CN113930520B - SNP molecular marker related to grass carp characters and application thereof - Google Patents

Abstract

This application is a divisional application of 201811648716.7. The application belongs to the field of molecular biology DNA marking technology and application, and discloses that SNP loci related to grass carp growth conditions are positioned at 374 rd base of gene SNP 37; when the 374 th base of the SNP37 of the gene is TT genotype, the weight is obviously better than that of CC genotype individuals. The molecular marker-assisted breeding method can be effectively used for molecular marker-assisted breeding of grass carp, and proper genotype grass carp parents are selected for breeding, so that grass carp fries with faster growth can be obtained, and the breeding process of grass carp is accelerated.

Description

SNP molecular marker related to grass carp characters and application thereof

The application is a divisional application of China application with the application number of 201811648716.7, the application date of 2018, 12 months and 30 days and the invention name of SNP molecular marker related to grass carp characters and application thereof.

Technical Field

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

Background

Grass carp (Ctenopharyngodon idella) belongs to the subfamily of Oncorhynchidae (Leuci), and genus of grass carp (Ctenopharyngodon), and has the advantages of high growth speed, delicious meat quality, high nutritive value, etc., and is the object for culturing the freshwater fish with the largest annual output in China, the grass carp output is increased from 356 ten thousand tons to 590 ten thousand tons from 2007 to 2016, and the ten-year output is increased by 65.73%. Therefore, the healthy benign development of 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 sales industries to be strong, and solves the employment problem of a large number of people. To date, although aquatic workers have made much effort, grass carp has not had improved variety of artificial breeding identified by the national institutional review board, most of actual production is directly domesticated by wild species, directional breeding is lacking, problems of slow growth speed, uneven specification, poor disease resistance and the like of offspring are often caused by poor parent quality in production, and farmers often face great economic loss, so that breeding grass carp improved variety with high yield and high quality is always a central target of grass carp breeding work.

The breeding of new grass carp varieties with higher growth speed can save the cost of feed, shorten the cultivation period and greatly reduce the cultivation cost of grass carp, so that the development of a rapid and reliable breeding technology is necessary for cultivating the grass carp quick-growing varieties (lines). The development and application of molecular marker assisted breeding technology can accelerate the breeding process of grass carp fine breed while developing conventional breeding research. SNPs refer to polymorphisms in genomic DNA sequences due to single nucleotide variations, and non-synonymous SNPs located in coding regions of genes can lead to amino acid changes, thereby affecting the function of proteins, especially SNPs occurring in structural functional regions are particularly important, ultimately leading to changes in biological phenotype.

Genome-wide association analysis (Genome-wide association study, GWAS) aims to screen Genome-wide for SNPs associated with traits. The cost of GWAS analysis is mainly 2: first, the number of samples used for analysis; second, the cost of genotyping. The greater the number, the higher the cost of typing and measuring the property. To reduce costs, GWAS methods have been developed for sequencing only Extreme samples, such as BSR-seq (RNA-seq based BSA), XP-GWAS (Extreme-phenoype genome-wide association study), and the like. Studies prove that XP-GWAS can effectively reduce the workload of genotyping, and SNP screening with low cost and high benefit can be performed. SNP typing technology also gradually develops from medium and low flux at early stage to high flux gene chip and resequencing technology. Wherein RAD-seq (Restriction Association site DNA sequencing) is an inexpensive and efficient SNP discovery and typing high throughput method. Considering the experimental cost and efficiency comprehensively, the RAD-seq technology and XP-GWAS strategy can be combined to search SNP loci related to grass carp growth traits in the whole genome range, so that new genetic markers are searched, and basic data is provided for molecular marker-assisted selection of new varieties of fast-growing grass carp.

Disclosure of Invention

The invention aims at providing 4 SNPs marks related to grass carp weight 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, 159 th Y of SEQ ID NO.1 is a base T or C, 410 th Y of SEQ ID NO.2 is a base C or T, 370 th Y of SEQ ID NO.3 is a base C or T, and 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 grass carp.

SNP loci related to growth speed of grass carp are 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.2, 370 th base of SEQ ID NO.3 and 374 th base of SEQ ID NO. 4.

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

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

A method for screening the growth speed of grass carp, which comprises the following steps:

detecting whether the SNP locus at the 159 th base of the grass carp gene sequence SEQ ID NO.1 is genotype CC, if so, obtaining grass carp with excellent weight and fast growth;

or/and detecting whether the SNP locus at the 410 th base of the grass carp gene SEQ ID NO.2 is genotype CC, if so, the grass carp is excellent in weight and fast in growth;

or/and detecting whether the SNP locus at the 370 th base of the grass carp gene SEQ ID NO.3 is genotype CC, if so, the grass carp is excellent in weight and fast in growth;

or/and detecting whether the SNP locus at the 374 rd base of the grass carp gene SEQ ID NO.4 is 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 grass carp;

(2) And (3) carrying out PCR amplification experiments by taking the extracted DNA as a template to obtain a target fragment containing SNP loci, and detecting whether the genotype of 159 th base of a grass carp gene SEQ ID NO.1, 410 th base of a gene SEQ ID NO.2 and 370 th base of a gene SEQ ID NO.3 is CC or whether the genotype of 374 th base of a gene SEQ ID NO.4 is TT.

Further, the step (2) of PCR amplification: PCR amplification was performed using SNP24-F, SNP24-R, SNP35-F, SNP35-R, SNP36-F, SNP-R, SNP37-F and SNP37-R primer pairs, resulting in 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, SNP-P and SNP37-P, and determining genotypes of 159 th base of a grass carp gene SEQ ID NO.1, 410 th base of a gene SEQ ID NO.2, 370 th base of a gene SEQ ID NO.3 and 374 th base of a 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 15s; annealing at 54 ℃ for 15s and extending at 72 ℃ for 30s,24 cycles.

The beneficial effects of the invention are as follows:

(1) The genotypes obtained by the invention are based on the base mutation generated inside the gene, so that no genetic exchange exists and no further verification of the phenotype is required.

(2) According to the invention, through detecting four SNP markers, SNP24, SNP35, SNP36 and SNP37 of the grass carp, the grass carp with larger weight can be effectively selected under the same culture condition, and the molecular marker-assisted breeding of the grass carp can be effectively used. And then can carry out genotype identification to the grass carp parent according to actual breeding demand, select suitable genotype grass carp parent and reproduce, can obtain the grass carp offspring (fry) that growth is faster, practice thrift breeding time, low cost, the accuracy is high, quickens grass carp breeding process.

Drawings

FIG. 1 is a diagram showing detection peaks after SNP extension reaction: wherein A is a detection peak diagram with a genotype of TC, B is a detection peak diagram with a genotype of TT, C is a detection peak diagram with a genotype of CC, and the colors and corresponding nucleotides of the peak diagram are as follows: green-a, red-T, black-C, blue-G.

Detailed Description

The invention is further illustrated below with reference to examples.

Example 1 acquisition of SNP markers

1. The sequence of the grass carp gene obtained by sequencing is shown as SEQ ID NO. 1-SEQ ID NO.4, and each SNP locus is found at 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.2, 370 th base of SEQ ID NO.3 and 374 th base of SEQ ID NO.4, and alleles C and T are found at 159 th base of SEQ ID NO.1, 410 th base of SEQ ID NO.2, 370 th base of SEQ ID NO.3 and 374 th base of SEQ ID NO.4, so that the genotypes of CC, TC and TT are formed.

The samples for analysis of the growth traits (body weights) are 25 month old grass carp populations which are bred in the same batch and bred in the same pond, 298 grass carp is randomly selected for correlation analysis of the growth traits (body weights), the 20 tails of individuals with extremely large body weights (average body weight is 2659+/-126.40 g) and the 20 tails of individuals with extremely small body weights (average body weight is 744.76 +/-73.35 g), and 2ml of venous blood of the parent and offspring tail respectively is extracted (ACD anticoagulant is added) 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 tag (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

And (5) carrying out preliminary verification on the 84 screened different SNPs marks by using constructed extreme large individual 20 tail and extreme small individual 20 tail samples. Of the 84 SNPs sites selected, 40 did not detect SNP mutation and 44 detected SNP mutation. Analysis of the difference approaches of the 44 SNPs with detected mutants in the extremely large and small groups showed that SNP24, SNP36 differed significantly (P < 0.05), SNP37, SNP38 differed extremely significantly (P < 0.01) (see table 1).

TABLE 1 markers for typing in extreme populations

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

Example 3 verification of weight-related SNP markers of grass carp weight

1. Sample DNA extraction

(1) 100ul of blood of the fish to be tested or 3mg of sheared finbar tissue are taken, 0.5mL of lysate (10 mmol/L Tris-HCl;0.1mol/L EDTA;0.5% SDS;30mg/L RNase;100mg/L proteinase K, pH 8.0) is added, and the mixture is digested at 55℃for 1 hour, with gentle shaking in between.

(2) Adding equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), mixing, standing at room temperature for 5min, centrifuging at 12000 rpm for 10 min, collecting supernatant, extracting with chloroform once, standing at room temperature for 5min, centrifuging at 12000 rpm for 10 min, and collecting supernatant.

(3) 2 volumes of absolute ethanol were added, and the mixture was allowed to stand at room temperature for 10 minutes to precipitate DNA, and the mixture was centrifuged at 12000 rpm for 10 minutes.

(4) Washing with 70% ethanol for 1 time, centrifuging at 12000 rpm for 2 min, sucking off the supernatant, standing at room temperature, drying for 10 min, adding 50 μl TE (10 mmol/L Tris-HCl;1mmol/L EDTA, pH 8.0) to dissolve DNA, and storing at 4deg.C for use.

2. Primer design and Synthesis

According to grass carp gene fragments (SEQ ID NO. 1-SEQ ID NO. 4), designing and synthesizing primer pairs and Snapshot primers of 4 SNP loci related to body weight, wherein 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)。

PCR amplification reaction system:

PCR amplification reaction conditions:

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

4. 3ul of PCR amplification reaction products were purified by ExoI and Sap, and primers and dNTPs in the PCR products were removed.

Purifying a reaction system:

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

5. The purified PCR product was subjected to single base extension.

The reaction system is as follows:

the extension primer sequences 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)。

extension reaction conditions:

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

6. Grass carp genotype analysis

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

Correlation analysis was performed on screened SNPs with significant differences and grass carp body weight using least squares, which were identical in random population as verified in extreme population, i.e. all 4 SNPs were significantly correlated with growth trait (P < 0.05). The weight average value of the CC types marked by SNP24, SNP35 and SNP36 is significantly higher than that of the TT type, while the weight average value of the TT type marked by SNP37 is significantly higher than that of the CC and TC genotypes (see Table 2).

Table 24 SNPs markers and weight trait association analysis

Note that: the same letter indicates that the difference between the two genotypes is not significant (P > 0.05), and the different letter indicates that the difference between the two genotypes is significant (P < 0.05).

As can be seen from Table 2, the SNP locus of 159 th base of grass carp gene SEQ ID NO.1, 410 th base of gene SEQ ID NO.2, 370 th base of gene SEQ ID NO.3, or/and 374 th base of gene SEQ ID NO.4 is closely related to the weight of grass carp. According to the invention, through detecting four SNP markers, SNP24, SNP35, SNP36 and SNP37 of grass carp, grass carp with larger weight can be effectively selected under the same culture condition. The invention discovers that the SNP marker can be closely related to the weight of the grass carp, and can be effectively used for molecular marker assisted breeding of the grass carp. Furthermore, the genotype identification can be carried out on grass carp parents according to actual breeding demands, proper genotype grass carp parents are selected for breeding, grass carp offspring (fries) which grow faster can be obtained, positive significance is brought to the production of the fast-growing grass carp fries, the breeding time can be greatly saved, the cost is low, the accuracy is high, and the grass carp breeding process is accelerated.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhujiang aquatic institute of China aquatic science institute

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

<130>

<150> 201811648716.7

<151> 2018-12-30

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 423

<212> DNA

<213> Ctenopharyngodon idella

<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

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<212> DNA

<213> Ctenopharyngodon idella

<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

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<212> DNA

<213> Ctenopharyngodon idella

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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

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<212> DNA

<213> Ctenopharyngodon idella

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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

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<400> 5

cgtagtcacg acaggacacg t 21

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<213> artificial sequence

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cagtgattct tctgttaatt ct 22

<210> 7

<211> 22

<212> DNA

<213> artificial sequence

<400> 7

gatatgggat gactgtctct cc 22

<210> 8

<211> 21

<212> DNA

<213> artificial sequence

<400> 8

caatccaggt cagatagtac a 21

<210> 9

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<213> artificial sequence

<400> 9

tcagggttca aagtttgagt g 21

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gtgcgaggtg attggtatct g 21

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<212> DNA

<213> artificial sequence

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gacaatgagt tcactattct 20

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<212> DNA

<213> artificial sequence

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caaggttctg gaatcattcc t 21

<210> 13

<211> 22

<212> DNA

<213> artificial sequence

<400> 13

tacgctgtga gaaactgtga gg 22

<210> 14

<211> 22

<212> DNA

<213> artificial sequence

<400> 14

gtgaaagggg gaatactata ac 22

<210> 15

<211> 32

<212> DNA

<213> artificial sequence

<400> 15

tttttttttt cggactcgga ggttgagagc ta 32

<210> 16

<211> 22

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<213> artificial sequence

<400> 16

agatctcaat ccaagcgagc ac 22

Claims (5)

1. The application of the primer for detecting SNP molecular markers in breeding grass carp varieties with excellent weight,

the sequence of the SNP molecular marker is shown as SEQ ID NO.4, the SNP locus is positioned at 374 rd position, and the polymorphism is C/T;

if the genotype of the SNP locus at the 374 rd base of the grass carp gene sequence SEQ ID NO.4 is detected as TT, breeding is selected.

2. A method for screening grass carp with excellent weight, comprising the steps of:

detecting whether SNP locus at 374 rd base of grass carp gene sequence SEQ ID NO.4 is genotype TT, if so, selecting reproduction.

3. The method according to claim 2, comprising the steps of:

(1) Extracting DNA of grass carp;

(2) And (3) carrying out PCR amplification experiments by taking the extracted DNA as a template to obtain a target fragment containing SNP loci, and detecting whether the genotype of the 374 rd base of the grass carp gene SEQ ID NO.4 is TT.

4. The method of claim 3, wherein the step of,

and (2) PCR amplification in the step (2): PCR amplification is carried out by using SNP37-F and SNP37-R primer pairs to obtain PCR products, wherein the nucleotide sequences of the primers are as follows:

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

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

performing PCR extension amplification on the PCR product by using an extension primer SNP37-P, and determining the genotype of the 374 rd base of the grass carp gene SEQ ID NO. 4; the nucleotide sequence of the extension primer is as follows:

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

5. the method of claim 4, wherein the PCR amplification system is:

DNA 1μl；

10×buffer 1.5μl；

25mM MgCl ₂ 1.5μl；

10mM dNTP 0.3 μl;

0.15. Mu.l each of the upstream primer and the downstream primer;

0.3 μl of Taq enzyme;

H ₂ o was added to 15. Mu.l;

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

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