CN106434909A - Molecular marker method for predicting and identifying duck growth and abdomen fat percentages and application - Google Patents

Abstract

The invention provides a molecular marker method for predicting and identifying duck growth and abdomen fat percentages and application. A pair of primers are designed according to sequences of a duck VLDLR gene 5'UTR and a signal peptide coding region; duck genome DNA is subjected to PCR amplification through the primers, the amplification length is 374 bp, and sequencing is carried out after agarose gel electrophoresis detection. The polymorphisms, haplotypes and diplotypes of the VLDLR gene 5'UTR and the signal peptide coding region are judged according to the sequencing result, and the 8 haplotypes and the 15 diplotypes are established based on the 4 SNPs. The average weight of the diplotype H1H1 is highest, the abdomen fat percentage of the diplotype H5H7 is highest, and the abdomen fat percentage of the diplotype H1H1 is lowest. The diplotype H1H1 has a positive effect on the body weight, the diplotype H5H8 has a positive effect on the abdomen fat percentages, and the diplotype H1H1 and the diplotype H5H8 can be used as auxiliary molecular markers for selecting meat duck new varieties.

Description

A molecular marker method and application for predicting and identifying duck growth and abdominal fat rate

technical field

The invention belongs to the technical field of water fowl molecular biology, and in particular relates to a molecular marker method for predicting and identifying duck growth and abdominal fat rate, and the molecular marker method can be applied to marker-assisted selection breeding of new strains of domestic duck meat ducks.

Background technique

my country is the country with the largest number of ducks in the world. According to reports, my country's duck stock accounts for about 60% of the world's total duck stock, and the annual meat duck slaughter accounts for about 75% of the world's total meat duck slaughter. Although the amount of meat duck raised is less than that of broiler chickens, it still occupies an important position in the world's meat and poultry industry. It has developed very fast in the past ten years, especially in developing countries such as China and Southeast Asia. Meat ducks, like other poultry, have the advantages of high fecundity and rapid multiplication. The breeding has developed from the original breed selection to the commercial matching line breeding. The meat duck matching lines bred abroad include British Cherry Valley meat duck, Australia's Tego meat duck, American maple duck, Denmark's Hager meat duck, Lijia meat duck, French Aubaixing meat duck, Muscovy duck and Japan's Osaka meat duck, etc. my country's meat duck breed improvement and genetic breeding work started relatively late, and the scientific and systematic selection and breeding was only paid attention to and carried out after the 1950s. At present, Peking duck breeding has reached the advanced level in the world. The breeding of fine meat duck breeds in my country mainly adopts conventional breeding methods combined with molecular breeding methods, that is, through closed-group family breeding supplemented by DNA molecular marker technology to select and breed new meat duck strains with certain target traits. The current application of DNA molecular marker technology in poultry genetics and breeding is mainly reflected in genetic diversity analysis, germplasm identification, genetic map construction for kinship research, QTL mapping and molecular marker-assisted breeding. The breeding of new strains for the target traits of meat ducks mainly focuses on growth rate, feed utilization efficiency, carcass quality and reproductive performance. The heritability of growth rate is high and easy to measure, so the effect of individual selection is better. However, if we only pay attention to the results of growth rate selection, while promoting the rapid growth of meat ducks, it is easy to cause excessive fat deposition, reduce feed utilization and affect carcass quality. The main goals of carcass quality in meat ducks are to increase breast and leg meat yield and reduce carcass fat. Therefore, reducing the fat deposition of meat ducks and cultivating low-fat lean meat strains have become important breeding contents. With the development of molecular biology techniques, people can implement early selection and indirect selection through molecular markers by looking for major genes that control fat deposition or molecular markers linked to them. At present, although some progress has been made in the identification and application of duck fat deposition candidate genes, there are very few that can really be used to select low abdominal fat rate for breeding practice, so it is very necessary to find effective candidates that can affect duck fat deposition Genes and molecular markers.

Very low-density lipoprotein receptor (VLDLR) is a protein on the surface of the cell membrane, consisting of 846 amino acids, and is a receptor for various ligands such as ApoE-containing lipoprotein particles. VLDL receptor has five functional domains: ligand binding domain, epidermal growth factor precursor domain, glycosyl domain, transmembrane domain and intracellular domain. It is primarily responsible for binding and inwardly mobilizing apolipoproteins, such as very low-density lipoprotein (VLDL), to provide triglycerides as an energy source to extrahepatic tissues. Many studies have shown that very low-density lipoprotein receptors are abundant in tissues with active fatty acid metabolism, such as heart, skeletal muscle, and adipose tissue, and can selectively bind to apolipoprotein E and triglyceride-rich lipoproteins . Peroxisome proliferator-activated receptor (PPARα) is a central node among candidate genes for fat deposition, and studies have shown that PPARα can regulate the expression of VLDLR genes. Most of the research on VLDLR focuses on humans and mice. Studies have found that VLDLR is related to obesity and body weight. Mutations in human VLDLR genes can also lead to blood lipid metabolism disorders. Dyslipidemia is closely related to atherosclerosis, which is a cardiovascular and cerebrovascular disease. Therefore It has received extensive attention and in-depth research.

Contents of the invention

The purpose of the present invention is to provide a molecular marker method and application for predicting and identifying duck growth and abdominal fat rate. In the present invention, the pure strain duck of Gaoyou Duck Breeding Farm is used as the experimental object, and the polymorphism of the 5'UTR and signal peptide coding region of VLDLR gene is detected by PCR sequencing method, and the correlation between different genotypes and growth traits and abdominal fat rate is analyzed , in order to discover new nucleotide polymorphisms (SNPs) sites and effective molecular marker information that can affect growth and fat deposition, and breed meat ducks with fast growth and low abdominal fat rate, in order to cultivate high-quality meat ducks Strain lays the foundation.

The technical scheme that realizes the object of the present invention is:

A molecular marker method for predicting and identifying duck growth and abdominal fat rate, comprising the following steps:

Using the whole genome DNA of the duck to be tested containing the VLDLR gene as a template, use Primer5.0 to design primers, the amplification length is 374bp, and the sequences of the upstream and downstream primers are:

Upstream 5'-ATTACACTGCCAAATGACC-3';

Downstream 5'-CGGGAACTGGGATTCTTC-3';

After the product was detected by agarose gel electrophoresis, it was purified and bidirectionally sequenced to identify polymorphisms in the 5'UTR and signal peptide coding regions of the domestic duck VLDLR gene. The association analysis between the VLDLR gene haplotype combination and the growth rate and abdominal fat rate was carried out to find effective molecular marker information that can affect the growth rate and abdominal fat rate, so as to assist molecular markers to breed high-quality meat duck strains.

Further, the PCR amplification reaction system was 25 μL, and the reaction program was pre-denaturation at 94°C for 10 minutes, followed by 35 cycles (denaturation at 94°C for 30 seconds, annealing at 53.5°C for 30 seconds, extension at 72°C for 30 seconds), extension at 72°C for 10 minutes, and storage at 4°C. .

Further, the purified bidirectional sequencing uses the primers of the PCR reaction as the sequencing primers, and the PCR products are purified and sequenced, and the haplotype and haplotype frequency of the SNP site are counted using PHASE software, and the haplotype combination is constructed.

The sequencing results were compared with the NCBI sequence (HQ446852.1), and four polymorphic sites and their typing were obtained:

At the g.151 site, the frequency of the GA genotype was the highest at 0.56, the frequency of the AA genotype was the lowest at 0.04, and the allele frequency was G>A;

At the g.170 locus, the CC genotype has the highest frequency of 0.50, the CT genotype has the lowest frequency of 0.22, and the allele frequency is C>T;

At the g.206 site, the frequency of the AG genotype was the highest at 0.50, the frequency of the GG genotype was the lowest at 0.17, and the allele frequency was A>G;

At the g.278-295 site, the frequency of the DD type was the highest at 0.65, and the frequency of the DB type was the lowest at 0.11, and the allele frequency was D>B.

Using PHASE2.0 software, the haplotypes and diplotypes of the four polymorphic sites were constructed. A total of 8 haplotypes were constructed at 4 polymorphic sites, namely H1 (GTAB), H2 (ATAD), H3 (GTGD), H4 (GCAD), H5 (GCGD), H6 (ATGD), H7 (ACAD ), H8 (ACGD). Among them, H8(A-C-G-D) was the most important haplotype, with a frequency as high as 29.81%, and the frequencies of H3(GTGD) and H7(ACAD) were the lowest, both at 0.96%. 15 diplotypes were constructed based on 8 haplotypes, and H4H8 had the highest frequency of 28.16%. Among the 15 diplotypes, except for H1H1, H5H8, H2H8, and H4H8, the frequencies of other diplotypes were all lower than 5%, so correlation analysis was not performed.

The VLDLR gene molecular marker related to duck growth and fat of the present invention can be applied to duck marker-assisted selection breeding.

Compared with the prior art, the present invention has the remarkable advantages of:

(1) The polymorphism of VLDLR was confirmed to be related to growth and fat deposition in ducks.

(2) Through the correlation analysis of polymorphisms in the 5'UTR and signal peptide coding regions of duck VLDLR gene and growth and abdominal fat rate, it laid the foundation for breeding high-quality meat duck strains.

Description of drawings

Figure 1 is the result of amplified fragments of duck VLDLR gene 5'UTR and signal peptide coding region.

Figure 2 is the genotype map of polymorphic sites in the 5'UTR and signal peptide coding regions of duck VLDLR gene.

detailed description

In order to better understand the present invention, the technical solution of the present invention will be specifically described below through specific examples.

Example 1

1 Experimental materials and methods

1.1 Experimental animals

The ducks used in the experiment were Gaoyou ducks, which were uniformly hatched and hatched in the Gaoyou duck breeding base, with a total of 267 ducks. The test ducks were fed, managed and immunized according to conventional methods. They were raised on the ground until the 10th week, and there were pools and playgrounds during the feeding period.

1.2 Determination of growth performance

At 0, 3, 4, 5, 6, 7, 8, 9, and 10 weeks of age, weigh on an empty stomach on the first morning of each age, and record the body weight of Gaoyou ducks at the age of one week. The blood samples of Gaoyou ducks were taken and preserved after anticoagulation, and slaughtered after measuring the live weight to measure the carcass performance indicators. Slaughter measurements were carried out in accordance with the uniform standards of the National Poultry Breeding Committee, and the eviscerated weight, half eviscerated weight, and abdominal fat weight were measured, and the abdominal fat rate was calculated. Abdominal fat rate=abdominal fat weight/(whole eviscerated weight+abdominal fat weight).

1.3 DNA extraction

Genomic DNA was extracted by conventional phenol/chloroform extraction. Use trace ultraviolet spectrophotometry to detect the concentration and purity of DNA, and the A260/A280 must be between 1.6 and 1.9.

1.4PCR amplification

According to the duck VLDLR gene sequence published in Genbank, Primer5.0 was used to design primers, the amplified length was 374bp, and the amplified fragment included the 5'UTR of VLDLR gene and all signal peptide regions.

The upstream and downstream primer sequences are:

Upstream 5'-ATTACACTGCCAAATGACC-3'

Downstream 5'-CGGGAACTGGGATTCTTC-3'

The PCR amplification reaction system was 25 μL, and the reaction program was pre-denaturation at 94°C for 10 min, followed by 35 cycles (denaturation at 94°C for 30 S, annealing at 53.5°C for 30 S, extension at 72°C for 30 S), extension at 72°C for 10 min, and storage at 4°C.

1.5 Sequencing and polymorphism detection

The PCR products were detected on a gel imager after 1% agarose gel electrophoresis and Goldview staining, and the high-yield and specific PCR products were sent to Huada Gene Co., Ltd. for purification and bidirectional sequencing; DNAMAN software was used to combine the sequencing images Perform polymorphism detection and analysis on the sequencing results to accurately determine SNPs.

1.6 Statistical methods

Allele frequencies were calculated using the chi-square test for allelic Hardy-Weinberg equilibrium.

According to the abdominal fat rate and the characteristics of the tested duck group, a linear regression model is established: Y=μ+G+e, wherein: Y is the measured value of the character; μ is the group mean; G is the genotype effect; e is the random residual .

PHASE2.0 software was used to construct haplotypes and diplotypes and calculate their frequency.

Popgen32 and PIC_CALC software were used to calculate genetic heterozygosity (He), effective allele number (Ne) and polymorphic information content (PIC).

Statistical analysis using SPSS15.0 software, each genotype body weight and abdominal fat rate expressed as mean ± standard error.

The linkage disequilibrium analysis of SNPs loci was analyzed by SHEsis software and r2 was calculated.

2 Results and Analysis

2.1 Amplification results

The designed primers were used to amplify Gaoyou duck genomic DNA by PCR, and the product was detected by 1% agarose gel electrophoresis, and a clear band appeared between 500bp-250bp, which was consistent with the expected fragment size (374bp), and there was no Non-specific amplification, the results are shown in Figure 1.

2.2 Calculation of allele frequencies

The genotype frequencies and allele frequencies of the 4 polymorphic sites in the 5'UTR and signal peptide coding regions of the VLDLR gene are shown in Table 1. It can be seen from Table 1 that at the g.151 site, the GA genotype has the highest frequency, and the allele frequency is G>A; at the g.170 site, the CC genotype has the highest frequency, and the allele frequency is C>T ; At the g.206 locus, the frequency of the AG genotype is the highest, and the allele frequency is A>G; at the g.278-295 locus, the frequency of the DD genotype is the highest, and the allele frequency is D>B. Genetic heterozygosity (He), effective number of alleles (Ne) and polymorphic information content (PIC) are important parameters to reflect the genetic characteristics of a population. As can be seen from Table 1, He (genetic heterozygosity) and Ne (effective number of alleles) at g. ) and Ne (effective number of alleles) were the lowest at 0.42 and 1.71; the PIC (polymorphic information content) at the g.206 site was the highest at 0.37, and the g.278-295 site was the lowest at 0.33. By Chi-square test, except the g.206 locus conformed to the Hardy-Weinberg genetic equilibrium state (p>0.05), the other three loci did not conform to the Hardy-Weinberg genetic equilibrium state (p<0.05).

Table 1 Gene frequency and balance test of 5'UTR and signal peptide coding region of VLDLR gene in Gaoyou duck population (n=267)

The expression method in the genotype frequency: frequency/number (genotype); the expression method in the allele frequency: allele frequency (allele)

2.3 Haplotype, diplotype construction and their frequency

Using PHASE2.0 software, haplotypes and diplotypes were constructed for the 4 polymorphic sites, and the corresponding frequencies were analyzed. The results are shown in Table 2 and Table 3. A total of 8 haplotypes were constructed for the 4 polymorphic sites Types are H1 (GTAB), H2 (ATAD), H3 (GTGD), H4 (GCAD), H5 (GCGD), H6 (ATGD), H7 (ACAD), H8 (ACGD). Among them, H8(A-C-G-D) was the most important haplotype, with a frequency as high as 29.81%, and the frequencies of H3(GTGD) and H7(ACAD) were the lowest, both at 0.96%. 15 diplotypes were constructed based on 8 haplotypes, and H4H8 had the highest frequency of 28.16%. Among the 15 diplotypes, except for H1H1, H5H8, H2H8, and H4H8, the frequency of other diplotypes was lower than 5%, and correlation analysis was not performed.

Table 2 Haplotypes and their frequencies of 4 polymorphic loci

Table 3 Partial diplotypes and their frequencies of the 4 polymorphic loci

2.4 Analysis of diplotype on growth performance and abdominal fat rate

Table 4 lists the correlations between some haplotype combinations with significant or extremely significant differences and growth traits and abdominal fat rate.

It can be seen from Table 4 that there was no significant difference in the body weight of each diplotype between 0 and 5 weeks of age (P>0.05); at 6 weeks of age, the body weight of the H1H1 diplotype was the highest, and that of the H2H8 diplotype was the lowest , the difference between the two was significant (P<0.05); at the age of 7 to 10 weeks, the body weight of the H1H1 diplotype was the highest and extremely significantly higher than that of the H2H8 diplotype (P<0.01) and significantly higher than that of the H4H8 diplotype ( P<0.05), but there was no significant difference between the body weight of H2H8 diplotype and H4H8 diplotype (P>0.05). There was no significant difference among the diplotypes for the slaughter rate, half-eviscerated rate and fully eviscerated rate, but the abdominal fat rate of the H5H8 type was significantly higher than that of the H1H1 diplotype and the H2H8 double-type Plotype (P<0.01), significantly higher than H4H8 diplotype (P<0.05), the abdominal fat rate of H4H8 diplotype was significantly higher than that of H1H1 diplotype (P<0.05), H1H1 diplotype and H2H8 diplotype There was no significant difference between the types (P>0.05). The results of the least square analysis showed that the average body weight of the H1H1 diplotype was the highest, and that of the H2H8 diplotype was the lowest; that of the H5H8 diplotype was the highest, followed by that of the H4H8 diplotype, and that of the H1H1 diplotype was the lowest. The diplotype H1H1 has a positive effect on body weight, and the H5H8 diplotype has a positive effect on abdominal fat rate, which can be used as auxiliary molecular markers for selecting new strains of meat ducks.

Table 4 Association analysis of diplotypes of 4 SNP loci with growth traits and abdominal fat rate

Claims (5)

1. a molecular marker method for predicting and identifying duck growth and abdominal fat rate, is characterized in that, comprises the following steps: Using the whole genome DNA of the duck to be tested containing the VLDLR gene as a template, use Primer5.0 to design primers, the amplification length is 374bp, and the sequences of the upstream and downstream primers are: Upstream 5'-ATTACACTGCCAAATGACC-3'; Downstream 5'-CGGGAACTGGGATTCTTC-3'; After the product was detected by agarose gel electrophoresis, it was purified and bidirectionally sequenced, and the polymorphisms in the 5'UTR and signal peptide coding regions of the duck VLDLR gene were identified by sequencing; The association analysis between duck VLDLR gene haplotype combinations and slaughter traits was carried out to find effective molecular marker information that can affect growth rate and abdominal fat rate, and then used to breed new strains of high-quality meat ducks. 2. The molecular marker method for detecting duck growth and abdominal fat rate according to claim 1, characterized in that, the PCR amplification reaction system is 25 μL, and the reaction program is 94 ° C pre-denaturation for 10 min, and then 35 cycles (denaturation at 94°C for 30S, annealing at 53.5°C for 30S, extension at 72°C for 30S), extension at 72°C for 10 min, and storage at 4°C. 3. the molecular marker method for detecting duck growth and abdominal fat rate according to claim 1, is characterized in that, described purifying two-way sequencing is with the primer of PCR reaction as sequencing primer, carries out purifying sequencing with PCR product, uses The PHASE software counts the haplotypes and haplotype frequencies of SNP sites and constructs haplotype combinations. 4. the molecular marker method for detecting duck growth and abdominal fat rate according to claim 1, is characterized in that, said duck VLDLR gene 5'UTR and signal peptide coding region polymorphism result this VLDLR gene has 4 Polymorphic sites: g.151, g.170, g.206, g.278-295; At the g.151 site, the frequency of the GA genotype was the highest at 0.56, the frequency of the AA genotype was the lowest at 0.04, and the allele frequency was G>A; At the g.170 locus, the CC genotype has the highest frequency of 0.50, the CT genotype has the lowest frequency of 0.22, and the allele frequency is C>T; At the g.206 site, the frequency of the AG genotype was the highest at 0.50, the frequency of the GG genotype was the lowest at 0.17, and the allele frequency was A>G; At the g.278-295 site, the frequency of the DD type was the highest at 0.65, and the frequency of the DB type was the lowest at 0.11, and the allele frequency was D>B. 5. The application of the molecular marker method for detecting duck growth and abdominal fat rate described in any one of claims 1-4 in the process of duck breeding for marker-assisted selection.