Molecular marker affecting growth performance of fine-wool sheep and application thereof

Abstract

The application provides a molecular marker affecting the growth performance of fine wool sheep, wherein the molecular marker is located at the 25th chromosome positive strand of ARS-UI_Ramb_v3.0 version sequence information of a sheep reference genome, and the base is C or T at the 6782144th position, or the molecular marker is located at the 25th chromosome positive strand of ARS-UI_Ramb_v3.0 version sequence information of a sheep reference genome, and the base is A or G at the 6784190th position. The application also provides a method for early selection of the growth performance of fine wool sheep by using the molecular marker. The method can perform early selection on the growth performance of fine wool sheep, and has great economic application value and scientific research value.

Description

Technical Field

[0001] This application pertains to the fields of gene testing and animal husbandry. Specifically, this application provides a molecular marker that affects the growth performance of fine-wool sheep and its application. Background Technology

[0002] Fine-wool sheep are an important livestock breed, and their growth performance and wool quality are of great significance to livestock production. Improving the growth performance of fine-wool sheep has always been one of the major challenges in fine-wool sheep breeding. Among these, the selection of height, body length, weight, chest girth, cannon bone girth, and wool length is particularly important, as these are some of the most important indicators in the measurement of fine-wool sheep growth performance.

[0003] While traditional breeding methods have made some progress in selecting for the growth performance of fine-wool sheep, problems remain, including long breeding cycles, low accuracy, and high costs, leaving significant room for improvement. Genome-wide association analysis (GWAS) is one of the most important methods for identifying genetic links between phenotypes and genotypes, facilitating early selection for various traits in fine-wool sheep, saving production costs, and accelerating genetic progress. Single nucleotide polymorphisms (SNPs) are among the most common forms of genetic variation in the genome. Analyzing the genetic information of fine-wool sheep can help identify more stable and efficient SNP loci associated with their growth performance, enabling early molecular marker selection for important traits at the molecular level, which is of great significance for the selection of fine-wool sheep for growth performance. Summary of the Invention

[0004] This invention aims to provide an accurate and efficient method for predicting the growth performance of fine-wool sheep by detecting the genotype of specific SNP loci, thus providing a scientific basis for the breeding and raising of fine-wool sheep.

[0005] The SNP1 and SNP2 molecular markers described in this application correspond to positions 6782144 and 6784190 of the positive strand of chromosome 25 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information published in NCBI, respectively. The former has a base of C or T, and the latter has a base of A or G.

[0006] On the one hand, this application provides a molecular marker that affects the growth performance of fine-wool sheep. The molecular marker is located at position 6782144 on the positive strand of chromosome 25 of the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and the base is C or T. Alternatively, the molecular marker is located at position 6784190 on the positive strand of chromosome 25 of the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and the base is A or G.

[0007] On the other hand, this application provides a method for early selection of growth performance in fine-wool sheep, the method comprising:

[0008] (1) Extract genomic DNA from the fine-wool sheep to be tested;

[0009] (2) Detect the genotype of the above SNP sites in the genomic DNA extracted in step (1);

[0010] (3) Early selection of fine-wool sheep growth performance based on the genotypes obtained in step (2).

[0011] Furthermore, the fine-wool sheep are Brura Merino sheep, Chinese Merino sheep, Dexin fine-wool sheep, German Merino sheep, superfine Merino sheep, or Yunnan semi-fine-wool sheep.

[0012] Furthermore, the growth performance is one or more of the following: body height, body length, body weight, chest circumference, canal circumference, and hair length.

[0013] Furthermore, the growth properties are body height, body length, body weight, chest circumference, canal circumference, and hair length.

[0014] Further, step (1) of extracting genomic DNA from the fine-wool sheep to be tested includes: collecting blood from the jugular vein of the fine-wool sheep to be tested, treating it with an anticoagulant, then lysing and digesting it with a protease, and then extracting the genomic DNA using the phenol-formaldehyde method and dissolving it in sterile double-distilled water.

[0015] Furthermore, in step (2), resequencing, SNP genotyping based on gene chips, or SNP genotyping based on PCR are used to determine the genotype.

[0016] PCR-based typing methods include, but are not limited to, Sanger sequencing, Taqman-PCR, RFLP, ARMS, CAPS, KASP, and high-resolution melting curves.

[0017] Furthermore, in step (3), if SNP1 is heterozygous for CT and SNP2 is heterozygous for AG, the fine-wool sheep being tested is judged to have good growth performance in terms of body height, body length, weight, chest circumference and / or cannon circumference; if SNP1 is homozygous for CC and SNP2 is homozygous for AA, the fine-wool sheep being tested is judged to have good growth performance in terms of wool length.

[0018] On the other hand, this application provides the application of reagents for detecting the genotypes of the above-mentioned SNP sites in the preparation of a kit for early selection of growth performance in fine-wool sheep.

[0019] Furthermore, the fine-wool sheep are Brura Merino sheep, Chinese Merino sheep, Dexin fine-wool sheep, German Merino sheep, superfine Merino sheep, or Yunnan semi-fine-wool sheep.

[0020] Furthermore, the growth performance is one or more of the following: body height, body length, body weight, chest circumference, canal circumference, and hair length.

[0021] Furthermore, the growth properties are body height, body length, body weight, chest circumference, canal circumference, and hair length.

[0022] Furthermore, the reagents for detecting the genotype of the above-mentioned SNP sites are reagents for determining the genotype using resequencing methods, SNP genotyping methods based on gene chips, or SNP genotyping methods based on PCR.

[0023] PCR-based typing methods include, but are not limited to, Sanger sequencing, Taqman-PCR, RFLP, ARMS, CAPS, KASP, and high-resolution melting curves.

[0024] On the other hand, this application provides the application of the above-mentioned molecular markers or methods in fine hair breeding.

[0025] The beneficial effects of this invention are as follows: The two SNP molecular markers of this invention are associated with the growth performance of fine-wool sheep and are novel molecular markers. By establishing a linear regression model, the phenotype and genotype of the fine-wool sheep population to be tested can be linked. Determining the genotype of the corresponding SNP loci in the selected fine-wool sheep allows for early selection of their growth performance, saving production costs and accelerating genetic progress, thus better serving the breeding of fine-wool sheep and possessing significant economic and scientific research value. Attached Figure Description

[0026] Figure 1 This study shows the relationship between body height (BH) and SNP1 genotypes CC (n=3827) and CT (n=14).

[0027] Figure 2 This study shows the relationship between body height (BH) and SNP2 locus genotypes AA (n=3827) and AG (n=14).

[0028] Figure 3 This shows the relationship between body length (BL) and SNP1 genotypes CC (n=3827) and CT (n=14).

[0029] Figure 4 This shows the relationship between body length (BL) and SNP2 locus genotypes AA (n=3827) and AG (n=14).

[0030] Figure 5 This study shows the relationship between body weight (BW) and SNP1 genotypes CC (n=3736) and CT (n=14).

[0031] Figure 6 This shows the relationship between body weight (BW) and SNP2 locus genotypes AA (n=3736) and AG (n=14).

[0032] Figure 7 This study shows the relationship between SNP1 locus genotypes CC (n=3826) and CT (n=14) and chest circumference (CC).

[0033] Figure 8 This study shows the relationship between SNP2 locus genotypes AA (n=3826) and AG (n=14) and chest circumference (CC).

[0034] Figure 9 This shows the relationship between the SNP1 locus genotypes CC (n=3826) and CT (n=14) and the tube circumference (GW).

[0035] Figure 10 This shows the relationship between the SNP2 locus genotypes AA (n=3826) and AG (n=14) and the tube perimeter (GW).

[0036] Figure 11 This shows the relationship between hair length (WL) and the SNP1 locus genotypes CC (n=2739) and CT (n=14).

[0037] Figure 12 This shows the relationship between hair length (WL) and SNP2 locus genotypes AA (n=2739) and AG (n=14). Detailed Implementation

[0038] Example 1: Determination of the fine-wool sheep population to be tested

[0039] This study investigated populations of six different sheep breeds: Brumla Merino (BMF), China Merino (CMF), Dexin Fine Wool (DXF), German Merino (GMF), Superfine Merino (SCM), and Yunnan Semi-Fine Wool (YSF). The age and sample size distributions for each breed are as follows:

[0040] Brussels Merino (BMF):

[0041] Sampling age: 1 year and 2 years.

[0042] Sample size: 397 (1-year-olds) and 113 (2-year-olds).

[0043] Chinese Merino sheep (CMF):

[0044] Sampling age: 1 year and 1.1 years.

[0045] Sample size: 738 (1 year old) and 364 (1.1 years old).

[0046] Dexin Fine Wool Sheep (DXF):

[0047] Sampling age: 1 year old.

[0048] Sample size: 746.

[0049] German Merino sheep (GMF):

[0050] Sampling age: from 0.5 years to 5 years.

[0051] Sample sizes: 43 (0.5 years), 34 (0.8 years), 20 (1 year), 165 (2 years), 38 (3 years), 27 (4 years) and 32 (5 years).

[0052] Superfine Merino Lamb (SCM):

[0053] Sampling age: from 0.75 years to 10 years.

[0054] Sample size: 178 (0.75 years old), 166 (0.83 years old), 173 (1 year old), 42 (2 years old), 117 (3 years old), 43 (4 years old), 132 (5 years old), 62 (6 years old), 24 (7 years old), 19 (8 years old), 16 (9 years old) and 6 (10 years old).

[0055] Yunnan semi-fine wool sheep (YSF):

[0056] Sampling age: 1 year and 2 years.

[0057] Sample size: 46 (1 year old) and 43 (2 years old).

[0058] Example 2: Measurement of body height, body length, weight, chest circumference, cannon bone circumference, and hair length

[0059] In this study, the height, length, weight, chest circumference, cannon bone circumference, and wool length of the fine-wool sheep population were measured. The specific measurement methods are as follows: First, the sheep were securely fixed in place, and their height, length, and wool length were measured using a steel ruler; chest circumference and cannon bone circumference were measured using a tape measure; finally, their weight was accurately measured using a weighing scale. These measurement methods ensured the accuracy and consistency of the data, providing a reliable basis for subsequent analysis.

[0060] Example 3: Detection of SNP molecular markers

[0061] 1. Genomic DNA extraction

[0062] Take 20 μl of ACD-anticoagulated chicken blood sample, add 500 μl of cell lysis buffer and incubate for 30 min, then add 10 μl of 10% proteinase K and incubate overnight at 55°C. ACD anticoagulant: 1.32% (m / v) sodium citrate, 0.48% (m / v) citric acid, 1.47% (m / v) glucose. Proteinase K: purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0063] Add an equal volume of Tris-saturated phenol (pH 8.0) to the digested blood, slowly invert the centrifuge tube for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and carefully aspirate the supernatant into a clean centrifuge tube using a wide-bore pipette tip.

[0064] Repeat the previous step. If the supernatant is clear and transparent, this step can be omitted.

[0065] Add equal amounts of phenol:chloroform (1:1), slowly invert the centrifuge tube for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and carefully aspirate the supernatant into a clean centrifuge tube using a wide-bore pipette tip.

[0066] Add an equal volume of chloroform, slowly invert the centrifuge tube for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and carefully aspirate the supernatant into a clean centrifuge tube using a wide-bore pipette tip.

[0067] Add twice the volume of anhydrous ethanol, gently shake the centrifuge tube, and stop shaking when a white, misty precipitate appears.

[0068] Carefully pick out the DNA precipitate with a pipette tip and place it in a centrifuge tube containing 75% ethanol.

[0069] Remove the ethanol completely and allow it to evaporate at room temperature. Then, add an appropriate amount of TE buffer to dissolve the DNA.

[0070] Store the dissolved DNA at -20°C.

[0071] 2. Genotyping test

[0072] Resequencing technology was used. High-throughput sequencing was performed directly on the target regions of the extracted sheep genomic DNA using resequencing. After rigorous quality control, the sequencing data was compared with the sheep reference genome ARS-UI_Ramb_v3.0 sequence information to obtain the precise base sequences containing SNP1 and SNP2 loci. Specifically, SNP1 is located at position 6782144 on chromosome 25 of the sheep genome, and SNP2 is located at position 6784190.

[0073] Example 3: Establishing the association between growth phenotypic and genotype in fine-wool sheep

[0074] The phenotypic data (body height, body length, weight, chest girth, cannon bone girth, and wool length) of each fine-wool sheep were compiled and matched with the corresponding SNP1 and SNP2 locus genotype data. Linear regression was used to determine the potential linear correlation between genotype and growth performance indicators, and the corresponding linear regression equation was derived. The linear regression equation, y = a + bx, represents this relationship, where y is the growth performance indicator, x is the genotype variable, a is the intercept, and b is the regression coefficient. R02 The p-value measures the goodness of fit of the linear regression model. The p-value of the Kruskal-Wallis test is used to determine whether there are significant differences in growth performance indicators between different genotype groups. If the p-value is less than 0.05, then there are significant differences in growth performance indicators between different genotype groups, meaning there is a significant correlation between genotype and growth performance.

[0075] The specific data is as follows:

[0076] Figure 1 The relationship between body height (BH) and SNP1 genotypes CC (n=3827) and CT (n=14) is shown. The figure indicates that the overall body height values ​​for the CT genotype are higher than those for the CC genotype. The Kruskal-Wallis test p-value is 0.00026, indicating a high degree of significance in body height differences between the different genotypes. R 2 =0.0035, indicating that while the SNP1 locus genotype has a limited explanatory power for body height, it does have some correlation. The linear regression equation, y = 66 + 5.4x, further confirms the positive correlation between the CT genotype and higher body height.

[0077] Figure 2 The relationship between body height (BH) and SNP2 genotypes AA (n=3827) and AG (n=14) was shown. The BH value corresponding to the AG genotype was higher than that of the AA genotype. The Kruskal-Wallis test p-value was 0.00022, indicating a significant difference in body height between the different genotypes. 2 =0.0036, meaning that the SNP2 locus genotype has some explanatory power for body height. The linear regression equation is y = 65 + 6.2x, indicating that the AG genotype is significantly associated with higher body height.

[0078] Figure 3 The relationship between body length (BL) and the SNP1 genotypes CC (n=3827) and CT (n=14) was shown. The body length corresponding to the CT genotype was significantly higher than that of the CC genotype. The Kruskal-Wallis test p-value was 6.6e-05, indicating a highly significant difference in body length between the different genotypes. R 2 =0.0041, indicating that the SNP1 locus genotype has some explanatory power for body length. The linear regression equation, y = 58 + 17x, confirms that the CT genotype is positively correlated with longer body length.

[0079] Figure 4The relationship between body length (BL) and SNP2 locus genotypes AA (n=3827) and AG (n=14) was shown. The body length corresponding to the AA genotype was higher than that of the AG genotype. The Kruskal-Wallis test p-value was 5.6e-05, and R0 was [not specified]. 2 =0.0042, and the linear regression equation is y = 57 + 17x, indicating that the AG genotype is significantly associated with longer body length.

[0080] Figure 5 This study demonstrates the relationship between body weight (BW) and SNP1 genotypes CC (n=3736) and CT (n=14). The BW value corresponding to the CT genotype is higher than that of the CC genotype. The Kruskal-Wallis test p-value is 0.00034, indicating a significant difference in body weight between the different genotypes. 2 =0.0034, indicating that the SNP1 locus genotype has some explanatory power for body weight. The linear regression equation is y = 32 + 18x, showing that the CT genotype is significantly associated with higher body weight.

[0081] Figure 6 This study demonstrates the relationship between body weight (BW) and SNP2 genotypes AA (n=3736) and AG (n=14). The BW value corresponding to the AG genotype is higher than that of the AA genotype. The Kruskal-Wallis test p-value is 0.00029, indicating a significant difference in body weight between the different genotypes. 2 =0.0035, indicating that the SNP2 locus genotype has some explanatory power for body weight. The linear regression equation is y = 30 + 20x, showing that the AG genotype is significantly associated with higher body weight.

[0082] Figure 7 The relationship between chest circumference (CC) and the SNP1 genotypes of CC (n=3826) and CT (n=14) is shown. The figure indicates that the chest circumference corresponding to the CT genotype is greater than that of the CC genotype. The Kruskal-Wallis test p-value is 1.0e-06, showing that the difference in chest circumference between the different genotypes is highly significant. 2 =0.0062, indicating that the SNP1 locus genotype has some explanatory power for chest circumference. The linear regression equation, y = 87 + 17x, confirms that the CT genotype is positively correlated with larger chest circumference.

[0083] Figure 8 The relationship between SNP2 genotypes AA (n=3826) and AG (n=14) and chest circumference (CC) was shown. The chest circumference values ​​corresponding to the AG genotype were higher than those corresponding to the AA genotype. The Kruskal-Wallis test p-value was 6.9e-07, indicating a highly significant difference in chest circumference between the different genotypes.2 =0.0064, indicating that the SNP2 locus genotype has some explanatory power for chest circumference. The linear regression equation is y = 85 + 19x, showing that the AG genotype is significantly associated with a larger chest circumference.

[0084] Figure 9 The relationship between the SNP1 genotypes CC (n=3826) and CT (n=14) and canal circumference (GW) was shown. The GW value corresponding to the CT genotype was higher than that of the CC genotype. The Kruskal-Wallis test p-value was 3.4e-05, indicating that the difference in canal circumference between the different genotypes was highly significant. 2 =0.0045, indicating that the SNP1 locus genotype has some explanatory power for canal circumference. The linear regression equation is y = 7.3 + 2.8x, indicating that the CT genotype is significantly associated with larger canal circumference.

[0085] Figure 10 The relationship between the SNP2 locus genotypes AA (n=3826) and AG (n=14) and tube circumference (GW) was shown. The GW value corresponding to the AG genotype was higher than that of the AA genotype. The Kruskal-Wallis test p-value was 2.8e-05, indicating that the differences in tube circumference between the different genotypes were highly significant. 2 =0.0046, indicating that the SNP2 locus genotype has some explanatory power for tube circumference. The linear regression equation is y = 7 + 3x, showing that the AG genotype is significantly associated with a larger tube circumference.

[0086] Figure 11 This study demonstrates the relationship between hair length (WL) and SNP1 genotypes CC (n=2739) and CT (n=14). The hair length corresponding to the CT genotype is lower than that of the CC genotype. The Kruskal-Wallis test p-value is 0.0015, indicating a certain difference in hair length between different genotypes. 2 =0.0037, indicating that the SNP1 genotype has some explanatory power for hair length. The linear regression equation is y = 12 - 1.5x, showing that the CC genotype is significantly associated with longer hair length.

[0087] Figure 12 This study demonstrates the relationship between hair length (WL) and SNP2 genotypes AA (n=2739) and AG (n=14). The hair length corresponding to the AG genotype is lower than that of the AA genotype. The Kruskal-Wallis test p-value is 0.0022, indicating a certain difference in hair length between different genotypes. 2 =0.0034, indicating that the SNP2 locus genotype has some explanatory power for hair length. The linear regression equation is y = 11 - 1.4x, showing that the AA genotype is significantly associated with longer hair length.

[0088] Subsequently, the applicant randomly selected hundreds of individuals from various fine-wool sheep populations, including BMF, CMF, DXF, GMF, SCM, and / or YSF, for verification. Phenotypic determination and genotyping were performed using the methods described above. It was found that when the genotype based on SNP1 was CT heterozygous and the genotype based on SNP2 was AG heterozygous in the genomes of the tested fine-wool sheep populations, the height, length, weight, chest circumference, and cannon bone circumference of the fine-wool sheep were significantly increased. When the genotype based on SNP1 was CC homozygous and the genotype based on SNP2 was AA homozygous in the genomes of the tested fine-wool sheep individuals, the wool length of the fine-wool sheep was significantly increased.

[0089] Example 4: Application of early molecular marker-assisted selection of fine-wool sheep individuals

[0090] In this study, selected fine-wool sheep individuals were screened using early molecular marker-assisted selection technology. Specifically, during the embryonic stage of the fine-wool sheep, genomic DNA was extracted from their blood using the phenol-chloroform method, and gene chip technology was used to detect the genotypes of SNP1 and SNP2 loci. The detection steps were consistent with the previously described method. The screening criteria are as follows:

[0091] If the genotype of SNP1 in the genome of a fine-wool sheep is CT heterozygous and the genotype of SNP2 is AG heterozygous, then the phenotypic traits of the fine-wool sheep, such as height, length, weight, chest circumference, and cannon bone circumference, will be significantly improved.

[0092] If the genotype of SNP1 in the genome of a fine-wool sheep is homozygous (CC) and the genotype of SNP2 is homozygous (AA), then the wool length phenotype of that fine-wool sheep is significantly enhanced.

[0093] By using early molecular marker-assisted selection, the association between SNP locus genotypes and important economic traits in fine-wool sheep was clarified, providing a scientific basis for early trait prediction and selection of high-quality individuals in fine-wool sheep.

Claims

1. A method for early selection of fine wool or fine-wool crossbred sheep for growth performance, characterised by, The method comprises: (1) extracting genomic DNA of the fine-wool sheep or semi-fine-wool sheep to be tested; (2) detecting the genotypes of SNP sites in the genomic DNA extracted in step (1); the SNP sites are SNP1 located at the 25th chromosome of the positive strand of the ARS-UI_Ramb_v3.0 version sequence information of the sheep reference genome, and the base is C or T, and SNP2 located at the 25th chromosome of the positive strand of the ARS-UI_Ramb_v3.0 version sequence information of the sheep reference genome, and the base is A or G; (3) early selecting the growth performance of the fine-wool sheep or semi-fine-wool sheep based on the genotypes obtained in step (2); The fine-wool sheep or semi-fine-wool sheep is a Bluefaced Leicester sheep, a Chinese Merino sheep, a De Xin fine-wool sheep, a German Merino sheep, a super-fine Merino sheep or a Yunnan semi-fine-wool sheep; The growth performance is body height, body length, body weight, chest circumference, girth and wool length; In step (3), when SNP1 is CT heterozygous and SNP2 is AG heterozygous, it is judged that the fine-wool sheep or semi-fine-wool sheep to be tested has better growth performance in body height, body length, body weight, chest circumference and girth; if SNP1 is CC homozygous and SNP2 is AA homozygous, it is judged that the fine-wool sheep or semi-fine-wool sheep to be tested has better growth performance in wool length.

2. The method of claim 1, wherein the step (1) of extracting the genomic DNA of the fine-wool sheep or semi-fine-wool sheep to be tested comprises: The fine-wool sheep or semi-fine-wool sheep to be tested is subjected to jugular vein blood collection, and after anticoagulation treatment with an anticoagulant, lysis and protease digestion treatment, the genomic DNA is extracted by the phenol chloroform method and dissolved with sterilized double distilled water.

3. The method according to claim 1, wherein in step (2), the genotypes are determined by a resequencing method, a SNP typing method based on a gene chip or a SNP typing method based on PCR.

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