CN111304338A - SNP molecular marker linked with sex of channel catfish and genetic sex identification method - Google Patents
SNP molecular marker linked with sex of channel catfish and genetic sex identification method Download PDFInfo
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Abstract
The invention belongs to the technical field of fish genetic breeding, and relates to the technical field of SNP molecular markers. The invention discloses an SNP molecular marker linked with the sex of Ictalurus punctatus and a genetic sex identification method, which comprises the screening and verification of the SNP marker linked with the sex of the Ictalurus punctatus, the cloning of a male specific DNA sequence, sequence analysis, specific primer design and a PCR method for identifying the genetic sex; according to the obtained SNP marker primer linked with the sex of the channel catfish and the male specific DNA sequence, the PCR identification method of the genetic sex of the channel catfish is created. The invention screens SNP markers linked with the sex of the channel catfish and male specific sequences for the first time, and establishes a method for identifying the genetic sex of the channel catfish based on PCR and Sanger sequencing technologies. The method has the characteristics of high efficiency, accuracy and stability, has important application value in the aspects of culture of the parthenocarpic fry of the channel catfish and the like, and also has important application prospect in the research of positioning sex-determining genes and sex-determining mechanisms.
Description
Technical Field
The invention belongs to the technical field of molecular markers for fish genetic breeding, and particularly relates to an SNP molecular marker for identifying the sex of channel catfish and an identification method thereof.
Background
Ictalurus punctatus (Ictalures puncatus) is also called catus americanus and catus sulcus, belongs to the order of catiformes and Ictaluridae, is native to the United states, and has the characteristics of wide temperature range, superior meat quality, few thorns, high growth speed, strong disease resistance, poor feeding quality, high feed conversion rate, easy formation of seedling raising, breeding, feed, processing industrial production and the like. Since the introduction of the seeds in the 80 th generation of the 20 th century, China is popularized to 20 provinces and cities such as Hubei, Hunan, Sichuan and Jiangsu, the culture scale and the yield are increased year by year, and the annual yield is maintained to be about 25 ten thousand tons at present. The channel catfish is a diploid organism, the chromosomes of which consist of 28 pairs of autosomes and 1 pair of sex chromosomes (XX and XY chromosomes respectively), and belong to an XY/XX sex determination system, male heterozygosis and female homozygosis. The male and female growth of the channel catfish presents obvious allelopathy, namely the growth speed of a male individual and the quality of a mature body are higher than those of a female individual. Therefore, molecular markers linked with the sex of the channel catfish are actively excavated, and the method has important theoretical significance and application value on the research of sex-determining key genes and control mechanisms of the channel catfish, parthenocarpy fry breeding and the like.
SNP (Single Nucleotide polymorphism) refers to variation of a single Nucleotide in the genome, including substitution, transversion, deletion and insertion. The method has the characteristics of strong stability, high specificity, co-dominant inheritance, and a large number of markers which can be obtained, and is easy to realize high-throughput screening and typing. The genetic sex determination technology based on the SNP marker is one of the most widely and reliably applied means for sex determination of aquatic animals at present. In the process of various fish sex reversal researches, the genetic sex can be accurately identified by utilizing sex-linked SNP markers. Therefore, a set of method for rapidly and accurately identifying the genetic sex of the channel catfish based on SNP molecular markers is developed, and the method has important significance for developing the channel catfish full-male breeding work and researching sex determining genes and mechanisms.
Disclosure of Invention
The invention aims to provide an SNP molecular marker for identifying the genetic sex of channel catfish and an identification method thereof. The method provided by the invention can be used for rapidly and accurately identifying the genetic sex of the channel catfish. Mainly realized by carrying out PCR amplification on sex-linked SNP marker loci of the channel catfish, sequencing amplification products and identifying sequencing peak graphs.
The purpose of the invention can be realized by the following technical scheme:
an SNP molecular marker linked with the sex of channel catfish, which has the polymorphisms of g.43A > C, g.192G > A and g.197T > A3 at the 43 th, 192 th and 197 th positions of the nucleotide sequence shown in the specification. The genotype of an individual with male genetic sex at three positions is AC, GA and TA; individuals with female genetic sex have genotypes AA, GG, TT at three locations.
GTCCCCCTGACTCCACATCTATGCTGCAGTCTTCATGCTCAAR(A/C)TTCAGCCTTGTGTT CTGCACCCGACTTGTTGCCTCCACCTGAGATTTTGCCCCCACAGGATGATGATCCGCCTT CCTTAAGTCCTGAAGATGTACCAACATCTACAACTCACCGATGCCAGCAATGTCCTGAG GCCTTCAGTAGTTCAM(G/A)CACTN(T/A)CTTGCTGTCCATATGCAAAGCCACAAGAGAA GATTTGTCAGTCATCTGTTTTGCAAGTACTGCAATAAAAAGTTCATGCACCTGAAACGAT TGCGC。
Specific primers for detecting the SNP molecular markers are SEQ ID NO.31 and SEQ ID NO. 32.
A kit for detecting the SNP molecular marker, which comprises the specific primer.
The SNP molecular marker, the specific primer or the kit can be applied to identification of the genetic sex of the channel catfish.
A method for identifying the genetic sex of channel catfish comprises the steps of extracting channel catfish genomic DNA of the genetic sex to be detected, carrying out PCR amplification on the channel catfish genomic DNA by adopting specific primers of SEQ ID NO.31 and SEQ ID NO.32, carrying out bidirectional Sanger sequencing on an amplification product after purification, reading a Sanger sequencing peak diagram by using Chromas software, and judging the base sequencing peak diagram types of the sequence of the amplification product at the 43 th, 192 th and 197 th positions; the genotype of an individual with male genetic sex at three positions is AC, GA and TA, namely a sequencing peak graph is a set peak; individuals with female genetic sex have AA, GG and TT genotypes at three positions, namely a sequencing peak image is a single peak.
The invention relates to a sex-linked SNP marker screening and genetic sex identification method for channel catfish, which comprises the following steps:
1. sex-linked SNP marker screening of channel catfish and primer sequence thereof
1.1 selection of sex-linked SNP markers
And (3) screening high-quality SNP markers, and performing sex linkage analysis in the channel catfish holomorphic family to obtain 16 candidate sex linkage SNP markers.
1.2. Design of primers for amplifying SNP markers
The obtained 16 SNP markers are accurately positioned to the genome of the channel catfish, and the 150bp (301 bp) sequence of the upstream and downstream candidate sex linked SNP markers is intercepted. Primer 3 online primer design software is used for designing PCR amplification primers according to the flanking sequences of the SNP markers.
The nucleotide sequences for amplifying the 16 SNP markers are respectively as follows:
the 1 st pair: the forward primer is shown as SEQ ID NO. 1; the reverse primer is shown as SEQ ID NO. 2;
and 2, pair: the forward primer is shown as SEQ ID NO. 3; the reverse primer is shown as SEQ ID NO. 4;
and the 3 rd pair: the forward primer is shown as SEQ ID NO. 5; the reverse primer is shown as SEQ ID NO. 6;
and 4, pair: the forward primer is shown as SEQ ID NO. 7; the reverse primer is shown as SEQ ID NO. 8;
the 5 th pair: the forward primer is shown as SEQ ID NO. 9; the reverse primer is shown as SEQ ID NO. 10;
and 6 th pair: the forward primer is shown as SEQ ID NO. 11; the reverse primer is shown as SEQ ID NO. 12;
and 7, pair: the forward primer is shown as SEQ ID NO. 13; the reverse primer is shown as SEQ ID NO. 14;
and the 8 th pair: the forward primer is shown as SEQ ID NO. 15; the reverse primer is shown as SEQ ID NO. 16;
the 9 th pair: the forward primer is shown as SEQ ID NO. 17; the reverse primer is shown as SEQ ID NO. 18;
the 10 th pair: the forward primer is shown as SEQ ID NO. 19; the reverse primer is shown as SEQ ID NO. 20;
the 11 th pair: the forward primer is shown as SEQ ID NO. 21; the reverse primer is shown as SEQ ID NO. 22;
the 12 th pair: the forward primer is shown as SEQ ID NO. 23; the reverse primer is shown as SEQ ID NO. 24;
the 13 th pair: the forward primer is shown as SEQ ID NO. 25; the reverse primer is shown as SEQ ID NO. 26;
the 14 th pair: the forward primer is shown as SEQ ID NO. 27; the reverse primer is shown as SEQ ID NO. 28;
the 15 th pair: the forward primer is shown as SEQ ID NO. 29; the reverse primer is shown as SEQ ID NO. 30;
the 16 th pair: the forward primer is shown as SEQ ID NO. 31; the reverse primer is shown as SEQ ID NO. 32;
2. sex-linked SNP marker verification of channel catfish
2.1. Amplification of sex-linked SNP markers
Genomic DNAs of male and female individual samples of the 8-channel catfish are respectively extracted, and PCR amplification is respectively carried out by using 16 pairs of designed primers. PCR amplification was performed using 2 XTaq Plus Master Mix (Dye Plus) kit (Biotech, Inc. of Nanjing Novowed) in the following reaction system:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
2.2. Agarose electrophoresis
The PCR amplification product was electrophoresed on a 1% agarose gel. And after the electrophoresis is finished, placing the gel on a gel imaging system to observe the electrophoresis result, and determining whether the DNA fragment containing the SNP marker to be detected is successfully amplified, wherein the successfully amplified SNP marker has a clear band with the size equivalent to that of the target.
Purification and sequencing of PCR products
And (3) cutting gel of the amplification product, recovering and purifying, and performing bidirectional Sanger sequencing, wherein the primer used for sequencing is the same as the PCR amplification primer. Sanger sequencing peak plots were read using Chromas software.
The primer combinations amplify DNA fragments with target sizes, sequencing results prove that the amplified DNA sequences are target sequences, target SNP markers are also proved to exist really, but only the SNP markers amplified by the primer combinations of the SEQ ID NO.31 sequence and the SEQ ID NO.32 sequence have the characteristic of male sex linkage. In addition, another two SNP markers exist at the 149 th position and the 5 th position upstream and downstream of the marker, and the three SNP markers are named g.43A > C, g.192G > A and g.197T > A respectively. The 3 SNP markers are heterozygous (namely AC, GA and TA) in a male individual, and the sequencing map is a set of peaks at three positions; homozygous in female (i.e., AA, GG, TT), sequencing maps are unimodal at three positions. Therefore, the g.43A > C, g.192G > A and g.197T > A markers are possible to be channel catfish male specific SNP markers.
2.4. Identification of male specific SNP marker of channel catfish
Randomly selecting 96 individuals from the channel catfish breeding population, and performing PCR amplification by using primer combinations of SEQ ID NO.31 sequence and SEQ ID NO.32 sequence. And performing agarose gel electrophoresis and purification on the amplification product, and performing bidirectional Sanger sequencing.
PCR amplification was performed using 2 XTaq Plus Master Mix (Dye Plus) kit (Biotech, Inc. of Nanjing Novowed) in the following reaction system:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
Comparing the sequencing and typing results of 96 individuals with the sex recorded by corresponding samples in a database, which shows that all amplification products of male individuals are heterozygotic (namely AC, GA and TA) at 43, 192 and 197 and homozygotic (namely AA, GG and TT) in female individuals, and shows that the genetic sex of channel catfish can be accurately identified by sequencing and typing 3 sites g.43A > C, g.192G > A and g.197T > A.
3. Application method of channel catfish genetic sex identification based on SNP marker
The genetic sex identification of ictalurus punctatus was performed according to the following 2 primer combinations.
PCR amplification
Primers with a sequence of SEQ ID NO.31 and a sequence of SEQ ID NO.32 are used for amplifying genomic DNA of channel catfish to be detected in a combined mode, a 2 xTaq Plus Master Mix (Dye Plus) kit (Nanjing Nuojingzau Biotechnology Co., Ltd) is used for PCR amplification, and the reaction system is as follows:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
SNP site typing
And (3) performing agarose gel electrophoresis and purification on the amplification product, and performing bidirectional Sanger sequencing on the amplification product on an ABI 3730XL sequencer, wherein the sequencing primer is the same as the PCR amplification primer.
The chromatas software was used to read the genotype of the amplification products at 3 sites g.43A > C, g.192G > A, g.197T > A. Individuals at positions 43, 192, 197 that are all heterozygous (i.e., AC, GA, TA) are males, and individuals that are all homozygous (i.e., AA, GG, TT) are females.
The invention has the advantages that:
SNP markers linked with the male sex of the channel catfish are screened for the first time, male specific mutation sites are found, and a genetic sex identification method based on the SNP marker technology is established. The invention has the characteristics of high efficiency, accuracy and stability.
Drawings
FIG. 1 shows the result of female and male individual typing of SNP markers linked to channel catfish 3.
Wherein, males have mantle peaks at positions 43, 192 and 197 of the sequence of the amplification product, and females have a single peak at three positions.
Detailed Description
The invention will be described in detail in the following with reference to the following embodiments and accompanying drawings, which include three aspects: (1) screening sex-linked SNP markers of channel catfish and designing amplification primers thereof; (2) verifying sex-linked SNP markers of the channel catfish; (3) establishing a method for identifying the genetic sex of channel catfish based on SNP markers.
Example 1
1. Screening of sex-linked SNP markers of channel catfish and primer sequences thereof
1.1. Selection of sex-linked SNP markers
156 individuals of channel catfish holosible line and parent DNA samples are collected to construct 19 libraries, sequencing data are 147Gb, parent sequencing data are 3Gb, the coverage genome is about 3 x, the average data volume of each sample of filial generation sequencing is 900Mb, and the coverage genome is 1 x. And (3) carrying out genetic linkage map construction by using 10661 preliminarily screened SNPs, wherein the LOD is more than 10, and 29 linkage groups are separated. The number of SNP markers finally used for constructing the genetic linkage map is 5017, the genetic distance is 3147.3cM, and the average length is 0.63 cM. And carrying out sex linkage analysis on 5017 SNP markers for constructing the linkage map to obtain 16 candidate sex linkage SNP markers in total.
1.2. Design of primers for amplifying SNP markers
The obtained 16 SNP markers are accurately positioned to the positions of the genome, and the sequences of the upper upstream and the lower downstream 150bp (301 bp in total) of the candidate sex linked SNP markers are intercepted. Primer 3 online primer design software is used to design primers for the flanking sequences of the SNP markers, and primer synthesis is carried out by the Shanghai organism.
TABLE 1 sex-linked SNP sites and primer information of Ictalurus punctatus
2. Verification of sex-linked SNP markers
2.1. Genomic DNA extraction
Selecting 8 male and female individuals of the channel catfish with known sex, shearing about 25mg of tail fin sample of each channel catfish, grinding the channel catfish sample into powder by using liquid nitrogen, and transferring the powder into a 1.5ml centrifuge tube. mu.L STE DNA extraction buffer (10mmol/L Tris-HCl, pH 8.0; 1mmol/L EDTA, pH 8.0), 35. mu.L SDS (10%, g/100ml) were added successively, and finally 15. mu.L proteinase K (0.2%, g/100ml) was added and mixed by inversion. Adding 1 μ L RNaseA, and placing in a water bath kettle at 55 deg.C for 1 h. An equal volume (about 700. mu.L) of Tris saturated phenol was added, mixed by shaking on a DNA mixer, centrifuged at 12000rpm for 10min at 4 ℃ and after completion the supernatant was transferred to another clean 1.5ml centrifuge tube using a pipette. Adding equal volume of phenol-ethanol mixture (phenol, chloroform, isoamyl alcohol ratio is 25:24:1) into the supernatant, and placing on a DNA mixer to uniformly mix for 15min by shaking. About 500. mu.L of chloroform with the same volume was added to the supernatant, and the mixture was shaken and mixed for 15min on a DNA mixer. 1mL of dehydrated ethanol precooled at-20 ℃ is added into the supernatant to precipitate DNA, and the DNA is centrifuged by a centrifuge at 12000rpm for 5min, and then the supernatant is discarded. Adding 70% ethanol for washing twice, adding absolute ethanol for washing once, drying, adding 200 μ L TE, and dissolving completely. The concentration was measured with a NanoDrop ND-1000 spectrophotometer and each DNA sample was diluted to 100 ng/. mu.L of working solution.
PCR amplification
Each pair of SNP amplification primers performs PCR amplification on 16 male and female samples respectively. PCR amplification was performed using 2 XTaqPlus Master Mix (Dye Plus) kit (Biotech, Inc. of Nanjing Novowed) in the following reaction scheme:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
2.3. Agarose electrophoresis
The PCR amplification product was electrophoresed on a 1% agarose gel. And after the electrophoresis is finished, placing the gel on an ultraviolet projection detector to observe the electrophoresis effect, and determining whether the SNP marker to be detected is successfully amplified, wherein the successfully amplified SNP marker has a clear band with the length equivalent to the designed amplification length.
Purification and sequencing of PCR products
And (3) cutting gel of the amplification product, recovering and purifying, and performing bidirectional Sanger sequencing, wherein the primer used for sequencing is the same as the PCR amplification primer. Sanger sequencing peak plots were read using Chromas software. The result shows that the above primer combinations all amplified DNA fragments with target size, the sequencing result proves that the amplified DNA sequence is the target sequence, the target SNP marker is also proved to exist really, but only the SNP markers amplified by the primer combinations of the SEQ ID NO.31 sequence and the SEQ ID NO.32 sequence have sex linkage characteristics, in addition, another two SNP markers exist at 149 th position and 5 th position upstream and downstream of the marker, and the three SNP markers are named as g.43A > C, g.192G > A and g.197T > A respectively. The 3 SNP markers are all heterozygous (i.e. AC, GA, TA) in male individuals and homozygous (i.e. AA, GG, TT) in female individuals. Therefore, the g.43A > C, g.192G > A and g.197T > A markers are possible to be channel catfish male specific SNP markers.
2.4 SNP marker sex linkage verification
Further using primers of SEQ ID No.31 and SEQ ID No.32PCR to amplify the genome DNA of the sex-matured channel catfish 20 female fish and 20 male fish, using 2 XTaq Plus Master Mix (Dye Plus) kit (Nanjing Nodezakstan Biotech Co., Ltd.) for PCR amplification, and using the reaction system:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
The amplification product was recovered and purified by cutting gel and ligated with PMD-18T vector (Takara) for 2h at 16 ℃. The ligation products were added to competent cells for cloning and spread on LB plates containing ampicillin, and cultured upside down at 37 ℃ overnight. 10 positive clones were selected, shake-cultured at 37 ℃ for 5 hours in a liquid medium containing ampicillin, and Sanger sequencing was performed after extracting plasmids using a plasmid extraction kit. Sanger sequencing results were read using Chromas software.
The primers SEQ ID No.31 and SEQ ID No.32 amplified 2 sequences (SEQ ID No.33 and SEQ ID No.34) in 20 male samples and only one sequence (SEQ ID No.34) in female. The first sequence of SEQ ID NO.33 in males is C, A, A at bases 43, 192, 197, respectively, and is unique to males; the second sequence of SEQ ID NO.34 has A, G, T bases at the three aforementioned positions, respectively, and is identical to the sequence amplified in females. Thus, SEQ ID NO.33 is probably a sequence unique to the male of Ictalurus punctatus, with base C, A, A closely linked to the male sex at positions 43, 192, and 197. The g.43A > C, g.192G > A and g.197T > A markers can be male specific SNP markers of channel catfish.
3. Application method of channel catfish genetic sex identification based on SNP marker
3.1. Genomic DNA extraction
And randomly selecting 96 parent samples from the breeding population of the channel catfish, and extracting the same 2.1 of genome DNA.
3.2. Fluorescent primer synthesis
Primer sequences of SEQ ID NO.31 and SEQ ID NO.32 were synthesized, and the synthesis of the primers was entrusted to Shanghai Biotech.
PCR amplification
The 96-channel catfish genome DNA with unknown gender is used as a template, primers of SEQ ID NO.31 and SEQ ID NO.32 are used for PCR amplification, 2 xTaq Plus Master Mix (Dye Plus) kit (Nanjing Nozam Biotech Co., Ltd.) is used for PCR amplification, and the reaction system is as follows:
the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60-51 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
3.4. Genotyping
And (3) performing bidirectional Sanger sequencing after the amplification product is purified, wherein the primers used for sequencing are the same as the PCR amplification primers. Sanger sequencing peak patterns were read using Chromas software and base sequencing peak pattern types at 43, 192, 197 were determined.
3.5. Analysis of results
Judging the base sequencing peak pattern types of the channel catfish of 96 channel catfish at 43 th, 192 th and 197 th base sequencing peak patterns of the amplified product sequences, and then carrying out genetic sex estimation. The results show that: the genetic sex is a male sample with 53 tails, the genotypes at three positions are AC, GA and TA, namely, the peak image is a set peak; sample 43 tail of female genetic sex, genotype AA, GG, TT at three positions, namely peak pattern is single peak. The peak patterns of male and female channel catfish at 43, 192, 197 are shown in figure 1. And (3) checking the sex of the channel catfish sample presumed according to the genotyping with the sex recorded by the corresponding sample in the database, wherein the result is 100% matching.
Sequence listing
<110> research institute for fresh water and aquatic products in Jiangsu province
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tgcagtatga acctgtgtct tga 23
<210>14
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
acagttgagc gagcattctg a 21
<210>15
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
caaggagagc agcgtaaaac c 21
<210>16
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
cacactcact cctctgctgt c 21
<210>17
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
ggtggatccg aaaactatcc ca 22
<210>18
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gttttgttgt gtcccacgct 20
<210>19
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tcaaatttca cgcttgccga a 21
<210>20
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
cagtcactga gagagcaaaa caa 23
<210>21
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
ggtctcatcc ctcaccaaaa c 21
<210>22
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
tgttcatgag tccaccacca 20
<210>23
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
caatcccgaa cccttgacac 20
<210>24
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
caggtctttc tttcatgctt gt 22
<210>25
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
actgctttct cctggtcaca 20
<210>26
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
ccgtattggt gctagagaaa gac 23
<210>27
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
atgcagtcgt ggtaaagtgg 20
<210>28
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
caaatccaaa gcctcgtaaa gc 22
<210>29
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
ctcattaggg cgttagtggc 20
<210>30
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
tacacagcaa ccttgaccac 20
<210>31
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
gtccccctga ctccacatct at 22
<210>32
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
gcgcaatcgt ttcaggtgc 19
<210>33
<211>293
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gtccccctga ctccacatct atgctgcagt cttcatgctc aacttcagcc ttgtgttctg 60
cacccgactt gttgcctcca cctgagattt tgcccccaca ggatgatgat ccgccttcct 120
taagtcctga agatgtacca acatctacaa ctcaccgatg ccagcaatgt cctgaggcct 180
tcagtagttc aacactactt gctgtccata tgcaaagcca caagagaaga tttgtcagtc 240
atctgttttg caagtactgc aataaaaagt tcatgcacct gaaacgattg cgc 293
<210>34
<211>293
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
gtccccctga ctccacatct atgctgcagt cttcatgctc aaattcagcc ttgtgttctg 60
cacccgactt gttgcctcca cctgagattt tgcccccaca ggatgatgat ccgccttcct 120
taagtcctga agatgtacca acatctacaa ctcaccgatg ccagcaatgt cctgaggcct 180
tcagtagttc agcacttctt gctgtccata tgcaaagcca caagagaaga tttgtcagtc 240
atctgttttg caagtactgc aataaaaagt tcatgcacct gaaacgattg cgc 293
Claims (6)
1. An SNP molecular marker linked with the sex of channel catfish is characterized in that SNP molecular markers with g.43A > C, g.192G > A and g.197T > A3 polymorphisms respectively exist at the 43 th, 192 th and 197 th positions of a nucleotide sequence shown as the specification;
GTCCCCCTGACTCCACATCTATGCTGCAGTCTTCATGCTCAAR(A/C)TTCAGCCTTGTGTTCTGCACCCGACTTGTTGCCTCCACCTGAGATTTTGCCCCCACAGGATGATGATCCGCCTTCCTTAAGTCCTGAAGATGTACCAACATCTACAACTCACCGATGCCAGCAATGTCCTGAGGCCTTCAGTAGTTCAM(G/A)CACTN(T/A)CTTGCTGTCCATATGCAAAGCCACAAGAGAAGATTTGTCAGTCATCTGTTTTGCAAGTACTGCAATAAAAAGTTCATGCACCTGAAACGATTGCGC。
2. the SNP molecular marker according to claim 1, wherein individuals of male genetic sex are genotyped AC, GA, TA at three positions; individuals with female genetic sex have genotypes AA, GG, TT at three locations.
3. A specific primer for detecting the SNP molecular marker of claim 1 or 2, wherein the specific primer is SEQ ID NO.31 or SEQ ID NO. 32.
4. A kit for detecting the SNP molecular marker according to claim 1 or 2, wherein the kit comprises the specific primer according to claim 3.
5. Use of the SNP molecular marker according to claim 1 or 2, the specific primer according to claim 3 or the kit according to claim 4 for identifying the genetic sex of Ictalurus punctatus.
6. The method for identifying the genetic sex of the channel catfish is characterized in that the method comprises the steps of extracting channel catfish genomic DNA of the genetic sex to be detected, carrying out PCR amplification on the channel catfish genomic DNA by adopting specific primers SEQ ID NO.31 and SEQ ID NO.32, carrying out bidirectional Sanger sequencing on an amplification product after purification, reading a Sanger sequencing peak diagram by using Chromas software, and judging the base sequencing peak diagram types of the sequence of the amplification product at positions 43, 192 and 197; the genotype of an individual with male genetic sex at three positions is AC, GA and TA, namely a sequencing peak graph is a set peak; individuals with female genetic sex have AA, GG and TT genotypes at three positions, namely a sequencing peak image is a single peak.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186307A (en) * | 2021-06-21 | 2021-07-30 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method based on channel catfish male specific gene zbtb38-Y and application |
| CN113293218A (en) * | 2021-06-21 | 2021-08-24 | 江苏省淡水水产研究所 | SNP molecular marker for selecting weight gain character of channel catfish and application |
| CN113373244A (en) * | 2021-07-14 | 2021-09-10 | 江苏省淡水水产研究所 | Method for rapidly detecting genetic sex of channel catfish based on SNP site-specific primer extension reaction |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040055029A1 (en) * | 2002-09-18 | 2004-03-18 | Wolters William R. | Genetically distinct strain of channel catfish designated NWAC103, with improved growth performance |
| WO2010115275A1 (en) * | 2009-04-09 | 2010-10-14 | Genome Atlantic | Genetic marker identification in atlantic cod |
| CN104419758A (en) * | 2013-09-05 | 2015-03-18 | 江苏省淡水水产研究所 | Molecular genetics identification method for channel catfishes and flathead catfishes |
| CN107502663A (en) * | 2017-09-19 | 2017-12-22 | 江苏省淡水水产研究所 | A kind of channel catfish microsatellite Parentage determination method |
| CN109161603A (en) * | 2018-10-31 | 2019-01-08 | 江苏省淡水水产研究所 | The microsatellite marker primer and genetic sex identification method of channel catfish gunther sex-linked |
-
2020
- 2020-03-13 CN CN202010177166.6A patent/CN111304338B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040055029A1 (en) * | 2002-09-18 | 2004-03-18 | Wolters William R. | Genetically distinct strain of channel catfish designated NWAC103, with improved growth performance |
| WO2010115275A1 (en) * | 2009-04-09 | 2010-10-14 | Genome Atlantic | Genetic marker identification in atlantic cod |
| US20120100996A1 (en) * | 2009-04-09 | 2012-04-26 | Sophie Hubert | Genetic marker identification in atlantic cod |
| CN104419758A (en) * | 2013-09-05 | 2015-03-18 | 江苏省淡水水产研究所 | Molecular genetics identification method for channel catfishes and flathead catfishes |
| CN107502663A (en) * | 2017-09-19 | 2017-12-22 | 江苏省淡水水产研究所 | A kind of channel catfish microsatellite Parentage determination method |
| CN109161603A (en) * | 2018-10-31 | 2019-01-08 | 江苏省淡水水产研究所 | The microsatellite marker primer and genetic sex identification method of channel catfish gunther sex-linked |
Non-Patent Citations (5)
| Title |
|---|
| NAIRONG PAN等: "Identification of male-specific SNP markers and development of rapid PCR-based genetic sex identification method in channel catfish (Ictalurus punctatus)" * |
| SHIYONG ZHANG等: "Construction of a High-Density Linkage Map and QTL Fine Mapping for Growth- and Sex-Related Traits in Channel Catfish (Ictalurus punctatus)" * |
| 张世勇;王明华;钟立强;秦钦;潘建林;陈校辉;边文冀;: "斑点叉尾鮰MSTN基因4个SNP位点及其与生长性状的相关性" * |
| 张世勇;钟立强;秦钦;王明华;潘建林;陈校辉;边文冀;: "斑点叉尾鮰GHRH基因3个SNPs位点及其单倍型组合与生长性状的关联分析" * |
| 赫崇波,周遵春,刘卫东: "斑点叉尾基因组的研究" * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186307A (en) * | 2021-06-21 | 2021-07-30 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method based on channel catfish male specific gene zbtb38-Y and application |
| CN113293218A (en) * | 2021-06-21 | 2021-08-24 | 江苏省淡水水产研究所 | SNP molecular marker for selecting weight gain character of channel catfish and application |
| CN113186307B (en) * | 2021-06-21 | 2023-08-01 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method and application based on channel catfish male specific gene zbtb38-Y |
| CN113293218B (en) * | 2021-06-21 | 2024-02-06 | 江苏省淡水水产研究所 | SNP molecular marker for selecting weight gain character of channel catfish and application |
| CN113373244A (en) * | 2021-07-14 | 2021-09-10 | 江苏省淡水水产研究所 | Method for rapidly detecting genetic sex of channel catfish based on SNP site-specific primer extension reaction |
| CN113373244B (en) * | 2021-07-14 | 2023-05-23 | 江苏省淡水水产研究所 | Method for rapidly detecting genetic sex of channel catfish based on SNP locus specific primer extension reaction |
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