CN116516024A - DNA fingerprint of pelteobagrus fulvidraco and application thereof - Google Patents

DNA fingerprint of pelteobagrus fulvidraco and application thereof Download PDF

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CN116516024A
CN116516024A CN202310666892.8A CN202310666892A CN116516024A CN 116516024 A CN116516024 A CN 116516024A CN 202310666892 A CN202310666892 A CN 202310666892A CN 116516024 A CN116516024 A CN 116516024A
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pelteobagrus fulvidraco
dna
dna fingerprint
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王涛
商如华
尹绍武
张莹
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Nanjing Normal University
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Abstract

The invention discloses a DNA fingerprint of pelteobagrus fulvidraco and application thereof. The DNA fingerprint of pelteobagrus fulvidraco provided by the invention comprises 106 SNP loci, and is obtained by re-sequencing pelteobagrus fulvidraco in four different areas (Jiangsu Yangtze Nanjing section, heilongsu Lijiang, hubei Honghu lake and inner Mongolian Goujia temple). When the method is used for identifying pelteobagrus fulvidraco, compared with the traditional group identification method, the method is more objective, accurate and reliable.

Description

DNA fingerprint of pelteobagrus fulvidraco and application thereof
Technical Field
The invention relates to a DNA fingerprint of pelteobagrus fulvidraco and application thereof, belonging to the field of molecular biology.
Background
The pelteobagrus fulvidraco belongs to catfish order, the family of the bagrus, the genus pelteobagrus, is widely distributed fish, and is common economic fish in rivers and lakes in the middle and downstream of the Yangtze river. The pelteobagrus fulvidraco is smaller, but the meat is delicious, and the demand of the pelteobagrus fulvidraco is continuously increased in recent years. According to the year identification of 2022 fishery statistics, the culture amount of pelteobagrus fulvidraco in 2021 is 587,822 tons, which is 3.95 percent higher than that in 2020. In order to understand the relation among the genetic background, the genetic structure and the populations of pelteobagrus fulvidraco, the populations are distinguished. This is difficult to perform only from the aspect of appearance, etc., and therefore, it is necessary to develop a more accurate identification method.
Identification technology based on DNA molecular level is rapidly developing, and at the DNA molecular level, different species and even different individuals of the same species can be well distinguished, and at present, the DNA molecular technology has been widely applied to classification identification and kindred relation research among genus, species and varieties. The DNA molecular technology for fish identification mainly comprises an SSR marking technology and an SNP marking technology. The SNP marker technology has the advantages of wide distribution, high accuracy and the like.
At present, no related report of establishing pelteobagrus fulvidraco DNA fingerprint by SNP (single nucleotide polymorphism) marker technology exists.
Disclosure of Invention
The invention aims to: the invention aims to provide a DNA fingerprint spectrum for identifying pelteobagrus fulvidraco; the invention further aims to provide an identification method for identifying pelteobagrus fulvidraco populations by using the DNA fingerprint.
The technical scheme is as follows: the DNA fingerprint of pelteobagrus fulvidraco provided by the invention comprises 106 SNP loci, wherein the SNP loci and specific nucleotides in the SNP loci are as follows:
the invention discloses application of the DNA fingerprint spectrum of pelteobagrus fulvidraco in identification of pelteobagrus fulvidraco.
The invention discloses a pelteobagrus fulvidraco identification method, which comprises the following steps: detecting the nucleotide at 106 SNP loci in the genome DNA of the fish sample to be detected, comparing the nucleotide with the specific nucleotide at 106 SNP loci corresponding to the DNA fingerprint spectrum of pelteobagrus fulvidraco in claim 1, and identifying the sample to be detected as pelteobagrus fulvidraco population when the coincidence rate is more than or equal to 95%.
Further, the fish sample to be tested is tail fin tissue of the fish. Specifically, the genomic DNA of the fish to be tested is obtained from fresh skein tissue of the fish to be tested.
Further, the authentication method includes the steps of:
(1) Extracting DNA from the fish sample to be detected and resequencing;
(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 pelteobagrus fulvidraco defined in claim 1, and identifying the sample to be tested as pelteobagrus fulvidraco colony when the coincidence rate is more than or equal to 95%.
Further, after the fish sample to be detected is identified as the pelteobagrus fulvidraco population, the method further comprises the step of identifying the specific living area of the pelteobagrus fulvidraco.
Further, the step of identifying the pelteobagrus fulvidraco specific living area is to calculate genetic distance between samples, compare and analyze the genetic relationship with the samples in the invention, and finally identify the pelteobagrus fulvidraco specific living area as one of Jiangsu Yangtze Nanjing section, heilongjiang Wusu Lijiang, hubei Honghu lake or inner Mongolin Gudu temple.
The 4 areas of Jiangsu Yangtze river Nanjing section, heilongjiang Wusu Lijiang, hubei Honghu and inner Mongolian Du temple have high-quality pelteobagrus fulvidraco germplasm resources, and can provide good breeding materials for creating new pelteobagrus fulvidraco germplasm in China. Therefore, the DNA fingerprint of the pelteobagrus fulvidraco is established by using the DNA of the pelteobagrus fulvidraco of the 4 geographical groups.
Further, the DNA fingerprint of pelteobagrus fulvidraco in the living area of Jiangsu Yangtze river Nanjing section is:
。
further, the DNA fingerprint of pelteobagrus fulvidraco in the living area of Souzuril river of Heilongjiang is:
。
further, the DNA fingerprint of pelteobagrus fulvidraco in the living area of Hubei Honghu lake is:
。
further, the DNA fingerprint of pelteobagrus fulvidraco in the domestic area of inner Mongolicum temple is:
。
the beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the method, the pelteobagrus fulvidraco in four different areas (Yangtze river Nanjing section, heilongjiang Wu Zuijiang, hubei Honghu and Nemongolic Duxi temple) is re-sequenced, the constructed DNA fingerprint of the pelteobagrus fulvidraco is more comprehensive, pelteobagrus fulvidraco germplasm identification can be more effectively carried out, and compared with a traditional group identification method, the method is more objective, accurate and reliable, and can realize accurate management and efficient utilization of pelteobagrus fulvidraco germplasm resources.
Drawings
FIG. 1 is a mass distribution diagram of sample sequencing;
FIG. 2 is a plot of sequencing base content;
FIG. 3 shows the presence of 106 SNPs on the chromosome;
FIG. 4 is a genetic distance relationship of four different geographic populations of pelteobagrus fulvidraco;
FIG. 5 is a genetic distance relationship of example 2.
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 pelteobagrus fulvidraco in different geographic groups, constructing a pelteobagrus fulvidraco DNA database based on re-sequencing, screening and filtering pelteobagrus fulvidraco specific sites in the database, calculating the relationships among samples based on the specific sites, and making a phylogenetic tree. The method comprises the following specific steps: 1. collecting yellow catfish of Nanjing section of Yangtze river, wusu Li river, hebei Honghu lake and inner Mongolian Du temple group; 2. respectively extracting DNA of the collected pelteobagrus fulvidraco of different geographical groups, and carrying out quality 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 pelteobagrus fulvidraco; 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 SNP molecular identity card of pelteobagrus fulvidraco.
Example 1 establishment of DNA finger print of pelteobagrus fulvidraco
1. Tail fins of pelteobagrus fulvidraco in different geographic groups are widely collected, wherein 16 tail fins in Jiangsu Yangtze Nanjing section are marked as A1-A16; 16 Heilongjiang Wusu Li Jiang strips, marked as B1-B16; 13 lagoons from Hubei province, noted C1-C13; the inner Mongolian Goodyear temple 11 is designated as D1-D11.
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) from Norvezan, OD value and concentration were measured according to the instructions, and the samples were used after electrophoresis detection of quality. Wherein OD 260 /OD 280 Should be between 1.8 and 2.0; when the sample amount is 2. Mu.l, the sample concentration is more than or equal to 50 ng/. Mu.l. Rejecting unqualified samples of DNA quality detection: a2 (OD) 260 /OD 280 ：0.854)、A8(OD 260 /OD 280 ：1.274)、A9(OD 260 /OD 280 ：1.112)、A12(OD 260 /OD 280 ：1.09)、A15(OD 260 /OD 280 ：1.556)、B15(OD 260 /OD 280 ：4.791)、C3(OD 260 /OD 280 :0.203 And C7 (OD) 260 /OD 280 ：0.562)。
3. And (5) resequencing the fragments after quality control is qualified.
Sequencing by using an MGISEQ T7 sequencer, randomly breaking the DNA sample which is qualified in detection, and screening the DNA fragments which meet the requirements and are suitable for the 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. Sample sequencing data yield and quality summary results are shown in table 1.
Table 1 simplified sequencing data statistics for pelteobagrus fulvidraco samples
。
And detecting the quality distribution of sequencing data. The sequencing error rate of each base is obtained by converting a sequencing value (Qphred) by a corresponding formula, and the sequencing quality value is obtained by calculating a predicted base discrimination error rate model in the base recognition process, and the corresponding relation is shown in table 2.
TABLE 2 concise correspondence between base correct recognition rate and Phred score
Phred score Incorrect base recognition Base correct recognition rate Q-score
10 1/10 90％ Q10
20 1/100 99％ Q20
30 1/1000 99.9％ Q30
40 1/10000 99.99％ Q40
。
In order to reflect the stability of the sequencing quality in the sequencing data process, the base position of clear Reads is taken as an abscissa, and the average sequencing quality value of each position is taken as an ordinate, so that a 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 the clear Reads as the abscissa and the proportion of the ATCCN base at each position as the ordinate. The results are shown in FIG. 2.
Sequencing data contamination detection contamination assessment was performed by randomly selecting 10,000 reads from fastq files for each sample and having blastn compare them to the NT database. The results showed that all samples did not have contamination problems. The results are shown in Table 3.
TABLE 3 data pollution statistics
。
5. The published pelteobagrus fulvidraco genome was selected as the reference genome, and the genome used in the invention was Tachysurus fulvidraco (GCF-022655615.1, genome size 712 MB). Comparing the simplified clear Reads to a reference genome, sequencing the positions 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. The results are shown in Table 4.
Table 4 data alignment statistics table
。
6. And (5) performing mutation detection and statistics on SNP loci. Mutation site detection was performed on each sample using Sentieon to obtain gccf for each sample. In the subsequent joint-rolling step, gVCF of all samples is subjected to joint analysis to obtain a variation 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 >. 9,984,114SNPs were obtained.
7. And screening the DNA fingerprint specificity locus of the pelteobagrus fulvidraco. Based on the 9,984,114SNP loci obtained, further filtration was performed by deletion rate, MAF value, single copy, site heterozygosity, site depth. Firstly, filtering according to the site deletion rate of 0 and the MAF value of more than or equal to 0.1 to obtain 2,291,131 SNP sites; 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 1,848,795 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, 106 SNP loci as set forth in Table 5 were obtained.
TABLE 5 DNA finger print of pelteobagrus fulvidraco
。
8. And calculating the relationships among all samples.
Based on the 106 SNP loci of Table 6, the genetic distances between samples are calculated to make sample discrimination. For 106 sites, the genetic distance between samples is calculated by using the 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 DNA finger print of pelteobagrus fulvidraco of different geographic groups.
And constructing DNA finger-prints of pelteobagrus fulvidraco in different geographic groups according to the screened SNP loci. The DNA finger print of pelteobagrus fulvidraco in different areas is as follows:
table 6 DNA finger print of Pelteobagrus fulvidraco in Nanjing section of Yangtze river Jiangsu
TABLE 7 DNA finger print of Heilongjiang Wusu Li river pelteobagrus fulvidraco
TABLE 8 DNA finger print of Pelteobagrus fulvidraco in Honghu lake of Hubei province
Table 9 DNA finger print of Fulteobagrus fulvidraco in Mongolian Ducheni
Example 2 actual sample identification
Taking Jiangsu Changjiang Nanjing section; heilongjiang Wusu Li Jiang; 3 tail of each sample of yellow catfish in Honghu lake and inner Mongolian Duxi temple. Sample numbers of Jiangsu Yangtze Nanjing section are E1, E2 and E3; the sample numbers of the Heilongjiang Wusu Li river are E4, E5 and E6; the sample numbers of the Hubei Honghu lake are E7, E8 and E9; the inner Mongolian Temple sample numbers are E10, E11 and E12. The rest steps are the same as in example 1, and the screened nucleotides at 106 SNP loci are compared with Table 6, so that the coincidence rate is more than 95%. The relation of the genetic distances is shown in figure 5, and the identification result is in accordance with the actual result, so that the pelteobagrus fulvidraco DNA finger print of the invention patent can be used for effectively distinguishing Jiangsu Yangtze Nanjing sections; heilongjiang Wusu Li Jiang; hubei Honghu and inner Mongolian Duxi temple yellow catfish group.

Claims (10)

1. The DNA fingerprint of pelteobagrus fulvidraco is characterized by comprising 106 SNP loci, wherein the SNP loci and specific nucleotides thereof are as follows:
。
2. use of a DNA fingerprint of pelteobagrus fulvidraco according to claim 1 for identifying pelteobagrus fulvidraco.
3. The identification method of pelteobagrus fulvidraco is characterized by comprising the following steps: detecting the nucleotide at 106 SNP loci in the genome DNA of the fish sample to be detected, comparing the nucleotide with the specific nucleotide at 106 SNP loci corresponding to the DNA fingerprint spectrum of pelteobagrus fulvidraco in claim 1, and identifying the fish sample to be detected as pelteobagrus fulvidraco colony when the coincidence rate is more than or equal to 95%.
4. The identification method of pelteobagrus fulvidraco according to claim 2, wherein the fish sample to be tested is the tail fin tissue of fish.
5. Identification method of pelteobagrus fulvidraco according to claim 2, characterized in that it comprises the following steps:
(1) Extracting DNA from the fish sample to be detected and resequencing;
(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 pelteobagrus fulvidraco defined in claim 1, and identifying the sample to be tested as pelteobagrus fulvidraco population when the coincidence rate is more than or equal to 95%.
6. The method for identifying pelteobagrus fulvidraco according to claim 5, further comprising the step of identifying a particular living region of pelteobagrus fulvidraco after identifying the sample of fish to be tested as a pelteobagrus fulvidraco population: and calculating genetic distance between samples, and comparing and analyzing the genetic relationship with the samples in the invention to finally identify that the specific living area of pelteobagrus fulvidraco is one of Jiangsu Yangtze Nanjing section, heilongjiang Wusu Lijiang, hubei Honghu or inner Mongolin Gou Simiao.
7. The identification method of pelteobagrus fulvidraco according to claim 6, wherein the DNA fingerprint of pelteobagrus fulvidraco in the south of the Yangtze river in the living area is:
。
8. the identification method of pelteobagrus fulvidraco according to claim 6, wherein the DNA fingerprint of pelteobagrus fulvidraco in the living area of black longriver, urst river is:
。
9. the identification method of pelteobagrus fulvidraco according to claim 6, wherein the DNA fingerprint of pelteobagrus fulvidraco in the living area of lagoons Hubei province is:
。
10. the identification method of pelteobagrus fulvidraco according to claim 6, wherein the DNA fingerprint of pelteobagrus fulvidraco in which the living area is the inner mongolica temple is:
。
CN202310666892.8A 2023-06-07 2023-06-07 DNA fingerprint of pelteobagrus fulvidraco and application thereof Pending CN116516024A (en)

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