CN117418023B - Double-band onyx and imogolix species-specific markers, specific primers, application and identification method and kit - Google Patents
Double-band onyx and imogolix species-specific markers, specific primers, application and identification method and kit Download PDFInfo
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Abstract
The invention relates to a qualitative detection method for species germplasm identification by a molecular biology technology, in particular to a species specific marker, a specific primer, an application and a rapid identification method and a kit in species identification of double-belonged onyx and imogolix. The nucleotide sequences of the specific markers of the mitochondrial genomes of double-belted gobies and imogolides are the bases shown in SEQ ID NO. 1 (Xtbi ID Nm: 1) and SEQ ID NO. 2 (Ytti ID Nm: 1). The method utilizes a pair of primers to amplify 497bp and 644bp DNA fragments which are different by 147bp in the individual double-belted goby and the individual imogolian goby, only 497bp single DNA fragments are amplified in the individual double-belted goby and only 644bp single DNA fragments are amplified in the individual imogolian goby, and the single DNA fragments can be resolved by agarose gel electrophoresis, thereby shortening the time for accurately identifying the species of the double-belted goby and the imogolian goby and improving the species identification and detection efficiency.
Description
Technical Field
The invention relates to a qualitative detection method for species germplasm identification by a molecular biology technology, in particular to a species specific marker, a specific primer, an application and a rapid identification method and a kit in species identification of double-belonged onyx and imogolix.
Background
The double-belted goby and the imogolian goby are belonging to the order Perciformes (Perciformes), the family Gekkonidae (Gobiidae), the genus onyxTridentige) Is warm temperature near-shore benthonic fish, and mainly inhabits the tropical, subtropical and temperate fresh water or brackish water environments of the western Pacific ocean. Both have fused ventral fin features, and the formed suction cups make them very easy to adapt to offshore benthonic lives; is a dominant species in the intertidal zone and mainly ingests benthic small fish, juvenile shrimp, pelleted animals and other benthic invertebrates. The double-belted onyx and the imogolin are simultaneously bait organisms of rare protective species such as Acipenser sinensis and the like, are positioned at the middle and downstream of a food chain, are one of key species of estuary ecosystems, play an important role in mass transfer and energy flow, and are very important in ecological status.
The taxonomic study shows that the double-belted onyx and the onyx are of the same genus and closely related species, the external morphology is very similar, the morphological characteristic difference is very weak, and the morphological difference is mainly concentrated on whether the uppermost fin of the pectoral fin is free, whether the hip fin has red longitudinal bands in life, whether the sensory tube holes at the top of the head are thick and the like. Because of the small size of the double-belted onyx and the amox, the rapid on-site species identification and the fixed sample classification identification cannot be performed based on the weak morphological characteristics. Studies have also found that the range of movement of double and imogolins in habitat is limited, and that perching and foraging activities tend to occur in fixed areas, with both being in mixed mode. This severely restricts the accurate assessment of the fish resources of the double belted onyx and the imogolix and the progress of biodiversity protection. In addition, the research also finds that two kinds of gobies begin to mature in 1 year, and the spawning period is usually 4 months to 5 months, and the problems that whether the gobies are in similar spawning habitats, have similar adhesive spawn adhesion matrixes and the like are unknown when the gobies are spawned due to the fact that the gobies with double belongings and the line gobies are in a mixed life style and form adhesive spawns. This also restricts the awareness of people to the sustainable replenishment of the amount of dominant fish species resources in such intertidal zones, resulting in a lack of basis for the formulation of protection strategies for their species biodiversity.
At present, no report about species identification of double-belted onyx and line onyx is seen, and the ecological position variation of double-belted onyx and line onyx is considered as baits of numerous economic fishes and endangered species, so that the important supporting effect on the stabilization of an ecological system of fishery is realized, and the molecular technology for establishing high-efficiency and rapid identification between double-belted onyx and line onyx by mining the specific molecular markers of double-belted onyx and line onyx is a technical problem to be solved in the aspects of accurate evaluation and biodiversity protection of the fishing industry resources of double-belted onyx and line onyx.
Disclosure of Invention
The invention aims to overcome the defects of the existing species identification and detection technology of double-belted onyx and imogolin, and provides a species specific marker, a specific primer, an application and a rapid identification method and a kit in species identification of double-belted onyx and imogolin.
In order to achieve the above purpose, the invention adopts the technical scheme that:
the nucleotide sequence of the intergerm line specific markers of the double-band onyx and the line onyx is a base shown as SEQ ID NO:1 (Xtbi ID Nm: 1) and/or SEQ ID NO:2 (Ytti ID Nm: 1).
The base shown in SEQ ID NO. 1 of the specific markers of the double-belted onyx and the imogolian is a double-belted onyx and imogolian onyx mitochondrial genome homologous fragment, and the specific markers of the double-belted onyx and the imogolian onyx mitochondrial genome share a DNA characteristic marker;
the base shown in the specific markers SEQ ID NO. 2 of the double-band onyx sinensis and the onyx sinensis is an insert sequence segment between the 100 th site, the 110 th site, the 135 th site and the 143 th site of the base sequence shown in the specific marker SEQ ID NO. 1, namely the DNA marker special for the onyx sinensis mitochondrial genome.
Use of a specific marker between the germ cells of the germ cell line of the germline of the double belonged and the imogolian goldens, for the identification of the species of double belonged and imogolian goldens.
A species identification specific primer for detecting the specific markers between the germ cells of the perigeda and amogolian goldens of the claim:
MTrb_F:1 : 5’- CTAGCTCCCAAAGCTAGCATTC-3’；
MTrb_R:2 : 5’- AACTCCACCAATAACTTATGCC-3’。
a method for identifying species of double-belted onyx and imogolin, goby,
1) And (3) PCR amplification: extracting genome DNA from the double-belted onyx and the tridentiger; performing PCR amplification by using the obtained genome DNA as a template and adopting the species-specific primer pair of the double-band onyx sinensis and the imogolin scales, and comparing the obtained PCR amplification product with the species-specific markers of the double-band onyx sinensis and the imogolin scales;
2) And (3) judging results: amplifying 497bp single DNA fragment (base shown in SEQ ID NO: 1) from the genome DNA of the double-band goby and the imogolian goby to be detected, namely judging that the imogolian goby to be detected is double-band goby individual Kazakhan gobyTridentiger bifasciatus）；
Or amplifying only 644bp single DNA fragment (base shown as SEQ ID NO: 2) in the genomic DNA of the measured goby, namely judging that the measured goby is individual gobyTridentiger trigonocephalus）。
A kit for identifying species of both belonged onyx and amox comprising said species identification specific primer.
The invention has the advantages that:
the invention obtains the sequence of the mitochondrial control region of the double-band onyx sinensis and the sequence of the line onyx sinensis through the whole genome sequence and the structure of the mitochondrial control region of the double-band onyx sinensis and the line onyx sinensis, has a significant DNA fragment insertion region, discovers the DNA markers shared by the double-band onyx sinensis and the line onyx sinensis and unique to the line onyx sinensis, and applies the fragments to species identification of the double-band onyx sinensis and the line onyx sinensis;
the method can rapidly, accurately and efficiently distinguish the species of the double-band onyx and the tridentate, and because the mitochondrial genome is a haploid genome, the method only amplifies one specific target band (497 bp) in the double-band onyx, only amplifies one specific target band (644 bp) in the tridentate, the respective specific bands of the double-band onyx (497 bp) and the tridentate (644 bp) are extremely easy to distinguish from the primer dimer, and meanwhile, the difference between the specific bands of the two species is 147bp, the agarose gel electrophoresis can be adopted to rapidly and accurately distinguish the band types of the double-band onyx and the tridentate, so that the method is extremely convenient to be applied to rapid species identification operation in the field of fishery resource investigation experiments and in indoor laboratories. The method for efficiently, rapidly and accurately identifying the species of the double-belt onyx and the imogolin, disclosed by the invention, has important significance and application value for enhancing the accurate evaluation and biodiversity protection of the fishery resources of the double-belt onyx and the imogolin, and improving the cognition of sustainable supplement of the two resources.
Drawings
Fig. 1: the nucleotide sequence comparison diagram of the mtDNA control region Tb of the double-belted onyx goby and the mtDNA control region Tt of the onyx goby is obtained, and the positions of the primers at the two ends are indicated by black thick underlines; i (L): represents Tt and Tb sequence identity; … … …: representing the base inconsistent sequences of the two; - - - - - - - -: representing an indel sequence; the black underline represents the region in which the deleted sequence is located.
Fig. 2: the nucleotide sequence homology of the Tb of the double-band onyx gobius mitochondrial control region and the Tt of the onyx gobius mitochondrial control region is inserted into a differential DNA fragment pattern diagram, and the primer positions are represented by F and R; the same blank color region represents the homologous region, and the black region represents the insertion site information.
Fig. 3: the invention discloses a PCR product 1.5% agarose gel electrophoresis result diagram of individual double-belted onyx and imogolix, M: DL 2000 DNA Make; the individual showing one band (497 bp) is a double-band onyx species, the uppermost fin of the pectoral fin is not dissociated, and the hip fin is red in life; the individual showing one band (644 bp) is the imogolian goby species, the uppermost fin of the pectoral fin is free, and the hip fin has two red longitudinal bands in life.
Detailed Description
The following description of the embodiments of the present invention is further provided in connection with the accompanying examples, and it should be noted that the embodiments described herein are for the purpose of illustration and explanation only, and are not limiting of the invention.
The present invention determines species classification based on the unique DNA markers of the imogolite mtDNA control region and the DNA signature common to the double-belted onyx mtDNA control region:
firstly, acquiring the whole genome sequences of the double-belted onyx sinensis and the ooonyx sinensis mitochondria from a NCBI (National Center of Biotechnology Information) database by adopting an information acquisition method; obtaining the insertion and deletion characteristics of large fragments of double-band onyx gobius and imogolicus in the homologous region of a mitochondrial genome control region through a genome bioinformatics analysis method, wherein the DNA fragment length of the double-band onyx gobius mtDNA control region is 497bp, the sequence of the double-band onyx gobius mtDNA control region is Tb, the sequence of the double-band onyx gobius mtDNA control region is Xtbi ID Nm 1, the DNA fragment length of the imogolicus mtDNA control region is 644bp, and the double-band onyx gobius mtDNA control region homologous sequence and insertion sequence are contained, and the sequence of the double-band onyx gobius mtDNA control region is named Tt, and the sequence of the double-band onyx gobius mtDNA control region is Ytti ID Nm 1; four large fragment insertion sequences are found among Tb 100 th site, tb 110 th site, tb 135 th site and Tb 143 th site which are homologous to Tb, wherein the size of each insertion sequence is 51bp, 24bp, 18bp and 54bp, the DNA sequence of an mtDNA control region (Tt) of the amomum morifolium is more than that of a homologous DNA region (Tb) of the mtDNA control region of the amomum morifolium, the fragment is a DNA marker specific to the mtDNA control region of the amomum morifolium, the Tb is a DNA sequence shared between the amomum morifolium and the amomum morifolium, and the fragment is a DNA characteristic marker of the amomum morifolium.
The invention relates to a method for rapidly identifying the species of double-belted onyx and imogolix; the specific detection method is to determine whether the to-be-detected onyx gobi has an Xtbi ID Nm 1 nucleotide fragment (497 bp) and a Ytti ID Nm 1 nucleotide fragment (644 bp); carrying out rapid identification through PCR primers; the precise identification of the species of the double-belonyx and the imogolix is realized, and the sequences of the upstream primer and the downstream primer are respectively as follows:
MTrb_F:1 : 5’- CTAGCTCCCAAAGCTAGCATTC-3’；(Xtbtt ID Nm:2)
MTrb_R:2 : 5’- AACTCCACCAATAACTTATGCC-3’ (Xtbtt ID Nm:3)
wherein 497bp single DNA fragment is amplified in the individual double-belted onyx gobius, and 497bp fragment is a specific marker fragment of double-belted onyx gobius species; and a single DNA fragment of 644bp is amplified in the individual imogolian goby, and the 644bp fragment is a labeling fragment specific to the imogolian goby species.
Example 1 screening and validation of DNA markers specific for double-belted onyx and imogolix species
Discovery of the mitochondrial genome of double and imogolides, and containing large fragment insertion into the target DNA sequence:
the whole genome sequence of the double-belted goby and the imogolian goby used in the invention is obtained from NCBI (National Center of Biotechnology Information) international database, the NCBI registration number of the double-belted goby is NC015992, and the NCBI registration number of the imogolian goby is NC029738. Bioinformatics analysis of the mitochondrial genome sequences of double-belted onyx and imogolian onyx using genomic sequence homology alignment is visible (see fig. 1), the mitochondrial genome control regions of double-belted onyx and imogolian onyx are homologous and have large fragment DNA fragments inserted, wherein the DNA fragment length on the mtDNA control region of double-belted onyx is 497bp, designated Tb, and the sequence of SEQ ID NO:1 is Xtbi ID Nm:1:
SEQ ID NO:1（Xtbi ID Nm:1）
CTAGCTCCCAAAGCTAGCATTCTAAATTTAACTATTCTTTGTTATAAACATATATGTAATATCACCATATATATATATCGAACATATATATTAATGTTTACTAACACATATATGTATAATAACCATTCAGTTACTTCGACCATTCATTCATCAACATTCATCCAAGAATTACAAATTGCATCTCTCTAGATTTAACATAACTGCACACTAACAAAATATATTAATGCTCAAAGACAAGAATGAACACTCAATATATATATATATAAAACAATAAGCCATCACCACAAAATTTAAGTACACATTTTTGGGCCACATCACCCGTAAACACGACCATCTATTCGTATTTAATAGAAATTACCCAATGATAGTGATGTACCCTAATTGTGAGGGTCGCTCACAGTGAATTTTTACAGACATTTGGCTCTACATCTCAAGGCCATTACCTGCAAGTCTTCCCCACACTTTCACTGGCCCTGGCATAAGTTATTGGTGGAGTT。
the length of the DNA fragment on the mtDNA control region of the imogolides is 644bp, the fragment comprises a homologous sequence and an insertion sequence of the mtDNA control region of the double-banded imogolides, the fragment is named as Tt, and the sequence of SEQ ID NO:2 is Ytti ID Nm:1:
SEQ ID NO:2（Ytti ID Nm:1）：
CTAGCTCCCAAAGCTAGCATTCTAAATTTAACTATTCTTTGTACATATATGTAATTAAACCATTAATATATCTAGACCATATATATTAATGTTTATAAGATATATTATGTATAATAACCATCTATTTCTTTAAACCATTAATATATCTAGACCATATATATTAATGTTTATAAGATATATTATGTATAATAACCATCTATTTCTTTAAACCATTAATATATCTAGACCATATATATTAATGTTTATAAGATATATTATGTATAATAACCATTTATCTCTTTTAACCACTCAGGAATTAACATTCATCCAAGAAAAACAAATTACATCATCTGTAAATGAACACAATTGCACACTAAAAAGAAATATTAATGCTTTAAGACAATAATCTTAAAATTCAAAATAATATGATATAATACAATAACCCATCACCATTAAACTTAAACTTACATTTCTCAGCCACATCAACCGTAAACACGACCATCTATTCGCATATAATAGAAATTACCCAATGATAGTGATGTACCCTACTCGTGAGGGTCGCTCATAGTGAATTTTTACAGACATTTGGCTCTACATCTCAAGGCCAATACCTGCAAGTCTTCCCCACACTTTCACTGGCCCTGGCATAAGTTATTGGTGGAGTT。
alignment of mitochondrial genome DNA sequences of double-banded gobies and imogolides shows that four large fragments of insertion sequences are found among 100 th site, 110 th site, 135 th site and 143 th site of double-banded gobies Tb, the sizes are respectively 51bp, 24bp, 18bp and 54bp, wherein the DNA sequence of an mtDNA control region (Tt) of the imogolides is more inserted into a 147bp DNA sequence than that of a homologous DNA region (Tb) of the mtDNA control region of the double-banded gobies, and the fragments are DNA markers special for the mtDNA control region of the imogolides, and the existence or absence of the fragments can be used for identifying the species of the imogolides; tb is a DNA sequence shared between the species of the double-belted onyx and the species of the imogolix, and is a DNA signature of the double-belted onyx species, the presence or absence of which can be used to identify the double-belted onyx species, as shown in FIG. 2.
Sequence verification of DNA markers specific for the species of double and imogolides: two primers were designed based on the nucleotide sequence characteristics of the Ytti ID Nm 1 of the mtDNA control region of the imogolides and the Xtbi ID Nm 1 of the mtDNA homologous control region of the double-belted onyx. The PCR amplification was performed using two primers, MTrb_F1 and MTrb_R:2, simultaneously with the selection of the double-belonyx and amoonyx of known species, under the following reaction conditions and procedures: PCR reaction system is 20 mu L, including 5.0 mu L10Buffer;4.0 [ mu ] L dNTP (2.5 mmol/L); 1.0 [ mu ] L rTaq enzyme (5U/[ mu ] L)The method comprises the steps of carrying out a first treatment on the surface of the 1.0 [ mu ] L MTrb_F:1;1.0 2 is [ mu ] L MTrb_R; 1.0 [ mu ] L DNA template, 7.0 [ mu ] L ddH 2 O; and (5) uniformly mixing and centrifuging. PCR amplification reaction procedure: 96 ℃ for 4mins;96℃60s,58℃30s,72℃1min,30 cycles; storing at 72deg.C for 10min and 4deg.C. The individual differences of the double-belted onyx and the line onyx can be distinguished by agarose gel electrophoresis of the PCR product with 1.5 percent. The differential fragment products of double-belted onyx and imogolix are recovered by tapping and transformed into competent cells by PMD18-T vector, and positive clones are picked and sent to Qingdao Paeno Biotechnology Co. Sequencing results confirm that the mtDNA control regions of the double and the line onyx are homologous and contain large fragments inserted into the target DNA sequence fragments.
Example 2 establishment and use of the identification technique of the species of the double-belted onyx sinensis and the imogolix sinensis
Interspecific species identification was performed on 12 tails of double-belated onyx gobies and 12 tails of line onyx gobies from the Qingdao, spring bay:
high quality DNA extraction: extracting double-band onyx gobius and tridentatus fin DNA by using a marine animal DNA extraction kit, identifying the integrity of genome DNA by 1.0% agarose gel electrophoresis, measuring the OD value of DNA supernatant by using an ultraviolet spectrophotometer, adjusting the DNA concentration to 70 ng/mu L, and freezing at-20 ℃ for later use.
PCR reaction system and PCR amplification identification: the species assignment of the double belongings of the imogolides was detected by PCR method using the primers MTrb_F1 and MTrb_R2 of DNA fragments specific to the double belongings of the imogolides and the imogolides. 20 mu L of a PCR reaction system: 10Buffer 5.0 [ mu ] L, dNTP 4.0.0 [ mu ] L, rTaq, 1.0 [ mu ] L of enzyme (5U/[ mu ] L), and 1.0 [ mu ] L, ddH of each of the upstream and downstream primers (MTrb_F: 1 and MTrb_R: 2) and template 1.0 [ mu ] L, DNA 2 O7.0 [ mu ] L. PCR amplification reaction procedure: 96 ℃ for 4mins;96℃60s,58℃30s,72℃1min,30 cycles; storing at 72deg.C for 10min and 4deg.C. 10 +.>Loading Buffer 2.0 μL using 1.5% agarose gel electrophoresisGel imaging clearly resolved the belted onyx and amox species assignment at a constant voltage of 110V for 30 minutes during electrophoresis (see fig. 3).
As shown in FIG. 3, only one specific target band (497 bp) was amplified in the individual double-belted goby, only one specific target band (644 bp) was also amplified in the individual line goby, and the species-specific bands of double-belted goby (497 bp) and line goby (644 bp) were both extremely distinguishable from the primer dimer, so that the species-specific molecular markers developed by the present invention were applicable to rapid identification of the species attribution of wild double-belted goby and line goby, with universality.
SEQ ID NO:3
22
DNA
Artificial sequence (Artificial Sequence)
CTAGCTCCCAAAGCTAGCATTC
SEQ ID NO: 4
22
DNA
Artificial sequence (Artificial Sequence)
AACTCCACCAATAACTTATGCC 。
Claims (4)
1. An intergerm specific marker of a perigeum of a double-belted onyx and a line onyx, characterized in that: the nucleotide sequences of the specific markers of the double-band onyx and the imogolix are shown as SEQ ID NO. 1 and SEQ ID NO. 2.
2. The specific marker between species of double belted gobies and line gobies according to claim 1, characterized in that: the nucleotide shown in SEQ ID NO. 1 is a specific marker fragment of a double-belt onyx gobius species, the nucleotide shown in SEQ ID NO. 2 is a specific marker DNA marker of the specific DNA markers of the onyx gobius mitochondrial genome, wherein the insertion sequence fragments are arranged among the 100 th site, the 110 th site, the 135 th site and the 143 th site of the nucleotide sequence shown in SEQ ID NO. 1.
3. Use of a specific marker between germ cells of the germ cell line of the allied double and line of the imogolides of claim 1, characterized in that: the use of said specific markers for the identification of the species of the double and line onyx.
4. A method for identifying species of double-belted onyx and imogolin, which is characterized in that,
1) And (3) PCR amplification: extracting genome DNA from the double-belted onyx and the tridentiger; performing PCR amplification by using the obtained genomic DNA as a template and using a pair of species-specific primers of the double-belated goby and the imogolian goby, and comparing the obtained PCR amplification product with the species-specific markers of the double-belated goby and the imogolian goby according to claim 1; the species-specific primer pairs are:
MTrb_F:1 : 5’- CTAGCTCCCAAAGCTAGCATTC-3’；
MTrb_R:2 : 5’- AACTCCACCAATAACTTATGCC-3’
2) And (3) judging results: amplifying single DNA fragment of 497bpSEQ ID NO:1 nucleotide from the genome DNA of the double-band goby and the imogolian goby to be detected, namely judging that the double-band goby to be detected is the individual imogolian gobyTridentiger bifasciatus）；
Or, only amplifying single DNA fragment of the nucleotide shown in 640 bpSEQ ID NO:2 in the genomic DNA of the onyx sinensis Wiegmann to be detected, namely judging that the onyx sinensis Wiegmann to be detected is the individual onyx sinensis WiegmannTridentiger trigonocephalus）。
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| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
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| WO2019135230A1 (en) * | 2018-01-04 | 2019-07-11 | Ramot At Tel-Aviv University Ltd. | Transgenic eukaryotic organisms and methods for gender selection |
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|---|---|---|---|---|
| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
| CN103074335A (en) * | 2012-11-22 | 2013-05-01 | 浙江海洋学院 | Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method |
| CN103074336A (en) * | 2012-11-22 | 2013-05-01 | 浙江海洋学院 | Goby mitochondrion ND1 gene complete sequence amplimer, design and amplification method |
| WO2019135230A1 (en) * | 2018-01-04 | 2019-07-11 | Ramot At Tel-Aviv University Ltd. | Transgenic eukaryotic organisms and methods for gender selection |
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| "Characteristic and phylogenetic analyses of mitochondrial genome for Rhinogobius filamentosus (Teleostei: Gobiidae: Gobionellinae), an endemic species in China";Xiao Jiang Chen et al.;《MITOCHONDRIAL DNA PART B》;20221231;第7卷(第9期);第1752-1755页 * |
| "浙江沿海刺虾虎鱼属（Acanthogobius）和缟虾虎鱼属（Tridentiger）分子分类研究";顾翠平;《硕士电子期刊》;20140715(第7期);第1-54页 * |
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