A molecular marker associated with chicken shank length and its application

Abstract

This invention discloses a molecular marker associated with the chicken shank length trait and its application, belonging to the field of poultry breeding technology. This invention selected 505 Ma Huang chickens as experimental subjects for genome-wide association analysis (GWAS). Shank length trait data were collected, and blood samples were collected from under the wings at 77 days of age to extract genomic DNA. The integrity, purity, and concentration of the extracted genomic DNA were tested. Qualified samples were resequencing, and then GWAS was performed on the shank length trait, detecting 458 SNPs significantly associated with shank length. Further validation analysis was conducted on some of these SNP sites and their association with the shank length trait, providing theoretical support for breeding chicken breeds associated with the shank length trait.

Description

Technical Field

[0001] This invention relates to the field of poultry breeding technology, and in particular to a molecular marker related to chicken shank length and its application. Background Technology

[0002] The development of poultry farming is of great significance to animal husbandry and agriculture. The shank length trait in chickens is an important indicator of skeletal development and a key parameter for measuring growth performance and slaughter rate. While some key genes and signaling pathways related to skeletal growth and development (such as BMP and Wnt) have been identified in poultry shank length research, their genetic mechanisms remain unclear. In particular, the relationship between polygenic inheritance and phenotype is complex, and genetic mapping and genome-wide association studies (GWAS) are lacking. GWAS, as an effective tool, is increasingly used in animal and plant breeding and even human disease research. It combines resequencing methods with statistical principles to identify candidate regions and genes associated with target traits across the entire genome. Currently, there are few reports on genes related to chicken shank length, both domestically and internationally, and there is still a significant research gap in the use of large-scale sequencing for studies of chicken shank length-related traits. With the development of genome sequencing technology, related technologies are being applied more widely and effectively in the study of quantitative traits in animals and plants. Through methods such as genome-wide association studies (GWAS), breeding efficiency can be significantly improved.

[0003] Genome-wide association studies (GWAS) can provide insights into the genetic mechanisms of shank length traits during breeding, and pinpoint key genes or regulatory regions, thus providing a scientific basis for marker-assisted selection (MAS) and genome selection (GS), accelerating the breeding process of high-yielding and high-quality chicken breeds. Therefore, identifying marker genes and SNPs for chicken shank length traits not only has urgent application needs but also opens up new directions for poultry genetic breeding research. In the future, combining multi-omics data (such as transcriptomics and epigenomics) will further promote precision breeding and genetic improvement of chicken shank length traits. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker related to chicken shank length and its application, in order to solve the problems existing in the prior art. By analyzing chicken genomic DNA through genome-wide association studies, candidate genes and SNP sites that are significantly related to chicken shank length can be screened out, providing theoretical support for the future breeding of high-quality chicken breeds.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] In a first aspect, the present invention provides a molecular marker associated with the shank length trait in chickens, wherein the SNP site of the molecular marker is located at the 7457474th base on the Z chromosome of the chicken genome, and a T / C mutation exists at this site; the accession number of the chicken genome in the Ensemble database is GRCg6a_release95.

[0007] Preferably, the genotype of the molecular marker is TT, CC, or TC.

[0008] Preferably, the shank length trait is that of a chicken.

[0009] Preferably, the molecular marker is related to tibia length.

[0010] Preferably, the chicken includes Jiangfeng Mahuang chicken.

[0011] Secondly, the present invention also provides the application of the said molecular marker in any of the following:

[0012] (1) Application in identifying chicken shank length trait;

[0013] (2) Application in the improvement of chicken shank length trait;

[0014] (3) Application in screening new breeds related to chicken shank length trait.

[0015] Thirdly, the present invention also provides an application for detecting products with the described molecular markers in any of the following:

[0016] (1) Application in identifying chicken shank length trait;

[0017] (2) Application in the improvement of chicken shank length trait;

[0018] (3) Application in screening new breeds related to chicken shank length trait

[0019] Fourthly, the present invention also provides a breeding method related to the chicken shank length trait, comprising the following steps:

[0020] Extract genomic DNA from the chicken to be tested, perform whole-genome sequencing or obtain 100kb sequences upstream and downstream of the SNP site of the molecular marker, determine the tibia length trait of the chicken to be tested based on the genotype of the SNP site, and select chicken breeds with excellent tibia length based on the determination results.

[0021] Preferably, the genotype of the SNP site of the molecular marker is used to determine the shank length of chickens, and the optimal genotype for shank length is TT.

[0022] Preferably, in breeding chickens for the shank length trait, chicken breeds with the TT genotype for shank length are retained.

[0023] The present invention discloses the following technical effects:

[0024] This invention selected the shank length phenotype of Jiangfeng Ephedra chicken for genome-wide association analysis. By selecting these traits, single nucleotide polymorphisms that are significantly associated with shank length were identified. As a result, 458 SNPs that are significantly associated with shank length were detected.

[0025] This invention also takes the ChrZ:7457474 locus as an example to analyze its association with the chicken shank length trait. The results show that the ChrZ:7457474 locus is significantly associated with chicken shank length, and the shank length of the TT genotype population is higher than that of the TC and CC populations. The phenotypic differences between TT and other genotypes are significant (P<0.05), while the phenotypic differences between TC and CC genotypes are not significant (P>0.05).

[0026] This invention has identified key genes and signaling pathways that influence shank length in poultry, revealing the underlying genetic mechanisms. It fills the gap in genome-wide association studies of shank length in Ma Huang chickens and provides theoretical support for the breeding of new high-quality chicken breeds. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a partial illustration of 1% agarose gel electrophoresis of the genomic DNA stock solution; where 456-472 represent sample numbers and M represents standard DNA molecules.

[0029] Figure 2 Manhattan plot of the whole-genome association analysis results for tibia length;

[0030] Figure 3 The result is a QQ graph of the tibia length. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1

[0037] 1. Laboratory animals

[0038] The experimental animals used were the 17th generation G strain of the Ma Huang chicken, produced by Guangdong Jiangfeng Industrial Co., Ltd. The chickens were cage-raised at 77 days old, managed according to the farm's feeding procedures. The chicken house was well-ventilated, and the ambient temperature, humidity, and lighting were suitable. Routine immunizations and disease prevention were administered.

[0039] 2. Sample collection and processing

[0040] When the rearing period was 77 days, 505 Jiangfeng Ma Huang chickens (170 roosters and 335 hens) were randomly selected from the caged and housed group for blood collection under the wings. 1 mL of blood was drawn with a syringe, injected into a blood collection tube containing heparin sodium, and mixed to prevent blood clotting. The individual wing number was marked on the blood collection tube and stored in a -80℃ refrigerator.

[0041] 3. Experimental Methods

[0042] 3.1 Phenotypic Data Determination

[0043] Twelve hours before slaughter, the experimental chickens were provided with drinking water. At slaughter, the Ma Huang chickens were bled through the carotid artery and then scalded in water at a temperature of about 60°C for 3 minutes to remove feathers. After the carcass surface was dried with kitchen paper, the appearance characteristics of the carcass were measured, and the shank length of each individual was measured and recorded using vernier calipers.

[0044] 3.2 Extraction and Detection of Genomic DNA

[0045] Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, following standard extraction procedures. The extracted genomic DNA was then tested for integrity, purity, and concentration. DNA that met the requirements was retained, while DNA that did not meet the requirements was discarded or re-extracted and re-tested.

[0046] 3.3 Sequencing data quality control and statistical comparison with reference genome

[0047] The experiment was conducted according to the standard protocol provided by the sequencing company. For qualified genomic DNA samples, appropriate fragment sizes were selected using gel electrophoresis, followed by PCR amplification and library construction. The constructed libraries underwent quality testing, and qualified libraries were then sequenced using the DNBSEQ-T7 sequencer, performed by BGI Genomics Co., Ltd. To ensure the quality of information analysis, base sequencing quality distribution analysis, base type distribution checks, insert fragment distribution statistics, depth distribution statistics, and filtering of the raw image data (Raw Reads) files obtained from high-throughput sequencing were performed during sequencing using the DNBSEQ-T7 system.

[0048] The final sequences obtained from sequencing are remapped onto a reference genome before further analysis. The percentage of clean reads that can be mapped onto the reference genome is called the alignment efficiency, or Mapped (%). The reference genome species is Gallus gallus, and the reference genome is GRCg6a_release95, sourced from the Ensemble database.

[0049] 4. Data processing and statistical analysis

[0050] 4.1 Descriptive statistical analysis of tibia length traits

[0051] The collected tibia length data were initially organized using Excel, and outliers were removed from the data of each trait according to the μ±3σ principle. Descriptive statistical analysis was then performed on the data using Excel. The results of the analysis included sample size, mean, standard deviation, and coefficient of variation.

[0052] 4.2 Genome-wide association analysis

[0053] The samples were sent to Beijing Biomarker Biotechnology Co., Ltd. for genome-wide association analysis. In order to screen for genes or molecular markers associated with tibia length traits across the entire genome, this invention performed low-coverage sequencing (6X) on 505 quality-tested samples.

[0054] Genome-wide association analysis was performed using the LMM model in GEMMA software, combining phenotypic and genotypic data. Considering fixed factors (SNP effects) and random effects (inter-individual kinship), the statistical model is as follows:

[0055] This invention is based on the developed high-density molecular marker data and uses GEMMA for association analysis. The formula for the linear mixture model (LMM) of GEMMA software is as follows (1):

[0056] y=Wα+xβ+μ+e (1)

[0057] Where y is the phenotypic vector; W is the indicator matrix of the fixed effects; α is the coefficient vector of the fixed effects; x is the genotype vector; β is the SNP effect; μ is the random effects vector; and e is the residual.

[0058] 4.3 Group Stratification

[0059] Population stratification refers to the difference in allele frequencies due to different ancestors. It has been proven to be a confounding factor that may lead to many false positive results. Therefore, when conducting association analysis on the shank length of Jiangfeng Mahuang chickens, a Quantile-Quantile Plot is plotted on the shank length of Jiangfeng Mahuang chickens to determine whether there is any bias in the association analysis and whether there is stratification in the sample population.

[0060] 4.4 Gene annotation of significant SNPs

[0061] After obtaining significant SNPs from genome-wide association analysis, genes within 50kb upstream and downstream of these sites were retrieved for gene annotation based on a reference genome.

[0062] 5. Results and Analysis

[0063] 5.1 Genomic DNA Detection Results

[0064] Genomic DNA extracted from 505 samples underwent quality testing via agarose gel electrophoresis. Some genomic DNA test results are shown below. Figure 1 As shown, the electrophoresis wells must be clean and uncontaminated, the main band must be clear, and there must be no tailing. Additionally, DNA purity must be measured at 1.6. <OD 260 / OD 280 <2.0, 1.8 <OD260 / OD 230 <2.1. Genomic DNA testing results must simultaneously meet the above requirements before library construction can proceed. Unqualified samples must be discarded or DNA extracted again. Of all samples tested in this invention, 505 met the requirements.

[0065] 5.2 Sequencing data quality control and alignment results with the reference genome

[0066] The sequencing data quality control results are shown in Table 1. Base type distribution detection was mainly used to check for AT and CG segregation, which may originate from sequencing or library construction. Significant segregation can affect subsequent analysis. The average percentage of G and C bases in the sample (GC(%)) was 41.36%, the average percentage of bases with a quality value greater than or equal to 20 (Q20(%)) was 98.03%, and the average percentage of bases with a quality value greater than or equal to 30 (Q30(%)) was 94.55%. The alignment efficiency between the sample genomic DNA and the reference genomic DNA was above 97.04%, with an average of 99.19%, indicating that the library construction and sequencing of this sample were normal.

[0067] Table 1. Evaluation Statistics of Sample Sequencing Data

[0068]

[0069] Note: Clean_Reads: Number of filtered reads; Clean_Base: Number of filtered bases, calculated by multiplying the number of Clean_Reads by the sequence length.

[0070] 5.3 Statistical analysis of SNP detection results between sample and reference genome

[0071] There are two main types of SNP mutations: transition (Ti, a variation between bases of the same type) and transversion (Tv, a variation between bases of different types). Generally, the probability of transition is higher than that of transversion, i.e., Ti / Tv is greater than 1. Table 2 shows that a total of 1,996,687,423 SNPs were detected in this experiment, and the heterozygous ratio (Het-ratio) was 48.27%.

[0072] Table 2. Statistical analysis of SNP detection results between the genome and the reference genome.

[0073]

[0074] Note: Heterozygosity Number (number of heterozygotes), Homozygosity Number (number of homozygotes), Het-ratio (percentage of heterozygotes).

[0075] 5.4 Descriptive statistical analysis results of tibia length traits

[0076] The results of the descriptive statistical analysis of shank length are shown in Table 3. The analysis revealed that a total of 505 chickens were measured (170 roosters and 335 hens). The coefficient of variation for shank length was 3.47% for roosters and 5.04% for hens. The difference in shank length between male and female Ma Huang chickens was highly significant (P<0.01).

[0077] Table 3. Descriptive statistical analysis of tibia length traits.

[0078]

[0079] Note: Different letters on the shoulder label indicate extremely significant differences (P<0.01), while identical letters or no letters indicate no significant differences (P>0.05).

[0080] 5.5 Results of Genome-wide Association Analysis

[0081] This study used Fastlmm, Emmax, and Gemma software to perform genome-wide association analysis on 505 Ma Huang chickens. Significantly related SNPs associated with shank length were identified across the entire genome. The SNP molecular markers were derived from the international chicken reference gene GRCg6a_release95, with the genome source being Ensemble.

[0082] Table 4 shows the partial results of the genome-wide association analysis of tibia length, displaying the information of the top 10 SNPs significantly associated with tibia length selected based on -log10(p)>5. The corresponding Manhattan plot is shown below. Figure 2 As shown, 458 SNPs were found to be significantly correlated with tibia length, located on chromosomes Chr 2, Chr 5, Chr 13 and sex chromosomes Z and W, respectively.

[0083] Table 4. Results of genome-wide association analysis of tibia length (top 10 SNPs)

[0084]

[0085]

[0086] Note: Chr: chromosome; Allele: allele; MAF: minor allele frequency.

[0087] 5.6 Group Stratification Assessment Results

[0088] Significant SNPs were found in tibia length analysis using different software and models. The QQ plot of tibia length trait is shown in the figure. Figure 3 As shown, the horizontal axis represents the expected value, and the vertical axis represents the observed value. The thin line in the graph represents the 45° line, which is the predicted threshold. The gray area represents the 95% confidence interval of the scatter plot. The greater the distance between the SNP and the solid line, the stronger the association. Figure 3 As can be seen, most of the loci in the lower left corner of the graph are on the diagonal, indicating that the model selection is reasonable. The loci in the upper right corner that extend beyond the diagonal and confidence interval represent high significance with the target trait. This indicates that there is no population stratification in the experimental population.

[0089] 5.7 Gene annotation of SNPs that are significantly associated at the whole genome level

[0090] Genome-wide association analysis identified 458 SNPs that were significantly associated with tibia length, located on chromosomes Chr 2, Chr 5, Chr 13, and sex chromosomes Z and W. Preliminary gene annotation of significant sites was performed using Ensembl, covering an upstream and downstream range of 100 kb. Some annotation results are shown in Table 5.

[0091] Table 5. Annotation results of genome-wide association analysis of tibial length (partial).

[0092]

[0093] 5.8 Association analysis between significant loci and tibia length trait

[0094] Association analysis of genotype and tibia length phenotype was performed using SPSS software. The results showed that there were three genotypes (TT, CT and CC) at the ChrZ:7457474 locus in the Ma Huang chicken population. The tibia length of the TT genotype population was higher than that of the TC and CC populations. The phenotypic differences between the TT and TC and CC genotypes were significant (P<0.05), while the phenotypic differences between the TC and CC genotypes were not significant (P>0.05), as shown in Table 6.

[0095] Table 6. SNPs significantly associated with tibia length traits.

[0096]

[0097] Note: * indicates a significant correlation at the 0.05 level; different letters on the superscript indicate significant differences.

[0098] The above results also show that the SNP marker at the ChrZ:7457474 locus provided by this invention indicates that the Ma Huang chickens corresponding to the TT genotype have higher shank lengths, those corresponding to the TC genotype have lower shank lengths, and those corresponding to the CC genotype have even lower shank lengths. Shank length is an important indicator of chicken skeletal and carcass development; chickens with higher shank lengths generally have better overall development. Therefore, selecting the TT genotype for shank length in chicken breeding can improve breeding efficiency and quickly select chicken breeds with superior shank length traits.

[0099] Genotyping of tibia length was performed using the SNP site ChrZ:7457474 provided above, and chicken breeds with the TT genotype for tibia length were retained, providing a new molecular marker for screening chickens for the tibia length trait.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of products that detect molecular markers related to the shank length trait of Ephedra chickens in any of the following: (1) Application in identifying the shank length trait of Ephedra in chickens; (2) Application in the improvement of shank length trait in Ephedra chickens; (3) Application in screening new breeds related to the shank length trait of Ephedra chicken; The SNP site of the molecular marker is located at the 7457474th base on chromosome Z of the chicken genome, where a T / C mutation exists; the accession number of the chicken genome in the Ensemble database is GRCg6a_release95; The genotypes of the molecular markers are TT, CC, and TC; The shank length of Ephedra chickens was determined by using the genotype of the SNP locus of the molecular marker, and the optimal genotype for shank length was TT.

2. A breeding method associated with the Ephedra equisetina chicken tibia length character, characterized by, Includes the following steps: Genomic DNA of the Ma Huang chicken to be tested was extracted, and whole-genome sequencing was performed to obtain 100kb sequences upstream and downstream of the SNP site of the molecular marker described in claim 1. The tibia length trait of the Ma Huang chicken to be tested was determined based on the genotype of the SNP site, and Ma Huang chicken breeds with excellent tibia length were selected based on the determination results. The shank length of Ephedra chickens was determined by using the genotype of the SNP locus of the molecular marker, and the optimal genotype for shank length was TT.

3. The breeding method according to claim 2, characterized in that, In the breeding of Ma Huang chickens for shank length, chicken breeds with the TT genotype for shank length are retained.

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