CN114150071B - Application of chicken TRIM13 gene SNP molecular marker in chicken growth and slaughter trait improvement breeding and breeding method - Google Patents
Application of chicken TRIM13 gene SNP molecular marker in chicken growth and slaughter trait improvement breeding and breeding method Download PDFInfo
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- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention relates to an application of chicken TRIM13 gene SNP molecular markers 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 researches, the SNP locus (T-C, rs 732405671) at the 1 st position of 87 th base in the 3 rd intron region of the chicken TRIM13 gene is obviously related to the chicken growth trait and slaughter trait. 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 at an intermediate value. The SNP molecular marker genotype is detected, so that the weight of the chicken in the mature period can be predicted early and effectively, and the SNP molecular marker genotype can be used as a molecular marker for genetic improvement of chicken varieties, thereby shortening the breeding time and accelerating the breeding process. The invention provides a chicken growth and slaughter character improvement breeding method based on chicken TRIM13 gene SNP molecular markers, which can be used for breeding high-weight or large-size chicken varieties.
Description
Technical Field
The invention relates to an application of chicken TRIM13 gene SNP molecular markers 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 livestock product, and even though meat consumption in developed countries is relatively fixed, meat consumption has doubled by people in developing countries since 1980. The increase in population and income, as well as the change in food preferences, have increased the demand for livestock products. Throughout the world, chicken is the second largest meat consumer next to pork, the proportion of chicken consumption is up to about 35% of the total meat consumption (37% pork), and the total chicken consumption rises annually by about 1.6% (greater than 1.1% pork) between 2012-2014 (http:// www.fao.org/ag/againfo/thes/en/meat/background. Html), increasing chicken weight and chicken yield are important tasks for modern breeding efforts. The growing characters such as weight are taken as main economic characters of livestock and poultry, and how to improve the economic characters of local livestock and poultry varieties under the condition that the germplasm characteristics are not changed, and the maintenance of the sustainable development of local livestock and poultry is an urgent problem to be solved. Therefore, it is necessary to develop new genetic markers, and to accelerate the progress of genetic improvement by means of molecular biology, thereby shortening the breeding time and accelerating the progress of breeding.
Disclosure of Invention
The invention aims to provide an application of chicken TRIM13 gene SNP molecular markers in chicken growth and slaughter trait improvement breeding.
The invention also aims to provide a method for improving and breeding chicken growth and slaughter traits based on chicken TRIM13 gene SNP molecular markers, which can be used for breeding high-weight or large-size chicken breeds.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides an application of chicken TRIM13 gene SNP molecular markers 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 and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual.
Specifically, the genotype of the SNP molecular marker of the TRIM13 gene of the chicken is detected by the kit.
The kit comprises a primer pair shown as SEQ ID NO.3 and SEQ ID NO.4, and restriction enzyme Bsp1286I.
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 character improvement breeding method based on chicken TRIM13 gene SNP molecular markers, which comprises the following steps: taking the genome DNA of a chicken to be detected as a template, carrying out PCR amplification by using a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, carrying out enzyme digestion reaction on a PCR product by using Bsp1286I endonuclease, 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 and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual; chicken individuals with CC genotype are selected and remained.
Preferably, the reaction system of PCR amplification is: 2X Taq PCR MasterMix. Mu.L, ddH 2 O6. Mu.L, upstream primer P-F1. Mu.L, downstream primer P-R1. Mu.L, and DNA template 2. Mu.L.
Preferably, the reaction procedure of the PCR amplification is as follows: 95 ℃ for 5min;95℃15s,60℃15s,72℃5s, 35 cycles total; stored at 72℃for 5min and at 4 ℃.
Preferably, the system of the cleavage reaction is 25uL, including PCR products 22.5uL, 10 XBuffer 2uL and Bsp1286I 0.5uL.
Preferably, the size of the cleavage product is detected by agarose gel electrophoresis to determine the genotype of the SNP molecular marker.
Preferably, the mass fraction of the agar used for agarose gel electrophoresis detection is 2.0%, the voltage is 120V, and the electrophoresis time is 30min.
The chicken growth and slaughter character improvement breeding method based on the chicken TRIM13 gene SNP molecular marker can be applied to breeding high-weight or large-size chicken seeds.
The invention has the beneficial effects that:
the invention provides an application of chicken TRIM13 gene SNP molecular markers in chicken growth and slaughter trait improvement breeding: first record complete solid-onset-Anka F with 766 phenotypes data 2 And carrying out genome-wide association analysis on the resource group chicken serving as a research object, and then carrying out large-scale SNP typing work on 87 th base of the 3 rd intron region of the F2 resource group TRIM13 gene in detail on the phenotype record so as to verify the accuracy of the SNP locus. The invention proves that the SNP molecular marker is positioned at the 1 st position of 87 th base in the 3 rd intron region of the chicken TRIM13 gene, 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; (T-C, rs 732405671) is significantly associated with chicken growth traits (body weight, shank length, shank circumference, chest depth, chest width, sternum length, oblique body length, pelvic bone width) and slaughter traits (full breech-free, liver weight, heart weight, spleen weight, paw weight). The allele T of the SNP locus is positively correlated with low body weight traits, the allele C is positively correlated with high body weight traits, and the phenotype traits of heterozygous allele CTAt an intermediate value. The weight is an important economic property of chickens, the properties such as shank circumference, sternum length and the like are strongly related to the body size of chickens, and the SNP locus lays a foundation for screening out homozygous high-quality local chicken groups. The SNP molecular marker genotype is detected, so that the weight of the chicken in the mature period can be predicted early and effectively, and the SNP molecular marker can be used as a molecular marker for genetic improvement of chicken varieties, thereby shortening the breeding time, accelerating the breeding process and having great economic application value and scientific research value.
The invention further provides a chicken growth and slaughter trait improvement breeding method based on chicken TRIM13 gene SNP molecular markers, which comprises the following steps: designing primers at the upstream and downstream of the 87 th base SNP locus of the 3 rd intron region of the chicken TRIM13 gene, and detecting the genotype of the SNP molecular marker by using a PCR-RFLP method 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 an enzyme digestion product electrophoresis condition, and the corresponding relation between SNP molecular marker genotype and chicken growth characteristics is defined, namely when the genotype of the SNP molecular marker is CC, the chicken to be detected is a high-weight and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual. Selecting and reserving chicken individuals with CC homozygous genotype for cultivation, discarding chicken individuals with TT and CT genotypes, and obtaining chicken germplasm resources with excellent growth characteristics of high weight and large size. The method has strong operability, does not need special instruments, is easy to popularize, can be used for predicting the weight and the body size of the chickens early, quickly and effectively at low cost, can be used for auxiliary selection and molecular breeding of the chickens, and has wide application prospect in the aspect of improving the germplasm resources of the chickens.
Drawings
FIG. 1 is a peak diagram of mutation position sequences of different genotypes;
FIG. 2 is a schematic diagram showing an analysis of Bsp1286I endonuclease;
FIG. 3 is a graph showing agarose electrophoresis results of three genotypes Bsp1286I after cleavage.
Detailed Description
The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples were all conventionally commercially available unless otherwise specified.
Example 1: acquisition of SNP molecular markers related to chicken growth and slaughter traits
Complete solid-Anka F was recorded with 766 phenotypic data 2 And carrying out whole genome association analysis on the resource group chicken serving as a study object. Established solid-Anka F by Henan province poultry germplasm resource innovation research center 2 The resource group consists of 4 orthogonal families (Anka cock x fixed-born hen) and 3 backcross families (fixed-born cock x Anka hen), and the economic character determination method is as follows: body weight was measured every 2 weeks, body size index was measured every 4 weeks, and slaughtered when fed to 12 weeks of age. The materials were weighed after 12 hours of pre-slaughter (without stopping water). The measured growth and slaughter indexes and meat quality indexes are 57. (cf. Han R L, li Z J, li M J, et al Novel9-bp indel in visfatin gene and its associations with chicken growth. Br Poult Sci,2011,52 (1): 52-57.). The study utilized simplified genome sequencing to genotype 766 chickens of the resource group, and after quality control and removal of SNPs on sex chromosomes, a total of 734 chickens had 321314 SNPs. Based on genotyping data and phenotype data, carrying out genome-wide association analysis on growth traits such as different week-old weights (2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks), 4 weeks, 8 weeks and 12 weeks shank length, shank circumference, body oblique length, sternum length and the like and 12 weeks slaughter traits, and researching to find that 1 SNP molecular marker exists in 87 th base of 3 rd intron region of chicken TRIM13 gene, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and 123 th base from the 5' end is C or T; (T-C, rs 732405671) was significantly associated with chicken 10 weeks of body weight, 12 weeks of body weight, 8 weeks of shin circumference, 12 weeks of sternum length, myogastric weight and paw weight (table 1).
TABLE 1 Whole genome correlation analysis of the SNP and fixed-Ankara chicken F 2 Multiple properties of resource group are obviously related
Note that: whole genome correlation analysis-log 10 (P)>5.72 indicated that significant levels were reached.
Record details F of the table 2 The 87 th base of the 3 rd intron region of the TRIM13 gene of the resource group carries out large-scale SNP enzyme cutting work to verify the accuracy of the SNP locus, and further carries out SNP typing results and F 2 The molecular markers were found to be closely related to multiple growth traits and slaughter traits of chickens by correlation analysis of economic traits of resource groups (table 2). The average body weight, the shank circumference and the 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 group. Weight is an important economical trait of chickens, while shank circumference and sternum length are strongly related to chicken body size; the average total bore-free weight, liver weight and paw weight of the CC genotype group are slightly higher than those of the CT group, and are obviously higher than those of the TT group. The total bore-free rate is an important slaughtering index of chickens and is a main index for measuring the meat production performance of livestock and poultry; the above results further confirm the above correlation analysis results: the SNP locus of the T-C mutation (figure 1) of the 87 th base in the 3 rd intron region of the chicken TRIM13 gene is obviously related to the chicken growth trait and slaughter trait, wherein the allele C is positively related to the high-weight trait, and the allele T is positively related to the low-weight trait. When the genotype of the SNP molecular marker is CC, the chicken to be detected is a high-weight and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual.
TABLE 2 fixed initial-Ankara F2 resource group phenotype statistics for different genotypes
Note that: a , b the same row of superscripts differ in that a very significant level (P<0.01)。
Example 2: kit for detecting SNP molecular marker genotype of TRIM13 gene of chicken
The kit in this example comprises primers for detecting SNP molecular markers of the TRIM13 gene of the chicken obtained in example 1 and restriction enzyme Bsp1286I, and the sequences of the primers are 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 character improvement breeding method based on chicken TRIM13 gene SNP molecular marker
Based on the genomic information of SNP molecular markers obtained in example 1 and the chicken TRIM13 gene sequence published by NCBI (ENSGALG 00000017011: ENSGALT 00000027478.6), primers were designed to amplify a partial sequence of the 3 rd intron region of the TRIM13 gene, and the nucleotide sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Firstly, taking the genome DNA of the chicken to be detected as a template, and carrying out PCR amplification by using a primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4:
the PCR amplification reaction system is as follows: 2X Taq PCR MasterMix. Mu.L, ddH 2 O6. Mu.L, upstream primer P-F1. Mu.L, downstream primer P-R1. Mu.L, DNA template 2. Mu.L;
the reaction procedure for PCR amplification was: 95 ℃ for 5min;95℃15s,60℃15s,72℃5s, 35 cycles total; stored at 72℃for 5min and at 4 ℃.
Next, the PCR product was identified by cleavage with Bsp1286I endonuclease using restriction endonuclease length polymorphism (RFLP, restriction Fragment Length Polymorphism): the base at this position was cleaved by Bsp1286I when it was C and not by Bsp1286I when it was T, thereby allowing typing (FIG. 2).
The system of the cleavage reaction was 25uL, including 22.5uL of PCR product, 10 XBuffer 2uL and Bsp1286I 0.5uL (see Table 4);
TABLE 4 TRIM13 cleavage reaction System
Finally, detecting the size of the enzyme digestion product through agarose gel electrophoresis to judge the genotype of the SNP molecular marker; the mass fraction of the agar adopted in the agarose gel electrophoresis detection is 2.0%, the voltage is 120V, and the electrophoresis time is 30min;
if the cleavage product is 189bp, the base at the mutation position is homozygous TT type, if the cleavage product is 122/67bp, the base at the mutation position is homozygous CC type, and if the cleavage 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 and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual; selecting and reserving chicken individuals with CC homozygous genotype for cultivation, discarding chicken individuals with TT and CT genotypes, and obtaining chicken germplasm resources with excellent growth characteristics of high weight and large size.
Agarose electrophoresis results after cleavage of the three genotypes Bsp1286I are shown in fig. 3, lane 7: DNA markers (700, 600, 500, 400, 300, 200 and 100 bp); lanes 1, 2 and 3 are two bands, the sizes of which are 122 and 67bp, and are named genotype homozygous CC type; lanes 4, 5 and 6 are bands with 189bp, and are named genotype homozygous TT type; lanes 7, 8, 9 are three bands, with sizes 189, 122 and 67bp, respectively, designated genotype heterozygous CT; as can be seen from FIG. 3, 1, 2 and 3 individuals are high-weight, large-size chickens, and need to be reserved for breeding; 4. 5, 6, 7, 8, 9 individuals are low-weight and small-size chickens, and need to be discarded; and (3) carrying out breeding of high-weight and large-size chicken breeds based on the genotyping result of the TRIM13 gene SNP molecular marker.
<110> Henan agricultural university
Application of <120> chicken TRIM13 gene SNP molecular marker 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> TRIM13 Gene PCR amplified sequence
<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
ctggccctg 189
<211> 1270
<212> DNA
<213> chicken
<221> sequence of 3 rd intron 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. Chicken with meatTRIM13The application of the gene SNP molecular marker in chicken growth and slaughter character 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. Use according to claim 1, characterized in that for chickensTRIM13Detecting the genotype of the SNP molecular marker of the gene, wherein when the genotype of the SNP molecular marker is thatAnd when the genotype of the SNP molecular marker is TT, the chicken to be tested is a low-weight and small-size individual.
3. Use according to claim 2, characterized in that the chicken is detected by a kitTRIM13The kit comprises a primer pair shown as SEQ ID NO.3 and SEQ ID NO.4 and restriction enzyme Bsp1286I.
4. The use according to claim 3, wherein the kit further comprises one or more of dNTPs, PCR reaction buffers, DNA polymerase.
5. Chicken-basedTRIM13The chicken growth and slaughter character improvement breeding method of the gene SNP molecular marker is characterized by comprising the following steps: 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 a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, carrying out enzyme digestion reaction on a PCR product by using Bsp1286I endonuclease, 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 and large-size individual, and when the genotype of the SNP molecular marker is TT, the chicken to be detected is a low-weight and small-size individual; selecting and reserving chicken individuals with CC homozygous genotype.
6. The method for improving chicken growth and slaughter characteristics and breeding according to claim 5, wherein: the PCR amplification reaction system comprises: 2X Taq PCR MasterMix. Mu.L, ddH 2 O6. Mu.L, upstream primer P-F1. Mu.L, downstream primer P-R1. Mu.L, and DNA template 2. Mu.L.
7. The method for improving chicken growth and slaughter characteristics and breeding according to claim 5, wherein: the system of the cleavage reaction was 25. Mu.L, including 22.5. Mu.L of PCR product, 10 XBuffer 2. Mu.L and Bsp1286I 0.5. Mu.L.
8. The method for improving chicken growth and slaughter characteristics and breeding according to claim 5, wherein: and detecting the size of the enzyme digestion product through agarose gel electrophoresis to judge the genotype of the SNP molecular marker.
9. The improved chicken growth and slaughter trait breeding method according to claim 8, wherein: the mass fraction of the agar used for agarose gel electrophoresis detection is 2.0%, the voltage is 120V, and the electrophoresis time is 30min.
10. The use of the improved chicken growth and slaughter trait breeding method according to claim 5 for breeding high body weight or large body size chickens.
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