CN101892318A - Method and kit for detecting correlated mutation allele of total farrow number of sow as well as application - Google Patents
Method and kit for detecting correlated mutation allele of total farrow number of sow as well as application Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting the correlated mutation allele of a total farrow number of a sow as well as the application. The method adopts a PCR (Polymerase Chain Reaction)-SSCP (Single-Strand Conformation Polymorphism) method, PCR augmentation comprises a section from 315-bit deoxyribonucleotide at the 5' terminal of Gen Bank Accession Number AX752829, PCR augmentation products are detected by gel electrophoresis, and the gene type of the sow to be detected is confirmed according to an electrophoresis result: the gene type of the sow to be detected can be GG or AA which means a homozygote if the electrophoresis result presents two straps, and the gene type of the sow to be detected can be GA which means a heterozygote if the electrophoresis result presents three straps. The detection method of the invention has simple operation, low expense and high accuracy, can realize the automatic direct detection and has higher practical breeding application values. The method and the kit of the invention can be applied to breed grices, early select grices to be selected, effectively solve the problem of long time for selecting excellent grices in practical production, decrease the breeding expense, increase the total sow farrow number and improve the economic benefits.
Description
Technical field
The present invention relates to a kind of method that detects correlated mutation allele of total farrow number of sow as, test kit and have application in the breeding of the pig of high total characters of number born and pig in cultivation.
Background technology
Reproductivity is important economic characters that influence pig industry, and the raising of reproductivity will bring huge economic benefit to pig industry production undoubtedly, but the breeding efficiency that how to improve pig reproductivity is the problem that receives much concern always.The breeding of pig mainly is divided into conventional breeding and molecular breeding two aspects, and in traditional breeding method, the foundation of selection is phenotype but not genotype normally, and this is because people can't directly know individual genotype, can only be inferred from phenotype.Owing to lack clear and definite corresponding relation between the phenotype of quantitative character and the genotype, make efficiency of selection not high.Improve the efficient of selection, optimal method should be directly to select genotype.The genotype of molecule marker can be discerned, for realize to genotypic direct selection provide may, this also be molecule marker in breeding, use main aspect.At present, utilizing cognation between objective trait and the marker gene type to carry out marker assisted selection is one of emphasis of research.
The FUT1 assignment of genes gene mapping is in No. 6 karyomit(e) 6q11, yet near this chromosomal region, be found and relevant (the Yasue H of pig farrowing proterties, et al. Analysis of allele segregation distortion in a swine reource family. Animal Biotechnology, 1999,10:147-152).FUT1 is a candidate gene of susceptibility or genetics of resistance proterties as the adhesion of F18 mediation, and with baby pig edema, disease-relateds such as diarrhoea, to influencing of animal health than can observedly Duoing several times of (Meijerink E, et al. Two alpha (1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci. Mammalian Genome, 1997,8:736-741).Because the very direct reproductive performance that influences of animal health, the FUT1 gene has been considered to be a candidate gene of litter size of pig.Buske or the like studies show that market pig: the ratio height of the genotypic sow of heterozygote AB in the high yield group, and the ratio height of the genotypic sow of BB in the low yield group, illustrate that the AB genotype helps litter size (Buske B, et al. Analysis of association of GPX5, FUT1 and ESR2 genotypes with litter size in a commercial pig cross population. Archives of Animal Breeding, 2006,49:259-268).Horak etc. discover FUT1 in Prestice piebald pig
G/ FUT1
GGenotypic sow obviously surpasses FUT1 on the total litter size of 1-6 tire
A/ FUT1
AGenotypic sow (Horak P, et al. The FUT1 and ESR genes-their variability and associations with reproduction in Prestice Black-Pied sows. Journal of Animal Breeding and Genetics, 2005,122:210-213).
Zhang Yinhong etc. detect the swinery FUT1 gene polymorphic of 26 kinds in China and foreign countries, and the result shows,
Hin6On the I site, 3 external pig kinds such as Large White, landrace and Du Luoke all exist polymorphic, and in the majority with GG type and AG type; All test sample of Shanxi Black pig, Taiyuan flower pig and 3 local pig kinds of length pig all show as the GG type.With the relation between variance analysis FUT1 genotype, kind and parity and the farrowing proterties, genotype and kind are remarkable to the influence of total litter size of pig, and how many 3 kinds of genotype litter size of pig averages are followed successively by AA〉AG〉GG, parity is not remarkable to total litter size influence; And genotype, kind and parity are all not remarkable to producing the young number influence of living.Employing PCR-RFLP methods such as Sun Pengxiang are analyzed the pig FUT1 gene pleiomorphism of reviving too.The result shows: the too pig FUT1 gene open reading frame 307 site warps of reviving
Hin6After the I enzyme is cut, produce GG type and AG type, there is not resistance homozygote AA type, its the 2 kinds of genotype and the correlation results between the pig production performance of reviving too show that the individual average production performance of AG type all is higher than GG type individuality, and the weight of weaning litter of AG type individuality, the tire digital display work of always farrowing is higher than GG type individuality (P＜0.05), and for some production traitss, the AG type is the beneficial gene type.Sieve still Zhuo waits discovery, in Pig Beijing Black multiparity sow, FUT1 gene A A genotype individuality is than the TNB obvious high 1.11 (P＜0.05) of GG type individuality, in TNB and NBA size general trend is AA〉AG〉sieve GG(is still tall and erect. the association analysis of the polymorphism of 10 genes of Pig Beijing Black and farrowing proterties. 2008, Shaanxi Yang Ling: Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology, master thesis).
In sum, the FUT1 gene has become a main candidate gene of imitating that can influence the total litter size of sow.
Summary of the invention
The objective of the invention is to: a kind of method that detects correlated mutation allele of total farrow number of sow as, test kit are provided and have application in the breeding of the pig of high total characters of number born and pig in cultivation.
The method of detection correlated mutation allele of total farrow number of sow as provided by the invention is: adopt PCR-SSCP method, pcr amplification comprises the fragment from 5 ' terminal the 315th deoxyribonucleotide of GenBank Accession Number AX752829, the detected through gel electrophoresis pcr amplification product, determine the genotype of sow to be measured according to electrophoresis result: if electrophoresis showed is two bands, the genotype of sow to be measured is GG or AA, is homozygote; If electrophoresis showed is three bands, the genotype of sow to be measured is GA, is heterozygote.
Aforementioned pcr amplification is a template with the genomic dna of sow to be measured; Used primer is to being sequence 1(5 '-ATCTCCCGTGGCCATATTCT-3 ') and sequence 2(5 '-ATGGCAGGCTGGATGAAG-3 ') primer formed is right.When adopting this primer when carrying out pcr amplification, according to the difference of the genomic dna of sow to be measured, the dna fragmentation that amplification obtains is the nucleotide sequence shown in nucleotide sequence shown in the sequence 3 or the sequence 4.
Carry out sow genotype to be measured and declare the method for type and be by detecting correlated mutation allele of total farrow number of sow as: the primer of forming with sequence 5 '-ATCTCCCGTGGCCATATTCT-3 ' and sequence 5 '-ATGGCAGGCTGGATGAAG-3 ' is to being primer, to genotype is GG, AA, three kinds of pig DNAs of GA are carried out pcr amplification respectively, amplified production reclaims test kit with sepharose respectively and reclaims purifying, PCR product behind the purifying adopts the SSCP gel electrophoresis method to carry out gel electrophoresis, and the band behind the electrophoresis is made as standard substance: S1 in test kit be GG genotype standard substance, S2 is AA genotype standard substance, and S3 is GA genotype standard substance; When declaring the genotype of type sow to be measured, adopt PCR-SSCP method, in same polyacrylamide gel, the PCR product of while three standard substance of electrophoresis and the sow gained to be measured that increases, the identical person with S1 of band is the GG genotype behind the electrophoresis, the identical person with S2 of band is the AA genotype, and the identical person with S3 of band is the GA genotype.
The present invention also provides a kind of test kit that detects correlated mutation allele of total farrow number of sow as, contains sequence 1(5 '-ATCTCCCGTGGCCATATTCT-3 ') and sequence 2(5 '-ATGGCAGGCTGGATGAAG-3 ') primer formed to and S1, S2, three standard substance of S3; S1 is GG genotype standard substance, and S2 is AA genotype standard substance, and S3 is GA genotype standard substance.
The aforementioned agents box can be used for preparing the test kit of total characters of number born of auxiliary detection sow.
The above method, described test kit all can be used for cultivating the pig with high total characters of number born, thereby are applied to the breeding of pig.
Sow to be measured of the present invention specifically can be Pig Beijing Black.
The difference (G/A) of pig GenBank Accession Number AX752829 from a deoxyribonucleotide of 5 ' terminal the 315th deoxyribonucleotide existence.Experiment shows that the litter size of the genotypic sow of AA is higher than the litter size of the genotypic sow of GG.In the multiparity stage, AA homozygous genotype colony is than high about 1.11 of the total litter size of GG homozygous genotype colony, so this mutation allele can be used as the total litter size trait molecular of pig breeding mark.
The present invention adopt the PCR-SSCP method detect pig GenBank Accession Number AX752829 from 5 ' terminal the 315th deoxyribonucleotide, it is very accurate that genotype is declared type, testing cost is cheap, has very high breeding practice using value.With the method for the invention the total characters of number born of pig is selected, can be made total litter size of swinery multiparity stage improve about 1.11, thereby obtain considerable economic.
Use method provided by the present invention pig is carried out breeding, can carry out early screening to pig to be selected, alleviate effectively and selected long problem of good boar time in the actual production, reduce the breeding cost, effectively increase the total litter size of the pig in the actual production, improved economic benefit.Detection method of the present invention is simple to operate, expense is cheap, accuracy is high, and can realize automatic direct detection.Method provided by the invention and test kit will be played a great role in the breeding work of pig.
Description of drawings
Fig. 1 be comprise pig GenBank Accession Number AX752829 from the pcr amplified fragment of 5 ' terminal the 315th deoxyribonucleotide and the SSCP electrophorogram of three standard substance.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Primer in following examples is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.
Embodiment 1: the selection of good boar in the breeding of pig
One, the foundation of PCR-SSCP detection method and pleomorphism site are determined
1, pcr amplification
According to pig GenBank Accession Number AX752829 sequences Design primer, primer is as follows:
The U(upstream primer): 5 '-ATCTCCCGTGGCCATATTCT-3 ' (sequence 1);
The D(downstream primer): 5 '-ATGGCAGGCTGGATGAAG-3 ' (sequence 2).
The selection Pig Beijing Black is an experiment material.Genomic dna with Pig Beijing Black is a template, uses primer U and D to carry out pcr amplification.
Amplification system is: genomic dna 200ng, 10 * pcr amplification damping fluid, 2.5 μ l, dNTPs 5mM, each 50ng of upstream and downstream primer, Taq archaeal dna polymerase 0.75U, Mg
2+2.5mmol/L, use ddH
2O postreaction system to 25 μ l.
The PCR reaction conditions is: 95 ℃ of sex change 5min; 95 ℃ of sex change 20s then, 52.4 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; Last 72 ℃ are extended 10min.
The PCR product after sepharose detects in-20 ℃ of preservations.
2, sscp analysis
Get each 1.5ul of PCR product of step 1, respectively with 8.5ul sample-loading buffer (98% methane amide, 0.09% tetrabromophenol sulfonphthalein, 0.09% dimethylbenzene cyanogen, 2% glycerine, 0.1mol/L EDTA) mix, 98 ℃ of sex change 10 minutes were put cooled on ice rapidly 10 minutes, carried out 12% polyacrylamide gel electrophoresis (12 hours, voltage 8V/cm), silver dyes colour developing, according to coloration result analyzing gene type.The result as shown in Figure 1, above-mentioned PCR product has three kinds of banding patterns, shows that there is pleomorphism site in extension increasing sequence, and homozygous genotype pig individuality produces 2 kinds of banding patterns (AA type, GG type) through amplification, produce two bands respectively, heterozygous genes type pig individuality produces three bands (GA type) through amplification.
3, cloning and sequencing and sequential analysis
Respectively the individual PCR product of two kinds of homozygous genotypes is reclaimed test kit (day root biochemical technology company limited) with sepharose and reclaim purifying, behind the dna fragmentation connection carrier pGEM-T (Promega company) that reclaims, to connect product transformed into escherichia coli DH5 α competent cell (proud (Beijing) Science and Technology Ltd. tomorrow hundred), according to the carboxylic Bian penicillin resistance label screening positive colony on the carrier, obtain containing the segmental recombinant plasmid of recovery.With T7 on this recombinant plasmid vector and SP6 promoter sequence is that primer carries out nucleotide sequencing (the excellent Bo Ao in Beijing Bioisystech Co., Ltd) to it.Sequencing result shows: product 1 is sequence 3, and product 2 is a sequence 4; The length of sequence 3 and sequence 4 is 201bp, the difference (G/A) that only has a deoxyribonucleotide, this polymorphic site (called after G315A) is arranged in sequence table from the 161st deoxyribonucleotide of 5 ' end, i.e. GenBank Accession Number AX752829's from 5 ' terminal the 315th deoxyribonucleotide.When if the G315A of sow to be measured is G, its homozygotic genotype is GG; When if the G315A of sow to be measured is A, its homozygotic genotype is AA; Their heterozygote genotype is GA.
4, three kinds of genotype standard substance designs
(the GG genotype is S1 to get in the step 2 three kinds of genotype PCR products, the AA genotype is S2, the GA genotype is S3) each 30ul, reclaim test kit (day root biochemical technology company limited) with sepharose respectively and reclaim purifying, the PCR product behind the purifying is carried out gel electrophoresis according to above-mentioned SSCP gel electrophoresis method.From Fig. 1 as a result as can be seen, behind S1, S2, the S3 electrophoresis band clear, do not have assorted band, to declare type clear, can be made as standard substance: S1 in this test kit is GG genotype standard substance, S2 is AA genotype standard substance, S3 is GA genotype standard substance.In same polyacrylamide gel, the PCR product of three standard substance of electrophoresis and primer amplification that this test kit provides pig gained to be detected as Fig. 1, can be differentiated the genotype of the pig of examining clear, accurately and quickly simultaneously.
Two, the correlation analysis of G315A polymorphic site and the total litter size of pig
For determining whether the G315A polymorphic site is relevant with the total litter size of pig, is experiment material with 314 Pig Beijing Black sows, carries out following test: extract genome and carry out PCR-SSCP analysis, the same step 1 of method; Write down parity, total litter size isophenous simultaneously, G315A site and total litter size are carried out the least square association analysis.The results are shown in following table 1.
Annotate: the same column subscript contains same letter represents difference not significantly (P〉0.05).
The result shows: in the multiparity parity, AA genotype individuality is being Duoed 1.11 than GG genotype individuality aspect total litter size respectively; G315A site and multiparity stage total farrowing digital display work related (P＜0.05).
So, in the pig breeding of reality, preferably select the genotypic pig of AA to carry out breeding.
More than used Pig Beijing Black all come from Beijing generation Xinhua and contain animal husbandry Science and Technology Ltd..
Sequence table
Sequence 1:ATCTCCCGTGGCCATATTCT
Sequence 2:ATGGCAGGCTGGATGAAG
Sequence 3(AA genotype sequence):
ATCTCCCGTGGCCATATTCTGCCTGGCGGGCACGCCGGTACACCCCAACGCCTCCGATTCCTGTCCCAAGCATCCTGCCTCCTTTTCCGGGACCTGGACTATTTACCCGGATGGCCGGTTTGGGAACCAGATGGGACAGTATGCCACGCTGCTGGCCCTGACGCAGCTCAACGGCCGCCAGGCCTTCATCCAGCCTGCCAT
Sequence 4(GG genotype sequence):
ATCTCCCGTGGCCATATTCTGCCTGGCGGGCACGCCGGTACACCCCAACGCCTCCGATTCCTGTCCCAAGCATCCTGCCTCCTTTTCCGGGACCTGGACTATTTACCCGGATGGCCGGTTTGGGAACCAGATGGGACAGTATGCCACGCTGCTGGCCCTGGCGCAGCTCAACGGCCGCCAGGCCTTCATCCAGCCTGCCAT
Claims (8)
1. method that detects correlated mutation allele of total farrow number of sow as, it is characterized in that: adopt PCR-SSCP method, pcr amplification comprises the fragment from 5 ' terminal the 315th deoxyribonucleotide of GenBank Accession Number AX752829, the detected through gel electrophoresis pcr amplification product, determine the genotype of sow to be measured according to electrophoresis result: if electrophoresis showed is two bands, the genotype of sow to be measured is GG or AA, is homozygote; If electrophoresis showed is three bands, the genotype of sow to be measured is GA, is heterozygote.
2. according to the method for the described detection correlated mutation allele of total farrow number of sow as of claim 1, it is characterized in that: the used primer of pcr amplification is right to the primer of forming for sequence 5 '-ATCTCCCGTGGCCATATTCT-3 ' and sequence 5 '-ATGGCAGGCTGGATGAAG-3 '.
3. according to the method for the described detection correlated mutation allele of total farrow number of sow as of claim 1, it is characterized in that: the genomic dna with sow to be measured is that template is carried out pcr amplification.
4. carry out the method that sow genotype to be measured is declared type by detecting correlated mutation allele of total farrow number of sow as, it is characterized in that: the primer of forming with sequence 5 '-ATCTCCCGTGGCCATATTCT-3 ' and sequence 5 '-ATGGCAGGCTGGATGAAG-3 ' is to being primer, to genotype is GG, AA, three kinds of pig DNAs of GA are carried out pcr amplification respectively, amplified production reclaims test kit with sepharose respectively and reclaims purifying, PCR product behind the purifying adopts the SSCP gel electrophoresis method to carry out gel electrophoresis, and the band behind the electrophoresis is made as standard substance: S1 in test kit be GG genotype standard substance, S2 is AA genotype standard substance, and S3 is GA genotype standard substance; When declaring the genotype of type sow to be measured, adopt PCR-SSCP method, in same polyacrylamide gel, the PCR product of while three standard substance of electrophoresis and the sow gained to be measured that increases, the identical person with S1 of band is the GG genotype behind the electrophoresis, the identical person with S2 of band is the AA genotype, and the identical person with S3 of band is the GA genotype.
5. test kit that detects correlated mutation allele of total farrow number of sow as is characterized in that: contain primer that sequence 5 '-ATCTCCCGTGGCCATATTCT-3 ' and sequence 5 '-ATGGCAGGCTGGATGAAG-3 ' forms to and S1, S2, three standard substance of S3; S1 is GG genotype standard substance, and S2 is AA genotype standard substance, and S3 is GA genotype standard substance.
6. the application of test kit as claimed in claim 5 in the test kit of total characters of number born of preparation auxiliary detection sow.
As among the claim 1-4 as described in each method have application in the breeding of the pig of high total characters of number born and pig in cultivation.
8. has application in the breeding of the pig of high total characters of number born and pig as test kit as described in claim 5 or 6 in cultivation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651526A (en) * | 2015-03-16 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and application of SNP marker |
| CN104651527A (en) * | 2015-03-17 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and primers of SNP marker |
| CN104694651A (en) * | 2015-03-16 | 2015-06-10 | 南京农业大学 | SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and detection method and application thereof |
| CN104694652A (en) * | 2015-03-16 | 2015-06-10 | 南京农业大学 | SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and primers and application thereof |
| CN114807143A (en) * | 2022-05-27 | 2022-07-29 | 南京农业大学 | IncRNA causal mutation site related to total litter size trait of pig and application thereof |
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| CN101724705A (en) * | 2010-01-19 | 2010-06-09 | 扬州大学 | Molecular detection kit for detecting resistance of pig Escherichia coli F18 and application thereof |
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Patent Citations (1)
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| CN101724705A (en) * | 2010-01-19 | 2010-06-09 | 扬州大学 | Molecular detection kit for detecting resistance of pig Escherichia coli F18 and application thereof |
Non-Patent Citations (2)
| Title |
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| 《中国畜牧杂志》 20090215 包文斌 等 杜洛克FUT1基因多态性及其与产仔性能的关联分析 5-7页 1-8 第45卷, 第3期 2 * |
| 《扬州大学学报( 农业与生命科学版)》 20060630 吴圣龙 等 猪alpha1岩藻糖转移酶基因点突变的PCR-SSCP检测 68-71页 1-8 第27卷, 第2期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651526A (en) * | 2015-03-16 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and application of SNP marker |
| CN104694651A (en) * | 2015-03-16 | 2015-06-10 | 南京农业大学 | SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and detection method and application thereof |
| CN104694652A (en) * | 2015-03-16 | 2015-06-10 | 南京农业大学 | SNP (single nucleotide polymorphism) marker related to Erhualian sow litter traits and primers and application thereof |
| CN104651527A (en) * | 2015-03-17 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and primers of SNP marker |
| CN114807143A (en) * | 2022-05-27 | 2022-07-29 | 南京农业大学 | IncRNA causal mutation site related to total litter size trait of pig and application thereof |
| CN114807143B (en) * | 2022-05-27 | 2023-04-28 | 南京农业大学 | lncRNA causal mutation site related to pig total litter size trait and application thereof |
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Application publication date: 20101124 |