CN114150071A - Application of SNP molecular marker of chicken TRIM13 gene in chicken growth and slaughter trait improvement breeding and breeding method - Google Patents
Application of SNP molecular marker of chicken TRIM13 gene in chicken growth and slaughter trait improvement breeding and breeding method Download PDFInfo
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Abstract
The invention relates to application of a chicken TRIM13 gene SNP molecular marker in chicken growth and slaughter trait improvement breeding and a breeding method, and belongs to the technical field of biological breeding. According to the invention, through research, 1 SNP locus (T-C, rs732405671) of 87 th base of 3 rd intron region of chicken TRIM13 gene is found to be obviously related to chicken growth traits and slaughter traits. The allele T of the SNP locus is positively correlated with the low weight trait, the allele C is positively correlated with the high weight trait, and the phenotype trait of the heterozygous allele CT is in an intermediate value. 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, and the breeding process is accelerated. The invention provides a chicken growth and slaughter trait improvement breeding method based on SNP molecular markers of a chicken TRIM13 gene, which can be used for breeding high-weight or large-size chicken varieties.
Description
Technical Field
The invention relates to application of a chicken TRIM13 gene SNP molecular marker in chicken growth and slaughter trait improvement breeding and a breeding method, 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 application of a chicken TRIM13 gene SNP molecular marker in chicken growth and slaughter trait improvement breeding.
Another purpose of the invention is to provide a method for improving breeding of chicken growth and slaughter traits based on SNP molecular markers of the TRIM13 gene, which can be used for breeding high-weight or large-size chicken species.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of SNP molecular markers of a TRIM13 gene of a chicken in chicken growth and slaughter trait improvement breeding, wherein the nucleotide sequence of the SNP molecular markers is shown as SEQ ID NO.1, and the 123 th base from the 5' end is C or T; the application is to detect the genotype of the SNP molecular marker of the TRIM13 gene of the chicken, when the genotype of the SNP molecular marker is CC, 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.
Specifically, the kit is used for detecting the genotype of the SNP molecular marker of the chicken TRIM13 gene.
The kit comprises primer pairs shown as SEQ ID NO.3 and SEQ ID NO.4 and restriction endonuclease Bsp 1286I.
Preferably, the kit further comprises one or more of dNTPs, PCR reaction buffer solution and DNA polymerase.
The invention provides a chicken growth and slaughter trait improvement breeding method based on SNP molecular markers of a chicken TRIM13 gene, 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.3 and SEQ ID No.4, carrying out enzyme digestion reaction on a PCR product by using Bsp1286I endonuclease, wherein if the enzyme digestion product is 189bp, the base at a mutation position is homozygous TT type, if the enzyme digestion product is 122/67bp, the base at the mutation position is homozygous CC type, and if the enzyme digestion product is 189/122/67bp, the base at the mutation position is heterozygous CT type; when the genotype of the SNP molecular marker is CC, 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 CC genotype.
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, 60 ℃ 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 Bsp1286I 0.5.5 uL.
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 and slaughter trait improvement breeding method based on the SNP molecular marker of the chicken TRIM13 gene can be applied to breeding high-weight or large-size chicken species.
The invention has the beneficial effects that:
the invention provides an application of SNP molecular markers of a chicken TRIM13 gene in chicken growth and slaughter trait improvement breeding, which comprises the following steps: first record complete with 766 phenotypical dataFixed origin-Anka F2The resource group chicken is taken as a research object to carry out whole genome association analysis, and then large-scale SNP typing work is carried out on 87 th base of 3 rd intron region of F2 resource group TRIM13 gene with detailed phenotype record to verify the accuracy of the SNP sites. The invention proves that 1 SNP molecular marker which is positioned at 87 th base of 3 rd intron region of chicken TRIM13 gene exists, the nucleotide sequence is shown as SEQ ID NO.1, and 123 th base from 5' end is C or T; (T-C, rs732405671), is significantly related to chicken growth traits (body weight, shin length, shin circumference, chest depth, chest width, sternum length, oblique body length, pelvic width) and slaughter traits (total bore, liver weight, heart weight, spleen weight, paw weight). The allele T of the SNP locus is positively correlated with the low weight trait, the allele C is positively correlated with the high weight trait, and the phenotype trait of the heterozygous allele CT is in an intermediate value. The body weight is an important economic character of the chicken, the characters such as the shank circumference, the breast bone length and the like are strongly related to the size of the chicken body, and the SNP locus lays a foundation for screening a homozygous high-quality local chicken group. 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 further provides a chicken growth and slaughter trait improvement breeding method based on the SNP molecular marker of the chicken TRIM13 gene, which comprises the following steps: primers are designed at the upstream and downstream of the 87 th base SNP site of the 3 rd intron region of the chicken TRIM13 gene, and the PCR-RFLP method is adopted to detect the genotype of the SNP molecular marker by using Bsp1286I enzyme. 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 CC, the chicken to be detected is a high-weight large-size individual, and when the SNP molecular marker genotype is TT, the chicken to be detected is a low-weight small-size individual. Selecting chicken individuals with CC homozygous genotype to breed, and discarding chicken individuals with TT and CT 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 schematic representation of the Bsp1286I endonuclease assay;
FIG. 3 shows the results of agarose electrophoresis after digestion with Bsp1286I of three genotypes.
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 records2And performing whole genome association analysis on the resource group chickens as research objects. 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.). In the research, after 766 chickens of the resource group are genotyped, quality control and removal of SNP on sex chromosome by simplified genome sequencing, the total amount of 321314 SNP of 734 chickens are obtained. The resource group was subjected to growth traits of different weeks of age, body weight (2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks), 4 weeks, 8 weeks and 12 weeks of shin length, shin circumference, oblique body length, sternum length, etc., and 12 weeks of slaughter trait based on genotyping data and phenotypic dataPerforming whole genome association analysis, and finding that the nucleotide sequence of 1 SNP molecular marker existing in 87 th base of 3 rd intron region of chicken TRIM13 gene is shown as SEQ ID NO.1, and 123 th base from 5' end is C or T; (T-C, rs732405671), was significantly correlated with chicken 10 weeks body weight, 12 weeks body weight, 8 weeks shin circumference, 12 weeks sternum length, myogastric weight and paw weight average (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 form2Carrying out large-scale SNP enzyme digestion typing work on 87 th base of 3 rd intron region of resource group TRIM13 gene to verify the accuracy of the SNP sites, and further carrying out SNP typing result and F2The economic traits of the resource group are subjected to correlation analysis to find that the molecular marker is closely related to a plurality of growth traits and slaughter traits of chickens (Table 2). The average body weight, shin girth and sternum length of the CC genotype group are slightly higher than those of the CT group, and are obviously higher than those of the TT genotype group. The body weight is an important economic character of the chicken, and the length of the shank and the sternum is strongly related to the size of the chicken body; the average total bore weight, liver weight and paw weight of the CC gene group are slightly higher than the average total bore weight, liver weight and paw weight of the CT group, and are obviously higher than the average total bore weight, liver weight and paw weight of the TT gene group. The full-bore rate is an important slaughtering index of the chicken and a main index for measuring the meat production performance of the livestock and poultry; the above results further confirm the correlation analysis results described above: the SNP locus of the T-C mutation (shown in figure 1) of the 87 th base of the 3 rd intron region of the chicken TRIM13 gene is obviously related to chicken growth traits and slaughter traits, wherein allele C is positively related to high weight traits, and allele T is positively related to low weight traits. When the genotype of the SNP molecular marker is CC, the chicken to be detected has high weight and large sizeAnd when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-body individual.
TABLE 2 phenotypic statistics for different genotypes of the FISH-Anka F2 resource pool
Note:a,bthe same row, different superscripts, indicates that a very significant level (P) is reached<0.01)。
Example 2: kit for detecting SNP molecular marker genotype of chicken TRIM13 gene
The kit in the embodiment comprises a primer for detecting the SNP molecular marker of the chicken TRIM13 gene obtained in the embodiment 1 and a restriction enzyme Bsp1286I, wherein the sequence of the primer is shown in Table 3:
TABLE 3TRIM13 Gene SNP detection primers
Also comprises one or more of dNTPs, PCR reaction buffer solution and DNA polymerase.
Example 3: chicken growth and slaughter trait improvement breeding method based on chicken TRIM13 gene SNP molecular marker
Based on the genomic information of the SNP molecular marker obtained in example 1 and the sequence of chicken TRIM13 gene published by NCBI (ENSGALG00000017011: ENSGALT00000027478.6), primers were designed to amplify a partial sequence of the 3 rd intron region of TRIM13 gene, and the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO. 4.
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.3 and SEQ ID NO. 4:
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 reaction procedure for PCR amplification was: 5min at 95 ℃; 35 cycles of 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 5 s; preserving at 72 deg.C for 5min and 4 deg.C.
Secondly, Restriction Fragment Length Polymorphism (RFLP) is utilized to carry out Restriction enzyme digestion identification on the PCR product by Bsp1286I endonuclease: the base at this position is cleaved with Bsp1286I when it is C and not with Bsp1286I when it is T, thereby being used for typing (see FIG. 2).
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 TRIM13 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 189bp, the base at the mutation position is homozygous TT type, if the enzyme digestion product is 122/67bp, the base at the mutation position is homozygous CC type, and if the enzyme digestion product is 189/122/67bp, the base at the mutation position is heterozygous CT type; when the genotype of the SNP molecular marker is CC, 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 CC homozygous genotype to breed, and discarding chicken individuals with TT and CT 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 genotypes of Bsp1286I are shown in FIG. 3, lane 7: DNA Marker (700, 600, 500, 400, 300, 200 and 100 bp); lanes 1, 2 and 3 are two bands, the band size is 122 and 67bp, and the lane is named as genotype homozygous CC type; lanes 4, 5 and 6 are single bands, the size of each band is 189bp, and the genotype homozygous TT type is named; lanes 7, 8 and 9 are three bands with sizes of 189, 122 and 67bp, respectively, and are named as genotype heterozygous CT types; as can be seen from FIG. 3, individuals 1, 2 and 3 were high-weight, large-sized chickens, which were reserved for breeding; 4. 5, 6, 7, 8 and 9 individuals are low-weight and small-size chickens and need to be discarded; based on the genotyping result of the TRIM13 gene SNP molecular marker, breeding of high-weight and large-size chicken species is carried out.
<110> Henan university of agriculture
Application of SNP molecular marker of TRIM13 gene of chicken in chicken growth and slaughter trait improvement breeding and breeding method
<160> 4
<170> PatentIn version 3.5
<211> 189
<212> DNA
<213> Chicken
<221> PCR amplification sequence of TRIM13 Gene
<222> 123 (n is t or c)
<400> 1
ggtgccttct tctgcctgat gaccttagtc ctcagggtga gtagaaaagt tggcaactct 60
ttcaaactcc cttatccaag caaacatggg gaattcccct cttccttgtt gctggaggag 120
cangggaact gtgaggtctc actgtgactg catctgtctg gttgtgaaag aaactcagga 180
<211> 1270
<212> DNA
<213> Chicken
<221> sequence of intron 3 of TRIM13 gene
<222> 87
<400> 2
gtgagtagaa aagttggcaa ctctttcaaa ctcccttatc caagcaaaca tggggaattc 60
ccctcttcct tgttgctgga ggagcatggg aactgtgagg tctcactgtg actgcatctg 120
tctggttgtg aaagaaactc aggactggcc ctgccattct tccctgtgct ttcctaggat 180
cagtggtatt ttattagtct tatcctttgc agcatgtacc tttccttgta gctcttctct 240
tggttctctt tctctgccat tttgtaattt ttagagggga caggacccat agcaacagtt 300
cagatgaagt catcagaacg agataaaaga gctcattcgt ctgctttgct tgttacaaga 360
ctactttgtt gctctttttg tgatacatct tttaatttgt aaaaggcatt ttattacatc 420
ctttaaaaca taatgtagat tgagtttata caagagaagc atctttttaa ttatagctca 480
aaactaagtg cgtaagagtt ttggctaata caagctggtg tttattagtg acttgtgttc 540
ttagagcctg ctctaatgac agggtgtctg agaacttggg ttagaggcac tgggaggatg 600
gaggaatgca tccctccact gagactttcc attacaaagg agtgtggtaa atccttttcc 660
tgtaggccct gtccacaaga agagactact gaggcattag tcctgtgaga attggagata 720
gaggtagtgg atgagtaaaa atagttaaca ggtttatttt gctctgaatt accaaaatta 780
tgggcagctg ttctttgaaa tatgctggct gtgtgctgaa tagctcgatg ataatgcgtt 840
cacttggtta tttacaaata tggtgggagc aggtgaaact ccttaagttc ttgtacgttt 900
ctgcatattg catcagtgca ggaagatgca agccttgaat tactagtaca tgagttattt 960
tcatttgaca tggaaagcct tccttggcga gcccttttcc tgcgtttcta tttgagagtg 1020
agaaaatggt tctgcctgga cccccgtcca ttcctgtgcc tgccatctgg tggccagatt 1080
ccacagctaa cttgatccct ttggggaaga tctgattctg ggctgggttg tcggagccca 1140
tctggggctc tgcctttggg gtccttgagg aaaaaaatag atcctcaggt ggagatcagc 1200
ttttgtgtct ttcctggaga agagaagcac attctgtaag ttctgttact gtctttgttc 1260
tctttcctag 1270
<211> 20
<212> DNA
<213> Artificial sequence
<221> upstream primer P-F
<400> 3
ggtgccttct tctgcctgat 20
<211> 20
<212> DNA
<213> Artificial sequence
<221> downstream primer P-R
<400> 4
cagggccagt cctgagtttc 20
Claims (10)
1. The application of the SNP molecular marker of the chicken TRIM13 gene in chicken growth and slaughter trait improvement breeding is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the 123 th base from the 5' end is C or T.
2. The use of claim 1, wherein the genotype of SNP molecular marker of TRIM13 gene of chicken is detected, when the genotype of SNP molecular marker is CC, the chicken to be detected is high-weight and large-size individual, and when the genotype of SNP molecular marker is TT, the chicken to be detected is low-weight and small-size individual.
3. The use according to claim 2, characterized in that the genotype of the SNP molecular marker of the TRIM13 gene of chicken is detected by a kit, which comprises a primer pair shown as SEQ ID No.3 and SEQ ID No.4, and a restriction enzyme Bsp 1286I.
4. The use of claim 3, wherein the kit further comprises one or more of dNTPs, PCR reaction buffer, and DNA polymerase.
5. A chicken growth and slaughter trait improvement breeding method based on chicken TRIM13 gene SNP molecular marker is characterized in that: the method 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.3 and SEQ ID No.4, carrying out enzyme digestion reaction on a PCR product by using Bsp1286I endonuclease, wherein if the enzyme digestion product is 189bp, the base at a mutation position is homozygous TT type, if the enzyme digestion product is 122/67bp, the base at the mutation position is homozygous CC type, and if the enzyme digestion product is 189/122/67bp, the base at the mutation position is heterozygous CT type; when the genotype of the SNP molecular marker is CC, 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 large-size individual; selecting and keeping chicken individuals with CC homozygous genotype.
6. The method for improving chicken growth and slaughter traits according to claim 5, characterized in that: 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.
7. The method for improving chicken growth and slaughter traits according to claim 5, characterized in that: the enzyme digestion reaction system is 25uL, and comprises 22.5uL of PCR product, 10 XBuffer 2uL and Bsp1286I 0.5.5 uL.
8. The method for improving chicken growth and slaughter traits according to claim 5, characterized in that: and detecting the size of the enzyme digestion product through agarose gel electrophoresis to judge the genotype of the SNP molecular marker.
9. The method for improving chicken growth and slaughter traits according to claim 8, characterized in that: the agarose gel electrophoresis detection adopts the agarose with the mass fraction of 2.0 percent, the voltage of 120V and the electrophoresis time of 30 min.
10. Use of the chicken growth and slaughter trait improved breeding method of claim 5 for breeding high-weight or large-size chicken species.
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| CN116837110A (en) * | 2023-07-07 | 2023-10-03 | 宁夏大学 | SNP locus on chromosome 7 and related to chicken growth traits and application thereof |
| CN116837110B (en) * | 2023-07-07 | 2024-04-30 | 宁夏大学 | SNP locus on chromosome 7 and related to chicken growth traits and application thereof |
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| CN114150071B (en) | 2023-07-25 |
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