A molecular marker for identifying chicken feed utilization traits based on the TGFBR3 gene, its identification method and application

Abstract

The present application relates to a kind of molecular marker based on TGFBR3 gene identification chicken feed utilization character and its identification method and application, the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1, wherein, the base of the nucleotide sequence in the 323th position is C or T.The present application establishes a kind of poultry feed utilization early selection breeding method by identifying the type of the molecular marker present in chicken genome, according to genotype, the feed utilization character of chicken is selected, the method is simple, fast, low cost, does not need special instrument, is suitable for the needs of molecular marker assisted breeding experiment.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a molecular marker for identifying chicken feed utilization traits based on the TGFBR3 gene, its identification method, and its application. Background Technology

[0002] Feed costs account for approximately 60%-70% of total costs in poultry production, while feed efficiency during poultry growth and development is only 65%-70%. Therefore, improving feed efficiency is a crucial way to save costs. Due to limited land resources, and the fact that grains such as corn and soybean meal are not only a source of human food but also a major component of livestock feed, feed costs continue to rise as the scale of livestock farming expands. Therefore, feed efficiency is a significant economic trait in livestock farming and needs to be maximized. Although feed conversion ratios have been significantly improved, only 65%-70% of the feed is used for maintenance and production, with the remainder being excreted as waste. Therefore, improving feed efficiency is essential for reducing production costs and saving expensive feed components for various other uses. Currently, the two key indicators for evaluating feed efficiency traits both domestically and internationally are feed conversion rate (FCR) and residual feed intake (RFI).

[0003] The TGFBR3 (Transforming Growth Factor Beta Receptor 3) gene, located on chromosome 8, has 19 exons and a coding region of 109,901 bp. It is a protein-coding gene that encodes the transforming growth factor (TGF)-βIII receptor. The encoded receptor is a membrane proteoglycan that typically functions as a co-receptor along with other members of the TGF-β receptor superfamily. Shedding of the extracellular domain yields soluble TGFBR3, which may inhibit TGFB signaling. Alternative splicing transcript variants encoding different isoforms have been identified for this gene. Associated pathways include apoptosis in synovial fibroblasts and negative regulation of FGFR3 signaling. Gene ontology (GO) annotations associated with this gene include heparin binding and SMAD binding. An important paralog of this gene is ENG.

[0004] TGFBR3, a key signal transduction molecule, plays a crucial role in cell proliferation, differentiation, and apoptosis. By participating in the signal transduction of the TGF-β superfamily, it profoundly influences animal growth and metabolism. Studies have shown that TGFBR3 may indirectly affect feed efficiency by regulating skeletal muscle growth and metabolism. Specifically, TGFBR3 can promote myoblast proliferation and differentiation, thereby increasing muscle mass, improving animal growth rate, and feed conversion ratio. Furthermore, TGFBR3 may also participate in regulating lipid 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*, through genome-wide association analysis (GWAS), found that the NCAPG I442M locus was significantly associated with feed efficiency, and TGFBR3, as an important signal transduction molecule, may participate 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 mechanisms of TGFBR3 and feed efficiency are not yet fully understood, further research and technological advancements promise to reveal more details about the role of TGFBR3 in feed efficiency regulation. This will provide new breeding strategies and nutritional regulation methods for livestock production, contributing to improved feed efficiency and overall livestock production profitability.

[0005] "Huaibei Ma Chicken" is an excellent local chicken breed in Suzhou City, Anhui Province, and an important raw material for producing Fuliji roasted chicken, a geographical indication product. "Huaibei Ma Chicken" is highly adaptable, tolerant of roughage, and can survive and reproduce under various environmental conditions. Based on the above, this invention proposes a molecular marker for identifying chicken feed utilization traits based on the TGFBR3 gene, as well as its identification method and application. Summary of the Invention

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

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

[0008] This invention provides a molecular marker for identifying chicken feed utilization traits 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] This invention also provides the application of a molecular marker based on the TGFBR3 gene for identifying chicken feed utilization traits in the identification of chicken feed utilization traits.

[0010] As a further optimization of the present invention, if the molecular marker type of the chicken to be tested is CC, the chicken has the best feed utilization trait; if the molecular marker type of the chicken to be tested is TT, the chicken has a moderate feed utilization trait; and if the molecular marker type of the chicken to be tested is CT, the chicken has a poor feed utilization trait.

[0011] This invention also provides a method for identifying chicken feed utilization traits using molecular markers, comprising the following steps:

[0012] (1) Extract total DNA from the venous blood of chicken wings;

[0013] (2) Design specific amplification primers with the molecular marker site and the sequence composed of its upstream and downstream bases as the target sequence, use the total DNA as a template, and perform PCR amplification using the specific amplification primers to obtain the amplification product;

[0014] (3) Genotyping and sequencing of the amplification products to obtain the molecular marker type of the chicken to be tested;

[0015] (4) Determine the chicken feed utilization characteristics based on molecular marker types;

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

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

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

[0019] As a further optimization of the present invention, the sequence of the specific amplification primer is as follows:

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

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

[0022] As a further optimization of the present invention, the genotyping detection method involves obtaining enzyme digestion products by digesting the amplification products, detecting the enzyme digestion products using agarose gel electrophoresis, and performing genotyping based on the images. If the enzyme digestion products:

[0023] If it contains one stripe, it is of type TT;

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

[0025] If it contains 3 bands, it is a CT type.

[0026] As a further optimization of the present invention, the enzyme digestion products are detected by agarose gel electrophoresis at a mass ratio of 1.5%-2.0%.

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

[0028] The nucleotide sequence of the molecular marker provided by this invention is shown in SEQ ID NO.1, wherein the 323rd base of the nucleotide sequence is C or T. This invention establishes a breeding method for early selection of poultry feed utilization by identifying the type of presence of the molecular marker in the chicken genome and selecting chickens based on genotype for feed utilization traits. This method is simple, rapid, low-cost, and does not require special instruments, making it suitable for the needs of molecular marker-assisted breeding experiments. Attached Figure Description

[0029] Figure 1 Agarose gel electrophoresis images of PCR amplification products from a portion of the samples;

[0030] Figure 2 Agarose gel electrophoresis image of the enzyme digestion products obtained by enzyme digestion of PCR amplification products from a portion of the samples;

[0031] Figure 3 This is the genotype verification sequencing result for the C323T site (site 323 in SEQ ID NO.1) in the chicken TGFBR3 gene. Detailed Implementation

[0032] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0033] 1. Materials

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

[0035] 2. Method

[0036] 2.1 Primer Design

[0037] The DNA sequence corresponding to the TGFBR3 gene shown in SEQ ID NO.1 was found in the chicken genome database. Using the partial DNA sequence of the TGFBR3 gene (containing the site of the polymorphic molecular marker of this invention and the sequence composed of its upstream and downstream bases) as a template, specific amplification primers were designed. The specific amplification primer sequences are shown below:

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

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

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

[0041] 2.2 Extraction of total DNA from blood

[0042] 450 Huaibei Ma chickens were selected, and blood was collected from the wing veins. Total DNA was extracted from the blood. The total DNA was extracted from the chicken wing vein blood samples using a blood DNA extraction kit produced by Tiangen Biotech Co., Ltd. The extraction steps were performed according to the kit instructions.

[0043] 2.3 PCR amplification

[0044] Using Mix, produced by Shanghai Yisheng Biotechnology Co., Ltd., PCR amplification of the target fragment of the TGFBR3 gene was performed using pre-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 were as follows: 94℃ pre-denaturation for 5 min; first step denaturation at 94℃ for 30 s; second step annealing at 53℃ for 30 s (annealing temperature is set according to the primers); third step extension at 72℃ for 30 s, with the second and third steps repeated 34 times for a total of 35 cycles; and a final extension at 72℃ for 10 min.

[0048] 2.4 Detection and sequencing of PCR amplification products

[0049] PCR amplification products were detected using 2% agarose gel electrophoresis, such as... Figure 1As shown, after imaging with a gel imaging system, a band of approximately 470 bp in length was obtained, which is consistent with the predicted length, indicating that the target fragment was obtained. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. (Nanjing) for sequencing, and the sequence is shown in SEQ ID NO.4, which is consistent with the predicted result.

[0050] 2.5 Genotyping

[0051] 2.5.1 Prepare the enzyme digestion system as shown in Table 2. The enzyme digestion conditions are 37℃ for 12-16 hours. Use HhaⅠ restriction endonuclease from Hefei Ruijie Biotechnology Co., Ltd. to digest the PCR amplification products.

[0052] Table 2 Enzyme digestion system

[0053]

[0054] 2.5.2 Detection was performed using 1.5% mass ratio low-voltage agarose gel electrophoresis to obtain results such as... Figure 2 The results shown are partial; among them, if the enzyme digestion product contains 1 band, it is of the TT type; contains 2 bands, it is of the CC type; and contains 3 bands, it is of the CT type.

[0055] 2.6 Enzyme digestion and sequencing verification

[0056] Statistical analysis of the gene genotyping agarose gel electrophoresis images yielded three genotypes: CC, TT, and CT. One individual was selected from each of these three genotypes for sequencing alignment. The sequencing alignment images are shown below. Figure 3 As shown in the sequencing results, C mutated to T, and the arrows indicate the mutation locations, which is consistent with the enzyme digestion typing results.

[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, 450 Huaibei Ma chickens from step 2.2 were used as experimental materials. Feed intake (ADFI) at 90–120 days of age, average daily gain (ADG), body weight at 90 days of age (BW90), feed conversion ratio (FCR), and residual feed intake (RFI) were recorded. The 2.5 genotyping method was used to genotype the 450 Huaibei Ma chickens, and the results are shown in Table 3.

[0059] Table 3. Genotype detection results for individuals with different phenotypes

[0060]

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

[0062] 2.8 Statistical Analysis

[0063] The association between the three genotypes and the chicken feed utilization trait was analyzed using the least squares analysis method in SAS 9.4 software. The association analysis results between different genotypes and each trait are shown in Table 4.

[0064] Table 4. Association analysis between chicken TGFBR3 genotype and chicken feed utilization rate trait

[0065]

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

[0067] Experimental conclusions: As shown in Table 4, for the C323T locus of the TGFBR3 gene, the 90-day body weight (BW90) of CC genotype individuals was significantly higher than that of CT genotype individuals. The residual feed intake (FCR) of CC genotype individuals was significantly lower than that of CT and TT genotype individuals. There were no significant differences among the three genotypes in terms of average daily gain (ADG), daily feed intake, and feed conversion ratio (FCR). Therefore, it can be concluded that CC genotype individuals have the best feed utilization trait, TT genotype individuals have a moderate feed utilization trait, and CT genotype individuals have a poor feed utilization trait.

[0068] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. Use of a molecular marker based on the TGFBR3 gene for identifying a feed utilization trait in a chicken, characterized in that, The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, wherein the base at position 323 of the nucleotide sequence is C or T. If the type of the molecular marker of the chicken to be tested is CC type, the chicken has the best feed utilization trait; if the type of the molecular marker of the chicken to be tested is TT type, the chicken has a medium feed utilization trait; and if the type of the molecular marker of the chicken to be tested is CT type, the chicken has a poor feed utilization trait. The chicken is Huaibei chicken, and the feed utilization trait is 90-day-old body weight and residual feed intake.

2. A method for identifying a chicken feed utilization trait based on a molecular marker of the TGFBR3 gene, characterized in that, The method comprises the following steps: (1) extracting total DNA of Huaibei chicken wing vein blood; (2) designing specific amplification primers with the sequence of the site where the molecular marker is located and the upstream and downstream bases thereof as a target sequence, using the total DNA as a template, and performing PCR amplification with the specific amplification primers to obtain an amplification product; The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, wherein the base at position 323 of the nucleotide sequence is C or T. (3) performing genotyping detection and sequencing on the amplification product to obtain the type of the molecular marker of the chicken to be tested; (4) judging the feed utilization trait of the chicken according to the type of the molecular marker; If the type of the molecular marker of the chicken to be tested is CC type, the chicken has the best feed utilization trait; If the type of the molecular marker of the chicken to be tested is TT type, the chicken has a medium feed utilization trait; If the type of the molecular marker of the chicken to be tested is CT type, the chicken has a poor feed utilization trait; The feed utilization trait is 90-day-old body weight and residual feed intake.

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

4. The method for identifying chicken feed utilization traits using molecular markers according to claim 3, wherein, The genotyping detection method is to obtain an enzyme digestion product by enzyme digestion of the amplification product with restriction enzyme Hha I, to detect the enzyme digestion product by agarose gel electrophoresis, to perform genotyping according to the image, and to determine the type of the molecular marker as follows: if the enzyme digestion product contains one band, it is TT type; if the enzyme digestion product contains two bands, it is CC type; if the enzyme digestion product contains three bands, it is CT type.

5. The method for identifying chicken feed utilization traits using molecular markers according to claim 4, wherein, The enzyme digestion product is detected by agarose gel electrophoresis with a mass ratio of 1.5%-2.0%.

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