CN118064607A - DNA fingerprint of leiocassis longirostris, construction method and application thereof - Google Patents
DNA fingerprint of leiocassis longirostris, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a leiocassis longirostris DNA fingerprint spectrum, a construction method and application thereof, wherein the leiocassis longirostris DNA fingerprint spectrum comprises 121 specific SNP loci, and the leiocassis longirostris DNA fingerprint spectrum can be used for identifying leiocassis longirostris of different populations. The DNA fingerprint spectrum library and the DNA fingerprint spectrum of the Leiocassis longirostris variety of specific population (Guangdong population, sichuan population, hubei population and Jiangsu population) are obtained based on the resequencing, and when the DNA fingerprint spectrum library is used for identifying Leiocassis longirostris variety, the traditional appearance assessment method is broken through, the population source and living area distribution of Leiocassis longirostris can be more accurate, and more objective and accurate identification results are provided.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a leiocassis longirostris DNA fingerprint and a construction method and application thereof.
Background
Leiocassis longirostris (L.longirostris) belongs to catfish (Siluriformes), leiodaceae (Bagridae) and Leiocassis (Leiocassis), and is deeply favored by consumers due to the advantages of less meat tenderness, high nutritive value and the like, thus being an important economic fish in China. The leiocassis longirostris is mainly distributed in water areas such as Yangtze river, huaihe river and Zhujiang river, and the genetic characters of the leiocassis longirostris at different geographic positions are different due to long-term geographic isolation, so that the accurate management and efficient utilization of the leiocassis longirostris germplasm resources are necessary for researching the relation among the genetic background, genetic structure and different geographic groups of the leiocassis longirostris.
At present, the traditional appearance assessment method is still mainly adopted for the identification of the leiocassis longirostris germplasm resources or the colony. The DNA fingerprint technology has little application in aquaculture varieties, and the construction of the DNA fingerprint of the Leiocassis longirostris by using SNP molecular markers is not reported, so that the construction of the DNA fingerprint of the Leiocassis longirostris is very necessary.
The main problems in the existing leiocassis longirostris variety identification and regional distribution technology are as follows: 1. the existing morphological method is used for identifying leiocassis longirostris germplasm resources and is greatly influenced by human factors and environmental conditions. 2. In the prior art, microsatellite marker technology (SSR) or random amplified polymorphic DNA technology (RAPD) is utilized to analyze the genetic diversity of different groups of leiocassis longirostris, and group identification is carried out according to the relativity, but the molecular marker technology has long required experimental period and low accuracy. 3. The third generation molecular marker technology is used for positioning insertion and deletion of the leiocassis longirostris and other varieties of fishes on the genome, so that whether a sample to be detected is a leiocassis longirostris population can be identified, but the regional distribution cannot be further identified by the method.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a DNA fingerprint spectrum for identifying leiocassis longirostris, and the DNA fingerprint spectrum can be used for rapidly, efficiently and accurately identifying whether an unknown population is leiocassis longirostris, and determining the population and specific living areas thereof. The invention also provides a construction method and application of the leiocassis longirostris DNA fingerprint. The invention provides the DNA fingerprint spectrum for identifying the leiocassis longirostris for the first time and has high accuracy.
The technical scheme is as follows: in order to achieve the above purpose, the DNA fingerprint of the leiocassis longirostris of the present invention includes 121 SNP sites, and the SNP sites and specific nucleotides thereof are as follows:
The DNA fingerprint of the leiocassis longirostris disclosed by the invention, the fingerprint of the leiocassis longirostris comprises a DNA fingerprint of the leiocassis longirostris of the Guangdong population, a DNA fingerprint of the leiocassis longirostris of the Hubei population, a DNA fingerprint of the leiocassis longirostris of the Sichuan population and a DNA fingerprint of the leiocassis longirostris of the Jiangsu population, the four DNA fingerprints comprise 121 SNP loci, the SNP loci and the specific nucleotide at the SNP loci are as follows, and the SNP loci and the specific nucleotide of the DNA fingerprint of the leiocassis longirostris of the Guangdong population are as follows:
SNP loci and specific nucleotides of DNA finger-print of Leiocassis longirostris of Hubei population are as follows:
SNP loci and specific nucleotides of DNA finger-print of leiocassis longirostris of Sichuan population are as follows:
the SNP locus and specific nucleotide of the DNA fingerprint of leiocassis longirostris of Jiangsu population are as follows:
The construction method of the DNA fingerprint of the leiocassis longirostris comprises the following steps:
(1) Collecting tail fins of leiocassis longirostris of different geographical groups widely;
(2) Extracting and detecting the quality of the collected tail fin tissues by using a DNA extraction kit;
(3) Resequencing the fragments with qualified quality control;
(4) Detecting and analyzing quality distribution detection, base distribution detection and pollution monitoring are carried out on the resequencing data; (5) Selecting a leiocassis longirostris whole genome as a reference genome, comparing the simplified CLEAN READS to the reference genome, repeatedly reading the position sequence and the mark, and finally counting various comparison indexes, wherein the comparison rate and the coverage rate index can reflect the quality of a sample, a library, sequencing and a reference sequence;
(6) Carrying out mutation site detection on each sample by using Sentieon to obtain gVCF of each sample, carrying out joint analysis on gVCF of all samples in the subsequent joint-calling step to obtain a mutation result of each individual in the population, and constructing a specific SNP site database of Leiocassis longirostris;
(7) And further filtering according to the deficiency rate, MAF value, single copy, site heterozygosity and depth, and screening high polymorphism SNP sites for constructing DNA fingerprint.
Wherein, in the step (2), the collected tail fin tissue is extracted by using a DNA extraction kit, and the quality detection is as follows: extracting DNA, measuring OD value and concentration, and performing electrophoresis to detect quality, wherein OD 260/OD280 should be 1.8-2.0; when the sample size is 2 mu l, the sample concentration is more than or equal to 50 ng/. Mu.l, and the DNA quality detection results are qualified; in the step (3), sequencing is carried out by using a MGISEQ T sequencer, enzyme slicing and sectioning are carried out on the DNA sample which is qualified in detection, the purified DNA fragment is connected with a sequencing joint, rolling circle amplification is carried out to prepare DNA, and then sequencing is carried out on an arrayed chip.
And (3) performing quality distribution detection, base distribution detection and pollution monitoring on the re-sequencing data in the step (4) to ensure the data quality.
Wherein, the reference genome in the step (5) is PRJNA692071 Leiocassis longirostris whole genome at NCBI.
In the step (6), the detection of the variant loci is required to be controlled in quality in order to ensure the accuracy of SNP, and the SNP loci obtained after combination are subjected to preliminary filtration through filtration parameters, wherein the filtration parameters are QD < 2.0I FS > 60.0I MQ < 40.0I SOR > 3.0I MQRankSum < -12.5I ReadPosRankSum < -8.0.
In the step (7), filtering is performed according to the site deletion rate of 0 and the MAF value of more than or equal to 0.1; extracting the sequence 100bp upstream and downstream of the locus to carry out copy number analysis, and reserving the locus of which the sequence upstream and downstream of the locus is unique on the genome; and secondly, screening sites according to the interval of more than 4Mb by filtering sites with the heterozygosity of the sites being less than 0.1 and the average depth being more than 5, ensuring the site spacing, and further filtering.
The construction method of the DNA fingerprint of the leiocassis longirostris is applied to the identification of the leiocassis longirostris of different populations and the identification of specific living areas of the leiocassis longirostris.
The application process comprises the following steps:
(1) Carrying out DNA extraction and resequencing on a tail fin tissue sample of the leiocassis longirostris to be detected;
(2) SNP locus screening is carried out on the re-sequenced data; the screening conditions are that the site deletion rate is set to be 0 and the MAF value is more than or equal to 0.1 for filtering; then extracting the sequence of 100bp upstream and downstream of the locus for copy number analysis, and reserving the locus of which the sequence upstream and downstream of the locus is unique on the genome; then filtering the loci with the heterozygosity rate of the loci less than 0.1 and the average depth more than 5; finally screening sites according to the interval of more than 4Mb to obtain SNP sites of the fish sample to be tested;
(3) And (3) comparing the nucleotide at the SNP locus obtained in the step (2) with the SNP locus in the DNA fingerprint of the leiocassis longirostris, and identifying the sample to be detected as the leiocassis longirostris population when the coincidence rate is more than or equal to 95%.
The invention provides an application of a DNA fingerprint spectrum of leiocassis longirostris in the identification of leiocassis longirostris and a specific identification method of leiocassis longirostris, which comprises the following steps: detecting the nucleotide at 121 SNP loci in the genome DNA of a fish sample to be detected, comparing the nucleotide with the specific nucleotide at 121 SNP loci corresponding to the DNA fingerprint spectrum of the leiocassis longirostris in claim 1, and identifying the fish sample to be detected as the leiocassis longirostris when the coincidence rate is more than or equal to 95%.
Preferably, the fish sample to be tested is tail fin tissue of fish.
Further, the leiocassis longirostris living areas include Guangdong, sichuan, hubei and Jiangsu.
Further, after the fish sample to be detected is identified as the leiocassis longirostris population, the method further comprises the step of identifying the specific living area of the leiocassis longirostris, wherein the step of identifying the specific living area of the leiocassis longirostris is to calculate the genetic distance between samples, and the genetic relationship comparison analysis is carried out on the sample in the invention, so that the specific living area of the leiocassis longirostris is finally identified as Guangdong, sichuan, hubei or Jiangsu. The four areas have high-quality leiocassis longirostris germplasm resources, and can provide good breeding materials for the creation of new leiocassis longirostris germplasm. Therefore, the invention selects the DNA fingerprint of the Leiocassis longirostris established by utilizing the DNA of the Leiocassis longirostris of the 4 geographical groups.
The invention constructs the leiocassis longirostris DNA fingerprint by utilizing the SNP locus with high polymorphism for the first time. The invention discloses a leiocassis longirostris DNA fingerprint and application thereof. The DNA fingerprint of the leiocassis longirostris comprises 121 specific SNP loci, the DNA fingerprint library and the DNA fingerprint of a specific population (Guangdong population, sichuan population, hubei population and Jiangsu population) leiocassis longirostris variety are obtained based on resequencing, and when the DNA fingerprint is used for leiocassis longirostris variety identification, the traditional appearance assessment method is broken through, the population source of leiocassis longirostris can be more accurate, and more objective and accurate identification results are provided.
The beneficial effects are that: compared with the prior art, the invention has the remarkable advantages that:
1. The invention constructs the DNA fingerprint of the leiocassis longirostris by using 121 high polymorphism SNP loci for the first time, and can identify the leiocassis longirostris of different populations and specific living areas of the leiocassis longirostris more effectively and accurately.
2. According to the invention, by resequencing leiocassis longirostris in four different areas (Guangdong, hubei, sichuan and Jiangsu), the constructed DNA fingerprint of the leiocassis longirostris is more comprehensive, and the leiocassis longirostris germplasm identification can be more effectively performed.
3. The method can simultaneously realize the identification of whether the unknown population is leiocassis longirostris and the definition of the area to which the leiocassis longirostris belongs, and is more efficient.
4. The invention uses high polymorphism SNP locus as identification index of different groups of leiocassis longirostris, SNP locus belongs to the third generation molecular marker technology, and compared with the first two generation molecular markers, the invention has the advantages of short sequencing time, high cost performance, high sequencing flux and the like.
Drawings
FIG. 1 is a base mass distribution diagram of sample sequencing data;
FIG. 2 is a plot of sample sequencing base content;
FIG. 3 shows the presence of 121 SNPs on the chromosome;
FIG. 4 is a genetic distance relationship of four different geographical populations of Leiocassis longirostris;
FIG. 5 is a graph showing the genetic distance relationship of the unknown geographic population Leiocassis longirostris.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The research process and principle of the invention are as follows: and (3) widely collecting leiocassis longirostris of different geographical populations, constructing a leiocassis longirostris DNA database based on resequencing, screening and filtering the leiocassis longirostris specific sites in the database, calculating the relationships among samples based on the specific sites, and making phylogenetic tree. The method comprises the following specific steps: 1. collecting Leiocassis longirostris of Guangdong, hubei, sichuan and Jiangsu population; 2. respectively extracting DNA of the collected leiocassis longirostris of different geographical groups, and carrying out mass analysis on the extracted DNA; 3. resequencing the fragments with qualified quality control; 4. detecting the quality distribution, the base distribution and the pollution of the re-sequencing data; 5. comparing reads of each sample with a reference genome by adopting software, setting reasonable screening and filtering parameters, and constructing a specific locus (SNP locus) database of the leiocassis longirostris; 6. and (5) performing mutation detection and statistics on SNP loci. 7. Screening finger print loci; 8. calculating the relationships among samples and constructing a phylogenetic tree; 9. constructing the leiocassis longirostris SNP molecular identity card.
Example 1
Establishment of DNA fingerprint of leiocassis longirostris
1. Collecting tail fins of leiocassis longirostris of different geographical populations, wherein 16 Guangdong population is marked as G1-G16; 20 Hubei colonies, marked as H1-H20; 20 Sichuan groups are marked as S1-S20; 20 leiocassis longirostris of Jiangsu population, which is marked as J1-J20.
2. And extracting the collected tail fin tissues by using a DNA extraction kit, and detecting the quality of the tail fin tissues.
DNA extraction was performed using a DNA extraction kit (FastPure Cell/Tissue DNA Isolation Mini Kit, DC 102-01) from Norvezan, OD value and concentration were measured according to the instructions, and the samples were used after running electrophoresis to detect quality. Wherein OD260/OD280 should be between 1.8-2.0; when the sample amount is 2. Mu.l, the sample concentration is more than or equal to 50 ng/. Mu.l. And the DNA quality detection results are all qualified.
3. And (5) resequencing the fragments after quality control is qualified.
Sequencing by MGISEQ T7 sequencer, randomly breaking the DNA sample, and screening the DNA fragment with proper size. The purified DNA fragments were ligated to sequencing adaptors, rolling circle amplified to prepare DNA, and then sequenced on an arrayed chip.
4. Detecting and analyzing the resequencing data, detecting the quality distribution, detecting the base distribution and monitoring the pollution. And the data quality is ensured, and the original data is filtered and evaluated before the information analysis.
In order to reflect the stability of the sequencing quality during the sequencing data process, the base position of CLEAN READS is taken as an abscissa, and the average sequencing quality value of each position is taken as an ordinate, so that the sequencing quality distribution diagram of each sample is obtained. From the graph, the quality values were all 30 or more, indicating good sequencing quality. The results are shown in FIG. 1.
The base distribution is used to detect the presence or absence of AT, GC separation, which may be a consequence of sequencing or library construction and may affect subsequent quantitative analysis. Theoretically, the contents of G and C bases and A and T bases should be equal in each sequencing cycle, and the whole sequencing process is stable and unchanged and is horizontal. The corresponding distribution map is obtained by taking the base position in CLEAN READS as the abscissa and the proportion of ATCGN bases at each position as the ordinate. The results are shown in FIG. 2.
5. The NCBI project PRJNA692071 Leiocassis longirostris whole genome was selected as the reference genome. And comparing the simplified CLEAN READS with a reference base group, performing position sequencing and marking repeated reads, and finally counting various comparison indexes. The comparison rate and coverage index can reflect the quality of samples, library construction, sequencing, reference sequences and the like.
6. And (5) performing mutation detection and statistics on SNP loci. Each sample was subjected to mutation site detection using Sentieon to obtain gVCF of each sample. In the subsequent joint-calling step, gVCF of all samples is subjected to joint analysis to obtain a mutation result of each individual in the population. In order to ensure SNP accuracy, quality control is required, and the SNP loci obtained after combination are subjected to preliminary filtration through the filtration parameters recommended by GATK: QD < 2.0 < FS > 60.0 < MQ < 40.0 > SOR > 3.0 < MQRankSum < -12.5 < ReadPosRankSum < -8.0, obtaining 5,023,513 SNPs.
7. Based on the SNP sites, filtration was further performed by deletion rate, MAF value, single copy, site heterozygosity rate, site depth. Firstly, filtering according to the site deletion rate of 0 and the MAF value of more than or equal to 0.1; and extracting the sequence 100bp upstream and downstream of the locus to perform copy number analysis, and reserving the locus of which the sequence upstream and downstream of the locus is unique on the genome to obtain 570,949 SNP loci. And secondly, reserving 600 sites through the sites with the heterozygosity rate of the filtering sites smaller than 0.1 and the average depth larger than 5. And then screening the sites according to the interval of more than 4Mb, and ensuring the site spacing. Finally, 121 SNP loci shown in Table 1 were obtained, and the presence of 121 SNPs on the chromosome is shown in FIG. 3.
TABLE 1 DNA fingerprint of Leiocassis longirostris
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8. And calculating the relationships among all samples.
Based on the 121 SNP sites of table 1, the inter-sample genetic distances were calculated to make sample discrimination. For 121 sites, the genetic distance between samples is calculated by using Plink software, all samples can be separated according to the genetic distance, the magnitude of the genetic distance matrix value represents the distance of the genetic distance, the smaller the value is, the closer the genetic relationship between the samples is, and the result is shown in fig. 4.
9. Constructing leiocassis longirostris DNA finger print of different geographical groups.
And constructing leiocassis longirostris DNA finger print of different geographical groups according to the screened SNP loci. Leiocassis longirostris DNA fingerprint patterns in different regions are shown in tables 2-5 below:
TABLE 2 DNA fingerprint of leiocassis longirostris of Guangdong population
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TABLE 3 DNA fingerprint of Leiocassis longirostris of Hubei population
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TABLE 4 DNA fingerprint of leiocassis longirostris of Sichuan population
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TABLE 5 DNA fingerprint of leiocassis longirostris of Jiangsu population
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Example 2
Taking a leiocassis longirostris sample (A1) in Jiangsu region, extracting DNA, taking tail fins, extracting DNA by using a DNA extraction kit (FastPure Cell/Tissue DNA Isolation Mini Kit) of Norvezan company, measuring OD value and concentration according to the specification, running electrophoresis to detect quality, and storing in a refrigerator at-20 ℃ for later use after meeting the requirements.
Sequencing by MGISEQ T7 sequencer, randomly breaking the DNA sample, and screening the DNA fragment with proper size. The purified DNA fragments were ligated to sequencing adaptors, rolling circle amplified to prepare DNA, and then sequenced on an arrayed chip.
Sequencing results show that 117 core SNP loci of the sample accord with the leiocassis longirostris fingerprint of Jiangsu population in Table 5, and the accord ratio is 96.7%. And for 121 loci, genetic distances between the unknown sample and four known leiocassis longirostris populations in the present invention were calculated using Plink software. The result of the clustering graph shows that the unknown sample and Jiangsu population are gathered into one type (figure 5), so that the sample is judged to be the Leiocassis longirostris of Jiangsu population, and the sampling result is met.
The feasibility and the accuracy of the invention are proved by examples 1 and 2, 4 tail fins of the Leiocassis longirostris in geographic population are widely collected in example 1, DNA is extracted, 121 high polymorphism SNP loci are obtained through a resequencing technology, and DNA fingerprint spectrums for identifying the Leiocassis longirostris in different populations are constructed. Example 2 is used for proving the feasibility of the DNA fingerprint constructed by the invention, randomly selecting a Jiangsu population sample, carrying out cluster analysis on genotypes of 121 high-polymorphism SNP loci, finding that the coincidence rate of the sample and the DNA fingerprint of the Jiangsu population reaches 96.7%, and identifying the sample as the Jiangsu population. Meanwhile, the population of Guangdong, hubei and Sichuan are randomly selected respectively, and the coincidence rate of the sample and the DNA fingerprint of the corresponding population is found to be more than or equal to 95 percent, so that the sample can be identified as the population of Guangdong, hubei or Sichuan. Namely, the DNA fingerprint in the invention can effectively identify the leiocassis longirostris of different populations of Jiangsu, guangdong, hubei and Sichuan, and has high accuracy.
Claims (10)
1. The DNA fingerprint of the leiocassis longirostris is characterized by comprising 121 SNP loci, wherein the SNP loci and specific nucleotides thereof are as follows:
2. The DNA fingerprint of the leiocassis longirostris is characterized by preferably comprising the DNA fingerprint of the leiocassis longirostris of the Guangdong population, the DNA fingerprint of the leiocassis longirostris of the Hubei population, the DNA fingerprint of the leiocassis longirostris of the Sichuan population and the DNA fingerprint of the leiocassis longirostris of the Jiangsu population, wherein the four DNA fingerprints comprise 121 SNP loci, the SNP loci and the specific nucleotides at the SNP loci are as follows, and the SNP loci and the specific nucleotides of the DNA fingerprint of the leiocassis longirostris of the Guangdong population are as follows:
SNP loci and specific nucleotides of DNA finger-print of Leiocassis longirostris of Hubei population are as follows:
SNP loci and specific nucleotides of DNA finger-print of leiocassis longirostris of Sichuan population are as follows:
the SNP locus and specific nucleotide of the DNA fingerprint of leiocassis longirostris of Jiangsu population are as follows:
3. A method for constructing a DNA fingerprint of leiocassis longirostris according to claim 1 or 2, comprising the following steps:
(1) Collecting tail fins of leiocassis longirostris of different geographical groups widely;
(2) Extracting and detecting the quality of the collected tail fin tissues by using a DNA extraction kit;
(3) Resequencing the fragments with qualified quality control;
(4) Detecting and analyzing quality distribution detection, base distribution detection and pollution monitoring are carried out on the resequencing data;
(5) Selecting a leiocassis longirostris whole genome as a reference genome, comparing the simplified CLEAN READS to the reference genome, repeatedly reading the position sequence and the mark, and finally counting various comparison indexes, wherein the comparison rate and the coverage rate index can reflect the quality of a sample, a library, sequencing and a reference sequence;
(6) Carrying out mutation site detection on each sample by using Sentieon to obtain gVCF of each sample, carrying out joint analysis on gVCF of all samples in the subsequent joint-calling step to obtain a mutation result of each individual in the population, and constructing a specific SNP site database of Leiocassis longirostris;
(7) And further filtering according to the deficiency rate, MAF value, single copy, site heterozygosity and depth, and screening high polymorphism SNP sites for constructing DNA fingerprint.
4. The method for constructing a DNA fingerprint of leiocassis longirostris according to claim 3, wherein the extracting and quality detecting of the collected tail fin tissue in the step (2) using a DNA extraction kit is as follows: extracting DNA, measuring OD value and concentration, and performing electrophoresis to detect quality, wherein OD 260/OD280 should be 1.8-2.0; when the sample size is 2 mu l, the sample concentration is more than or equal to 50 ng/. Mu.l, and the DNA quality detection results are qualified; and (3) re-sequencing in the step (3) by using a MGISEQ T sequencer, performing enzyme-sectional segmentation on the DNA sample which is qualified in detection, connecting the purified DNA fragments with a sequencing joint, performing rolling circle amplification to prepare DNA, and then performing sequencing on an arrayed chip.
5. The method for constructing a DNA fingerprint of leiocassis longirostris according to claim 3, wherein the quality of the re-sequenced data is ensured by performing quality distribution detection, base distribution detection and pollution monitoring on the re-sequenced data in the step (4).
6. The method for constructing a DNA fingerprint of leiocassis longirostris according to claim 3, wherein the reference genome in the step (5) is PRJNA692071 leiocassis longirostris whole genome on NCBI.
7. The method for constructing a DNA fingerprint of Leiocassis longirostris according to claim 3, wherein in step (6), the detection of variant sites is performed to ensure the accuracy of SNP, and the quality control is required, and the SNP sites obtained after combination are preliminarily filtered by a filtering parameter which is QD < 2.0 < FS > 60.0 < MQ < 40.0 < SOR > 3.0 < MQRankSum < -12.5 < ReadPosRankSum < -8.0.
8. The method for constructing a DNA fingerprint of leiocassis longirostris according to claim 3, wherein in the step (7), filtering is performed according to the site deletion rate of 0 and MAF value of 0.1 or more; extracting the sequence 100bp upstream and downstream of the locus to carry out copy number analysis, and reserving the locus of which the sequence upstream and downstream of the locus is unique on the genome; and secondly, screening sites according to the interval of more than 4Mb by filtering sites with the heterozygosity of the sites being less than 0.1 and the average depth being more than 5, ensuring the site spacing, and further filtering.
9. An application of a method for constructing a DNA fingerprint of leiocassis longirostris according to claim 1 in the identification of leiocassis longirostris of different populations and the identification of specific living areas of leiocassis longirostris.
10. The application according to claim 9, wherein the process of the application is:
(1) Carrying out DNA extraction and resequencing on a tail fin tissue sample of the leiocassis longirostris to be detected;
(2) SNP locus screening is carried out on the re-sequenced data; the screening conditions are that the site deletion rate is set to be 0 and the MAF value is more than or equal to 0.1 for filtering; then extracting the sequence of 100bp upstream and downstream of the locus for copy number analysis, and reserving the locus of which the sequence upstream and downstream of the locus is unique on the genome; then filtering the loci with the heterozygosity rate of the loci less than 0.1 and the average depth more than 5; finally screening sites according to the interval of more than 4Mb to obtain SNP sites of the fish sample to be tested;
(3) Comparing the nucleotide at the SNP locus obtained in the step (2) with the SNP locus in the DNA fingerprint of the leiocassis longirostris in claim 1 or 2, and identifying the sample to be detected as the leiocassis longirostris population when the coincidence rate is more than or equal to 95%, and further identifying the region distribution of the leiocassis longirostris population.
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