CN102220410B - Method for auxiliary identification of chickens with different weight properties - Google Patents
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Abstract
The invention discloses a method for auxiliary identification of chickens with different weight properties, which comprises the following steps: determining if the deoxyribonucleotide at the 101th locus from the 5' terminal of the sequence of a gene HMGA2 of chicken to be detected is G or T, and determining if the genotype of the chicken to be detected is GG, GT or TT, wherein GG genotype indicates that the deoxyribonucleotide at the 101th locus from the 5' terminal of the sequence of the gene HMGA2 is a homozygote of G, TT genotype indicates that the deoxyribonucleotide at the 101th locus from the 5' terminal of the sequence of the gene HMGA2 is a homozygote of T, GT genotype indicates that the deoxyribonucleotide at the 101th locus from the 5' terminal of the sequence of the gene HMGA2 is a heterozygote of G and T, chickens of TT genotype are heavier than those of GG genotype in statistics, and chickens of TT genotype are much heavier than those of GG and GT genotypes. With single nucleotide polymorphism (SNP) as a genetic marker of chickens, a molecular biological means can be adopted to detect the locus, so that early breeding can be realized while time and resources are greatly saved.
Description
Technical field
The present invention relates to a kind of method that assistant identification has the different weight characters chicken.
Background technology
SNP (single nucleotide polymorphism single nucleotide polymorphism) mainly refers in genomic level, and the polymorphism of the DNA sequence dna that is caused by the variation of single core thuja acid comprises conversion, transversion, insertion or disappearance.Densely distributed SNP in genome because it carries huge genetic information amount, is considered to the best genetic marker of application prospect, and the detection of mankind SNP mark has become the important means of gene diagnosis.The research of the genetic map of SNP mark has huge promoter action to marker assisted selection, and in agricultural animal, the gene of a lot of important characters is located.
SNP detects normal some existing mature technologies that adopt, and (the Sanger sequencing is connected with the tetra-sodium sequencing, single strand conformation polymorphism (SSCP), Restriction fragment length polymorphism (RFLP), the hybridization of allele specific oligonucleotide oligonucleotide (ASO), the special technique means such as connection, DNA chip and Taqman probe of oligonucleotide as DNA sequencing.
HMGA2 as structural transcription factor is a kind of nonhistones Chromatin Protein, contains 3 AT-hook in conjunction with the territory, can be rich in the ditch in AT zone to DNA by specific binding.It is generally acknowledged, HMGA2 works in Growth of Cells and metabolism of fat.HMGA2 mainly expresses the embryo period, expresses hardly in adult tissue, and expression can be activated in tumour cell.The mouse of HMGA2 afunction shows as nanism, and fatty tissue reduces in a large number, when having served as the HMGA2 of expression deletion C-terminal, mouse shows as gigantosoma and adipopexis, and can form tumour, therefore, HMGA2 studies mainly as the gene relevant to cancer.The nucleotide sequence of chicken HMGA2 gene sees that GENBANKACCESSION NO.:NC_006088 (Chr1) is from 5 ' end 36072025-36177364 position Nucleotide.
Body weight index in growth traits is one of economic characters important during livestock industry is produced always, is also the important indicator in the broiler chicken breed breeding.Compare with nineteen fifty-seven, broiler chicken present age calendar year 2001, the carcass output of strain improved 6 times, and wherein 85%-90% is given the credit to the genetics effect, and only having 10%-15% is that improvement due to the feed nutrition level causes.The research of poultry genetic marker assistant breeding is deepening continuously, and a large amount of proterties has been determined the candidate region on hereditary level.There is abundant native chicken breed resource in China, how to be problem in the urgent need to address to its assessment, protection and seed selection.
Summary of the invention
The purpose of this invention is to provide a kind of method that assistant identification has the different weight characters chicken.
The method (one) that assistant identification provided by the invention has a different weight characters chicken comprises the steps: to detect that in the HMGA2 gene of chicken to be measured, sequence 1 is G or T from the 101st deoxyribonucleotide of 5 ' end, and the genotype of determining chicken to be measured is GG or TT; Described GG genotype is that in the HMGA2 gene, sequence 1 is the homozygote of G from the 101st deoxyribonucleotide of 5 ' end; Described TT genotype is that in the HMGA2 gene, sequence 1 is the homozygote of T from the 101st deoxyribonucleotide of 5 ' end; The body weight of TT genotype chicken is statistically higher than the body weight of GG genotype chicken.
Described method (one) also can comprise the step of selecting chicken to be measured; Described chicken to be measured is the genotypic chicken of GG and the genotypic chicken of TT.
In described method (), determine that the genotypic method of chicken to be measured is specific as follows: detect with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company and the hybridization plate genomic dna to chicken to be measured; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: when a＞6b, the genotype of chicken to be measured is GG; When a＜1/6b, the genotype of chicken to be measured is TT.
Described method (one) specifically also comprises the step of following selection chicken to be measured: with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company with hybridize plate the genomic dna of chicken to be measured is detected; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: when a＞6b, the genotype of chicken to be measured is GG; When a＜1/6b, the genotype of chicken to be measured is TT; Select the genotypic chicken of GG and the genotypic chicken of TT as chicken to be measured.
The method (two) that assistant identification provided by the invention has a different weight characters chicken comprises the steps: to detect that in the HMGA2 gene of chicken to be measured, sequence 1 is G or T from the 101st deoxyribonucleotide of 5 ' end, and the genotype of determining chicken to be measured is GT or TT; Described GT genotype is that in the HMGA2 gene, sequence 1 is the heterozygote of G and T from the 101st deoxyribonucleotide of 5 ' end; Described TT genotype is that in the HMGA2 gene, sequence 1 is the homozygote of T from the 101st deoxyribonucleotide of 5 ' end; The body weight of TT genotype chicken is statistically higher than the body weight of GT genotype chicken.
Described method (two) also can comprise the step of selecting chicken to be measured; Described chicken to be measured is the genotypic chicken of GT and the genotypic chicken of TT.
In described method (two), determine that the genotypic method of chicken to be measured is as follows: detect with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company and the hybridization plate genomic dna to chicken to be measured; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: when 1/3b＜a＜3b, the genotype of chicken to be measured is GT; When a＜1/6b, the genotype of chicken to be measured is TT.
Described method (two) specifically also comprises the step of following selection chicken to be measured: with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company with hybridize plate the genomic dna of chicken to be measured is detected; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: when 1/3b＜a＜3b, the genotype of chicken to be measured is GT; When a＜1/6b, the genotype of chicken to be measured is TT; Select the genotypic chicken of GT and the genotypic chicken of TT as chicken to be measured.
In described method () and method (two): the cat. no of the SNPlex GenotypingSystem 48-plex kit of described U.S. applying biological company is 4375768; Described hybridization plate is the hybridization plate of U.S. applying biological company, and preferred cat. no is 4362933 hybridization plate; And/or; The nucleotide sequence of described HMGA2 gene is as shown in the sequence 6 of sequence table; And/or; Described chicken to be measured is the chicken of CAU resource family, and the F2 of preferred CAU resource family is for individuality; And/or; Described weight character for nascent body weight, 1 age in week body weight, 2 age in week body weight, 3 age in week body weight, 5 age in week body weight, 6 age in week body weight or 10 age in week body weight.
Above arbitrary described method all can be applicable to a breed of chicken.
The present invention finds that the SNP (single nucleotide polymorphism) in the HMGA2 gene of chicken has the characteristic of significant correlation with body weight, and the body weight of TT genotype chicken is significantly higher than GG genotype and GT genotype.This SNP can be used as the genetic marker of chicken, uses molecular biology method that this site is detected, and realizes early stage seed selection, greatly saves time and resource.
Description of drawings
Fig. 1 is each sample genotyping result in embodiment 2.
Fig. 2 is each sample genotyping result in embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
CAU resource family: locate for research the experimental population that affects chicken important economical trait QTL, seeks clone's major gene by what the Li Ning of China Agricultural University teach problem group was set up in 1998, the parent is French star's broiler chicken and calm and peaceful plumage Gallus Domesticus; The source is China Agricultural University's experiment pasture; CAU resource family reference: Deng, X.M., Li, J.Y., Li, N., and Wu, C.X. (2001) .[Genetic analysis of important growth traitbased on F-2 resource population in chicken] .Yi Chuan Xue Bao 28,801-807.; China Agricultural University guarantees to provide to the public.
The discovery of embodiment 1, rs13849381 site SNP and body weight dependency
Measure the body weight of F2 for individual 1 to 12 age in week in CAU resource family.Analyze near the HMGA2 gene and inner 14 SNP sites (rs15231375, rs15231403, rs13849241, rs14809326, rs15231448, rs15231464, rs15231472, rs13750437, rs14809399, rs13849381, rs15231566, rs10722387, SNP36178184) altogether, use the SNPlex technology to carry out genotype detection, determined that 1395 CAU resource family F2 are for the genotype in the above-mentioned SNP site of individuality.
Adopt the statistical study of JMP statistical analysis software, to carrying out the variance statistical study for examination chicken group's HMGA2 gene genotype and growth traits body weight, result shows rs13849381 site (G/T according to the least square analytical model; Chr1:36175314 is arranged in the intron 4 of gene) there is significant correlation with body weight.
The association analysis of rs13849381 G/T genotype and body weight in embodiment 2, CAU resource family
Experiment material: 1395 CAU resource family F2 are for individuality.
One, body weight determination
The body weight in each age in week in 0 age (birth weight) in week of each individuality of difference, 1 age in week age to 12 in week.
Two, the detection of SNP
1, the extraction of genomic dna
The wing venous blood collection, cracking after anti-freezing is processed is after protease K digesting, with the imitative extracting of phenol ,-20 ℃ of preservations after the TE dissolving.
2, genotype is determined
Get genomic dna, adopt SNPlex GenotypingSystem 48-plex kit (cat. no 4375768) and the hybridization plate (cat. no 4362933) of U.S. applying biological company (appliedbiosystems), according to the test kit specification sheets, individuality is carried out genotype detection.Negative control is dilution gene group DNA TE damping fluid used.
Three probes (two upstream probe and a downstream probe) sequence following (5 ' → 3 '):
Probe A (upstream probe): 5 '-CGTGGGCACTC-3 ' (sequence 3); Probe B (upstream probe): 5 '-CGTGGGCACTA-3 ' (sequence 4); Probe C (downstream probe): 5 '-CCTTTGTTTT-3 ' (sequence 5).
Sequenator is the 3730xl model, carries software genemapper4.0 to data analysis with instrument.
The genotype judging criterion: the fluorescence intensity of probe A is a, and the fluorescence intensity of probe B is b, and a+b＞1000RFU (fluorescent signal value unit) is if a+b≤1000RFU needs repeated experiments; When a＞6b, the genotype of sample is GG; When 1/3b＜a＜3b, the genotype of sample is GT; When a＜1/6b, the genotype of sample is TT.
The genotypic chicken of GG is the homozygote of DNA shown in sequence 1, and the genotypic chicken of TT is the homozygote of DNA shown in sequence 2, and the genotypic chicken of GT is the heterozygote of DNA shown in DNA shown in sequence 1 and sequence 2.Shown in sequence 1 and sequence 2, DNA only has the difference of a Nucleotide, sequence 1 is G from the 101st deoxyribonucleotide of 5 ' end, sequence 2 is T from the 101st deoxyribonucleotide of 5 ' end, is a mononucleotide polymorphic, and sequence 1 and sequence 2 are the partial sequence of HMGA2 gene.
The results are shown in Table 1 and Fig. 1.Fig. 1 is the dendrogram that analysis software provides, and each point represents body one by one;
X-coordinate: X=log
10(fluorescence intensity that the fluorescence intensity that probe A is corresponding+probe B is corresponding);
Ordinate zou: the Y=inverse sine (fluorescence intensity that probe A is corresponding/(fluorescence intensity that probe A is corresponding
2The fluorescence intensity that+probe B is corresponding
2)
0.5
The distribution of table 1 site rs13849381 genotype in F2 colony
From table 1 result, the GG type is for testing the preponderant genotype in colony used.
Three, association analysis
Adopt the Fit Model process of JMP (7.0 editions) statistics analysis software package to carry out statistical study, to carrying out the variance statistical study for examination chicken group's genotype and growth traits, P-value＜0.05 shows significant difference according to the least square analytical model.
Statistic analysis models:
Y＝μ+Sex
i+Family
j+Hatch
k+Genotype
l+e
The phenotypic character of Y individuality (body weight) observed value;
μ is the average of colony;
Sex is that sex is to the effect value of phenotypic character (body weight);
Family is that family is to the effect value of phenotypic character (body weight);
Hatch is that different batches is to the effect value of phenotypic character (body weight);
Genotype is that genotype is to the effect value of phenotypic character (body weight);
E is the random residual effect corresponding to observed value.
The results are shown in Table 2.
The correlation results of each week body weight in age (g) of table 2 and rs13849381
From table 2 result, the body weight of the chicken of TT genotype and two other genotype (GG/GT) has significant difference, has advantage aspect body weight.Birth weighs (birth weight), 1 age in week is heavy, 2 ages in week are heavy, 3 ages in week are heavy and weigh 5 ages in week, and each genotypic difference is (P＜0.01) extremely significantly; 6 ages in week are heavy and 10 ages in week are heavy, and each genotypic difference is (P＜0.05) significantly.
The application of embodiment 3, rs13849381 site SNP
Respectively the chicken of 16 kinds being carried out SNP measures: white eared pheasant, dark grey chicken, camellia chicken, Xianju Chicken, Henan cockfighting, Chongren Chicken, Xiaoshan chicken, the black Lang shan Mountain, limit chicken, deer park chicken, silk plumage Gallus Domesticus, hide that chicken, Shi Qi are assorted, (front 14 kinds are available from from Poultry Institute, Chinese Academy of Agricultural Science for peace card, the precious aa of section, the precious Am of section; Latter two kind is available from Beijing poultry breeding company).
One, the detection of SNP
1, the extraction of genomic dna
The wing venous blood collection, cracking after anti-freezing is processed, after protease K digesting, with the imitative extracting of phenol, TE dissolves-20 ℃ of preservations.
2, the genotype of each kind is determined
With embodiment 2.
The results are shown in Table 3 and Fig. 2.
The distribution of polymorphism in different standard-bred poultries in table 3 rs13849381 site
Result shows, the rs13849381 site T gene frequency of Ke Bao and An Ka significantly raises, and TT homozygous individual proportion is also higher.
Result shows, rs13849381 site TT homozygous individual has advantage aspect body weight, and in particularly early growth process (1-6 age in week) (general 6 weeks of commercial meat bird Time To Market average out to), difference is more obvious.The rs13849381 site can be used as a genetic marker, is applied to the molecular genetic marker assisted Selection of chicken growth traits, improves speed and the accuracy of broiler chicken seed selection.
Sequence table
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＜120〉a kind of assistant identification has the method for different weight characters chicken
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Claims (5)
1. an assistant identification has the method for different weight characters chicken, comprises the steps: to detect that in the HMGA2 gene of chicken to be measured, sequence 1 is G or T from the 101st deoxyribonucleotide of 5 ' end, and the genotype of determining chicken to be measured is GG or TT; Described GG genotype is that in the HMGA2 gene, sequence 1 is the homozygote of G from the 101st deoxyribonucleotide of 5 ' end; Described TT genotype is that in the HMGA2 gene, sequence 1 is the homozygote of T from the 101st deoxyribonucleotide of 5 ' end; The body weight of TT genotype chicken is statistically higher than the body weight of GG genotype chicken.
2. the method for claim 1 is characterized in that: describedly determine that the genotypic method of chicken to be measured is as follows: with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company with hybridize plate the genomic dna of chicken to be measured is detected; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: as a during 6b, the genotype of chicken to be measured is GG; When a＜1/6b, the genotype of chicken to be measured is TT; Wherein, the fluorescence intensity of DNA probe shown in sequence 3 is a, and the fluorescence intensity of DNA probe shown in sequence 4 is b.
3. an assistant identification has the method for different weight characters chicken, comprises the steps: to detect that in the HMGA2 gene of chicken to be measured, sequence 1 is G or T from the 101st deoxyribonucleotide of 5 ' end, and the genotype of determining chicken to be measured is GT or TT; Described GT genotype is that in the HMGA2 gene, sequence 1 is the heterozygote of G and T from the 101st deoxyribonucleotide of 5 ' end; Described TT genotype is that in the HMGA2 gene, sequence 1 is the homozygote of T from the 101st deoxyribonucleotide of 5 ' end; The body weight of TT genotype chicken is statistically higher than the body weight of GT genotype chicken.
4. method as claimed in claim 3 is characterized in that: describedly determine that the genotypic method of chicken to be measured is as follows: with the SNPlex Genotyping System 48-plex kit of 3730xl model sequenator, U.S. applying biological company with hybridize plate the genomic dna of chicken to be measured is detected; Carry out DNA shown in the sequence 5 of DNA shown in the sequence 4 of DNA, sequence table shown in sequence 3 that described detection probe used is sequence table and sequence table; Fluorescence data is analyzed: when 1/3b＜a＜3b, the genotype of chicken to be measured is GT; When a＜1/6b, the genotype of chicken to be measured is TT; Wherein, the fluorescence intensity of DNA probe shown in sequence 3 is a, and the fluorescence intensity of DNA probe shown in sequence 4 is b.
5. the application of arbitrary described method in a breed of chicken in claim 1 to 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102534006B (en) * | 2012-01-13 | 2013-08-14 | 中国农业大学 | Method for detecting homozygosis or heterozygosis of silkie fiber melanin gene |
| CN103276090B (en) * | 2013-05-30 | 2013-12-11 | 扬州大学 | Jinghai yellow chicken 16-week weight molecular genetic marker and application thereof |
| CN110592238B (en) * | 2019-10-16 | 2022-04-15 | 西北农林科技大学 | SNP (Single nucleotide polymorphism) marker related to growth traits of black-bone chickens and application thereof |
| CN114921572B (en) * | 2022-06-17 | 2023-05-05 | 江西农业大学 | SNP molecular marker for identifying Taihe black-bone chicken variety and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1643162A (en) * | 2002-03-15 | 2005-07-20 | 衣阿华州立大学研究基金公司 | Novel HMGA alleles and use of the same as genetic markers for growth, fatness, meat quality and feed efficiency traits |
| CN101307358A (en) * | 2008-06-20 | 2008-11-19 | 东北农业大学 | Haploid type marking method for predicting and identifying weight and metatarsal bone trait of chicken |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1643162A (en) * | 2002-03-15 | 2005-07-20 | 衣阿华州立大学研究基金公司 | Novel HMGA alleles and use of the same as genetic markers for growth, fatness, meat quality and feed efficiency traits |
| CN101307358A (en) * | 2008-06-20 | 2008-11-19 | 东北农业大学 | Haploid type marking method for predicting and identifying weight and metatarsal bone trait of chicken |
Non-Patent Citations (8)
| Title |
|---|
| A genome scan for quantitative trait loci associated with body weight at different developmental stages in chickens;Y. Gao等;《Animal Genetics》;20061231;第37卷;276-278 * |
| C. Song等.Evaluation of SNPs in the chicken HMGA2 gene as markers for body weight gain.《Animal Genetics》.2010,第42卷第333-336页. |
| Evaluation of SNPs in the chicken HMGA2 gene as markers for body weight gain;C. Song等;《Animal Genetics》;20101231;第42卷;333-336 * |
| Ex-FABP作为鸡腹脂性状主要候选基因的研究;仇雪梅等;《生物化学与生物物理进展》;20051231;第32卷(第5期);429-434 * |
| HMGA2基因与鸡体重周增重的相关性分析;宋迟等;《全球肉鸡产业论坛暨第二届中国白羽肉鸡产业发展大会会刊》;20100513;125-129 * |
| Y. Gao等.A genome scan for quantitative trait loci associated with body weight at different developmental stages in chickens.《Animal Genetics》.2006,第37卷276-278. |
| 仇雪梅等.Ex-FABP作为鸡腹脂性状主要候选基因的研究.《生物化学与生物物理进展》.2005,第32卷(第5期),429-434. |
| 宋迟等.HMGA2基因与鸡体重周增重的相关性分析.《全球肉鸡产业论坛暨第二届中国白羽肉鸡产业发展大会会刊》.2010,125-129. |
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| Publication number | Publication date |
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| CN102220410A (en) | 2011-10-19 |
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