Primer for detecting molecular marker of LCORL gene related to broiler growth traits and its application

Abstract

The present invention belongs to the technical field of molecular markers, and specifically discloses a primer and application for detecting a molecular marker of an LCORL gene related to broiler growth traits, including an upstream primer and a downstream primer; the molecular marker site is determined on the specific chromosome position of the genome after comparing the chicken 7.0 reference genome GRCg7b as the reference genome, and the molecular marker is Chr4_75408776, which is located at the 75408776th T / C mutation on chromosome 4 of the chicken genome. The present invention can be effectively used for molecular marker-assisted selection breeding of broiler growth traits, and individuals with TT genotypes at the molecular marker Chr4_75408776 site are selected as breeders, which can accelerate the genetic selection progress of yellow-feathered broiler growth traits, and at the same time improve the carcass uniformity of yellow-feathered broilers, effectively improving the industrial competitiveness of yellow-feathered broilers.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, in particular to a primer for detecting a molecular marker of an LCORL gene related to growth traits of broiler chickens and an application thereof. Background Art

[0002] The growth traits of broilers, including growth rate, shank length and shank circumference, are important economic traits of broilers. Yellow-feathered broilers are my country's national chickens, mainly bred from local Chinese breeds, or from hybrids of local Chinese breeds combined with foreign commercial breeds with high production performance. Compared with foreign commercial chicken breeds (white-feathered broilers), yellow-feathered broilers have the advantages of delicious meat and strong resistance to adversity, but their low production efficiency (slow growth rate and low reproductive performance) and poor carcass uniformity (weight, shank length and shank circumference are important factors affecting carcass uniformity) have always been the core factors restricting their industrial competitiveness.

[0003] At present, the selection of growth traits in the yellow-feathered broiler industry mainly relies on phenotypic selection, which is easily affected by environmental factors. The use of molecular marker-assisted selection of growth traits can effectively improve the genetic selection progress of growth traits. At present, there are many technologies for the selection of growth-related traits based on single or multiple (haplotype) SNPs sites as molecular markers in chickens, but most of these markers are not obtained by whole genome association analysis, and their significance with growth traits is not high. The significance of most sites is lower than 5.00E-05, and these sites cannot reach the significance of 5.00E-08 to 5.00E-09 (0.05 / 1000000SNPs-0.05 / 10000000SNPs) at the whole genome level, that is, the genetic effect of the marker site is not large.

[0004] Therefore, developing growth trait-related molecular markers with greater genetic effects for use in molecular marker-assisted selection of growth traits such as growth rate, shank length and shank circumference can accelerate the progress of genetic selection of growth traits of yellow-feathered broilers, while improving the carcass uniformity of yellow-feathered broilers, effectively enhancing the industrial competitiveness of yellow-feathered broilers. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a primer for detecting a molecular marker of the LCORL gene related to the growth traits of broiler chickens and its application.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the purposes of the present invention is to provide a primer for detecting a molecular marker of the LCORL gene associated with broiler growth traits, comprising:

[0008] Upstream primer, sequence F: 5′-CCTCTTGCCAGCTGAAGGAA-3′;

[0009] Downstream primer, sequence: R: 5′-GGAGGCACAATGTTGCGTTT-3′;

[0010] Among them, the specific chromosome position of the molecular marker site in the genome is determined after comparison with the chicken 7.0 reference genome GRCg7b as the reference genome. The molecular marker is Chr4_75408776, located at the 75408776th T / C mutation on chromosome 4 of the chicken genome.

[0011] The second object of the present invention is to provide an application of the above primers in assisted selection breeding for improving the growth traits of broiler chickens, comprising the following steps:

[0012] S1. Extracting genomic DNA from broiler chickens to be tested;

[0013] S2. Perform PCR amplification on the genomic DNA of the chicken to be tested using the primers. After the PCR amplification reaction program is completed, perform Sanger sequencing on the product; and select individuals with the TT genotype at the molecular marker Chr4_75408776 site as breeder chickens.

[0014] Furthermore, the PCR amplification reaction system is: 500 ng genomic DNA, 25 μL 2X Pro TaqMasterMix (dye plus), 1 μL upstream primer with a concentration of 0.2 μM, 1 μL downstream primer with a concentration of 0.2 μM, and enzyme-free sterile water is added to a total reaction system of 50 μl; the PCR reaction program is 94°C 30s; 98°C 10s, 60°C 30s, 72°C 1min, 35 cycles; 72°C 2min.

[0015] Preferably, the broiler is Qingyuan Ma chicken.

[0016] Preferably, the broiler growth traits include body weight, shank length and shank circumference.

[0017] Compared with the prior art, the present invention conducted phenotypic measurement related to growth traits (weight at 42 days of age, weight at 119 days of age, shank length at 119 days of age and shank circumference at 119 days of age) and whole genome resequencing (average sequencing depth of individuals>10×) on 1496 pure Qingyuan Ma chicken individuals, and carried out whole genome association analysis. The excavated Chr4:75408776 locus (GRCg7b) had a very high genetic correlation with weight at 42 days of age, shank circumference at 119 days of age and shank length at 119 days of age, and its significance with the three traits reached 9.37%, 9.74%, 9.63%, 9.59%, 9.97%, 9.87%, 9.76%, 9.96%, 9.84%, 9.76%, 9.87%, 9.96%, 9.84 ... 3.90E-13, 1.43E-26 and 2.53E-18, and the explained phenotypic variation variance reached 3.42%, 7.01% and 4.85% respectively. The genetic effect is very significant, so it can be effectively used in molecular marker-assisted selection breeding of broiler growth traits. Selecting individuals with the TT genotype at the molecular marker Chr4_75408776 locus as breeding chickens can accelerate the progress of genetic selection of growth traits of yellow-feathered broilers, while improving the carcass uniformity of yellow-feathered broilers, and effectively enhancing the industrial competitiveness of yellow-feathered broilers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the whole genome association analysis of body weight at 42 days of age in a group of 1496 purebred Qingyuan Ma chicken hens: a is the QQ plot; b is the Manhattan plot.

[0019] Figure 2 This is the whole genome association analysis of tibia circumference of 1496 purebred Qingyuan Ma chicken hens at 119 days of age: a is the QQ plot; b is the Manhattan plot.

[0020] Figure 3 This is the whole genome association analysis of tibia length in a population of 1496 purebred Qingyuan Ma chicken hens at 119 days of age: a is the QQ plot; b is the Manhattan plot.

[0021] Figure 4 For the genotyping of the molecular marker site Chr4:75408776. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0023] Example 1

[0024] Location of molecular marker site Chr4:75408776 (GRCg7b) of LCORL gene related to growth traits in broiler chickens

[0025] 1) Phenotypic measurements of body weight at 42 days of age, body weight at 119 days of age, shank circumference at 119 days of age, and shank length at 119 days of age were performed on 1501 purebred Qingyuan Ma chicken hens. Blood samples were collected from each individual for whole genome resequencing (average sequencing depth>10×). After excluding 5 individuals with individual locus deletion rates greater than 0.05, 1496 individual genome data were retained and population SNP quality control was performed using VCFtools. The quality control conditions were: "--not-chr W--not-chr Z--min-alleles 2--max-alleles 2--maf 0.05--max-missing0.95", and 11807487 high-quality autosomal SNPs were obtained. LD-pruning of autosomal SNPs was performed using Plink 1.9 (parameter "--indep-pairwise 5050.5”), and 2568220 low-linked autosomal SNPs were retained for genome-wide association analysis;

[0026] Based on the high-quality autosomal SNPs after LD-pruning, a genome-wide association analysis was performed on body weight at 42 days of age, body weight at 119 days of age, shank circumference at 119 days of age, and shank length at 119 days of age using the mixed linear model of GCTA (PC1-PC10 as quantitative covariates), and the phenotypic variance explained proportion (PVE) of each SNP was estimated. 3.8937E-07 (Bonferroni correction; 1 / 2568220) and 1.9469E-08 (Bonferroni correction; 0.05 / 2568220) were used as the genome-wide potential significance threshold and significance threshold, respectively. The results showed that 12, 0, 335, and 88 SNPs were associated with body weight at 42 days of age ( Figure 1 )、119-day-old body weight、119-day-old shank circumference( Figure 2 ) and shin length at 119 days of age ( Figure 3 ) are significantly correlated; specifically, Figure 1 This is a genome-wide association analysis of body weight at 42 days of age in a population of 1496 purebred Qingyuan Ma chicken hens; Figure 1 The a in is the QQ plot (Quantile-quantile plot), which represents the degree of fit between the actual observed value and the expected value; Figure 1The b in the figure is a Manhattan plot, which represents the correlation between all SNP sites on the autosomes of the whole genome (chromosomes 1-39) and the body weight at 42 days of age. The black dotted line is the potential significance threshold of the whole genome (-log10(1 / 2568220)=6.4096), and the red dotted line is the significance threshold of the whole genome (-log10(0.05 / 2568220)=7.7107). Figure 1 In the Manhattan plot b, the three most significant SNPs were Chr4:75408776 (p=3.90E-13; GRCg7b), Chr4:75422020 (p=2.01E-12; GRCg7b) and Chr4:75673592 (p=8.59E-12; GRCg7b), and the percentages of phenotypic variance explained were 3.42%, 3.22% and 3.04%, respectively. The Chr4:75408776 and Chr4:75422020 sites are both located in the LCORL gene region (Chr4:75407671-75427097; GRCg7b).

[0027] Figure 2 A genome-wide association analysis of shank circumference of 1496 purebred Qingyuan Ma chicken hens at 119 days of age; Figure 2 The a in is the QQ plot (Quantile-quantile plot), which represents the degree of fit between the actual observed value and the expected value; Figure 2 The b in the figure is a Manhattan plot, which represents the correlation between all SNP sites on the autosomes of the whole genome (chromosomes 1-39) and the tibia circumference at 119 days of age. The black dotted line is the potential significance threshold of the whole genome (-log10(1 / 2568220)=6.4096), and the red dotted line is the significance threshold of the whole genome (-log10(0.05 / 2568220)=7.7107). Figure 2 In the Manhattan plot b, the three most significant SNPs were Chr4:75402940 (p=2.47E-27; GRCg7b), Chr4:75408776 (p=1.43E-26; GRCg7b) and Chr4:75422020 (p=2.64E-25; GRCg7b), and the percentages of phenotypic variance explained were 7.27%, 7.07% and 6.74%, respectively. All three loci are located in the LCORL gene region (Chr4:75407671-75427097; GRCg7b).

[0028] Figure 3 A genome-wide association analysis of shank length in 1496 purebred Qingyuan Ma chicken hens at 119 days of age; Figure 3 The a in is the QQ plot (Quantile-quantile plot), which represents the degree of fit between the actual observed value and the expected value; Figure 3 The b in the figure is a Manhattan plot, which represents the correlation between all SNP sites on the autosomes of the whole genome (chromosomes 1-39) and tibia length and weight at 119 days of age. The black dotted line is the potential significance threshold of the whole genome (-log10(1 / 2568220)=6.4096), and the red dotted line is the significance threshold of the whole genome (-log10(0.05 / 2568220)=7.7107). Figure 3 In the Manhattan plot b, the three most significant SNPs were Chr4:75408776 (p=2.53E-18; GRCg7b), Chr4:75402940 (p=4.20E-17; GRCg7b) and Chr4:75422020 (p=1.44E-15; GRCg7b), and the percentages of phenotypic variance explained were 4.85%, 4.51% and 4.08%, respectively. All three loci are located in the LCORL gene region (Chr4:75407671-75427097; GRCg7b).

[0029] have Figure 1-Figure 3 It can be seen that the site Chr4:75408776 (GRCg7b) has a very high correlation with body weight at 42 days of age, shank circumference at 119 days of age and shank length at 119 days of age. Its significance with the three traits reached 3.90E-13, 1.43E-26 and 2.53E-18, respectively, and the phenotypic variation variance that can be explained reached 3.42%, 7.01% and 4.85%, respectively. The genetic effect of this site is a typical additive effect.

[0030] Then, the genotypes of the locus Chr4:75408776 corresponding to the body weight at 42 days of age, shank circumference at 119 days of age, and shank length at 119 days of age were counted respectively. The results are shown in Table 1.

[0031] Table 1 Phenotypes of body weight at 42 days, shank circumference at 119 days and shank length at 119 days corresponding to different genotypes of Chr4:75408776 (GRCg7b)

[0032]

[0033] Note: REF stands for reference allele and ALT stands for mutant allele.

[0034] As shown in Table 1, the genotype TT group > genotype TC group > genotype CC group. Individuals with the T allele have larger body weight, thicker shin circumference and longer shin length, which is a growth advantage allele.

[0035] Example 2: Design and synthesis of primers for detecting molecular markers of the LCORL gene associated with broiler growth traits

[0036] NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) was used to design the upstream and downstream primers for the amplification of site Chr4:75408776 (GRCg7b), where the upstream primer sequence is:

[0037] F: 5'-CCTCTTGCCAGCTGAAGGAA-3' (see SEQ ID NO. 1);

[0038] The downstream primer sequence is:

[0039] R: 5'-GGAGGCACAATGTTGCGTTT-3' (see SEQ ID NO. 2);

[0040] The upstream primers and downstream primers were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0041] Example 3: Application of primers for detecting molecular markers of LCORL genes associated with broiler growth traits in assisted selection breeding for improving broiler growth traits

[0042] 1) Extract blood samples from individual Qingyuan Ma chickens to be tested, and extract genomic DNA using the phenol-chloroform method;

[0043] 2) The above primers were used to perform PCR amplification on the genomic DNA of the chickens to be tested; the PCR reagents, methods and reaction procedures were all selected from Aikerui Biotechnology Co., Ltd. (Changsha, China), and the reaction system and reaction conditions are shown in Tables 2 and 3 below, respectively.

[0044] Table 2 PCR reaction system

[0045]

[0046] *1: When using 2X ProTaq Master Mix (dyeplus) for the first time, centrifuge it before use to avoid enzyme loss.

[0047] *2: Normally, the recommended template addition amount is no more than 500ng; the template amount can be adjusted according to actual needs.

[0048] *3: Primers are usually used at a final concentration of 0.2 μM, which can be adjusted within the range of 0.2 to 1.0 μM based on experimental results.

[0049] *4: The reaction system needs to be prepared on ice, and then placed in a PCR instrument for reaction.

[0050] Table 3 PCR reaction conditions

[0051]

[0052] After the PCR amplification reaction program was completed, the product was stored at 4°C for future use and sent to Shanghai Biotech Co., Ltd. for Sanger sequencing. The full length of the PCR amplification sequence is as follows:

[0053]

[0054] The 452nd base (shaded base Y; T / C mutation) is the site Chr4:75408776 (GRCg7b)-SNP marker; the genotyping of site Chr4:75408776 (GRCg7b) is as follows Figure 4 As shown in the figure, during Sanger sequencing, the red and blue peaks of TC heterozygotes may overlap, making it difficult to judge by the color of the peaks, but the overall peak height is only half of that of TT or CC homozygotes. Figure 4 As shown in a, when the individual Sanger sequencing peak graph is TT, the individual's weight at 42 days of age, shank circumference at 119 days of age, and shank length at 119 days of age are larger. Figure 4 As shown in b, when the individual Sanger sequencing peak is TC, the individual's weight at 42 days, shank circumference at 119 days, and shank length at 119 days are medium. Figure 4 As shown in c, when the individual Sanger sequencing peak is CC, the individual's 42-day-old weight, 119-day-old shank circumference and 119-day-old shank length are smaller. Therefore, in the breeding of yellow-feathered broilers, the genetic progress of early (42-day-old) weight, market-age (119-day-old) shank length and market-age (119-day-old) shank circumference selection can be accelerated by detecting this SNP marker and directional selection of TT genotype individuals as breeders, and at the same time, the group uniformity of 42-day-old weight, 119-day-old shank length and 119-day-old shank circumference of yellow-feathered broilers can be indirectly improved.

[0055] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for detecting the correlation between body weight, shin length and shin circumference of Qingyuan Ma chicken LCORL A primer for molecular marker of a gene, characterized in that include: Upstream primer, sequence F: 5′-CCTCTTGCCAGCTGAAGGAA-3′; Downstream primer, sequence: R: 5′-GGAGGCACAATGTTGCGTTT-3′; Among them, the specific chromosome position of the molecular marker site in the genome is determined after comparison with the chicken 7.0 reference genome GRCg7b as the reference genome. The molecular marker is Chr4_75408776, located at position 75408776 on chromosome 4 of the chicken genome, which is a T / C mutation.

2. An application of the primer according to claim 1 in assisted selection breeding for improving the weight, shank length and shank circumference of Qingyuan Ma chicken, characterized in that: The following steps are involved: S1. Extracting genomic DNA of the Qingyuan Ma chicken to be tested; S2. Perform PCR amplification on the genomic DNA of the chicken to be tested using the primers. After the PCR amplification reaction program is completed, perform Sanger sequencing on the product; and select individuals with the TT genotype at the molecular marker Chr4_75408776 site as breeder chickens.

3. The use according to claim 2, characterized in that: The PCR amplification reaction system is: 500 ng genomic DNA, 25 μL 2X Pro Taq Master Mix (dye plus), 1 μL upstream primer with a concentration of 0.2 μM, 1 μL downstream primer with a concentration of 0.2 μM, and enzyme-free sterile water is added to make the total reaction system 50 μl; the PCR reaction program is 94°C 30s; 98°C 10s, 60°C 30s, 72°C 1min, 35 cycles; 72°C 2min.

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