both meat and wool" is not only crucial to stabilizing the advantages of the fine-wool sheep industry, but also a question of the future development direction of my country's sheep industry.

[0003] In 2003, a company in Jilin Province used South African meat Merino sheep and hybridized them with Northeast fine-wool sheep with Australian blood, and formulated a breeding plan for a new breed of meat Merino sheep that can be used for both meat and wool. After 15 years of systematic breeding, it passed the national new livestock and poultry variety approval in 2018 and was named "Qianhua Meat Merino Sheep". The characteristics of this breed are that it produces both meat and wool, and its production performance has reached the international advanced level.

[0004] At the same time, whole-genome association analysis is to associate single nucleotide polymorphism markers covering the whole genome with phenotypes through statistical means to detect genetic variations that are most likely to affect the relevant phenotype. Whole-genome association analysis is a key technology for exploring the major effect genes of quantitative traits in sheep breeding at this stage, and it is also the main way to analyze the genetic mechanism of quantitative traits. Compared with traditional genomic positioning of quantitative traits, whole-genome association analysis based on SNPs and phenotypes has the advantages of being able to detect associated variations that have less impact on phenotypes and exploring associated variations in relatively narrow genomic regions.

[0005] With the advancement of genotyping technology and the development of genetic statistics, a large number of researchers have used whole genome association analysis to locate QTLs and identify candidate genes for sheep growth traits. Although whole genome association analysis related to sheep growth traits has been reported, wool traits are quantitative traits and are affected by micro-effect polygenes, so they are not suitable for the above analysis methods. Summary of the invention

[0006] To solve the above problems, the present invention provides a method for screening whole-genome SNPs associated with wool traits of Qianhua meat-producing Merino sheep. By performing association analysis between genotypes and wool traits of sheep, the genetic basis of high-yield and high-efficiency wool traits of sheep can be revealed, thereby accelerating the breeding process of ultra-fine wool strains and improving breeding stability.

[0007] To achieve the above object, the present invention provides a method for screening whole genome SNPs related to traits of dry Chinese meat Merino wool, comprising the following steps:

[0008] S1. Blood sample collection: draw blood from the vein of a Qianhua meat Merino sheep to obtain a whole blood sample, store the whole blood sample in liquid nitrogen, and then transfer it to a set temperature to obtain frozen anticoagulated blood for later use;

[0009] S2, extracting DNA from the frozen anticoagulated blood to obtain a solution containing free DNA;

[0010] S3, DNA quality control detection: respectively measure the concentration, purity and content of DNA in the solution in step S2. If the set requirements are met, proceed to step S4;

[0011] S4, using restriction endonucleases to completely digest the whole genome DNA, recovering the digested fragments with a set length, and building a library in the dd-RAD method to obtain a genome database;

[0012] S5. Perform double-end sequencing on the genome database using NGS technology based on the Illumina NovaSeq sequencing platform to obtain genome-wide SNPs.

[0013] S6. Analyze the GWAS between wool traits and genome-wide SNPs.

[0014] Preferably, the set temperature in step S1 is -80°C.

[0015] Preferably, step S2 specifically includes the following steps:

[0016] S21. Place the frozen anticoagulated blood at room temperature until it thaws;

[0017] S22, add 200 μl of anticoagulated blood to a 1.5 ml centrifuge tube, then add 20 μl of proteinase K, and mix well;

[0018] S23, add 200 μl of lysis buffer BLⅡ to a 1.5 ml centrifuge tube, vortex until mixed evenly, and then place in a water bath at 70°C for 10 min until the solution in the 1.5 ml centrifuge tube is clear;

[0019] S24, centrifuging after cooling to remove water droplets on the inner wall of the tube cover of the centrifuge tube;

[0020] S25, adding 100 μl of anhydrous ethanol to a 1.5 ml centrifuge tube, and vortexing until mixed uniformly to obtain a mixture;

[0021] S26, transferring the mixture to an adsorption column and centrifuging to discharge the waste liquid;

[0022] S27, add 500 μl of rinsing solution BWⅠ to the adsorption column, centrifuge, and discharge the waste liquid;

[0023] S28, add 600 μl of rinsing solution BWⅡ to the adsorption column, and discharge the waste liquid;

[0024] S29, repeat step S28;

[0025] S210, after leaving the room, open the lid and air dry at room temperature;

[0026] S211. Place the adsorption column in a new 1.5 ml centrifuge tube, vertically suspend it and drop 150 μl of elution solution BE. Let it stand at room temperature for 5 minutes and then centrifuge to obtain a solution containing free DNA.

[0027] Preferably, the centrifugation conditions of step S26 are: centrifugation at 12000 rpm for 60 s;

[0028] The centrifugation conditions of step S27 are: 12000 rpm for 30 s;

[0029] The centrifugation conditions of step S28 are: 12000 rpm for 30 s;

[0030] The centrifugation conditions of step S210 and step S211 are both: centrifugation at 12000 rpm for 2 min.

[0031] Preferably, in step S3, the concentration and purity of the DNA are detected by 0.7% agarose electrophoresis, and the DNA content is determined by ultraviolet spectrophotometer;

[0032] After the test, the collected DNA solution was stored at -20°C.

[0033] Preferably, step S4 specifically includes the following steps:

[0034] S41. Alignment to sheep genome: Use bwa mem program to align the data after restriction digestion and filtration to the reference genome, obtain a sam file recording the alignment results, and sort the sam file and convert it into a bam file;

[0035] S42, obtaining individual genotype information: performing genotype calling based on the comparison, and constructing an individual genotype data set;

[0036] S43, annotate SNPs: functionally annotate the variants based on the genotype information of the individuals in the genotype data set, and determine whether the SNPs are located in the coding region, non-coding region, or regulatory region of the gene;

[0037] S44. Population structure analysis: Perform model-based population admixture analysis to divide individuals into different genetic groups and determine the genetic relationships between and within groups to obtain a genome database.

[0038] Preferably, the parameters for comparison are based on the default parameters of bwa ​​mem;

[0039] The sam files were sorted and converted into bam files using picard 1.107 software.

[0040] Preferably, step S6 specifically includes the following steps:

[0041] S61. Establishment of GWAS analysis model:

[0042] Y=SNP+Gender+PCA+Kinship+e (1)

[0043] In the formula, SNP represents single nucleotide polymorphism, Gender represents gender, PCA represents principal component analysis, Kinship represents kinship, and e represents residual;

[0044] S62. Bonferroni was used to correct the P value of the significant difference level in the GWAS analysis results;

[0045] S63. Manhattan plots and QQ plots were drawn using qqman in R software to evaluate the association results.

[0046] The present invention has the following beneficial effects:

[0047] 1. Provide markers for the selection of traits for dry Chinese meat Merino wool;

[0048] 2. It can speed up the breeding process of ultra-fine wool strains and improve the stability of breeding.

[0049] 3. Revealing the genetic basis of high-yield and high-efficiency wool traits in sheep has important theoretical significance and reference value for exploring the functional genes of wool traits in Qianhua meat-producing Merino sheep.

[0050] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a flow chart of a method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool of the present invention;

[0052] Figure 2 This is a distribution diagram of SNPs on chromosomes in an embodiment of the present invention (the number of SNPs is counted in a 1Mb window);

[0053] Figure 3 It is a graph of the results of the capillary whole genome association analysis of an embodiment of the present invention;

[0054] Figure 4 This is a result diagram of the whole genome association analysis of hair length according to an embodiment of the present invention;

[0055] Figure 5 This is a diagram of the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis results of an embodiment of the present invention. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention are further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions.

[0057] It should be noted that the terms "include", "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or server comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or devices.

[0058] Like reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0059] In order to find relevant molecular markers, supplement the growth and development related QTL database of Qianhua meat Merino sheep, and mine the genetic markers and functional genes that affect the growth traits of Qianhua meat Merino sheep, the present invention is designed: as shown, a method for screening whole genome SNPs related to the wool traits of Qianhua meat Merino sheep, comprising the following steps:

[0060] S1. Blood sample collection: draw blood from the vein of a Qianhua meat Merino sheep to obtain a whole blood sample, store the whole blood sample in liquid nitrogen, and then transfer it to a set temperature to obtain frozen anticoagulated blood for later use;

[0061] The set temperature in step S1 is -80°C.

[0062] S2, extracting DNA from the frozen anticoagulated blood to obtain a solution containing free DNA;

[0063] Step S2 specifically includes the following steps:

[0064] S21. Place the frozen anticoagulated blood at room temperature until it thaws;

[0065] S22, add 200 μl of anticoagulated blood to a 1.5 ml centrifuge tube, then add 20 μl of proteinase K, and mix well;

[0066] S23, add 200 μl of lysis buffer BLⅡ to a 1.5 ml centrifuge tube, vortex until mixed evenly, and then place in a water bath at 70°C for 10 min until the solution in the 1.5 ml centrifuge tube is clear;

[0067] S24, centrifuging after cooling to remove water droplets on the inner wall of the tube cover of the centrifuge tube;

[0068] S25, adding 100 μl of anhydrous ethanol to a 1.5 ml centrifuge tube, and vortexing until mixed uniformly to obtain a mixture;

[0069] S26, transferring the mixture to an adsorption column and centrifuging to discharge the waste liquid;

[0070] The centrifugation conditions of step S26 are: 12000 rpm for 60 s;

[0071] S27, add 500 μl of rinsing solution BWⅠ to the adsorption column, centrifuge, and discharge the waste liquid;

[0072] The centrifugation conditions of step S27 are: 12000 rpm for 30 s;

[0073] S28, add 600 μl of rinsing solution BWⅡ to the adsorption column, and discharge the waste liquid;

[0074] The centrifugation conditions of step S28 are: 12000 rpm for 30 s;

[0075] S29, repeat step S28;

[0076] S210, after leaving the room, open the lid and air dry at room temperature;

[0077] S211. Place the adsorption column in a new 1.5 ml centrifuge tube, vertically suspend it and drop 150 μl of elution solution BE. Let it stand at room temperature for 5 minutes and then centrifuge to obtain a solution containing free DNA.

[0078] The centrifugation conditions of step S210 and step S211 are both: centrifugation at 12000 rpm for 2 min.

[0079] S3, DNA quality control detection: respectively measure the concentration, purity and content of DNA in the solution in step S2. If the set requirements are met, proceed to step S4;

[0080] In step S3, the concentration and purity of the DNA are detected by 0.7% agarose electrophoresis, and the DNA content is measured by ultraviolet spectrophotometer;

[0081] After the test, the collected DNA solution was stored at -20°C.

[0082] S4, using restriction endonucleases to completely digest the whole genome DNA, recovering the restriction fragments of the set length, and building a library according to the dd-RAD (digest restriction site-associated DNA sequencing) method to obtain a genome database;

[0083] Step S4 specifically includes the following steps:

[0084] S41. Alignment to the sheep genome: The data after restriction filtration were aligned to the reference genome using the bwa mem program to obtain a sam file recording the alignment results, and the sam file was sorted and converted into a bam file; the alignment parameters were all in accordance with the default parameters of bwa ​​mem; the picard1.107 software was used to sort the sam file and convert it into a bam file.

[0085] In this embodiment, the "FixMatelnformation" command is used to ensure the consistency between all paired-endreads information.

[0086] And because the reads near InDel are most prone to Mapping errors, in order to minimize SNPs caused by Mapping errors, the reads near InDel need to be realigned to improve the accuracy of SNP mapping. The IndelRealigner command in the GATK program is used to realign all reads near InDel to improve the accuracy of SNP prediction.

[0087] S42, obtaining individual genotype information: performing genotype calling based on the comparison, and constructing an individual genotype data set;

[0088] S43, annotate SNPs: functionally annotate the variants based on the genotype information of the individuals in the genotype data set, and determine whether the SNPs are located in the coding region, non-coding region, or regulatory region of the gene;

[0089] S44. Population structure analysis: Perform model-based population admixture analysis to divide individuals into different genetic groups and determine the genetic relationships between and within groups to obtain a genome database.

[0090] S5. Use NGS technology (Next-Generation Sequencing) based on the Illumina NovaSeq sequencing platform to perform double-end sequencing on the genome database to obtain genome-wide SNPs.

[0091] S6. Analyze the GWAS (genome-wide association study) between wool traits and genome-wide SNPs.

[0092] Step S6 specifically includes the following steps:

[0093] S61. Establishment of GWAS analysis model:

[0094] Y=SNP+Gender+PCA+Kinship+e (1)

[0095] In the formula, SNP represents single nucleotide polymorphism, Gender represents gender, PCA represents principal component analysis, Kinship represents kinship, and e represents residual;

[0096] S62. Bonferroni was used to correct the P value of the significant difference level in the GWAS analysis results;

[0097] S63. Manhattan plots and QQ plots were drawn using qqman in R software to evaluate the association results.

[0098] Example

[0099] In this embodiment, 200 individuals (55 rams and 145 ewes) born from January 2020 to August 2021 were randomly selected, and the hair length, capillary fineness, and dirty hair weight were measured, and blood samples were collected.

[0100] The above individuals were analyzed for screening of whole genome SNPs related to wool traits using the Qianhua meat Merino wool traits screening method of the present invention, and the screened SNPs were filtered using vcftools software according to MAF less than 0.05 and missing rate greater than 0.2. A total of 335,682 SNPs were obtained for whole genome association analysis of 11 phenotypic traits, and the model used was Y=SNP+Gender+PCA+Kinship+e; then, based on LD attenuation, the 500,000 bp region before and after the significant site was determined as the candidate gene region significantly associated with the trait, and the candidate gene was searched.

[0101] Table 1 Capillary screening results

[0102]

[0103]

[0104] Table 2 Hair length screening results

[0105]

[0106] Combining Table 1, Table 2 and Figure 2 - Figure 5 It can be seen that 5 significant SNPs were screened for capillary fineness, which were located on chromosomes 3, 9, 9, 13, and 19 respectively; 3 significant SNPs were screened for hair length, which were located on chromosomes 15, 18, and 25 respectively, and no significant SNPs were screened for dirty hair weight. A total of 47 genes related to hair quality traits were identified for fineness and hair length, including AVPR1A, RXYLT1, SRGAP1, LOC114116487 (GSDML), GSDMC, LOC11411645FAM49B, ASAP1, TRNAG-CCC-64, RIDA, RPL30, LOC114116536, ERICH5, LAPTM4B, MTDH, NIPAL2, POP1, and MATN2.

[0107] It should be noted that in Figure 5 In the , the degree of enrichment is measured by Rich factor, FDR value and the number of genes enriched in this pathway. Rich factor refers to the ratio of the number of candidate genes enriched in the pathway to the number of annotated genes. The larger the Rich factor, the greater the degree of enrichment. FDR (false discovery rate) generally ranges from 0 to 1. The closer it is to zero, the more significant the enrichment.

[0108] After selecting and breeding individuals carrying genes related to the above-mentioned wool traits, cross-breeding is carried out and individuals carrying the above-mentioned wool traits are used as the basic group to establish an ultra-fine wool breed.

[0109] Therefore, the present invention adopts the above-mentioned whole-genome SNPs screening method related to the wool traits of Qianhua meat-producing Merino sheep. By performing association analysis between the genotype and the wool traits of sheep, the genetic basis of high-yield and high-efficiency wool traits of sheep can be revealed, thereby accelerating the breeding process of ultra-fine wool strains and improving breeding stability.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool, characterized in that: The following steps are involved: S1. Blood sample collection: draw blood from the vein of a Qianhua meat Merino sheep to obtain a whole blood sample, store the whole blood sample in liquid nitrogen, and then transfer it to a set temperature to obtain frozen anticoagulated blood for later use; S2, extracting DNA from the frozen anticoagulated blood to obtain a solution containing free DNA; S3, DNA quality control detection: respectively measure the concentration, purity and content of DNA in the solution in step S2. If the set requirements are met, proceed to step S4; S4, using restriction endonucleases to completely digest the whole genome DNA, recovering the digested fragments with a set length, and building a library in the dd-RAD method to obtain a genome database; Step S4 specifically includes the following steps: S41, alignment to sheep genome: using bwa mem program to align the data after restriction digestion and filtration to the reference genome, obtaining a sam file recording the alignment results, and sorting the sam file and converting it into a bam file; S42, obtaining individual genotype information: performing genotype calling based on the comparison, and constructing an individual genotype data set; The parameters for the comparison were all based on the default parameters of bwa ​​mem; The sam files were sorted and converted into bam files using picard 1.107 software; S43, annotate SNPs: functionally annotate the variants based on the genotype information of the individuals in the genotype data set, and determine whether the SNPs are located in the coding region, non-coding region, or regulatory region of the gene; S44. Population structure analysis: Perform model-based population admixture analysis to divide individuals into different genetic groups and determine the genetic relationships between and within groups to obtain a genome database; S5. Perform double-end sequencing on the genome database using NGS technology based on the Illumina NovaSeq sequencing platform to obtain genome-wide SNPs. S6, GWAS analysis between wool traits and genome-wide SNPs; Step S6 specifically includes the following steps: S61. Establishment of GWAS analysis model: （1） In the formula, represents single nucleotide polymorphism, Indicates gender, represents principal component analysis, Indicates kinship, represents the residual; S62. Bonferroni was used to correct the P value of the significant difference level in the GWAS analysis results; S63. Manhattan plots and QQ plots were drawn using qqman in R software to evaluate the association results.

2. The method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool according to claim 1, characterized in that: The set temperature in step S1 is -80°C.

3. The method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool according to claim 1, characterized in that: Step S2 specifically includes the following steps: S21. Place the frozen anticoagulated blood at room temperature until it thaws; S22, add 200 μl of anticoagulated blood to a 1.5 ml centrifuge tube, then add 20 μl of proteinase K, and mix well; S23, add 200 μl of lysis buffer BLⅡ to a 1.5 ml centrifuge tube, vortex until mixed evenly, and then place in a water bath at 70°C for 10 min until the solution in the 1.5 ml centrifuge tube is clear; S24, centrifuging after cooling to remove water droplets on the inner wall of the tube cover of the centrifuge tube; S25, adding 100 μl of anhydrous ethanol to a 1.5 ml centrifuge tube, and vortexing until mixed uniformly to obtain a mixture; S26, transferring the mixture to an adsorption column and centrifuging to discharge the waste liquid; S27, add 500 μl of rinsing solution BWⅠ to the adsorption column, centrifuge, and discharge the waste liquid; S28, add 600 μl of rinsing solution BWⅡ to the adsorption column, and discharge the waste liquid; S29, repeat step S28; S210, after leaving the room, open the lid and air dry at room temperature; S211. Place the adsorption column in a new 1.5 ml centrifuge tube, vertically suspend it and drop 150 μl of elution solution BE. Let it stand at room temperature for 5 minutes and then centrifuge to obtain a solution containing free DNA.

4. The method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool according to claim 3, characterized in that: The centrifugation conditions of step S26 are: 12000 rpm for 60 s; The centrifugation conditions of step S27 are: 12000 rpm for 30 s; The centrifugation conditions of step S28 are: 12000 rpm for 30 s; The centrifugation conditions of step S210 and step S211 are both: centrifugation at 12000 rpm for 2 min.

5. The method for screening whole genome SNPs related to traits of dried Chinese meat Merino wool according to claim 1, characterized in that: In step S3, the concentration and purity of the DNA are detected by 0.7% agarose electrophoresis, and the DNA content is measured by ultraviolet spectrophotometer; After the test, the collected DNA solution was stored at -20°C.

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