Molecular marker for identifying early growth speed of ducks based on CTNND2 gene as well as identification method and application of molecular marker

Abstract

The invention relates to a molecular marker for identifying the early growth speed of ducks based on CTNND2 genes and an identification method and application thereof, and belongs to the technical field of molecular markers, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 201st basic group of the nucleotide sequence is G or A. If the duck molecular marker type to be detected is AA type, the duck growth speed is high, and if the duck growth speed is high, the duck early growth speed is high, the duck early growth speed is high. If the to-be-detected duck molecular marker type is a GG type, the duck growth speed is slow, and if the to-be-detected duck molecular marker type is a GA type, the duck growth speed is medium. The molecular marker is found on the basis of correlation research on the CTNND2 gene and the early-stage growth speed of the ducks, the ducks with the high early-stage growth speed can be screened out by identifying the types of the molecular marker in a duck genome, and a breeding method for selecting excellent duck varieties in the early stage is established; and a novel molecular marker assisted breeding method is provided for the detection of the growth traits of the ducks.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular markers, and particularly relates to a molecular marker for identifying early growth rate of ducks based on CTNND2 Gene, an identification method thereof and application thereof. BACKGROUND

[0002] In the breeding process, early growth rate is a core breeding trait with far-reaching influence, which can shorten the growth cycle of livestock and poultry, reduce feeding costs and improve output. Growth rate is usually highly correlated with feed conversion rate, especially in the rapid growth period, which is usually positively correlated with feed efficiency and carcass meat yield. Selecting individuals with fast early growth can screen genotypes that can more efficiently convert feed into muscle or weight gain, significantly reducing production costs. Young livestock with sufficient early nutrition and good growth have stronger immune organs and disease resistance, and have higher survival rates, especially in intensive high-density feeding environments. Early growth rate usually has moderate heritability (0.2-0.4) and responds well to direct selection.

[0003] CTNND2 As a member of the catenin family, it plays a key role in some mechanisms by regulating cell adhesion and Wnt signaling. CTNND2 The encoded delta-catenin is a core component of cell adhesion junctions, which maintains intercellular junctions by binding to cadherins and anchors the actin cytoskeleton. During muscle development, cell adhesion is crucial for myotube formation, myofiber fusion and muscle tissue integrity. It is speculated that CTNND2 may indirectly affect muscle growth rate by regulating the adhesion ability of muscle cells, affecting the number and arrangement of myofibers. CTNND2 is an important regulator of the Wnt signaling pathway, which inhibits the activation of the Wnt pathway by competing with beta-catenin to bind TCF / LEF transcription factors. Wnt signaling plays a key role in the proliferation and differentiation of muscle stem cells, which suggests that CTNND2 may affect muscle growth potential by inhibiting the Wnt pathway to regulate the resting state or activation of muscle stem cells. In the rapid growth period, CTNND2 up-regulated expression may limit excessive muscle proliferation and maintain energy balance; while in the embryonic period, its moderate expression may promote myotube formation.

[0004] Therefore, CTNND2 the gene may be an important candidate gene for regulating early growth rate of ducks. At present, the research on CTNND2 gene mainly focuses on mammals, and in the field of oviparous poultry, especially for ducks, there are few reports on CTNND2 gene. In view of this, the present application provides a molecular marker for identifying early growth rate of ducks based on CTNND2The application discloses a molecular marker for identifying early growth speed of ducks, and a method and application thereof. SUMMARY

[0005] The application aims to provide a method for identifying early growth speed of ducks based on CTNND2 The application discloses a molecular marker for identifying early growth speed of ducks, and a method and application thereof.

[0006] The application achieves the above object by the following technical scheme: The application provides a method for identifying early growth speed of ducks based on CTNND2 The application discloses a molecular marker for identifying early growth speed of ducks, and a method and application thereof.

[0007] The application also provides application of the molecular marker in identifying early growth speed of ducks. As a further optimization scheme of the application, the early growth speed of the duck refers to average daily weight gain per week within the first three weeks.

[0008] As a further optimization scheme of the application, if the molecular marker type of the to-be-tested duck is AA type, the growth speed of the duck is relatively fast. If the molecular marker type of the to-be-tested duck is GG type, the growth speed of the duck is relatively slow. If the molecular marker type of the to-be-tested duck is GA type, the growth speed of the duck is medium.

[0009] The application also provides a method for identifying early growth speed of ducks by using the molecular marker, comprising the following steps: (1) extracting total DNA of duck metatarsal medial vein blood; (2) taking the upstream and downstream CTNND2 gene nucleotide sequences of the site where the molecular marker is located as templates, designing specific amplification primers, taking the total DNA as a template, and performing PCR amplification by using the specific amplification primers to obtain an amplification product; (3) performing sequencing and genotyping detection on the amplification product to obtain the molecular marker type of the to-be-tested duck; (4) judging the feed utilization rate trait of the duck according to the molecular marker type.

[0010] As a further optimization scheme of the application, the sequence of the specific amplification primer is as follows: SEQ ID NO. 2: F: TGATAAAGGCAGCAAACAT; SEQ ID NO. 3: R: ATTCTGTTTTGTCCCCTGT.

[0011] As a further optimization scheme of the present invention, the genotyping detection method is to find the mutation site based on mixed pool sequencing, and then perform Sanger sequencing on the amplified product to determine the genotype of each individual.

[0012] The present invention also provides a method for screening duck breeds with fast early growth rate using molecular markers, which comprises extracting total DNA from duck blood, performing typing detection on the duck genotype using molecular markers, and selecting individuals with genotype AA, i.e., duck breeds with fast early growth rate.

[0013] The beneficial effects of the present invention are: The present invention is based on CTNND2 Research on the correlation between genes and early growth rate in ducks has led to the discovery of a molecular marker. By identifying the presence of this molecular marker in the duck genome, it is possible to screen for ducks with fast early growth rates, establish a breeding method for early selection of superior duck breeds, and provide a new molecular marker-assisted breeding method for measuring growth traits in ducks. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For Duck CTNND2 Genotype sequencing results of the G201A site in the gene (site 201 in SEQ ID NO.1). DETAILED DESCRIPTION

[0015] The present application is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0016] 1. Experimental Materials The Qiangying ducks used in this application were provided and raised by Anhui Huangshan Qiangying Duck Industry Co., Ltd. The methods used in the present invention are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0017] 2. CTNND2 Gene polymorphism detection 2.1、 CTNND2 Primer design at the mutation site From the duck genome database, we found the sequence shown in SEQ ID NO. CTNND2 The DNA sequence corresponding to the gene is expressed as CTNND2 The partial gene DNA sequence (the sequence of the site where the molecular marker is located and its upstream and downstream bases) is used as a template to design specific amplification primers. The specific amplification primer sequences are as follows: SEQ ID NO. 2: F: TGATAAAGGCAGCAAACAT; SEQ ID NO. 3: R: ATTCTGTTTTGTCCCCTGT.

[0018] The length of the amplifiable region of the primer is 97 bp, and the sequence is shown in SEQ ID NO. 4, which contains the molecular marker site of the G / A mutation of the application (at position 201 in SEQ ID NO. 1).

[0019] 2.2, Extraction of total DNA from blood Select 200 Qiangying ducks, collect blood from the medial metatarsal vein, extract total DNA from blood, and use the blood DNA extraction kit produced by Tiangeng Biological Technology Co., Ltd. to extract total DNA from the medial metatarsal vein blood sample of duck. The extraction steps are carried out according to the instructions of the kit.

[0020] 2.3, PCR amplification Use Mix produced by Shanghai Yisheng Biological Company to perform PCR amplification reaction on the target fragment of the gene by using the synthesized sequencing-specific primer pair CTNND2 The PCR amplification system is shown in Table 1. Table 1 PCR amplification system ; The PCR reaction conditions are as follows: 95℃ pre-denaturation for 5min; first step 95℃ denaturation for 45s; second step 64.8℃ annealing for 45s (annealing temperature is set according to the primer); third step 72℃ extension for 30s, wherein the second step to the third step is cycled for 31 times, a total of 32 cycles; 72℃ extension for 10min.

[0021] 2.4, Sequencing of PCR amplification product The PCR product is sequenced by Beijing Qikeng Biological Technology Co., Ltd. (Nanjing), and the sequencing results are compared with the sequence of the target gene searched in the reference genome using SnapGene software to find SNP sites.

[0022] 2.5, Genotyping According to the results of finding mutation sites by pooling sequencing, the blood DNA of the above 200 Qiangying ducks is amplified according to the PCR steps. The PCR product is subjected to Sanger sequencing by Beijing Qikeng Biological Technology Co., Ltd. (Nanjing), and the sequencing results are compared with the sequence of the target gene searched in the reference genome using SnapGene software to determine the genotype of each individual. Double peaks are observed at the mutation site for heterozygous mutation, and single peak is for homozygous mutation. The sequencing alignment chart is shown in Figure 1 , and the G mutation into A is shown by the arrow.

[0023] 3. CTNND2 Association analysis between gene polymorphism and average daily weight gain in ducks 3.1、 CTNND2 Gene and genotype frequencies and genetic diversity analysis To determine the duck CTNND2 The association between the G / A polymorphism at the G201A locus and important phenotypic traits of ducks was studied. The 200 Qiangying ducks in 2.2 were used as experimental materials. The genotyping method in 2.5 was used to genotype the 200 Qiangying ducks. The results are shown in Table 3: Table 3 Genotype detection results of individuals with different phenotypes ; Experimental conclusion: The chi-square test results showed that the genotype of the experimental duck population was in Hardy-Weinberg equilibrium ( P >0.05).

[0024] 3.2、 CTNND2 Association analysis between gene polymorphism and average daily weight gain The average daily gain (ADG) of 200 Qiangying ducks from 1 to 6 weeks was counted, and the association between the three genotypes and the average daily gain (ADG) of ducks from 1 to 6 weeks was analyzed using the least squares analysis method in SAS9.4 software. The results are shown in Table 4: Table 4 Duck CTNND2 Association analysis between genotype and average daily weight gain ; Note: Different lowercase letters in the same row indicate significant differences ( P <0.05), different capital letters in the same row indicate extremely significant differences ( P <0.01).

[0025] It can be seen from the table that: CTNND2 For this molecular marker site of the gene, in the first three weeks, the average daily weight gain of the AA duck was significantly higher than that of the other two genotypes. It is worth noting that the average daily weight gain of the GG duck was significantly lower than that of the GA duck in the first week, but from the second week on, the value of the GG duck gradually caught up with and exceeded that of the GA duck. By the sixth week, the weekly average daily weight gain of the GG duck even exceeded that of the GA duck. This result shows that the early growth rate of individuals with the AA genotype is the fastest, the early growth rate of individuals with the GA genotype is medium, and the early growth rate of individuals with the GG duck genotype is the slowest. In other words, the AA duck is the dominant genotype that promotes weight gain in the early stage, while the GG duck contributes more to weight gain as growth and development progresses, becoming the dominant genotype that promotes weight gain in the later stage.

[0026] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A method based on CTNND2 The invention relates to a molecular marker for the genetic identification of early growth rate in ducks, characterized by: The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, wherein the 201st base of the nucleotide sequence is G or A.

2. Use of the molecular marker according to claim 1 in identifying the early growth rate of ducks.

3. The use according to claim 2, characterized in that: The early growth rate of ducks refers to the average daily weight gain of ducks per week in the first three weeks.

4. The use according to claim 2, characterized in that: If the molecular marker type of the duck to be tested is AA type, the duck grows faster; If the molecular marker type of the duck to be tested is GG type, the duck grows slowly; If the molecular marker type of the duck to be tested is GA type, the duck has a medium growth rate.

5. A method for identifying the early growth rate of ducks using the molecular marker according to claim 1, characterized in that: The following steps are involved: (1) Extract total DNA from the medial metatarsal vein blood; (2) The upstream and downstream of the site where the molecular marker is located CTNND2 Using the gene nucleotide sequence as a template, designing specific amplification primers, and using the total DNA as a template, performing PCR amplification using the specific amplification primers to obtain an amplified product; (3) Sequencing and genotyping the amplified products to obtain the molecular marker type of the duck to be tested; (4) Determine the average daily weight gain of ducks based on the type of molecular markers.

6. The method for identifying the early growth rate of ducks using molecular markers according to claim 5, wherein: The sequences of the specific amplification primers are: SEQ ID NO.2: F: TGATAAAGGCAGCAAACAT; SEQ ID NO. 3: R:ATTCTGTTTGTCCCCTGT.

7. The method for identifying the early growth rate of ducks using molecular markers according to claim 5, wherein: Mutation sites were found by pooled sequencing, and the amplified products were then subjected to Sanger sequencing to determine the genotype of each individual.

8. A method for screening duck breeds with fast early growth rate using the molecular marker according to claim 1, characterized in that: The total DNA of the duck's blood is extracted, and the duck's genotype is detected by using the molecular markers, and individuals with genotype AA are selected, which are duck breeds with fast early growth rate.

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