CN109554486B - SNP molecular marker related to grass carp traits and application thereof - Google Patents

Abstract

The invention discloses SNP molecular markers related to grass carp traits and application thereof, wherein SNP loci related to grass carp growth conditions are located at 159 th base of grass carp gene SNP24, 410 th base of gene SNP25, 370 th base of gene SNP26 and 374 th base of gene SNP 27; if the 159 th base of the SNP24, the 410 th base of the SNP25 and the 370 th base of the SNP26 are CC genotypes, the weight is obviously better than that of TT genotypes, and if the 374 th base of the SNP27 is TT genotypes, the weight is obviously better than that of CC and TC genotypes. The invention can be effectively used for molecular marker-assisted breeding of grass carps, selects proper genotype grass carp parents to breed, can obtain grass carp fries with faster growth, and accelerates the breeding process of the grass carps.

Description

SNP molecular marker related to grass carp traits and application thereof

Technical Field

The invention belongs to the field of molecular biology DNA marking technology and application, and particularly relates to a SNP molecular marker related to grass carp traits and application thereof.

Background

Grass carp (Ctenophagogon idella) belongs to the subfamily of Octissicidae (Leuciscinae) and grass carp (Ctenophagon), has the advantages of high growth speed, delicious meat quality, high nutritional value and the like, is a culture object with the largest annual output of freshwater cultured fishes in China at present, and the annual output of the grass carp is increased from 356 ten thousand tons to 590 ten thousand tons from 2007 to 2016, and the annual output is increased by 65.73%. Therefore, the healthy and benign development of the grass carp breeding industry not only provides a large amount of high-quality animal protein for people in China and even developing countries, but also drives the development of related industries such as feed processing, transportation and sale industries to be strong, and solves the employment problem of a large number of people. Until now, although a lot of efforts have been made by fishermen, grass carp still has no improved variety of artificial breeding identified by the national improved variety approval committee, in practice, the grass carp is mostly formed by directly domesticating wild species, directional breeding is lacked, problems of slow growth speed, irregular specification, poor disease resistance and the like of offspring due to poor parent quality often occur in production, and farmers often face great economic losses, so breeding high-yield and high-quality grass carp improved variety is always the central target of grass carp breeding work.

The method for breeding the new grass carp species with higher growth speed can save feed cost, shorten breeding period and further greatly reduce the breeding cost of the grass carp, so that the development of a rapid and reliable breeding technology is necessary for breeding the fast-growing grass carp species (line). When developing conventional breeding research, the development and application of molecular marker assisted breeding technology can accelerate the breeding process of grass carp fine breeds. SNPs refer to polymorphisms in genomic DNA sequences due to single nucleotide variations, and non-synonymous SNPs located in coding regions of genes may cause amino acid changes, thereby affecting the function of proteins, and particularly SNPs occurring in structural functional regions are particularly important, ultimately causing changes in biological phenotypes.

Genome-wide association analysis (GWAS) aims at screening SNPs associated with traits from a Genome-wide range. The cost of GWAS analysis is mainly 2, first, the number of samples used for analysis; second, the cost of genotyping. The larger the amount, the higher the typing cost and the cost for measuring the traits. To reduce the cost, GWAS methods have been developed that only sequence Extreme samples, such as BSR-seq (RNA-seq based BSA), XP-GWAS (Extreme-genotype-with-association study), etc. Researches prove that XP-GWAS can effectively reduce the workload of genotyping and can carry out SNP screening with low cost and high benefit. SNP typing techniques are also gradually developed from low and medium throughput in early stages to high throughput gene chip, re-sequencing techniques. Wherein RAD-seq (restriction Association site DNA sequencing) is a cheap and efficient SNP discovery and typing high-throughput method. By comprehensively considering the experiment cost and efficiency, the RAD-seq technology and the XP-GWAS strategy can be adopted to search SNP sites related to the growth traits of the grass carps in the whole genome range, so as to search new genetic markers and provide basic data for molecular marker-assisted breeding of new varieties of fast-growing grass carps.

Disclosure of Invention

The invention aims to provide a marker of 4 SNPs related to body weight of grass carp and application thereof.

The technical scheme adopted by the invention is as follows:

the gene sequence related to grass carp growth is shown in SEQ ID NO. 1-SEQ ID NO. 4.

In the gene sequence, the 159 th Y of SEQ ID NO.1 is a base T or C, the 410 th Y of SEQ ID NO.2 is a base C or T, the 370 th Y of SEQ ID NO.3 is a base C or T, and the 374 th Y of SEQ ID NO.4 is a base C or T.

The gene sequences SEQ ID NO. 1-SEQ ID NO.4 are applied to judging the growth speed of the grass carps.

And SNP sites related to the growth speed of the grass carp are 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.1, 370 th base of SEQ ID NO.1 and 374 th base of SEQ ID NO. 1.

The SNP locus is applied to judging or identifying the growth speed of the grass carp.

The SNP locus is applied to breeding of grass carp varieties with excellent growth.

A method for screening the growth speed of grass carps comprises the following steps:

detecting whether the SNP locus at the 159 th base position of the grass carp gene sequence SEQ ID NO.1 is genotype CC or not, if so, determining that the grass carp is excellent in weight and fast in growth;

or/and detecting whether the SNP locus at the 410 th base position of the grass carp gene SEQ ID NO.2 is a genotype CC or not, if so, determining that the grass carp is excellent in weight and fast in growth;

or/and detecting whether the SNP locus at the 370 th base position of the grass carp gene SEQ ID NO.3 is a genotype CC or not, if so, determining that the grass carp is excellent in weight and fast in growth;

or/and detecting whether the SNP locus at the 374 th base position of the grass carp gene SEQ ID NO.1 is the genotype TT, if so, the grass carp is excellent in weight and fast in growth.

Further, the method comprises the following steps:

(1) extracting DNA of the grass carp;

(2) carrying out PCR amplification experiment by taking the extracted DNA as a template to obtain a target fragment containing SNP sites, and detecting whether the genotype of the 159 th base of the grass carp gene SEQ ID No.1, the 410 th base of the gene SEQ ID No.2 and the 370 th base of the gene SEQ ID No.3 is CC or not or whether the genotype of the 374 th base of the gene SEQ ID No.4 is TT or not.

Further, the PCR amplification of the step (2): PCR amplification was performed using SNP24-F, SNP24-R, SNP35-F, SNP35-R, SNP36-F, SNP36-R, SNP37-F and SNP37-R primer pairs to obtain PCR products, the nucleotide sequences of the primers were as follows:

SNP24-F：5'-CGTAGTCACGACAGGACACGT-3'；(SEQ ID NO.5)

SNP24-R：5'-CAGTGATTCTTCTGTTAATTCT-3'；(SEQ ID NO.6)

SNP35-F：5'-GATATGGATGACTGTCTCTCC-3'；(SEQ ID NO.7)

SNP35-R：5'-CAATCCAGGTCAGATAGTACA-3'；(SEQ ID NO.8)

SNP36-F：5'-TCAGGGTTCAAAGTTTGAGTG-3'；(SEQ ID NO.9)

SNP36-R：5'-GTGCGAGGTGATTGGTATCTG-3'；(SEQ ID NO.10)

SNP37-F：5'-GACAATGAGTTCACTATTCT-3'；(SEQ ID NO.11)

SNP37-R：5'-CAAGGTTCTGGAATCATTCCT-3'(SEQ ID NO.12)；

then carrying out PCR extension amplification on the PCR product by using extension primers SNP24-P, SNP35-P, SNP36-P and SNP37-P to determine the genotype of the 159 th base of the grass carp gene SEQ ID No.1, the 410 th base of the gene SEQ ID No.2, the 370 th base of the gene SEQ ID No.3 and the 374 th base of the gene SEQ ID No. 4; the nucleotide sequence of the extension primer is as follows:

SNP24-P：5'-TACGCTGTGAGAAACTGTGAGG-3'；(SEQ ID NO.13)

SNP35-P：5'-GTGAAAGGGGGAATACTATAAC-3'；(SEQ ID NO.14)

SNP36-P：5'-TTTTTTTTTTCGGACTCGGAGGTTGAGAGCTA-3'；(SEQ ID NO.15)

SNP37-P：5'-AGATCTCAATCCAAGCGAGCAC-3'(SEQ ID NO.16)。

further, the PCR amplification system is as follows:

the PCR amplification reaction conditions are as follows: denaturation at 94 ℃ for 15 s; annealing at 54 ℃ for 15s, and extension at 72 ℃ for 30s, and 24 cycles.

The invention has the beneficial effects that:

(1) the genotype obtained by the invention is based on base mutation generated in the gene, so that genetic exchange does not exist, and further verification of phenotype is not required.

(2) According to the invention, by detecting four SNP markers of the grass carp, namely SNP24, SNP35, SNP36 and SNP37, the grass carp with larger weight can be effectively selected under the same breeding condition, and the method can be effectively used for molecular marker assisted breeding of the grass carp. Furthermore, the method can identify the genotype of grass carp parents according to actual breeding requirements, select the proper genotype grass carp parents for breeding, obtain grass carp offspring (fry) which grow faster, save breeding time, have low cost and high accuracy, and accelerate the breeding process of grass carp.

Drawings

FIG. 1 is a detection peak diagram after SNP extension reaction, wherein the colors and corresponding nucleotides of the peak diagram are as follows: green-a, red-T, black-C, blue-G.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1 acquisition of SNP marker

1. The grass carp gene sequence obtained by sequencing is shown as SEQ ID NO. 1-SEQ ID NO.4, SNP loci are respectively found at 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.1, 370 th base of SEQ ID NO.1 and 374 th base of SEQ ID NO.1, allele C and allele T are arranged at 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.1, 370 th base of SEQ ID NO.1 and 374 th base of SEQ ID NO.1, and CC, TC and TT genotypes are formed.

The sample for growth character (weight) analysis is a 25-month-old grass carp colony which is bred in the same batch and raised in the same pond, 298 grass carps are randomly selected for growth character (weight) correlation analysis, 20 individual tails with extremely large weight (average weight of 2659 +/-126.40 g) and 20 individual tails with extremely small weight (average weight of 744.76 +/-73.35 g) are selected, and 2ml of each of parent and offspring tail vein blood (added with ACD anticoagulant for anticoagulation) is respectively extracted for subsequent experiments.

In the following sequenceYRepresentative are mutated bases.

The gene sequence fragment of SNP24 is shown as SEQ ID NO.1, RAD library tag (Ctg294657.0), and the mutation position is T159C:

ATCGGTCCACGTAACTCTCCCGAAAGCCTTTCCGCACCGCGCGGGCCATACTGACCACCGTAGTCACGACAGGACACGTGCTGAAACCCAAGAGGACGGGATGATGGTCTTTAATTCATTAACATGACGAGTGCTGTGTGTGTGTGTGTGTGTTTGTTYCCTCACAGTTTCTCACAGCGTATGCCGCTCGTCCCCGTCGGACACACACAGGTGTTCAGAGGGCCGCAGGCAGCACCATGTTGGCAGGGAGGAACACACGGTGTAGTGACAGCTGCACACACACACACAATTACATGATCACACACTACATCAAAACACCACAGAATTAACAGAAGAATCACTGCAAATGAAACTGAAACTGAAAAAATGAATGTCATATGAATGATTCAATGATTAATGATGTCATAATGAAAGGATTATAAT(SEQ ID NO.1)；

SNP35 sequence fragment, RAD library tag (ctg355219.0), mutation position C410T:

ATTACAACACACCAGTGGTGAGGTCAGCTGGTGGTAGCATGTGTTTCCTGATGTTGATTAGTTTGATTTTGTCTAGTATAAGTGCATTCTTTTTCTTTGGAGAACCCACATCTGCACTTTGCCTCCTGCGAAATGCCATATTTGCATTTTTCTTCACTGTCTGTATTTCCTGTTTGACTGTCCGTTCCTTTCAAATTGTTTGTGTTTTTAAAATGGCTGCTCAGTTCCCTAAGGTGCACAGCCTTTGGGTAAAGCACAATGGCCAGTGGCTCTTCATTGCATTTACTTCTGTCATTCATTTAATTTCTTGTGTGATATGGATGACTGTCTCTCCTGTCAAAGTCACGGCTGACTGGTGGACTTATACAGATCAAATTATGCTCGTCTGTGAAAGGGGGAATACTATAACYTTAACCATAGTTGTGTTCATAGGTTGGTTTCTTGGTTTCCTGTGTCTCCTGTTTTCCTACATGGGAAGAGATCTGCCGAAAAATTACAATGAGGCCAAATCAATAACCTTCAGTCTAACTTTGTACTATCTGACCTGGATTGGATATTTCACAGCATACCTCTCTTTCAAAAGCAAATACATCATCCTTTTGAATGCACTGGCTCAAATATCCAGTATAAATGGAATt(SEQ ID NO.2)；

SNP36 sequence fragment, RAD library signature (ctg36503.0), mutation position C370T:

AGTGTTCCGTGAGGAAATGGTGCGTTGACCACAGGGCTTTGATTACAGAAGCGGCTGGTGTAATGGAGACGGAGGACTCTCTGTTCTCCTGAGGCCCCCAGACGCTTCCCGCATGTGTTTACTTAACGCAGTTAGGGCTTACGCCATGCATGTGATTACCAAGTGGCTGACACTTCATATCGCAAATAGACAAACTAAACAGAGAGGCCACACAACTTCACCCTAATTTTTTTTTGGAGGGAAATTCTGTGGTTTGGATGTGAAATTCTGTGAATGGTCCCTGTAGAGTTTAAAATCTTCTTTTTTTTAATATCTTCCCCCATGTATCAGGGTTCAAAGTTTGAGTGCGGACTCGGAGGTTGAGAGCTAYGCGTCCTTCATAGCCCAGGCTCTAGAAAAGACGCGTGGCCGTGAGTGTGTGCCCTCTTGGGAGGAGATCCAGGGGCTGATGGGAAGGCAGGAGATACTGTGTGCTGTGCACTACCCTGGACCGGGCTGCTGCCAGATACCAATCACCTCGCACACTACGGCTAATGAGGTATTGCATCAGTCGTTTCCTCTACAGCAGGGATTGGGAAc(SEQ ID NO.3)；

SNP37 sequence fragment, RAD library tag (ctg378190.0), mutation position C374T: :

AATCGACTCTAAAGTCTGGATCGGATGGACGTGTTCGAATGCACACCTGTGACCACATATAACTATAATAATAAAAAAATAAAAAAATAATTTAGGGCAAGTATTATGTTTTTTTTTTTGTTTTTTTGGCTGTTGGCAATTCAGCTTTGCAGTTTTAAATTGCAATATTTCACAATATGTCTGCTTTTACTGTATTTTTGATCAAATAAATGCAGCATTGGTGAGACTTTACCAACCCTAAACTTTTGGCCCTAAACCAGCAGGATAACGCCACAAAGCTTAAATCATCTCAAACTGGTTTTTTAAACAAGACAATGAGTTCACTATTCTCAAATGGCCTCCACAGTCTCCAGATCTCAATCCAAGCGAGCACYTTTGGGATGTGGTGCAACAGGAGATTTGCATAATGGATGTGCAGCTGACAAATCTGCAGCAACTGTGTGATGCCATCATGATAATATGGACCAAACTCTCTGAGGAATGATTCCAGAACCTTGTTGAATTTATGCCACTATGAATTAAGGCAGTTCTGAAATCAACTTGGTACTAGAAAGATGTACAGTACCTAATAAAGTGGCCGGTTAGTgtatgtatacacacacacacacacacacacacacacac(SEQ ID NO.4).

example 2 screening for growth differential markers

The constructed 20-tailed samples of the extremely large individuals and 20-tailed samples of the extremely small individuals are used for carrying out primary verification on the screened 84 differential SNPs markers. Of the 84 selected SNPs sites, 40 were not detected and 44 were detected SNP mutations. The close analysis of the difference of the 44 SNPs detected with the mutant in the extremely large individual group and the extremely small individual group shows that the difference of SNP24, SNP36 (P <0.05), SNP37 and SNP38 (P <0.01) is very significant (see Table 2).

TABLE 2 markers for extreme population typing

Note: "_" indicates that this position P < 0.05.

Example 3 verification of correlation of SNP marker associated with grass carp body weight and body weight

1. Sample DNA extraction

(1) 100ul of blood of the fish to be tested or 3mg of fin tissue after cutting is taken, 0.5mL of lysate (10mmol/L Tris-HCl; 0.1mol/L EDTA; 0.5% SDS; 30mg/L RNase; 100mg/L proteinase K, pH8.0) is added, and the fish is digested at 55 ℃ for 1 hour while gently shaking occasionally.

(2) Adding phenol/chloroform/isoamyl alcohol (25: 24: 1) with the same volume, evenly mixing by inversion, standing for 5 minutes at room temperature, centrifuging for 10 minutes at 12000 r/min, taking supernatant, extracting once by using chloroform, standing for 5 minutes at room temperature, centrifuging for 10 minutes at 12000 r/min, and taking supernatant.

(3) 2 times of volume of absolute ethanol is added, the mixture is kept stand at room temperature for 10 minutes to precipitate DNA, and the mixture is centrifuged at 12000 rpm for 10 minutes.

(4) Washing with 70% ethanol for 1 time, centrifuging at 12000 rpm for 2 minutes, removing the supernatant, standing at room temperature for drying for 10 minutes, adding 50. mu.l of TE (10mmol/L Tris-HCl; 1mmol/L EDTA, pH8.0) to dissolve DNA, and storing at 4 ℃ for further use.

2. Primer design and Synthesis

A primer pair and Snapshot primers of 4 SNP loci related to body weight are designed and synthesized according to grass carp gene fragments (SEQ ID NO. 1-SEQ ID NO.4), and the primer sequences are as follows:

SNP24-F：5'-CGTAGTCACGACAGGACACGT-3'；(SEQ ID NO.5)

SNP24-R：5'-CAGTGATTCTTCTGTTAATTCT-3'；(SEQ ID NO.6)

SNP35-F：5'-GATATGGGATGACTGTCTCTCC-3'；(SEQ ID NO.7)

SNP35-R：5'-CAATCCAGGTCAGATAGTACA-3'；(SEQ ID NO.8)

SNP36-F：5'-TCAGGGTTCAAAGTTTGAGTG-3'；(SEQ ID NO.9)

SNP36-R：5'-GTGCGAGGTGATTGGTATCTG-3'；(SEQ ID NO.10)

SNP37-F：5'-GACAATGAGTTCACTATTCT-3'；(SEQ ID NO.11)

SNP37-R：5'-CAAGGTTCTGGAATCATTCCT-3'(SEQ ID NO.12)

3, PCR amplification reaction system:

PCR amplification reaction conditions:

denaturation at 94 ℃ for 15 s; annealing at 54 ℃ for 15s, extending at 72 ℃ for 30s, and performing 24 cycles; extension at 72 ℃ for 3 min. Obtaining PCR amplification reaction products. The amplification can obtain bands with band sizes of 423bp, 638bp, 579bp or 624 bp.

4. 3ul of PCR amplification reaction product was purified by ExoI and Sap, and the primers and dNTP in the PCR product were removed.

Purifying the reaction system:

purification reaction conditions: the purified PCR product is obtained at 37 ℃ for 45min and 80 ℃ for 15 min.

5. Single base extension of purified PCR products

The reaction system is as follows:

the primer sequences for extension were as follows:

SNP24-P：5'-TACGCTGTGAGAAACTGTGAGG-3'；(SEQ ID NO.13)

SNP35-P：5'-GTGAAAGGGGGAATACTATAAC-3'；(SEQ ID NO.14)

SNP36-P：5'-TTTTTTTTTTCGGACTCGGAGGTTGAGAGCTA-3'；(SEQ ID NO.15)

SNP37-P：5'-AGATCTCAATCCAAGCGAGCAC-3'；(SEQ ID NO.16)

and (3) extension reaction conditions:

5. mu.l of the extension product was taken, added with 8. mu.l of loading, denatured at 95 ℃ for 3min, and immediately subjected to ice-water bath.

6. Grass carp genotype analysis

Performing SNaPshot typing on the PCR product by using a sequencer (the sequencer model is ABI 3730XL), judging the genotype of each sample grass carp according to the color of a peak diagram of a sequencing result, judging whether an SNP locus at the 159 th base position of the grass carp gene SEQ ID NO.1 is a CC genotype, and if the SNP locus is the CC genotype, judging that the grass carp is heavier and grows fast; whether the SNP locus at the 410 th base position of the gene SEQ ID NO.2 is a CC genotype or not, if the SNP locus is the CC genotype, the grass carp with heavier weight and rapidly growing weight is obtained; whether the SNP locus at the 370 th base position of the gene SEQ ID NO.3 is a CC genotype or not, if the SNP locus is the CC genotype, the grass carp with heavier weight and rapidly growing weight is obtained; or/and whether the SNP locus at the 374 th base position of the gene SEQ ID NO.4 is TT genotype or not, if so, the grass carp with heavier weight and fast growth is obtained.

The correlation analysis of the screened SNPs with significant differences and the body weight of the grass carp is carried out by using a least square method, and the results in a random population are the same as those in an extreme population verification, namely the 4 SNPs are all significantly related to growth traits (P < 0.05). The weight average values of the CC type marked by the marker SNP24, the SNP35 and the SNP36 are all obviously higher than that of the TT type, while the weight average value of the TT type marked by the SNP37 is obviously higher than that of the CC and TC genotypes (see Table 3).

TABLE 34 analysis of association of SNPs markers with body weight traits

Note: the same letters indicate no significant difference between the two genotypes (P >0.05) and different letters indicate significant difference between the two genotypes (P < 0.05).

As can be seen from Table 3, the 159 th base of the grass carp gene SEQ ID NO.1, the 410 th base of the gene SEQ ID NO.2, the 370 th base of the gene SEQ ID NO.3, and/or the 374 th base of the gene SEQ ID NO.4 are closely related to the weight of the grass carp. According to the invention, by detecting four SNP markers of the grass carp, namely SNP24, SNP35, SNP36 and SNP37, the grass carp with larger weight can be effectively selected under the same culture condition. The invention finds that the SNP marker can be closely related to the body weight of the grass carp, and can be effectively used for molecular marker-assisted breeding of the grass carp. Furthermore, the method can identify the genotype of grass carp parents according to actual breeding requirements, select the appropriate genotype grass carp parents for breeding, obtain grass carp offspring (fry) with faster growth, has positive significance on the production of fast-growing grass carp fry, can greatly save breeding time, has low cost and high accuracy, and accelerates the breeding process of grass carp.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Zhujiang aquatic research institute of Chinese aquatic science research institute

<120> SNP molecular marker related to grass carp traits and application thereof

<130>

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 423

<212> DNA

<213> Artificial sequence

<400> 1

atcggtccac gtaactctcc cgaaagcctt tccgcaccgc gcgggccata ctgaccaccg 60

tagtcacgac aggacacgtg ctgaaaccca agaggacggg atgatggtct ttaattcatt 120

aacatgacga gtgctgtgtg tgtgtgtgtg tgtttgttyc ctcacagttt ctcacagcgt 180

atgccgctcg tccccgtcgg acacacacag gtgttcagag ggccgcaggc agcaccatgt 240

tggcagggag gaacacacgg tgtagtgaca gctgcacaca cacacacaat tacatgatca 300

cacactacat caaaacacca cagaattaac agaagaatca ctgcaaatga aactgaaact 360

gaaaaaatga atgtcatatg aatgattcaa tgattaatga tgtcataatg aaaggattat 420

aat 423

<210> 2

<211> 638

<212> DNA

<213> Artificial sequence

<400> 2

attacaacac accagtggtg aggtcagctg gtggtagcat gtgtttcctg atgttgatta 60

gtttgatttt gtctagtata agtgcattct ttttctttgg agaacccaca tctgcacttt 120

gcctcctgcg aaatgccata tttgcatttt tcttcactgt ctgtatttcc tgtttgactg 180

tccgttcctt tcaaattgtt tgtgttttta aaatggctgc tcagttccct aaggtgcaca 240

gcctttgggt aaagcacaat ggccagtggc tcttcattgc atttacttct gtcattcatt 300

taatttcttg tgtgatatgg atgactgtct ctcctgtcaa agtcacggct gactggtgga 360

cttatacaga tcaaattatg ctcgtctgtg aaagggggaa tactataacy ttaaccatag 420

ttgtgttcat aggttggttt cttggtttcc tgtgtctcct gttttcctac atgggaagag 480

atctgccgaa aaattacaat gaggccaaat caataacctt cagtctaact ttgtactatc 540

tgacctggat tggatatttc acagcatacc tctctttcaa aagcaaatac atcatccttt 600

tgaatgcact ggctcaaata tccagtataa atggaatt 638

<210> 3

<211> 579

<212> DNA

<213> Artificial sequence

<400> 3

agtgttccgt gaggaaatgg tgcgttgacc acagggcttt gattacagaa gcggctggtg 60

taatggagac ggaggactct ctgttctcct gaggccccca gacgcttccc gcatgtgttt 120

acttaacgca gttagggctt acgccatgca tgtgattacc aagtggctga cacttcatat 180

cgcaaataga caaactaaac agagaggcca cacaacttca ccctaatttt tttttggagg 240

gaaattctgt ggtttggatg tgaaattctg tgaatggtcc ctgtagagtt taaaatcttc 300

ttttttttaa tatcttcccc catgtatcag ggttcaaagt ttgagtgcgg actcggaggt 360

tgagagctay gcgtccttca tagcccaggc tctagaaaag acgcgtggcc gtgagtgtgt 420

gccctcttgg gaggagatcc aggggctgat gggaaggcag gagatactgt gtgctgtgca 480

ctaccctgga ccgggctgct gccagatacc aatcacctcg cacactacgg ctaatgaggt 540

attgcatcag tcgtttcctc tacagcaggg attgggaac 579

<210> 4

<211> 624

<212> DNA

<213> Artificial sequence

<400> 4

aatcgactct aaagtctgga tcggatggac gtgttcgaat gcacacctgt gaccacatat 60

aactataata ataaaaaaat aaaaaaataa tttagggcaa gtattatgtt tttttttttg 120

tttttttggc tgttggcaat tcagctttgc agttttaaat tgcaatattt cacaatatgt 180

ctgcttttac tgtatttttg atcaaataaa tgcagcattg gtgagacttt accaacccta 240

aacttttggc cctaaaccag caggataacg ccacaaagct taaatcatct caaactggtt 300

ttttaaacaa gacaatgagt tcactattct caaatggcct ccacagtctc cagatctcaa 360

tccaagcgag cacytttggg atgtggtgca acaggagatt tgcataatgg atgtgcagct 420

gacaaatctg cagcaactgt gtgatgccat catgataata tggaccaaac tctctgagga 480

atgattccag aaccttgttg aatttatgcc actatgaatt aaggcagttc tgaaatcaac 540

ttggtactag aaagatgtac agtacctaat aaagtggccg gttagtgtat gtatacacac 600

acacacacac acacacacac acac 624

<210> 5

<211> 21

<212> DNA

<213> Artificial primer

<400> 5

cgtagtcacg acaggacacg t 21

<210> 6

<211> 22

<212> DNA

<213> Artificial primer

<400> 6

cagtgattct tctgttaatt ct 22

<210> 7

<211> 22

<212> DNA

<213> Artificial primer

<400> 7

gatatgggat gactgtctct cc 22

<210> 8

<211> 21

<212> DNA

<213> Artificial primer

<400> 8

caatccaggt cagatagtac a 21

<210> 9

<211> 21

<212> DNA

<213> Artificial primer

<400> 9

tcagggttca aagtttgagt g 21

<210> 10

<211> 21

<212> DNA

<213> Artificial primer

<400> 10

gtgcgaggtg attggtatct g 21

<210> 11

<211> 20

<212> DNA

<213> Artificial primer

<400> 11

gacaatgagt tcactattct 20

<210> 12

<211> 21

<212> DNA

<213> Artificial primer

<400> 12

caaggttctg gaatcattcc t 21

<210> 13

<211> 22

<212> DNA

<213> Artificial primer

<400> 13

tacgctgtga gaaactgtga gg 22

<210> 14

<211> 22

<212> DNA

<213> Artificial primer

<400> 14

gtgaaagggg gaatactata ac 22

<210> 15

<211> 32

<212> DNA

<213> Artificial primer

<400> 15

tttttttttt cggactcgga ggttgagagc ta 32

<210> 16

<211> 22

<212> DNA

<213> Artificial primer

<400> 16

agatctcaat ccaagcgagc ac 22

Claims (6)

1. The SNP molecular marker related to the grass carp body weight has a sequence shown as SEQ ID No.1, the SNP locus is located at the 159 th site, and the polymorphism is T/C.

2. The application of the primer for detecting the SNP molecular marker of claim 1 in the breeding of grass carp varieties,

and selecting and breeding if the genotype of the SNP site at the 159 th base of the grass carp gene sequence SEQ ID NO.1 is CC.

3. A method for screening grass carp, which comprises the following steps:

detecting whether the SNP locus at the 159 th base position of the grass carp gene sequence SEQ ID NO.1 is a genotype CC or not, and if so, selecting and breeding.

4. A method according to claim 3, characterized by the steps of:

(1) extracting DNA of the grass carp;

(2) and (3) carrying out a PCR amplification experiment by taking the extracted DNA as a template to obtain a target fragment containing the SNP locus, and detecting whether the genotype of the 159 th base of the grass carp gene SEQ ID NO.1 is CC.

5. The method of claim 4,

the PCR amplification in the step (2): carrying out PCR amplification by using SNP24-F and SNP24-R primer pairs to obtain a PCR product, wherein the nucleotide sequences of the primers are as follows:

SNP24-F：5'-CGTAGTCACGACAGGACACGT-3' (SEQ ID NO.5)；

SNP24-R：5'-CAGTGATTCTTCTGTTAATTCT-3' (SEQ ID NO.6)；

performing PCR extension amplification on the PCR product by using an extension primer SNP24-P to determine the genotype of the 159 th base of the grass carp gene SEQ ID NO. 1; the nucleotide sequence of the extension primer is as follows:

SNP24-P：5'-TACGCTGTGAGAAACTGTGAGG-3' (SEQ ID NO.13)。

6. the method of claim 5, wherein the PCR amplification system is:

DNA 1μl；

10×buffer 1.5μl；

25mM MgCl₂ 1.5μl；

0.3. mu.l of 10mM dNTP;

upstream primer and downstream primer 0.15 μ l;

taq enzyme 0.3. mu.l;

H₂supplementing O to 15 μ l;

the PCR amplification reaction conditions are as follows: denaturation at 94 ℃ for 15 s; annealing at 54 ℃ for 15s, and extension at 72 ℃ for 30s, and 24 cycles.

Images