CN115011701A - SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker - Google Patents
SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker of cryptocaryon irritans disease resistance related traits of trachinotus ovatus, which is cloned from trachinotus ovatus LAAO gene and is positioned in LAAO gene exon, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1, two allelic gene mutations (C/T) and (G/A) exist at the base positions 6499 and 6535 of the sequence respectively, and also discloses a primer for amplifying the SNP molecular marker and application of the SNP molecular marker or the primer in identifying or breeding the cryptocaryon irritans disease resistant trachinotus ovatus variety. The molecular markers screened by the invention are used for performing correlation analysis on the cryptocaryon irritans resistant disease of trachinotus ovatus, and the two markers can be applied to early screening of trachinotus ovatus breeding materials with disease resistance as a target, so that the breeding efficiency can be effectively improved, and the breeding period can be shortened.
Description
Technical Field
The invention belongs to the technical field of molecular marker assisted breeding of fish genetic breeding, and particularly relates to an SNP molecular marker for cryptocaryon irritans disease resistance of trachinotus ovatus and application thereof.
Background
Trachinotus ovatus (Trachinotus ovatus) belongs to Perciformes (Perciformes), Carangidae (Carangidae) and Trachinotus (Trachinotus), commonly known as golden pompanus, short pompano and the like in taxonomy and is mainly distributed in southern sea areas of China. The trachinotus ovatus is thick, solid and tender in meat quality, delicious in taste and high in growth speed, and is edible seawater teleosts with both nutritional value and economic value. However, due to the influence of factors such as overlarge culture density and deterioration of water quality in sea areas, various diseases can be caused in the culture sea areas of the trachinotus ovatus to cause huge economic loss, Cryptocaryon irritans (Cryptocaryon irritans) disease is one of the diseases which can cause serious influence on the culture industry of the trachinotus ovatus, and the death rate of the trachinotus ovatus infected by the Cryptocaryon irritans is extremely high, so that the research on how to improve the resistance of the trachinotus ovatus to the Cryptocaryon irritans is very necessary.
In recent years, due to environmental pollution, frequent disease and the like, the natural population of trachinotus ovatus is seriously degenerated, the germplasm is seriously degenerated and the like; meanwhile, due to close breeding of trachinotus ovatus culture colonies, artificial breeding of small-sized parents and the like, trachinotus ovatus culture germplasm is seriously degraded, disease resistance is weakened, culture performance is reduced and the like. The continuous and healthy development of the trachinotus ovatus breeding industry is severely restricted by the problems, so that the fine breed breeding work of the trachinotus ovatus is urgently needed to be carried out. The molecular marker assisted selective breeding is one of the improved variety breeding methods, the molecular marker tightly linked with the target character gene can be used for carrying out correlation analysis on the target character, the close correlation between the candidate molecular marker and the individual phenotype can be determined, and the breeding period can be further shortened.
Single Nucleotide Polymorphism (SNP) is a genetic marker, and is regarded as a molecular marker with the most development potential due to the characteristics of large quantity, wide distribution, high representativeness, high genetic stability and easy realization of automatic analysis. The SNP is also widely applied and can be used in the aspects of genetic map construction, trait gene association analysis, germplasm identification, genetic relationship analysis, auxiliary breeding and the like, wherein the SNP and trait gene association analysis can not only reveal the connection between the SNP and the genetic traits, but also carry out molecular breeding according to SNP sites and actual breeding requirements, and has a great benefit. The polymorphism of susceptibility/resistance of the trachinotus ovatus MHC II beta gene and the photobacterium mermairei of the trachinotus ovatus is analyzed by the Kyochich et al, and the 3 polymorphic loci of TO-DAB-04, TO-DAB-05 and TO-DAB-10 in the trachinotus ovatus MHC II beta gene are found TO be obviously related TO the susceptibility of the photobacterium mermairei, the TO-DAB-01 locus has obvious relevance TO the photobacterium mermairei resistance, and the TO-DAB-01 locus can provide reference for the practical application of disease-resistant breeding.
At present, less work is carried out on cryptocaryon irritans disease resistant genetic breeding of trachinotus ovatus, SNPs have the advantages of high polymorphism, wide distribution and the like, and have wide application prospect in fish genetic breeding. At present, no report of the gene locus related to the cryptocaryon irritans resistant character of trachinotus ovatus is found.
Disclosure of Invention
The invention aims to provide an SNP molecular marker of properties related to cryptocaryon irritans resistance of trachinotus ovatus and a primer for amplifying the SNP molecular marker.
The invention also aims to provide application of the SNP molecular marker or the primer in identifying or breeding cryptocaryon irritans resistant trachinotus ovatus variety.
The last purpose of the invention is to provide a method for acquiring the SNP molecular marker and a method for identifying the cryptocaryon irritans disease resistant Trachinotus ovatus.
The first object of the present invention can be achieved by the following technical solutions: an SNP molecular marker of cryptocaryon irritans disease-resistant related traits of trachinotus ovatus, wherein the SNP molecular marker is SNP6499 or SNP6535, the SNP6499 is located in the LAAO gene as shown in SEQ ID NO: 1, the base of the 6499 th site from the 5' end of the base sequence is C or T, and the SNP6535 is located in the LAAO gene as shown in SEQ ID NO: 1 at position 6535 from the 5' end, wherein the base is G or A.
The SNP molecular marker is obtained by cloning in a trachinotus ovatus LAAO gene, is positioned in an LAAO gene exon, and has a nucleotide sequence shown as SEQ ID NO: 1, two allelic mutations (C/T) and (G/A) exist at the base positions 6499 and 6535 of the sequence respectively, and the two allelic mutations respectively cause tyrosine silence and glycine to leucine.
In the SNP molecular marker, TT genotype individuals at the 6499 th position are more resistant than CC genotype individuals, namely the ability of resisting cryptocaryon irritans disease of the TT genotype individuals at the 6499 th position is higher than that of the CC genotype individuals; the AA genotype individuals at the 6535 th site are more resistant to the infection than the GG genotype individuals, namely the AA genotype individuals at the 6535 th site have higher cryptocaryon irritans disease resistance than the GG genotype individuals.
The invention also provides a primer for amplifying the SNP molecular marker, the primer is a primer pair SNP6499 or SNP6535, and the base sequences of the upstream primer and the downstream primer of the primer pair SNP6499 are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3, the base sequences of the upstream primer and the downstream primer of the primer pair SNP6535 are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
The primer pair SNP6499 is used for amplifying the 6499 th SNP molecular marker, the primer pair SNP6535 is used for amplifying the 6535 th SNP molecular marker, and in addition, the primer pair SNP6535 can also simultaneously amplify the 6499 th SNP molecular marker.
Specifically, the method comprises the following steps:
the base sequence of the primer pair SNP6499 upstream primer To __ LAAO-G-F1 for amplifying SNP6499 molecular marker is as follows:
5’-TGAAACGGTAAGACGCTC-3’，
the base sequence of the primer pair SNP6499 downstream primer To _ LAAO-G-R1 for amplifying SNP6499 molecular marker is as follows:
5'-GTCAGACCAGGTGTAGGAA-3' (PCR amplified length 3841 bp);
the base sequence of the upstream primer To _ LAAO-G-F2 of the SNP6535 of the primer pair SNP6535 for amplifying the SNP6535 molecular marker (or simultaneously amplifying the SNP6499 and the SNP6535 molecular marker) is as follows:
5’-CAGCCAGTCAGATAAAGG-3’；
the base sequence of the downstream primer To _ LAAO-G-R2 of the SNP6535 of the primer pair for amplifying the SNP6535 molecular marker (or simultaneously amplifying the SNP6499 and the SNP6535 molecular marker) is as follows:
5'-CCATAATACCCAAGAACC-3' (PCR amplified length of 926 bp).
The second objective of the present invention can be achieved by the following technical solutions: the SNP molecular marker or the primer is applied to the identification or breeding of cryptocaryon irritans resistant Trachinotus ovatus strains.
The last object of the present invention can be achieved by the following technical solutions: the method for acquiring the SNP molecular marker comprises the following steps:
(1) extracting the genome DNA of the trachinotus ovatus individual;
(2) designing the primer pair SNP6499/SNP6535 according to the trachinotus ovatus LAAO genome sequence, and carrying out PCR amplification;
(3) detecting by using agarose gel electrophoresis after amplification, sequencing a sample with a uniform band in a gel image, and judging potential SNPs sites according to a sequencing peak image;
(4) SNPs loci are subjected to genotyping, and two SNP loci exist in an exon of the LAAO gene, and are named as SNP6499 locus and SNP6535 locus respectively.
Wherein:
preferably, in the step (1), a MagenDNA extraction kit (D6310-03B) is used for extracting the genomic DNA of the trachinotus ovatus individual.
Preferably, during the PCR amplification in the step (2), the total volume of the PCR reaction system is 100 μ L: 76 μ L of double distilled water, 10 μ L of 10 XPCR (TaKaRa) buffer (containing Mg) 2+ ) 10mmol/L dNTPs 1.5. mu.L, rTaqDNA polymerase (TaKaRa) 1.5. mu.L, forward and reverse primers 3. mu.L each, 100 ng/. mu.L genomic DNA 5. mu.L.
Preferably, in the PCR amplification in step (2), the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 40s, 30 cycles (long fragment >3kb: denaturation at 94 ℃ for 40s, annealing at 56 ℃ for 40s, extension at 72 ℃ for 90s, 30 cycles); extending at 72 deg.C for 10min, and storing at 10 deg.C.
Preferably, 1% (mass percentage) agarose gel electrophoresis is used for detection in step (3).
The invention also provides an identification method of cryptocaryon irritans disease resistant trachinotus ovatus, which comprises the following steps:
(1) extracting the genome DNA of the trachinotus ovatus;
(2) carrying out PCR amplification by adopting the primer pair SNP6499 or the primer pair SNP6535 to obtain a PCR product;
(3) and carrying out genotyping on the amplified product, carrying out statistics on the genotype of the SNP locus and the frequency distribution of allele, and then testing the association degree of the SNP locus and the cryptocaryon irritans resistance character to obtain whether the significant difference exists between the SNP locus and the cryptocaryon irritans resistance character.
In the identification method of cryptocaryon irritans disease resistant Trachinotus ovatus, the identification method comprises the following steps:
preferably, in the step (3), the genotype of the SNP site and the frequency distribution of the allele are counted by using Excel 2016, and then the association degree of the SNP site and the cryptocaryon irritans resistance character is subjected to chi-square test by using SPSS Statistics 17.0 software to obtain whether the significant difference exists between the SNP site and the cryptocaryon irritans resistance character.
Preferably, in the step (3), the TT genotype individual at the SNP6499 site is more resistant to the CC genotype individual (P <0.05), and the AA genotype individual at the SNP6535 site is more resistant to the GG genotype individual (P < 0.05).
Preferably, the TT genotype individual at the SNP6499 locus and the AA genotype individual at the SNP6535 locus in the step (3) are anti-cryptocaryon irritans disease trachinotus ovatus strains.
The invention has the following beneficial effects:
(1) the invention obtains the SNP molecular marker of the character related to the cryptocaryon irritans resistance of the trachinotus ovatus from the LAAO gene screening, and provides a useful genetic marker for the cultivation of disease-resistant varieties of the trachinotus ovatus;
(2) the molecular markers screened by the invention are used for performing correlation analysis on the cryptocaryon irritans resistant disease of trachinotus ovatus, and the two markers can be applied to early screening of trachinotus ovatus breeding materials with disease resistance as a target, so that the breeding efficiency can be effectively improved, and the breeding period can be shortened.
Drawings
FIG. 1 is the sequencing peak diagram of SNP6499 of Trachinotus ovatus in example 1, wherein (a) is TT genotype, (b) shows heterozygosity, TC genotype, n represents C or T, and (C) is CC genotype;
FIG. 2 shows the sequencing peaks of Trachinotus ovatus SNP6535 in example 1, wherein (a) shows AA genotype, (b) shows heterozygosity, GA genotype, n represents G or A, and (C) shows GG genotype.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to specific examples so that those skilled in the art can better understand and implement the technical solutions of the present invention. Reagents or materials used in the examples were commercially available, unless otherwise specified.
Example 1
The SNP molecular marker of the behavior related to cryptocaryon irritans resistance of trachinotus ovatus, provided by the embodiment, is SNP6499 or SNP6535, wherein the SNP6499 is located in the LAAO gene as shown in SEQ ID NO: 1, the base of the nucleotide sequence is C or T at the 6499 th site from the 5' end, and the SNP6535 is located in the LAAO gene as shown in SEQ ID NO: 1 at position 6535 from the 5' end, wherein the base is G or A.
The LAAO gene is shown as SEQ ID NO: 1, the TT genotype individual at position 6499 of the base sequence shown in the specification is more resistant to the CC genotype individual, namely the TT genotype individual at position 6499 has higher cryptocaryon irritans disease resistance than the CC genotype individual; the AA genotype individuals at 6535 are more resistant to the infection than the GG genotype individuals, namely the AA genotype individuals at 6535 have higher cryptocaryon irritans disease resistance than the GG genotype individuals.
The primer for amplifying the SNP molecular marker is a primer pair SNP6499 or SNP6535, and the base sequences of the upstream primer and the downstream primer of the primer pair SNP6499 are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3, the base sequences of the upstream primer and the downstream primer of the primer pair SNP6535 are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
The SNP molecular marker is obtained by the following steps:
(1) randomly and respectively selecting 10 genome DNAs of trachinotus ovatus individuals, and adopting a MagenDNA extraction kit (D6310-03B);
(2) according To the document Zhang et al, (2019) the trachinotus ovatus LAAO genome sequence is adjusted, the genome sequence primer for amplifying the gene is designed, and the primer is To _ LAAO-G-F1 (the upstream primer for amplifying SNP6499 molecular marker) 5'-TGAAACGGTAAGACGCTC-3' and To _ LAAO-G-R1 (the downstream primer for amplifying SNP6499 molecular marker), 5'-GTCAGACCAGGTGTAGGAA-3' (3841bp, the length of the primer pair SNP6499 or the primer pair SNP 6535) or To _ LAAO-G-F2 (the upstream primer for amplifying SNP6535 molecular marker or simultaneously amplifying SNP6499 molecular marker and SNP6535 molecular marker), 5'-CAGCCAGTCAGATAAAGG-3', To _ LAAO-G-R2 (the downstream primer for amplifying SNP6535 molecular marker or simultaneously amplifying SNP6499 molecular marker and SNP6535 molecular marker) 5'-CCATAATACCCAAGAACC-3' (926bp, length of primer pair SNP6535 amplification);
(3) using 100. mu.L of a reaction system (76. mu.L of double distilled water, 10 XPCR (TaKaRa)) 10. mu.L of a buffer solution (containing Mg) 2+ ) 10mmol/L dNTPs 1.5. mu.L, rTaq DNA polymerase (T)aKaRa)1.5 μ L, forward and reverse primers 3 μ L each, and 100ng/μ L genomic DNA 5 μ L) were subjected to PCR amplification under the following conditions: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles (long fragment)>3kb, 94 ℃ denaturation for 40s, 56 ℃ annealing for 40s, 72 ℃ extension for 90s, 30 cycles); extending at 72 deg.C for 10min, and storing at 10 deg.C;
(4) detecting by using 1% agarose gel electrophoresis after amplification, sending a sample with a uniform band in a gel image to Guangzhou Rui Boxing Ke biotechnology limited for sequencing, and judging potential SNPs sites according to a sequencing peak image;
the sequencing peak map of the Trachinotus ovatus SNP6499 is shown in figure 1;
the sequencing peak of Trachinotus ovatus SNP6535 is shown in figure 2;
(5) the SNPs loci were genotyped in the above 10 individuals by methods such as PCR sequencing, and as a result, 2 SNP loci were found in the exons of the LAAO gene, which were designated as SNP6499 and SNP6535, respectively.
The nucleotide sequence of the LAAO gene and 2 SNP sites are shown below:
wherein the underlined and shaded portions are the two allelic mutations (C/T) and (G/A) present at bases 6499 and 6535, respectively, of the sequence.
Example 2
The method for identifying the cryptocaryon irritans resistance of the trachinotus ovatus by using the SNP molecular marker or the primer comprises the following steps:
(1) selecting the genome DNA of trachinotus ovatus;
(2) carrying out PCR amplification on SNP6499 or SNP6535 by using the primers in the embodiment 1 to obtain a PCR product;
(3) sequencing the PCR product, analyzing the genotype, and judging whether the trachinotus ovatus belongs to the cryptocaryon irritans resistant disease or not according to the difference of the genotypes.
The method specifically comprises the following steps:
selecting healthy trachinotus ovatus 1600 tail, randomly and evenly dividing into a control group and an experimental group, and respectively placing the control group and the experimental group at two 20m positions 3 The pond is cultured in live seawater, cryptocaryon irritans (600 trophosome/tail) is used for infecting trachinotus ovatus in an experimental group, and the rest culture conditions are consistent with those of a control group. Defining 50 fish died firstly as susceptible group, randomly selecting 50 fish from later alive fish as resistant group, and genotyping the 100 fish.
The genotype and the frequency distribution of the allele of the SNP locus are counted by using Excel 2016, and then the relevance between the SNP locus and the cryptocaryon irritans resistance character is subjected to chi-square test by using SPSS staticiscs 17.0 software to obtain whether the significant difference exists between the SNP locus and the cryptocaryon irritans resistance character.
As can be seen from the results of association analysis in Table 1 below, TT genotype individuals at SNP6499 site were more resistant than CC genotype individuals (P < 0.05); the AA genotype individual at SNP6535 site is more resistant than the GG genotype individual (P < 0.05).
Therefore, the TT genotype individual at SNP6499 site and the AA genotype individual at SNP6535 site.
TABLE 1 correlation analysis of different genotypes and disease resistance traits of Trachinotus ovatus LAAO gene
Note: different letters indicate significance of difference, P <0.05 indicates significance of difference.
The above embodiments are only used for illustrating the present invention, and the scope of the present invention is not limited to the above embodiments. The object of the present invention can be achieved by those skilled in the art based on the above disclosure, and any modifications and variations based on the concept of the present invention are included in the scope of the present invention, and the specific scope of the protection is subject to the claims.
Sequence listing
<110> research institute for aquatic products in south China sea
<120> SNP molecular marker for cryptocaryon irritans disease resistance related traits of trachinotus ovatus and application thereof
<150> 2021104469901
<151> 2021-04-25
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7320
<212> DNA
<213> Trachinotus ovatus (Trachinotus ovatus)
<400> 1
gtgaaaattt aaaaattaat ttgcgtctga agtgaaaact tgtgtaactg catgcaaaca 60
ttagccaatt cgcacaaaat acatagtaca actgaggctg attggaatat cattaatttt 120
gcatttattt ggttttaaac ccaagtattg gacacataaa gagtttgacc tgatgttggc 180
actggatgaa tgaaaagtca gaggataacc aaaatcacta gggttcattc aggaaaggtt 240
gaatatcacg gtcaatttaa acactgaata acatcacctt ttcttcatca ttacctcttt 300
tacaaatgtt cacttcaagc agacccaagc ctccgtcctc ctgttggtgg aggaggtggt 360
gtgtcagagt ccagcttcac gtctgtctct gcatctccac tgtagaaaaa ctgtcaggcc 420
atgaacggaa accttgcatc ggtccacagt tttgacgagt atcaacacat tcagtgggtt 480
gtacggtcaa gtgctcagga tgatacgata acttggattg gaggctctga ctgtcaacag 540
gtaatttatc actaaagctc tggtgttaca gagagaacta cacttattga cacatcggtt 600
ctgtcctctc ttgtcctcta ggagagtgct tggctcgact atgatatagt gacccttgac 660
ttcaaactgt agtatgcatt gttgaccagc tgtcccactg aaagattttg taataagttc 720
atgaaaatga taccagaata agttgtttga tatgatttaa acatcagtta cagcagatag 780
tagcagatag ttggttcaca cacagtatca caccacctac agggtttcat acttttattg 840
tctggtaaca ttttcttgaa aatcacaaat cagatcaata ttaaaagaaa aatttgaaat 900
atctgttgaa aatcaaagaa aaaaaattat gtttcagaca agttatatca gcaaacgtat 960
gttcaactca aatagattga gtcaagttct ttatgtgtgt atgtgtgttt acatgtgtgc 1020
gtatgaatga gaaaaaaggg ccagagaaca aatggtcttc ttcaaaaaat gtccctacca 1080
cagataaaaa taaacctgct cccttcatac aagtggtcca ctggacattt gatccataaa 1140
acttgattag tgaaacttca tttaactaag tgtgtgggtg gctgctcatg tcggccatat 1200
ctaaatccac cattgggtgt ggtgttctgt ctcacactaa tgaaatgtat gaagccctta 1260
ctcactttta aagcattgag caaatgtttc cttcagcgat catcagagtt tactttgact 1320
gcttgactac tggctttggg cagtagctga tgtgaaactg gataaagtgg cgttgttagg 1380
ccgacatcat ccttagataa ataccagtga aaacaagtaa tgagttattt ctctcaacat 1440
gaaaaccagg atctgccttt taatatttgg aaatcattta ttgtttgact ttctgtctgt 1500
gtgttgtcac tgtggccttt gttcctggtt ttgtccctgt ttgtcagagt atttaataac 1560
ttttcctatt tctctctgtg tatgtttcag tgacttccac agtcttgggt gttttgtgga 1620
ggaaaaccca gcaggaatta tgaattaaat cctcagaccc catttatcct tgtgttacaa 1680
tcatcagaaa tttgttgttt aatgataacc agaaacatct cagccaagaa gtatcatgtc 1740
aaacaggcaa gagatacttg gggggttgtt atatatatat atgacttata tgacttatgt 1800
cccctgtagt agcctatgac atcatgctac gctatcttat gctatgttat ggtttgttat 1860
cactttgttt gcctttatct tgtttcacta gataaataga aagaaagtca catagagcat 1920
caccgtcaac ttccattgag ggtggggact tgaaacaatg ttctaaaaaa atcatgatga 1980
agagataatt cacttaaaca tttcaagagg ggaattctcc agtggccctg agagctcaat 2040
atactgcaac ctaagaaaac atgcaaatag acaaaaaaca aacaaattaa gaaaaaacct 2100
tcatcagtct gacaacatat gatgcacaac accaaataag aaataaaacc ttatttcctc 2160
aattcagttc attttttaac aggtttctct tgaacacaca gccttgttga tgaagataat 2220
gaagatgatg tcttgttgca ggtgtttttc tggtcatgag acatgaccag ttactcttga 2280
tcatgacagt cagacactga gaaatagtga ttatgatgac cgaaccctga tttaaaaaaa 2340
aaaaacaggg caggaagtca tgcgggcaag ctggatagat aaaagaaatt ccagcacttc 2400
acctcaggtt gatgtgactg caggttttga aacggtaaga cgctctcttt ttatttgaca 2460
ctttatctta gacaatgatt aaaatttcat gatgactaaa taagcataac caagttttag 2520
tattttcttt aactttatga attagttcaa tttctaataa atgtttagat agatagatgg 2580
atgaataagg aacaaaacta attacagata ttacagataa gattataata cagaaactgt 2640
atatctcatc aaagttttca aaatgacatg attacatttg atttccttag atcagctttt 2700
gtgttgttga attcattctg tatttgtttg aatgatatca tcagcaaatt gcatcaaatg 2760
taaatgtatg actttagtat agacattagt gaaacactta tattgttttg tcagaaagag 2820
aagtctggtc tttccaggat tgaaacatgt ctaatatatt ttctcttcct cttcatcctc 2880
ctcatgacac caaagaggga gggaacgtat acctcgtaac tgaaatggag gcgatatgga 2940
aattatgtgg gtatttctaa aaaacaaata ttgtatatgt cagatagata gatagataga 3000
tactttaagg ccagtgacag gacactttaa gcagctgtga ggttggatat ctgtatgaat 3060
gaaacacagt gactgtataa tactgtataa tactgtaagt actgtaagtt gacacttcct 3120
ctcatcagtt ccttcatctc ttccctgtct gtgattaaca tccgctgaat aatgttcaag 3180
ttctgcttta gtcttctagt ctaggtgaag ctggattgcc tgatggtatg atgaaatgag 3240
gagaggtcaa tttttgcggc acttttacag gaatatatgt gacgctgtgt ctagaggaac 3300
ttctgaggaa ctgattgttt ttcgaagcat accttcatgt cagcttcagt tgtctttaaa 3360
cgtgatgtgg acaaggtcaa atacaatcac gtgtctcacg tctagttgct tcacttggct 3420
tttaatctgt caaacactgg caactgtgca acaagtgtac tcaatttaat atccgtggat 3480
atttaacagt taaaagtctt ttggtttaaa ttccttgtcc caataattcc gttctttttc 3540
cttttgttgt gcctccagtt gccgtcaatg tgctgctgct cacactgtac catggccacg 3600
cagccgcagt cagcatgaag gcccatctgg ctgattgtct ggaggacaaa gactacgatg 3660
tgctgctgca cactgtggag accggcctcc cacacctaga gacatctcat catgttgtca 3720
ttgttggagc tggcatggct ggactgacgg ccgccaaact actacaagat gcaggacaca 3780
aggtaacaaa gaggaagaga ttttgacaaa cggctcttat acaatatatt ccctcaaaat 3840
ctgctaaaaa tttcaggagc cacaccaagc actaaccagt tcagttcatg acagtacaaa 3900
gttaaaatta atcaaatcac cacaacataa ttgccatatg ggggccttag gcttcctggc 3960
ttcctttttt tttttttggt atgaatgcaa gtagcagtaa ttctagacag ccagaggtaa 4020
cacatgtaaa acattacaag actggtttta ctcgtattta tccatttgta cttatgtgtt 4080
tgaataaggt aaccatcgta gaggctaata gtcgtgttgg aggacgtgtg gagacataca 4140
ggaatgaaaa agagggctgg tatgctgaac tgggggccat gaggatccca gattctcacc 4200
agtaagttgg agagtttaat ttaaacatgt atatgcagtc tttcgcctct cagactaaca 4260
tgattgacct gcttaacagg attgcccttt ggtttgctaa aaagcttgga gtgaagttga 4320
ataaattcat catggatgac ccgcagacct ttttggttaa tgggcggcgg gagcggacat 4380
acacagtgaa agcaaaccct gatgtcctcg ggtacaaggt agacatagaa tgagaaaggg 4440
aagtcggccg accagctgct acagcaagct ttgcagaagg tacttgagac aaaggaaagg 4500
atggaggtgt caaaaaacac tggaaataaa accacaaacc tactgtttca gcaaagcttc 4560
acattaaacg gctggtcaca caaaaaagtt gcacaactac cctaaggggg ggaaggagct 4620
gagaagaaag tgtgagggag gaatggagag atgggcgaag ggtgattgag ggtgagcttc 4680
atctagtgac aatgaattcc atatcaaatt tcatgacagg caatacaata cctcactgtg 4740
gatgaaaatt ttgaatttta ccgaccgagt ggttttgctt tttgggggct tcagttttct 4800
cttcagtgtt ttgtctgtgc gctccaacct ccaagacctt ccactggttt gtgttttgtg 4860
tcaggcttgg gcatacagta gagttacaca ctcttggttt attgtagaca gatgtcagag 4920
ctcagtctgt gactactcac catagcagca ccagctcaag agataagttg acacatttct 4980
attttctttg ttgtaggtga aagatgaaat ggaggctcat ggctgccagg ctgcgttcaa 5040
aaaatatgac cattattctg tgaaggtaaa gcacaatgtg atccaatgga atgttgtcca 5100
atgagaacac tgtgtgttga ataaatatgt gcatcactac cagtattttg aaacatatca 5160
aataacacaa gttgggtgag ccattttcct tagaaacagt cacctaatgg ttaacttgat 5220
ggatatcata ggacacactt gcttcagctt aaggttttgt ttgcggaacg attttttctg 5280
catgtatctt aatattgact attgtgagtc acataacgtg acctcgaaaa gtacatcaaa 5340
tcagtgttaa aaattaacac aacttttcaa atattagatt tagagaaaag tgttctatgt 5400
cttgtcacat aaaaggccca tattttactc ttattttcca actctgtttt catattttgt 5460
aaaatgctac acaaactgtc aaactattag aactggttga gccatttggc aacctgacta 5520
gtgaaaacat ttcatcaaaa atgtggtttt aacaaatctt tgtttaatag gagtatctga 5580
aagaagaagg acatttgagt tcagaagcag tgagaatgat tggagatctg ctaaatgaac 5640
agagcctcat gtacacagcg ctgactgaga tgctctatga ccaggctgac atcaatgaca 5700
acaccatgta aggcctgaat tctcacaatg aaaactattt aaaatgtaca gaacttcact 5760
tcacattaaa ttggttgctt gaaaaatgca taaaacaatt ctatttctct ttctgttttg 5820
taggtactat gaagtgactg gtgggtcgga ccttctcccc aaagcttttc ttaacgtact 5880
cgatgtccct attttcttga actccaaggt caggcgcatc agccagtcag ataaaggtgt 5940
cgttgtatca taccagacgg gtgaagagtc ttctctgacg aacattcagg cagacgttgt 6000
cctggtaaca accacagcca aagcagccct gttcattgac tttgatccac ctctctctat 6060
cagaaagatg gaggcgctga ggtcagtcca ctatgacagc tccactaaaa tcatcctcac 6120
cttcaacgag acgttctgga cggaggacgg catcaaagga ggaaagagca tcaccgacag 6180
gccctctcat ttcatctact accccagcca cagtttccca aagaacaaga ccatcggcgt 6240
ccttctggct tcctacacct ggtctgacga ctccctcctc ttccaaggag caagtgatga 6300
ggacctgaaa gagctcgttc tgagagattt ggtgaagatc cacggtgagc gggtcaggtc 6360
tcgctgcaca ggggtggtgg tgaagaggtg gacctcagat cctcacagtt tgggcgcctt 6420
tgctctgttc acaccctacc aacatttaga gtactcaagg gagctcttta aacatgaagg 6480
cagggttcac ttcgctggcg aacacacagc cttccctcac gcttggatcg aaacgtctat 6540
gaaatctgcg atcagggctg ctaccaacat caacaaagag gcgcacaaag cagcagctaa 6600
acaccgaggg gagctctagg ctgttaaaat ttaaaaatta cataaaagta ttgtattttt 6660
tcttgttgat tgctttgctt taaggttttt ttttattact gattaatcta cagattattt 6720
gggctgcaag acatctatga aattcaaaat cagagctgtt acaaatatta gcgtgtggtg 6780
gcattgaact cagagtcatt caaatatcca gtgcaagatg agctctaggt tcttgggtat 6840
tatggtatta ataaaagaat aacatttcat tgaatattaa tgtaacctgt ttgagcaata 6900
taaagtgctt gtttcacatt gacttaacaa agaaagataa atgtctccct aaaaatctcc 6960
caaacaactt cttttacata tacattcaca atttacattt ttcctccatc catccattat 7020
ttatatctat ccatctagtg cgtggttgca ggaaactgga gactaacatt gcctggtcac 7080
aaagtctatc acagaggtga catatagaca gacaatcatt cgagctcaca ttaacaccta 7140
caggtcattt taaagtcagg gttgaaccta acagcctgca gatttgaacc cagaacgttt 7200
ttgctgcaaa gtgacaatct tttcttcttg ccttgtttgt ttctacccca gcacctctgt 7260
atcatgcata ataaatcaat gtgatatctg aatcaagctg tgacattttg ttaagtaatg 7320
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgaaacggta agacgctc 18
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtcagaccag gtgtaggaa 19
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cagccagtca gataaagg 18
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccataatacc caagaacc 18
Claims (9)
1. An SNP molecular marker of properties related to cryptocaryon irritans resistance of trachinotus ovatus, which is characterized in that: the SNP molecular marker is SNP6499 or SNP6535, and the SNP6499 is located in the LAAO gene as shown in SEQ ID NO: 1, the base of the nucleotide sequence is C or T at the 6499 th site from the 5' end, and the SNP6535 is located in the LAAO gene as shown in SEQ ID NO: 1 at position 6535 from the 5' end, wherein the base is G or A.
2. The SNP molecular marker according to claim 1, which is characterized in that: the TT genotype individual at the 6499 th site is more resistant than the CC genotype individual, namely the ability of resisting the cryptocaryon irritans disease of the TT genotype individual at the 6499 th site is higher than that of the CC genotype individual; the AA genotype individuals at 6535 are more resistant to the infection than the GG genotype individuals, namely the AA genotype individuals at 6535 have higher cryptocaryon irritans disease resistance than the GG genotype individuals.
3. The primer for amplifying the SNP molecular marker of claim 1, which is characterized in that: the primer is a primer pair SNP6499 or SNP6535, and the base sequences of the upstream primer and the downstream primer of the primer pair SNP6499 are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3, the base sequences of the upstream primer and the downstream primer of the primer pair SNP6535 are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
4. The use of the SNP molecular marker of claim 1 or 2 or the primer of claim 3 for identifying or breeding strains of Trachinotus ovatus which are resistant to cryptocaryon irritans.
5. The method for obtaining the SNP molecular marker according to claim 2, comprising the steps of:
(1) extracting genome DNA of trachinotus ovatus individuals;
(2) designing a primer pair SNP6499 or a primer pair SNP6535 according to the LAAO genome sequence of the trachinotus ovatus, and carrying out PCR amplification;
(3) detecting by using agarose gel electrophoresis after amplification, sequencing a sample with a uniform band in a gel image, and judging potential SNPs sites according to a sequencing peak image;
(4) SNPs loci are subjected to genotyping, and two SNP loci exist in an exon of the LAAO gene, and are named as SNP6499 locus and SNP6535 locus respectively.
6. An identification method of cryptocaryon irritans disease resistant Trachinotus ovatus is characterized by comprising the following steps:
(1) extracting the genome DNA of trachinotus ovatus;
(2) carrying out PCR amplification by using the primer pair SNP6499 or the primer pair SNP6535 in the claim 3 to obtain a PCR product;
(3) and carrying out genotyping on the amplified product, carrying out statistics on the genotype of the SNP locus and the frequency distribution of allele, and then testing the association degree of the SNP locus and the cryptocaryon irritans resistance character to obtain whether the significant difference exists between the SNP locus and the cryptocaryon irritans resistance character.
7. The authentication method as claimed in claim 6, wherein: and (3) counting the genotype of the SNP site and the frequency distribution of the allele by using Excel 2016 in the step (3), and then carrying out chi-square test on the association degree of the SNP site and the cryptocaryon irritans resistance by using SPSS staticiscs 17.0 software to obtain whether a significant difference exists between the SNP site and the cryptocaryon irritans resistance.
8. The authentication method as claimed in claim 6, wherein: in the step (3), the TT genotype individual at the SNP6499 site is more resistant to the CC genotype individual (P <0.05), and the AA genotype individual at the SNP6535 site is more resistant to the GG genotype individual (P < 0.05).
9. The authentication method as claimed in claim 6, wherein: and (4) the TT genotype individual at the SNP6499 locus and the AA genotype individual at the SNP6535 locus in the step (3) are cryptocaryon irritant disease resistant trachinotus ovatus varieties.
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| CN202110446990.1A CN113322328A (en) | 2021-04-25 | 2021-04-25 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
| CN2021104469901 | 2021-04-25 |
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| CN202111129290.6A Pending CN113667764A (en) | 2021-04-25 | 2021-09-26 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
| CN202210434077.4A Active CN115011701B (en) | 2021-04-25 | 2022-04-24 | SNP molecular marker of character related to anti-cryptocaryon irritans of trachinotus ovatus and application thereof |
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| CN202111129290.6A Pending CN113667764A (en) | 2021-04-25 | 2021-09-26 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105994021A (en) * | 2016-04-08 | 2016-10-12 | 华南农业大学 | Production method for cryptocaryon irritant prevention trachinotus ovatus seedling |
| CN110106256A (en) * | 2019-04-18 | 2019-08-09 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN112501317A (en) * | 2020-12-28 | 2021-03-16 | 厦门大学 | SNP (Single nucleotide polymorphism) markers applicable to Cryptocaryon irritans resistant breeding of large yellow croakers |
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| CN108411007B (en) * | 2018-05-25 | 2019-02-26 | 海南晨海水产有限公司 | SNP marker and its application |
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2021
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105994021A (en) * | 2016-04-08 | 2016-10-12 | 华南农业大学 | Production method for cryptocaryon irritant prevention trachinotus ovatus seedling |
| CN110106256A (en) * | 2019-04-18 | 2019-08-09 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN112501317A (en) * | 2020-12-28 | 2021-03-16 | 厦门大学 | SNP (Single nucleotide polymorphism) markers applicable to Cryptocaryon irritans resistant breeding of large yellow croakers |
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| CN115011701B (en) | 2024-03-19 |
| CN113322328A (en) | 2021-08-31 |
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