CN114525345A - SSR molecular marker of castor silkworm and application thereof - Google Patents
SSR molecular marker of castor silkworm and application thereof Download PDFInfo
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Abstract
The invention discloses a SSR molecular marker of a castor silkworm and application thereof, belonging to the technical field of molecular biology and silkworm variety identification. The invention obtains the information of genome SSR molecular marker loci by analyzing the genome sequence of the ricinus communis, and designs and synthesizes 15 pairs of SSR primers with high amplification efficiency and rich polymorphism. 20 parts of castor silkworm germplasm are taken as materials to construct the SSR fingerprint of the castor silkworms. According to the method established by the invention, the detection of the SSR marker can be completed by utilizing PCR amplification and electrophoresis detection technologies, the method can be used for identifying the castor silkworm germplasm resources, revealing the genetic variation of each strain at the DNA level, and can also be used for developing and applying a specific marker.
Description
Technical Field
The invention belongs to the technical field of molecular biology and silkworm variety identification, and particularly relates to a ricinus communis SSR molecular marker and application thereof, which are suitable for genetic diversity research, germplasm identification and genetic relationship analysis of ricinus communis.
Background
The SSR molecular marker technology is applied to the present day, and has become the most popular and mature molecular marker technology by virtue of high polymorphism, abundant marker quantity and low cost. Nowadays, the method is widely applied to many research fields such as biological genetic diversity, genetic map construction, genetic analysis, molecular marker-assisted breeding and the like. The traditional method for establishing the SSR marker by constructing a genome library is time-consuming, labor-consuming and low in efficiency, so that the application of the SSR marker is limited. At present, the search of SSR loci from a large number of DNA sequences obtained by gene sequencing by utilizing a microsatellite search tool has become the most convenient and effective way for developing microsatellite markers.
The castor silkworm (Philosamia cynthia ricini), also known as cassava silkworm and Indian silkworm, is a spinning economic insect with diversification and no diapause period under proper environmental conditions. The domestication and breeding of Indian people are carried out in the 16 th century, more than 20 countries and regions are introduced before and after the 20 th century for breeding, and the 40 th century is introduced into the northeast, east and south China. The castor silkworm is a euryphagic insect, and can eat cassava, ailanthus altissima, coriaria sinica and other leaves besides the castor leaves. The castor silkworm has the advantages of fast growth, easy breeding, strong disease resistance, strong silkworm body, backlight clustering and the like, is the third economic insect next to the silkworm and the tussah in China, and can also be used as a good genetic research material. Researches on the castor silkworm are relatively few, and only the analysis of detecting genetic diversity in a plurality of varieties by adopting an ISSR method is reported, but the ISSR marker has the defects of poor repeatability and the like, and no report of developing the SSR marker by using the DNA sequence of the castor silkworm is found at present. The method aims at marking and selecting the castor silkworms, is used for variety molecular screening, and particularly has urgent need in the application of producing excellent varieties.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of screening SSR molecular markers on the genome level based on the complete sequence information of the castor silkworm genome and provide technical support for germplasm resource preservation and molecular marker-assisted breeding. The invention firstly utilizes a bioinformatics method to detect the SSR locus of the ricinus communis, develops the SSR molecular marker of the ricinus communis with good amplification and rich polymorphism, establishes the SSR amplification technical system of the ricinus communis, and applies the SSR molecular marker to the genetic diversity research of the ricinus communis germplasm resources and the construction of a DNA fingerprint.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:
the SSR molecular marker of the ricinus communis has a nucleotide sequence shown as SEQ ID NO of 1-15.
Further, the primer of the SSR molecular marker is shown as SEQ ID NO: 16-45.
The SSR molecular marker of the ricinus communis can be applied to variety identification, germplasm resource diversity analysis, core germplasm establishment and molecular marker-assisted breeding of the ricinus communis.
The PCR reaction system and the PCR reaction program of the SSR molecular marker of the castor silkworm are as follows:
PCR reaction Total 25. mu.L: mu.L of 10 XPCR buffer, 2. mu.L of dNTP (2.5mmol/L), 1. mu.L of each of the upstream and downstream primers (10mmol/L), 0.3. mu.L of DNA polymerase, 1. mu.L of DNA template (500 ng/. mu.L), ddH2O was added to bring the volume to a final reaction volume of 25. mu.L.
PCR amplification was performed on a BIO RAD T100 Thermal Cycler under conditions of 95 ℃ pre-denaturation for 3min, 95 ℃ denaturation for 30s, annealing for 30s (the annealing temperature depends on the primer), and 72 ℃ extension for 30 s; for a total of 35 cycles, an additional 5min extension at 72 ℃ was carried out and the reaction was finally stopped at 12 ℃.5 μ L of PCR amplification product was subjected to 1% agarose gel electrophoresis and primary screening.
Primer sequences, repeat motifs, fragment sizes and annealing temperatures are detailed in Table 1.
TABLE 1 SSR molecular marker primer sequence, annealing temperature and amplification band
The castor silkworm genome has 155 Scaffolds, the total base length is 450479495 bp, the maximum Scaffold length is 33970159 bp, the average length is 2906319 bp, and the GC content is 34.3%. The MISA software searches in genome sequence to find 186790 SSR loci, gSSR loci in genome total frequency of 0.04%, average 1 gSSR per 2.4 kb. In order to improve the accuracy and the universality of gSSR marker development, homology comparison is carried out on the sequence containing the gSSR loci obtained by searching and the group data of the castor silkworm variety B7, and 7036 gSSR loci with polymorphism are screened. And (3) randomly selecting 28 pairs of SSRs in a segmentation manner, evaluating sequences before and after an SSR repeated motif by using Primer Premier 6.0 software, and designing conservative primers. The primers were synthesized by Biotechnology engineering (Shanghai) Co., Ltd, and amplification detection was carried out in 20 parts of the silkworm germ resource of Ricinus communis. Agarose gel electrophoresis detection shows that 3 of 28 primers in the SSR have no amplification band or non-specific band, and the other 25 pairs of Polymerase Chain Reaction (PCR) amplification products are consistent with a target band and can be used as candidate markers for further experiments, and the effective amplification rate reaches 89.3%. Selecting a specific primer for stable amplification, adding a fluorescent label, and performing polymorphism detection in 20 parts of sericulture resources of the ricinus communis by using a full-automatic capillary nucleic acid sequencer to finally obtain 15 pairs of SSR primers with high amplification efficiency and rich polymorphism, gSSR _ P61, gSSR _ P173, gSSR _ P3547, gSSR _ P507, gSSR _ P3446, gSSR _ P1556, gSSR _ P3555, gSSR _ P3410, gSSR _ P3729, gSSR _ P3411, gSSR _ P3888, gSSR _ P3840, gSSR _ P6165, gSSR _ P3765 and gSSR _ P6101.
Has the advantages that:
the 15 pairs of SSR primers disclosed by the invention have the advantages of high amplification efficiency and rich polymorphism, and are beneficial to application of germplasm resource identification, gene marker development and the like.
Drawings
FIG. 1: 6101 amplification peak profile in B1;
FIG. 2: 6101 amplification peak profile in B13;
FIG. 3: 3888 peak amplification in B13;
FIG. 4: 3888 amplification peak profile in B1;
FIG. 5: 6165 amplification peak pattern in B1;
FIG. 6: 6165 amplification peak pattern in B2.
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
In order to explain the technical content and structural characteristics of the SSR marker of the ricinus communis of the invention in detail, the following embodiments are further described.
1. Method for developing castor silkworm molecular marker
1.1 screening of SSR sites in DNA sequence of Ricinus communis
The MISA software is used for searching SSR loci, and the searching standard parameters are set to be single nucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum repetition times are respectively 10, 6, 5 and 5. Through searching, the sequence containing SSR locus is obtained.
1.2 design and Synthesis of SSR primers for Ricinus communis
Sequences around 100bp around the SSR repeat motif were evaluated and primers were designed using Primer Premier 6.0 software. The distance between the SSR locus and the flanking sequence is about 50-300bp, the length of the primer sequence is 18-27bp, the GC content is 40-60%, the annealing temperature is 50-65 ℃, the length of the amplification product is 80-300bp, and the occurrence of dimer, hairpin structure, mismatch and the like is avoided as much as possible. After the primer design is finished, the primer is synthesized by the committee bioengineering (Shanghai) Limited liability company.
1.3 screening of SSR primers from Ricinus communis
1.3.1 general PCR reaction
PCR reaction Total 25. mu.L: 2.5. mu.L of 10 XPCR buffer, 2. mu.L of dNTP (2.5mmol/L), 1. mu.L of each of the upstream and downstream primers (10mmol/L), 0.3. mu.L of DNA polymerase, 1. mu.L of DNA template (500 ng/. mu.L), ddH2O was added to make a volume of 25. mu.L to the final reaction volume. PCR amplification was performed on a BIO RAD T100 Thermal Cycler. The reaction conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 30s (the annealing temperature depends on the primer), and extension at 72 ℃ for 30 s; for a total of 35 cycles, an additional 5min extension at 72 ℃ was carried out and the reaction was finally stopped at 12 ℃.5 μ L of PCR amplification product was subjected to 1% agarose gel electrophoresis and primary screening.
1.3.2 fluorescent primer PCR amplification
The total PCR reaction was 25 μ L: 2.5 uL 10 XTaq Buffer (with MgCl2), 1 uL dNTP (mix,10 uM), 1 uL upstream and downstream primers (10 uM, HEX or 6-FAM fluorescein at the 5' end of the primers), 0.5 uL Taq enzyme (5U/. mu.L), 1 uL DNA template (20-50 ng/. mu.L), and ddH2O to a final reaction volume of 25 uL. PCR amplification was performed on a VeritiTM 96well PCR instrument of ABI, USA, under conditions of pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s (the annealing temperature depends on the primers), extension at 72 ℃ for 30s, and 10 cycles first; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s (annealing temperature depends on the primer), extension at 72 ℃ for 30s, 35 cycles, re-extension at 72 ℃ for 5min, and final reaction termination at 12 ℃.
1.3.3 full-automatic capillary accounting analyzer detection
A96-well reaction plate is taken, a marker pen is used for marking the plate name and the experiment date, an electronic Short repeat (STR) detection table is manufactured, and an on-line table is automatically generated. Using a continuous applicator, a mixture of 990. mu.L HIDI and 10. mu.L LIZ500 was pipetted into a 96-well reaction plate at 10. mu.L per well, and the 96-well plate was placed in a plate centrifuge and centrifuged at 1200rmp for 15 s. Using 12 rows of 10. mu.L rowbars, 1. mu.L of sample was added to the corresponding wells of the 96-well plate against the STR detection table, and the 96-well plate was placed in a plate centrifuge and centrifuged at 1200rmp for 15 s. The 96-well plate was sealed with a sealing membrane, shaken, and the 96-well plate was placed in a plate centrifuge, centrifuged at 1200rmp for 30s, and placed in a PCR instrument. The denaturation procedure was 98 ℃ for 5min, the hot lid was not heated, and the 96-well plate was rapidly cooled on an ice-water mixture immediately after the procedure was completed. The STR samples were tested in 96-well plates placed in a plate centrifuge, centrifuged at 1200rmp for 15s, using an ABI 3730xl apparatus, usa. And a profile file for each site on each sample was automatically generated by GeneMapper v3.7 software and the product fragment size was obtained from the peak profile.
1.4 data statistics and analysis
Converting the data format by using CONVERT (version 1.31) software, calculating indexes such as allele factors (Na), effective allele factors (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Polymorphic Information Content (PIC), inbreeding coefficient (Fis) and Shannon (Shannon) information index (I) of each locus by using POP-GENE (version 1.32) and PIC-CALC software, and testing whether the allele frequency of the polymorphic locus deviates from the Hardy-Weinberg equilibrium (HWE). Bayesian clustering analysis is carried out on the genotypes of the population by using the Structrue 2.3.4 software, and the potential genetic structure of the ricinus communis population is detected. Genetic distance (Nei,1983) and cluster analysis (1000 Bootstrap methods) was performed on the germplasm of ricinus armeniaca using PowerMarker V3.25 software.
The 15 SSR markers can be used for genetic diversity analysis, molecular fingerprint map construction and the like of the castor silkworm germplasm resources, have good polymorphism and repeatability, and are reliable and effective molecular markers.
The above description is only an example of the preferred embodiment of the present invention, and should not be taken as limiting the scope of the present invention, so that the equivalent changes made in the claims of the present invention will still fall within the scope of the present invention.
TABLE 1.15 SSR primers polymorphic band amplification
TABLE 2 genetic diversity of microsatellite loci and Hardy-Welnberg equilibrium test
The allelic factors are observed.
② effective allelic factors.
And thirdly, observing the heterozygosity.
And fourthly, expecting the heterozygosity.
Polymorphic information content.
And sixthly, the inbreeding coefficient is determined.
Seventhly, the shannon index.
-Hardy-Weinberg equilibrium chi-square test P-value: p < 0.050,: p is less than 0.010.
Sequence listing
<110> university of Jiangsu science and technology
SSR molecular marker of <120> castor silkworm and application thereof
<160> 45
<170> SIPOSequenceListing 1.0
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<211> 427
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 1
atattcttat ccgatgtttg accaaaattc agagagtttg cttgtgtaaa attacagtaa 60
atgtaccagt aatttgtgta attacggttc accgagctgg catcattatg cagggtaggg 120
gtagtggggt aggtagagtc gggcagtcgg gcggagcggt acgcgcgctc gtcgtcgtag 180
gatatatgga tgaatttata aagaagaaga agaagaagaa gaagaagaaa aagaaagaaa 240
aaacagacac aaaagtgaca tcaatcaatc gatcaaccaa cacatgatgg ataattaatt 300
aattacgggg ggtaattttt gggtgttttg tagggggtaa ttaattattg tgtgtgagat 360
aaatcgtcgc gacactagtg gtgtagcagt agtaataata tagtagtagt ataattatcg 420
acattcg 427
<210> 2
<211> 427
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 2
tacattttga tgggtactcg acacattaaa ctaacagatg atatcaaatt aattacaatt 60
aatataatat atgtttcaat aatccttgat tcaaatgtac atatgataat taaagagaat 120
cgtgcacttt aactttttga tttatgaaac tgatctaata tgtatcttat aaaaatttta 180
gtgcagtgaa agaaagtaac aataataata ataataataa taataataaa gaagtaaaag 240
acgaatccga aagaaccgtc tatttgttac ataggtaacc gctgacatac attaagtggt 300
acaattcaaa cagtcaaccg gacaaatgcc ccgcaagtac ggagcttaaa tttattaccg 360
ctatctgtta ctccctccaa gatggacacg gacactggac atttctaaag ttataatatt 420
ttctttg 427
<210> 3
<211> 424
<212> DNA
<213> Artificial sequence (artifical sequence)
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tgcaggagga gggcggcggt gcgggcggcg cggtcgccgc ggaggaggcg ctggcggcgc 60
tgacgcggcg cgagggctcg gcgcgtgcgc tggcggcggc gcacgaggtg ctcgtggcgg 120
agcgcgaccg cctgcgggac tcgctggccg agctgcacgc gcagctcgac gactatcagg 180
taccacacgc cataacccgt gcacgcacgc acgcacgcac gcacatatct gccggattcc 240
acgtcaactc cacctggtga aggaaaccat agcatggtgg ttgaggattt tttaatgatg 300
cctgtctggg tagctaccac cctcaaagtt gaaaacggta acatcacttg agaatctcag 360
tacctaggct taatgtgctt atcataataa accatatcgt cgcttaacat cagctgagat 420
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<210> 4
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<212> DNA
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actagatcct attgaaattg aaattgctcg gtttttaatc cgaatgattt gaccaatgga 60
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caaccaggtg acgatgatgg aaaacctcgc atagaaacca gaaatgtgta tgaggagtcc 180
ttgtttgtac tgtcacaatg acacacacac acacacacat atatatatta gaaataccaa 240
atcgtcaata cgtcgtgatc tggagatccc tagccgtaag attaataaac ccgttctttc 300
cgttgggttt acccatcact tgaacaaaat tgtttttaat tattctgtgt atcatcatct 360
taaccttgtc ctacaataag ttgtctcaat cccgtgtcgc cccttctatg aggctgat 418
<210> 5
<211> 416
<212> DNA
<213> Artificial sequence (artifical sequence)
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gtaatttatt tagttgtgtt aaatgacatg tttttatgta attttaggta gcataaagtc 60
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atcatgcaaa catgcaaggc tacaaaataa taacaacaaa gttgcatatt tatcatctat 180
accaaataat gaaaatggtg gagagagaga gagagataca gaaaggtaat taagaggaac 240
cgagcaccag tgaatagaag aaattaacgt tagctgcctt tagccgttat tgtttacagt 300
aaggtacaca tgcgtcgaaa tacgagcgtc ggtttttacg cccgtaacct gcgacattta 360
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<210> 6
<211> 458
<212> DNA
<213> Artificial sequence (artifical sequence)
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aacaaataaa acccatcgtg attcggtttt tggctcgatg gaggaaggat gaaatcctga 60
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aaataacatt tattgtaatg atcatttaac cagtcgcaac aagctctctt acaaaccgcc 180
aaaacacatg ttaaagataa atatatatat atatatatat atatatatat atatatatat 240
atatatatat atatatatga gcaaaaaact attctataat ggttcgtcag tcagtcacta 300
gccctgtttt acatatatca agcgaatgtg attaaaaaaa atgtgtagga ttcacttggc 360
tttcccttat tatatagttt gatacctttg atatagttat tatatttaca atatattact 420
tttgtatatt cgtaaacttt ggcattacct ttaggttc 458
<210> 7
<211> 433
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 7
taattgcgac atcatcgtcg caaatgttag tgcggatgaa ttccgtattg atgtccacct 60
gttcgaacct caccaattaa gaattaaagc atgacgaaac agtgaaagta atgagtttga 120
attgaaaggt tttacatgta tattaaagga tttggtcggt gcggtgtgtg tgtgcgtgtc 180
gctgagtatc gcggtgtaca gcggcggcgg cggcggcggc ggcggcggcg gcgctgtggc 240
gggtctctcc ggtgcgtacg cactgttgct acccgcgtac ttggcgcacc tcgccaagtg 300
ccgcgccgac ctcgaccaac agctcgctgc cctacaacgc gtgcatacag atacgaactt 360
gcctcaagaa gattacagag aatattgtaa gtttcatcca tcatcatcat catcatcatc 420
tcagccaaaa tac 433
<210> 8
<211> 428
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 8
taatacacaa ctgcgccacc gagataatcc tatattaatg tgcgttgttt taatgaacca 60
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tgcatatcct tatttgaaac gcattttacg tcacgtcacg attttgaata aagaacggat 180
aggaagtgcg cagagagagt gagagagaga gagagagaga gagagagaaa cgagacaatc 240
tgttaatacg tgaaagtaca aactttgtac ctctttttac gaaaattgta aggcggatgg 300
agtatgaaat ttcacactta tagtttatgc acttatactt aaaattatgc ataagataca 360
ttaaatcaat aaaaaaaaac ataacacaca gacactagca tgtatttaac acaacacacg 420
tctatttt 428
<210> 9
<211> 418
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 9
atcccttatg tcataagtaa atttcttgca aaacgtcaag tacatataag tacttccgac 60
aacactaagt acccgttacg ctcactcata atgtatgtag acgaaatatc cgcaatacaa 120
ctttgcgatt tgttttaata acaataaatt cacgaacttt tcaattcaaa acccgaggcg 180
gtattatctg agcagtgtgc gtgtgtgtgt gtgtgtgtat gtaacgagtt aaaaacatcg 240
tgcgcaaata aagttcacct aacgagcctc gcttcgaaaa caaggttatt cgtaaacctc 300
gaagtgctta gagtcaagcg ataatataga aactatgtta cgtgttcaaa acgatgatga 360
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<210> 10
<211> 430
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 10
gaagataact ttaaaagata gatgaataaa tatgattaca tatttgaaag tgataacgac 60
acgtcgcggc cgcgggttcg attcccgggc gaatcagaaa tgtttacgat ctaataatga 120
cattaaatat tgtttgatta aaccatttta tatgttatta ttgtaacgtt tagtttatta 180
tctggtgatg ctgtgagtgt gagagagaga gagagagaga gagagagaga gtgtaggttc 240
agtagtgagc cttgtcgggt ccctccatgt cctcgctgct cttcagcatc aggtagtagc 300
tgttgaccgt caggatgcag tacgcgacaa agcctgaccc cgcgaaaaca ttgtacacaa 360
tattaatata ttgtctgtat gtttgttact acacatttaa cttgttattt caattctctg 420
aacatgtttt 430
<210> 11
<211> 422
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 11
gattttgacg ggcctttttt tggcagatag ctgaagttac tcctcgtaac ttaggctact 60
ttaatttttg aaaataatta gagttccatg taaaaaaaat taaaactcat gaaatcgtga 120
ataagatccg attcgttgca caatgcacag ttttactgac aggtatttga ttttttttta 180
aatatctatt tctcaaactc tatatatata tatatatata tatctctttc tattccgcag 240
attacccgga acaatcagac aaacattttt tttaaattgt atttttggtt tacgtatata 300
cgatacgata gattcatata tatttagtaa aaaaatgaga ttttgatttt acaaacagac 360
acttaaattt tatttacaac tagtatagat ttgtatactt atgtttagct tagtaatgag 420
gt 422
<210> 12
<211> 420
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 12
tcctgcaaaa ctgaaccgat ttataatttt cggcttcggc tatgcaagac gttttccttt 60
tttcatcaaa tttatttaac gaacgtctga aaatgacggt aaaagtcaag cattcttttc 120
attttcaaag gtttctagtt tctacaatag ctaatttatg gaaaaaatca aaacttacag 180
gcatggtgtt gatatatatg tatatatata tatatatata ttatttggta taaaaagagg 240
aaacagggga ctacgtttgt atggaataaa taccactata atatcttctt aattcattgt 300
gttgtttagc tagttcgctt ttttattttg tactcttatc aacaataact ttcaattcca 360
tttcattcat gtcttctcat tcctgcaata actggtcaca tatgtagctc tttcgatgct 420
<210> 13
<211> 415
<212> DNA
<213> Artificial sequence (artifical sequence)
<400> 13
agaggagtta aattgaaaca actacataaa tttaacgcat tttaaaatta aattttatct 60
ttaattcatg tttaatgcat tatatgaaac agcactgttc tagatagtgc atagtggaat 120
gggaatgtat ggagtatttt caaccttcta ctgtaatctg taccgtacaa ttagaattaa 180
ttacttataa ttatttttag tattattatt attattaaag tttttactat acagcgcata 240
aaaaagcatg aggtcttttc gacctctccc attaaaggtt tcaaatttta atttgatttc 300
gtttactaaa gtaaaattta tattcactca ggtaccgcat cgcatttcgc gagaccctct 360
gctgcgagaa agttggaaag cggaagagcc agtataaaga ccattcgagc ataag 415
<210> 14
<211> 420
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgaacccgt tatctgcaat attagaggga gcttccttta tgaacaggga tcaaacttaa 60
tagtacgaac tttagaccgg tctctataat agacatgtct atactataca gcgatataag 120
tcgagtaact tggcttttta attaactaaa tattgtttta tgttttctac ctataacgtt 180
tatgttatct ttctatctcc tatatatata tatatatata tgtatatata aggaaagttt 240
gttatctttg tccgaggtaa actcgaaaac tactggaccg atcagcatga aaccacaacc 300
attcgacgcg gaattaatcg tagctggtta taggctataa attgttcaaa ttctattata 360
aacttttaat ataaagaatg aatatagtaa ataataatag aatggatcta ttataaagaa 420
<210> 15
<211> 421
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttttcacaat tttttattat atttgtctca gcacgcgcag actctgggtc tgcgtgaaat 60
ttaaaatggt ttacaatttc aatattatag ttttaatata accaccatat caggtggacc 120
attacaaaat atttgcgggc cgtggattga gtatcactga tttaaagcta cttattccga 180
aactatattc aatcttcgtg taataataat aataataata acctatctaa tcgtttagaa 240
ttaaacacag aatagtgaat gtatgtatat attcaaggta attctgaata tattgagtgt 300
gcgtcgacgt tataagcaac ttctgtctgg ccaataaact agaggtcaag taacagatag 360
attatgtggt cagcgtccga gtattgtgta ctgtacggac ttgagtatgt ttttgaattt 420
t 421
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggtaggtaga gtcgggcagt 20
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ccatcatgtg ttggttgatc ga 22
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gagaatcgtg cactttaact 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acggttcttt cggattcgtc 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gcagctcgac gactatcagg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttcaccaggt ggagttgacg 20
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
caaccaggtg acgatgatgg a 21
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acggctaggg atctccagat 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tgcaaacatg caaggctaca 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
attcactggt gctcggttcc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaccagtcgc aacaagctct 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gggctagtga ctgactgacg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gctgagtatc gcggtgtaca 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctgtatgcac gcgttgtagg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cacaatatgg cgtgggtcca 20
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ccatccgcct tacaattttc gt 22
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcaaaacccg aggcggtatt 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ttcgaagcga ggctcgttag 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gattcccggg cgaatcagaa 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cccgacaagg ctcactactg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tccgattcgt tgcacaatgc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gttccgggta atctgcggaa 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tgacggtaaa agtcaagcat 20
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
acgtagtccc ctgtttcctc t 21
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
accttctact gtaatctgta ccgt 24
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cgatgcggta cctgagtgaa 20
<210> 42
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gcgatataag tcgagtaact tggc 24
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcgtcgaatg gttgtggttt 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
atatttgcgg gccgtggatt 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
taacgtcgac gcacactcaa 20
Claims (3)
1. The SSR molecular marker of the ricinus communis is characterized in that the nucleotide sequence of the SSR molecular marker is shown as SEQ ID NO 1-15.
2. The SSR molecular marker of the ricinus communis according to claim 1, wherein the primer of the SSR molecular marker is shown in SEQ ID NO 16-45.
3. The SSR molecular marker of the ricinus communis Linn of claim 1 or 2, and can be used for variety identification, germplasm resource diversity analysis, core germplasm establishment and molecular marker-assisted breeding of the ricinus communis Linn.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030194730A1 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics (Cdfd) | Novel FISSR-PCR primers and methods of identifying genotyping diverse genomes of plant and animal systems including rice varieties, a kit thereof |
| CN1970792A (en) * | 2005-10-14 | 2007-05-30 | 中国科学院上海生命科学研究院 | SSR marker for domestic silkworm and application thereof |
| CN104328197A (en) * | 2014-11-14 | 2015-02-04 | 福建农林大学 | Hibiscus cannabinus L. expression sequence tag SSR (Simple Sequence Repeat) DNA markers |
| CN106755328A (en) * | 2016-11-25 | 2017-05-31 | 中国农业科学院作物科学研究所 | A kind of construction method of broad bean SSR finger-prints |
| CN109517925A (en) * | 2019-01-24 | 2019-03-26 | 中国农业科学院麻类研究所 | Flax SSR molecular marker and its application |
-
2022
- 2022-02-14 CN CN202210134182.6A patent/CN114525345B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030194730A1 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics (Cdfd) | Novel FISSR-PCR primers and methods of identifying genotyping diverse genomes of plant and animal systems including rice varieties, a kit thereof |
| CN1970792A (en) * | 2005-10-14 | 2007-05-30 | 中国科学院上海生命科学研究院 | SSR marker for domestic silkworm and application thereof |
| CN104328197A (en) * | 2014-11-14 | 2015-02-04 | 福建农林大学 | Hibiscus cannabinus L. expression sequence tag SSR (Simple Sequence Repeat) DNA markers |
| CN106755328A (en) * | 2016-11-25 | 2017-05-31 | 中国农业科学院作物科学研究所 | A kind of construction method of broad bean SSR finger-prints |
| CN109517925A (en) * | 2019-01-24 | 2019-03-26 | 中国农业科学院麻类研究所 | Flax SSR molecular marker and its application |
Non-Patent Citations (2)
| Title |
|---|
| 李刚等: "基于16SrDNA序列对蓖麻蚕肠道细菌多样性的研究" * |
| 郭秋红等: "利用SSR标记鉴定家蚕不同系统的品种初探" * |
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| CN114525345B (en) | 2023-04-21 |
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