Fgf2 gene snps marker for detecting milk production traits of sheep and application thereof

Abstract

The application discloses a kind of for detecting sheep milk production character FGF2 Gene SNPs marker and its application belong to the field of biotechnology.The present application takes east frier milk sheep as research object, and the nucleotide sequence variation site of gene is detected by applying PARMS, Sanger and other technologies FGF2 General linear mixed effect model is constructed, the correlation between sequence variation and sheep lactation character is researched, and molecular genetic marker for regulating sheep lactation character is excavated.The present application has achieved some positive effects, and indeed has great advantages compared with prior art;in FGF2 Gene discovers SNPs site c.178+11694 T / C;The milk production character of test sheep of SNP site 1 c.178+11694 T / C genotype CC or CT is better than or auxiliary better than the test sheep of SNP site 1: c.178+11694 T / C genotype TT.

Description

TECHNICAL FIELD

[0001] The present application relates to a FGF2 gene SNP marker for detecting the milk production traits of sheep and its application, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Sheep milk is widely recognized and welcomed due to its unique nutritional ingredients, easy digestion and high nutritional value, and is known as the "King of Milk" in the international nutrition community. The milk protein, casein, calcium content, part of the trace element content and conjugated linoleic acid content in sheep milk are higher than those in 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 lactation is an extremely important factor to ensure 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 heritability of lactose content is 0.25), and it is difficult to achieve good breeding effect by using conventional breeding methods. Molecular marker assisted selection can effectively shorten the generation interval of selection and improve the selection accuracy of low heritability traits, but the prerequisite is to find effective molecular genetic markers.

[0003] Fibroblast growth factor 2 (FGF2) is a member of the fibroblast growth factor family, which is an effective mitogen for epithelial cells. The signaling pathways in which FGF2 is involved are closely related to breast cancer and breast development. Jeong et al. found that FGF2 gene induces mammary epithelial cell proliferation and cell cycle progression by activating PI3K / AKT, ERK1 / 2 and JNK MAPK signaling cascades, and protects mammary epithelial cells from endoplasmic reticulum stress, thereby increasing milk production in dairy cows. In addition, 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 leads to defects in the branching morphogenesis of mouse mammary glands. Yu Guangpu et al. found that FGF2 can successfully stimulate the branching morphogenesis of bovine mammary gland organoids in type I collagen. Previous studies have found that nucleotide sequence variations in 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 of dairy cows is significantly related to milk fat content, percentage and somatic cell score, which is consistent with the results of Wang Xi et al. At the same time, Brzakova et al. found that this SNP site also affects milk production in dairy cows. Marete et al. found that FGF2 gene is a candidate gene for bovine mammary gland development, and the SNP site rs41609100 is significantly related to the pre-udder attachment and pre-teat position. Abdolreza et al. found that g.11863T>C in the first intron of the FGF2 gene of dairy cows can be used as a candidate SNP site affecting milk production traits. At present, FGF2 gene related research has been carried out in many species such as mice, rabbits, frogs, pigs, goats and dairy cows, but there are few reports on the effect of FGF2 gene on the development of sheep mammary glands and lactation performance.

[0004] Therefore, in the present study, the nucleotide sequence variation sites of FGF2 gene were detected by PARMS, Sanger and other technologies, and a general linear mixed effect model was constructed to study the correlation between nucleotide sequence variation and sheep lactation traits, in order to provide a theoretical basis for mining molecular genetic markers for regulating sheep lactation traits. SUMMARY

[0005] The application provides a use of a genotype of the following SNP site for identifying or assisting in identifying a milk production trait of a sheep.

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

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

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

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

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

[0011] The SNP site 1: a test sheep with a genotype of the SNP site 1: c. 178+11694 T / C being CC or CT is superior or assisted in being superior in a milk fat percentage and a dry matter content to a test sheep with a genotype of the SNP site 1: c. 178+11694 T / C being TT.

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

[0013] The present application also provides application of the above method in screening of a milk sheep or breeding of a milk sheep.

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

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

[0016] The application further provides a method for detecting a molecular marker related to milk fat percentage and dry matter content of sheep, comprising the following steps:

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

[0018] b) performing pool sequencing and sequence analysis on the PCR amplification product, obtaining a variation site and determining a variation type;

[0019] c) performing FGF2 gene typing on an identified individual of milk sheep, and identifying the genotype of a SNP site in a detection region of a sheep FGF2 gene according to a typing result.

[0020] The application further provides a kit for detecting a molecular marker, wherein the kit contains the primer pair.

[0021] The application further provides application of the primer pair, which is any one of the following (a)-(f):

[0022] (a) identifying or assisting in identifying milk fat percentage and dry matter content in sheep;

[0023] (b) sheep screening;

[0024] (c) sheep breeding;

[0025] (d) preparing a 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) preparing a kit for sheep breeding.

[0028] Beneficial effects

[0029] The application takes Dongfali raw milk sheep as a research object, detects nucleotide sequence variation sites of an FGF2 gene by using PARMS, Sanger and other technologies, constructs a general linear mixed effect model, studies the correlation between nucleotide sequence variation and sheep lactation traits, and mines a molecular genetic marker for regulating sheep lactation traits. The application has achieved some positive effects and has great advantages compared with prior art, and the advantages are as follows:

[0030] (1) One SNP site c. 178+11694 T / C was found in the first intron of FGF2 gene; three genotypes CC, CT and TT were detected at the site c. 178+11694 T / C; at the SNP1 site, the CC genotype and CT genotype individuals increased the dry matter content by 1.467% and 0.940% (P=0.019) respectively compared with the TT genotype individuals, and their milk fat rates increased by 1.594% and 1.173% (P=0.005) respectively compared with the TT genotype individuals.

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

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

[0033] Figure 2 Figure 3: Genotype PARMS typing map of sheep FGF2 gene; FAM: wild type; HEX: mutant type; FAM / HEX: hybrid type; negative control: no DNA template added. DETAILED DESCRIPTION

[0034] The application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0035] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0036] In the quantitative experiments in the following examples, three repeated experiments were set up, and the results were averaged, unless otherwise specified.

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

[0038] The following indexes were determined using a UL40BC milk component analyzer:

[0039] Fat percentage: the percentage of fat contained in milk.

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

[0041] Ash content: the content of minerals in milk.

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

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

[0044] Acidity: the pH value of milk, which is an important physicochemical index for measuring the freshness of sheep milk.

[0045] Example 1: Gene sequencing and analysis of SNPs related to dry matter content and fat percentage of sheep milk

[0046] 1. Test animals and collection of samples

[0047] (1) Collection of blood

[0048] At Gansu Yuansheng Farming and Stockbreeding Technology Co., Ltd., 449 East Fleece milk sheep ewes with complete pedigree information records of age, lambing number, and parity were selected. In the middle of lactation, 5 mL of blood sample was collected from the jugular vein of each test 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 Fleece milk sheep ewes with blood samples collected above, 50 mL of milk sample was collected from each of them once a day in the morning, noon, and evening during the middle of lactation, and mixed and measured for milk component indexes such as fat percentage, protein percentage, ash content, non-fat solid content, dry matter content, and acidity of each sample. The daily milk yield of each test sheep was determined using a Lelyva 9JP-2X24 side-by-side milking machine.

[0051] 2. Extraction of sheep blood genomic DNA

[0052] The specific steps are as follows:

[0053] Take 0.8 mL of blood sample and drop it onto an FTA card. After natural air drying, the genomic DNA in the FTA card is extracted using a two-step method for PCR amplification.

[0054] The specific operation steps are as follows:

[0055] 1) Take a 1.2 mm diameter disc from the FTA paper with a swab, and put it into a 0.2 ml PCR tube;

[0056] 2) Add 200 μL of 20 mM NaOH to the PCR tube containing the disc, and incubate at room temperature for 30 minutes, or at 50°C for 30 minutes if the blood sample has been stored for a long time. The PCR tube should be inverted from time to time during the incubation period;

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

[0058] 4) Discard the TE buffer in the tube, and air dry the disc at room temperature, and store it for later use.

[0059] 3. Design, amplification, and sequencing of FGF2 gene primers

[0060] The specific steps are as follows:

[0061] (1) Design of FGF2 gene primers

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

[0063] Table 1: Primer sequence information

[0064]

[0065] (2) PCR amplification

[0066] The PCR amplification used a 20 μL reaction system, which contained 1.2 mm DNA card, 0.8 μL of each of the upstream and downstream primers, 10.4 μL of Taq DNA polymerase, and 8.0 μL of ddH2O. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing for 30 s (the annealing temperature is shown in Table 1), extension at 72°C for 30 s, a total of 30 cycles, and extension at 72°C for 7 min. The PCR amplification results were detected by 1.5% agarose gel electrophoresis.

[0067] From the above amplification products, 30 specific PCR stock solutions of unrelated samples were randomly selected, mixed according to the equal principle, and sent to Tianrun Aoke Biotechnology Co., Ltd. for Sanger sequencing. SNPs sites were screened using Chromas software.

[0068] (3) Experimental results

[0069] Through Blast comparison of the sanger sequencing results, combined with Chromas software observation and analysis results, two SNPs sites were detected in the first intron region of FGF2 gene, namely SNP1 and SNP2, which occurred T / C and C / T mutations respectively.

[0070] Two SNPs sites were found in the first intron of FGF2 gene, which were c. 178+11694 T / C (named SNP1) and c. 178+26124 C / T (named SNP2). Figure 1 Figure 1 As shown in the following table:

[0071] SNP1: represents single nucleotide polymorphism site 1, which is c. 178+11694 T / C, indicating that the FGF2 gene sequence on sheep chromosome 17 at c. 178+11694 bp exists base T / C mutation;

[0072] SNP2: represents single nucleotide polymorphism site 2, which is c. 178+26124 C / T, indicating that the FGF2 gene sequence on sheep chromosome 17 at c. 178+26124 bp exists base C / T mutation.

[0073] Based on the two SNPs found, primers for amplifying the above SNPs can be designed, including forward primers and reverse primers, which are located upstream and downstream of the above two single nucleotide polymorphism sites. Based on the primers of the above SNPs, reagents for detecting the above SNPs can also be designed using existing technology.

[0074] Example 2: Analysis of the correlation between genotyping and the dry matter content and milk fat rate of sheep milk

[0075] 1. Experimental method

[0076] (1) According to the sequencing results in Example 1, the SNPs sites that occur mutation in the first intron region of FGF2 gene were counted. For all 449 blood DNA samples extracted from the blood of East Fleece milk sheep, the information of FGF2 gene SNPs sites was corresponded, and then the PARMS (Penta-primer amplification refractory mutation system) technology combined with GeneMatrix high-throughput genotyping system was used for genotyping analysis. The SNPs sites screened were sent to Wuhan Jingpei Biological Technology Co., Ltd. for allele and genotype typing.

[0077] (2) Gene frequency, genotype frequency, and Hardy-Weinberg law calculation ​

[0078] The allele frequency, genotype frequency, homozygosity (Ho), heterozygosity (He), effective number of alleles (Ne) and polymorphism information content (PIC) of the SNP site were calculated according to the online website http: / / www.msrcall.com / Gdicall.aspx.

[0079] (3) Correlation analysis of lactation traits

[0080] Application of the molecular marker in the correlation analysis of milk fat percentage and dry matter content in sheep milk

[0081] The polymorphism of 449 East Fleece ewes was detected, the genotype was determined, and the correlation between the genotype / allele of the FGF2 SNP site and the lactation traits of the sheep was analyzed by using the general linear mixed effect model of SPSS 16.0, and the analysis result was expressed by mean value ± standard error.

[0082] In order to determine the fixed effects and random effects included in the model, the influence of age, lambing number and parity on the lactation traits of the sheep was analyzed, and the result shows that the lambing number and the parity have no significant influence on the lactation traits, but the age has a very significant influence on the lactation traits. Therefore, the genotype (allele) and the age are added to the general linear mixed effect model.

[0083] The model is Y = μ + Genotype (Allele) + Age + e, wherein Y represents the lactation traits, µ is the population mean, Genotype is the genotype, Allele is the allele, Age is the age, and e is the random residual error.

[0084] 2, Experimental results

[0085] The embodiments of the present application have achieved some positive effects in the research and development or use process, and indeed have great advantages compared with the prior art, and the following contents are described in combination with the data, charts and the like in the experimental process.

[0086] (1) The genotype detection result of the sheep FGF2 is shown in Tables 2-3:

[0087] Table 2: Genotype of the SNP1 site of the sheep FGF2 gene

[0088]

[0089] Table 3: Genotype of the SNP2 site of the sheep FGF2 gene

[0090]

[0091] Results showed that:

[0092] Three genotypes CC, CT and TT were detected at c. 178+11694 T / C locus, and three genotypes DD, DE and EE were detected at c. 178+26124 C / T locus. Figure 2

[0093] (2) Analysis of genetic polymorphism of FGF2 gene in sheep

[0094] At SNP1 locus, 180, 50 and 219 individuals were CC, TT and CT genotypes, respectively, 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 polymorphic information content PIC of this population were 0.458, 1.845 and 0.353, respectively.

[0095] At SNP2 locus, 7, 320 and 122 individuals were DD, EE and DE genotypes, respectively, 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 this population were 0.257, 1.346 and 0.224, respectively.

[0096] (3) Association analysis of sheep FGF2 genotypes with lactation traits of sheep

[0097] At SNP1 locus, the dry matter content of individuals with CC genotype and CT genotype was increased by 1.467% and 0.940% compared with individuals with TT genotype (P = 0.019), and their milk fat rate was increased by 1.594% and 1.173% compared with individuals with TT genotype (P = 0.005), and the genotype of SNP1 locus had no significant effect on other lactation traits (P > 0.05) (Table 4).

[0098] Table 4: Association analysis of SNP1 genotype of FGF2 with lactation traits

[0099]

[0100] ​In SNP2, the effect of the gene on the lactation traits was not analyzed due to the small number of DD genotypes (7) and the low frequency (1.6%). Only the effect of EE and DE genotypes on the traits was analyzed. The results showed that the SNP2 genotypes had no significant effect on all the lactation traits (P>0.05) (Table 5).

[0101] Table 5: Association analysis of SNP2 genotypes of FGF2 with lactation traits in sheep

[0102]

[0103] (4) Effect of FGF2 alleles on lactation traits in sheep

[0104] At SNP1 locus, the presence of allele C was associated with higher dry matter content (present: 16.990 ± 0.186; absent: 15.906 ± 0.449) (P = 0.021) and higher milk fat percentage (present: 5.465 ± 0.172; absent: 4.172 ± 0.417) (P = 0.003). In addition, the presence of allele T was associated with lower dry matter content (present: 16.574 ± 0.228; absent: 17.225 ± 0.252) (P = 0.044) (Table 6).

[0105] Table 6: Effect of alleles of SNP1 on lactation traits in sheep FGF2 SNP1 of the alleles of the lactation traits in sheep

[0106] At SNP2 locus, the status (present vs. absent) of alleles D and E had no significant effect on all the lactation traits (P>0.05) (Table 7).

[0107] Table 7: Effect of alleles of SNP2 on lactation traits in sheep FGF2 SNP2 of the alleles of the lactation traits in sheep

[0108] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the application, and any person skilled in the art, without departing from the spirit and scope of the application, can make various modifications and modifications, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. Use of a reagent for detecting the genotype of the following SNP site in identifying or assisting in identifying the milk production trait of sheep; the SNP site is the thymine T at position c. 178+11694 of the gene on the positive strand of chromosome 17 corresponding to the sequence information of the sheep reference genome Oar_v4.0 version FGF2 thymine T at position c. 178+11694 of the gene is replaced by cytosine C, the sheep is an East Friesian dairy sheep, and the milk fat percentage and dry matter content of the test sheep with the SNP site: c. 178+11694 T / C genotype of CC or CT are better than or assist in being better than the test sheep with the SNP site: c. 178+11694 T / C genotype of TT. The milk production trait is milk fat percentage or dry matter content.

2. Application of the following SNP site as a detection target in identifying or assisting in identifying the milk production trait of sheep; the SNP site is, corresponding to the sequence information of the positive strand of chromosome 17 of the sheep reference genome Oar_v4.0 version FGF2 thymine T at position c. 178+11694 of the gene is replaced by cytosine C, the sheep is an East Fleece dairy sheep, and the milk fat percentage and dry matter content of the test sheep with the SNP site: c. 178+11694 T / C genotype of CC or CT are better than or assist in being better than the test sheep with the SNP site: c. 178+11694 T / C genotype of TT. The milk production trait is milk fat percentage or dry matter content.

3. A method of identifying or assisting in the identification of a ewe's milk production trait, characterised in that, comprising the steps of: detecting a sequence information of a positive strand of chromosome 17 corresponding to a reference genome of sheep Oar_v4.0 version of a test sheep FGF2 a genotype of a 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, and the sheep is an East Friesian dairy sheep; The milk production trait is milk fat percentage or dry matter content.

4. Use of the method according to claim 3 for the selection of milk sheep for milk fat percentage or dry matter content or for breeding milk sheep for milk fat percentage or dry matter content, characterized in that, The test sheep described in claim 3, which corresponds to the SNP site c. 178+11694 T / C genotype of CC or CT in the positive strand of chromosome 17 of the sheep reference genome Oar_v4.0 version sequence information, is used for milk production or breeding, and the milk sheep is East Fleece milk sheep. FGF2 The test sheep described in claim 3, which corresponds to the SNP site c. 178+11694 T / C genotype of CC or CT in the positive strand of chromosome 17 of the sheep reference genome Oar_v4.0 version sequence information, is used for milk production or breeding, and the milk sheep is East Fleece milk sheep.

5. Use of a primer pair for detecting the SNP of claim 1, characterized in that, The milk production trait is milk fat percentage or dry matter content. FGF2 The nucleotide sequences of the primer pair upstream and downstream of the SNP site are shown in SEQ ID NO. 1~2, and the application is any one of the following (a)-(f): (a) identifying or assisting in identifying the milk fat percentage and dry matter content in sheep milk; (b) screening of sheep milk fat percentage or dry matter content; (c) breeding of sheep milk fat percentage or dry matter content; (d) preparing a kit for identifying or assisting in identifying the milk fat percentage and dry matter content in sheep milk; (e) preparing a kit for screening of sheep milk fat percentage or dry matter content; (f) preparing a kit for breeding of sheep milk fat percentage or dry matter content; In (a)-(f), the sheep is East Fleece dairy sheep.

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