Molecular marker for analyzing breeding traits of laying hens and application of molecular marker

Abstract

The invention relates to the field of animal breeding, in particular to a molecular marker for analyzing breeding traits of laying hens and application, and provides a specific primer pair for analyzing the breeding traits of the laying hens, so that early-stage precise breeding of the laying hens is realized. The technical scheme has the advantages of simplicity and convenience in operation, low cost, accuracy in detection and the like, and has an important application value in molecular precise breeding of laying hen breeding traits.

Description

Technical Field

[0001] The present invention belongs to the field of animal breeding, specifically relates to the field of biological detection technology, and more specifically relates to molecular markers for analyzing the reproductive traits of laying hens and applications thereof. Background Art

[0002] In the laying hen industry, reproductive traits are extremely important and have relatively low heritability. Key reproductive traits include age at first laying, hen weight, egg weight, and egg number. Studies have shown that the heritability of first-laid hen weight is approximately 0.28-0.68, the heritability of first-laid egg weight is approximately 0.06-0.38, and the heritability of egg number is approximately 0.05-0.44. Therefore, molecular marker-assisted selection can be used in breeding to improve genetic progress. Recent studies have shown that introns can act as regulatory elements to regulate gene expression, which has greatly promoted the research progress of marker-assisted selection.

[0003] Single nucleotide polymorphisms (SNPs) refer to variations in a single nucleotide in the genome. These variations are stably inherited by offspring during genetic transmission, and therefore can be used as molecular markers for population selection and seed conservation. SNPs can be used in marker-assisted selection (MAS), enabling early selection, improving selection efficiency, reducing breeding costs, and enhancing selection accuracy. Summary of the Invention

[0004] This invention addresses the low efficiency and poor reliability of existing breeding methods by proposing a molecular marker for analyzing the reproductive traits of laying hens and its application. This method enables early, precise breeding of laying hens, featuring ease of operation, low cost, and accurate detection. It holds significant application value in molecular precision breeding for reproductive traits in laying hens.

[0005] To achieve the technical purpose of the present invention, the first aspect of the present invention provides a molecular marker for detecting reproductive traits of laying hens, characterized in that the molecular marker is as shown in the following 1), 2), 3) or 4):

[0006] 1) Located at position 107114231 of chromosome 3 of laying hens, the polymorphic base is G / A. Laying hens with genotype AA have better initial egg weight than those with genotype GG.

[0007] 2) Located at position 107114820 of chromosome 3 of laying hens, the polymorphic base is G / A, and genotype AA has a better total egg production than genotype AG or genotype GG;

[0008] 3) Located at intron 107117405 in laying hens, the polymorphic base is A / G; laying hens with genotype AA have better initial egg weight than those with genotype AG;

[0009] 4) Located at intron 107117682 in laying hens, the polymorphic base is C / T; genotype CC has a better total egg production than genotype CT;

[0010] The above molecular marker loci were determined based on the comparison of the Gallus_gallus_5.0 genome of the red jungle fowl.

[0011] To achieve the technical purpose of the present invention, the present invention provides a primer combination for detecting reproductive traits of laying hens, wherein the primer sequences are SEQ ID NO.1-SEQ ID NO.2 and / or SEQ ID NO.3-SEQ ID NO.4 and / or SEQ ID NO.5-SEQ ID NO.6 and / or SEQ ID NO.7-SEQ ID NO.8.

[0012] To achieve the technical purpose of the present invention, the present invention also provides a kit for detecting reproductive traits of laying hens, which has the primer combination described above.

[0013] To achieve the technical purpose of the present invention, the present invention further provides a method for detecting the reproductive traits of laying hens, by using the above-mentioned primer combination or kit to detect the sample to be tested, and judging the reproductive traits of the sample to be tested based on the test results.

[0014] In particular, the reproductive traits of the sample to be tested are determined based on the test results. When there is a mutation in the chromosome of the sample to be tested, it is determined that the sample to be tested can increase the population base of excellent reproductive traits of laying hens in the group and improve the reproductive capacity of the group.

[0015] Specifically, when the genotype detected at position 107114231 of chromosome 3 is AA or / and the genotype at position 107114820 of chromosome 3 is AA or / and the genotype at position 107117405 of intron is AA or / and 4) the genotype at position 107117682 of intron is CC, its reproductive ability is better.

[0016] In summary, the beneficial effects of the present invention are:

[0017] The analytical markers provided by the present invention can quickly and accurately identify laying hens with better reproductive capacity, improve the reproductive capacity of laying hen groups, increase breeding efficiency, and thus improve the production performance and economic benefits of the group; the molecular markers, primers, and kits provided by the present invention can be used to complete the detection of laying hen reproductive capacity under ordinary laboratory conditions, with simple operation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison of three genotypes CC, CT, and TT at Chr3.107114119 (CT);

[0019] Figure 2 Comparison of three genotypes CC, CT, and TT of Chr3.107114188 (TC);

[0020] Figure 3 Comparison of three genotypes of Chr3.107114231 (GA): AA, AG, and GG;

[0021] Figure 4 Comparison of three genotypes CC, CT, and TT of Chr3.107114463 (CT);

[0022] Figure 5 Comparison of three genotypes AA, AG, and GG of Chr3.107114820 (GA);

[0023] Figure 6 Comparison of three genotypes CC, CT, and TT of Chr3.107117348 (TC);

[0024] Figure 7 Comparison of three genotypes of Chr3.107117355 (AG): AA, AG, and GG;

[0025] Figure 8 Comparison of three genotypes of Chr3.107117356 (GT): GG, GT, and TT;

[0026] Figure 9 Comparison of three genotypes of Chr3.107117405 (AG): AA, AG, and GG;

[0027] Figure 10 Comparison of three genotypes of Chr3.107117493 (AG): AA, AG, and GG;

[0028] Figure 11 Comparison of three genotypes CC, CT, and TT of Chr3.107117497 (CT);

[0029] Figure 12 Comparison of three genotypes of Chr3.107117645 (GA): AA, AG, and GG;

[0030] Figure 13 Comparison of three genotypes CC, CT, and TT of Chr3.107117682 (CT). DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The bioinformatics software and products used in the present invention are all commercially available. Various processes and methods that are not described in detail adopt conventional methods known to those skilled in the art. The sources of materials used, trade names, and if it is necessary to list their components, are all indicated when they first appear. Unless otherwise specified, the same reagents used thereafter are the same as those first indicated.

[0032] Example 1 Acquisition of molecular markers for egg-laying performance of chickens

[0033] 1. Phenotype collection and DNA extraction

[0034] 500 laying hens were selected and raised for 300 days. Phenotypic data were recorded, including the total number of eggs laid (n), first-laid chicken weight (kg), 300-day chicken weight (kg), first-laid egg weight (g), and 300-day egg weight (g) for each laying hen during the 300-day period. DNA was also collected from the blood of the 300-day-old chickens.

[0035] The phenotypic statistical method and DNA extraction method all adopt conventional techniques in the field and will not be described in detail in the present invention.

[0036] Since sequencing has certain requirements for DNA quality, low DNA quality will affect the sequencing results. Therefore, in one embodiment of the present invention, 2% agarose gel electrophoresis is used to detect whether the extracted DNA has been degraded. The electrophoresis results show that the DNA sample bands are clear, bright, complete, and without tailing, indicating that the DNA quality of the test population is good and meets the sequencing requirements.

[0037] 2. DNA data processing

[0038] 500 DNA samples of acceptable quality were sequenced on the BGI sequencing platform. The raw sequencing data were quality-controlled using fastp (v0.23.4). The resulting sequencing data were then aligned to the chicken reference genome (Gallus_gallus_6.0) using BWA software. The resulting bam files were then sorted, deduplicated, de-redundant, and indexed using SAMtools to generate pre-processed bam files.

[0039] 3. Genome-wide SNP detection

[0040] The bam file was input into GATK for variant detection, and the obtained raw SNP data was strictly filtered with the following filtering parameters: QD < 2.0, QUAL < 30.0, SOR > 3.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0. Variants with coverage > 30% and minor allele frequency (MAF) > 0.01 were retained, and SNPs within 5 bp of the InDel were removed. Preliminary quality control of the SNP sites was performed using vcftools. The quality control criteria were: the call rate of SNPs at a single site reached above 95%; the call rate of individual SNPs reached above 90%; the minimum allele frequency MAF threshold was set to 0.01, and the Hardy-Weinberg equilibrium P value was < 10 -5 Finally, 15,473,778 high-quality SNPs were obtained for subsequent analysis. The ANNOVAR software package was used to classify SNPs into different genomic regions (i.e., exons, introns, splice sites, upstream and downstream of genes, and intergenic regions).

[0041] 4. Screening of SNPs related to reproductive traits of laying hens

[0042] GWAS analysis based on sequencing results and phenotypic data for reproductive traits such as total egg production, first-laid chick weight, first-laid egg weight, and egg weight identified 13 SNPs associated with total egg production, first-laid chick weight, first-laid egg weight, and egg weight. Thirteen SNPs were detected in the egg defensin gene, including 12 introns of chromosome 13 and one in exon 2. Genotype frequencies at each locus ranged from 9% to 59%, and allele frequencies ranged from 23% to 77%. Hardy-Weinberg tests showed no statistically significant differences between the measured and expected genotype frequencies at each locus (P>0.05), indicating Hardy-Weinberg equilibrium and population representativeness. The specific SNP sites are Chr3.107114119 (CT), Chr3.107114188 (TC), Chr3.107114231 (GA), Chr3.107114463 (CT), Chr3.107114820 (GA), Chr3.107117348 (TC), Chr3.107117355 (AG), Chr3.107117356 (GT), Chr3.107117405 (AG), Chr3.107117493 (AG), Chr3.107117497 (CT), Chr3.107117645 (GA), Chr3.107117682 (CT). The genotype comparison results are as follows. Figure 1-13 shown.

[0043] Further association analysis of the 13 initially screened SNPs with phenotypic data revealed that only four SNPs were significantly (P<0.05) or extremely significantly (P<0.01) associated with reproductive traits. Specifically, SNP 107114231 (GA) in the intron of chromosome 13 was significantly associated with first egg weight (P<0.05), SNP 107114820 (GA) in the exon was extremely significantly associated with total egg production at 300 days (P<0.01), SNP 107117405 (AG) was significantly associated with first egg weight (P<0.05), and SNP 107117682 (CT) was significantly associated with total egg production at 300 days (P<0.05).

[0044] Finally, the four variant sites provided by the present invention were confirmed, specifically: located at position 107114231 of chromosome 3 of laying hens, with a polymorphic base of G / A; located at position 107114820 of chromosome 3 of laying hens, with a polymorphic base of G / A; located at position 107117405 of the intron of laying hens, with a polymorphic base of A / G; located at position 107117682 of the intron of laying hens, with a polymorphic base of C / T.

[0045] Specifically, the genotype AA located at position 107114231 of chromosome 3 of laying hens has a better initial egg weight than that of laying hens with genotype GG; the genotype AA located at position 107114820 of chromosome 3 of laying hens has a better total egg production than that of genotype AG or genotype GG; the genotype AA located at position 107117405 of the intron of laying hens has a better initial egg weight than that of laying hens with genotype AG; the genotype CC located at position 107117682 of the intron of laying hens has a better total egg production than that of genotype CT.

[0046] Example 2 Acquisition of a primer set for chicken egg-laying performance

[0047] Primers were designed based on the four sites obtained in Example 1 using conventional methods. The primers finally obtained were:

[0048]

[0049] Application Examples

[0050] The four SNP sites obtained in Example 1 or the primers obtained in Example 2 were used to analyze the reproductive capacity of a laying hen population. The specific method is as follows:

[0051] 1. Sample selection and phenotypic data collection

[0052] 248 newly hatched laying hens were raised for 300 days, and the total number of eggs laid in 300 days (pieces), weight of first-laid chickens (kg), weight of chickens at 300 days (kg), weight of first-laid eggs (g), and weight of eggs at 300 days (g) were recorded.

[0053] 2. Genomic DNA extraction and primer design

[0054] The blood DNA of each sample in the chicken flock was extracted using a traditional method. The genetic diversity of 248 laying hens was directly detected using primers designed according to the loci provided in Example 1 or using the primers provided in Example 2.

[0055] 3. PCR amplification and sequencing

[0056] A 50 μL PCR system was used: 1 μL DNA template, 2 μL each of upstream and downstream primers, 25 μL Taq PCR Master Mix, and dd H₂O added to 50 μL. PCR reaction parameters included: initial denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, 35 cycles of annealing at a specific annealing temperature (see Table 1) for 30 seconds, and extension at 72°C for 1 minute; followed by a final extension at 72°C for 10 minutes. PCR amplification products were verified by electrophoresis on a 1% agarose gel. Amplified products were sent to a company for sequencing.

[0057] 4. Data statistical processing

[0058] Seqman in DNAstar software was used to detect and count SNP sites in the sequencing results, and EXCEL tools were used to count the genotypes of 248 chickens.

[0059] 4.1 SNP site detection

[0060] SNP loci were detected in 248 DNA samples. The test results showed that the genotype frequencies of each locus ranged from 9% to 59%; the allele frequencies ranged from 23% to 77%. The Hardy-Weinberg test showed that there was no statistical difference between the measured and expected genotype frequencies of each locus (P>0.05), which was consistent with the Hardy-Weinberg genetic equilibrium and representative of the population, as shown in Table 1.

[0061] Table 1 Results of SNP detection of 248 DNA samples

[0062]

[0063] 4.2 SNPs and phenotypic data analysis results

[0064] The phenotypic data of reproductive traits were statistically analyzed based on the SNP analysis results, as shown in Table 2

[0065] Table 2 Correlation between polymorphic sites in egg defensin gene and different egg production traits

[0066]

[0067]

[0068] Note: The same letters or no letters in the shoulders indicate no significant difference (P>0.05), the letters a and b indicate significant difference (P<0.05), and the letters A and B indicate extremely significant difference (P<0.01).

[0069] According to the data in the table, it can be seen that Chr3.107114231 (GA) is significantly correlated with the initial egg weight (P<0.05), Chr3.107114820 (GA) is extremely significantly correlated with the total number of eggs produced (P<0.01), 107117405 (AG) is significantly correlated with the initial egg weight (P<0.05), and 107117682 (CT) is significantly correlated with the number of eggs produced at 300 days (P<0.05).

[0070] At Chr3.107114231 (GA), the first egg weight of AA-type individuals was 1.73 g higher than that of GG-type individuals, with a significant difference between AA and GG types (P<0.05). A missense mutation (GCT-ACT) occurred at Chr3.107114820 (GA), resulting in AA-type individuals laying 23.96 and 23.93 more eggs than AG-type and GG-type individuals, respectively, with extremely significant differences between AA, AG, and GG types (P<0.01). At Chr3107117405 (AG), the first egg weight of AA-type individuals was 1.4 g higher than that of AG-type individuals, with a significant difference between AA and AG types (P<0.05). At Chr3 107117682 (CT), the total number of eggs laid by CC-type individuals was 9.45 more than that of CT-type individuals, with a significant difference between CC and CT types (P<0.05).

[0071] This application is the first to explore the relationship between SNPs in laying hens and reproductive traits such as total egg production, initial egg weight, and 300-day egg weight. This can be used for rapid testing of laying hen reproductive performance. Selecting superior genotypes in actual breeding can improve the production performance and economic benefits of laying hen populations.

[0072] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A molecular marker for detecting reproductive traits of laying hens, characterized in that: The molecular markers are shown in 1), 2), 3) or 4) below: 1) Located at position 107114231 of chromosome 3 of laying hens, the polymorphic base is G / A. Laying hens with genotype AA have better initial egg weight than those with genotype GG. 2) Located at position 107114820 of chromosome 3 of laying hens, the polymorphic base is G / A, and genotype AA has a better total egg production than genotype AG or genotype GG; 3) Located at intron 107117405 in laying hens, the polymorphic base is A / G; laying hens with genotype AA have better initial egg weight than those with genotype AG; 4) Located at intron 107117682 in laying hens, the polymorphic base is C / T; genotype CC has a better total egg production than genotype CT; The above molecular marker loci were determined based on the comparison of the Gallus_gallus_5.0 genome of the red jungle fowl.

2. A primer combination for detecting reproductive traits of laying hens, the primer sequences of which are shown as SEQ ID NO.1-SEQ ID NO.

6.

3. A kit for detecting reproductive traits of laying hens, comprising the primer combination according to claim 2.

4. A method for detecting reproductive traits of laying hens, comprising: detecting a sample using the primer combination of claim 2 or the kit of claim 3.

5. The method of claim 4, comprising: The sample to be tested is tested using the primer combination provided in claim 3 or the kit provided in claim 4, and the reproductive traits of the sample to be tested are determined based on the test results.

6. The method of claim 5, wherein determining the reproductive traits of the sample to be tested based on the test results is that when there is a mutation in the chromosome of the sample to be tested, it is determined that the sample to be tested can increase the population base of excellent reproductive traits of laying hens in the population and improve the reproductive capacity of the population.

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