CN109628606B - SNP molecular marker capable of tracing source by using double umbilical snails and application thereof - Google Patents
SNP molecular marker capable of tracing source by using double umbilical snails and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker for tracing a double umbilical snail and application thereof. The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1 and is 857bp in total, the 305 th base position of the SNP molecular marker has a base mutation 305G → 305A, and the SNP molecular marker is specifically identified by BstUI restriction endonuclease. By detecting and identifying the distribution condition of the alleles of the SNP molecular marker in 90 double-umbilical snail test populations from 3 different places, the sample alleles of the SNP molecular marker at different places are found to have close frequency, rich polymorphism, small distribution difference of the allele frequencies and a heterozygosity of more than 0.3, and the SNP molecular marker is preliminarily judged to be used as the DNA tracing of the double-umbilical snails.
Description
Technical Field
The invention belongs to the technical field of molecular detection. More particularly, relates to a double umbilical snail SNP molecular marker and application thereof in tracing.
Background
The double-navel snail belongs to the phylum mollusca, spirochaeta and family of flat snail, and 18 kinds of the known double-navel snails such as smooth double-navel snail, ligusticum double-navel snail, shallow double-navel snail, purchaser double-navel snail, Asian double-navel snail and the like can be used as intermediate hosts of schistosoma mansoni to transmit schistosomiasis, thereby causing serious disease burden to the world, particularly to regions of Africa, south America, Asia and the like and seriously hindering the development of society and economy. Wherein the Chinese ligusticum double navel snails are mainly distributed in fresh water areas of northeast regions of the Saururus of the Caribbean sea and the coastal sea of Brazil, have higher susceptibility to the schistosoma mansoni, and are important vectors for spreading local schistosomiasis mansoni. In the same species, the Chinese ligusticum double-navel snails have strong long-distance diffusion capacity and colonization capacity due to strong drying tolerance and high reproductive capacity. The original double navel-free snail in China is bred, and then the snail is found in hong Kong, Shenzhen and Dongguan; and surveys show that the Shenzhen river water body double navel snails present a wide high-density distribution state, and as foreign species, have formed the dominant population of the freshwater snails in the local, so mansoni schistosomiasis has the risk of spreading and epidemic in the local, and has aroused high importance of the disease prevention and control department in China. In order to achieve the purposes of controlling vectors, monitoring epidemic situations and reducing occurrence and spread of schistosomiasis mansoni, one of the key problems to be solved is to know the genetic variation condition and the population genetic structure of the binavel snails after invading China and find out geographical specific molecular markers of the binavel snails, thereby providing theoretical basis for scientific prevention and control of zoonosis.
In recent years, the emergence of molecular marker technologies based on DNA polymorphism provides effective detection means for traceability research, mainly including AFLP (amplified fragment length polymorphism) markers, RFLP (restriction endonuclease fragment length polymorphism), RAPD (random amplified polymorphic DNA) markers, SSR (microsatellite) markers, SNP (single nucleotide polymorphism) markers, and the like.
The AFLP marker is a first-generation molecular marker method, has rich polymorphism, strong genetic stability and high reliability, but has higher requirements on the quality and purity of a DNA template and the level of operators, higher difficulty in statistics and relatively higher cost; SSR markers are second-generation molecular marker methods, have the advantages of high polymorphism, low mutation rate and less required template amount, but are complex in marked banding pattern, complicated in process and not suitable for automatic typing; and SNP is used as a third-generation molecular marker, refers to the variation of a single nucleotide on the same site of a genome, has the advantages of wide distribution, high density, strong representativeness, good genetic stability and the like, has low requirements on the quality of a template, generally represents a bi-allele, presents the characteristics of being not the same, is easy to determine, is suitable for high-throughput automatic detection and typing, and is the most effective method for tracing the molecular marker at present.
However, unlike general SNP molecular markers, SNP molecular markers for traceability must have at least the following characteristics: (1) the variation degree is high, and the allele frequencies are close; (2) the distribution frequency difference of the allelic genes among the populations is small; (3) the heterozygosity is greater than 0.3. Regarding the SNP molecular marker of tracing, patent CN201710607015.8 discloses a SNP marker combination for tracing identification of beef cattle individuals and meat products and application thereof, and patents CN201510785854.X, CN201510785895.9 and CN201510783449.4 all disclose a SNP molecular marker for tracing on No. 6 pig chromosome and application thereof; patent CN201210529628.1 discloses a group of geographic specificity plasmodium vivax molecular markers and application thereof in insect strain traceability; at present, no related report of SNP sites for tracing the navel snail exists.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art, and to provide a double umbilical snail SNP molecular marker which can be used for identifying double umbilical snails from different sites.
The first purpose of the invention is to provide a SNP molecular marker which can be used for tracing the double umbilical cord snail.
The second purpose of the invention is to provide the application of the SNP molecular marker in the tracing of the double umbilical cord snail.
The above object of the present invention is achieved by the following technical solutions:
a double umbilical snail SNP molecular marker has a nucleotide sequence shown as SEQ ID NO.1, wherein the nucleotide sequence is 857bp in total, a base substitution is carried out at a 305 th base position, the base substitution is 305G or 305A, and the position is indicated by n in the SEQ ID NO.1 sequence.
Specifically, the SNP molecular marker is a fragment on a Lonicera bicolor LOC106060343 gene.
Further, the SNP molecular marker is specifically recognized by BstUI restriction enzyme. The invention discovers that the BstUI-RFLP polymorphism is caused by the base mutation (305G → 305A) existing at the 305 th base of the SNP molecular marker described in SEQ ID NO. 1. The locus is controlled by two alleles, wherein the A allele only has a 857bp fragment, the G allele has a 305bp fragment and a 552bp fragment, and the two alleles can form two genotypes of GG and GA.
The SNP molecular marker is obtained by sequencing a DNA pool (pool), the distribution condition of alleles of the SNP molecular marker in a test population (total 90 individuals) comprising 3 places is analyzed, the allele frequencies of the SNP molecular marker in different populations are found to be close, the polymorphism is rich, the allele frequency distribution difference is small, the heterozygosity is more than 0.3, and the SNP molecular marker is preliminarily judged to be used as the DNA tracing source of the dicentra.
Therefore, the invention also requests to protect the application of the SNP molecular marker in the DNA tracing of the double umbilical snail or in the preparation of the DNA tracing product of the double umbilical snail.
A method for detecting and identifying the SNP molecular markers in the double navel snails specifically comprises the following steps:
(1) extracting genome DNA of the double umbilical cord snail;
(2) and (2) using the DNA in the step (1) as a template, carrying out PCR amplification on a DNA fragment (SEQ ID NO.1) of the LOC106060343 gene of the double navel snail, sequencing and analyzing the SNP locus genotyping condition.
Preferably, the PCR amplification primers in the step (2) comprise an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are sequentially shown in SEQ ID NO. 2-3.
Preferably, in the step (2), after obtaining the DNA fragment of the LOC106060343 gene, the genotyping condition of the SNP site in the hemifusus dyumbilicus can be rapidly detected and identified by a PCR-BstUI-RFLP method.
The invention also requests to protect the application of the primer pair in the DNA tracing of the double-navel snail or in the preparation of the DNA tracing test product of the double-navel snail.
A product for tracing the DNA of the double umbilical snails comprises a primer pair for detecting and identifying the SNP molecular marker.
Preferably, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are sequentially shown in SEQ ID No. 2-3.
Preferably, the kit also comprises a restriction enzyme BstUI, and the genotype of the dicentra sinensis can be quickly detected and identified through enzyme digestion.
Preferably, the product is a bisumbilical snail DNA traceability kit.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.1 and is 857bp in total, the 305 th base position of which has a base mutation 305G → 305A and is specifically recognized by BstUI restriction enzyme. In 90 navel snail test populations from 3 different places, the sample allele frequencies of the SNP molecular marker at the different places are found to be close, the polymorphism is rich, the allele frequency distribution difference is small, the heterozygosity is more than 0.3, and the SNP molecular marker is preliminarily judged to be used as the DNA tracing of the navel snails.
Drawings
FIG. 1 is a BstUI-RFLP enzyme map of the LOC106060343 gene fragment of the hemifusus umbiliciformis. In FIG. 1, the lanes include DNA marker, GG genotype individual and GA genotype individual in the order from left to right.
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.
Example 1 search of SNP molecular markers
(1) Construction of DNA pool (pool)
Respectively and randomly collecting 2 Shenzhen, Dongguan and the double navel snails in Hongkong regions, extracting the genome DNA, equivalently absorbing the DNA of 6 individuals, putting the DNA into the same centrifugal tube, and uniformly mixing for later use.
(2) The primer design takes the DNA sequence (Genbank: NW _013232000.1) of the LOC106060343 gene of the hemifusus umbilicalis as a template, and the primer design separates the DNA fragment of the LOC106060343 gene of the hemifusus umbilicalis as follows:
an upstream primer: 5'-GCTCTGTCTCTTCTTCAGGC-3' (shown as SEQ ID NO. 2)
A downstream primer: 5'-GTGTTGATAGGGGTGAAAACGA-3' (shown as SEQ ID NO. 3)
The total volume of the PCR reaction was 50. mu.L, with approximately 100ng of the duplex umbilical snail genomic DNA, 1.1 XPCR mix 45. mu.L, 2. mu.L of each of the 10. mu. mol/L upstream and downstream primers. The PCR amplification procedure was: pre-denaturation at 98 ℃ for 2min, (denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 15s) were cycled 35 times, and finally extension at 72 ℃ for 3 min. The PCR reaction product is detected by 2% agarose gel electrophoresis, and the band is required to be clear and has no impurity band.
(3) Clone sequencing analysis
Purifying the obtained DNA fragment of the LocCloupia utilis LOC106060343 gene by using a PCR product purification kit, then connecting the DNA fragment with a pMD18-T vector, recovering after transformation, uniformly coating a recovering bacterium solution on an LB culture medium, and inversely placing a flat plate in an incubator at 37 ℃ for overnight culture. The bacterial strain identified by colony PCR is cultured in LB culture medium at 37 ℃ overnight, and a plurality of clones are randomly picked for sequencing.
Sequencing analysis shows that: the DNA sequence amplified by the primer is 857bp in length, the sequence is shown as SEQ ID NO.1, and a base mutation exists at the 305 th base (305G → 305A). The base mutation at the 305 th base was found to cause a BstUI-RFLP (Restriction Fragment Length Polymorphism, BstUI-RFLP site) Polymorphism by analysis of molecular biology software.
(4) Establishment of PCR-BstUI-RFLP detection method and detection of mutation sites
The total volume of the enzyme digestion reaction is 50 mu L, wherein 10 multiplied by 5 mu L of buffer, 15-20 mu L of PCR product, 1 mu L (10U) of restriction enzyme BstUI, and H is used2And supplementing 50 mu L of O, incubating for 15min at 60 ℃, and detecting the enzyme digestion result by using 2% agarose gel electrophoresis.
The results are shown in FIG. 1: in the sequence of SEQ ID NO.1, the A allele has only one 857bp fragment, the G allele has two fragments of 305bp and 552bp, and the two alleles can form two genotypes of GG and GA.
Example 2 allele distribution
(1) Design of test populations
Test groups: collecting 90 double navel snails in total from Shenzhen (40), Dongguan (24) and hong Kong (26), and extracting genome DNA. The purpose of the test population is to detect the distribution of SNP molecular markers in different populations.
(2) Genotype detection
The use of an upstream primer: 5'-GCTCTGTCTCTTCTTCAGGC-3' (shown as SEQ ID NO. 2)
A downstream primer: 5'-GTGTTGATAGGGGTGAAAACGA-3' (shown in SEQ ID NO. 3) were amplified (same amplification conditions as in example 1) and all genotypes of the test population were examined by the same PCR-BstUI-RFLP method.
(3) Statistical analysis
The genotypes of all individuals of the test population were recorded and the allele frequencies and heterozygosity were calculated, with the results shown in table 1 below.
TABLE 1
Analyzing the distribution of the SNP locus alleles to find that the allele frequencies of the SNP molecular markers in various populations are close and the polymorphisms are rich; the distribution difference of allele frequencies G and A among populations is small, and the heterozygosity H is larger than 0.3, so that the SNP molecular marker is preliminarily considered to be used for DNA tracing detection of the double umbilical snails.
Sequence listing
<110> Zhongshan university
<120> SNP molecular marker capable of tracing source by using double umbilical snails and application thereof
<130> YG18108945AA042
<141> 2018-12-14
<160> 3
<170> SIPOSequenceListing 1.0
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<213> Bauscula (Biomphalalia)
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gctctgtctc ttcttcaggc atgtcctcag gtattgcaaa tggcaccggc agtgttaaat 60
catcagaaga caatagattc aaaagatcat caagtgtccc catatctttc caaaatgctg 120
caatgacagt gtccagttac aatgagaaga aaagcaatgc cttggtcaca agtgaagagg 180
attttcagaa ttctctcaaa gacattgacg ctgaaatagc caaaaatcct actgctcttg 240
tctcaaaaat aaaagagatc ttgaaagtca ggcctcggaa ggatgaaaat tctgaaatcc 300
caacncgtat tcctgggcca tcaagcttga gtagaagtag ccggtcaaag tctgtgacta 360
atttgtacaa caacaggttg agtagctgtg acgaggatga ggaggccatt tctccatccg 420
caaaaataaa aatgatggaa aggccaccag tggaaatatt gcctacactg agttcatgta 480
ccaaaacagt caggaggaaa ttcctgagct tagccgcacc tggtcatgca ggtctgattc 540
tagtgatggc cagagcagag tggagactcc cgctcccaga ctggccaccc ctctcacaac 600
tggccgctcc actttgatcc tgaagaagcc agaaaagcta ggtcgaagcc tgtctcatga 660
aaaggattct aattcacaaa caaacatttc aagaggacat gttttctcta ctggtgaaga 720
ggatgctgat tttgtctaac aacgaccatt tttttttgtg tcaggaattt aactttgaat 780
ggcttttccc aatgttttta cataatgtat ttatagatca aaaactattt tatgatcgtt 840
ttcaccccta tcaacac 857
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Claims (6)
1. A SNP molecular marker applicable to the tracing of the navel snail is characterized in that the nucleotide sequence is shown as SEQ ID NO.1, and one base substitution is carried out at the 305 th base, namely 305G or 305A.
2. The application of the reagent for detecting the SNP molecular marker according to claim 1 in the DNA tracing of the double umbilical snails or the preparation of products for the DNA tracing of the double umbilical snails.
3. A group of primer pairs for amplifying the SNP molecular marker in claim 1 is characterized by comprising an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are sequentially shown as SEQ ID NO. 2-3.
4. The primer pair of claim 3 is applied to the DNA tracing of the double umbilical snails or the preparation of the DNA tracing product of the double umbilical snails.
5. A method for detecting and identifying the SNP molecular marker of claim 1 in the hemifusus dybowskii is characterized in that the SNP genotype at the 305 th site of the sequence SEQ ID NO.1 is obtained by the method of PCR-BstUI-RFLP through the primer pair and BstUI endonuclease of claim 3.
6. A kit for tracing the DNA of the double umbilical snail, which is characterized by comprising a primer pair for detecting and identifying the SNP molecular marker of claim 1 and a restriction endonuclease BstUI; the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are sequentially shown in SEQ ID No. 2-3.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255877A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
| KR20170053283A (en) * | 2015-11-06 | 2017-05-16 | 영남대학교 산학협력단 | Composition for identifying short hair spepcies in Sapsaree comprising single nucleotide polymorphism markers |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170053283A (en) * | 2015-11-06 | 2017-05-16 | 영남대학교 산학협력단 | Composition for identifying short hair spepcies in Sapsaree comprising single nucleotide polymorphism markers |
| CN105255877A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae) and Schistosoma mansoni (Platyhelminthes: Trematoda) in Molluscs by PCR-RFLP;Roberta Lima Caldeira等;《Biomed Res Int.》;20161117;第1-5页 * |
| 基于RFLP-PCR的猪肉溯源技术研究;吴华莉等;《上海农业学报》;20171231;第33卷(第6期);第57-62页 * |
| 基于线粒体16S rDNA序列的深圳水库区双脐螺分子鉴定;高世同等;《中华地方病学杂志》;20161231;第35卷(第9期);第636-639页 * |
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