STAT3 gene SNPs (Single Nucleotide Polymorphism) marker for detecting milk production character of sheep and application of STAT3 gene SNPs marker

Abstract

The invention discloses an STAT3 gene SNPs marker for detecting sheep milk production traits and application of the STAT3 gene SNPs marker, and belongs to the technical field of biology. According to the present invention, the milk sheep is adopted as the research object, the expression quantity of the STAT3 gene in seven tissues such as mammary gland is researched, the SNPs site of the gene is detected, and the influence of the nucleotide sequence variation on the sheep lactation performance is analyzed so as to provide the effective molecular marker for improving the sheep lactation performance; five SNPs (single nucleotide polymorphisms) loci, namely c.22 + 537C / T, c.22 + 658C / T, c.318 + 126C / G, c.417 + 360G / A and c.417 + 395T / C, are found on the STAT3 gene of the sheep; compared with a traditional seed selection method, the lambs can be selected when the lambs are newly born, and the selection accuracy is higher than that of a traditional breeding method. Therefore, 'early selection and accurate selection 'of the milk production performance of the milk sheep can be realized.

Description

Technical Field

[0001] The invention relates to a STAT3 gene SNPs marker for detecting sheep milk production traits and application thereof, belonging to the field of biotechnology. Background Art

[0002] Signal transducers and activators of transcription 3 (STAT3) is one of the important members of the STAT family, which has the dual role of intracytoplasmic signal transduction and intranuclear transcriptional activation. As intracellular effector molecules, the STAT protein family mainly participates in various biological processes such as cell proliferation, survival, differentiation and angiogenesis through the JAK / STAT signaling pathway. Studies have shown that most members of the STAT family are related to mammary gland development and lactation. For example, growth hormone and prolactin activate the tyrosine kinase of the JAK family after binding to their receptors, and then bind to STAT1 to affect the lactation performance of female livestock. STAT4 is expressed in mammary tissue during pregnancy. When interleukin-12 (IL-12) binds to the surface receptor of mammary epithelial cells, it activates the JAK / STAT4 signaling pathway and regulates the differentiation of mammary cells. STAT5 is known as a mammary factor. It was first discovered in goat mammary glands. It mainly affects the vitality, proliferation, milk fat synthesis, casein synthesis and mammary gland development of mammary epithelial cells through the JAK / STAT5 pathway. STAT6 is also known as a regulator of mammary gland differentiation. In early pregnancy, it can induce the expression of GATA binding protein 3 (GATA3), thereby stimulating acinar differentiation and proliferation.

[0003] As a nuclear transcription factor and cytoplasmic signaling factor, STAT3 protein is widely expressed in various tissues of livestock, such as mammary gland, liver and kidney, and regulates physiological processes such as cell growth, migration, proliferation and transformation. Studies have found that STAT3 is a key regulator of programmed cell death mediated by lysosomes during mammary gland involution and a key mediator of mammary gland involution. Knockout of STAT3 inhibits apoptosis of mammary epithelial cells in mice and delays mammary gland involution. Further studies have found that STAT3 induces mammary gland involution by affecting the expression of insulin-like growth factor binding protein-5 (IGFBP-5) in mammary epithelial cells. However, the effect of STAT3 gene polymorphism on sheep lactation traits has not been reported. Summary of the invention

[0004] In this study, 414 dairy sheep were selected as the research subjects. Real-time fluorescence quantitative PCR (reverse transcriptase-quantitative-PCR, RT-qPCR), Sanger sequencing and penta-primer amplification refractory mutation system (PARMS) were used to study the expression level of STAT3 gene in seven tissues including mammary gland, detect the SNPs sites of this gene, and analyze the effect of its nucleotide sequence variation on the lactation performance of sheep, in order to provide effective molecular markers for improving the lactation performance of sheep.

[0005] The present invention provides a single nucleotide polymorphism genetic molecular marker related to sheep milk production traits, wherein the single nucleotide polymorphism genetic marker is located in the sheep STAT3 gene, and the single nucleotide polymorphism genetic molecular marker is any one or more combinations of the following (a) to (e);

[0006] (a) The single nucleotide polymorphism genetic marker is located at the c.22+537bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a C / T base mutation;

[0007] (b) The single nucleotide polymorphism genetic marker is located at the c.22+658bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a C / T base mutation;

[0008] (c) The single nucleotide polymorphism genetic marker is located at the c.318+126bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a C / G base mutation;

[0009] (d) the single nucleotide polymorphism genetic marker is located at the c.417+360bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a G / A base mutation;

[0010] (e) The single nucleotide polymorphism genetic marker is located at the c.417+395bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a T / C base mutation.

[0011] The present invention also provides the use of at least one of the following five SNP sites in identifying or assisting in identifying sheep milk production traits; the SNP site is a cytosine C at position c.22+537 of the STAT3 gene on the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, which is replaced by thymine T;

[0012] The SNP site is corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, where the cytosine C at position c.22+658 of the STAT3 gene on the positive strand of chromosome 11 is replaced by thymine T;

[0013] The SNP site is corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, in which the cytosine C at position c.318+126 of the STAT3 gene on the positive strand of chromosome 11 is replaced by guanine G;

[0014] The SNP site is that the guanine G at position c.417+360 of the STAT3 gene in the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version is replaced by adenine A;

[0015] The SNP site is that the thymine T at c.417+395 of the STAT3 gene on the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version is replaced by cytosine C.

[0016] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0017] The present invention also provides a reagent for detecting the genotype of at least one of the following five SNP sites for use in identifying or assisting in identifying sheep milk production traits; the SNP site is a cytosine C at position c.22+537 of the STAT3 gene on the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, which is replaced by thymine T;

[0018] The SNP site is corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, where the cytosine C at position c.22+658 of the STAT3 gene on the positive strand of chromosome 11 is replaced by thymine T;

[0019] The SNP site is corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, in which the cytosine C at position c.318+126 of the STAT3 gene on the positive strand of chromosome 11 is replaced by guanine G;

[0020] The SNP site is that the guanine G at position c.417+360 of the STAT3 gene in the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version is replaced by adenine A;

[0021] The SNP site is that the thymine T at c.417+395 of the STAT3 gene on the positive strand of chromosome 11 corresponding to the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version is replaced by cytosine C.

[0022] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0023] The present invention also provides an application of a haplotype combination sequence of a single nucleotide polymorphism molecular marker associated with sheep milk production traits in improving sheep milk fat content and / or dry matter content and / or ash content and / or average daily milk production, wherein the haplotype combination is the haplotype combination of the single nucleotide polymorphism genetic marker associated with sheep milk production traits according to claim 1, and the haplotype combination sequence tag is CCGGTTTT or CTGCTCTC or CTGCTTTT or CTGCTCTT or CTGGTCTC;

[0024] The single nucleotide polymorphism genetic marker is located at the c.22+537bp position of the STAT3 gene on the positive strand of chromosome 11 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / T base mutation;

[0025] Or located at c.22+658bp of the STAT3 gene on the positive strand of chromosome 11 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / T base mutation;

[0026] Or located at c.318+126bp of the STAT3 gene on the positive strand of chromosome 11 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a C / G base mutation;

[0027] Or located at c.417+360bp of the STAT3 gene on the positive strand of chromosome 11 in the sheep reference genome ARS-UI_Ramb_v3.0 version sequence information, and there is a G / A base mutation;

[0028] Or it is located at the c.417+395bp position of the STAT3 gene on the positive strand of chromosome 11 in the sequence information of the sheep reference genome ARS-UI_Ramb_v3.0 version, and there is a T / C base mutation.

[0029] The present invention also provides a method for identifying or assisting in identifying sheep milk production traits, the method being any one of the following 1)-5):

[0030] 1) comprising the following steps: detecting the genotype of the SNP site c.22+537C / T in the STAT3 gene of the tested sheep; the genotype of the SNP site c.22+537C / T is CC, CT or TT;

[0031] The milk fat rate and dry matter content of the test sheep whose SNP site c.22+537C / T genotype in the STAT3 gene is CC are better or auxiliary better than those of the test sheep whose SNP site c.22+537C / T genotype is CT and TT;

[0032] 2) comprising the following steps: detecting the genotype of the SNP site c.22+658C / T in the STAT3 gene of the tested sheep; the genotype of the SNP site c.22+658C / T is CC, CT or TT;

[0033] The milk fat rate and dry matter content of the test sheep whose SNP site c.22+658C / T genotype in the STAT3 gene is CC are better than or auxiliary better than those of the test sheep whose SNP site c.22+658C / T genotype is TT.

[0034] 3) comprising the following steps: detecting the genotype of the SNP site c.318+126C / G in the STAT3 gene of the tested sheep; the genotype of the SNP site c.318+126C / G is CC, CG or GG;

[0035] The milk fat rate of the test sheep whose genotype of the SNP site c.318+126C / G in the STAT3 gene is CC is better than or auxiliary better than that of the test sheep whose genotype of the SNP site c.318+126C / G is GG;

[0036] 4) comprising the following steps: detecting the genotype of the SNP site c.417+360G / A in the STAT3 gene of the tested sheep; the genotype of the SNP site c.417+360G / A is GG, GA or AA;

[0037] The milk fat rate and dry matter content of the test sheep whose genotype of the SNP site c.417+360G / A in the STAT3 gene is GG are better than or auxiliary better than those of the test sheep whose genotype of the SNP site c.417+360G / A is GA and AA;

[0038] 5) comprising the following steps: detecting the genotype of the SNP site c.417+395T / C in the STAT3 gene of the tested sheep; the genotype of the SNP site c.417+395T / C is TT, TC or CC;

[0039] The milk fat rate and dry matter content of the test sheep whose genotype of the SNP site c.417+395T / C in the STAT3 gene is TT are better than or auxiliary better than those of the test sheep whose genotype of the SNP site c.417+395T / C is CC.

[0040] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0041] The invention also provides application of the method in sheep screening or sheep breeding.

[0042] In one embodiment of the present invention, in the application, any of the following sheep are selected for milk production or breeding:

[0043] The test sheep whose genotype of the SNP site c.22+537C / T in the STAT3 gene is CC;

[0044] or a test sheep whose genotype of the SNP site c.22+658C / T in the STAT3 gene is CC;

[0045] or a test sheep whose genotype of the SNP site c.318+126C / G in the STAT3 gene is CC;

[0046] or a test sheep whose genotype in the SNP site c.417+360G / A in the STAT3 gene is GG;

[0047] Or the test sheep whose genotype at the SNP site c.417+395T / C in the STAT3 gene is TT.

[0048] The present invention also provides a primer pair for detecting mutations in the STAT3 gene, wherein the upstream and downstream nucleotide sequences of the primer pair are respectively shown as SEQ ID NOs.1-2; or the upstream and downstream nucleotide sequences of the primer pair are respectively shown as SEQ ID NOs.3-4; or the upstream and downstream nucleotide sequences of the primer pair are respectively shown as SEQ ID NOs.5-6.

[0049] In one embodiment of the present invention, the present invention amplifies the first intron region by using primers shown in SEQ ID NOs. 1-2, and detects c.22+537C / T (SNP1) and c.22+658C / T (SNP2) in the first intron region;

[0050] The present invention amplifies the third intron region by using primers shown in SEQ ID NO.3-4, and detects c.318+126C / G (SNP3) in the third intron region;

[0051] The present invention amplifies the 4th intron region by using primers shown in SEQ ID NO. 5-6, and c.417+360G / A (SNP4) and c.417+395T / C (SNP5) are detected in the 4th intron region.

[0052] The present invention also provides a method for detecting molecular markers related to sheep milk production traits, the method comprising the following steps:

[0053] a) extracting genomic DNA from sheep blood as a sample, and performing PCR amplification on the sheep STAT3 gene using primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6;

[0054] b) performing mixed pool sequencing and sequence analysis on the above PCR amplification products to obtain the mutation site and determine the mutation type;

[0055] c) Performing STAT3 genotyping on the identified sheep individuals, and identifying the genotype and haplotype of the SNP site in the sheep STAT3 gene detection region according to the typing results.

[0056] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0057] The present invention also provides a kit for detecting the above-mentioned molecular markers related to sheep milk production traits, wherein the kit comprises the following primer pairs. The nucleotide sequences of the upstream and downstream of the primer pair are respectively shown in SEQ ID NO.1-2; or the nucleotide sequences of the upstream and downstream of the primer pair are respectively shown in SEQ ID NO.3-4; or the nucleotide sequences of the upstream and downstream of the primer pair are respectively shown in SEQ ID NO.5-6.

[0058] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0059] The present invention also provides an application of a primer pair or a primer combination, wherein the application is any one of the following (a)-(f):

[0060] (a) Identification or assistance in the identification of milk production traits in sheep;

[0061] (b) sheep screening;

[0062] (c) sheep breeding;

[0063] (d) preparing a kit for identifying or assisting in identifying sheep milk production traits;

[0064] (e) preparing a kit for sheep screening;

[0065] (f) Preparation of a kit for sheep breeding.

[0066] The upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 1 to 2, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 3 to 4, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 5 to 6, respectively.

[0067] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

[0068] Beneficial Effects

[0069] The present invention uses lactating sheep as the research object, applies Sanger and other technologies to detect the nucleotide sequence variation sites of the STAT3 gene, and constructs a general linear mixed effects model to study the correlation between nucleotide sequence variation and sheep lactation traits, and explores molecular genetic markers that regulate sheep lactation traits; the present invention has achieved some positive effects and indeed has great advantages compared with the prior art, as follows:

[0070] (1) A total of five SNPs were found in the sheep STAT3 gene. Among them, c.22+537C / T (SNP1) and c.22+658C / T (SNP2) were detected in the first intron, c.318+126C / G (SNP3) was detected in the third intron, and c.417+360G / A (SNP4) and c.417+395T / C (SNP5) were detected in the fourth intron.

[0071] (2) At SNP1, the milk fat rate of CC type individuals was 0.724% and 1.262% higher than that of CT and TT type individuals, and the dry matter content was 0.924% and 1.584% higher than that of TT type individuals (P<0.05). At SNP2, the milk fat rate and dry matter content of CC type individuals were 0.828% and 1.041% higher than those of TT type individuals (P<0.05). At SNP3, the milk fat rate of CC type individuals was significantly higher than that of GG type individuals. The milk fat rate of GG type individuals at SNP4 was 0.734% and 1.062% higher than that of GA and AA type individuals, and the dry matter content was 0.939% and 1.299% higher than that of CC type individuals (P<0.05). The milk fat rate of TT type individuals at SNP5 was significantly higher than that of CC type individuals by 0.968%, and the dry matter content was 0.756% and 1.194% higher than that of CC and TC type individuals (P<0.05).

[0072] (3) In the traditional selection method, individual selection must be carried out after the sheep have lactated based on their milk production performance. At this time, the sheep are about 1.5 years old, and due to the low heritability of some traits, the selection accuracy of this method is not high. If the progeny selection method with higher selection accuracy is used, the generation interval is too long (about 3 years old), and early selection cannot be achieved.

[0073] Therefore, compared with the traditional breeding method, the present invention can select lambs when they are just born, and the selection accuracy is higher than that of the traditional breeding method. Therefore, the present invention can achieve "early selection and accurate selection" of milk production performance of dairy sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 :The expression levels of STAT3 gene in seven tissues of sheep; Note: Different lowercase letters represent significant differences (P<0.05).

[0075] Figure 2 :SNPs sites of sheep STAT3 gene; Note: The arrows indicate the SNPs positions.

[0076] Figure 3 : PARMS phenotyping diagram of sheep STAT3; Note: The X-axis represents the FAM fluorescence signal intensity, and the Y-axis represents the HEX fluorescence signal intensity. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0078] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0079] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0080] The detection methods involved in the following embodiments are as follows:

[0081] The following indicators were measured using the UL40BC milk composition analyzer:

[0082] Milk fat content: the percentage of fat in milk.

[0083] Milk protein rate: the percentage of protein contained in milk.

[0084] Ash content: The amount of minerals in milk.

[0085] Non-fat solids content: the content of nutrients in milk other than fat and water.

[0086] Dry matter content: the content of other nutrients in milk after removing water.

[0087] Lactose: The amount of lactose in sheep's milk.

[0088] The daily milk production of each test sheep was measured using a DeLaval 9JP-2X24 parallel milking machine.

[0089] Example 1: Gene sequencing and analysis of SNPs associated with dry matter content and milk fat rate in sheep milk

[0090] 1. Collection of experimental animals and samples

[0091] (1) Blood collection and breast milk collection

[0092] A total of 414 sheep in lactation period with complete records of parity, lambing number, age, etc. were selected from Gansu Yuansheng Agriculture and Animal Husbandry Technology Co., Ltd., including 35 Hu sheep (H), 17 East Friensian sheep (EF), 193 East Friensian sheep ♂×Hu sheep♀ hybrid first generation sheep (F1) and 169 East Friensian sheep ♂×Hu sheep♀ progressive second generation sheep (F2). During the lactation period (around 30 days after lambing), the daily milk production of the experimental sheep was measured using a DeLaval 9JP-2X24 parallel milking machine (DeLaval, Tianjin, China). 50 mL of milk sample was collected from each experimental sheep, and milk components such as milk fat rate, milk protein rate, ash content, non-fat milk solids content, dry matter content and lactose rate were measured using a UL40BC milk composition analyzer (Uchuang, Hangzhou, China). At the same time, 1 mL of blood was collected from each test sample, dropped on the FTA card and dried for genomic DNA extraction. In addition, three F1 sheep in lactation were selected, and their hearts, livers, spleens, lungs, kidneys, ovaries and mammary gland tissues were collected after slaughter, immediately stored in liquid nitrogen and brought back to the laboratory as soon as possible, and frozen in a -80℃ refrigerator for total RNA extraction.

[0093] 2. Extraction of genomic DNA from sheep blood and RNA from tissues

[0094] The specific steps are as follows:

[0095] 800 μL of blood was dropped onto the FTA card, dried at room temperature, and blood genomic DNA was extracted from the FTA card according to the method described by Zhou et al. (2006) for screening STAT3 gene SNPs sites. The specific method is to use a sampler to take a 1.2 mm diameter disc on the FTA card, place it in a 1.5 mL centrifuge tube, add 200 μL of 20 mmol / L NaOH to the centrifuge tube, and incubate at room temperature for 30 minutes; if the blood sample is stored for a long time, heat it in a 50°C water bath for 25 minutes, and turn the centrifuge tube from time to time during the incubation period; discard the NaOH solution in the tube. Add 200 μL TE solution and let it stand for 2 minutes; discard the TE buffer solution, air-dry the disc at room temperature, and set aside.

[0096] Total RNA from heart, liver, spleen, lung, kidney, ovary and breast tissues was extracted by Rrizol method, and the concentration and purity of RNA were detected by NanoDrop8000 spectrophotometer (Thermo Fisher Scientific, New York, USA). Then, the qualified RNA was reverse transcribed into cDNA using Evo M-LV reverse transcription kit (Acori Biotech, Changsha, China) for detecting the expression of STAT3 gene.

[0097] 3. STAT3 gene PCR primer design, amplification and sequencing

[0098] The specific steps are as follows:

[0099] (1) Primer design for STAT3 gene

[0100] By comparing the SNPs region of the STAT3 gene found in goats and cattle by previous researchers, and referring to the SNPs information predicted on the AOD website and the sheep STAT3 gene sequence on NCBI (XM_060395625), 7 pairs of specific primers P1~P7 were designed using Primer 5.0 software to amplify the 8th exon, 9th exon, 1st intron, 3rd intron, 4th intron, 11th intron and 3'UTR region of the STAT3 gene. At the same time, a pair of RT-qPCR primers were designed in the CDS region to detect the expression level of the STAT3 gene (Table 1).

[0101] Table 1: Primer sequences, product lengths and annealing temperatures

[0102] (2) PCR amplification and SNPs site detection

[0103] Thirty samples were randomly selected from the test sheep, and 20 μL PCR reaction system was used to amplify 7 regions of STAT3 gene, including one 1.2mm DNA card, 0.8 μL of upstream and downstream primers (10 μmol / L), 10.4 μL of TaqDNA polymerase, and 8.0 μL of ddH2O. The amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56-62℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles in total; 72℃ final extension for 7 min; 4℃ storage. 2 μL of PCR amplification products of 30 samples were taken from each sample, mixed according to the principle of equal amount, and sent to Yangling Tianrun Aoke Biotechnology Co., Ltd. for Sanger sequencing. Finally, Chromas software was used to compare and screen SNPs sites.

[0104] (3) Analysis of STAT3 gene expression characteristics

[0105] The qualified cDNA was diluted to a concentration of 30 ng / μL, and β-actin was used as the internal reference gene. RT-qPCR was performed on an Applied Biosystems QuantStudio®6 Flex qPCR instrument (Thermo Fisher Scientific, New York, USA) using a 20 μL system, including 10 μL SYBR Green qPCR Master Mix (Sevier, Wuhan, China), 7.2 μL deionized water, 0.8 μL primers, and 2 μL cDNA. Three biological replicates and four technical replicates were performed for each sample, and 2 were used for final analysis. -ΔΔCtThe relative expression of STAT3 gene in different tissues of sheep was calculated by this method.

[0106] (4) Experimental results

[0107] RT-qPCR results showed that STAT3 gene was expressed in 7 tissues of sheep, with the highest expression in lung, followed by liver and mammary gland, but significantly higher than that in heart, spleen, kidney and ovary tissues (P<0.05), and the lowest expression in spleen ( Figure 1 ).

[0108] The results showed that a total of 5 SNPs were found in the sheep STAT3 gene ( Figure 2 ). Among them, c.22+537C / T (SNP1) and c.22+658C / T (SNP2) were detected in the first intron region, c.318+126C / G (SNP3) was detected in the third intron region, and c.417+360G / A (SNP4) and c.417+395T / C (SNP5) were detected in the fourth intron region.

[0109] SNP1: single nucleotide polymorphism site 1, which is: c.22+537C / T, indicating that the STAT3 gene sequence located on sheep chromosome 11 has a base C / T mutation at c.22+537bp;

[0110] SNP2: indicates single nucleotide polymorphism site 2, which is: c.22+658C / T indicates that the STAT3 gene sequence located on sheep chromosome 11 has a base C / T mutation at c.22+658bp;

[0111] SNP3: single nucleotide polymorphism site 3, which is: c.318+126C / G, indicating that the STAT3 gene sequence located on sheep chromosome 11 has a base C / G mutation at c.318+126bp;

[0112] SNP4: single nucleotide polymorphism site 4, which is: c.417+360G / A, indicating that the STAT3 gene sequence located on sheep chromosome 11 has a base G / A mutation at c.417+360bp;

[0113] SNP5: represents single nucleotide polymorphism site 5, which is: c.417+395T / C, indicating that the STAT3 gene sequence located on sheep chromosome 11 has a base T / C mutation at c.417+395bp;

[0114] Based on the 5 SNPs found, primers for amplifying the above SNPs can be designed, including a forward primer and a reverse primer, wherein the forward primer and the reverse primer are respectively located upstream and downstream of the above 5 single nucleotide polymorphism sites. Based on the primers of the above SNPs, a kit for detecting the above SNPs can also be designed using existing technology.

[0115] Example 2: Association between genotyping and milk production traits in sheep

[0116] 1. Experimental methods

[0117] (1) Genotype determination and haplotype construction

[0118] According to the sequencing results in Example 1, the SNPs sites where mutations occurred in the 1st intron region, the 3rd intron region, and the 4th intron region of the STAT3 gene were counted, and the blood DNA extracted from the blood of 414 lactating ewes selected in Example 1 was sent to Wuhan Jingpeptide Biological Co., Ltd. for PARMS genotyping. The specific method is as follows:

[0119] Five primers were used for PCR reaction, including two common fluorescent primers (CFPs) labeled with fluorophores FAM and HEX, two allele-specific primers (ASPs), and one locus specific primer (LSP). ASPs were combined with LSP to amplify the target site, and CFP recognized the linker sequence and combined with LSP to amplify the corresponding DNA template. The genotype was detected by competitive allele-specific polymerase chain reaction (AS-PCR). Finally, the scatter plot was drawn using Excel 2016 software according to the fluorescence signal value. The specific information of PARMS typing primers is shown in Table 2. Haploview v4.2 software was used to construct the haplotype of STAT3 gene.

[0120] Table 2: PARMS genotyping primer information

[0121] (2) Correlation analysis of lactation traits

[0122] Application of molecular markers of the present invention in association analysis of sheep milk production traits

[0123] A total of 414 lactating ewes were tested for polymorphism, their genotypes were determined, and the general linear mixed effects model of SPSS27.0 was used to analyze the effects of STAT3 gene nucleotide sequence variation (genotype and haplotype combination) on sheep lactation traits. The analysis results were expressed as mean ± standard error.

[0124] In order to determine the fixed and random effects included in the model, we analyzed the effects of age, lambing number and parity on sheep lactation traits. The results showed that lambing number and parity had no significant effect on lactation traits, but age had a very significant effect on lactation traits, so age was added to the model.

[0125] The analysis model is Y = μ + Genotype (Haplotype) + Population + Age + e, where Y represents lactation traits, μ is the population mean, Genotype is the genotype, Haplotype is the haplotype, Population is the experimental population, Age is age, and e is the random effect.

[0126] 2. Experimental results

[0127] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the test process.

[0128] (1) Identification of genotype

[0129] Three genotypes were detected for each SNPs site. Figure 3 ), respectively, CC, CT, TT genotypes in SNP1 and SNP2, CC, CG, GG genotypes in SNP3, GG, GA, AA genotypes in SNP4, and TT, TC, CC genotypes in SNP5;

[0130] No SNPs were detected in other regions.

[0131] (2) Association analysis between sheep STAT3 genotype and lactation traits

[0132] As shown in Tables 3 and 4, the genotypes of the four loci, SNP1, SNP2, SNP4 and SNP5, have significant effects on both milk fat rate and dry matter content, while the genotype of the SNP3 locus only significantly affects the milk fat rate. At SNP1, the milk fat rate of CC type individuals was 0.724% and 1.262% higher than that of CT and TT type individuals, and the dry matter content was 0.924% and 1.584% higher (P<0.05). At SNP2, the milk fat rate and dry matter content of CC type individuals were 0.828% and 1.041% higher than those of TT type individuals, respectively (P<0.05). At SNP3, the milk fat rate of CC type individuals was significantly higher than that of GG type individuals by 0.263% (P<0.05). At SNP4, the milk fat rate of GG type individuals was 0.734% and 1.062% higher than that of GA and AA type individuals, and the dry matter content was 0.939% and 1.299% higher (P<0.05). At SNP5, the milk fat rate of TT type individuals was significantly higher than that of CC type individuals by 0.968%, and the dry matter content was 0.756% and 1.194% higher than that of CC and TC type individuals (P<0.05).

[0133] Table 3: Association analysis between STAT3 genotype and sheep lactation traits

[0134] Table 4: Association analysis between STAT3 genotype and sheep lactation traits

[0135] (3) Effect of sheep STAT3 gene haplotype on sheep lactation traits

[0136] The results of Haploview v4.2 software analysis showed that there was no linkage relationship between SNP4 and the other four SNPs, so the haplotype of SNP4 and other SNPs was not constructed. SNP1, SNP2, SNP3, and SNP5 were in a state of strong linkage disequilibrium (r 2 >0.66), a total of 7 haplotypes with frequencies greater than 1% were constructed, namely H1~H7. A total of 28 haplotype combinations were constructed with the 7 haplotypes, and the 7 haplotype combinations with frequencies greater than 5% are shown in Table 5;

[0137] Table 5: STAT3 gene haplotype combination frequencies

[0138] For haplotype combinations with a frequency greater than 5%, their correlation with sheep lactation traits was further analyzed (Table 6). The milk fat rate and dry matter content of haplotype combination H1H7 were significantly higher than those of H1H1 and H1H3 (P < 0.05), and the ash content of H1H7 was significantly higher than that of H1H1 (P < 0.05). The average daily milk yield of H1H2 was 0.266 kg higher than that of H1H7.

[0139] Table 6: Correlation analysis between STAT3 gene haplotype combinations and sheep lactation traits

[0140] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A single nucleotide polymorphism genetic molecular marker associated with sheep milk production traits, characterized in that: The single nucleotide polymorphism genetic marker is located in sheep STAT3 In the gene, the single nucleotide polymorphism genetic molecular marker is any one or more combinations of the following (a) to (e); (a) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.22+537bp, and there is a C / T base mutation; (b) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.22+658bp, and there is a C / T base mutation; (c) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.318+126bp, and there is a C / G base mutation; (d) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.417+360bp, and there is a G / A base mutation; (e) The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.417+395bp, and there is a T / C base mutation.

2. Use of a reagent for detecting the genotype of at least one of the following five SNP sites in identifying or assisting in identifying the milk production trait of sheep; the SNP site is the positive strand of chromosome 11 corresponding to the sheep genome sequence STAT3 The cytosine C at position c.22+537 of the gene was replaced by thymine T; The SNP site corresponds to the positive strand of chromosome 11 in the sheep genome sequence. STAT3 The cytosine C at position c.22+658 of the gene was replaced by thymine T; The SNP site corresponds to the positive strand of chromosome 11 in the sheep genome sequence. STAT3 The cytosine C at position c.318+126 of the gene was replaced by guanine G; The SNP site corresponds to the positive strand of chromosome 11 in the sheep genome sequence. STAT3 The guanine G at position c.417+360 of the gene was replaced by adenine A; The SNP site corresponds to the positive strand of chromosome 11 in the sheep genome sequence. STAT3 Thymine T at gene c.417+395 was replaced by cytosine C; Preferably, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

3. An application of a haplotype combination sequence of a single nucleotide polymorphism molecular marker associated with sheep milk production traits in improving sheep milk fat content and / or dry matter content and / or ash content and / or average daily milk production, characterized in that: The haplotype combination is the haplotype combination of the single nucleotide polymorphism genetic marker associated with the sheep milk production trait according to claim 1, and the haplotype combination sequence tag is CCGGTTTT or CTGCTCTC or CTGCTTTT or CTGCTCTT or CTGGTCTC; The single nucleotide polymorphism genetic marker is located on the positive strand of chromosome 11 of the sheep genome sequence STAT3 The gene is located at position c.22+537bp, and there is a C / T base mutation; Or located on the positive strand of chromosome 11 in the sheep genome sequence STAT3 The gene is located at position c.22+658bp, and there is a C / T base mutation; Or located on the positive strand of chromosome 11 in the sheep genome sequence STAT3 The gene is located at position c.318+126bp, and there is a C / G base mutation; Or located on the positive strand of chromosome 11 in the sheep genome sequence STAT3 The c.417+360bp position of the gene has a G / A base mutation; or is located on the positive strand of chromosome 11 in the sheep genome sequence STAT3 The gene is located at position c.417+395bp, and there is a T / C base mutation.

4. A method for identifying or assisting in identifying the milk production traits of sheep, characterized in that: The method is any one of the following 1)-5): 1) The following steps are included: testing the sheep STAT3 The genotype of the SNP site c.22+537C / T in the gene; the genotype of the SNP site c.22+537C / T is CC, CT or TT ; Said STAT3 The genotype of the SNP site c.22+537C / T in the gene is CC The milk fat rate and dry matter content of the tested sheep are better or better than the SNP site c.22+537C / T genotype. CT and TT of the tested sheep; 2) The following steps are included: testing the test sheep STAT3 The genotype of the SNP site c.22+658C / T in the gene; the genotype of the SNP site c.22+658C / T is CC, CT or TT ; Said STAT3 The genotype of the SNP site c.22+658C / T in the gene is CC The milk fat rate and dry matter content of the tested sheep are better or better than the SNP site c.22+658C / T genotype. TT of the tested sheep; 3) comprising the following steps: testing the sheep STAT3 The genotype of the SNP site c.318+126C / G in the gene; the genotype of the SNP site c.318+126C / G is CC, CG or GG ; Said STAT3 The genotype of the SNP site c.318+126C / G in the gene is CC The milk fat rate of the tested sheep is better than or assisted by the SNP site c.318+126C / G genotype is GG of the tested sheep; 4) comprising the following steps: testing the sheep STAT3 The genotype of the SNP site c.417+360G / A in the gene; the genotype of the SNP site c.417+360G / A is GG, GA or AA ; Said STAT3 The genotype of the SNP site c.417+360G / A in the gene is GG The milk fat rate and dry matter content of the tested sheep are better or better than the SNP site c.417+360G / A genotype. GA and AA of the tested sheep; 5) comprising the following steps: testing the sheep STAT3 The genotype of the SNP site c.417+395T / C in the gene; the genotype of the SNP site c.417+395T / C is TT, TC or CC ; Said STAT3 The genotype of the SNP site c.417+395T / C in the gene is TT The milk fat rate and dry matter content of the tested sheep are better or better than the SNP site c.417+395T / C genotype. CC of the tested sheep; Preferably, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

5. Use of the method according to claim 4 in sheep screening or sheep breeding.

6. The use according to claim 5, characterized in that: In the application, any of the following sheep in claim 4 is selected for milk production or breeding: Said STAT3 The genotype of the SNP site c.22+537C / T in the gene is CC of the tested sheep; or STAT3 The genotype of the SNP site c.22+658C / T in the gene is CC of the tested sheep; or STAT3 The genotype of the SNP site c.318+126C / G in the gene is CC of the tested sheep; or STAT3 The genotype of the SNP site c.417+360G / A in the gene is GG of the tested sheep; or STAT3 The genotype of the SNP site c.417+395T / C in the gene is TT of the tested sheep.

7. A detection STAT3 A primer pair for detecting a mutation in a gene, characterized in that The upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 1 to 2, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 3 to 4, respectively; or the upstream and downstream nucleotide sequences of the primer pair are shown as SEQ ID NOs. 5 to 6, respectively.

8. A method for detecting the molecular markers related to sheep milk production traits according to claim 1, characterized in that: The steps include: a) Extracting genomic DNA from sheep blood as a sample, using the primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 to identify sheep STAT3 The gene was amplified by PCR; b) performing mixed pool sequencing and sequence analysis on the above PCR amplification products to obtain the mutation site and determine the mutation type; c) by identifying individual sheep STAT3 Genotyping, identification of sheep based on genotyping results STAT3 The genotype and haplotype of the SNP loci in the gene detection region.

9. A kit for detecting molecular markers related to sheep milk production traits, characterized in that: The kit comprises the primer pair according to claim 7.

10. Use of the primer pair or primer combination according to claim 7, wherein the use is any one of the following (a)-(f): (a) Identification or assistance in the identification of milk production traits in sheep; (b) sheep screening; (c) sheep breeding; (d) preparing a kit for identifying or assisting in identifying sheep milk production traits; (e) preparing a kit for sheep screening; (f) preparing a kit for sheep breeding; Preferably, the milk production trait is milk fat percentage and / or ash content and / or dry matter content and / or average daily milk yield.

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