FGF2 gene SNPs marker for detecting sheep milk production character and application of FGF2 gene SNPs marker

Abstract

The invention discloses an FGF2 gene SNPs marker for detecting sheep milk production traits and application of the FGF2 gene SNPs marker, and belongs to the technical field of biology. According to the invention, Dongfenglian milk sheep is taken as a research object, PARMS, Sanger and other technologies are applied to detect a nucleotide sequence variation site of an FGF2 gene, a general linear mixing effect model is constructed, the correlation between sequence variation and sheep lactation traits is researched, and a molecular genetic marker for regulating and controlling the sheep lactation traits is excavated; the method has certain positive effects, and has great advantages compared with the prior art; an SNPs (single nucleotide polymorphisms) site c. 178 + 11694 T / C is found in the FGF2 gene; the milk production character of the test sheep with the genotype of the SNP site 1 c.178 + 11694 T / C being CC or CT is superior to or assisted superior to that of the test sheep with the genotype of the SNP site 1 c.178 + 11694 T / C being TT.

Description

Technical Field

[0001] The invention relates to an FGF2 gene SNPs marker for detecting sheep milk production traits and application thereof, belonging to the field of biotechnology. Background Art

[0002] Sheep milk has been widely recognized and welcomed due to its unique nutritional ingredients, easy digestibility and high nutritional value. It is known as the "king of milk" in the international nutrition community. The content of milk protein, casein, calcium, some trace elements and conjugated linoleic acid in sheep milk is higher than that of cow milk and goat milk, and it has low allergenicity. In addition, sheep milk is the main source of nutrition for the survival, growth and normal development of newborn lambs. Therefore, sufficient milk production is an extremely important factor in ensuring the survival rate and growth rate of offspring lambs. Lactation traits are important economic traits, but the heritability of some traits is low (such as the heritability of milk production is 0.20~0.25, and the lactose content is 0.25), and it is difficult to achieve good breeding results using conventional breeding methods. Molecular marker-assisted selection can effectively shorten the selection generation interval and improve the selection accuracy of low-heritability traits, but the premise is to find effective molecular genetic markers.

[0003] Fibroblast growth factor 2 (FGF2) is a member of the fibroblast growth factor family and an effective epithelial cell mitogen. The signal transduction pathways involved are closely related to breast cancer and mammary gland development. Jeong et al. found that the FGF2 gene induced mammary epithelial cell proliferation and cell cycle progression by activating the PI3K / AKT, ERK1 / 2 and JNK MAPK signaling cascades in dairy cows, and protected mammary epithelial cells from endoplasmic reticulum stress, thereby increasing the milk production of dairy cows. In addition, the FGF2 gene can also regulate the expression of interferon-τ (IFNT), a key member of the signal transduction pathway involved in milk synthesis. Chodosh et al. found that the absence of FGF receptors would lead to defects in the branching morphology of the mouse mammary gland. Yu Guangpu et al. found that FGF2 can successfully stimulate the branching morphology of cow mammary gland organoids in type I collagen. Previous studies have found that nucleotide sequence variations of the FGF2 gene significantly affect the lactation performance of livestock. For example, Wang et al. found that the g.11646 A>G site in the first intron of the FGF2 gene in dairy cows was significantly correlated with the milk fat content, percentage and somatic cell score of milk, which is consistent with the results of Wang Xin et al. At the same time, Brzáková et al. found that this SNP site also affects the milk production of dairy cows. Marete et al. found that the FGF2 gene is a candidate gene for mammary gland development in dairy cows, and its SNPs site rs41609100 is significantly correlated with the front udder attachment and front nipple position. Abdolreza et al. found that g.11863T>C at the first intron 1 of the FGF2 gene in dairy cows can be used as a candidate SNPs site affecting milk production traits. At present, relevant research on the FGF2 gene has been carried out in many species such as mice, rabbits, frogs, pigs, goats and dairy cows, but there are few reports on its effects on mammary gland development and lactation performance in sheep.

[0004] Therefore, this study took East Fry dairy sheep as the research object, applied PARMS, Sanger and other technologies to detect the nucleotide sequence variation sites of the FGF2 gene, and constructed a general linear mixed effects model to study the correlation between nucleotide sequence variation and sheep lactation traits, in order to provide a theoretical basis for exploring molecular genetic markers that regulate sheep lactation traits. Summary of the invention

[0005] The present invention provides an application of detecting the genotype of the following SNP loci in identifying or assisting in identifying the milk production trait of sheep;

[0006] The SNP site is that the thymine T at position c. 178+11694 of the FGF2 gene on the positive strand of chromosome 17 in the sequence information of the sheep reference genome Oar_v4.0 version is replaced by cytosine C.

[0007] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or milk protein percentage and / or ash content and / or non-fat milk solids content and / or dry matter content and / or acidity and / or daily milk yield.

[0008] The present invention provides an application of the following SNP site as a detection target in identifying or assisting in identifying sheep milk production traits; the SNP site is that thymine T at position c. 178+11694 of the FGF2 gene on the positive strand of chromosome 17 corresponding to the sequence information of the sheep reference genome Oar_v4.0 version is replaced by cytosine C.

[0009] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or milk protein percentage and / or ash content and / or non-fat milk solids content and / or dry matter content and / or acidity and / or daily milk yield.

[0010] The present invention also provides a method for identifying or assisting in identifying sheep milk production traits, the method comprising the following steps: detecting the genotype of the SNP site c. 178+11694 T / C in the FGF2 gene of the tested sheep; the genotype of the SNP site c. 178+11694 T / C is CC, TT or CT;

[0011] The milk fat rate and dry matter content of the test sheep whose SNP site 1: c. 178+11694 T / C genotype is CC or CT are better than or auxiliary better than those of the test sheep whose SNP site 1: c. 178+11694 T / C genotype is TT.

[0012] In one embodiment of the present invention, the milk production trait is milk fat percentage and / or milk protein percentage and / or ash content and / or non-fat milk solids content and / or dry matter content and / or acidity and / or daily milk yield.

[0013] The invention also provides application of the method in dairy sheep screening or dairy sheep breeding.

[0014] In one embodiment of the present invention, in the application, any of the above-mentioned test sheep is selected for milk production or breeding; the SNP site 1: c. 178+11694 T / C genotype is CC or CT test sheep;

[0015] The present invention also provides a primer pair for detecting FGF2 gene mutation, wherein the upstream and downstream nucleotide sequences of the primer pair are shown in SEQ ID NO.1-2 respectively.

[0016] The present invention also provides a method for detecting molecular markers related to milk fat rate and dry matter content of sheep milk, the method comprising the following steps:

[0017] a) extracting genomic DNA from sheep blood as a sample, and performing PCR amplification on the sheep FGF2 gene using the primers shown in SEQ ID NO.1-2;

[0018] b) performing mixed pool sequencing and sequence analysis on the above PCR amplification products to obtain the mutation site and determine the mutation type;

[0019] c) Performing FGF2 genotyping on the identified dairy sheep individuals, and identifying the genotype of the SNP site in the sheep FGF2 gene detection region according to the typing results.

[0020] The present invention also provides a kit for detecting molecular markers, wherein the kit comprises the above primer pair.

[0021] The present invention also provides an application of the above primer pair, wherein the application is any one of the following (a)-(f):

[0022] (a) To determine or assist in the determination of milk fat content and dry matter content in sheep;

[0023] (b) sheep screening;

[0024] (c) sheep breeding;

[0025] (d) Preparation of a test kit for identifying or assisting in identifying milk fat percentage and dry matter content in sheep;

[0026] (e) preparing a kit for sheep screening;

[0027] (f) Preparation of a kit for sheep breeding.

[0028] Beneficial Effects

[0029] The present invention uses East Fry raw milk sheep as the research object, applies PARMS, Sanger and other technologies to detect the nucleotide sequence variation sites of the FGF2 gene, and constructs a general linear mixed effect 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:

[0030] (1) A SNP site c. 178+11694 T / C was found in the first intron of the FGF2 gene. Three genotypes, CC, CT and TT, were detected at the c. 178+11694 T / C site. At the SNP1 site, the dry matter content of CC and CT genotype individuals was 1.467% and 0.940% higher than that of TT genotype individuals (P=0.019), and their milk fat content was 1.594% and 1.173% higher than that of TT genotype individuals (P=0.005).

[0031] (2) At the SNP1 locus (c. 178+11694 T / C), the presence of allele C was associated with higher dry matter content (presence: 16.990±0.186; absence: 15.906±0.449) (P=0.021) and higher milk fat percentage (presence: 5.465±0.172; absence: 4.172±0.417) (P=0.003). In addition, the presence of allele T was associated with lower dry matter content (presence: 16.574±0.228; absence: 17.225±0.252) (P=0.044). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Sequencing peaks of sheep FGF2 gene SNP1 (A) and SNP2 (B).

[0033] Figure 2 : PARMS genotype of sheep FGF2; FAM: wild type; HEX: mutant; FAMHEX: heterozygous; negative control: no DNA template added. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0037] The detection methods involved in the following embodiments are as follows:

[0038] The following indicators were measured using the UL40BC milk composition analyzer:

[0039] Milk fat content: the percentage of fat in milk.

[0040] Milk protein rate: the percentage of protein contained in milk.

[0041] Ash content: The amount of minerals in milk.

[0042] Non-fat solids content: the content of nutrients in milk other than fat and water.

[0043] Dry matter content: the content of other nutrients in milk after removing water.

[0044] Acidity: The pH value of milk is an important physical and chemical indicator to measure the freshness of sheep milk.

[0045] Example 1: Gene sequencing and analysis of SNPs associated with dry matter content and milk fat rate in sheep milk

[0046] 1. Collection of experimental animals and samples

[0047] (1) Blood collection

[0048] A total of 449 East Fry raw-milk sheep ewes with complete pedigree information such as age, lambing number, and parity were selected from Gansu Yuansheng Agriculture and Animal Husbandry Technology Co., Ltd. In the mid-lactation period of the experimental sheep, 5 mL of blood samples were collected from the jugular vein of each experimental sheep in a vacuum blood collection tube (containing EDTA anticoagulant) and stored in a -20°C refrigerator.

[0049] (2) Collection of milk

[0050] For all 449 East Friesian raw milk ewes from which blood samples were collected, 50 mL of milk samples were collected once in the morning, afternoon and evening every day during their mid-lactation period. After mixing, the milk fat rate, milk protein rate, ash content, non-fat milk solids content, dry matter content, acidity and other milk component indicators of each sample were measured. The daily milk production of each test sheep was measured using a DeLaval 9JP-2X24 parallel milking machine.

[0051] 2. Extraction of genomic DNA from sheep blood

[0052] The specific steps are as follows:

[0053] Take 0.8 mL of blood sample and drop it onto the FTA card. After air drying, use a two-step method to extract genomic DNA from the FTA card for PCR amplification.

[0054] The specific steps are as follows:

[0055] 1) Use a sampler to take a 1.2 mm diameter disc from the FTA test paper and place it in a 0.2 ml PCR tube;

[0056] 2) Add 200 μL of 20 mM NaOH to the PCR tube containing the disk and incubate at room temperature for 30 minutes. If the blood sample is stored for a long time, incubate at 50°C for 30 minutes. During the incubation period, the PCR tube should be turned over from time to time.

[0057] 3) Discard the liquid in the tube, add 200 μL TE solution, and let it stand for 2 minutes;

[0058] 4) Discard the TE buffer and air-dry the disk at room temperature for later use.

[0059] 3. FGF2 gene PCR primer design, amplification and sequencing

[0060] The specific steps are as follows:

[0061] (1) Primer design of FGF2 gene

[0062] Using the sheep FGF2 sequence (Gene ID: 443306) in NCBI as a template, two pairs of primers were designed using Prime3.0 to amplify the first intron region of the FGF2 gene. The primers were synthesized by Yangling Tianrun Aoke Biotechnology Co., Ltd. The specific information is shown in Table 1.

[0063] Table 1: Primer sequence information

[0064] (2) PCR amplification

[0065] PCR amplification used a 20 μL reaction system, including one 1.2 mm DNA card, 0.8 μL of upstream and downstream primers, 10.4 μL of Taq DNA polymerase, and 8.0 μL of ddH2O. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, denaturation at 94°C for 30 s, annealing for 30 s (annealing temperature see Table 1), extension at 72°C for 30 s, a total of 30 cycles, and extension at 72°C for 7 min. 1.5% agarose gel electrophoresis was used to detect the PCR amplification results.

[0066] From the above amplification products, specific PCR stock solutions of 30 unrelated samples were randomly selected, mixed according to the principle of equal amount, and sent to Tianrun Aoke Biotechnology Co., Ltd. for Sanger sequencing, and Chromas software was used to screen SNPs sites.

[0067] (3) Experimental results

[0068] By performing Blast alignment on the Sanger sequencing results and combining the Chromas software to observe and analyze the results, a total of two SNPs sites were detected in the first intron region of the FGF2 gene, namely: SNP1 and SNP2 had T / C and C / T mutations, respectively.

[0069] Two SNPs were found in the first intron of the FGF2 gene, namely c. 178+11694 T / C (named SNP1) and c.178+26124 C / T (named SNP2) ( Figure 1 ).like Figure 1 As shown:

[0070] SNP1: single nucleotide polymorphism site 1, which is: c. 178+11694 T / C, indicating that the FGF2 gene sequence located on sheep chromosome 17 has a base T / C mutation at c. 178+11694 bp;

[0071] SNP2: indicates single nucleotide polymorphism site 2, which is: c.178+26124 C / T, indicating that the FGF2 gene sequence located on sheep chromosome 17 has a base C / T mutation at c.178+26124 bp;

[0072] Based on the two 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 two 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.

[0073] Example 2: Correlation analysis between genotyping and dry matter content and milk fat percentage of sheep milk

[0074] 1. Experimental methods

[0075] (1) According to the sequencing results in Example 1, the SNPs sites where mutations occurred in the first intron region of the FGF2 gene were counted, and the blood DNA samples extracted from the blood of all 449 East Friesian raw milk sheep were matched with the SNPs site information of the FGF2 gene. Then, genotyping analysis was performed using PARMS (Penta-primer amplification refractory mutation system) technology combined with GeneMatrix high-throughput genotyping system, and the samples were sent to Wuhan Jingpeptide Biotechnology Co., Ltd. for allele and genotyping of the screened SNPs sites.

[0076] (2) Calculation of gene frequency, genotype frequency, and Hardy-Weinberg law

[0077] The allele frequency, genotype frequency, homozygosity (Ho), heterozygosity (He), effective number of alleles (Ne) and polymorphism information content (PIC) of SNPs loci were calculated according to the online website http: / / www.msrcall.com / Gdicall.aspx.

[0078] (3) Correlation analysis of lactation traits

[0079] Application of the molecular marker of the present invention in the correlation analysis of milk fat rate and dry matter content in sheep milk

[0080] A total of 449 East Fry dairy sheep were tested for polymorphism and their genotypes were determined. The general linear mixed effects model of SPSS16.0 was used to analyze the correlation between the genotype / allele of the SNPs locus of FGF2 and the lactation traits of sheep. The analysis results were expressed as mean ± standard error.

[0081] In order to determine the fixed and random effects included in the model, we analyzed the effects of age, lambing number and parity on lactation traits in sheep. 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. Therefore, we added genotype (allele) and age to the general linear mixed effects model.

[0082] The model is Y = μ + Genotype (Allele) + Age + e, where Y represents the lactation trait, μ is the population mean, Genotype is the genotype, Allele is the allele, Age is the age, and e is the random residual.

[0083] 2. Experimental results

[0084] 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.

[0085] (1) The results of sheep FGF2 genotype detection are shown in 2-3:

[0086] Table 2: Genotype of SNP1 site of sheep FGF2 gene

[0087] Table 3: Genotype of SNP2 locus of sheep FGF2 gene

[0088] The results show:

[0089] Three genotypes, CC, CT and TT, were detected at the c.178+11694 T / C locus, and three genotypes, DD, DE and EE, were detected at the c.178+26124 C / T locus ( Figure 2 ).

[0090] (2) Analysis of genetic polymorphism of sheep FGF2 gene

[0091] At the SNP1 locus, 180, 50 and 219 individuals were CC, TT and CT genotypes, and the frequencies of the three genotypes were 0.401, 0.111 and 0.488, respectively. The frequencies of alleles C and T were 0.645 and 0.355, respectively. The genetic heterozygosity He, effective allele number Ne and polymorphism information content PIC of the population were 0.458, 1.845 and 0.353, respectively.

[0092] At the SNP2 locus, 7, 320 and 122 individuals were DD, EE and DE genotypes, and the frequencies of the three genotypes were 0.016, 0.713 and 0.271, respectively. The frequencies of alleles D and E were 0.151 and 0.849, respectively. The genetic heterozygosity He, effective allele number Ne and polymorphic information content PIC of the population were 0.257, 1.346 and 0.224, respectively.

[0093] (3) Analysis of the association between sheep FGF2 genotype and sheep lactation traits

[0094] At the SNP1 locus, the dry matter content of CC and CT genotype individuals was 1.467% and 0.940% higher than that of TT genotype individuals (P=0.019), and their milk fat percentage was 1.594% and 1.173% higher than that of TT genotype individuals (P=0.005). The genotype of SNP1 locus had no significant effect on other lactation traits (P>0.05) (Table 4).

[0095] Table 4: Association analysis between SNP1 genotype of sheep FGF2 and lactation traits

[0096] In SNP2, the number of DD genotypes was too small (7) and the frequency was only 1.6%, so the effect of this gene on lactation traits was not analyzed. Only the effects of EE and DE genotypes on traits were analyzed. The results showed that the SNP2 genotype had no significant effect on all lactation traits (P>0.05) (Table 5).

[0097] Table 5: Association analysis between SNP2 genotype of sheep FGF2 and lactation traits

[0098] (4) Effects of FGF2 alleles on sheep lactation traits

[0099] At the SNP1 locus, the presence of allele C was associated with higher dry matter content (presence: 16.990 ± 0.186; absence: 15.906 ± 0.449) (P = 0.021) and higher milk fat percentage (presence: 5.465 ± 0.172; absence: 4.172 ± 0.417) (P = 0.003). In addition, the presence of allele T was associated with lower dry matter content (presence: 16.574 ± 0.228; absence: 17.225 ± 0.252) (P = 0.044) (Table 6)

[0100] Table 6: Sheep FGF2 Effects of SNP1 alleles on lactation traits in sheep

[0101] At the SNP2 locus, the status (presence or absence) of alleles D and E had no significant effect on all lactation traits (P>0.05) (Table 7).

[0102] Table 7: Sheep FGF2 Effects of SNP2 alleles on lactation traits in sheep

[0103] 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. Use of the genotype of the following SNP site in identifying or assisting in identifying the milk production trait of sheep; the SNP site is the positive strand of chromosome 17 corresponding to the sequence information of the sheep reference genome Oar_v4.0 version FGF2 The thymine T at position c. 178+11694 of the gene was replaced by cytosine C.

2. Application of the following SNP loci as detection targets in identifying or assisting in identifying sheep milk production traits; the SNP loci correspond to the positive strand of chromosome 17 in the sequence information of the sheep reference genome Oar_v4.0 version FGF2 The thymine T at position c. 178+11694 of the gene was replaced by cytosine C.

3. The use according to claim 1 or 2, characterized in that: The milk production traits are milk fat rate and / or milk protein rate and / or ash content and / or non-fat milk solids content and / or dry matter content and / or acidity and / or daily milk yield.

4. A method for identifying or assisting in identifying the milk production traits of sheep, characterized in that: The following steps are included: testing the test sheep FGF2 The genotype of the SNP site c. 178+11694 T / C in the gene; The genotype of the SNP site c. 178+11694 T / C is CC, TT or CT; The milk fat rate and dry matter content of the test sheep whose SNP site 1: c. 178+11694 T / C genotype is CC or CT are better or auxiliary better than those of the test sheep whose SNP site 1: c. 178+11694 T / C genotype is TT; Preferably, the milk production trait is milk fat percentage and / or milk protein percentage and / or ash content and / or non-fat milk solids content and / or dry matter content and / or acidity and / or daily milk yield.

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

6. The use according to claim 5, characterized in that: In the application, the test sheep described in claim 4 is selected for milk production or breeding; the test sheep FGF2 The genotype of SNP site c. 178+11694 T / C in the gene is CC or CT.

7. A detection FGF2 A primer pair for detecting gene mutation, characterized in that: The nucleotide sequences upstream and downstream of the primer pair are shown in SEQ ID NO.1~2 respectively.

8. A method for detecting molecular markers related to milk fat content and dry matter content of sheep milk, characterized in that: The steps include: a) Extract genomic DNA from sheep blood as a sample, and use the primers shown in SEQ ID NO.1~2 to PCR amplify the FGF2 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 dairy sheep FGF2 Genotyping, identification of sheep based on genotyping results FGF2 The genotype of the SNP site in the gene detection region.

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

10. The use of the primer according to claim 7, wherein the use is any one of the following (a)-(f): (a) To identify or assist in the identification of milk fat content and dry matter content in sheep milk; (b) sheep screening; (c) sheep breeding; (d) preparing a kit for identifying or assisting in identifying the milk fat rate and dry matter content in sheep milk; (e) preparing a kit for sheep screening; (f) Preparation of a kit for sheep breeding.

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