CN101956009A - Method for detecting number of littermate and characters of sow - Google Patents
Method for detecting number of littermate and characters of sow Download PDFInfo
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Abstract
The invention discloses a method for detecting number of littermates and characters of a sow, which comprises the steps of: 1, detecting whether the 71th nucleotide from 5' end of a DNA segment shown in SEQ ID NO:1 in a genome of a sow to be detected is G or A; detecting whether the 81th nucleotide from 5' end of a DNA segment shown in SEQ ID NO:2 in the genome of the sow to be detected is C or A; and determining the haplotype of the sow to be detected, determining the number of the littermates and the characters of the sow to be detected according to the haplotype. The method can be used for detecting reproduction characters of the sow in a development early stage and knowing the genetic differentiation degree of the sow, provides a useful molecular marker for genotyping, screening and auxiliary breeding of different haplotypes of the sow, and has important meanings on improving the number of the littermates of the sow. Therefore, the method is beneficial to the quickening of the breeding speed and the reducing of the breeding cost, has the advantages of low cost, simple operation and short period, and is suitable for popularization and application.
Description
Technical field
The present invention relates to a kind of method that pigsty produces the young number proterties of living that detects.
Background technology
(luteinizing hormone is that molecular weight is 28000D by a kind of glycoprotein of adenohypophysis basophilic cell excretory LH) to prolan B.LH and FSH, TSH, CG etc. are made up of through non-conjugated combination α and β two subunits, utilize different processing to separate at an easy rate.Wherein the α subunit is identical in four kinds of hormones, and the β subunit has determined the exceptional function of various hormones.The effect of LH in buck mainly is to stimulate interstitial glands to produce male sex hormone, and influences the production of sperm process of convoluted seminiferous tubule by male sex hormone.In jenny, LH is that interior testosterone of ovary and estradiol are synthetic necessary, also can induce the synthetic of the interior PgR of progesterone and granulosa cell simultaneously.In the blood, the appearance of LH peak value induces mature follicle to discharge ovum in oestrus cycle of adult animals or people's menstrual cycle, and the luteinization and the corpus luteum that start ovarian follicle form.
Mammiferous LH subunit is made up of 100-130 amino acid.Have very high homology between different plant species β subunit, the gene of its coding also all contains 3 exons and 2 introns.
Japan Orita etc. discovers, the single stranded DNA fragment is complicated space folded conformation, this three-dimensional arrangement is mainly kept by interaction force in its inner base pairing equimolecular, when a base changes, can influence its space conformation more or less, conformation is changed, and the discrepant single strand dna of space conformation is varied in size by exclusion in polyacrylamide gel.Therefore by native polyacrylamide gel electrophoresis (PAGE), can very observantly discrepant molecular separation on the conformation be opened, this method be single strand conformation polymorphism (Single-Strand Conformation Polymorphism,, SSCP) analyze.SSCP is used to check the transgenation of pcr amplification product, has set up the PCR-SSCP technology, further improved the simplicity and the susceptibility that detect mutation method.Its primary process is: 1. pcr amplification target DNA; 2. with special pcr amplification product sex change, then snapback makes it to become the single strand dna with certain space structure; 3. an amount of single stranded DNA is carried out native polyacrylamide gel electrophoresis; 4. dye by radioactive automatic developing, silver at last or ethidium bromide chromogenic assay result.If discovery single stranded DNA band mobility is compared with normal control change, just can judge that this chain conformation changes, and then infer in this dna fragmentation that base mutation is arranged.This method is easy, quick, sensitive, does not need special instrument, is fit to the needs of clinical experiment.But it also has weak point.For example, can only to determine the position and the type of sudden change at last, also need further order-checking as a kind of sudden change detection method; Deposition condition requires strict; In addition, because SSCP causes that according to point mutation the change of single strand dna three-dimensional conformation realizes electrophoretic separation, in the time of point mutation when some position so just may occurring inoperative or effect be very little to the change of single strand dna three-dimensional conformation, add other condition effect, polyacrylamide gel electrophoresis can't be differentiated cause omission.However this method is compared the higher detection rate that still has with additive method.At first, it can find the base mutation of unknown position in the target dna fragment.Takao the experiment proved that less than the single base mutation in the dna fragmentation of 300bp 90% can be found by SSCP, and he thinks that most available these methods of all single sequence changes that it is now know that detect.In addition, the SSCP method can be separated by the sudden change single stranded DNA of polyacrylamide gel electrophoresis with different mobilities, and can further purify.Can finally differentiate the mutant DNA fragment in this way from the dna sequence dna level.
It is one of leading indicator of estimating swine reproduction performance that pigsty produces the young number of living, and is a quantitative character that heritability is very low, is difficult to improve nest with conventional breeding technique and produces the young number proterties of living.
Summary of the invention
An object of the present invention is to provide the method that a kind of auxiliary detection pigsty produces the young number proterties of living.
The nest of auxiliary discriminating pig provided by the present invention produces the method for the young number proterties of living, and is following 1) or 2) shown in:
What 1) comprise the steps: to detect dna fragmentation shown in the SEQ ID NO:1 in the genome of pig to be measured is G or A from 5 ' terminal the 71st Nucleotide; What detect dna fragmentation shown in the SEQ ID NO:2 in the genome of pig to be measured is C or A from 5 ' terminal the 81st Nucleotide; Determine the haplotype of described pig to be measured, determine that according to described haplotype the nest of described pig to be measured produces the young number proterties of living;
2) introne 1 that comprises the steps: to detect the LH subunit gene of pig to be measured is G or A from the base of 5 ' terminal the 67th Nucleotide, the intron 2 that detects the LH beta subunit gene of described pig to be measured is C or A from the base of 5 ' terminal the 30th Nucleotide, determine the haplotype of described pig to be measured, determine that according to described haplotype the nest of described pig to be measured produces the young number proterties of living;
Described 1) in, the method for the haplotype of described definite described pig to be measured is:
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACC;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AADD;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCC;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBDD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCC;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABDD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCD;
Described 2) in, the method for the haplotype of described definite described pig to be measured is:
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AADD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCD;
The nest of described haplotype correspondence produces the young number of living and is followed successively by AACC＞BBDD＞ABCC＞AADD=ABCD＞BBCC＞ABDD＞BBCD＞AACD from high to low.
In the aforesaid method, dna fragmentation shown in the SEQ ID NO:1 is that G or the method for A comprise the steps: with primer I to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 71st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is G or A according to dna fragmentation shown in the SEQ ID NO:1 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 71st Nucleotide;
In the aforesaid method, dna fragmentation shown in the SEQ ID NO:2 is that C or the method for A comprise the steps: with primer II to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 81st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is C or A according to dna fragmentation shown in the SEQ ID NO:2 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 81st Nucleotide;
In the aforesaid method, described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4;
In the aforesaid method, described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
It is right that another object of the present invention provides a kind of primer that is used for assisting the nest of differentiating pig to produce the young number proterties of living.
The primer that the nest that is used for auxiliary discriminating pig provided by the present invention produces the young number proterties of living is right, primer I and primer is made of II; Described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4; Described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
Another object of the present invention provides a kind of method of cultivating pig.
The method of cultivation pig provided by the present invention is following 1) or 2) shown in:
What 1) comprise the steps: to detect dna fragmentation shown in the SEQ ID NO:1 in the genome of pig to be measured is G or A from 5 ' terminal the 71st Nucleotide; What detect dna fragmentation shown in the SEQ ID NO:2 in the genome of pig to be measured is C or A from 5 ' terminal the 81st Nucleotide; Determine the haplotype of described pig to be measured, select the pig of the haplotype of AACC to carry out breeding;
2) introne 1 that comprises the steps: to detect the LH beta subunit gene of pig to be measured is G or A from the base of 5 ' terminal the 67th Nucleotide, the intron 2 that detects the LH beta subunit gene of described pig to be measured is C or A from the base of 5 ' terminal the 30th Nucleotide, determine the haplotype of described pig to be measured, select the pig of the haplotype of AACC to carry out breeding;
Described 1) in, the method for the haplotype of described definite described pig to be measured is:
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACC;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AADD;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCC;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBDD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCC;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABDD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCD;
Described 2) in, the method for the haplotype of described definite described pig to be measured is:
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AADD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCD;
In the method for above-mentioned cultivation pig, dna fragmentation shown in the SEQ ID NO:1 is that G or the method for A comprise the steps: with primer I to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 71st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is G or A according to dna fragmentation shown in the SEQ ID NO:1 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 71st Nucleotide;
In the method for above-mentioned cultivation pig, dna fragmentation shown in the SEQ ID NO:2 is that C or the method for A comprise the steps: with primer II to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 81st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is C or A according to dna fragmentation shown in the SEQ ID NO:2 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 81st Nucleotide;
In the method for above-mentioned cultivation pig, described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4;
In the method for above-mentioned cultivation pig, described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
Result of study of the present invention shows that LH β gene may be that the control pigsty produces a major gene of the young number of living or has genetic linkage closely with it.Therefore can utilize 67 and 30 these two mutational sites of intron 2 of LH β gene intron 1, with its haplotype nest be produced the young number proterties of living and select.The inventive method can be at the reproductive trait of growing the early detection pig, understand the genetic variation and genetic differentiation degree of pig, somatotype, screening and assistant breeding for the different genotype of pig provide useful molecule marker, its selection, change that to produce the young number of living significant for improving the sow nest.Therefore, the present invention will play a great role in the breeding of pig, for the marker assisted selection and the breeding of pig prolificacy provides the theory and practice basis, can shorten breeding cycle, improve breeding efficiency, and will bring considerable economic to the producer.
Description of drawings
Fig. 1 is the primer location of pig LH β gene.
Fig. 2 is the SSCP electrophorogram of primer P1.
Fig. 3 is the SSCP electrophorogram of primer P3.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can be bought from biotech firm among the following embodiment.
The single nucleotide polymorphism analysis of embodiment 1, LH β gene
Laboratory animal blood that exsomatizes or tissue samples are planted pig farm, kind pig farm, long river, Tianjin, benefit non-hibernating eggs pig farm, Yantai and Changshu, Jiangsu seed stock breeding station from Jiangxi Province.
1, the extraction of genomic dna:
Get 500 μ l cracking blood samples and add 350 μ l blood extracting solutions, mixing; Add Proteinase K (20mg/ml) 4 μ l, to final concentration 200 μ g/ml; After 50-55 ℃ of digestion is spent the night, add the 5MNacl 173ml of preheating, to final concentration 1.5M, mixing; With the extracting of equal-volume chloroform; Get supernatant, use saturated phenol: chloroform (1: 1) extracting; Water adds the dehydrated alcohol of 2 times of volumes.Centrifugal then, remove supernatant, precipitate washing with alcohol with 70%, centrifugal final vacuum or air drying, precipitation is dissolved in the aseptic double-distilled water.
2, PCR-SSCP process:
(1) design primer: according to pig LH β gene complete sequence design primer, the 1296-3335 fragment in this gene 3335bp complete sequence that increases, primer location is as shown in Figure 1.Primer sequence is as shown in table 1.
The primer sequence of table 1, amplification pig LH beta subunit gene
(2) pcr amplification
PCR reaction system: 10 * amplification buffer 1.5ul
4dNTP mixture (4 * 2.5mmol/L) 1.2ul
Primer (10pmol/ul) 1.5ul * 2
Template DNA (100ng/ul) 1ul
Taq archaeal dna polymerase (3u/ul) 1ul
ddH
2O 7.3ul
Cumulative volume 15ul
Reaction conditions: 94 ℃ of pre-sex change 5 minutes; Cycling condition (94 ℃ of sex change, 30 seconds; 51-63 ℃ of annealing (P1:54 ℃, P2:55 ℃, P3:51 ℃, P4:52 ℃, P5:57 ℃, P6:56 ℃, P7:58 ℃, P8:60 ℃, P9:63 ℃), 30 seconds; 72 ℃ of extensions, 1 minute); 30 circulations; 72 ℃ were extended 7 minutes; 4 ℃ of preservations.
(3) 14% non-denaturing polyacrylamide gels (30ml volume 0.1cm adhesive tape) electrophoresis:
Get 1.5 μ lPCR products and place the PCR pipe, add 5 μ l sex change Buffer, 98 ℃ of sex change 10 minutes; Ice bath is 10 minutes rapidly, uses the microsyringe point sample; 140 volts of opening power, electrophoresis 14-16 hour; Silver dyes colour developing.
The electrophorogram of the product that primer P1 amplification obtains as shown in Figure 2, the electrophorogram of the product that primer P3 amplification obtains as shown in Figure 3, the product band that utilizes primer P1 and P3 amplification to obtain there are differences.Among Fig. 2, for homozygote, the contained base difference of its dna double chain, its space conformation is also inequality after the sex change, therefore obtains two bands; Only under situation once in a while, its double-stranded space conformation possibility difference is little, so electrophoresis is inseparable, as the homozygote of Fig. 3.Generally speaking, homozygote is the 1-2 band, and heterozygote is the 2-4 band.
The product that primer P1 (SEQ ID NO:3 and SEQ ID NO:4) and P3 (SEQ ID NO:5 and SEQ ID NO:6) amplification is obtained checks order respectively, and the nucleotide sequence of the product that primer P1 amplification obtains is shown in sequence in the sequence table 1; The nucleotide sequence of the product that primer P3 amplification obtains is shown in sequence in the sequence table 2; Order-checking shows that there is 1 single nucleotide variation respectively in the product that primer P1 and P3 amplification obtain.There is single nucleotide mutation in primer P1 amplified production fragment, the mutational site be arranged in sequence table sequence 1 from 5 ' terminal the 71st Nucleotide (sequence of introne 1 that is pig LH beta subunit gene is from 5 ' terminal the 67th Nucleotide), be that G → A suddenlys change; There is single nucleotide mutation in primer P3 amplified production fragment, the mutational site be arranged in sequence table sequence 2 from 5 ' terminal the 81st Nucleotide (sequence of intron 2 that is pig LH beta subunit gene is from 5 ' terminal the 30th Nucleotide), be that C → A suddenlys change.
When the 67th bit base of introne 1 was G, the genotype of its homozygote pig note was made AA; When the 67th bit base of introne 1 was A, the genotype of its homozygote pig note was made BB; When the 67th bit base of introne 1 had plenty of A and has plenty of G, the genotype note of its heterozygote pig was made AB; When the 30th bit base of intron 2 was C, the genotype of its homozygote pig note was made CC; When the 30th bit base of intron 2 was A, the genotype of its homozygote pig note was made DD; When the 30th bit base of intron 2 had plenty of C and has plenty of A, the genotype note of its heterozygote pig was made CD;
Altogether 329 pigs on 101 pigs on 78 pigs planting the pig farm from Jiangxi Province, kind pig farm, long river, Tianjin, benefit non-hibernating eggs pig farm, Yantai and 120 pigs of Changshu, Jiangsu seed stock breeding station have been carried out single nucleotide polymorphism analysis.The genotype of the individual LH beta subunit gene introne 1 of pig and 2 is merged into haplotype, obtain following haplotype: AACC, AADD, AACD, BBCC, BBDD, BBCD, ABCC, ABDD, ABCD altogether.
The single nucleotide polymorphism of embodiment 2, pig LH beta subunit gene and the correlation analysis of reproductive trait
It is one of leading indicator of estimating swine reproduction performance that pigsty produces the young number of living, and it is meant in the sow pig that every nest is farrowed, the young number of work that can survive and.The number that its recording method can be survived and for record wean back piglet.
Determine the haplotype of pig individuality according to the method among the embodiment 1.
The pigsty of the multiparity pig of benefit non-hibernating eggs pig farm, record Yantai and Changshu, Jiangsu seed stock breeding station produces the young number of living, detect the haplotype of each pig, utilize the least square analytical procedure that the haplotype of pig and the relation of multiparity pigsty product young number proterties alive have been carried out statistical study.Statistic analysis models:
y
ijkl＝μ+B
i+HYS
j+G
k+e
ijkl
Y wherein
IjklBe i kind, in the production performance observed value of the k kind haplotype individuality of j field-year-Ji; μ is the least square average of production performance; B
iBe i level of kind effect value to production performance; HYS
jBe j the level of field-year-Ji effect value to production performance; G
kBe k level of haplotype effect value to production performance; e
IjklBe random residual effect corresponding to observed value.
Utilize the PROC-ANOVA process of SAS statistical package to carry out statistical study, result such as table 2.
Table 2, nest produce the least square analysis of the young number of living
By in the table as can be seen each haplotype produce to live aspect the young number proterties at nest, least square average maximum be AACC haplotype individuality, minimum is AACD haplotype individuality, the two differs 4.71.The P value of the test of difference of AACD and ABCD, AACD and AADD and AACD and AACC haplotype is respectively 0.0634,0.0661 and 0.0594 all near 0.05 significance level in addition.The nest of each haplotype correspondence produces the young number of living and is followed successively by AACC＞BBDD＞ABCC＞AADD=ABCD＞BBCC＞ABDD＞BBCD＞AACD from high to low.
The selection haplotype is that the pig of AACC carries out breeding.
Can detect 9 kinds of all haplotypes in the colony of reproductive trait record is arranged, the result that least square is analyzed shows: the P value of the test of difference of AACD and ABCD, AACD and AADD, AACD and AACC haplotype is all near 0.05 significance level.This shows that the effect of haplotype is not the simple addition of genotype effect separately, but want a little higher than single best genotype effect, this conclusion has special significance for utilizing dna marker to carry out assisted Selection, shows that the genetic improvement of assessing colony must be as the criterion with the influence of haplotype.
Claims (5)
1. the auxiliary nest of pig of differentiating produces the method for the young number proterties of living, and is following 1) or 2) shown in:
What 1) comprise the steps: to detect dna fragmentation shown in the SEQ ID NO:1 in the genome of pig to be measured is G or A from 5 ' terminal the 71st Nucleotide; What detect dna fragmentation shown in the SEQ ID NO:2 in the genome of pig to be measured is C or A from 5 ' terminal the 81st Nucleotide; Determine the haplotype of described pig to be measured, determine that according to described haplotype the nest of described pig to be measured produces the young number proterties of living;
2) introne 1 that comprises the steps: to detect the LH beta subunit gene of pig to be measured is G or A from the base of 5 ' terminal the 67th Nucleotide, the intron 2 that detects the LH beta subunit gene of described pig to be measured is C or A from the base of 5 ' terminal the 30th Nucleotide, determine the haplotype of described pig to be measured, determine that according to described haplotype the nest of described pig to be measured produces the young number proterties of living;
Described 1) in, the method for the haplotype of described definite described pig to be measured is:
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACC;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AADD;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCC;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBDD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCC;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABDD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCD;
Described 2) in, the method for the haplotype of described definite described pig to be measured is:
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AADD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCD;
The nest of described haplotype correspondence produces the young number of living and is followed successively by AACC＞BBDD＞ABCC＞AADD=ABCD＞BBCC＞ABDD＞BBCD＞AACD from high to low.
2. method according to claim 1 is characterized in that:
Dna fragmentation shown in the SEQ ID NO:1 is that G or the method for A comprise the steps: with primer I to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 71st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is G or A according to dna fragmentation shown in the SEQ ID NO:1 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 71st Nucleotide;
Dna fragmentation shown in the SEQ ID NO:2 is that C or the method for A comprise the steps: with primer II to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 81st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is C or A according to dna fragmentation shown in the SEQ ID NO:2 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 81st Nucleotide;
Described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4;
Described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
3. it is right that the nest that is used for auxiliary discriminating pig produces the primer of the young number proterties of living, and primer I and primer is made of II; Described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4; Described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
4. a method of cultivating pig is following 1) or 2) shown in:
What 1) comprise the steps: to detect dna fragmentation shown in the SEQ ID NO:1 in the genome of pig to be measured is G or A from 5 ' terminal the 71st Nucleotide; What detect dna fragmentation shown in the SEQ ID NO:2 in the genome of pig to be measured is C or A from 5 ' terminal the 81st Nucleotide; Determine the haplotype of described pig to be measured, select the pig of the haplotype of AACC to carry out breeding;
2) introne 1 that comprises the steps: to detect the LH beta subunit gene of pig to be measured is G or A from the base of 5 ' terminal the 67th Nucleotide, the intron 2 that detects the LH beta subunit gene of described pig to be measured is C or A from the base of 5 ' terminal the 30th Nucleotide, determine the haplotype of described pig to be measured, select the pig of the haplotype of AACC to carry out breeding;
Described 1) in, the method for the haplotype of described definite described pig to be measured is:
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACC;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AADD;
If dna fragmentation shown in the SEQ ID NO:1 is G from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made AACD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCC;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBDD;
If dna fragmentation shown in the SEQ ID NO:1 is A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made BBCD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is C from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCC;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 is A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABDD;
If dna fragmentation shown in the SEQ ID NO:1 has plenty of G, has plenty of A from 5 ' terminal the 71st Nucleotide in the genome of described pig to be measured, dna fragmentation shown in the SEQ ID NO:2 has plenty of C, has plenty of A from 5 ' terminal the 81st Nucleotide in the genome, and then the haplotype meter of described pig to be measured is made ABCD;
Described 2) in, the method for the haplotype of described definite described pig to be measured is:
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AADD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is G from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made AACD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured is A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made BBCD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is C from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCC;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 is A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABDD;
If the introne 1 of the LH beta subunit gene of described pig to be measured has plenty of G, has plenty of A from the base of 5 ' terminal the 67th Nucleotide, intron 2 has plenty of C, has plenty of A from the base of 5 ' terminal the 30th Nucleotide, and then the haplotype meter of described pig to be measured is made ABCD;
5. method according to claim 4 is characterized in that:
Dna fragmentation shown in the SEQ ID NO:1 is that G or the method for A comprise the steps: with primer I to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 71st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is G or A according to dna fragmentation shown in the SEQ ID NO:1 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 71st Nucleotide;
Dna fragmentation shown in the SEQ ID NO:2 is that C or the method for A comprise the steps: with primer II to be carried out pcr amplification to described pig to be measured from 5 ' terminal the 81st Nucleotide in the genome of described detection pig to be measured, obtains pcr amplification product; Is C or A according to dna fragmentation shown in the SEQ ID NO:2 in the genome of the sequential detection pig to be measured of described pcr amplification product from 5 ' terminal the 81st Nucleotide;
Described primer to the nucleotide sequence of I shown in SEQ ID NO:3 and SEQ ID NO:4;
Described primer to the nucleotide sequence of II shown in SEQ ID NO:5 and SEQ ID NO:6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010289445 CN101956009A (en) | 2010-09-21 | 2010-09-21 | Method for detecting number of littermate and characters of sow |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201010289445 CN101956009A (en) | 2010-09-21 | 2010-09-21 | Method for detecting number of littermate and characters of sow |
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| CN101956009A true CN101956009A (en) | 2011-01-26 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923912A (en) * | 2014-04-20 | 2014-07-16 | 华中农业大学 | Molecular marker related to pig mummy embryo number character and application thereof |
| CN108796093A (en) * | 2018-06-26 | 2018-11-13 | 华中农业大学 | With the molecular labeling and application of sow number born alive and the young number trait associations of 5 ages in days work |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238388A (en) * | 1998-06-10 | 1999-12-15 | Pic国际集团公开有限公司 | DNA markers for pig litter size |
| CN1357625A (en) * | 2000-12-08 | 2002-07-10 | 李宁 | Partial ESR gene sequence, swine farrowing characteristic related ESR gene and polymorphic FSH-Beta gene determination technology |
| CN1733938A (en) * | 2005-08-31 | 2006-02-15 | 中国农业大学 | Method for detecting pig number born character |
-
2010
- 2010-09-21 CN CN 201010289445 patent/CN101956009A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238388A (en) * | 1998-06-10 | 1999-12-15 | Pic国际集团公开有限公司 | DNA markers for pig litter size |
| CN1357625A (en) * | 2000-12-08 | 2002-07-10 | 李宁 | Partial ESR gene sequence, swine farrowing characteristic related ESR gene and polymorphic FSH-Beta gene determination technology |
| CN1733938A (en) * | 2005-08-31 | 2006-02-15 | 中国农业大学 | Method for detecting pig number born character |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923912A (en) * | 2014-04-20 | 2014-07-16 | 华中农业大学 | Molecular marker related to pig mummy embryo number character and application thereof |
| CN103923912B (en) * | 2014-04-20 | 2016-01-13 | 华中农业大学 | A kind of molecule marker relevant to pig mummy tire number proterties and application thereof |
| CN108796093A (en) * | 2018-06-26 | 2018-11-13 | 华中农业大学 | With the molecular labeling and application of sow number born alive and the young number trait associations of 5 ages in days work |
| CN108796093B (en) * | 2018-06-26 | 2021-01-12 | 华中农业大学 | Molecular marker associated with characters of number born alive piglets and number born alive piglets at 5 days of age and application of molecular marker |
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