Application of PXYLP1 gene SNP molecular marker in identifying, evaluating or assisting in breeding super-long reproductive cycle lo island red chicken with high eggshell strength trait

Abstract

The application discloses application of a PXYLP1 gene SNP molecular marker in identifying, evaluating or assisting in breeding super-long reproductive cycle Los Island red chickens with high eggshell strength characteristics, and belongs to the field of molecular biology. The SNP molecular marker provided by the application is located at positions 6550013 and 6550240 of chicken chromosome 9, corresponds to positions 274 and 501 of the nucleotide sequence shown in SEQ ID No. 1, and has C / A and C / T polymorphism. When the genotype of the position 6550013 site is mutated from CC to AA and the genotype of the position 6550240 site is mutated from CC to TT, the eggshell strength can be significantly improved. The application further provides a primer pair for detecting the SNP molecular marker and a detection method thereof. The SNP molecular marker can be used for assisted breeding of super-long reproductive cycle Los Island red chickens with eggshell strength characteristics, realizes early screening of excellent individuals, and accelerates the breeding process.

Description

Technical Field

[0001] This invention relates to the field of chicken genetics and breeding technology, and more specifically to the application of the PXYLP1 gene SNP molecular marker in the identification, evaluation or assisted selection of long-breeding-cycle Rhodes Island Red chickens with high eggshell strength traits. Background Technology

[0002] Poultry eggs are an important food resource that is affordable and rich in protein, and the poultry industry plays a central role in ensuring the supply of basic necessities. Eggshell strength is an important indicator of eggshell quality. It not only reduces industrial losses by minimizing breakage but also creates a stable microenvironment for embryonic development, thereby improving the hatchability of fertilized eggs. However, the formation mechanism of eggshell strength is not yet clearly and systematically understood, which poses difficulties for improving eggshell strength through genetic breeding.

[0003] The ROK Red, with its exceptionally long breeding cycle, is a breed of egg-laying chicken with unique reproductive characteristics. Its egg-laying cycle is significantly longer than that of ordinary egg-laying chickens, maintaining high egg production performance even at 90 weeks of age. However, with the extended breeding cycle, the decline in eggshell strength becomes particularly prominent, leading to an increased breakage rate and severely impacting farming efficiency. Studies have shown that from 43 to 90 weeks of age, both eggshell thickness and toughness in ROK Reds significantly decrease (P<0.05), with the heritability of eggshell toughness ranging from 0.37 to 0.58. Therefore, screening for molecular markers related to eggshell strength in ROK Reds with exceptionally long breeding cycles and conducting marker-assisted breeding is an effective way to improve eggshell quality.

[0004] In recent years, researchers have begun to use techniques such as genome-wide association analysis (GWAS) to identify molecular markers associated with eggshell strength in ROD Red chickens. Xiaoman Chen et al. (2024) conducted a GWAS study on eggshell quality in 3,261 ROD Red chickens, measuring traits such as eggshell strength at seven time points from 36 to 90 weeks of age. They found two significantly associated genomic regions located on chromosome 1 and screened out three candidate genes: FRY, PCNX2, and ENSGALG00000052468 (Geneticpatterns and genome-wide association analysis of eggshell quality traits of egg-type chicken across an extended laying period 2024). However, this study did not involve associated sites on chromosome 9. In addition, a study used single-cell transcriptomics technology to construct a single-cell atlas of the oviduct and uterus of Rhodes Island Red chickens, identifying six gene families related to eggshell strength (collagen family, solute carrier family, ATPase family, calcium voltage-gated channel family, annexin family, and integrin subunit family). However, this study also failed to uncover specific SNP molecular markers.

[0005] Regarding PXYLP1 gene research, existing technologies include a patent for molecular markers related to the PXYLP1 gene (CN118979114B). However, this patent discloses a combination of PXYLP1 gene SNP molecular markers related to the ratio of muscle fiber types in chicken leg muscles. Furthermore, studies have reported SNP molecular markers related to eggshell strength in other laying hen breeds (such as the Nandan Yao chicken), and there are also patents for general eggshell strength molecular markers (such as CN107338317A, with the SNP site located on chromosome 1). However, none of these involve the Rhodes Island Red breed, let alone the PXYLP1 gene SNP markers in the specific Rhodes Island Red population with its extended breeding cycle. In summary, there are currently no reports of applying PXYLP1 gene SNP molecular markers to the identification, evaluation, or assisted breeding of Rhodes Island Red eggs with extended breeding cycles.

[0006] Therefore, developing a PXYLP1 gene SNP molecular marker associated with the shell strength trait of Loch Ness Red eggs with an ultra-long breeding cycle, and applying it to the identification, evaluation, or assisted breeding of eggshell strength, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides the application of the PXYLP1 gene SNP molecular marker in the identification, evaluation or assisted breeding of long-breeding-cycle Rhodes Island Red chickens with high eggshell strength traits.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] The primary objective of this application is to provide the application of the PXYLP1 gene SNP molecular marker in the identification, evaluation, or assisted selection of Rhodes Island Red chickens with a long breeding cycle and high eggshell strength trait. The SNP molecular marker is located at positions 6550013 and 6550240 on chicken chromosome 9, and the genomic version is bGalGal1.mat.broiler.GRCg7b. The 6550013 position represents a C / A polymorphism, and the 6550240 position represents a C / T polymorphism.

[0010] As a preferred technical solution, when the genotype at locus 6550013 is detected as AA and the genotype at locus 6550240 is detected as TT, the chicken under test is determined to have high eggshell strength.

[0011] Another object of this application is to provide a primer pair for detecting the SNP molecular marker, the nucleotide sequences of the primer pair being shown in SEQ ID No. 2 and SEQ ID No. 3.

[0012] Another object of this application is to provide a kit comprising the primer pair described above.

[0013] Another object of this application is to provide the application of the primer pair or the kit described herein in the identification, evaluation or assisted selection of long-breeding-cycle Rhodes Island Red chickens with high eggshell strength traits.

[0014] Another object of this application is to provide: a method for assisting in the breeding of Loch Ness Red chickens with a high eggshell strength trait and an extra-long breeding cycle, the method comprising: (1) Extract genomic DNA from the Rhodes Island Red Chicken with the longest breeding cycle to be tested; (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 2 and SEQ ID No. 3; (3) Sequencing the amplified products and analyzing the sequencing results to determine the genotypes at positions 6550013 and 6550240 on chromosome 9 of the Rhodes Island Red chicken with the long breeding cycle to be tested. When the genotype at position 6550013 of the chicken to be tested is AA and the genotype at position 6550240 is TT, then the chicken to be tested has the performance of high eggshell strength.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention utilizes GWAS to genotype 428 RODBA individuals with recorded eggshell strength in an extended breeding cycle. Combining pedigree information and phenotypic data, statistical association analysis was performed, revealing for the first time that two SNP loci on the PXYLP1 gene (locus 6550013 and 6550240 on chromosome 9) are significantly associated with eggshell strength. These two loci exhibit C / A and C / T polymorphisms. When the genotype at locus 6550013 mutates from CC to AA, and the genotype at locus 6550240 mutates from CC to TT, the eggshell strength of RODBA chickens in the extended breeding cycle significantly increases. Therefore, this SNP molecular marker can be used for assisted breeding of RODBA chickens with eggshell strength in the extended breeding cycle, enabling early screening and accelerating the breeding process. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 The image shows a gel electrophoresis diagram of PCR amplification products containing the target SNP locus; bands 1-2 represent the CC / CC genotype; bands 3 and 6 represent the AA / TT genotype; and bands 4-5 represent the CA / CT genotype.

[0018] Figure 2 The sequencing peaks of the PCR amplification product fragments are shown in the Sanger sequencing results for three genotypes; where a represents genotype 6550013 and b represents genotype 6550240. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Discovery of SNP molecular markers in the PXYLP1 gene This invention used 428 Rhode Island Red chickens with an extended breeding cycle as research material. Based on genome-wide association analysis (GWAS), combined with pedigree and phenotypic data, two SNP loci in the PXYLP1 gene significantly associated with eggshell strength were identified: loci 6550013 and 6550240 on chromosome 9 (bGalGal1.mat.broiler.GRCg7b published in NCBI genome version), with C / A and C / T polymorphisms at these two loci, respectively. These two loci also correspond to positions 274 and 501 of SEQ ID No. 1. Association analysis results showed that when the genotype at locus 6550013 mutated from CC to AA, and the genotype at locus 6550240 mutated from CC to TT, the eggshell strength of Rhode Island Red chickens was significantly improved. Therefore, these two SNP loci can serve as molecular markers for the eggshell strength trait in Rhode Island Red chickens with an extended breeding cycle., SEQ ID No.1.

[0021] Example 2 Extracting chicken genomic DNA Blood samples were collected from 428 Rhode Island Red chickens with an extended breeding cycle, one sample from each bird, for DNA extraction. The extraction steps were performed according to the instructions of the blood DNA extraction kit from Tiangen Biotech Co., Ltd.: (1) Add 180 μL of GS buffer and 15 μL of blood sample to a new 1.5 mL centrifuge tube and mix by pipetting.

[0022] (2) Add 4 μL of Rnase A solution (100 mg / mL) to the centrifuge tube, shake for 15 seconds, and let stand at room temperature for 5 minutes.

[0023] (3) Add 20 μL of proteinase K solution to the centrifuge tube, and then add 200 μL of GB buffer to the centrifuge tube. Invert the centrifuge tube repeatedly to mix the liquid in the tube thoroughly.

[0024] (4) Set the temperature of the metal bath to 56°C and put the centrifuge tube in. Every 5 minutes, take out the centrifuge tube to observe the state of the solution, invert and mix it, and put it back until the solution in the centrifuge tube is clear and transparent. Then let the centrifuge tube stand at room temperature for 5 minutes.

[0025] (5) Add 350 μL of BD buffer to a centrifuge tube, mix it with a vortex mixer, add the mixture to the adsorption column with a pipette, put the adsorption column into a collection tube, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the tube after centrifugation.

[0026] (6) Add 500 μL of buffer GDB into the adsorption column, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the tube after centrifugation.

[0027] (7) Add anhydrous ethanol to PWB according to the operation requirements, add 600 μL of the prepared PWB solution to the adsorption column, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the tube.

[0028] (8) Repeat step (7) once.

[0029] (9) Centrifuge the adsorption column at 12,000 rpm for 2 minutes, and discard the waste liquid in the collection tube; place the adsorption column at room temperature for 2 minutes to allow the residual rinsing liquid to dry thoroughly.

[0030] (10) Add 50 μL of TB (elution buffer) to the adsorption column, place the adsorption column in a centrifuge tube, and centrifuge at 12000 rpm for 2 minutes to collect the eluted solution into the centrifuge tube.

[0031] (11) The concentration and quality of the extracted DNA can be detected by a micro spectrophotometer. If there is no abnormality in A280, the DNA quality is qualified. Then, the qualified DNA solution is stored in a -20℃ refrigerator for subsequent use.

[0032] Example 3 Detection of SNP molecular markers (1) Primer design: Based on the upstream and downstream sequences containing the SNP site (the SNP is located at positions 274 and 501 of the nucleotide sequence shown in SEQ ID No. 1), specific amplification primers were designed: Upstream primer F1: 5'-AGGGAGAGGGAGGTGATTGT-3', SEQ ID No. 2; Downstream primer R1: 5'-GCAGTCTTACTGTTAAAAGCTCTGT-3', SEQ ID No. 3.

[0033] (2) PCR amplification: DNA samples from 428 Rhodes Island Red chickens with an extended breeding cycle were amplified by PCR using the specific primers from (1). The amplification system is shown in Table 1. The amplified product containing the mutation region was obtained, and the amplified product was 631 bp. Figure 1 ).

[0034] Table 1 PXYLP1 Amplification System

[0035] The amplification program was as follows: First stage: pre-denaturation at 95℃ for 3 min; Second stage: denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 40 s for a total of 35 cycles; Third stage: final extension at 72℃ for 5 min, and storage at 4℃.

[0036] (3) Genotype determination: The amplified products are subjected to Sanger sequencing to obtain the genotype of the SNP locus. Figure 2 ), and compared the quality traits of eggs from different genotypes (Table 2, Table 3).

[0037] Table 2. Eggshell strength, egg weight, and egg shape index of different genotypes of Loch Ness Red eggs

[0038] Note: Duncan's method was used for multiple comparisons. Data in the same column marked with different lowercase letters indicate significant differences. P <0.05, marked with the same letter or not marked, indicates that the difference is not significant ( P >0.05).

[0039] Table 3 Egg production of Loch Ness Red chickens of different genotypes

[0040] Note: Duncan's method was used for multiple comparisons. Data in the same column are marked with different lowercase letters to indicate significant differences. P <0.05, marked with the same letter or not marked, indicates that the difference is not significant ( P >0.05).

[0041] Results Analysis: Tables 2 and 3 show that loci 6550013 and 6550240 exhibit C / A and C / T polymorphisms, respectively. Mutations from C to A and C to T significantly enhanced eggshell strength, but had no significant effect on egg weight, egg shape index, or egg production. These results indicate that the SNP molecular markers provided by this invention can be used for efficient and accurate screening of LOT Red chicken individuals with high eggshell strength and long breeding cycles.

[0042] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the PXYLP1 gene SNP molecular marker in the identification, evaluation, or assisted breeding of Rhode Island Red chickens with a long breeding cycle and high eggshell strength, characterized in that... The SNP molecular markers are located at positions 6550013 and 6550240 on chicken chromosome 9, and the genome version is bGalGal1.mat.broiler.GRCg7b; wherein position 6550013 is a C / A polymorphism and position 6550240 is a C / T polymorphism.

2. The application according to claim 1, characterized in that, When the genotype at locus 6550013 is AA and the genotype at locus 6550240 is TT, the chicken under test is determined to have high eggshell strength.

3. A primer pair for detecting the SNP molecular marker of claim 1 or 2, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID No. 2 and SEQ ID No.

3.

4. A reagent kit, characterized in that, The kit contains the primer pair as described in claim 3.

5. The application of the primer pair of claim 3 or the kit of claim 4 in the identification, evaluation or assisted selection of long-breeding-cycle Rhodes Red chickens with high eggshell strength trait.

6. A method for assisted breeding of Rhode Island Red chickens with an extra-long breeding cycle and high eggshell strength, characterized in that, The method includes: (1) Extract genomic DNA from the Rhodes Island Red Chicken with the longest breeding cycle to be tested; (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 2 and SEQ ID No. 3; (3) Sequencing the amplified products and analyzing the sequencing results to determine the genotypes at positions 6550013 and 6550240 on chromosome 9 of the Rhodes Island Red chicken with the long breeding cycle to be tested. When the genotype at position 6550013 of the chicken to be tested is AA and the genotype at position 6550240 is TT, then the chicken to be tested has the performance of high eggshell strength.

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