CN114150070A - SNP molecular marker related to chicken growth and slaughter traits, detection primer, kit and breeding method - Google Patents
SNP molecular marker related to chicken growth and slaughter traits, detection primer, kit and breeding method Download PDFInfo
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Abstract
The invention relates to an SNP molecular marker, a detection primer, a kit and a breeding method related to chicken growth and slaughter traits. According to the invention, through research, 1 SNP locus (A-T, Chr 4: 76224904, IVS2-21661) on the 50139 th (namely-21661) th base of the second intron region of the chicken LDB2 gene is found to be obviously related to chicken growth and slaughter traits. The allele A of the SNP locus is positively correlated with the high weight trait, the allele T is positively correlated with the low weight trait, and the phenotype trait of the heterozygous allele AT is in an intermediate value. The SNP locus can be used as a molecular marker for genetic improvement of chicken varieties, the genotype of the SNP molecular marker is detected, and the weight of the chicken in the mature period can be effectively predicted in an early stage, so that the breeding time is shortened, and the breeding process is accelerated. The invention provides a primer and a kit for detecting SNP molecular markers of LDB2 genes, and provides a chicken growth and slaughter trait improvement breeding method based on the SNP molecular markers of LDB2 genes, which can be used for breeding high-weight or large-size chicken varieties.
Description
Technical Field
The invention relates to SNP molecular markers, detection primers, a kit and a breeding method related to chicken growth and slaughter traits, and belongs to the technical field of biological breeding.
Background
Meat is the most valuable animal product, and although the consumption of meat in developed countries is relatively constant, the annual consumption of meat in the developing countries has doubled since 1980. The growth of populations and income, as well as the change in food preferences, has increased the demand for animal products. Throughout the world, the chicken is the second meat consumer in the world next to pork, the proportion of the chicken consumption in the total meat consumption is about 35% at most (37% of pork), and the total chicken consumption is still increased by about 1.6% (more than 1.1% of pork) every year in the period of 2012 and 2014 (http:// www.fao.org/ag/againfo/the mes/en/mean/background). The growth traits such as body weight are the main economic traits of livestock and poultry, and how to improve the economic traits of local livestock and poultry varieties under the condition of not changing the germplasm characteristics is an urgent problem to be solved for maintaining the sustainable development of local livestock and poultry. Therefore, there is a need to develop new genetic markers to accelerate the genetic improvement process by means of molecular biology, thereby shortening the breeding time and accelerating the breeding process.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to chicken growth and slaughter traits.
Secondly, the invention aims to provide the application of the SNP molecular marker in chicken growth and slaughter trait improvement breeding.
Meanwhile, the invention aims to provide a primer and a kit for detecting the genotype of the SNP molecular marker.
Finally, the invention aims to provide a method for improving breeding of chicken growth and slaughter traits.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an SNP molecular marker related to chicken growth and slaughter traits, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.1, and the 140 th base from the 5' end is A or T.
The invention provides application of the SNP molecular marker in chicken growth and slaughter trait improvement breeding, wherein the genotype of the SNP molecular marker is detected, when the genotype of the SNP molecular marker is AA, the chicken to be detected is a high-weight large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight small-size individual.
The invention provides a primer for detecting the genotype of the SNP molecular marker, and the nucleotide sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.
The invention provides a kit for detecting the genotype of the SNP molecular marker, which comprises a primer pair shown as SEQ ID NO.2 and SEQ ID NO.3 and a restriction endonuclease ApoI.
Preferably, the kit further comprises one or more of dNTPs, PCR reaction buffer solution and DNA polymerase.
The detection primer and the kit can be used for improving chicken germplasm resources.
The invention provides a method for improving breeding of chicken growth and slaughter traits, which comprises the following steps: taking the genome DNA of a chicken to be detected as a template, carrying out PCR amplification by using primers shown in SEQ ID NO.2 and SEQ ID NO.3, carrying out enzyme digestion reaction on a PCR product by using ApoI endonuclease, wherein if the enzyme digestion product is 256bp, the base AT a mutation position is homozygous AA type, if the enzyme digestion product is 136/120bp, the base AT the mutation position is homozygous TT type, and if the enzyme digestion product is 256/136/120bp, the base AT the mutation position is heterozygous AT type; when the genotype of the SNP molecular marker is AA, the chicken to be detected is a high-weight large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight small-size individual; selecting chicken individuals with AA genotypes.
Preferably, the reaction system for PCR amplification is: 2 × Taq PCR MasterMix 10. mu.L, ddH2O6. mu.L, upstream primer P-F1. mu.L, downstream primer P-R1. mu.L, DNA template 2. mu.L.
Preferably, the reaction procedure of the PCR amplification is as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 15s, 65 ℃ for 15s and 72 ℃ for 5 s; preserving at 72 deg.C for 5min and 4 deg.C.
Preferably, the system of the enzyme digestion reaction is 25uL, and comprises 22.5uL of PCR products, 10 xBuffer 2uL and 0.5uL of ApoI.
Preferably, the size of the enzyme-digested product is detected by agarose gel electrophoresis to determine the genotype of the SNP molecular marker.
Preferably, the agarose gel electrophoresis detection adopts agar with the mass fraction of 2.0%, the voltage of 120V and the electrophoresis time of 30 min.
The chicken growth character improvement breeding method based on the SNP molecular marker of the chicken LDB2 gene can be applied to breeding high-weight or large-size chicken species.
The invention has the beneficial effects that:
the invention provides an SNP molecular marker related to chicken growth and slaughter traits. First, 766 phenotypic data were recorded for complete solid-ampere card F2Performing genome-wide association analysis on resource group chickens as research objects, and recording detailed F of phenotype2Large-scale SNP typing work was performed on the 50139 (i.e., -21661) th base of the 2 nd intron of the LDB2 gene of the resource group to verify the accuracy of the above SNP sites. The invention proves that 1 SNP molecular marker which is located in 50139 (namely-21661) th base of the 2 nd intron region of chicken LDB2 gene has a nucleotide sequence shown as SEQ ID NO.1, and the 140 th base from the 5' end is A or T; (A-T, Chr 4: 76224904, IVS2-21661), is significantly associated with chicken growth traits (body weight, shank length, oblique body length). The allele T of the SNP locus is positively correlated with the low weight trait, the allele A is positively correlated with the high weight trait, and the phenotype trait of the heterozygous allele AT is in an intermediate value. The weight is an important economic character of the chicken, the length of the shank and the body slant length are strongly related to the size of the chicken body, and the SNP locus lays a foundation for screening out homozygous high-quality local chicken groups.
The SNP molecular marker genotype is detected, the weight of the chicken in the mature period can be effectively predicted in an early stage, and the SNP molecular marker can be used as a molecular marker for genetic improvement of chicken varieties, so that the breeding time is shortened, the breeding process is accelerated, and the SNP molecular marker has great economic application value and scientific research value. The invention provides application of the SNP molecular marker in chicken growth and slaughter trait improvement breeding.
The invention provides a primer and a kit for detecting the genotype of the SNP molecular marker, which can be used for improving chicken germplasm resources.
The invention further provides a chicken growth character improvement breeding method based on the SNP molecular marker of the chicken LDB2 gene, which comprises the following steps: primers are designed at the upstream and downstream of a 50139 (namely-21661) th base SNP site of a 2 nd intron region of the chicken LDB2 gene, and the genotype of the SNP molecular marker is detected by using ApoI enzyme and a PCR-RFLP method. The invention provides a primer nucleotide sequence, a PCR amplification reaction system and reaction conditions, a PCR amplification program, an enzyme digestion reaction system and enzyme digestion product electrophoresis conditions, and defines the corresponding relation between the SNP molecular marker genotype and the chicken growth traits, namely when the SNP molecular marker genotype is AA, the chicken to be detected is an individual with high weight, long shin length and oblique body length, and when the SNP molecular marker genotype is TT, the chicken to be detected is an individual with low weight, long shin length and oblique body length. Selecting chicken individuals with AA homozygous genotype to breed, and discarding chicken individuals with TT and AT genotypes to obtain chicken germplasm resources with excellent growth characteristics of high weight and large body type. The method has strong operability, does not need special instruments, is easy to popularize, can predict the weight and the body size of the chicken quickly and effectively at early stage with low cost, can be used for auxiliary selection and molecular breeding of the chicken, and has wide application prospect in the aspect of improving the germplasm resources of the chicken.
Drawings
FIG. 1 is a diagram of the sequence peaks at the positions of mutations of different genotypes;
FIG. 2 is a diagram showing the result of agarose electrophoresis after PCR amplification of LDB2 gene containing SNP molecular marker;
FIG. 3 is a schematic diagram of an ApoI endonuclease assay;
FIG. 4 is a diagram of the results of agarose electrophoresis after digestion with three genotype ApoI enzymes.
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples are, unless otherwise specified, conventionally available commercially.
Example 1: acquisition of SNP molecular marker related to chicken growth and slaughter traits
Complete Coumat-Anka F with 766 phenotypic data records2Whole genome association of resource group chickens as research objectsAnd (6) analyzing. Gushi-Anka F constructed by research center for germplasm resource innovation of poultry in Henan province2The resource group consists of 4 orthogonal families (Anka cock multiplied by the fixed hen) and 3 reverse families (fixed cock multiplied by the Anka hen), and the economic character determination method comprises the following steps: body weight was measured every 2 weeks, body ruler indices were measured every 4 weeks, and slaughter was given to 12 weeks of age. The material is stopped for 12 hours before slaughtering (without stopping water), and then weighed. The measured growth, slaughtering index and meat index totally have 57 indexes. (see Han R L, Li Z J, Li M J, et al. Novel9-bp index in visfatin gene and its associations with a chip growth. Br Poult Sci,2011,52(1): 52-57.). The present study utilized GBS-double enzyme simplified genome sequencing to genotype 766 chickens of this resource group, quality control and remove SNPs on the sex chromosome, with a total of 321314 SNPs for 734 chickens. Based on genotyping data and phenotypic data, carrying out whole genome correlation analysis on the body weights (2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks) of different weeks of age of the resource group, the growth traits such as shin length, shin circumference, oblique body length and sternum length of 4 weeks, 8 weeks and 12 weeks and the slaughter trait of 12 weeks, and finding that 1 SNP molecular marker exists in the 50139 (namely-21661) th base of the 2 nd intron region of the chicken LDB2 gene, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the 140 th base from the 5' end is A or T; (A-T, Chr 4: 76224904, IVS2-21661), which are significantly related to the growth and slaughter traits of chickens such as 12 weeks body weight, 12 weeks shank length, 12 weeks shank circumference, 12 weeks oblique body length, 12 weeks carcass weight, half bore weight, full bore weight, leg weight, etc. (Table 1).
TABLE 1 Whole genome Association of this SNP with Guart-Anka Chicken F2Multiple traits of resource groups are obviously related
Note: log in Whole genome Association analysis10(P)>5.72 indicates that significant levels were achieved.
Record a detailed F for the form2The 50139 (i.e., -21661) position of the 2 nd intron of the LDB2 gene of the resource group is subjected to large-scale SNP enzyme digestion typing work to verify the accuracy of the SNP sites, and thenOne-step SNP typing results and F2The correlation analysis of economic traits of the resource group shows that the molecular marker is closely related to a plurality of growth traits of chickens (Table 2). The average body weight, shin length, shin girth, oblique body length, carcass weight, half-bore weight and full-bore weight of the AA-gene type population are slightly higher than those of the AT population, and are obviously higher than those of the TT type population, such as the shin length, the shin girth, the body oblique length, the carcass weight, the half-bore weight and the full-bore weight. The weight is an important economic character of the chicken, the shank length and the body slant length are strongly related to the size of the chicken body, the dressing percentage and the total bore ratio are main indexes for measuring the meat production performance of the livestock and poultry, and the results further prove the correlation analysis results: the SNP locus of the A-T mutation (shown in figure 1) of the 50139 (namely-21661) th base of the 2 nd intron region of the chicken LDB2 gene is obviously related to the chicken growth traits and slaughter traits, wherein allele A is positively related to the high weight traits, and allele T is positively related to the low weight traits. When the genotype of the SNP molecular marker is AA, the chicken to be detected is a high-weight large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight small-size long individual.
TABLE 2A begin-an card F2Phenotypic statistics corresponding to different genotypes of a resource population
TABLE 2B begin-Anka F2Phenotypic statistics corresponding to different genotypes of a resource population
Note: p <0.01 indicates that a very significant level was reached.
Example 2: primer for detecting SNP molecular marker genotype of chicken LDB2 gene
This example provides primers for detecting SNP molecular markers of the chicken LDB2 gene obtained in example 1, and the sequences of the primers are shown in Table 3:
TABLE 3 SNP detection primers for LDB2 gene
Example 3: kit for detecting SNP molecular marker genotype of chicken LDB2 gene
The kit in the embodiment comprises a primer for detecting the SNP molecular marker of the chicken LDB2 gene obtained in the embodiment 1 and a restriction endonuclease ApoI, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3. In addition, the kit also comprises one or more of dNTPs, PCR reaction buffer solution and DNA polymerase.
Example 4: chicken growth and slaughter trait improving breeding method
Based on the genomic information of the SNP molecular marker obtained in example 1 and the chicken LDB2 gene sequence published by NCBI (ENSGALG00000014485), primers were designed to amplify a partial sequence of the 2 nd intron region of LDB2 gene, and the nucleotide sequences of the primers are shown as SEQ ID NO.2 and SEQ ID NO. 3.
Firstly, taking the genome DNA of a chicken to be detected as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO.2 and SEQ ID NO. 3:
the reaction system of PCR amplification is as follows: 2 × Taq PCR MasterMix 10. mu.L, ddH2O6 mu L, upstream primer P-F1 mu L, downstream primer P-R1 mu L and DNA template 2 mu L; the PCR amplification product fragment size was 256bp (FIG. 2).
The reaction procedure for PCR amplification was: 5min at 95 ℃; 35 cycles of 95 ℃ for 15s, 65 ℃ for 15s and 72 ℃ for 5 s; preserving at 72 deg.C for 5min and 4 deg.C.
Secondly, using Restriction Fragment Length Polymorphism (RFLP) to perform enzyme digestion identification on the PCR product by using ApoI endonuclease: the base at the position can be cut by ApoI when being T, and can not be cut by ApoI when being A, thereby being used for typing (as shown in FIG. 3).
The system of the enzyme digestion reaction is 25uL, and comprises 22.5uL of PCR products, 10 XBuffer 2uL and Bsp1286I 0.5.5 uL (see Table 4);
TABLE 4 LDB2 digestion system
Finally, detecting the size of the enzyme digestion product through agarose gel electrophoresis to judge the genotype of the SNP molecular marker; the agarose gel electrophoresis detection adopts 2.0% of agar by mass, the voltage is 120V, and the electrophoresis time is 30 min;
if the enzyme digestion product is 256bp, the base AT the mutation position is homozygous AA type, if the enzyme digestion product is 136/120bp, the base AT the mutation position is homozygous TT type, and if the enzyme digestion product is 256/136/120bp, the base AT the mutation position is heterozygous AT type; when the genotype of the SNP molecular marker is AA, the chicken to be detected is a high-weight large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight small-size individual; selecting chicken individuals with AA homozygous genotype to breed, and discarding chicken individuals with TT and AT genotypes to obtain chicken germplasm resources with excellent growth characteristics of high weight and large body type.
The results of agarose electrophoresis after digestion with three genotype ApoI are shown in FIG. 4, in lane 1: DNA Marker (700, 600, 500, 400, 300, 200 and 100 bp); lanes 2, 3 and 4 are a band with a size of 256bp, and the result is named genotype homozygous AA type; lanes 5, 6 and 7 are two bands with sizes of 136 and 120bp, and are named as genotype homozygous TT type; lanes 8, 9 and 10 are three bands with band sizes of 256, 136 and 120bp, respectively, and are designated as genotype heterozygous AT types; as can be seen from FIG. 3, individuals 2, 3 and 4 were high-weight, large-sized chickens, which were reserved for breeding; 5. 6, 7, 8, 9 and 10 individuals are low-weight and small-size chickens and need to be discarded; based on the gene typing result of the LDB2 gene SNP molecular marker, breeding high-weight and large-size chicken breeds.
<110> Henan university of agriculture
<120> SNP molecular marker, detection primer, kit and breeding method related to chicken growth and slaughter traits
<160> 3
<170> PatentIn version 3.5
<211> 256
<212> DNA
<213> Chicken
<221> PCR amplification sequence of LDB2 Gene
<222> 140 (n is a or t)
<400> 1
ttgttggctg tagaaggagt gtttctagta tttgaagcat attgtgtaca tcattattta 60
agacagatca accaatttcc aatatgtttg tttccgaaag aatgagtcct atagctttaa 120
aaagcaagct ggtgaaaatn ctaataattt gcagtgctgc tgaaaataaa gaggtctttt 180
ggttgcatca ttagaaagac tgttgtcaca tagctagact gatgagttat tgcatggagt 240
gctgtttgct gtttac 256
<211> 23
<212> DNA
<213> Artificial sequence
<221> upstream primer P-F
<400> 2
ttgttggctg tagaaggagt gtt 23
<211> 23
<212> DNA
<213> Artificial sequence
<221> downstream primer P-R
<400> 3
gtaaacagca aacagcactc cat 23
Claims (10)
1. An SNP molecular marker related to chicken growth and slaughter traits is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.1, and the 140 th base from the 5' end is A or T.
2. The use of the SNP molecular markers according to claim 1 for chicken growth and slaughter trait improvement breeding.
3. The use of claim 2, wherein the genotype of the SNP molecular marker is detected, and when the genotype of the SNP molecular marker is AA, the chicken to be tested is a high-weight and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be tested is a low-weight and small-size individual.
4. The primer for detecting the genotype of the SNP molecular marker according to claim 1, wherein the nucleotide sequence is shown as SEQ ID No.2 and SEQ ID No. 3.
5. Kit for detecting the genotype of the SNP molecular marker according to claim 1, comprising a primer pair shown as SEQ ID No.2 and SEQ ID No.3, and an ApoI restriction endonuclease.
6. The kit of claim 5, wherein the kit further comprises one or more of dNTPs, PCR reaction buffer, and DNA polymerase.
7. Use of the primer according to claim 4 or the kit according to claim 5 for improving chicken germplasm resources.
8. A chicken growth and slaughter trait improvement breeding method is characterized by comprising the following steps: taking the genome DNA of a chicken to be detected as a template, carrying out PCR amplification by using primers shown in SEQ ID NO.2 and SEQ ID NO.3, carrying out enzyme digestion reaction on a PCR product by using ApoI endonuclease, wherein if the enzyme digestion product is 256bp, the base AT a mutation position is homozygous AA type, if the enzyme digestion product is 136/120bp, the base AT the mutation position is homozygous TT type, and if the enzyme digestion product is 256/136/120bp, the base AT the mutation position is heterozygous AT type; when the genotype of the SNP molecular marker is AA, the chicken to be detected is a high-weight large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight small-size individual; selecting and keeping the chicken individuals with the AA homozygous genotype.
9. The method for improving and breeding chicken growth traits of claim 8, which is characterized by comprising the following steps: the reaction system of the PCR amplification is as follows: 2 × Taq PCR MasterMix 10. mu.L, ddH2O6. mu.L, upstream primer P-F1. mu.L, downstream primer P-R1. mu.L, DNA template 2. mu.L.
10. The method for improving and breeding chicken growth traits of claim 8, which is characterized by comprising the following steps: the enzyme digestion reaction system is 25uL, and comprises 22.5uL of PCR products, 10 XBuffer 2uL and 0.5uL of ApoI.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838809A (en) * | 2022-09-19 | 2023-03-24 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken slaughter trait and method for breeding slaughter-processing new strain |
| CN116179714A (en) * | 2022-09-19 | 2023-05-30 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken slaughtering and meat quality characteristics and breeding method of high-quality slaughtering and processing type novel variety |
| CN115838809B (en) * | 2022-09-19 | 2023-12-15 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken slaughter traits and breeding method of new slaughter processing type strain |
| CN116179714B (en) * | 2022-09-19 | 2023-12-15 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken slaughtering and meat quality characteristics and breeding method of high-quality slaughtering and processing type novel variety |
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