Molecular marker for identifying chicken feed utilization rate character based on TGFBR3 gene and identification method and application thereof

Abstract

The invention relates to a molecular marker for identifying the chicken feed utilization rate character based on a TGFBR3 gene and an identification method and application of the molecular marker, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the 323rd base of the nucleotide sequence is C or T. The breeding method for early selection of the poultry feed utilization rate is established by identifying the type of the molecular marker in the chicken genome and selecting the feed utilization rate character of the chicken according to the genotype, and the method is simple, rapid and low in cost, does not need special instruments, and is suitable for the needs of molecular marker-assisted breeding experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and specifically relates to a molecular marker for identifying the feed utilization rate trait of chickens based on the TGFBR3 gene, and an identification method and application thereof. Background Art

[0002] The feed cost in the poultry production process accounts for about 60%-70% of the total cost, and the feed efficiency of poultry during growth and development is only 65%-70%. Therefore, improving the feed efficiency of poultry is an important way to save costs. Due to limited land resources, and grains such as corn and soybean meal are not only one of the food sources for humans, but also the main components of livestock and poultry feed. Therefore, with the continuous expansion of the scale of the livestock industry, the feed cost has also been continuously increasing. Therefore, for livestock and poultry breeding, the feed efficiency trait is an economic trait with a greater impact, and the feed efficiency needs to be maximized. Although the feed conversion rate has been greatly improved, at most 65%-70% of the feed is used for maintenance and production, and the rest is excreted as waste. Therefore, improving the feed efficiency is crucial for reducing production costs and saving expensive feed ingredients for various other uses. At present, the two key indicators for evaluating the feed efficiency trait at home and abroad are the feed conversion rate (FCR) and the residual feed intake (RFI).

[0003] The TGFBR3 (Transforming Growth Factor Beta Receptor 3) gene is located on chromosome 8, has 19 exons, and the coding region length is 109901 bp. It is a protein-coding gene that encodes the transforming growth factor (TGF)-β type III receptor. The encoded receptor is a membrane proteoglycan that usually functions as a co-receptor together with other members of the TGF-β receptor superfamily. The extracellular domain shedding generates soluble TGFBR3, which may inhibit TGFB signaling. Alternative spliced transcript variants encoding different subtypes have been identified for this gene. Its related pathways include the apoptosis pathway in synovial fibroblasts and the negative regulation of FGFR3 signaling. The gene ontology (GO) annotations related to this gene include heparin binding and SMAD binding. An important paralog of this gene is ENG.

[0004] As a key signal transduction molecule, TGFBR3 plays an important role in processes such as cell proliferation, differentiation, and apoptosis. By participating in the signal transduction of the TGF-β superfamily, it has a profound impact on the growth and metabolism of animals. Research shows that TGFBR3 may indirectly affect feed efficiency by regulating the growth and metabolism of skeletal muscle. Specifically, TGFBR3 can promote the proliferation and differentiation of myoblasts, thereby increasing muscle mass and improving the growth rate and feed conversion rate of animals. In addition, TGFBR3 may also be involved in regulating fat metabolism, further affecting feed efficiency. In recent years, with the development of genomics and metabolomics technologies, researchers have begun to focus on the mechanism of action of TGFBR3 in feed efficiency regulation. For example, a study published in "PLOS ONE" found through genome-wide association analysis (GWAS) that the NCAPG I442M locus is significantly associated with feed efficiency, and TGFBR3, as an important signal transduction molecule, may be involved in this regulatory process. In addition, some studies have constructed gene networks to search for candidate genes related to feed efficiency, including TGFBR3. Although the specific regulatory mechanism of TGFBR3 and feed efficiency is not yet fully understood, with the in-depth research and technological progress, we are expected to reveal more details about the role of TGFBR3 in feed efficiency regulation. This will provide new breeding strategies and nutritional regulation means for the livestock industry, helping to improve feed efficiency and livestock production benefits.

[0005] "Huaibei Partridge Chicken" is an excellent local chicken breed in Suzhou City, Anhui Province, and is also an important raw material for producing the geographical indication product Fuliji Braised Chicken. "Huaibei Partridge Chicken" has strong adaptability and can tolerate rough feeding, and can survive and reproduce under various environmental conditions. Based on the above content, the present invention proposes a molecular marker for identifying the feed utilization trait of chickens based on the TGFBR3 gene, and its identification method and application. Summary of the Invention

[0006] The purpose of the present invention is to provide a molecular marker for identifying the feed utilization trait of chickens based on the TGFBR3 gene, and its identification method and application. Compared with the prior art, SNP (single nucleotide polymorphism) molecular markers have been developed for the candidate gene (TGFBR3 gene) related to the feed utilization trait of chickens, so as to solve the problem of slow progress in conventional phenotypic breeding and achieve the early identification of the feed utilization trait.

[0007] The present invention achieves the above object through the following technical solutions:

[0008] The present invention provides a molecular marker for identifying the feed utilization trait of chickens based on the TGFBR3 gene. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 323rd base of the nucleotide sequence is C or T.

[0009] The present invention also provides an application of a molecular marker for identifying the feed utilization trait of chickens based on the TGFBR3 gene in identifying the feed utilization trait of chickens.

[0010] As a further optimized solution of the present invention, if the molecular marker type of the chicken to be tested is the CC type, the feed utilization trait of this chicken is the best; if the molecular marker type of the chicken to be tested is the TT type, the feed utilization trait of this chicken is medium; if the molecular marker type of the chicken to be tested is the CT type, the feed utilization trait of this chicken is poor.

[0011] The present invention also provides a method for identifying the feed utilization trait of chickens using molecular markers, comprising the following steps:

[0012] (1) Extract the total DNA from the venous blood of the chicken wing.

[0013] (2) Design specific amplification primers with the sequence composed of the locus where the molecular marker is located and its upstream and downstream bases as the target sequence. Using the total DNA as a template, perform PCR amplification with the specific amplification primers to obtain an amplification product.

[0014] (3) Perform genotyping detection and sequencing on the amplification product to obtain the molecular marker type of the chicken to be tested.

[0015] (4) Judge the feed utilization trait of the chicken according to the molecular marker type.

[0016] If the molecular marker type of the chicken to be tested is the CC type, the feed utilization trait of this chicken is the best;

[0017] If the molecular marker type of the chicken to be tested is the TT type, the feed utilization trait of this chicken is medium;

[0018] If the molecular marker type of the chicken to be tested is the CT type, the feed utilization trait of this chicken is poor.

[0019] As a further optimized solution of the present invention, the sequences of the specific amplification primers are:

[0020] SEQ ID NO.2: Forward primer: GATGATGCTTTGTGGATGT;

[0021] SEQ ID NO.3: Reverse primer: TGAGGATAAGAGACCATACC.

[0022] As a further optimized solution of the present invention, the genotyping detection method is to obtain a digestion product by digesting the amplification product, detect the digestion product by agarose gel electrophoresis, and perform genotyping according to the image. If the digestion product:

[0023] contains 1 band, it is the TT type;

[0024] If it contains two bands, it is of the CC type;

[0025] If it contains three bands, it is of the CT type.

[0026] As a further optimization scheme of the present invention, the digested product is detected by agarose gel electrophoresis with a concentration of 1.5%-2.0% in mass ratio.

[0027] The present invention has the following beneficial effects:

[0028] The nucleotide sequence of the molecular marker provided by the present invention is as shown in SEQ ID NO.1, wherein the 323rd base of the nucleotide sequence is C or T. The present invention identifies the type of the molecular marker existing in the chicken genome, selects the chicken feed utilization trait according to the genotype, and establishes a breeding method for early selection of poultry feed utilization rate. This method is simple, fast, low-cost, does not require special instruments, and meets the needs of molecular marker-assisted breeding experiments. Description of the Drawings

[0029] Figure 1 It is the agarose gel electrophoresis pattern of the PCR amplification products of some samples;

[0030] Figure 2 It is the agarose gel electrophoresis pattern of the digested products obtained by digesting the PCR amplification products of some samples;

[0031] Figure 3 It is the genotype verification sequencing result of the C323T locus (the 323rd locus in SEQ ID NO.1) in the chicken TGFBR3 gene. Detailed Embodiments

[0032] The following further describes the present application in detail with reference to the drawings. It is necessary to point out here that the following detailed embodiments are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0033] 1. Materials

[0034] The methods used in this embodiment are all conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are all commercially available products unless otherwise specified.

[0035] 2. Methods

[0036] 2.1 Primer Design

[0037] Find the DNA sequence corresponding to the TGFBR3 gene shown in SEQ ID NO.1 from the chicken genome database, and use the DNA partial sequence of the TGFBR3 gene (the sequence composed of the locus where the polymorphic molecular marker of the present invention is located and its upstream and downstream bases) as a template to design specific amplification primers. The sequences of the specific amplification primers are as follows:

[0038] SEQ ID NO.2: Forward primer: GATGATGCTTTGTGGATGT;

[0039] SEQ ID NO.3: Reverse primer: TGAGGATAAGAGACCATACC.

[0040] The length of the amplifiable region of this primer is 470bp, and the sequence is as shown in SEQ ID NO.4, which contains the molecular marker of the C / T mutation at the C323T locus (the 323rd locus in SEQ ID NO.1).

[0041] 2.2 Extraction of total blood DNA

[0042] Select 450 Huaibei partridge chickens, collect blood from the wing vein, extract the total blood DNA, and use the blood DNA extraction kit produced by Tiangen Biotech Co., Ltd. to extract the total DNA in the wing vein blood sample of the chicken. The extraction steps can be carried out according to the kit instructions.

[0043] 2.3 PCR amplification

[0044] Use the Mix produced by Shanghai Yisheng Biotech Co., Ltd. to perform a PCR amplification reaction on the target fragment of the TGFBR3 gene through the synthesized sequencing-specific primers. The PCR amplification system is shown in Table 1:

[0045] Table 1 PCR amplification system

[0046]

[0047] The PCR reaction conditions are: pre-denaturation at 94°C for 5 min; the first step, denaturation at 94°C for 30 s; the second step, annealing at 53°C for 30 s (the annealing temperature is set according to the primer); the third step, extension at 72°C for 30 s. Among them, the second step to the third step are cycled 34 times, for a total of 35 cycles; extension at 72°C for 10 min.

[0048] 2.4 Detection and sequencing of PCR amplification products

[0049] Use 2% mass ratio agarose gel electrophoresis to detect the PCR amplification products, as Figure 1As shown, a band with an approximate length of 470 bp was obtained after imaging with a gel imager, which was consistent with the predicted length, indicating that the target fragment was obtained. The PCR product was sent to Beijing Tsingke Biotechnology Co., Ltd. (Nanjing) for sequencing, and the sequence was as shown in SEQ ID NO.4, which was consistent with the predicted result.

[0050] 2.5 Genotyping

[0051] 2.5.1 Prepare the restriction enzyme digestion system as shown in Table 2, and the restriction enzyme digestion condition is to keep at a constant temperature of 37°C for 12 - 16 hours. Use the HhaⅠ restriction enzyme of Hefei Ruijie Biotechnology Co., Ltd. to digest the PCR amplification product;

[0052] Table 2 Restriction Enzyme Digestion System

[0053]

[0054] 2.5.2 Use 1.5% mass ratio low-voltage agarose gel electrophoresis for detection to obtain the results (partial results) as shown in Figure 2 ; among them, if the restriction enzyme digestion product contains 1 band, it is the TT type; if it contains 2 bands, it is the CC type; if it contains 3 bands, it is the CT type.

[0055] 2.6 Verification by Restriction Enzyme Digestion and Sequencing

[0056] Statistically analyze the agarose gel electrophoresis map of gene restriction enzyme digestion genotyping to obtain three genotypes: CC, TT, and CT. Select one individual from each of these three genotypes for sequencing and comparison. The sequencing comparison map is as shown in Figure 3 ; in the sequencing result, C mutated to T, and the mutation position is marked by an arrow, which is consistent with the restriction enzyme digestion genotyping result.

[0057] 2.7 Effect Verification

[0058] To determine the association between the C / T polymorphism at the C323T locus of the chicken TGFBR3 gene and important phenotypic traits of chickens, 450 Huaibei partridge chickens in step 2.2 were used as experimental materials, and the feed intake (ADFI), average daily gain (ADG), body weight at 90 days of age (BW90), feed conversion ratio (FCR), and residual feed intake (RFI) at 90 - 120 days of age were statistically analyzed. Using the 2.5 genotyping method, 450 Huaibei partridge chickens were genotyped, and the results are shown in Table 3:

[0059] Table 3 Genotype Detection Results of Individuals with Different Phenotypes

[0060]

[0061] Experimental conclusion: The chi-square test results showed that the genotype of the experimental chicken population was in Hardy-Weinberg equilibrium (P > 0.05).

[0062] 2.8 Statistical analysis

[0063] The least squares analysis method in SAS 9.4 software was used to analyze the correlation between the three genotypes and the chicken feed utilization traits. The correlation analysis results between different genotypes and each trait are shown in Table 4:

[0064] Table 4 Correlation analysis of chicken TGFBR3 genotypes and chicken feed utilization traits

[0065]

[0066] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05), and different capital letters in the same row indicate extremely significant differences (P < 0.01).

[0067] Experimental conclusion: As can be seen from Table 4, for the C323T locus of the TGFBR3 gene, the body weight at 90 days (BW90) of CC individuals is significantly higher than that of CT individuals, and the residual feed intake (FCR) of CC individuals is extremely significantly lower than that of CT and TT individuals. There are no significant differences in average daily gain (ADG), daily feed intake, and feed conversion ratio (FCR) among the three genotypes. Thus, it can be concluded that CC genotype individuals have the best feed utilization traits, TT genotype individuals have medium feed utilization traits, and CT genotype individuals have poor feed utilization traits.

[0068] The above embodiments only express several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A molecular marker for identifying chicken feed utilization traits based on the TGFBR3 gene, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein the 323rd base of the nucleotide sequence is C or T.

2. Use of the molecular marker as claimed in claim 1 in identifying feed utilization traits in chickens.

3. The use according to claim 2, characterized in that: If the molecular marker type of the chicken to be tested is CC type, the chicken has the best feed utilization trait; if the molecular marker type of the chicken to be tested is TT type, the chicken has a medium feed utilization trait; if the molecular marker type of the chicken to be tested is CT type, the chicken has a poor feed utilization trait.

4. A method for identifying the trait of chicken feed utilization using the molecular marker according to claim 1, characterized in that: The following steps are involved: (1) Extracting total DNA from chicken wing venous blood; (2) designing specific amplification primers based on the sequence of the site where the molecular marker is located and its upstream and downstream bases as the target sequence, using the total DNA as a template, and performing PCR amplification using the specific amplification primers to obtain an amplified product; (3) performing genotyping detection and sequencing on the amplified product to obtain the molecular marker type of the chicken to be tested; (4) Determine the feed utilization trait of chickens based on the type of molecular markers; If the molecular marker type of the chicken to be tested is CC type, the feed utilization trait of this chicken is the best; If the molecular marker type of the chicken to be tested is TT type, the feed utilization trait of the chicken is medium; If the molecular marker type of the chicken to be tested is CT type, the feed utilization trait of the chicken is poor.

5. The method for identifying chicken feed utilization traits using molecular markers according to claim 4, characterized in that: The sequence of the specific amplification primer is: SEQ ID NO.2: Forward primer: GATGATGCTTTGTGGATGT; SEQ ID NO.3: Reverse primer: TGAGGATAAGAGACCATACC.

6. The method for identifying chicken feed utilization traits using molecular markers according to claim 5, characterized in that: The genotyping detection method is to obtain a digestion product by digesting the amplified product, detect the digestion product by agarose gel electrophoresis, and perform genotyping based on the image. If the digestion product: If it contains 1 band, it is TT type; If it contains 2 bands, it is CC type; If it contains 3 bands, it is CT type.

7. The method for identifying chicken feed utilization traits using molecular markers according to claim 6, characterized in that: The enzyme digestion products were detected by agarose gel electrophoresis with a mass ratio of 1.5%-2.0%.

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