CN104630210A - GLI2 gene as porcine newborn weight trait related molecular marker as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a GLI2 gene as a porcine newborn weight trait related molecular marker as well as a preparation method and application thereof. The preparation method comprises the following steps: A, extracting porcine genome DNA (Deoxyribonucleic Acid) by using a porcine sequence as a template design primer; 1), grinding a porcine muscular tissue in liquid nitrogen; 2), adding a digested tissue sample into Tris saturated phenol in equal volume, and freezing; 3), adding phenol/chloroform/isoamylol in equal volume, and transferring into another centrifugal tube; 4), adding chloroform/isoamylol in equal volume, freezing, and centrifuging; 5), absorbing supernate in the marked centrifugal tube, and adding absolute ethyl alcohol; 6), picking out DNA precipitation by using a gun head, and dissolving DNA by adding pure water; 7), measuring the concentration of the DNA on a DNA concentration measuring instrument, and detecting the quality of the extracted DNA under an ultraviolet lamp; and B, designing a primer so as to obtain a represented nucleotide acid sequence. The result is reliable; transfer of a target gene can be selected in the early period of breeding; the breeding period is greatly shortened; the breeding efficiency is increased by 2-3 times; and therefore, the GLI2 gene has obvious superiority.

Description

GLI2 gene is as pig birth weight proterties related molecular marker and preparation method and application

Technical field

The invention belongs to the Molecular Marker Assisted Selection Technology field of pig, be specifically related to a kind of as pig marker assisted selection with birth weight proterties related molecular marker, also relate to simultaneously a kind of as pig marker assisted selection with the preparation method of birth weight proterties related molecular marker, also relate to a kind of as pig marker assisted selection with the purposes of birth weight proterties related molecular marker.

Background technology

Pig is important economic animal, and pork, due to the tender delicious favor being extensively subject to human consumer for a long time of its meat, is one of main source of our people's animal protein.In recent years, the consumption of people to pork grows with each passing day, and how to improve production performance, reduction production cost has become one of focus of pig breeding worker.

Protocols in Molecular Biology develop rapidly as herein is provided important opportunity, by means of this technique means, scientists excavates out birth weight that is large quantities of and pig, weaning weight, the molecule marker that the important reproductive trait such as litter size significantly associates.This production performance for raising pig provides theoretical foundation, and from making it in fact to be greatly improved.The birth weight of pig, as an important reproductive trait, has indivisible close ties with the postnatal speed of growth of pig and incubation rate.There is a lot of bibliographical information birth weight recently to the impact of the speed of growth, Lu Wei (Lu Wei, Liu Chong, in high official position, Liu Hang, Kong Junling, Wang Zirong. the comparison of different birth weight piglet 0 ~ 60 age in days growth performance. raise pigs [J] .2010.6) research discovery raising piglet birth weight, be conducive to ensureing that the piglet later stage grows fast.Li Jianhao (Li Jianhao. landrace birth weight is on the impact of the speed of growth and nurture rate. piglet produces [J] .2006.18) research finds that landrace 60 age in days is heavy and to be proportionate with 35 age in days weight averages and birth weight and birth weight and the speed of growth are proportionate.(the Beaulieu AD such as Beaulieu, Aalhus JL, Williams NH, Patience JF.Impact of piglet birth weight, birth order and litter size on subsequent growth performance, carcass quality, muscle composition and eating quality of pork [J] .J Anim Sci.2010) research discovery, the growth speed of pigs that birth weight is lower is comparatively slow, thus the time making it come into the market increases.Separately there are some researches show that the birth weight of piglet and incubation rate are also known relevant, birth weight piglet incubation rate when below 1.0Kg rises along with the increase of birth weight; Birth weight piglet incubation rate when more than 1.0Kg does not have obvious rule.When birth weight is less than or equal to 0.5Kg, piglet incubation rate is only 55.56%(height and founds the state. and Erhualian birth weight is on the impact [J] of the speed of growth and incubation rate. herding and animal doctor, 1992,24(1): 26).Therefore, the cloning and identification of pig birth weight genes involved be can be and explain that the genetic mechanism that pig and other mammalian fetal grow provides important clue, and provide theoretical foundation for the reproductive trait genetic improvement of pig.

GLI2 is the main transcription activator regulating Hh (Hedgehog) signal path.Hedgehog (Hh) family secretory signaling molecule plays important effect (reviewed by Ingham and McMahon, 2001) in the growth of the multinomial embryonic structure from fruit bat to the mankind is graphic.Its function is suppressed, the transmembrane protein Smoothened (Smo) that release suppresses by it after Hh signaling molecule and Patched (Ptc) receptors bind, and then a series of signal cascade reaction in activating cells.The body size of an animal reflects the number of cell number, the balance (Raff, M.C.1996.Size control:the regulation of cell numbers in animal.Devel173-175) of namely cell proliferation and necrocytosis usually.Research before shows that the reduction of Hh signal activity can cause the form of mouse short and small.In Various Tissues, Shh has mitogenetic effect, as somite mesoderm, retina and cerebellum.Another kind of Hh protein I hh controls the growth of cartilage, Ihh deficient mice presents short-limb dwarfism phenotype (Chuang, P.T.and McMahon, A.P.1999.Vertebrate Hedgehog signaling modulated by induction of Hedgehog-binding protein.Nature397:617-621).Thus affect fetation, and then affect the birth weight of piglet.Therefore the present invention intends the relation inquiring into GLI2 gene and pig birth weight, seeks to obtain a kind of molecule marker relevant to pig birth weight proterties.

Summary of the invention

The object of the invention is to there are provided a kind of as pig marker assisted selection with birth weight proterties related molecular marker, DNA marker assistant breeding utilizes a kind of modern breeding with objective trait gene closely linked DNA molecular marker, objective trait being carried out to indirect selections, the method directly selects genotype, compared with the traditional breeding way indirectly genotype selected by phenotype, it is not affected by environment, do not show hidden relation by allelotrope to disturb, reliable results; In addition, it can be selected in breeding in early days to the transfer of target gene, thus substantially reduces breeding cycle, can improve breeding efficiency and reach 2-3 doubly, have obvious superiority compared with conventional breeding.

Another object of the present invention be there are provided a kind of as pig marker assisted selection with the preparation method of birth weight proterties related molecular marker, CAPs (cleaved amplification polymorphism sequence-tagged sites) CAPs technology is also called PCR-RFLP, restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) technology.When being with the PCR primer of special design amplification target material, because the base mutation of specific site, insertion or missing number are little, so that without polymorphic appearance, often need that enzyme is carried out to corresponding pcr amplified fragment and cut process, to detect its polymorphism.CAPs is marked in diplont research can play huge effect, is that the strong of PCR mark supplements.This method, compared with RFLP, instead of enzyme cut unlike with amplification, avoids the steps such as the loaded down with trivial details DNA enzymatic of RFLP is cut, shifted, hybridization.The present invention is that Gli2 gene has the base mutation of a G446-A446 at the 446bp place of sequence table SEQ ID NO:1, cause NcoI-RFLP polymorphism, for provide a kind of as pig marker assisted selection with the preparation method of birth weight proterties related molecular marker.

Another object of the present invention be there are provided a kind of as pig marker assisted selection with the application of birth weight proterties related molecular marker, DNA molecular marker extensively exists in animal body, there is the specificity of each level of individuality, kind and kind, in simple Mendelian inheritance, not by the restriction of Crossing system, have no side effect between the non-allelic genes of mark, noiseless between mark, result is reliable and stable, and repeatability is strong, can be widely applied in real breeding work.

In order to realize above-mentioned object, the present invention adopts following technical measures:

Applicant obtains the partial dna sequence with pig birth weight trait related gene from report pig gene GLI2 clone, and its nucleotide sequence is as described in sequence table SEQ ID NO:1 and Fig. 2.There is the base mutation of a G446-A446 at the 446bp place of sequence table SEQ ID NO:1, cause NcoI-RFLP polymorphism.This base mutation is arranged in GLI2 gene the 8th exon.

As pig marker assisted selection with the preparation method of birth weight proterties related molecular marker, the steps include:

A, be template design primer by pig sequence (the ensemble number of including: ENSSSCG00000015733), extract pig genomic dna.

1) by Large White (from Wuhan City, Hubei Province Hua Zhong Agriculture University fine work pig farm, the bacon hogs kind of external blood relationship for routine is promoted) muscle tissue grind in liquid nitrogen, add equal-volume 1 × SET solution (1ml), Proteinase K (10mg/mL) is to final concentration 200 μ g/mL, sodium lauryl sulphate (i.e. SDS, 10%) to final concentration 0.5%, shake up.Be incubated overnight in 55 DEG C of shaking baths digestion.

2) postdigestive sample of organizing is added the saturated phenol of isopyknic Tris, slowly put upside down centrifuge tube 15min, 4 DEG C, the centrifugal 10min of 11000rpm in cryogenic freezing whizzer, careful Aspirate supernatant is transferred in another centrifuge tube, notes putting on corresponding mark.

3) isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1) is added, slowly put upside down centrifuge tube 10min, in low temperature (-9 DEG C to+40 DEG C) whizzer, 4 DEG C, the centrifugal 10min of 11000rpm, carefully draw supernatant, is transferred in another clean centrifuge tube.

4) add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24:1) and slowly put upside down centrifuge tube 10min, 4 DEG C, the centrifugal 10min of 11000rpm in low temperature (-9 DEG C to+40 DEG C) refrigerated centrifuge.

5) by supernatant liquor suck marked centrifuge tube in, add the precooling dehydrated alcohol of 2.5 times of volumes, namely can see white flock DNA.

6) with rifle head, DNA precipitation is chosen, be placed in the EP pipe that corresponding number is housed, allow ethanol volatilize under room temperature (20-25 DEG C, identical below) clean, add appropriate ultrapure water (general about 300ul) dissolving DNA.

7) on DNA concentration determination instrument, measure its concentration and purity, and be about 2h at (1%, m/V) sepharose 80 volts of electrophoresis, the DNA quality of Detection and Extraction under ultraviolet lamp.

B, design primer, rule is as follows: sequence is chosen should at the conservative section of gene; Avoid primer self or and primer between form more than 4 or 4 and match continuously, avoid primer self to form pili annulati card structure; Typical primer 18 to 24 nucleosides are long, and primer needs sufficiently long, ensure that sequence is unique, and reduce the possibility that sequence is present in non-aim sequence site.But length is greater than the primer of 24 nucleosides and does not mean that higher specificity.Longer sequence may be hybridized with wrong matched sequence, reduces specificity, and slower than short data records hybridization, thus reduces output; Tm value is 55-65 DEG C (because 60 DEG C of exonuclease activities are the highest), and GC content is at 40%-60%; TM difference between primer avoids exceeding 2 DEG C; 3 ' end of primer is avoided using base A, and 3 ' of primer holds the base avoiding appearance more than 3 or 3 consecutive identical; For the amplification avoided, design of primers preferably can across two exons; Prime end (last 5 Nucleotide) can not have G and C more than 2.

The DNA sequence dna of this primer is as follows: forward primer 5'-ATGTCCTAAGGAACCAAGCT-3', reverse primer 5'-TCCTGCCTTCTTTTGCTC-3';

C, by pcr amplification, PCR primer purifying and order-checking, obtain the nucleotide sequence as shown in sequence table SEQ ID NO:1 and Fig. 2.

One, pcr amplification: reaction cumulative volume is 10 μ l, the wherein above-mentioned pig DNA template 0.5 μ l prepared, distilled water 7.0 μ l, buffer1 μ l, Mg ²⁺0.6 μ l, 10mM forward primer and reverse primer each 0.3 μ l, dNTP0.2 μ l, Taq enzyme 0.1 μ l(10U/ μ l).PCR reaction conditions is: after 94 DEG C of denaturation 5min, and circulate 35 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 25s, and last 72 DEG C extend 5min.PCR primer detects through (2%, m/V) agarose gel electrophoresis.

Program: 94 degrees Celsius of denaturation 5min

72 degrees Celsius extend 5min

Preserve 3min for 15 degrees Celsius

Two, PCR primer purifying: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5ml centrifuge tube, then use PCR primer purification kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd., operate according to the specification sheets of this test kit) purified pcr product, concrete steps are in blob of viscose, add equimultiple volume PC solution, put 50 DEG C of incubation 10min, agarose blob of viscose is dissolved completely, within every two minutes, put upside down mixing once; The glue dissolved is transferred to centrifugal adsorbing column, and centrifugal adsorbing column is placed in waste collection pipe, the centrifugal 60s of 12000rpm, discards waste liquid; Put back by centrifugal adsorbing column in waste collection pipe, add 600 μ l rinsing liquid PW in centrifugal adsorbing column, the centrifugal 60s of 12000rpm, abandons filtrate.Wash once with 600 μ l rinsing liquid PW more in the same way; Centrifugal adsorbing column is put in meeting waste collection pipe, the centrifugal 2min of top speed; Careful taking-up centrifugal adsorbing column, is inserted in an aseptic 1.5ml centrifuge tube, is added 30 μ l distilled waters in adsorption film central authorities, after room temperature leaves standstill 2-10min, and the centrifugal 1min of top speed; Take out centrifugal adsorbing column, 1.5ml centrifuge tube (DNA solution) is placed in-20 DEG C and saves backup.

Three, determined dna sequence: sequencing is completed by Beijing Qing Kexin industry Bioisystech Co., Ltd, gene fragment surveys positive and negative two reactions.

A kind of molecule marker (SEQ ID NO:1) application in the association analysis of pig reproductive trait, the steps include:

1, the method for the PCR-RFLP that application is conventional detects the 446th bit base sudden change shown in sequence SEQ ID NO:1 and Fig. 2.

(1) PCR-RFLP primer sequence (this primer is also the primer of amplification GLI2 gene the 8th exon), the sequence of this primer pair is as follows:

GLI2SNP: forward primer 5'-ATGTCCTAAGGAACCAAGCT-3', (the 1-20 position corresponding to sequence shown in Fig. 2),

Reverse primer 5'-TCCTGCCTTCTTTTGCTC-3'.(the 702-719 position corresponding to sequence shown in Fig. 2);

This primer amplification fragment length is that 719bp(is shown in Fig. 2 and sequence table SEQ ID NO:1).

(2) pcr amplification condition:

PCR reacts cumulative volume 10 μ l, the pig genomic DNA template 1 μ l of wherein above-mentioned preparation, distilled water 7.1 μ l, 10 × damping fluid 1 μ l, 10mM forward primer and reverse primer each 0.3 μ l, dNTP0.2 μ l, Taq enzyme 0.1 μ l(5U/ μ l).PCR reaction conditions is: after 94 DEG C of denaturation 5min, and circulate 35 94 DEG C of sex change 20s, 57 DEG C of annealing 30s, 72 DEG C of extension 20s, and last 72 DEG C extend 5min.PCR primer detects through 2% agarose gel electrophoresis.

(3) RFLP detects:

PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 4 μ l, distilled water 4.9 μ l, 10 × damping fluid 1 μ l, restriction enzyme NcoI are 0.1 μ l (10U/ μ l), by centrifugal after sample blending, 4h placed by 37 DEG C of incubators, detect enzyme with 4% agarose gel electrophoresis and cut result, record genotype, takes pictures under ultraviolet lamp.

(4) gene type: enzyme cuts generation three kinds of genotype, AA genotype has 164bp and 555bp two band, GG genotype has 164bp, 277bp and 278bp tri-band, because 277bp and 278bp only differs 1bp, agarose gel electrophoresis can not be differentiated, two bands only having about 164bp and 277bp that therefore can identify, heterozygote AG genotype has 164bp, four bands of 277bp, 278bp and 555bp, but in like manner can tell three bands, i.e. about 164bp, 555bp and 277bp, as described in Figure 3.

The application of the association analysis between genotype and pig birth weight proterties, the molecular marker assisted selection for pig provides a new molecule marker.

The application of described a kind of primer in pig pig birth weight trait associations is analyzed, the steps include:

(1) PCR-RFLP primer sequence (this primer is also the primer of amplification GLI2 gene the 8th exon), the sequence of this primer pair is as follows:

GLI2SNP: forward primer 5'-ATGTCCTAAGGAACCAAGCT-3', (the 1-20 position corresponding to sequence shown in Fig. 2),

Reverse primer 5'-TCCTGCCTTCTTTTGCTC-3'.(the 702-719 position corresponding to sequence shown in Fig. 2);

This primer amplification fragment length is that 719bp(is shown in Fig. 2 and sequence table SEQ ID NO:1).

(2) pcr amplification condition:

PCR reacts cumulative volume 10 μ l, the pig genomic DNA template 1 μ l of wherein above-mentioned preparation, distilled water 7.1 μ l, 10 × damping fluid 1 μ l, 10mM forward primer and reverse primer each 0.3 μ l, dNTP0.2 μ l, Taq enzyme 0.1 μ l(5U/ μ l).PCR reaction conditions is: after 94 DEG C of denaturation 5min, and circulate 35 94 DEG C of sex change 20s, 57 DEG C of annealing 30s, 72 DEG C of extension 20s, and last 72 DEG C extend 5min.PCR primer detects through 2% agarose gel electrophoresis.

(3) RFLP detects:

PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 4 μ l, distilled water 4.9 μ l, 10 × damping fluid 1 μ l, restriction enzyme NcoI are 0.1 μ l (10U/ μ l), by centrifugal after sample blending, 4h placed by 37 DEG C of incubators, detect enzyme with 4% agarose gel electrophoresis and cut result, record genotype, takes pictures under ultraviolet lamp.

(4) gene type: enzyme cuts generation three kinds of genotype, AA genotype has 164bp and 555bp two band, GG genotype has 164bp, 277bp and 278bp tri-band, because 277bp and 278bp only differs 1bp, agarose gel electrophoresis can not be differentiated, two bands only having about 164bp and 277bp that therefore can identify, heterozygote AG genotype has 164bp, four bands of 277bp, 278bp and 555bp, but in like manner can tell three bands, i.e. about 164bp, 555bp and 277bp, as described in Figure 3.

The present invention compared with prior art, has the following advantages and effect:

The method directly selects genotype, and compared with the traditional breeding way indirectly selected genotype by phenotype, it is not affected by environment, does not show hidden relation and disturbs, reliable results by allelotrope; In addition, it can be selected in breeding in early days to the transfer of target gene, thus substantially reduces breeding cycle, can improve breeding efficiency and reach 2-3 doubly, have obvious superiority compared with conventional breeding.

The association analysis result (p<0.05) of table 3.GLI2 gene NCOI-RFLP genotype and Traits and birth weight proterties

Note: ^*represent P<0.01, ^*represent P<0.05.

Accompanying drawing explanation

Fig. 1 be a kind of as pig marker assisted selection with the schema of the preparation method of birth weight proterties related molecular marker.

The DNA sequence dna (answering with the sequence pair shown in sequence table 1) that Fig. 2 is a kind of clone pig GLI2 gene.

446bp place existence allelic sudden change of sequence shown in Fig. 2, be designated as " R ", bracket after " R " i.e. (G/A) is mutational site (the overstriking italics display with indicating underscore), primer sequence italic overstriking and the shade display of the head and the tail display of this amplified fragments.

Fig. 3 is three kinds of genotype (AA AG GG) and genome amplification electrophoresis result (P) schematic diagram of NcoI-RFLP in boar GLI2 gene the 8th exon.

M:DNA molecular weight standard (DL2000ladder) in figure.

Fig. 4 is the G-A sudden change schematic diagram that a boar GLI2 gene forward order-checking finds.

Embodiment

Embodiment 1:

As pig marker assisted selection with the preparation method of birth weight proterties related molecular marker, the steps include:

(1) phenol extraction method is utilized to extract Large White (landrace) muscle tissue STb gene:

1) by Large White (from Wuhan City, Hubei Province Hua Zhong Agriculture University fine work pig farm, the bacon hogs kind of external blood relationship for routine is promoted) muscle tissue grind in liquid nitrogen, add equal-volume 1 × SET solution (1ml), Proteinase K (10mg/mL) is to final concentration 200 μ g/mL, sodium lauryl sulphate (i.e. SDS, 10%) to final concentration 0.5%, shake up.Be incubated overnight in 55 DEG C of shaking baths digestion.

2) postdigestive sample of organizing is added the saturated phenol of isopyknic Tris, slowly put upside down centrifuge tube 15min, 4 DEG C, the centrifugal 10min of 11000rpm in cryogenic freezing whizzer, careful Aspirate supernatant is transferred in another centrifuge tube, notes putting on corresponding mark.

3) add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1), slowly put upside down centrifuge tube 10min, in refrigerated centrifuge, 4 DEG C, the centrifugal 10min of 11000rpm, carefully draw supernatant, is transferred in another clean centrifuge tube.

4) add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24:1) and slowly put upside down centrifuge tube 10min, 4 DEG C, the centrifugal 10min of 11000rpm in cryogenic freezing whizzer.

5) by supernatant liquor suck marked centrifuge tube in, add the precooling dehydrated alcohol of 2.5 times of volumes, namely can see white flock DNA.

6) with rifle head, DNA precipitation is chosen, be placed in the EP pipe that corresponding number is housed, allow ethanol volatilize under room temperature (20-25 DEG C, identical below) clean, add appropriate ultrapure water (general about 300ul) dissolving DNA.

7) on DNA concentration determination instrument, measure its concentration and purity, and be about 2h at (1%, m/V) sepharose 80 volts of electrophoresis, the DNA quality of Detection and Extraction under ultraviolet lamp.

(2) design of primers:

With the pig genome sequence (the ensemble number of including: ENSSSCG00000015733) of report for template, utilize biology primer-design software Primer Premier5.0, the primer of design amplification GLI2 gene the 8th exon, the DNA sequence dna of this primer pair is as follows:

GLI2: forward primer: 5'-ATGTCCTAAGGAACCAAGCT-3', (the 1-20 position corresponding to sequence table SEQ ID NO:1),

Reverse primer: 5'-TCCTGCCTTCTTTTGCTC-3'(corresponds to the 702-719 position of sequence table SEQ ID NO:1);

The DNA sequence dna expanding fragment length of this primer is 719bp.

(3) pcr amplification reaction:

PCR reacts: reaction cumulative volume is 10 μ l, the wherein above-mentioned pig DNA template 0.5 μ l prepared, distilled water 7.0 μ l, buffer1 μ l, Mg ²⁺0.6 μ l, 10mM forward primer and reverse primer each 0.3 μ l, dNTP0.2 μ l, Taq enzyme 0.1 μ l(10U/ μ l).PCR reaction conditions is: after 94 DEG C of denaturation 5min, and circulate 35 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 25s, and last 72 DEG C extend 5min.PCR primer detects through (2%, m/V) agarose gel electrophoresis.

(4) purifying of PCR primer and order-checking

The purifying of PCR primer: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5ml centrifuge tube, then use PCR primer purification kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd., operate according to the specification sheets of this test kit) purified pcr product, concrete steps are in blob of viscose, add equimultiple volume PC solution, put 50 DEG C of incubation 10min, agarose blob of viscose is dissolved completely, within every two minutes, put upside down mixing once; The glue dissolved is transferred to centrifugal adsorbing column, and centrifugal adsorbing column is placed in waste collection pipe, the centrifugal 60s of 12000rpm, discards waste liquid; Put back by centrifugal adsorbing column in waste collection pipe, add 600 μ l rinsing liquid PW in centrifugal adsorbing column, the centrifugal 60s of 12000rpm, abandons filtrate.Wash once with 600 μ l rinsing liquid PW more in the same way; Centrifugal adsorbing column is put in meeting waste collection pipe, the centrifugal 2min of top speed; Careful taking-up centrifugal adsorbing column, is inserted in an aseptic 1.5ml centrifuge tube, is added 30 μ l distilled waters in adsorption film central authorities, after room temperature leaves standstill 2-10min, and the centrifugal 1min of top speed; Take out centrifugal adsorbing column, 1.5ml centrifuge tube (DNA solution) is placed in-20 DEG C and saves backup.

(5) determined dna sequence: sequencing is completed by Beijing Qing Kexin industry Bioisystech Co., Ltd, gene fragment surveys positive and negative two reactions.

Embodiment 2:

PCR-RFLP detection method is set up:

(1) PCR-RFLP primer sequence (this primer is also the primer of amplification GLI2 gene the 8th exon), the sequence of this primer pair is as follows:

GLI2SNP: forward primer 5'-ATGTCCTAAGGAACCAAGCT-3', (the 1-20 position corresponding to sequence shown in Fig. 2),

Reverse primer 5'-TCCTGCCTTCTTTTGCTC-3'.(the 702-719 position corresponding to sequence shown in Fig. 2);

This primer amplification fragment length is that 719bp(is shown in Fig. 2 and sequence table SEQ ID NO:1).

(2) pcr amplification condition:

PCR reacts cumulative volume 10 μ l, the pig genomic DNA template 1 μ l of wherein above-mentioned preparation, distilled water 7.1 μ l, 10 × damping fluid 1 μ l, 10mM forward primer and reverse primer each 0.3 μ l, dNTP0.2 μ l, Taq enzyme 0.1 μ l(5U/ μ l).PCR reaction conditions is: after 94 DEG C of denaturation 5min, and circulate 35 94 DEG C of sex change 20s, 57 DEG C of annealing 30s, 72 DEG C of extension 20s, and last 72 DEG C extend 5min.PCR primer detects through 2% agarose gel electrophoresis.

(3) RFLP detects:

PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 4 μ l, distilled water 4.9 μ l, 10 × damping fluid 1 μ l, restriction enzyme NcoI are 0.1 μ l (10U/ μ l), by centrifugal after sample blending, 4h placed by 37 DEG C of incubators, detect enzyme with 4% agarose gel electrophoresis and cut result, record genotype, takes pictures under ultraviolet lamp.

Obtain 719bp specific amplification fragment with primer amplification pig genomic dna, sequencing result finds that this fragment exists G-A conversion at the 446th, is positioned at the 8th exon 1 (as shown in Figure 4), cause a NcoI restriction enzyme site ( , there is 1 NcoI restriction enzyme site all the time in polymorphism CATGG), in addition this fragment 164bp place.Therefore, enzyme cuts generation three kinds of genotype, and AA genotype has 164bp and 555bp two band, GG genotype has 164bp, 277bp and 278bp tri-band, because 277bp and 278bp only differs 1bp, agarose gel electrophoresis can not be differentiated, two bands only having about 164bp and 277bp that therefore can identify, heterozygote AG genotype has 164bp, 277bp, four bands of 278bp and 555bp, but in like manner can tell three bands, i.e. 164bp, about 555bp and 277bp, as described in Figure 3.Other implementation step is identical with embodiment 1.

Embodiment 3:

The present embodiment swinery is from purebred Large White (for the conventional kind applied) group on gold woods original seed herding company limited's original seed pig farm, Hubei, and offspring born measures body weight in 0 day.

According to the group structure of collected specimens, applicant uses mixture model to come the genotype effects of statistical study GLI2 gene the 8th exon 1 G/A pleomorphism site and the relation with birth weight proterties thereof, wherein fixed effect comprises the genotype effects of sex-effects, parity effect, the other effect of line, candidate gene, and stochastic effect comprise dam effect in male animal effect, male animal; MIXED MODELS program in SAS (Version8.0) software is adopted to carry out least squares means estimation (Breslow, N.E.; Clayton, D.G.Approximate Inference in Generalized Linear Mixed Models [J] .Journal of the American Statistical Association, 1993,88 (421): 9 – 25) and statistical study.

Include subarea NcoI-RFLP pleomorphism site to pig GLI2 gene the 7th and birth weight proterties carries out association analysis, concrete outcome is in table 1.As shown in Table 1: in 329 individualities of the purebred Large White group on gold woods original seed herding company limited's original seed pig farm, Hubei, AA genotype has 10, and AG genotype has 136, and GG genotype has 183, illustrates that G allelotrope is preponderated in this colony.Between different genotype, the result display pig GLI2 gene the 7th of Traits change significance includes each genotype of subarea NcoI-RFLP pleomorphism site with Large White birth weight, number of white blood cells, lymphocyte ratios and basophilic granulocyte ratio in significantly associating (P<0.05).By comparing between two the least squares means value of AA, AG and GG genotype individuals, result shows that the birth weight of AG genotype individuals is significantly higher than GG type individuality (P=0.005).

The association analysis result (p<0.05) of table 3.GLI2 gene NCOI-RFLP genotype and Traits and birth weight proterties

Note: ^*represent P<0.01, ^*represent P<0.05.

SEQUENCE LISTING

 

<110> Hua Zhong Agriculture University

 

<120> GLI2 gene is as pig birth weight proterties related molecular marker and preparation method and application

 

<130> r is a or g

 

<160> 1

 

<170> PatentIn version 3.1

 

<210> 1

<211> 719

<212> DNA

<213> pig

 

<400> 1

atgtcctaag gaaccaagct tttctgtcct gctccctggg ctgaccaggc cctgttatcc 60

 

ttagacccag agccagccct gccctggagg gctgaggttg agaccactga gtcaggagtg 120

 

aggggcccca gtatttctct aagagagggt cccacttcat ctccatggaa gtgggggctg 180

 

ctcctgtgag agggaggcgc tgatcagcag agctccgggc tctctcctgg ccatgcccct 240

 

ggccagagga ccaagcaagg ggactcattt tctgaaactg cagcacaagc gttggcccag 300

 

acggtctatg tgggaagagt cccagaggta aagtcccaat ttggggtgtc cctgtctgcc 360

 

cccatgtgat gactcccccc catgctcctt tctcccagaa caagcagagc agcgagtcag 420

 

ccgtgagcag caccatcaac cccatrgtca ttcacaagcg cagcaaggtc aagacagagg 480

 

ctgagggcct gcggccagcc tccccactga ctctgacaca ggtaacttgt cagctctccc 540

 

caccgcggct ctccggctag gactggggcc cggccgcgac tcctgggtca gctgcctagg 600

 

ggccttgacc ttgttggcgg atggatgaca gcctgcgtgt gtgaatgagc gaatgatggc 660

 

gtgagagaat gatggagcgc acatgctcct caaccactgc cgagcaaaag aaggcagga 719

 

 

Claims (4)

1. GLI2 gene is as a pig birth weight proterties related molecular marker, it is characterized in that: the molecule marker relevant to pig birth weight proterties, and its sequence is for shown in SEQ ID NO:1.

2. a kind of GLI2 gene according to claim 1 is as pig birth weight proterties related molecular marker, and it is characterized in that: the primer pair of molecule marker, its nucleotide sequence is as follows:

Forward primer: 5 ＇-ATGTCCTAAGGAACCAAGCT-3 ＇,

Reverse primer: 5 ＇-TCCTGCCTTCTTTTGCTC-3 ＇.

3. a kind of GLI2 gene according to claim 1 is as the application of pig birth weight proterties related molecular marker in pig birth weight trait associations is analyzed.

4. a kind of GLI2 gene described in claim 2 is as pig birth weight proterties related molecular marker, it is characterized in that: the application of primer pair in pig pig birth weight trait associations is analyzed of molecule marker.