CN109897903A - Molecular labeling and application based on FSH β identified for genes Large White reproductive trait - Google Patents

Abstract

The invention discloses a molecular marker and application for identifying reproductive traits of large white pigs based on FSH beta gene. The molecular marker is located on the exon No. 3 of the porcine FSHβ gene, that is, at 511, 617, 630, 652, 678, 735, 746, and 921 in the No. 3 exon region, denoted as g.511A>G, g. 617A>G, g.630C>T, g.652C>T, g.678C>T, g.735C>T, g.746A>G and g.921A>G. The invention also discloses the application of the above-mentioned molecular marker in the selection and breeding of pig breeds. The invention provides a new marker method for molecular marker-assisted selection of pigs, and provides a reference for molecular breeding of pigs.

Description

Molecular labeling and application based on FSH β identified for genes Large White reproductive trait

Technical field

The invention belongs to animal molecular marker breeding fields, specifically, being related to a kind of based on FSH β identified for genes great Bai The molecular labeling of pig reproductive trait and application.

Background technique

Pork is one of most important meat economic animal in the world today, and China consumes big country as pork, and pig is deposited Column amount ranks first in the world.To improve productivity effect, the reproductive performance of pig be always the concern of major breeding enterprise important indicator it One, wherein the total yield coefficient of pig and litter size living are particularly critical.

Molecular marker assisted selection is effectively prevented from tradition as one of modern domestic animal breeding programs important means The drawbacks of breeding technique based on phenotype, shorten genetic interval phase, the selection accuracy of raising.Molecular labeling auxiliary choosing The premise selected is to determine relevant to required phenotypic character candidate gene, purposefully these genes are detected so that it is determined that Breeding direction.

The follicular stimulating hormone (FSH) of pig is a kind of glycoprotein hormones of anterior pituitary basophil cell secretion, it and sexual gland Target cell combines, by secondary courier Ca²⁺And cAMP causes a series of biological respinses, promotes granular cell hyperplasia, endo cell Differentiation and the secretion of ovum vacuole, the receptor generation and the generation of aromatizing enzyme of induction metakentrin, prolactin, and stimulate female two The synthesis and release of alcohol, so that the development and maturation of coordinated control gametid, there is critically important work during animal reproduction With.Follicular stimulating hormone (FSH) is made of two subunits of α and β.Wherein α subunit is follicular stimulating hormone, luteotropin and thyroid Common to element, same interior or even in all mammals, α subunit is extremely conservative；Biology of the β subunit to FSH Characteristic is learned to play a leading role.

Follicular stimulating hormone β subunit (follicle-stimulating hormone beta subunit, FSH β) base of pig Because being located on No. 2 chromosomes of pig, overall length 10.16kb is made of 3 exons and 2 intrones.Follitropin beta subunit (FSH β) is mainly the growth and development for stimulating ovarian follicle, influences the quantity of growing follicle, and under the synergistic effect of luteotropin, The last maturation of ovarian follicle is excited, ovulation is induced.

Summary of the invention

In view of this, the present invention provides a kind of molecular labeling based on FSH β identified for genes Large White reproductive trait and answering With.

In order to solve the above-mentioned technical problem, the invention discloses a kind of based on FSH β identified for genes Large White reproductive trait Molecular labeling, the molecular labeling are located on the 3rd exon of pig FSH β gene, i.e., 3 exon regions 511,617, 630, at 652,678,735,746,921, be denoted as g.511A > G, g.617A > G, g.630C > T, g.652C > T, g.678C > T, G.735C > T, g.746A > G and g.921A > G.

The invention also discloses a kind of for detecting the primer pair of above-mentioned molecular labeling, including FSH β-E3-F and FSH β- E3-R, nucleotide sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.

The invention also discloses a kind of reagent or kit containing above-mentioned primer pair.

The invention also discloses a kind of methods for detecting above-mentioned molecular labeling, which comprises with Large White gene Group DNA is that template utilizes above-mentioned primer pair progress PCR amplification acquisition PCR product；Ago-Gel is carried out to the PCR product Electrophoresis.

Optionally, pcr amplification reaction system are as follows: 2 × Taq Master Mix, 15 1.5 μ L of μ L, DNA, 10 μM of upstreams are drawn Object 1.5 μ L, 10 μM of downstream primers 1.5 μ L, ddH₂O 10.5μL。

Optionally, pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 1.5min；94 DEG C of denaturation 20s, 56.4 DEG C of annealing 20s, 72 DEG C of extension 60s are recycled 30 times；Last 72 DEG C re-extend 5min, 4 DEG C of preservations.

The invention also discloses a kind of application of above-mentioned molecular labeling in pig kind breeding.

Compared with prior art, the present invention can be obtained including following technical effect:

There are 8 mutational sites, the breedings in this 8 mutational sites and pig on 3rd exon of FSH β gene of the present invention There are correlations for character especially total yield coefficient and litter size living, and are easy detection.Therefore the present invention provides one to be based on FSH The molecular labeling relevant to the reproductive trait of pig of β gene, and provide this molecular labeling detection method and its Application in reproductive trait selection provides a kind of new labeling method for the molecular marker assisted selection of pig, is the molecule of pig Breeding provides reference.

Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.

Detailed description of the invention

The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:

Fig. 1 is the 3 exon region mutational site A511G sequencer map of pig FSH β gene of the present invention；

Fig. 2 is the 3 exon region mutational site A617G sequencer map of pig FSH β gene of the present invention；

Fig. 3 is the 3 exon region mutational site C630T sequencer map of pig FSH β gene of the present invention；

Fig. 4 is the 3 exon region mutational site C652T sequencer map of pig FSH β gene of the present invention；

Fig. 5 is the 3 exon region mutational site C678T sequencer map of pig FSH β gene of the present invention；

Fig. 6 is the 3 exon region mutational site C735T sequencer map of pig FSH β gene of the present invention；

Fig. 7 is the 3 exon region mutational site A746G sequencer map of pig FSH β gene of the present invention；

Fig. 8 is the 3 exon region mutational site A921G sequencer map of pig FSH β gene of the present invention；

Fig. 9 is 3 exon pcr amplification product gel electrophoresis figure of pig FSH β gene of the present invention；Wherein, the hole 1-7 and The hole 9-15 is the 3 exon region amplified production of FSH β gene of Large White, and clip size 1022bp, the 8th hole is 2000 DNA marker；

Figure 10 is the 3 exon nucleotide sequence of pig FSH β gene that the present invention expands, and clip size is 1022bp, dash area is intron sequences in figure, and shown in box is base mutation, and underscore part is primer sequence Column.

Specific embodiment

Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.

Embodiment 1: the acquisition of pig ESR gene segment and SNPs detection

FSH β gene 3 exon nucleosides relevant to pig reproductive trait of the present invention as molecular labeling application Acid sequence, nucleotide sequence as shown in SEQ ID NO.1, wherein 511bp, 617bp of sequence, 630bp, 652bp, There are gene mutations at 678bp, 735bp, 746bp, 921bp.(total yield coefficient, life birth are young for the above SNPs and the reproductive trait of pig Number) there is certain correlation, and be easy to detect, it is selected using the above site as molecule aid mark relevant to pig reproductive trait It selects.

201 different lines (U.S. system, pellet are plus are) Large White is collected from Sichuan Province Leshan Tian Ren agriculture and animal husbandry farm Coat sample and the breeding record of production (primiparity and total yield coefficient and litter size living through producing), and 201 are extracted with kit The complete genome DNA of Large White.

It is online to compare FSH β according to the FSH β gene nucleotide series (NM_213875.1) announced in GenBank database 3 exon region in gene carries out specific primer design to three above region using Primer5.0 software and (is detailed in table 1), and by template of genomic DNA pcr amplification reaction is carried out.Reaction system are as follows: 2 × Taq Master Mix 15 μ L, DNA 1.5 μ L, 1.5 μ L of upstream primer (10 μM), downstream primer (10 μM) 1.5 μ L, ddH₂O 10.5μL.Response procedures: 94 DEG C of pre- changes Property 1.5min；94 DEG C of denaturation 20s, 56.4 DEG C of annealing 20s, 72 DEG C of extension 60s are recycled 30 times；Last 72 DEG C re-extend 5min, and 4 DEG C save amplified production.

1 FSH β gene primer information table of table

It takes 4 μ L to carry out agarose gel electrophoresis in every pipe PCR product, send the PCR product containing single band according to after glue To Chengdu, Qing Ke biotech firm is sequenced.Sequencing result is compared respectively using BioEdit software, finds purpose piece Section present in mutational site, and record it is each individual present in corresponding site (A511G, A617G, C630T, C652T, C678T, C735T, A746G, A921G) mutation type (mutational site corresponds to sequencer map as shown in figures 1-8).FSH β gene 3 The information such as exon nucleotide sequence, corresponding primer and mutational site difference is as shown in Figure 10.

The genotype frequency and gene frequency in 8 sites SNPs in 2 FSH β gene of table

As shown in Table 2,8 mutational sites of FSH β gene show as three kinds of genotype respectively in 201 Large Whites.Its In, the genotype frequency in the site A511G is that GG > AG > AA trend is showed in great Bai in U.S.A, and two kinds are presented in pellet system great Bai Genotype GG > AA is that a kind of genotype GG is presented in great Bai adding, and G gene frequency is all larger than A in three kinds of strain Large Whites Gene frequency；The genotype frequency in the site A617G is great Bai in U.S.A and pellet is that AA > AG > GG trend is showed in great Bai, It is that AA > GG > AG trend is showed in great Bai adding, A gene frequency is all larger than G equipotential base in three kinds of strain Large Whites Because of frequency；The genotype frequency in the site C630T is great Bai in U.S.A and pellet is that CC > CT > TT trend is showed in great Bai, is adding CC > TT > CT trend is showed in great Bai, C gene frequency is all larger than T allele frequency in three kinds of strain Large Whites Rate；The genotype frequency in the site C652T is great Bai in U.S.A and pellet is that TT > CT > CC trend is showed in great Bai, is great Bai adding In show TT > CC > CT trend, T gene frequency is all larger than C gene frequency in three kinds of strain Large Whites； The genotype frequency of C678T is in U.S. system, red system and plus is that big Bai Zhongjun shows CC > CT > TT trend, and C allele frequency Rate is all larger than T gene frequency；The genotype frequency in the site C735T U.S.A be showed in great Bai and pellet system great Bai TT > CT > The trend of CC is that TT > CC > CT trend is showed in great Bai adding, and T gene frequency is big in three kinds of strain Large Whites In C gene frequency；The genotype frequency in the site A746G is great Bai in U.S.A and pellet is to show AA > AG > GG in great Bai to become Gesture is that AA > GG > AG trend is showed in great Bai adding, and A gene frequency is all larger than G etc. in three kinds of strain Large Whites Position gene frequency；The genotype frequency of A921G is in U.S. system, red system and plus is that big Bai Zhongjun shows AA > AG > GG trend, and A Gene frequency is all larger than G gene frequency.

Embodiment 2: application of the molecular labeling in Large White reproductive trait

For the relationship established between ESR gene and Large White reproductive trait, chooses 201 and producing Large White (Leshan sichuan day People's animal husbandry) it is used as experimental material, 201 Large White Reproduction records are acquired, and using the SNPs detection mentioned in embodiment 1 Mode detects experimental population.Using the GLM model in SAS8.0 software to existing in the FSH β gene of every Large White 8 sites SNPs and its being associated property of reproductive trait analysis, as a result indicated with LSM ± standard error.

To in FSH β gene there are the site SNPs and reproductive trait (primiparity and total yield coefficient and litter size living) through producing into Row association analysis, using model are as follows: Y_ij=μ+G_i+P_j+e_ij, wherein Y_ijFor reproductive trait record value, μ is group's mean value, G_iFor Genotype effects, P_jFor parity effect, e_ijFor random residual effect.

Influence of the 3 FSH β gene different loci genotype of table to reproductive trait

As shown in Table 3, A617G, A746G, A921G in 201 Large White groups, in 3 exon of FSH β gene At site, produced life birth son number of the GG genotype in U.S. system individual is substantially less than AG genotype and AA genotype (p < 0.05)； At the site C630T, TT genotype in U.S. system individual produced life birth son number be substantially less than CT genotype and CC genotype (p < 0.05)；At the site C652T, C735T, produced life birth son number of the CC genotype in U.S. system individual is substantially less than CT genotype With TT genotype (p < 0.05)；At the site C678T, produced life birth son number of the CT genotype in U.S. system individual is significantly higher than CT Genotype (p < 0.05)；And difference is not significant (p > 0.05) between other characters.

Embodiment 3: haplotype analysis

Haplotype is carried out to from 8 SNP in 3 exon of FSH β gene that detected in 201 Large White individuals Analysis, obtains following result:

4 FSH β gene of table, 3 exon haplotype type and frequency

As shown in Table 4,8 mutational sites detected constitute 12 kinds of haplotypes, wherein highest three lists of frequency times Type is H1, H2 and H3, respectively 0.642,0.124 and 0.086.

Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention In the protection scope that benefit requires.

Sequence table

<110>Sichuan Agricultural University

<120>molecular labeling and application based on FSH β identified for genes Large White reproductive trait

<130> 2019

<141> 2019-04-26

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1022

<212> DNA

<213>follicular stimulating hormone β subunit (follicle-stimulating hormone beta subunit, FSH β)

<400> 1

attcaatgcc tgtctcattt tgattaaata gaaacttctg taatacttta acctaactct 60

ctctctctcc cctgaatccc ttaggacctg gtatacaagg acccagccag gcccaacatc 120

cagaaaacat gtaccttcaa ggagctggtg tacgagaccg tgaaagtacc tggctgtgct 180

caccatgcag actccctgta tacgtatcca gtagccactg aatgtcactg tggcaagtgt 240

gacagtgaca gtactgactg caccgtgaga ggcctggggc ccagctactg ctccttcagt 300

gaaatgaaag aataaagagc agtggacatt tcatgcttcc tacccttgtc tgaaggacca 360

agacgtccaa gaagtttgtg tgtacatgtg cccaggctgc aaaccactat gagagacccc 420

actgatccct gctgtcctgt ggaggaggag ctccaggaat gcagagtgct ggggcctcag 480

tcctatcacc actcaaccct gtattctggg tctggttcca taagttttat tcggtctttt 540

ttttttttaa attactcaat gaattttatt acatttataa ttgtacaatg atcatcacag 600

cccaatttta taggatttcc atcccaaacc cccagcatag acccccatct cccaatctgt 660

ctcatttgga aaccataagt ttttcaaagt ccgtgagtca gtatctactc agtcttatta 720

ccttaaagac atgtgggtgt tttctgttta ataatcttag aaatcctctc aagacaggga 780

tatggaccca gaggaaggaa atgggctaag aatgggtgaa aggactaaat gcagcattct 840

cccactagac acagaagcct acaagagcag ggccagtctc tttgtcatga gtgtggcctc 900

aatacctagc acagtgacta gaattcagta agaaactcaa gaatggcttc cttaaggaaa 960

gtaagattgg aaatgtaggg ggtaggaaaa tactgaaaga agatgttgga ggctatgtga 1020

tg 1022

<210> 2

<211> 17

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 2

attcaatgcc tgtctca 17

<210> 3

<211> 17

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 3

catcacatag cctccaa 17

Claims (7)

1. one kind is based on FSH β gene molecular labeling relevant to Large White reproductive trait, which is characterized in that the molecule mark Note is located on the 3rd exon of pig FSH β gene, i.e., 3 exon regions 511,617,630,652,678,735,746, At 921, be denoted as g.511A > G, g.617A > G, g.630C > T, g.652C > T, g.678C > T, g.735C > T, g.746A > G and g.921A>G。

2. the primer pair for detecting molecular labeling described in claim 1, which is characterized in that including FSH β-E3-F and FSH β- E3-R, nucleotide sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.

3. reagent or kit containing primer pair described in claim 2.

4. a kind of method for detecting molecular labeling described in claim 1, which is characterized in that the described method includes: with Large White Genomic DNA is that template utilizes primer pair progress PCR amplification acquisition PCR product as claimed in claim 2；To the PCR product Carry out agarose gel electrophoresis.

5. according to the method described in claim 4, it is characterized in that, pcr amplification reaction system are as follows: 2 × Taq Master Mix 15 μ L, DNA 1.5 μ L, 10 μM of upstream primers 1.5 μ L, 10 μM of downstream primers 1.5 μ L, ddH₂O 10.5μL。

6. according to the method described in claim 4, it is characterized in that, pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 1.5min； 94 DEG C of denaturation 20s, 56.4 DEG C of annealing 20s, 72 DEG C of extension 60s are recycled 30 times；Last 72 DEG C re-extend 5min, 4 DEG C of preservations.

7. application of the molecular labeling described in claim 1 in pig kind breeding.