Molecular marker related to early life weight of Tan sheep on chromosome 2 and application of molecular marker

Abstract

The invention provides a molecular marker site related to an initial life heavy character on a Tan sheep chromosome 2, wherein the molecular marker site is SNPs mutation on the Tan sheep chromosome 2. By optimizing the dominant alleles of the markers, the frequency of the dominant alleles can be increased generation by generation, the primary living weight character of the Tan sheep is improved, and the genetic improvement process of the Tan sheep is accelerated, so that the economic benefit of Tan sheep breeding is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology breeding, specifically involving molecular markers on the chromosomes of Tan sheep that are related to the primary life traits of Tan sheep and their applications. Background Technology

[0002] Tan sheep are a superior local sheep breed in my country, mainly distributed in the central and northern parts of Ningxia Hui Autonomous Region, as well as neighboring areas such as Inner Mongolia, Gansu, and Shaanxi. They are named after the Tan grasslands on the southern slopes of the Helan Mountains in Ningxia, where they inhabit high plains and low hills at altitudes of 1000-2000 meters. Adapted to arid and desertified environments, they possess a robust physique and are resistant to roughage, grazing year-round. Tan sheep meat is tender, with evenly distributed fat and almost no gamey odor, making it a highly sought-after culinary ingredient.

[0003] Early birth weight is a key indicator of the early growth and development of Tan sheep, directly affecting lamb survival rate, health status, and later growth performance and production potential. Lambs with higher early birth weight generally have stronger environmental adaptability, higher disease resistance, and faster growth rates, effectively reducing mortality and rearing costs during the breeding process and significantly improving the economic benefits of sheep farming. Conversely, lambs with excessively low early birth weight often face a higher risk of mortality and stunted growth, resulting in significant losses in sheep farming. Therefore, improving the early birth weight trait of Tan sheep is of great significance for improving the overall efficiency of Tan sheep farming and the sustainable development of the industry. Traditional Tan sheep breeding relies on phenotypic selection, which is susceptible to environmental factors, leading to low selection accuracy and a long breeding cycle, making it difficult to meet the efficiency requirements of modern animal husbandry. With the rapid development of molecular biology techniques, molecular marker-assisted breeding technology has provided a new approach to livestock and poultry breeding. By identifying molecular markers associated with target traits, early and precise selection of these traits can be achieved, unaffected by environmental factors, significantly shortening the breeding cycle and improving breeding efficiency. Currently, while some molecular marker studies related to growth traits have been conducted in sheep breeding, research on molecular markers for the initial life weight trait of the Tan sheep breed is relatively scarce. Furthermore, the genetic backgrounds of different breeds vary, limiting the applicability of these molecular markers to Tan sheep. Therefore, it is urgent to identify molecular markers closely related to the initial life weight trait of Tan sheep, and to utilize marker-assisted breeding techniques to eliminate genotypes detrimental to this trait, thereby improving the initial life weight trait. This has significant theoretical and practical implications for improving the efficiency of Tan sheep farming and protecting and utilizing Tan sheep genetic resources. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides molecular markers that are significantly associated with the early life weight traits of Tan sheep through genotyping and correlation analysis strategies. These markers are then used in marker-assisted selection and genomic selection to select favorable genotypes for breeding, thereby increasing the gene frequency of dominant alleles generation by generation. This accelerates the process of genetic breeding improvement of Tan sheep and brings significant economic benefits to local Tan sheep farming.

[0005] The primary objective of this invention is to identify the SNPs (species nuclei) that influence the initial life weight trait of Tan sheep. These molecular markers are located on chromosome 2 of the Tan sheep genome (reference sequence NC_056055.1), and the specific SNP information is as follows: Table 1 SNP locus information The molecular marker SNP1 C>T is located at position 201 of the sequence in SEQ ID NO.1, the molecular marker SNP2 A>G is located at position 202 of SEQ ID NO.1, and the molecular marker SNP3 C>G is located at position 201 of the sequence in SEQ ID NO.2. SEQ ID NO.1 SEQ ID NO.2 Another object of the present invention is to provide a chip for detecting the initial birth weight of Tan sheep, the chip comprising primers and probes for amplifying and detecting the aforementioned molecular markers.

[0006] Another object of the present invention is to provide a kit for detecting the initial birth weight of Tan sheep, the kit comprising primers and probes for amplifying and detecting the aforementioned molecular markers.

[0007] Another object of the present invention is to provide the application of the aforementioned molecular markers and / or the aforementioned chips and / or the aforementioned kits in improving the birth weight trait of Tan sheep or in breeding Tan sheep for the purpose of improving birth weight.

[0008] In a preferred embodiment, the specific method for applying the above-mentioned molecular markers and / or chips and / or kits in improving the initial life weight trait of Tan sheep or in breeding for the initial life weight of Tan sheep is as described below in the method for improving the initial life weight trait of Tan sheep.

[0009] This invention also provides a method for improving the initial weight trait of Tan sheep, characterized in that the method includes the following steps: 1) Determine the genotypes of the aforementioned SNP loci in the Tan sheep population; 2) Make appropriate selections based on the genotype of the SNP locus and the breeding objectives.

[0010] In a preferred embodiment, step 1) includes the following steps: 1.1) Extract genomic DNA from the sheep to be tested; 1.2) Genotyping of the Tan sheep to be tested; 1.3) Based on the detection results, determine the genotype of the aforementioned SNP sites in the Tan sheep resource population to be tested.

[0011] In a preferred embodiment, step 2) includes any one of steps 2.1)-2.3): 2.1) In a Tan sheep resource population, individuals with the heterozygous TC type at the SNP1 locus on chromosome 2 are culled to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep. 2.2) In one of the Tan sheep resource populations, individuals with a heterozygous GA type at the SNP2 locus on chromosome 2 are culled to increase the frequency of the AA genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep; 2.3) In one of the Tan sheep resource populations, individuals with a heterozygous GC genotype at the SNP3 locus on chromosome 2 are culled to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep.

[0012] In a preferred embodiment, step 2) includes: In a Tan sheep population, individuals with the genotype TC_GA on chromosome 2 containing the SNP1 and SNP2 combination were culled to increase the frequency of the CC_AA genotype, thereby increasing the initial life weight of the offspring Tan sheep. Alternatively, individuals with the genotype GC_TC on chromosome 2 of Tan sheep with the combination of SNP3 and SNP1 can be culled in a Tan sheep population to increase the frequency of the GC_CC, CC_TC, or CC_CC genotypes, thereby increasing the initial life weight of the offspring Tan sheep. Alternatively, individuals with the genotype GC_GA on chromosome 2 of Tan sheep with the combination of SNP3 and SNP2 can be culled in a Tan sheep population to increase the frequency of the GC_AA, CC_GA, or CC_AA genotypes, thereby increasing the initial life weight of the offspring Tan sheep. Alternatively, individuals with the genotype GC_TC_GA (SNP3, SNP1, and SNP2 combination on chromosome 2) can be culled in a Tan sheep population to increase the frequency of the GC_CC_AA, CC_TC_GA, or CC_CC_AA genotypes, thereby increasing the initial life weight of offspring Tan sheep.

[0013] In a preferred embodiment, step 2) includes: In a Tan sheep population, individuals with the genotype CC_AA for the SNP1 and SNP2 combination on chromosome 2 and / or the genotype CC_CC for the SNP3 and SNP1 combination and / or the genotype CC_AA for the SNP3 and SNP2 combination, and / or the genotype CC_CC_AA for the SNP3, SNP1 and SNP2 combination, are selected to improve the initial life weight of the offspring Tan sheep.

[0014] In a preferred embodiment, the aforementioned Tan sheep population includes Tan sheep and / or its synthetic lines.

[0015] The present invention has the following advantages and effects compared with the prior art: First, new molecular markers related to the weight trait of Tan sheep at birth were identified. Through marker-assisted selection, the breeding efficiency of improving the birth weight trait of Tan sheep was significantly improved.

[0016] Secondly, the molecular markers identified in this invention can be applied to the genetic improvement of the initial life weight trait of Tan sheep, thereby improving the initial life weight trait of offspring Tan sheep, optimizing the genetic structure of the Tan sheep population, and thus increasing the market competitiveness of breeding enterprises.

[0017] Third, the three SNP molecular markers identified in this invention can be used alone or in combination to screen and improve the initial life weight trait of offspring Tan sheep. Attached Figure Description

[0018] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 The figures show the initial life weights of different genotypes at the SNP 1 locus in Tan sheep; Figure A shows the overall statistical results, Figure B shows the statistical results of male individuals, and Figure C shows the statistical results of female individuals.

[0020] Figure 2 The figures show the initial life weights of different genotypes at the SNP 2 locus in Tan sheep; Figure A shows the overall statistical results, Figure B shows the statistical results of male individuals, and Figure C shows the statistical results of female individuals.

[0021] Figure 3 The figures show the initial life weights of different genotypes at the SNP 3 loci in Tan sheep; Figure A shows the overall statistical results, Figure B shows the statistical results of male individuals, and Figure C shows the statistical results of female individuals. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] Example 1: Detection of Variant Sites experimental animals The GWAS experimental group consisted of 256 Tan sheep from the Sujing Station of the Ningxia Tan Sheep Breeding Farm. Ningxia Tan The experimental sheep were all healthy and born between 2022 and 2023, with similar body conditions. Their initial live weight was measured at birth. The feeding conditions and environment of all sheep remained basically unchanged. The breeding location was above 1000 meters above sea level, characterized by windy and sandy springs, mild summers, early autumn coolness, long cold winters, little snow and rain, abundant sunshine, and strong evaporation. The average annual precipitation was about 300 mm.

[0025] 2. Extraction of blood DNA Five mL of blood was collected from the jugular vein of each sheep, anticoagulated with ACD, and stored frozen at -20 °C. DNA was extracted using a blood extraction kit.

[0026] 3. Genome resequencing After passing DNA quality control, the samples were sent to a sequencing company for Illumina next-generation resequencing. VCFtools software was used to perform quality control on all genotyping data, removing sites with an average sequencing depth below 5X, SNP qvalue below 30, genotyping deletions >5%, minimum allele frequency below 0.05, and Hardy-Weinberg test p-values ​​<0.000001.

[0027] 4. Genome-wide association study (GWAS) The GWAS analysis used in this invention employs a mixed linear model. The mixed linear model was first published by Yu et al. in Nature Genetics and is widely recognized as the best GWAS analysis model to date because it can effectively correct for population structure and complex kinship relationships within populations.

[0028] y is the phenotypic vector, and Xβ represents the population structure effect and gender. The labeling effect to be tested. It is a polygenic effect. This represents the residual effect. In polygenic effects, K represents the marker-inferred kinship matrix.

[0029] The results showed that three loci on chromosome 2 (NC_056055.1) had a significant impact on birth weight at birth, as shown in Table 2: Note: P The value represents the degree of association between each SNP and the initial life weight trait. The molecular marker SNP1 C>T is located at position 201 of the sequence in SEQ ID NO.1 below, the molecular marker SNP2 A>G is located at position 202 of SEQ ID NO.1, and the molecular marker SNP3 C>G is located at position 201 of the sequence in SEQ ID NO.2 below.

[0030] SEQ ID NO.1 SEQ ID NO.2 Example 2: Analysis of differences in initial life weight among individuals with different genotypes The above three loci were detected in a population of 252 samples, and the initial live weights of different genotypes were statistically analyzed and subjected to t-tests. The results are shown in Table 3 and... Figures 1-3 As shown: Table 3 Mutation sites and initial life weight traits Note: P The value indicates the significance of differences between groups, and the statistical results exclude individuals whose genotypes were not detected.

[0031] As shown in Table 3, the birth weight of the CC genotype at the SNP1 locus was significantly greater than that of the TC genotype. P <0.01), the birth weight of the AA genotype at SNP2 locus was significantly greater than that of the GA genotype. P <0.01. At the SNP3 locus, the CC genotype had a significantly greater initial birth weight than the GC genotype ( P <0.01). Undetected genotypes may be combinations of genotypes indicating fertilization failure or embryo lethality.

[0032] Further statistical analysis and t-tests were performed on the initial life weights of individuals with different genotype combinations at the three loci. The results are shown in Table 4: Table 4. Life weight traits of different genotype combinations Note: Different letters indicate significant differences between groups, uppercase letters indicate extremely significant differences (p<=0.01), and the same letters indicate no significant differences.

[0033] Example 3: Method for genetic improvement of birth weight trait in Tan sheep A method for genetic improvement of early life traits in Tan sheep, wherein the Tan sheep population includes Tan sheep and / or its synthetic lines, the method comprising the following steps: 1) Determine the genotypes of molecular marker loci associated with the initial weight traits of Tan sheep in the Tan sheep resource population, wherein the molecular marker loci are the loci in the aforementioned embodiments.

[0034] 2) Make appropriate selections based on the genotype of the molecular marker and the breeding objectives.

[0035] Step 1) includes the following steps: 1.1) Extract genomic DNA from the sheep to be tested; 1.2) Genotyping of the Tan sheep to be tested; 1.3) Based on the detection results, determine the genotypes of the Tan sheep to be tested at SNP1, SNP2 and SNP3 loci on chromosome 2 of the reference genome; Step 2) includes at least one of the following steps: 2.1) In a Tan sheep resource population, individuals with the heterozygous TC type at the SNP1 locus on chromosome 2 are culled to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep.

[0036] 2.2) In one of the Tan sheep resource populations, individuals with a heterozygous GA type at the SNP2 locus on chromosome 2 are culled to increase the frequency of the AA genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep; 2.3) In one of the Tan sheep resource populations, individuals with a heterozygous GC genotype at the SNP3 locus on chromosome 2 are culled to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the initial life weight of the offspring Tan sheep.

[0037] Preferably, individuals with the TC_GA genotype on chromosome 2 of Tan sheep that have the SNP1 and SNP2 combination on a core Tan sheep population are culled to increase the frequency of the CC_AA genotype, thereby increasing the initial life weight of the offspring Tan sheep.

[0038] Preferably, individuals with the genotype GC_TC on chromosome 2 of Tan sheep with the combination of SNP3 and SNP1 on a core Tan sheep population are culled to increase the frequency of the GC_CC, CC_TC, or CC_CC genotypes, thereby increasing the initial life weight of the offspring Tan sheep.

[0039] Preferably, individuals with the genotype GC_GA combining SNP3 and SNP2 on chromosome 2 of a core Tan sheep population are culled to increase the frequency of the GC_AA, CC_GA, or CC_AA genotypes, thereby increasing the initial life weight of the offspring Tan sheep.

[0040] Preferably, individuals with the genotype GC_TC_GA combining SNP3, SNP1, and SNP2 on chromosome 2 of a core Tan sheep population are culled to increase the frequency of the GC_CC_AA, CC_TC_GA, or CC_CC_AA genotypes, thereby increasing the initial life weight of the offspring Tan sheep.

[0041] More preferably, considering the proportion of each genotype in the population, in a core Tan sheep resource population, individuals with the SNP1 and SNP2 combination genotype CC_AA and / or the SNP3 and SNP1 combination genotype CC_CC and / or the SNP3 and SNP2 combination genotype CC_AA, and / or the SNP3, SNP1 and SNP2 combination genotype CC_CC_AA are selected and retained on chromosome 2, thereby improving the initial life weight of the offspring Tan sheep.

[0042] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A molecular marker associated with the initial live weight traits of Tan sheep, characterized in that, The molecular marker comprises at least one of SNP1, SNP2 or SNP3, the SNP1 corresponds to a C>T mutation at position 41735643 on chromosome 2 (NC_056055.1) of the sheep genome (ARS-UI_Ramb_v3.0), the nucleotide sequence of which is shown as SEQ ID NO. 1; The SNP2 corresponds to a A>G mutation at position 41735644 on chromosome 2 (NC_056055.1) of the sheep genome (ARS-UI_Ramb_v3.0), the nucleotide sequence of which is shown as SEQ ID NO. 1; The SNP3 corresponds to a C>G mutation at position 41740324 on chromosome 2 (NC_056055.1) of the sheep genome (ARS-UI_Ramb_v3.0), the nucleotide sequence of which is shown as SEQ ID NO.

2.

2. A chip for detecting the initial live weight of Tan sheep, the chip comprising primers and probes for amplifying and detecting the molecular marker of claim 1.

3. A kit for detecting the initial live weight of Tan sheep, the kit comprising primers and probes for amplifying and detecting the molecular marker of claim 1.

4. Use of the molecular marker of claim 1 and / or the chip of claim 2 and / or the kit of claim 3 in improving the initial live weight trait of Tan sheep or breeding for the initial live weight of Tan sheep.

5. A method for improving the initial live weight traits of Tan sheep, characterized in that, The method comprises the following steps: 1) determining the genotype of the SNP site of claim 1 in a Tan sheep resource population; 2) making a corresponding selection according to the genotype of the SNP site and the breeding goal.

6. The method of claim 5, wherein, Wherein step 1) comprises the following steps: 1.1) extracting the genomic DNA of the Tan sheep to be tested; 1.2) detecting the genotype of the Tan sheep to be tested; 1.3) determining the genotype of the SNP site of claim 1 in the Tan sheep resource population to be tested based on the detection result.

7. The method of claim 5, wherein, Wherein step 2) comprises any one of steps 2.1) to 2.3): 2.1) eliminating individuals with a heterozygous TC genotype at the SNP1 site on chromosome 2 of Tan sheep in a Tan sheep resource population to increase the frequency of the CC genotype at this site from generation to generation, thereby increasing the initial live weight of the offspring of Tan sheep; 2.2) eliminating individuals with a heterozygous GA genotype at the SNP2 site on chromosome 2 of Tan sheep in a Tan sheep resource population to increase the frequency of the AA genotype at this site from generation to generation, thereby increasing the initial live weight of the offspring of Tan sheep; 2.3) eliminating individuals with a heterozygous GC genotype at the SNP3 site on chromosome 2 of Tan sheep in a Tan sheep resource population to increase the frequency of the CC genotype at this site from generation to generation, thereby increasing the initial live weight of the offspring of Tan sheep.

8. The method of claim 5, wherein, Wherein step 2) comprises: eliminating individuals with a combined genotype of TC_GA at the SNP1 and SNP2 sites on chromosome 2 of Tan sheep in a Tan sheep resource population to increase the frequency of the CC_AA genotype, thereby increasing the initial live weight of the offspring of Tan sheep; or eliminating the individual with the combined genotype of GC_TC of SNP3 and SNP1 on chromosome 2 of Tan sheep in a Tan sheep resource population, to increase the genotype frequency of GC_CC, CC_TC or CC_CC, so as to improve the initial live weight of the offspring Tan sheep; or eliminating the individual with the combined genotype of GC_GA of SNP3 and SNP2 on chromosome 2 of Tan sheep in a Tan sheep resource population, to increase the genotype frequency of GC_AA, CC_GA or CC_AA, so as to improve the initial live weight of the offspring Tan sheep; or eliminating the individual with the combined genotype of GC_TC_GA of SNP3, SNP1 and SNP2 on chromosome 2 of Tan sheep in a Tan sheep resource population, to increase the genotype frequency of GC_CC_AA, CC_TC_GA or CC_CC_AA, so as to improve the initial live weight of the offspring Tan sheep.

9. The method of claim 5, wherein, The step 2) comprises: In a Tan sheep resource population, the individual with the combined genotype of CC_AA of SNP1 and SNP2 and / or the combined genotype of CC_CC of SNP3 and SNP1 and / or the combined genotype of CC_AA of SNP3 and SNP2 and / or the combined genotype of CC_CC_AA of SNP3, SNP1 and SNP2 on chromosome 2 of Tan sheep is selected and reserved, so as to improve the initial live weight of the offspring Tan sheep.

10. The method of claim 5, wherein, The Tan sheep population comprises Tan sheep and / or a synthetic line thereof.

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