CN114891894A - SSR molecular marker primer group for identifying Guangxi \/28064;, Zhongdao leopard gill and acantho salmoides protospecies and application thereof - Google Patents
SSR molecular marker primer group for identifying Guangxi \/28064;, Zhongdao leopard gill and acantho salmoides protospecies and application thereof Download PDFInfo
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Abstract
The invention provides an SSR molecular marker primer group for identifying Guangxi \/28064;, an original strain of a stringy gill and an echinoderma maculata and application thereof. The invention obtains a group of SSR molecular markers which can identify Guangxi \/28064Theobroma island leopard gill sea weever original strains through research: the nucleotide sequences of the SSR molecular marker are respectively shown as SEQ ID NO. 1-6 in the fields of PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06, and the SSR molecular marker has the characteristics of high polymorphism but low polymorphism in Guangxi 28064and Zhouzi island primary population; obtaining a primer group for amplifying SSR molecular markers by screening, wherein the primer group is stable in amplification and good in repeatability; meanwhile, a method for identifying Guangxi \28064;, the original variety of the gill and sea bass of the Zodiac island is established, and the problems that the geographic population identification of the existing gill and sea bass of the Leopard island is unclear and the original variety parent of the breeding and releasing seed seedlings is difficult to identify are solved.
Description
Technical Field
The invention belongs to the technical field of molecular biology. More particularly, relates to an SSR molecular marker primer group for identifying Guangxi \/28064;, an original strain of a stringy gill and a sea bass in an island and an application thereof.
Background
Plectropomus Leopardus (Plectropomus Leopadus), commonly known as "east star spot", belongs to Perciformes (Perciformes), Serratidae (Serranidae), Epinephilidae (Epinepineae), gill-spine Perch (Plectropomus), belongs to warm water island marine fishes, and is mainly distributed in the western pacific to the Indian ocean areas, and is an important cultivated fish in southern China. The plectropomus leopardus meat is fresh and tender, has delicious taste, has the characteristics of low fat, low cholesterol, high protein and the like, belongs to high-grade edible fishes, is deeply loved by consumers and has wide market prospect.
28064The Zhou island is located in the middle of the northern gulf sea area of northern sea city of Zhuang nationality of Guangxi, North Lin Guangxi North sea city, Dong Wang Leizhou peninsula, the southeast is adjacent to the oblique-yang island, the south is separated from the south island of Hainan, and the west faces Vietnam. 28064The northern edge of tropical coral in continental island and the region specificity make it have very high ecological value. 28064The fish in the sea area of the coral reef of the island is mainly of the order Perciformes, and the amount of the Perciformes accounts for 74.56% of the total number of collected species. Wherein the leopard gill and thorn perch is 28064a characteristic fish in the island of continental province. However, with the promotion of the construction of the northern gulf economic area in Guangxi and the aggravation of human activities such as tourism resource and ocean resource development, the pleopodes leopardus resource in the sea area is sharply reduced.
The method is an effective method for recovering or increasing natural population by putting artificially propagated and cultured plectropomus leopardus seedlings into the region of \28064H/A. However, leopard gill sea weever from different sea areas has differences in color, taste and appearance, and the leopard gill sea weever cultured from other sea area parents is thrown into the land area of \28064, which causes pollution to germplasm resources of the sea area. The plectropomus leopardus parent is very difficult to obtain, and enough parent cannot be obtained by a field capture method to be used as propagation releasing seedling culture. Meanwhile, the original land of the plectropomus leopardus parent is very difficult to judge by naked eyes. At present, a microsatellite molecular marker method is adopted for researching the population genetic structure and genetic variation of plectropomus leopardus, for example, the prior art discloses a construction method of a microsatellite DNA molecular marker of plectropomus leopardus, a microsatellite marker with rich polymorphism of plectropomus leopardus is developed by designing a specific primer of the microsatellite marker, and the microsatellite marker is applied to research on the population genetic structure, genetic breeding and the like of the plectropomus leopardus. However, the method only provides and verifies 7 pairs of primers, and 14649 sites can be detected in plectropomus leopardus, if the method is used for analyzing and verifying genetic structures of different populations, thousands of specific primers are designed, the operation is complex and time-consuming and labor-consuming, and meanwhile, microsatellite marker primers designed by the method cannot identify and distinguish the plectropomus leopardus primitive species. Therefore, a reliable molecular biological technology needs to be established for identifying the original strain of the gill leopard and spine weever in Guangxi \, 28064Haizia island.
Disclosure of Invention
The invention aims to provide an SSR molecular marker primer group for identifying Guangxi \28064;, a stringy gill-spiny bass protospecies in the island and an application thereof, and solves the problems that the existing geographic species identification of the stringy gill-spiny bass is unclear and the protospecies parent of the breeding and releasing seed seedlings is difficult to identify.
The invention aims to provide an SSR molecular marker for identifying Guangxi \/28064and original strains of gill and sea weever in the island leopard.
The second purpose of the invention is to provide a primer group for amplifying the SSR molecular marker.
The third purpose of the invention is to provide the application of the SSR molecular marker or the primer group.
The fourth purpose of the invention is to provide a detection kit for identifying Guangxi \/28064Theobroma island leopard gill sea weever protogenesis.
The fifth purpose of the invention is to provide a method for identifying Guangxi \/28064;, island leopard gill sea weever protogenic species.
The sixth purpose of the invention is to provide the application of the method for identifying the Guangxi \/28064;, the island leopard gill-sea weever protogenic species.
The above purpose of the invention is realized by the following technical scheme:
the invention designs an SSR molecular marker by a gill sea bass genome (with a delivery number of PRJDB9369, the total length of 748.23Mb, the GC content of 39.5%, the length of scaffold N50 of 30.01Mb and the length of contig N50 of 1.08 Mb.), wherein the SSR molecular marker is PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06, and the nucleotide sequences of the SSR molecular marker are respectively shown in SEQ ID No. 1-6. The SSR molecular markers are verified to show that the 6 SSR loci provided by the invention have the characteristics of high polymorphism but low polymorphism in Guangxi 28064Haizhou island primary population; meanwhile, a primer group which can be used for amplifying the SSR molecular marker is obtained by screening:
the sequences of the primers for amplifying the PLWZ-01 are respectively shown as SEQ ID NO. 7-8;
the primer sequences of the amplified PLWZ-02 are respectively shown as SEQ ID NO. 9-10;
the primer sequences of the amplified PLWZ-03 are respectively shown as SEQ ID NO. 11-12;
the primer sequences of the amplified PLWZ-04 are respectively shown as SEQ ID NO. 13-14;
the primer sequences of the amplified PLWZ-05 are respectively shown as SEQ ID NO. 15-16;
the primer sequences of the amplified PLWZ-06 are respectively shown in SEQ ID NO. 17-18.
The research of the invention shows that the primer group can be used for identifying Guangxi \ (28064); Zhongdao sea area leopard gill sea bass original strains, and the primer has the advantages of stable amplification and good repeatability; meanwhile, the method for identifying the original variety of Guangxi \28064;, the Pleuropterus striatus and the spinal weever can directly identify whether the geographic population of the Pleuropterus striatus and the spinal weever belongs to the original variety of Guangxi \28064;, the Pleuropterus striatus and the spinal weever can be constructed by the unique allele of the original variety of Guangxi \28064;, and UPGMA cluster analysis.
Therefore, the invention provides the application of the SSR molecular marker or the primer group in identifying Guangxi \/28064;, Ledebouriella parva gill sea weever primary strains or preparing a detection kit for identifying Guangxi \/28064;, Ledebouriella parva gill sea weever primary strains.
The invention provides a detection kit for identifying Guangxi \/28064;, island leopard gill and sea weever protogenesis, which contains the SSR molecular marker or the primer group.
The invention provides a method for identifying Guangxi \/28064;, an island leopard gill sea weever original strain, which comprises the following steps:
s1, collecting a sample of a plectropomus leopardus population to be identified, and extracting sample DNA;
s2, respectively carrying out PCR amplification by using the DNA extracted in the step S1 as a template;
s3, performing capillary electrophoresis typing on the PCR product amplified in the step S2;
s4, performing peak image interpretation on the typing result obtained in the step S3 and constructing an allele matrix;
s5, comparing the allele of the gill and sea weever to be detected obtained in the step S4 with the allele of the known Guangxi \28064, original variety of the gill and sea weever to calculate the genetic distance between samples;
s6, performing cluster analysis on the genetic distance obtained in the step S5, constructing a cluster tree, and judging whether the plectropomus leopardus to be detected is 2806416, namely the original variety of the continental island.
Preferably, the reaction system for PCR amplification in step S2 is:not containing Mg 2+ 10 XPCR buffer 2.5L, 25mM MgCl 2 2.0L, 10mM dNTP 0.5L, high fidelity PCR enzyme 1U, 10M forward primer 0.5L, 10M reverse primer 0.5L, DNA template 12.5ng, and the balance is made up to 25L by sterile double distilled water.
Preferably, the reaction procedure of the PCR amplification in step S2 is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
Preferably, the 5' end of the primer set used in step S2 is labeled with FAM fluorophore in step S3 for capillary electrophoresis typing, and capillary electrophoresis typing is performed using an ABI 3730XL genotyping sequencer.
Preferably, in step S5, the alleles of the known Guangxi \28064Theobromaisland leopard gill-spine weever protoplasm include:
the PLWZ-01 site is: 258. 261, 267;
the PLWZ-02 site is: 195. 279, 282, 288;
the PLWZ-03 site is: 238. 251, 256, 279;
the PLWZ-04 site is: 237. 252, 255, 258;
the PLWZ-05 site is: 257. 272, 291;
the PLWZ-06 sites are: 178. 199, 215, 219.
The invention also provides application of the method in identification of geographic populations of plectropomus leopardus or identification of original seed parents of breeding and releasing seed seedlings.
The invention has the following beneficial effects:
the invention designs an SSR molecular marker for identifying Guangxi \/28064;, the original strain of the island leopard gill sea weever: the nucleotide sequences of the SSR molecular markers are shown in SEQ ID No. 1-6 respectively, and the SSR molecular markers have the characteristics of high polymorphism but low polymorphism in Guangxi \28064andisland primary populations, and can effectively distinguish Guangxi 28064and island seal leopard gill sea weever primary populations from other populations. A primer group which can be used for amplifying the SSR molecular marker is obtained by screening, and the primer group has stable amplification and good repeatability; meanwhile, a method capable of identifying Guangxi \28064;, the original species of the Plectropomus leopardus is established, whether the Plectropomus leopardus to be identified is the original species or not is judged through a specific allele and a cluster tree, and the problems that the geographical population identification of the existing Plectropomus leopardus is not clear, and the original species parent of the breeding and releasing seed seedlings is difficult to distinguish are solved.
Drawings
FIG. 1 is a genetic relationship diagram for 173 panthering gill-thorn weever, including 36 panthering gill-thorn weever from Guangxi 28064, Haizhihai area (GX-WZ), gray marker region indicated Guangxi 28064, continental protist branch, source of panthering gill-thorn weever including: china Guangxi 28064%, Zhou (GX-WZ), Hainan east (HN-DF), Hainan tri (HN-SY), Hainan Wenchang (HN-WC), Guangdong ZJ (GD-ZJ), Malaysia (GW-Ma), Indonesia (GW-In), Philippines (GW-Ph) and Australia (GW-Au);
FIG. 2 shows the identification of whether the capture of the Plectropomus leopardus 1 tail in the North sea area of Guangxi 28064the Zhou island breeder's seed, wherein T is the test sample, and the rest of the reference samples include: guangxi 28064%, Zhou (GX-WZ), Hainan east (HN-DF), Hainan Sanju (HN-SY), Hainan Wenchang (HN-WC), Guangdong ZJ.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Primer synthesis and sequencing was performed by Wuhan Tianyihui Limited.
Example 1 screening of SSR markers for Primary species of Plectropomus leopardus
The Plectropomus leopardus genome is downloaded from Genbank, the submission number is PRJDB9369, the total length is 748.23Mb, the GC content is 39.5%, the length of scaffold N50 is 30.01Mb, and the length of contig N50 is 1.08 Mb. And predicting the SSR sequence by using misa software, selecting SSRs with 3-4 repeating units, and calculating the SSR locus with the highest expected heterozygosity.
The screened SSR locus marker is numbered as follows: the PLWZ-01, the PLWZ-02, the PLWZ-03, the PLWZ-04, the PLWZ-05 and the PLWZ-06 are successfully amplified in a plectropomus leopardus sample, have good polymorphism and can clearly distinguish alleles.
Primer3 software is used for designing primers with the length of PCR amplification products of 200-300 bp for the screened SSR batches. The primers were filtered, requiring a free energy of greater than 30 (no hairpin structure). And (3) carrying out genome Blast comparison by using the filtered primers, selecting specific primers, and theoretically amplifying no sequence in a non-SSR region and amplifying the sequence in the SSR region. The SSR amplified fragments have high selection polymorphism, good and stable amplification, high heterozygosity, different amplified fragments and the same or similar annealing temperature (58-60 ℃) and GC value controlled between 50-60%.
The primers obtained by screening are shown in the following table 1, and the PCR reaction system is 25 mu L through PCR amplification detection, and comprises the following steps: not containing Mg 2+ 10 XPCR buffer 2.5. mu.L, 25mM MgCl 2 2.0. mu.L, 10mM dNTP 0.5. mu.L, high fidelity PCR enzyme 1U, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder made up to 25. mu.L with sterile double distilled water. The reaction procedure for the PCR amplification is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
TABLE 1 primer set sequence Listing of SSR marker loci
The PCR verification result shows that the primers in the table 1 can be successfully amplified in a plectropomus leopardus sample, and the primers have clear bands, stable amplification and good repeatability.
Example 2 validation of SSR marker site polymorphisms
The method comprises the steps of collecting 173 total leopard gill sea weever from south China sea, Philippines, Malaysia, Indonesia and Australian sea areas, wherein the 173 total leopard gill sea weever specifically comprises Guangxi 28064of China, China (GX-WZ), Hainan east (HN-DF), Hainan Sanchi (HN-SY), Hainan Wenchang (HN-WC), Guangdong ZJ (GD-ZJ), Malaysia (GW-Ma), Indonesia (GW-In), Philippines (GW-Ph) and Australia (GW-Au). Wherein the feed is collected from 36 tails of Guangxi \/28064;, Haizian sea area Leopard gill sea weever. Extracting plectropomus leopardus sample DNA by adopting a tiangen marine animal tissue genome DNA extraction kit; using the extracted genomic DNA as a template, 173 samples were PCR-amplified using 6 pairs of primers shown in Table 1 in example 1 in a reaction system of 25. mu.L, including: not containing Mg 2+ 10 XPCR buffer 2.5. mu.L, 25mM MgCl 2 2.0. mu.L, 10mM dNTP 0.5. mu.L, high fidelity PCR enzyme 1U, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder was made up to 25. mu.L by sterile double distilled water.
The reaction procedure for amplification by PCR is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
After PCR amplification is finished, performing capillary electrophoresis typing, marking FAM fluorescent groups at the 5' ends of the 6 pairs of polymorphic SSR marker primers in the table 1, and performing capillary electrophoresis typing by using an ABI 3730XL genotype typing sequencer.
The capillary electrophoresis results of the 173 samples were subjected to peak plot interpretation and allele matrix construction using genemarker4.0, genetic distance calculation using GenALEx, and cluster tree construction using MEGA6.0 in the UPGMA method. The results are shown in FIG. 1, which indicates that a total of 39 samples including 36 samples collected from Guangxi of 2806460, the continent, were clustered in one clade, indicating that this one was 28064, the continent island protist.
The verification result of SSR marker site polymorphism is shown in the following table 2, which indicates that the 6 SSR sites adopted by the invention have the characteristics of high polymorphism but low polymorphism in Guangxi 28064Zhonghama primordial population. Wherein the allele of PLWZ-01 locus of 36 samples of Guangxi \, 28064and Odonopod striatellus primary population is 258/261/267; the allele at position PLWZ-02 is 195/279/282/288; the allele at PLWZ-03 locus is 238/251/256/279; the allele at position PLWZ-04 is 237/252/255/258; the allele at position PLWZ-05 is 257/272/291; the allele at PLWZ-06 locus is 178/199/215/219.
TABLE 2 Guangxi (Guangxi) \ 28064Theobroma island leopard gill and spine weever protospecies comparison with other species genetic diversity
Note: n: number of samples, Na: allelic factor, Ne: effective allele factor, I: shannon information index, Ho: observation of heterozygosity, He: desired heterozygosity, F: fixed index, Pop: population, Mean: average value, SE: standard error.
Example 3A method for identifying Guangxi \, 28064Theobroma island seal area Leopard gill sea weever original strains
Collecting 5 tails of plectropomus leopardus parent fish in a plectropomus leopardus culture farm, Zhanjiang, Guangdong, and slowly smelling the tails, numbering the tails, wherein the sampling part is a dorsal fin or a tail fin, the fish is not obviously damaged during sampling, and extracting leopard gill sea bass sample DNA by adopting a Tiangen marine animal tissue genome DNA extraction kit; using the extracted genomic DNA as a template, PCR amplification was performed on PLWZ-01-F/R, PLWZ-02-F/R, PLWZ-03-F/R, PLWZ-04-F/R, PLWZ-05-F/R and PLWZ-06-F/R using the primers for the different SSR marker sites PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06-F/R shown in example 1, respectively.
The reaction system for PCR amplification is 25. mu.L, and comprises: not containing Mg 2+ 10 XPCR buffer 2.5. mu.L, 25mM MgCl 2 2.0. mu.L, 10mM dNTP 0.5. mu.L, high fidelity PCR enzyme 1U, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder made up to 25. mu.L with sterile double distilled water.
The reaction procedure for the PCR amplification is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
After PCR amplification is finished, performing capillary electrophoresis typing, marking FAM fluorescent groups at the 5' ends of the 6 pairs of polymorphic SSR marker primers in the table 1, and performing capillary electrophoresis typing by using an ABI 3730XL genotype typing sequencer.
And performing peak image interpretation and allele matrix construction on the capillary electrophoresis result by using GeneMarker4.0, performing genetic distance calculation by using GenALEx, performing clustering tree construction by using MEGA6.0 and performing peak image interpretation on the obtained typing result by using a UPGMA method.
The typing and identification results are shown in the following table 3, and compared with the specific allele of the primordial population of Guangxi \28064;, Lebanhui gill and sea perch in example 2, the results show that the parent fish No.1 and 3 are Guangxi 28064;, the parent fish No. 2, 4 and 5 are not Guangxi \28064, and the parent fish in the island are. Therefore, the parent fish No.1 and 3 can be used as the parent and the female parent for Guangxi \28064andartificial propagation and releasing of fry in Zhongdao island.
TABLE 3 comparison of SSR locus characteristics of Choerospondias leopard gill sea weever and 28064Hainan original species in Xuwen farm
Example 4 identification of Ongxi (Guangxi) \ 28064;, Leibo island Leporis striolata and spinal weever original species
Capturing plectropomus leopardus 1 tail in the northern sea area of Guangxi, without knowing the species source, extracting plectropomus leopardus sample DNA, and performing PCR amplification on PLWZ-01-F/R, PLWZ-02-F/R, PLWZ-03-F/R, PLWZ-04-F/R, PLWZ-05-F/R and PLWZ-06-F/R by respectively using primers aiming at different SSR marker sites PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06-F/R shown in example 1 by taking the extracted genomic DNA as a template.
The reaction system for PCR amplification is 25. mu.L, and comprises: not containing Mg 2+ 10 XPCR buffer 2.5. mu.L, 25mM MgCl 2 2.0. mu.L, 10mM dNTP 0.5. mu.L, high fidelity PCR enzyme 1U, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder made up to 25. mu.L with sterile double distilled water.
The reaction procedure for the PCR amplification is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
After PCR amplification is finished, performing capillary electrophoresis typing, marking FAM fluorescent groups at the 5' ends of the 6 pairs of polymorphic SSR marking primers in the table 1, performing capillary electrophoresis typing by using an ABI 3730XL genotyping sequencer, merging and analyzing data of 5 tails of Zhongxi island samples, 5 tails of Guangdong Zhanjiang samples, 10 tails of Hainan Wenchang samples, 5 tails of Hainan east Oriental samples, 5 tails of Hainan Mitsui samples and 5 tails of Hainan Wenchang samples, performing genetic distance calculation by using GenALEx, performing cluster tree construction by using MEGA6.0 and an UPGMA method, and performing peak plot interpretation on obtained typing results.
The results are shown in FIG. 2, and the leopard gill-spiny bass sample was clustered with Guangxi 280648164Chuanma sample, and the sample was identified as Guangxi 28064Chuanma protospecies.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangdong ocean university
South China Sea Institute of Oceanography, Chinese Academy of Sciences
<120> SSR molecular marker primer group for identifying Guangxi \, 28064Theoblepis schrencki original strains and application thereof
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> DNA
<213> PLWZ-01(SIPOSequenceListing 1.0)
<400> 1
gctgaccggt gagtctcaac ttttgttctc aacttttatt tatttattta taataataat 60
aataataata ataataataa taataataat aataataata ataataataa taataataat 120
aataattgtg aataattaat ttacataaca ataataataa atattattat tattaattaa 180
tgtattaatg ttgtttggtg actacactaa agtttaaagt ctcagttcgg gttataaagg 240
ttgttttttt gtttttctca cagcggtcag gccgtggccg ccatcatctt cgtgctcggt 300
<210> 2
<211> 233
<212> DNA
<213> PLWZ-02(SIPOSequenceListing 1.0)
<400> 2
gtgctttgct cagcttgctt accccacaac ccgactccgg caaagtggat gaaatggatg 60
gctggatgga tggatggata aaaataataa taataataat aataataata ataataataa 120
taataataat aataataata ataataataa taataataag catacacagt tagaaagtac 180
acactcacac tcacacaccc aagtaacgat aagtgtgtcc ctgagcaaag gtc 233
<210> 3
<211> 258
<212> DNA
<213> PLWZ-03(SIPOSequenceListing 1.0)
<400> 3
gctgtgtttg agcaaagcca aaataaaatt tccactgagc tggacaataa agtctatcta 60
tctatctatc tatctatcta tctatctaat aacaataata ataataataa taataataat 120
aataataata ataataataa taataataat aataataata ataataatag taataataat 180
gatgataata ataataataa taataataat aataataata aattctattt attagcgcct 240
ttccggacac cagaggtc 258
<210> 4
<211> 288
<212> DNA
<213> PLWZ-04(SIPOSequenceListing 1.0)
<400> 4
ggtgaccttg agtgtcctga aaggtgccaa taaataacat tttataataa taataataat 60
aataataata ataataataa taataataat aataataatt attattatta ttattattgt 120
tgttgttgtt gttgttattg ttgttattgt taatattatt attattatta ttattattat 180
tattattatt attattatta ttattattat tattattata aatagggata caatgaaggt 240
atattggttt gcatgataat aattgacagt gtgtacaaca gctggagc 288
<210> 5
<211> 296
<212> DNA
<213> PLWZ-05(SIPOSequenceListing 1.0)
<400> 5
tggctttggc tcaaatgaca tttttctaca gctaatgaac tgatgcacct gcaatgctaa 60
taataataat aataataata ataataataa taataataat aataataata ataataataa 120
taataataat aataataata ataacaacaa taaagttgtt tctttttttt taaaaaagtc 180
cagttgctat ttatatttcg ttaaccacaa tgttgtgggg ttttttcttt taccacaaat 240
agaaagaaga ttcagaattt tacttccctc atttattccc tgtcagtttc cttcca 296
<210> 6
<211> 217
<212> DNA
<213> PLWZ-06(SIPOSequenceListing 1.0)
<400> 6
gccctacatt ctcatcgcag agttaataaa ttgcatatta ttattattat tattattatt 60
attattatta ttattattat tattattatt attattatta tctctgacat gaagactggg 120
cagctgaaac ccgatctctg tcaacccaaa catttccaag gcatttgttt ttctggatag 180
tatttgattt ccccacattc cctgaacgta gcagcag 217
<210> 7
<211> 20
<212> DNA
<213> PLWZ-01-F(SIPOSequenceListing 1.0)
<400> 7
gctgaccggt gagtctcaac 20
<210> 8
<211> 20
<212> DNA
<213> PLWZ-01-R(SIPOSequenceListing 1.0)
<400> 8
accgagcacg aagatgatgg 20
<210> 9
<211> 20
<212> DNA
<213> PLWZ-02-F(SIPOSequenceListing 1.0)
<400> 9
gtgctttgct cagcttgctt 20
<210> 10
<211> 20
<212> DNA
<213> PLWZ-02-R(SIPOSequenceListing 1.0)
<400> 10
gacctttgct cagggacaca 20
<210> 11
<211> 20
<212> DNA
<213> PLWZ-03-F(SIPOSequenceListing 1.0)
<400> 11
gctgtgtttg agcaaagcca 20
<210> 12
<211> 20
<212> DNA
<213> PLWZ-03-R(SIPOSequenceListing 1.0)
<400> 12
gacctctggt gtccggaaag 20
<210> 13
<211> 20
<212> DNA
<213> PLWZ-04-F(SIPOSequenceListing 1.0)
<400> 13
gcattgagca tcctggcaag 20
<210> 14
<211> 20
<212> DNA
<213> PLWZ-04-R(SIPOSequenceListing 1.0)
<400> 14
cagatcagga cagcactccg 20
<210> 15
<211> 20
<212> DNA
<213> PLWZ-05-F(SIPOSequenceListing 1.0)
<400> 15
tggctttggc tcaaatgaca 20
<210> 16
<211> 20
<212> DNA
<213> PLWZ-05-R(SIPOSequenceListing 1.0)
<400> 16
tggaaggaaa ctgacaggga 20
<210> 17
<211> 21
<212> DNA
<213> PLWZ-06-F(SIPOSequenceListing 1.0)
<400> 17
gccctacatt ctcatcgcag a 21
<210> 18
<211> 20
<212> DNA
<213> PLWZ-06-R(SIPOSequenceListing 1.0)
<400> 18
ctgctgctac gttcagggaa 20
Claims (10)
1. The SSR molecular marker for identifying Guangxi \/28064;, the original strain of the gill and sea bass of the island leopard is characterized in that the SSR molecular marker is PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06, and the nucleotide sequences of the SSR molecular marker are respectively shown in SEQ ID No. 1-6.
2. A primer set for amplifying the SSR molecular marker of claim 1, wherein said primer set is:
the sequences of the primers for amplifying the PLWZ-01 are respectively shown as SEQ ID NO. 7-8;
the primer sequences of the amplified PLWZ-02 are respectively shown as SEQ ID NO. 9-10;
the primer sequences of the amplified PLWZ-03 are respectively shown as SEQ ID NO. 11-12;
the primer sequences of the amplified PLWZ-04 are respectively shown as SEQ ID NO. 13-14;
the primer sequences of the amplified PLWZ-05 are respectively shown as SEQ ID NO. 15-16;
the primer sequences of the amplified PLWZ-06 are respectively shown in SEQ ID NO. 17-18.
3. The SSR molecular marker of claim 1 or the primer group of claim 2 is applied to identification of Guangxi \28064;, island leopard gill sea weever primary strains or preparation of a detection kit for identifying Guangxi \28064;, island leopard gill sea weever primary strains.
4. A detection kit for identifying Guangxi \28064;, island leopard gill and sea weever original strains is characterized by comprising the SSR molecular marker in claim 1 or the primer group in claim 2.
5. A method for identifying Guangxi \/28064;, Zhonghua island leopard gill sea weever original strains is characterized by comprising the following steps:
s1, collecting a sample of a plectropomus leopardus population to be identified, and extracting sample DNA;
s2, respectively carrying out PCR amplification by using the DNA extracted in the step S1 as a template and using the primer group in claim 2;
s3, performing capillary electrophoresis typing on the PCR product amplified in the step S2;
s4, performing peak image interpretation on the typing result obtained in the step S3 and constructing an allele matrix;
s5, comparing the allele of the plectropomus leopardus to be detected obtained in the step S4 with the allele of the known Guangxi \28064theoriginal strain of plectropomus leopardus, and calculating the genetic distance between samples;
s6, performing cluster analysis on the genetic distance obtained in the step S5, constructing a cluster tree, and judging whether the plectropomus leopardus to be detected is 2806416, namely the original variety of the continental island.
6. The method of claim 5, wherein the reaction system of the PCR amplification in step S2 is: not containing Mg 2+ 10 XPCR buffer 2.5. mu.L, 25mM MgCl 2 2.0. mu.L, 10mM dNTP 0.5. mu.L, high fidelity PCR enzyme 1U, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, DNA template 12.5ng, the remainder made up to 25. mu.L with sterile double distilled water.
7. The method of claim 5, wherein the reaction procedure of the PCR amplification in step S2 is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was carried out at 72 ℃ for 6 minutes.
8. The method of claim 5, wherein the step S3 is performed by labeling FAM fluorophore at 5' end of the primer set used in the step S2, and performing capillary electrophoresis typing using ABI 3730XL genotyping sequencer.
9. The method according to claim 5, wherein the allele of the original species Guangxi \28064Theobromafischeri is known in step S5 as:
the PLWZ-01 site is: 258. 261, 267;
the PLWZ-02 site is: 195. 279, 282, 288;
the PLWZ-03 site is: 238. 251, 256, 279;
the PLWZ-04 site is: 237. 252, 255, 258;
the PLWZ-05 site is: 257. 272, 291;
the PLWZ-06 sites are: 178. 199, 215, 219.
10. Use of the method according to any one of claims 5 to 9 for geographic population of gill leopard sea weever or for identifying the parent of the original species of the breeding and releasing seed.
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| CN116004848A (en) * | 2022-09-20 | 2023-04-25 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer and application thereof |
| CN116516028A (en) * | 2023-06-27 | 2023-08-01 | 中国海洋大学三亚海洋研究院 | SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof |
| CN117448465A (en) * | 2023-12-25 | 2024-01-26 | 中国水产科学研究院黄海水产研究所 | Germplasm identification primer and molecular identification method for red nine-thorn bass |
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| CN116004848A (en) * | 2022-09-20 | 2023-04-25 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer and application thereof |
| CN116004848B (en) * | 2022-09-20 | 2024-02-23 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer and application thereof |
| CN116516028A (en) * | 2023-06-27 | 2023-08-01 | 中国海洋大学三亚海洋研究院 | SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof |
| CN116516028B (en) * | 2023-06-27 | 2023-09-15 | 中国海洋大学三亚海洋研究院 | SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof |
| CN117448465A (en) * | 2023-12-25 | 2024-01-26 | 中国水产科学研究院黄海水产研究所 | Germplasm identification primer and molecular identification method for red nine-thorn bass |
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