CN110669846B - Dantope crane SNP marker and screening method and application thereof - Google Patents
Dantope crane SNP marker and screening method and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a red-crowned crane SNP marker and a screening method and application thereof, belonging to the technical field of molecular biology. The method comprises the steps of collecting blood samples of 10 Dandelion cranes, extracting genome DNA, constructing and sequencing a GBS library on the genome DNA by using a GBS-seq method, detecting group SNP by using SAMTOOLS software, selecting 72 SNP suitable for designing a primer, designing a corresponding primer pair according to the SNP sequence, collecting the genome DNA of another 30 Dandelion cranes, carrying out PCR amplification and sequencing by using the primer pair, and finally screening to obtain 33 high-quality public SNP sites. The 33 SNP loci serving as the molecular markers of the red-rooted crane can be applied to aspects of assisted propagation, genetic diversity research, SNP-based association analysis, resource identification and classification and the like, and have important application value for red-rooted crane population management.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a red-crowned crane SNP marker and a screening method and application thereof.
Background
Red-rooted crane (Grus japonens) also known as red-rooted crane, belonging to the order of the agrimoniales (Gruiformes) and the genus of the agrimonia (Grus), is named because the head of the red-rooted crane is naked and red. Mainly distributed in the far east of Russia, northeast of China, Mongolia, Korean peninsula, and North sea of Japan, and inhabiting in inland wetlands, coastal mudflats and other areas. The red-crowned crane is generally regarded as symbol of happiness, luckiness, longevity and faithfulness in east Asia region, and is well liked by people. The red-rooted crane needs a clean and open wetland environment as a habitat and is the indicator organism most sensitive to the change of the wetland environment. The red-rooted crane is the first-class protective animal in China and is rated as an endangered species in the IUCN red directory. In order to maintain the population quantity of the red-rooted cranes, the red-rooted crane protection area is established in salt cities, zalong and other places in China. The red-rooted crane faces the threat of habitat loss and fragmentation under the influence of habitat climate and environment change, water and soil pollution and human activities, and the wild population quantity of the red-rooted crane is still reduced.
Genetic diversity is a core concept of the evolutionary biology, is related to biological complexity, ecosystem restoration and the capability of species to react to environmental changes, and is a basic and important index of potential capability and long-term development of species to adapt to external adverse environments. Particularly, in the genetic diversity research of rare or endangered species, the current genetic variation level and genetic structure of the species are mastered, the problems of inbreeding depression or abnormal breeding and the like caused by undersize or over-dispersion of population scale are avoided, different management units are divided according to the current situation, corresponding protection and management measures are taken according to the actual situation, and the method has important significance for protecting endangered wild animals.
Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence Polymorphism caused by variation of a Single Nucleotide at the genome level, and is widely used in genetic analysis and the like as a means of molecular labeling due to abundant sites, wide distribution and convenient and fast detection. GBS (Genotyping-by-sequencing) refers to a sequencing method for obtaining a large number of genetic polymorphism tag sequences of a target species by breaking genomic DNA with restriction endonuclease and performing high-throughput sequencing on a specific fragment, and is also a commonly used SNP (single nucleotide polymorphism) screening mode. In the process of screening SNP, proper restriction enzyme is selected firstly, and genotyping is carried out by combining high-throughput colony sequencing to construct SNP molecular markers. Subsequently, specific primers are designed by using the discovered mutation sites to perform PCR amplification on the genomic DNA, and specific polymorphic products based on the SNP sites are obtained. Finally, the polymorphism of the product is analyzed by methods such as sequencing and dissolution curves. The SNP marker has high genetic stability and representativeness, the allele frequency is easy to estimate, and the mode of taking the SNP as the genetic research marker of the red-rooted crane can strengthen people's understanding of the genetic structure of the red-rooted crane, thereby better managing the red-rooted crane population.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a method for screening an SNP marker of red-rooted crane. Another object of the present invention is to provide a method for screening a SNP marker of the Eragrostis red-rooted salvia, which comprises screening the SNP marker of the Eragrostis red-rooted salvia. The problem to be solved finally by the invention is to provide the application of the Dantope crane SNP marker.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a screening method of an SNP marker of red-crowned crane comprises the following specific steps:
1) collecting a plurality of red-crowned crane blood samples, and extracting genome DNA;
2) carrying out enzyme digestion on the genomic DNA obtained in the step 1) by using restriction enzyme, adding a joint, then amplifying each sample, then mixing the samples, and selecting fragments for library construction; and performing double-end sequencing by using an Illumina HiSeq sequencing platform, performing data statistics and quality detection on the original data obtained by sequencing, and obtaining a sequence to be analyzed.
3) Carrying out group SNP detection on the sequence to be analyzed obtained in the step 2) by adopting software SAMTOOLS to obtain SNP sequences, obtaining public SNP sequences by comparison, designing corresponding primer pairs according to the obtained SNP sites, carrying out PCR amplification and sequencing on DNA of a plurality of red-crowned cranes which are additionally collected and extracted by using the designed primer pairs, and selecting sequences of 33 public SNP sites according to a sequencing result, wherein the primer pairs of the 33 public SNP sites are as follows:
preferably, the number of the blood samples of the red-rooted crane in the step 1) is 10, and the number of the additionally collected red-rooted crane DNA in the step 3) is 30.
Preferably, the main parameters for primer design in step 3) are: the length of the primer is 18-20 bp; a single primer is complementary by less than 3 nucleotides; ensuring that the difference of Tm values between the same pair of primers is not more than 2 ℃; there is less than 3 consecutive nucleotide pairings between the two primers.
Preferably, the PCR system in step 3) is 25 μ L: 12.5 μ L2 XTaq Master Mix Plus (Dye Plus), 10.5 μ L ddH2O, 1 mu L of each of the upstream and downstream primers, and 1 mu L of DNA; the procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 50-65 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; finally, the extension is carried out for 5min at 72 ℃.
The red-crowned crane 33 public SNP loci are as follows:
the application of the 33 public SNP loci of the red-rooted crane in the molecular marker assisted reproduction or genetic diversity research of the red-rooted crane.
Has the advantages that: compared with the prior art, the invention has the advantages that:
(1) the screening method of the Dandelion SNP marker provided by the invention comprises the steps of collecting 10 blood samples of the Dandelion, extracting genome DNA, constructing a GBS library by using a GBS-seq method and sequencing, carrying out group SNP detection by using software SAMTOOLS, selecting 72 SNP sequences, designing corresponding primer pairs according to the SNP sequences, collecting the genome DNA of another 30 Dandelion cranes, carrying out PCR amplification and sanger sequencing by using the primer pairs, and finally screening to obtain 33 high-quality public SNP sites.
(2) The method for developing 33 SNP sites of the red-rooted crane can be applied to aspects of assisted reproduction, genetic diversity research, SNP-based association analysis, resource identification and classification and the like, and has important application value for red-rooted crane population management.
Drawings
FIG. 1 is a diagram showing detection peaks after 3 SNP extension reactions.
Detailed Description
The invention is further described with reference to specific examples.
The invention uses DNAiso Reagent to extract the DNA of the red-crowned crane blood.
The invention adopts a GBS-seq method and uses an Illumina HiSeq PE150 platform for sequencing.
Example 1:
a method for Dandelion SNP screening and genetic polymorphism evaluation comprises the following steps:
step one, extracting DNA of red-rooted crane:
10 Danding crane blood samples were collected from the Hongshan forest zoo in Nanjing, and the samples were stored in sterile test tubes containing anticoagulant and stored at-80 ℃. The DNA extraction of the blood sample adopts DNAiso Reagent extraction Reagent, and the specific steps are as follows:
(1) mu.L of the blood sample was placed in a homogenizer and 1mL of DNAiso Reagent was added. The homogenization was repeated using a homogenizer until no significant clump tissue precipitates. Transfer the lysate to a centrifuge tube.
(2) The lysate was centrifuged at 10,000 g for 10 minutes at 4 ℃ and the supernatant was transferred to a new centrifuge tube.
(3) To the lysate was added 0.5mL of absolute ethanol. Mix for 1 minute by repeated inversion, use the tip to entangle out the DNA and transfer to a new centrifuge tube, or decant the supernatant and leave the DNA pellet at the bottom of the centrifuge tube.
(4) 1mL of 75% ethanol was slowly added along the walls of the centrifuge tube, the tube walls were washed gently upside down, centrifuged at 12,000g for 5 minutes at 4 ℃ and carefully discarded.
(5) The ethanol-removed genomic DNA precipitate was dried at room temperature for 10 seconds, and an appropriate amount of TE Buffer (pH8.0) was slowly added. The DNA was stored in a freezer at-20 ℃ until use.
Step two, GBS-seq genome DNA sequencing and Dandelion crane SNP site screening:
the method comprises the steps of carrying out enzyme digestion on genomic DNA by using restriction endonuclease to obtain a proper marker density, adding P1 and P2 linkers (which can be complementary with enzyme digestion DNA gaps) at two ends of a fragment subjected to enzyme digestion, carrying out PCR amplification to obtain marker sequences respectively containing P1 and P2 linkers at two ends to form a DNA fragment mixing pool, carrying out electrophoresis recovery on DNA in a required interval, establishing a DNA library, carrying out preliminary quantification by using Qubit 2.0 after the library construction is finished, diluting the library to the ng/mu l, detecting an insert fragment of the library by using Agilent 2100, and accurately quantifying the effective concentration of the library by using a Q-PCR method after the insert fragment accords with the expected value (the effective concentration of the library is more than 2nM) to ensure the quality of the library. And after the library is qualified, mixing different libraries in a pool according to the requirements of effective concentration and target offline data volume, and then carrying out Illumina HiSeq PE150 sequencing. Original image data obtained by sequencing is converted into sequence data (raw reads) through base recognition analysis, and sequencing sequences containing linker sequences are filtered; removing pairs of sequences containing more than 10% of N in the single-ended sequencing sequence and more than 50% of the low-mass (< ═ 5) bases; the sequencing data are strictly filtered to obtain a high-quality sequence to be analyzed, the stated data in the sample are counted, the result shows that the sequencing quality is high (Q20 is more than or equal to 94.67 percent, Q30 is more than or equal to 86.28 percent), the GC distribution is normal, the sample is not polluted, and the library building and sequencing are successful. Counting the sequence number captured by MseI at the two ends of the analysis sequence and the ratio of the captured sequence number to the analysis sequence number, namely enzyme capture rate, so as to evaluate the enzyme digestion efficiency; effective high-quality sequencing data are compared to a reference genome through BWA software (parameter: mem-t 4-k 32-M), the comparison result is sequenced through SAMTOOLS, and the result shows that the similarity of the data and the reference genome meets the requirement of re-sequencing analysis and has good coverage depth and coverage. The above experimental operations were performed by the Biotech Co., Ltd, kindred, Oenothera.
SAMTOOLS software was used to perform population SNP detection. For ensuring the reliability of SNP in a sample, the SNP detected in a sampleSequence support number, quality value (QUAL) and distance statistical cumulative distribution of neighboring SNPs. After filtration, 72 high quality public SNP sites of 10 Dantope cranes suitable for designing primers were obtained for subsequent analysis. And (3) designing 72 pairs of primers by using Primer Premier 5 according to 72 SNP sites, wherein the main parameters of Primer design are as follows: the length of the primer is 18-20 bp; a single primer is complementary by less than 3 nucleotides; ensuring that the difference of Tm values between the same pair of primers is not more than 2 ℃; there is less than 3 consecutive nucleotide pairings between the two primers. The PCR amplification was performed on DNA extracted from the blood of 30 Erythrosepalae in saline city using primers. The PCR amplification system is 25 mu L: 12.5 μ L2 XTaq Master Mix Plus (Dye Plus), 10.5 μ L ddH2O, 1 mu L of each of the upstream and downstream primers, and 1 mu L of DNA; the procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 50-65 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; finally, the extension is carried out for 5min at 72 ℃. 2. mu.L of the PCR product was subjected to agarose gel electrophoresis (1% concentration), and the result showed that the quality of the DNA extracted was excellent. The DNA was subjected to Sanger sequencing. Finally, 33 public SNP sites are selected, and the primer and site information are shown in Table 1. Sanger sequencing was performed by Biotech, Inc., of Ongzhi, Nanjing.
Step three, evaluating genetic polymorphism of germplasm resources:
the genetic polymorphism of the SNP typing results was calculated using Cervus V3.0.7, and expected heterozygosity (He), Polymorphic Information Content (PIC), and the results showed that the range of expected heterozygosity was 0.066-0.508, the average value was 0.329, the range of polymorphic information content was 0.062-0.375, and the average value was 0.261: using Genepop V4 Hardy Winberg Equilibrium (HWE), the results showed that the HWE had P values ranging from 0.012 to 1.000, with an average of 0.687, and only two sites, GjSNP14 and GjSNP22, deviated from the HWE, and the details are shown in Table 1, indicating that this group of sites can be applied to the evaluation of the Dandelion crane genetic resource polymorphisms.
TABLE 1 SNP sites, primers and genetic diversity analysis thereof
Sequence listing
<110> Nanjing university of forestry
<120> red-crowned crane SNP marker and screening method and application thereof
<130> 100
<160> 51
<170> SIPOSequenceListing 1.0
<210> 1
<211> 514
<212> DNA
<213> Grus japonensis
<400> 1
gctgtgcctt aatgtcactg ctacctgttt cacacatggt cttgcactgt aataaataga 60
gctcacgtta cagagttgag ctaggtgtgg tggaatagca tgtgagaatg caaacgcact 120
tttgcatata tactatagaa tgttttgggt tctcatcatg atgaggctat gggcaaagga 180
tgaaaactgc ctcaggtgac ctgaagggac agaggatgac cccataacca aggggaggga 240
gccaacaggc tgaagggaag gtgaggacaa aatggggtag caaggtttag ctgggaaaag 300
cagatgcaag accttaatag agtggcttac caagtcatga ctggagggcc agctgtggga 360
aggtgctgaa ggtggcagtg gtgaacaaat gggacactgt gaaggatttg ctaaggcata 420
tctattaaaa tccaagcctt cttcaggccc tcctgtatca gagatgggag cgagggcctg 480
ctcctgcact ttcaccctca cttacacctg tctg 514
<210> 2
<211> 570
<212> DNA
<213> Grus japonensis
<400> 2
ctgcagctta aaccataact attgtaatag gcttcaagaa atgtctgcat tgacatttct 60
gtctcccagg gagggggcaa tggatggaaa tcatcttatg ctacatctga gtaagtcact 120
caagattcct ttaacagtca gtggagattc atcgctccct aaagcaggta tttaaaacag 180
gtctggtgat tcatgctcta gaagtgccta tgtttcttct tgactagaaa gggatagctg 240
aaaggtatag gtgaaatgaa gctatatatg cagtcaggtg agatggatcc taccacaggg 300
tattactatg acctgagctc cttccagaaa ataaaagaga aattttacct tcacacacat 360
tacccacgca cacgcaaaaa tctaggagcc aaattcaatc tcttgttcct ttactgagcc 420
tagcatatta aaatcctagt ttcagtgaga attgctggta gttttatcaa accaaaaaga 480
aaatgctgaa ggcaaaagag ggaagaatac acgttttcat ttttattgtg catttcttct 540
caaggtagaa atcctagttg gagttaagcc 570
<210> 3
<211> 560
<212> DNA
<213> Grus japonensis
<400> 3
cagagcttta cttgtaaggt tatcatcatc atcacaagag attgccaaga atccccaaac 60
aaaaccccaa ggaaaagcaa gagttagccc agacaacagc gtgaccgaag ctcacatttt 120
aatgtcaaga atatgaattt aaaactgaat atgtctgctg tccagctgag caatgggtct 180
gaaaactgat tagctgtgtt tagggcaagg acattgtgct gcctaaaaat gtcagaggat 240
gctcagatga gaacgagcaa aaggtgaaag gacttaatgc cagccttggc tgcggacgtc 300
actcagccga ggagctcagc tttccccaaa gaagagcaca ctgaaacttc ttacaggacc 360
acacagaaca agacacaaca tagaacattt tcacagtcag ctgctactct gcaaatgggt 420
atagaagtaa aatcataaat aaaaagaatc agtggttgtg aaggttttgg atttttcaat 480
agaatataaa ccaagtacat aacattcact acttccaaag gagagtcact ggagaaaatg 540
tgaagtaacc gtgactggga 560
<210> 4
<211> 557
<212> DNA
<213> Grus japonensis
<400> 4
gtgtagatta ctgcattctc taactgtatt tataatgtgt atttatttca atttcttctt 60
ttagatttgg gcgtaagtta attatatcag acttgtataa actaacggat acggtggcaa 120
tccgtgagca agggagcggg cggctggtgt gccttttacc cagcagccaa gcccgacaga 180
gtccgttggg atcttcacag tcacatgatg gttcatcagc aaactgtagc cctataatat 240
tcgaagagtt ggaatatcac gagcctgttt gtaagcagca ttgcctgaat aaagacttta 300
tgtaagctgt gtatatacta ctataaaatc ttacaaaata tttgaacagc atttaacact 360
tgcaggtata ttaaagttga ggtatatatg ttcatatagc atgctccagt cactgtttat 420
atgtttacga gaataagtca ggcagaaaat agagagatgg cagagtgaat tcttatggat 480
caggtgtgat ccattatgat tggtcatgga ggcagttctc atggatcagg ctgtttgggt 540
aaggttattg tattcta 557
<210> 5
<211> 444
<212> DNA
<213> Grus japonensis
<400> 5
caaccagctg acatgctcag cagctagcgc ttgatgaatt gatggtctta gctcaattcc 60
agggtgactg ttaggcatga ccagaagcac agaccagaac ctctgcacgg accaagactc 120
aatggggatg ggaggcatta acaatgttct gttctggaag tacctccact ggatgcaaga 180
gtctgtgtta gcagagtttt cctgctgtag tcctatgtga acatatggtg tagagtgtaa 240
gtcttggctc ttcaggaatt gccagtcagc agcttcccat tgctctcagt gagtaataaa 300
aatgtaaata aagaagctgt gttatgtctc agaattactc agtggcagtt gtctgaacag 360
ttgtgcttat gctagaatct cagcaagtgc aagtgcaaac actcagacat ctggttgttt 420
cacacttgcc tagtctgctt gtgt 444
<210> 6
<211> 490
<212> DNA
<213> Grus japonensis
<400> 6
ggcatagcca ctgcttctag tgctacaatt ctgagagaca ttgtgatctc aagtctttct 60
tttggacatg gcgatccacc atctcaaatg ctttaaactt gaaatctcta tcagacttga 120
catccttccc aggtacctca ataggctttc tcagcacacg ccactagtgc tgcaattctc 180
actttaactg tttgaccacg gcagtggaat gacaaagcaa gcaacgcagg cttcacatag 240
gaatttctaa accacagcgc ttttgttctt gatgcccttg gcagctactg aactttggag 300
gcagcaaaaa cactgagaag tacattccag aaactgagtc tatcccattg caaactcata 360
gtttttcaag ctttctacat gcttcctact cctccttgcc aataaacgca acttgcttgt 420
gacaaaatcc agctcccttc caaagagcaa aatcctattc ctaaataaag ccttaatctc 480
aatgccactg 490
<210> 7
<211> 480
<212> DNA
<213> Grus japonensis
<400> 7
tggaagaggc cagtttatta taagaactgt gccagacagg aagtgtggac caggacttgt 60
tccagtatgt tgacataaaa gttgacccat aggaaaaaca gcacaggttg gtttaatttt 120
actggacaat acaataaaca agtaaagaac tattattccc tgtgccataa tgatgataag 180
cagaaaataa tagtgatttc tattctgctc aagtaacaag cagaacatac agaaatcagg 240
tgcctggcaa actcagatgc ctctttttcc ccagtccccc tgtaggtctt ccagtcacaa 300
aatgaaaaga acatgtctac gtgactggat cactgaaaac ataactcatg agtcagactt 360
ctgaccaggt ggggaaagtg aaagatgact ccttgatgat tgctttagtt aaaaagaaag 420
catcaaagtg ctcaggctat tactctctct agcaccttgg aactcaatga ctgctgaagg 480
<210> 8
<211> 560
<212> DNA
<213> Grus japonensis
<400> 8
gtgatatgat ataatggtgg cagagacatt cagatcatga atgcctgaga atatgcaacg 60
cagagaactt cccagaacct ctcttttcta agatgaacag tgactctgtt ctcactttaa 120
ttagaaatta tccaacccct gtgctgctta ttccccttcc ttcttcccta cctcctcatc 180
ccaatggctg ttctgatata atttgtctgg ctcaaactgt tcctcattta cagtgtggtt 240
ttgcaaaacc cctgattgat cgcctgaggt tttgcaaagc ccaactcagc cgctagccct 300
gccgatactc aaagttgttg tctcagctgt ggctacgtac atgaggaccc gcctgactct 360
aggctcctgt ggctgtgggg ctctaaagag tattaaaaaa acattaaaaa aaaagaaaaa 420
gcagaggtca acccaagtac agcaacttac agtaacataa aaattccctc caacaggtct 480
ctcttcacca cgagctgcct ctttaccagc atggaagtga ccatacaaat gcattaagcc 540
cctaagcaca caggggttga 560
<210> 9
<211> 499
<212> DNA
<213> Grus japonensis
<400> 9
acagcagcat cacaaaaccc tcataaatgc acacctagaa aatcttatca cagccaacct 60
ggcaccaggc agcaccccag gactggctaa gacaatctca acagcaatga atgatccaac 120
cccaaaacca acaccacatc caaagcacag aatcacactg catccacgct gaacgtagca 180
ttgtctgtgt ctgaggtact atgccaaaag cagcaaatgg agacaggtgc tcaaggggat 240
gctctgcaat ccatatgtct atgggacaag tctccaaaca ctggagggga tttggaggct 300
gcagcagaag cagcctttag cacagctgct tgtgcagcac caactgcatt gaagcaggga 360
atgggattaa cacccctggc tggtcttgca agcagcgctg tattgcactt tcaccatagc 420
cctttggaga aacacatcct tgttcagaaa ctcagcacaa cctggggtct gacagccttg 480
cgtagggcta tgggagctc 499
<210> 10
<211> 560
<212> DNA
<213> Grus japonensis
<400> 10
cggggcatgt tttgaagctg taagttattt attattttga actgggacaa aagcagtctc 60
aaggaaagta cgtttactaa gtatctgact ccaaggatga agtctgtcct gccagggagc 120
agctgctgcc ctaccagtct aaacactgta tttcaaagac aggggactaa ggctggacca 180
tacccacgca agtgtctgag aaaaataact tacaaaataa tagcttactg atcagatctg 240
ccaaagtatc caagcctgtg ggtaagttca agcataataa aattccttag attgtagagc 300
ataagcagaa aataaaccat caagctagat tttaacttgt gataaaatgc tgctgcagaa 360
tggcataact tgtagctgga gcggaggtga aagatcagca agaccaacca ctgcattgct 420
aacgctttag agctcctcag aagagaagga taaaactgca aatttaaatc aagagcattt 480
tattttgaaa ttgctttcca aagcacgata gttctaaaag taacgacttc aaagagtaaa 540
gaatgcagaa agtggcttat 560
<210> 11
<211> 502
<212> DNA
<213> Grus japonensis
<400> 11
atgagcttat gataggcaac actatgtcta tctcacatgt caacctcgct atgtatcact 60
tatcccccga gatttgaatc gggcacggat gcattttaac gttattttgg actttggaaa 120
gcagctgtgg aaacagttca ttgcagctga ttttcacttt gaaattttgg actgattcaa 180
agctcagagc tgcttcttcc ttgacaacct gccaaaaggg acatagcttg atcctccctg 240
aacctcccta ttttccattt gcagccacgt ttcaagtttt gctgaccatg aacgcagcat 300
ctaacttcta ttgcacccaa agaaaagaca tgaagcattt gtgccctgat cctgcaggca 360
ttaatcatct ctacaacttt gctcatgtga atatttctac cgatttcaac gagactaata 420
acacaatgca gtagatgcaa tagcgtctaa aggattggga ccttaaattg catctctaaa 480
ataccttatt tcccctgaga tt 502
<210> 12
<211> 496
<212> DNA
<213> Grus japonensis
<400> 12
tctgcagcaa ctgagctttg gttttgagct ctctgtctac agtctgcccc agaccagctt 60
gggtacctgg ataaaagaat tgtggcagta tgtgagggta tggccaatat cactttgtct 120
taatatcatt ttttttgtgc ttttacataa ttttttgttt catgccggtt ttagtattct 180
gctaggtcag atgaagtcag gggtcagggc tgtgcttgat ttaatacaac tggatggttt 240
gcctggtttt tgtctgttcc ttttggtgtc ctgtcatttg aattttatgt atttgtcttt 300
atttcttgag acgacttttc tgggtatcat tttctgtgct gtgttggtcc tttatgctgg 360
tacagattgc atttgttcat ctaaaatgcc tgtcctgcac aatttccatc atttcacagt 420
cttacatttt gtcattttct gcttgagttt ggttgtggtt tgttatgttt tgacagtggt 480
ttgtcataca gctgtg 496
<210> 13
<211> 511
<212> DNA
<213> Grus japonensis
<400> 13
ctcagctgca ctgaaattct tgcaaatgca gtcctgcatc tacatgtcct gtgcctgcag 60
cactagatca gacaccgtct gcatcttgtc acttacccgc tcctgaagga agggttttgc 120
cggccgataa catggcacag attcctccac ttttcatagg cagactgggt gccaatggct 180
tggacctaga gcctctccgt tgctgtcctt ttctcaatgc agagtcagaa ttgcttaatg 240
ttttatgctg tgacagatta gacaggagaa accttctgtt gtctgataac ctctttggat 300
gacaagtttg tatcattgga gctaaatcca tatgacagct actcgtcttg ctttgtctgt 360
tttgcttggg ctttttgagc tctgatacgt gtgaaagatg ttgacttgaa tatgccttgc 420
attgttgatt tttgtagatg aagatacagc ctaacaatct gggaaaatat gagttgtggt 480
tctacctgtc atgccagaat tgttggcaat c 511
<210> 14
<211> 601
<212> DNA
<213> Grus japonensis
<400> 14
gctatcacag cttctccatc gctttcctat ccctatttgg agggaatggg aactttccaa 60
atatctgcat gcaggagagg ctgtaggtct gaaacctacg tcagcaatct gcactcttgc 120
ccgctgccct gccagacaca acctctgcct ggtgggaagc gagctgttgt aggacctgcc 180
gagtttgtta tttgcaatgt agtgtttaaa actgctgcaa ggaaacccag gggatactaa 240
gcagagctgt tcattccctg tcaaaatgtg taaacatgaa caaatatggg gtctgtctgg 300
tgaacatcaa agtcaggtaa cacttagccc agtgcaacat cccatagctg tggggggact 360
aaagtgtgaa agagttcttg ttttacatag ctgctgtgaa acgtcacgca gcctggggtt 420
agagcatgtg ttggctgtgt ctccacacca tgaacctaag tcctccttct tccagctgcc 480
tggagccaga ttgtttcaac cttttgtttg ctttaaagtg tccttctgtc ctgcttttct 540
tgaaacatca cacaccctgc atgccagatg ctgctaatct agcactgctg acttgataca 600
t 601
<210> 15
<211> 468
<212> DNA
<213> Grus japonensis
<400> 15
ttcccattgt cacatcagtc cccaaaggac cataattaga ctgaatatta aagcatattt 60
tgactgaata ttttgactgg agggagctgc cgccgcttgc tctaatgacc actgagcact 120
tgcctgaagc ctgtactgct ctttttttct gaactggtgg aaaatttggg aagctggctt 180
gaagctgaga gaccacatgt gggtaccaaa gccagatgag gtacaaagca accgtttgtt 240
gagctgtcct gtgattggtg gaccaaggtt agtcttcatc tctgctgcag ggtacgtact 300
gcaacatatt ccagccaact ggtaagatat ttattttttg ataagtggat gtgtgggtta 360
aatgtctcat gttaggcttg cagccctcag taacttaaat atagaaataa gaagaaaaaa 420
gtcttataca aattcgtatc caagagaaaa tactacagag gttggaag 468
<210> 16
<211> 505
<212> DNA
<213> Grus japonensis
<400> 16
ctaacgactc tggttcagcc agagatgtac attgtcccac tactatgcac ctccacttaa 60
aggatcatct ctgtttcata gtaacctacc agtaagaatc ggatgcagta agatggaaga 120
gtactgcatg tggtttcaga tccttgggag aatgtaggaa catagtggac aaattccagg 180
tatgtagaag ggctccagaa gtaagccttt agatactcaa ggctgtggag ccctcagcag 240
tctgagcctt cccctgacct caggagagca tgttcgctag gcacagcaca gcgtgtctgg 300
ttattactgc ccacgtctct ttgtatgaat gcgctccaat ctacagcgta caatttaagt 360
cgcctgttgt taaagtcaaa tggctcatga tagtctgtaa tgcaatatag ataataacag 420
agtgtatgtg aaaatatttg ggaaaacatt aagagcctgt aatttgttct ccagccctca 480
gatgcatagg cctgttgcaa gatca 505
<210> 17
<211> 501
<212> DNA
<213> Grus japonensis
<400> 17
gatttgtgtg gaacaggagc ccttgctggc agaggactct agtaggaaac tgtctttgca 60
aaacagttag gcctgctggt tgcctgaatg cccatagctt atttagatga ctaaacccta 120
gaatttaaca gcagttactc aaaacagtag caaaatcact gaaaacatta ttcacctttt 180
gggaatacat ctacgacata ggtttctctt ccatacagct cctaatctga gaatgggatt 240
cagttgccct cacattgcca ttttatgcat atctgctgct ggtttaggta ctgtggacta 300
tcacacgcag ctttcagatg ccgctccagg acagcatgat cttcagtagc acctgactca 360
catctgccag ccttgcatca gcttgagtag cactagatat ttctgttaaa gcaactgaac 420
tgatgcctaa tacccaacgc ggtgcatagt tcagtgacaa agtaagtggg acttttcata 480
ggaaacctgt ggcatcagca c 501
<210> 18
<211> 18
<212> DNA
<213> Gjpr 1F primer sequence (Artificial)
<400> 18
gctgtgcctt aatgtcac 18
<210> 19
<211> 18
<212> DNA
<213> Gjpr 1R primer sequence (Artificial)
<400> 19
cagacaggtg taagtgag 18
<210> 20
<211> 19
<212> DNA
<213> Gjpr 2F primer sequence (Artificial)
<400> 20
ctgcagctta aaccataac 19
<210> 21
<211> 19
<212> DNA
<213> Gjpr 2R primer sequence (Artificial)
<400> 21
ggcttaactc caactagga 19
<210> 22
<211> 19
<212> DNA
<213> Gjpr 3F primer sequence (Artificial)
<400> 22
cagagcttta cttgtaagg 19
<210> 23
<211> 19
<212> DNA
<213> Gjpr 3R primer sequence (Artificial)
<400> 23
tcccagtcac ggttacttc 19
<210> 24
<211> 20
<212> DNA
<213> Gjpr 4F primer sequence (Artificial)
<400> 24
gtgtagatta ctgcattctc 20
<210> 25
<211> 20
<212> DNA
<213> Gjpr 4R primer sequence (Artificial)
<400> 25
tagaatacaa taaccttacc 20
<210> 26
<211> 18
<212> DNA
<213> Gjpr 5F primer sequence (Artificial)
<400> 26
caaccagctg acatgctc 18
<210> 27
<211> 18
<212> DNA
<213> Gjpr 5R primer sequence (Artificial)
<400> 27
cacaagcaga ctaggcaa 18
<210> 28
<211> 18
<212> DNA
<213> Gjpr 6F primer sequence (Artificial)
<400> 28
ggcatagcca ctgcttct 18
<210> 29
<211> 18
<212> DNA
<213> Gjpr 6R primer sequence (Artificial)
<400> 29
cagtggcatt gagattaa 18
<210> 30
<211> 18
<212> DNA
<213> Gjpr 7F primer sequence (Artificial)
<400> 30
tggaagaggc cagtttat 18
<210> 31
<211> 18
<212> DNA
<213> Gjpr 7R primer sequence (Artificial)
<400> 31
ccttcagcag tcattgag 18
<210> 32
<211> 20
<212> DNA
<213> Gjpr 8F primer sequence (Artificial)
<400> 32
gtgatatgat ataatggtgg 20
<210> 33
<211> 20
<212> DNA
<213> Gjpr 8R primer sequence (Artificial)
<400> 33
tcaacccctg tgtgcttagg 20
<210> 34
<211> 20
<212> DNA
<213> Gjpr 9F primer sequence (Artificial)
<400> 34
acagcagcat cacaaaaccc 20
<210> 35
<211> 20
<212> DNA
<213> Gjpr 9R primer sequence (Artificial)
<400> 35
gagctcccat agccctacgc 20
<210> 36
<211> 20
<212> DNA
<213> Gjpr 10F primer sequence (Artificial)
<400> 36
cggggcatgt tttgaagctg 20
<210> 37
<211> 20
<212> DNA
<213> Gjpr 10R primer sequence (Artificial)
<400> 37
ataagccact ttctgcattc 20
<210> 38
<211> 20
<212> DNA
<213> Gjpr 11F primer sequence (Artificial)
<400> 38
atgagcttat gataggcaac 20
<210> 39
<211> 20
<212> DNA
<213> Gjpr 11R primer sequence (Artificial)
<400> 39
aatctcaggg gaaataaggt 20
<210> 40
<211> 18
<212> DNA
<213> Gjpr 12F primer sequence (Artificial)
<400> 40
tctgcagcaa ctgagctt 18
<210> 41
<211> 18
<212> DNA
<213> Gjpr 12R primer sequence (Artificial)
<400> 41
cacagctgta tgacaaac 18
<210> 42
<211> 19
<212> DNA
<213> Gjpr 13F primer sequence (Artificial)
<400> 42
ctcagctgca ctgaaattc 19
<210> 43
<211> 19
<212> DNA
<213> Gjpr 13R primer sequence (Artificial)
<400> 43
gattgccaac aattctggc 19
<210> 44
<211> 20
<212> DNA
<213> Gjpr 14F primer sequence (Artificial)
<400> 44
gctatcacag cttctccatc 20
<210> 45
<211> 20
<212> DNA
<213> Gjpr 14R primer sequence (Artificial)
<400> 45
atgtatcaag tcagcagtgc 20
<210> 46
<211> 18
<212> DNA
<213> Gjpr 15F primer sequence (Artificial)
<400> 46
ttcccattgt cacatcag 18
<210> 47
<211> 18
<212> DNA
<213> Gjpr 15R primer sequence (Artificial)
<400> 47
cttccaacct ctgtagta 18
<210> 48
<211> 18
<212> DNA
<213> Gjpr 16F primer sequence (Artificial)
<400> 48
ctaacgactc tggttcag 18
<210> 49
<211> 18
<212> DNA
<213> Gjpr 16R primer sequence (Artificial)
<400> 49
tgatcttgca acaggcct 18
<210> 50
<211> 18
<212> DNA
<213> Gjpr 17F primer sequence (Artificial)
<400> 50
gatttgtgtg gaacagga 18
<210> 51
<211> 18
<212> DNA
<213> Gjpr 17R primer sequence (Artificial)
<400> 51
gtgctgatgc cacaggtt 18
Claims (2)
1. The red-crowned crane 33 public SNP loci, which are characterized by the following:
2. the use of 33 public SNP sites of the red-rooted crane according to claim 1 for the molecular marker assisted reproduction or genetic diversity studies of red-rooted crane.
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Citations (4)
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|---|---|---|---|---|
| WO2012162621A1 (en) * | 2011-05-26 | 2012-11-29 | Brandeis University | Methods for suppression pcr |
| CN108998538A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | A kind of spot Ji SNP marker and its screening technique and application |
| CN109439739A (en) * | 2018-08-16 | 2019-03-08 | 浙江海洋大学 | Yellow crucian carp high density SNP marker screening technique and application |
| CN109457022A (en) * | 2018-08-16 | 2019-03-12 | 浙江海洋大学 | Chinese herring SNP marker development approach and application based on high-flux sequence |
-
2019
- 2019-11-04 CN CN201911068399.6A patent/CN110669846B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012162621A1 (en) * | 2011-05-26 | 2012-11-29 | Brandeis University | Methods for suppression pcr |
| CN108998538A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | A kind of spot Ji SNP marker and its screening technique and application |
| CN109439739A (en) * | 2018-08-16 | 2019-03-08 | 浙江海洋大学 | Yellow crucian carp high density SNP marker screening technique and application |
| CN109457022A (en) * | 2018-08-16 | 2019-03-12 | 浙江海洋大学 | Chinese herring SNP marker development approach and application based on high-flux sequence |
Non-Patent Citations (2)
| Title |
|---|
| Development and preliminary evaluation of a genomewide single nucleotide polymorphisms resource generated by RAD-seq for the small yellow croaker (Larimichthys polyactis);Bai-Dong Zhang等;《Molecular Ecology Resources》;20160331;第16卷(第3期);第755-768页 * |
| 日本鳗鲡离散SNP标记筛选及群体遗传多样性分析;于磊等;《中国海洋大学学报》;20180430;第48卷(第4期);摘要，第38页左栏第2段，右栏第1-4段，第44页左栏第2-3段，表2 * |
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