CN114525350A - SNP (Single nucleotide polymorphism) marker related to Hu sheep melatonin character and application thereof - Google Patents

Abstract

The invention relates to an SNP marker related to Hu sheep melatonin character, a primer pair for specifically amplifying the SNP marker, a kit for specifically amplifying an SNP locus, a method for identifying the SNP marker of the Hu sheep, a method for predicting Hu sheep melatonin expression, and application of the primer pair or the kit.

Description

SNP (Single nucleotide polymorphism) marker related to Hu sheep melatonin character and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an SNP marker related to Hu sheep melatonin character, a primer pair of a specific amplification SNP marker, a kit of the specific amplification SNP marker, a method for identifying the Hu sheep SNP marker, a method for predicting Hu sheep melatonin expression, and application of the primer pair or the kit.

Background

China is a big breed country but not a strong breed country, and sheep varieties are abundant in resources but lack the same variety. Hu sheep (Husheet) genus sheep (Hu sheep) ((Hu sheep))Ovis aries) The sheep is a unique sheep variety in China and is mainly bred in southern areas, and also bred in inner Mongolia autonomous regions. The Hu sheep has early sexual maturity, is estrus in four seasons, can be bred in the year, has strong multiparous property, can produce 2-3 lambs in one birth under good feeding conditions, has the average lambing rate of 256 percent, can breed at a high reproduction rate of more than 300 percent, and has strong fecundity which is rare in world sheep varieties. The study on the mechanism of the Hu sheep multiple births mainly discusses the influence of reproductive endocrine, follicular development, ovulation rate, intrauterine environment, ecological environment of the producing area and the like on the Hu sheep multiple births, and the leading factors of the multiple births are not determined yet. At present, the molecular genetic mechanism is discussed to a small extent in Hu sheep polyembryony mechanism in China, and the Hu sheep polyembryony gene FecB is proved to exist in Hu sheep and is a highly purified high-yield variety.

Melatonin (Melatonin, MT) is chemically known as N-acetyl-5-hydroxytryptamine, a hormone secreted mainly by the pineal gland. In animals, melatonin regulates reproductive activity directly through the hypothalamic-pituitary-gonadal axis. Studies now prove that various tissues and organs such as the reproductive system, the digestive system, the immune system and the like can secrete melatonin. In most mammals, melatonin synthesis is largely divided into three major steps: 1) tryptophan is catalyzed by tryptophan hydroxylase (TPH) and L-Aromatic Amino Acid Decarboxylase (AAAD) to generate 5-hydroxytryptamine (serotonin). 2) The 5-hydroxytryptamine generates N-acetyl-5-hydroxytryptamine (N-actylserotonin) under the action of Arylalkylamine N-Acetyltransferase (AANAT). 3) N-acetyl-5-hydroxytryptamine (N-actylserotonin) is methylated under the catalytic action of oxindole-O-methyl transferase (ASMT) to generate N-acetyl-5-methoxytryptamine (N-acetyl-5-methoxytryptamine), namely melatonin. Melatonin plays its biological role, mainly through the MT1, MT2 pathways. In recent years, a plurality of researches prove that the application of melatonin can improve the production performance of mutton sheep. Therefore, the breeding of the Hu sheep individuals with high melatonin content for variety cultivation is beneficial to the development of the industry of mutton sheep and the development of the strategy of the industry development.

Single Nucleotide Polymorphism (SNP) refers to a DNA sequence polymorphism caused by variation of a single nucleotide at the chromosomal genome level, and the frequency of one allele in a population is not less than 1%. It is a restriction fragment length polymorphism, a microsatellite marker followed by a 3 rd generation molecular marker. With the rapid development of SNP, SNP has become an important research tool for sheep genome analysis, and is widely applied to positioning of economic trait QTL sites and breeding of excellent traits. Is currently widely applied to the field of sheep molecular breeding.

Disclosure of Invention

The invention aims to provide a SNP marker related to the Hu sheep melatonin character, a primer pair of a specific amplification SNP marker, a kit of the specific amplification SNP marker, a method for identifying the Hu sheep SNP marker, a method for predicting Hu sheep melatonin expression and application of the primer pair or the kit, and provides technical support for Hu sheep breeding.

In a first aspect, the present invention provides a SNP marker associated with the melatonin trait of Hu sheep. The SNP markers include:

the 7981372 th and/or 7981866 th base of the genome NC _ 040262.1;

17355171 th base of genome NC-040277.1.

Genomic NC-040262.1 is located on chromosome 11 in the coding region of the arylalkyl amine-N-acetyltransferase (AANAT) gene.

The genome NC _040277.1 is located on chromosome 26 in the coding region of the melatonin type I receptor (MTNR 1A) gene.

In some embodiments, the SNP markers include: the 7981372 th base of the genome NC-040262.1 is G or C.

In some embodiments, the SNP markers include: the 7981866 th base of the genome NC-040262.1 is G or A.

In some embodiments, the SNP markers include: the 17355171 th base of the genome NC-040277.1 is G or T.

In some embodiments, the amino acid corresponding to base 7981372 of genomic NC _040262.1 is mutated.

In some embodiments, the amino acid corresponding to base 7981372 of the genome NC _040262.1 is mutated from E to D.

In some embodiments, the amino acid corresponding to base 7981866 of genomic NC _040262.1 is mutated.

In some embodiments, the amino acid corresponding to base 7981866 of the genome NC _040262.1 is mutated from R to Q.

In some embodiments, the amino acid corresponding to base 17355171 of genomic NC _040277.1 is mutated.

In some embodiments, the amino acid corresponding to base 17355171 of the genome NC _040277.1 is mutated from a to D.

In a second aspect, the present invention provides a primer pair for specifically amplifying the SNP marker provided in the first aspect of the present invention. The primer pair comprises;

a primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2, which is used for specifically amplifying the SNP marker at the 7981372 th site and/or the SNP marker at the 7981866 th site; and/or

A primer pair shown as SEQ ID NO. 9 and SEQ ID NO. 10, which is used for specifically amplifying the SNP marker at position 17355171.

The nucleotide sequence shown in SEQ ID NO. 1 is CATCTCTGTCTCCGGCAACT.

The nucleotide sequence shown in SEQ ID NO. 2 is GAGTCAGCGGTCACTGTTCC.

The nucleotide sequence shown in SEQ ID NO. 9 is CTGACAGCACATTAGCTCAGACAT.

The nucleotide sequence shown in SEQ ID NO. 10 is CCTCTGCTACGTGTTCCTGATCT.

In a third aspect, the invention provides a kit. The kit comprises a primer pair provided by the second aspect of the invention.

In a fourth aspect, the invention provides a method for identifying a Hu sheep SNP marker. The method comprises the following steps:

extracting genomic DNA of Hu sheep;

carrying out PCR amplification reaction by using the genomic DNA as a template and the primer pair provided by the second aspect of the invention to obtain an amplification product;

analyzing the amplification product.

In some embodiments, the method further comprises: when the 7981372 genotype of the genome NC _040262.1 is CG or CC, the 7981866 genotype is GA or AA, and/or the 17355171 genotype of the genome NC _040277.1 is GG, the corresponding Hu sheep has high melatonin expression level.

In some embodiments, the method further comprises: when the 7981372 genotype of the genome NC-040262.1 is CC and/or the 7981866 genotype is AA, the melatonin expression level of the corresponding Hu sheep is higher.

In a fifth aspect, the invention provides a method for predicting melatonin expression in a Hu sheep. The method comprises the following steps:

extracting genomic DNA of Hu sheep;

carrying out PCR amplification reaction by using the genomic DNA as a template and the primer pair provided by the second aspect of the invention to obtain an amplification product;

analyzing the amplification product.

In some embodiments, the method further comprises: when the 7981372 genotype of the genome NC _040262.1 is CG or CC, the 7981866 genotype is GA or AA, and/or the 17355171 genotype of the genome NC _040277.1 is GG, the melatonin expression correlation of the corresponding Hu sheep is high.

In some embodiments, the method further comprises: when the 7981372 genotype of the genome NC-040262.1 is CC and/or the 7981866 genotype is AA, the melatonin expression level of the corresponding Hu sheep is higher.

In a sixth aspect, the invention provides the use of the primer pair provided in the second aspect of the invention in breeding of Hu sheep (Hu sheep).

In a seventh aspect, the invention provides the use of the kit provided in the third aspect in breeding of Hu sheep (Hu sheep).

The SNP marker provided by the invention is obviously related to the property expression of the melatonin of the Hu sheep. When the 7981372 genotype of the genome NC-040262.1 is CG or CC, the 7981866 genotype is GA or AA, and the 17355171 genotype of the genome NC-040277.1 is GG, the melatonin expression level of the Hu sheep is remarkably increased, and technical support is provided for breeding the Hu sheep.

Drawings

FIG. 1 shows the cumulative SNP distance distribution curve.

FIG. 2 shows the result of SNP analysis of AANAT gene.

FIG. 3 shows the result of SNP analysis of ASMT gene.

FIG. 4 shows the result of SNP analysis of MTNR1A gene.

FIG. 5 shows the result of SNP analysis of MTNR1B gene.

Fig. 6 shows SNP site genotypes significantly associated with the melatonin trait.

Detailed Description

The present invention is further illustrated by the following examples, which should be understood as being merely illustrative of the present invention and not limiting thereof, and all simple modifications thereof which are within the spirit of the invention are intended to be covered by the scope of the claims.

Genomic NC-040262.1 is located on chromosome 11 in the coding region of the arylalkyl amine-N-acetyltransferase (AANAT) gene.

Genomic NC-040278.1 is located in the coding region of the N-acetyl-5-hydroxytryptamine-methyltransferase (ASMT) gene on the X chromosome.

The genome NC _040277.1 is located on chromosome 26 in the coding region of the melatonin type I receptor (MTNR 1A) gene.

The genome NC _040272.1 is located on chromosome 21 in the coding region of the melatonin type II receptor (MTNR 1B) gene.

Example (b):

1. melatonin detection (high performance liquid chromatography)

Preparing a melatonin methanol chromatographic grade solution of 1mg/mL, sequentially diluting methanol to be 100, 50, 20, 10 and 5ng/L of detection peak area, and drawing a standard curve. Collecting 200 μ L blood, adding 800 μ L methanol, vortex vibrating for 30min, centrifuging at 4 deg.C and 14000r/min for 20min, sucking supernatant, filtering, storing in brown sample bottle, and storing at-20 deg.C with filter specification of 0.22 μm. And after the sample is detected, calculating the melatonin concentration according to the peak area of the sample.

2. High melatonin Hu sheep individual selection

In higher animals, melatonin is produced from the pineal gland. Melatonin produced by pineal gland is an endocrine hormone and enters blood. Therefore, the study uses high performance liquid chromatography to determine melatonin, and high melatonin Hu sheep individuals are selected according to the detection result of the melatonin in blood.

3. Whole genome detection

The genome re-sequencing comprises the steps of extracting and detecting the quality of the genome DNA of a sample, constructing a sequencing library and performing on-machine sequencing. And after the quality control in the experimental process is qualified, further adjusting the volume of the library by taking the target data amount as a reference, and performing Illumina HiSeq sequencing after mixing a plurality of libraries. The specific experimental steps are as follows:

(1) and extracting and detecting the quality of the genome DNA.

(2) The genomic DNA is disrupted.

(3) The cohesive ends of the DNA fragments were filled in and repaired, and the 3' ends were converted to cohesive ends using a repair method in which a base "A" was added.

(4) Index sequence DNA linkers were added to both sides of the sticky ends.

(5) And (4) performing screening by using magnetic beads, and collecting target fragments with proper lengths.

(6) And adding Index to the end of the target fragment by PCR amplification to construct a sequencing library.

(7) The sequencing library was bound to the sequencing chip by bridge PCR.

(8) Sequencing on Illumina HiSeq.

4. Biological information analysis process

(1) And performing image Base recognition (Base labeling) primary quality analysis on the sequencing result raw image Data by using software bcf2FASTQ (version 2.17.1.14) to obtain raw Data (Pass Filter Data, PF) of a sequencing sample, wherein the sequencing Data is stored in a FASTQ (fq for short) file format.

(2) The joint and low-quality sequence of the sequencing original Data (Pass Filter Data) are removed by using the second generation sequencing Data quality statistical software cutadapt (version 1.9.1) to obtain Clean Data for subsequent information analysis.

(3) And (3) comparing clean data to a reference Genome sequence by using Dragen Genome Pipline, and counting comparison results, including the number of reads of the reference sequence, the average depth, the coverage rate and the like.

(4) Based on the alignment results, SNPs (detection of single base mutation, detection of each site of the reference Genome, detection of the presence or absence of SNPs at the site, and saving the detection results in a Variant Call Format (VCF) Format file) were performed using Dragen Genome Pipline.

(5) The genome of a species is divided into different functional units according to the known genes of the species. Then according to the position where mutation occurs, each mutation site is classified into a functional region, and classification statistics is carried out on the mutation sites detected by each sample according to the functional regions.

5. Non-synonymous mutation site validation

The length of the primer is 15bp-30bp, the effective length is generally not more than 38bp, otherwise, the optimal extension temperature of PCR can exceed the optimal action temperature of Taq enzyme, thereby reducing the specificity of the product. The GC content should be between 40% and 60%, and the optimal Tm value is between 58 ℃ and 60 ℃. The primer itself cannot contain self-complementary sequences, otherwise a hairpin-like secondary structure is formed. There should be no more than 4 complementary or homologous bases between primers, which would otherwise form primer dimers, and 3' complementary overlap should be avoided. See table 1 for nucleotide sequences of primer pairs. In Table 1, bases 7981745, 7981372, 7980810, and 7981866 are located on genomic NC 040262.1 aralkylamine-N-acetyltransferase (AANAT); the 604657 th base is located on the genome NC-040278.1 (ASMT); the 17354935 th, 17355358 th, 17354943 th, 17377872 th and 17355171 th bases are located on the genome NC _040277.1 (MTNR 1A); the 1684394 th, 1684464 th, 1684557 th and 1684269 th bases were located on the genome NC-040272.1 (MTNR 1B).

TABLE 1 nucleotide sequences of primer pairs

6. SNP locus and character association analysis

The primer length adopts R language software to analyze the relevance between the SNP locus and the phenotype, and the used models are as follows: yij = μ + Gi + Pj + eij, where Yij is a trait observed value; mu is the total average value of the characters; gi is the genotype effect; pj is the fixation effect; eij is the random error.

7. Results of the experiment

7.1 Hu sheep melatonin concentration result detection

The melatonin detection is carried out on 195 Hu sheep sera, wherein the content of the melatonin is 51.3 percent in total of 100 percent in 0-0.5ng/mL, 21.5 percent in total of 42 percent in total of 0.5-1ng/mL, and 27.2 percent in total of 53 percent above 1 ng/mL. 3 individuals of high melatonin hu sheep (numbers B1-1 to B1-3 in Table 2) and 3 individuals of low melatonin hu sheep (numbers B2-1 to B2-3 in Table 2) were determined to be subjected to melatonin concentration rechecking and whole genome sequencing analysis, and the results are shown in Table 2:

TABLE 2 Whole genome sequencing analysis samples

7.2 sequencing raw data quality control

Whole genome re-sequencing was performed on 6 Hu sheep blood samples and the raw Data (Pass Filter Data, PF) are shown in Table 3. The result of removing the low-quality sequence from the original data is shown in table 4, clear Date is obtained, the clear data after QC is more than 99% in PF data, and statistics are shown in table 5. The sequencing data obtained by the experiment is good, and the subsequent related credit generation analysis can be carried out.

TABLE 3 PF data statistics

Note: q20 (%): respectively calculating the percentage of the base with the Phred value more than 20 to the total base; q30 (%): respectively calculating the percentage of the base with the Phred value more than 30 to the total base; GC (%): calculating the percentage of the sum of the number of bases G and C to the total number of bases; n (ppm): the content of the base N which cannot be judged by sequencing is in each million bases.

TABLE 4 Clean data statistics

Note: q20 (%): respectively calculating the percentage of the base with the Phred value more than 20 to the total base; q30 (%): respectively calculating the percentage of the base with the Phred value more than 30 to the total base; GC (%): calculating the percentage of the sum of the number of bases G and C to the total number of bases; n (ppm): the content of the base N which cannot be judged by sequencing is in each million bases.

TABLE 5 Clean data ratio statistics

7.3 sequencing filtration data quality control statistics

Clear data and a reference Genome sequence are compared by using Dragen Genome Pipline, the average comparison ratio reaches 99.89%, and the comparison result shows that the number of unique reads of the reference Genome accounts for 81.12% of all the reference genomes on comparison, which is shown in Table 6. The average coverage rate is 96.415%, and the average depth of coverage is 29.105%, as shown in table 7.

TABLE 6 sequencing alignment statistics

TABLE 7 genome coverage statistics

7.4 SNP detection and Annotation

Based on the alignment results, SNP (single base mutation) detection was performed using Dragen Genome Pipline. Statistics of genome-wide sequencing SNPs for 6 samples are shown in table 8. SNPs were classified into various types according to functional changes caused by the mutation sites, and the statistical results of different types of SNPs are shown in Table 9.

TABLE 8 genome-wide SNP/InDel statistics

TABLE 9 Whole genome SNP type statistics

The base types are four types of A, T, C and G, taking the example that the positive strand A of a reference genome is mutated into C, combining the positive and negative strand relation of the genome, the actual mutation mode is A: T paired base, the mutation is C: G paired base, and the mutation type in the negative strand is T > G, so the A > C and T > G type mutations can be classified into a type of mutation and marked as A: T > C: G. Therefore, SNP mutation patterns can be divided into 6 classes, namely A: T > T: A, A: T > C: G, A: T > G: C, C: G > T: A, C: G > G: C, and C: G > A: T. As shown in FIG. 1, genome-wide SNP mutations are mainly concentrated in two types, C: G > T: A and A: T > C: G. In FIG. 1, the abscissa is the number of SNP sites of different mutation types; the ordinate is the classification of 6 mutation types.

7.5 AANAT Gene SNP analysis

And carrying out mutation functional region classification on SNP sites of the AANAT genes of different individuals of the high melatonin Hu sheep group and the low melatonin Hu sheep group. The significant difference exists in the number of the SNPs between the high melatonin Hu sheep group and the low melatonin Hu sheep group, the mutation of the SNPs between different individuals is counted, the mutation mainly exists in two types of C: G > T: A and A: T > G: C, the difference SNP sites are 23, and the non-synonymous mutation is 4, wherein 2 of the mutation are significantly related to the melatonin, as shown in a figure 2 (A: SNPvenn analysis; B: SNP-mutant type).

7.6 ASMT Gene SNP analysis

And carrying out mutation functional region classification on SNP sites of ASMT genes of different individuals of the high melatonin Hu sheep group and the low melatonin Hu sheep group. The significant difference exists in the number of SNPs between the high melatonin Hu sheep group and the low melatonin Hu sheep group, the mutation of the SNPs between different individuals is counted, the mutation mainly exists in two types of C: G > T: A and A: T > G: C, the difference SNP sites are 168, and the non-synonymous mutation is 1, as shown in figure 3 (A: SNP-venn analysis; B: SNP-mutant type).

7.7 SNP analysis of the MTNR1A Gene

And carrying out mutation functional region classification on SNP sites of different individual MTNR1A genes of the high melatonin Hu sheep group and the low melatonin Hu sheep group. No significant difference exists in the quantity of SNPs between the high melatonin Hu sheep group and the low melatonin Hu sheep group, mutation of the SNPs between different individuals is counted, the mutation mainly exists in two types of C: G > T: A and A: T > G: C, 89 different mutation SNP sites exist, 5 non-synonymous mutations exist, and 1 of the mutations is significantly related to melatonin expression as shown in figure 4 (A: SNPvenn analysis; B: SNP-mutant type).

7.8 SNP analysis of the MTNR1B Gene

And carrying out mutation functional region classification on SNP sites of different individual MTNRA1B genes of the high melatonin Hu sheep group and the low melatonin Hu sheep group. The statistics of SNP mutations between different individuals were mainly found in two types, C: G > T: A and A: T > G: C, with 5 non-synonymous mutations, as shown in FIG. 5 (A: SNPvenn analysis; B: SNP-mutant type).

7.9 correlation analysis of non-synonymous mutation sites of melatonin synthetase and receptor genes

To further verify the correlation of the found SNP sites with melatonin, statistics were made on the SNP mutation sites of the melatonin synthase and receptor genes of high and low melatonin lakes using 195 lakes mentioned in 7.1, of which 14 non-synonymous sites having amino acid mutations in the exon regions were located (table 10). Adopting R language software to analyze the relevance between SNP locus and phenotype,P<0.05 indicated that the SNP sites were significantly associated with melatonin expression. The results are shown in table 11, where it can be seen that the 3 sites exhibit a significant correlation with the expression of the sheepfold melatonin, P02 (corresponding to position 7981372 of genome NC _040262.1 (AANAT)), P04 (corresponding to position 7981866 of genome NC _040262.1 (AANAT)) and P14 (corresponding to position 17355171 of genome NC _040277.1 (MTNR 1A)), respectively.

The melatonin concentration of the Hu sheep of the 7981372 th genotype CG of the genome NC-040262.1, the 7981866 th genotype GA and the 17355171 th genotype GG of the genome NC-040277.1 is higher than the average concentration of the melatonin in the population by 1.64 ng/mL. As shown in FIG. 6, the genotype of the 7981372 th SNP marker is GG, the correlation is 89.4%, and the correlation of the genotype CG is 10.6%; the genotype of the 7981866 th SNP marker is GG, the correlation is 86.8 percent, and the genotype GA correlation is 13.4 percent; the genotype of the 17355171 th SNP marker was GG, the correlation was 84.1%, and the correlation of genotype GT was 15.6%.

TABLE 10 summary of non-synonymous mutations

TABLE 11 test for significance of mutation sites

Note:P<0.05 means significant correlation at the 0.05 level (bilateral),P<0.01 indicates significant correlation at the 0.01 level (bilateral), with P01-P14 in Table 11 corresponding in sequence to P01-P14 in Table 10.

TABLE 12 significance site genotype corresponds to melatonin concentration

Therefore, the invention provides the SNP marker related to the Hu sheep melatonin character. When the 7981372 locus genotype of the genome NC-040262.1 is CG, the 7981866 locus genotype is GA and the 17355171 locus genotype of the genome NC-040277.1 is GG, the melatonin expression level of the Hu sheep is remarkably increased, and technical support is provided for breeding the Hu sheep.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

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Claims (10)

1. An SNP marker associated with a melatonin trait of Hu sheep, wherein the SNP marker comprises:

the 7981372 th and/or 7981866 th base of the genome NC _ 040262.1;

17355171 th base of genome NC-040277.1.

2. The SNP marker according to claim 1, comprising: the 7981372 th base of the genome NC-040262.1 is G or C, and/or the 7981866 th base is G or A; and/or

The 17355171 th base of the genomic NC-040277.1 is G or T.

3. A primer pair for specifically amplifying the SNP marker of claim 1 or 2, comprising;

a primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2, which is used for specifically amplifying the SNP marker at the 7981372 th site and/or the SNP marker at the 7981866 th site; and/or

A primer pair shown as SEQ ID NO. 9 and SEQ ID NO. 10, which is used for specifically amplifying the SNP marker at the 17355171 th position.

4. A kit comprising the primer pair of claim 3.

5. A method for identifying a SNP marker of a hu sheep, comprising the steps of:

extracting genomic DNA of Hu sheep;

performing a PCR amplification reaction using the genomic DNA as a template and the primer pair of claim 3 to obtain an amplification product;

analyzing the amplification product.

6. The method of claim 5, further comprising:

when the 7981372 genotype of the genome NC _040262.1 is CG or CC, the 7981866 genotype is GA or AA, and/or the 17355171 genotype of the genome NC _040277.1 is GG, the corresponding Hu sheep has high melatonin expression level.

7. A method of predicting melatonin expression in a hu sheep, the method comprising the steps of:

extracting genomic DNA of Hu sheep;

performing a PCR amplification reaction using the genomic DNA as a template and the primer pair of claim 4 to obtain an amplification product;

analyzing the amplification product.

8. The method of claim 7, further comprising:

when the 7981372 genotype of the genome NC _040262.1 is CG or CC, the 7981866 genotype is GA or AA, and/or the 17355171 genotype of the genome NC _040277.1 is GG, the corresponding Hu sheep has high melatonin expression level.

9. The use of the primer pair of claim 3 in breeding Hu sheep.

10. Use of the kit of claim 4 in breeding Hu sheep.

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