CN116445632A - Fugu obscurus DNA finger print and application thereof - Google Patents

Fugu obscurus DNA finger print and application thereof Download PDF

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CN116445632A
CN116445632A CN202310666880.5A CN202310666880A CN116445632A CN 116445632 A CN116445632 A CN 116445632A CN 202310666880 A CN202310666880 A CN 202310666880A CN 116445632 A CN116445632 A CN 116445632A
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fugu obscurus
obscurus
dna
fugu
identifying
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王涛
张莹
尹绍武
商如华
褚鹏
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Nanjing Normal University
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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Abstract

The invention discloses a DNA fingerprint spectrum of Fugu obscurus and application thereof. The DNA finger print of the fugu obscurus comprises 84 specific SNP loci. The invention also discloses application of the DNA finger print of the fugu obscurus in identifying the fugu obscurus. The DNA fingerprint library and the DNA fingerprint of the Fugu obscurus varieties of specific populations (Jiangsu population, guangdong population and Japanese population) are obtained based on the resequencing, and are used for identifying the Fugu obscurus pure varieties, so that the traditional appearance assessment method is broken through, the population sources of the Fugu obscurus can be more accurately identified, and more objective and accurate identification results are provided.

Description

Fugu obscurus DNA finger print and application thereof
Technical Field
The invention relates to the field of molecular biology research, in particular to a Fugu obscurus DNA fingerprint and application thereof.
Background
The fugu obscurus is an important economic cultivation fugu obscurus variety in China, and is deeply favored by consumers due to delicious meat and high nutritive value. Fugu obscurus is mainly distributed in the coast of China (east, yellow, bohai sea) and downstream of the Yangtze river. Because of long-term geographic isolation, genetic characters of different geographic groups are different, and in order to know the genetic background, genetic structure and relationship among groups of the fugu obscurus, the groups 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.
DNA fingerprinting refers to the collective term that a DNA sample is treated with a specific molecular marker technique to reveal a specific DNA fragment. With the progress and development of biotechnology, the DNA fingerprint technology is widely applied to variety resource diversity and variety identification research. The SNP molecular marker for constructing the DNA fingerprint has the advantages of high polymorphism detection rate, wide distribution in genome, stable and reliable result and the like. The construction of the DNA finger-print of the fugu obscurus based on the SNP markers provides accurate and efficient basis for distinguishing and identifying the varieties of the fugu obscurus.
At present, the application of the DNA fingerprint technology to aquaculture varieties is not much, and the construction of the DNA fingerprint of the Fugu obscurus by using SNP molecular markers is not reported.
Disclosure of Invention
The invention aims to: the invention aims at providing a DNA fingerprint spectrum for identifying fugu obscurus; the invention also aims at providing an identification method for identifying the fugu obscurus population by using the DNA fingerprint.
The technical scheme is as follows: the DNA fingerprint of the fugu obscurus comprises 84 SNP loci, and the SNP loci and specific nucleotides thereof are as follows:
。
the invention relates to an application of the DNA finger print of the fugu obscurus in identifying the fugu obscurus.
The invention relates to an identification method of Fugu obscurus, which comprises the following steps: detecting the nucleotide at 84 SNP loci in the genome DNA of the fish sample to be detected, comparing the nucleotide with the specific nucleotide at 84 SNP loci corresponding to the DNA finger print of the fugu obscurus in claim 1, and identifying the sample to be detected as the fugu obscurus 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 spectrum of the fugu obscurus defined in claim 1, and identifying the sample to be detected as the fugu obscurus population when the coincidence rate is more than or equal to 95%.
Further, after the fish sample to be tested is identified as the fugu obscurus population, the method further comprises the step of identifying the specific living area of the fugu obscurus.
Further, the step of identifying specific living areas of the fugu obscurus is to calculate genetic distances among samples, compare and analyze the genetic relationship with the samples in the invention, and finally identify the specific living areas of the fugu obscurus as Jiangsu, guangdong or Japan.
The 3 areas of Jiangsu population, guangdong population and Japanese population have high-quality fugu obscurus fish germplasm resources, and can provide good breeding materials for the creation of new fugu obscurus germplasm. Thus, the application selects the DNA fingerprint of the Fugu obscurus by using the DNA of the Fugu obscurus of the 3 geographical groups.
Further, the DNA fingerprint of Fugu obscurus in the living area is:
。
further, the DNA fingerprint of Fugu obscurus in the living area is that:
。
further, the DNA fingerprint of Fugu obscurus in the living area is Japanese is:
。
the beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the method, the Fugu obscurus in three different areas (Jiangsu, guangdong and Japan) is subjected to resequencing, the constructed DNA fingerprint of the Fugu obscurus is more comprehensive, the Fugu obscurus germplasm identification can be more effectively performed, and compared with a traditional group identification method, the method is more objective, accurate and reliable, and the precise management and the efficient utilization of Fugu obscurus germplasm resources can be realized.
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 84 SNPs on the chromosome;
FIG. 4 shows the genetic distance relationship of three different geographic groups of Fugu obscurus;
FIG. 5 is a graph showing the genetic distance relationship of Fugu obscurus of unknown geographic population.
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 the fugu obscurus of different geographical groups, constructing a DNA database based on the resequenced fugu obscurus, screening and filtering specific sites of the fugu obscurus in the database, calculating the genetic relationship among samples based on the specific sites, and making a phylogenetic tree. The method comprises the following specific steps: 1. collecting Fugu obscurus of Jiangsu, guangdong group; 2. respectively extracting DNA of the collected Fugu obscurus 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 the fugu obscurus; 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 the fugu obscurus.
Example 1 establishment of DNA finger print of Fugu obscurus
1. Collecting tail fins of Fugu obscurus in different geographic groups, wherein 6 pieces of Fugu obscurus are marked as A1-A6; 6 Guangdong groups, designated B1-B6; the re-sequencing data of the Fugu obscurus of Japanese population is derived from NCBI project PRJDB3165 and marked as C1-C6, and is directly used for mutation detection and statistics of SNP loci without quality control and screening.
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 electrophoresis to detect 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. And the DNA quality detection results are all qualified.
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 Fugu obscurus samples
Sample numbering Reads Bases Q20(％) Q30(％)
A1 34,511,956 5,176,793,400 34,044,726 5,056,305,081
A2 32,620,908 4,893,136,200 31,954,462 4,741,595,554
A3 43,286,974 6,493,046,100 42,515,014 6,293,434,075
A4 25,523,952 3,828,592,800 25,061,444 3,710,914,104
A5 36,972,204 5,545,830,600 36,402,666 5,398,669,921
A6 31,625,072 4,743,760,800 31,162,222 4,626,516,931
B1 44,520,760 6,678,114,000 42,506,084 6,232,327,480
B2 43,944,518 6,591,677,700 41,873,094 6,140,828,898
B3 37,018,790 5,552,818,500 35,274,754 5,181,125,884
B4 38,145,524 5,721,828,600 36,446,800 5,364,630,016
B5 33,011,580 4,951,737,000 31,736,592 4,670,895,747
B6 38,676,986 5,801,547,900 36,734,852 5,362,112,155
。
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 NCBI project PRJNA449558 Fugu obscurus whole genome was selected as the reference genome. 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, obtaining 12,861,472SNP.
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 1,088,678 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, 84 SNP loci as set forth in Table 5 were obtained.
TABLE 5 DNA finger print of Fugu obscurus
8. And calculating the relationships among all samples.
Based on the 84 SNP loci of Table 5, the genetic distances between samples were calculated to make sample discrimination. For 84 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 Fugu obscurus of different geographical groups.
And constructing the DNA finger print of the fugu obscurus of different geographical groups according to the screened SNP loci. The DNA finger print of the fugu obscurus in different areas is as follows:
TABLE 6 DNA finger print of Fugu obscurus population
/>
TABLE 7 DNA finger print of Fugu obscurus of Guangdong population
/>
TABLE 8 DNA finger print of Fugu obscurus of Japanese population
/>
。
Example 2 actual sample detection
Taking a Fugu obscurus sample (D1) provided by a Jiangsu ocean group in Jiangsu, extracting DNA, taking a tail fin, extracting DNA by using a DNA extraction kit (FastPure Cell/Tissue DNA Isolation Mini Kit) of Novain company, measuring OD value and concentration according to a specification, running electrophoresis detection quality, and storing in a refrigerator at-20 ℃ for later use after meeting the requirements.
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.
Sequencing results show that the 84 core SNP loci of the sample have 81 finger prints which accord with Fugu obscurus in Jiangsu population, and the accord ratio is 96%. And for 84 loci, the genetic distances between the unknown samples and the three populations of Fugu obscurus known in the present invention were calculated using Plink software. The result of the cluster map shows that the unknown sample and Jiangsu groups are gathered into one type (figure 5), so that the sample is judged to be Fugu obscurus of the Jiangsu groups, and the sampling result is met.

Claims (10)

1. A DNA fingerprint of fugu obscurus, said DNA fingerprint comprising 84 SNP sites, said SNP sites and specific nucleotides therein being as follows:
。
2. use of the DNA fingerprint of takifugu obscurus according to claim 1 for identifying takifugu obscurus.
3. The identification method of the fugu obscurus is characterized by comprising the following steps of: detecting the nucleotide at 84 SNP loci in the genome DNA of the fish sample to be detected, comparing the nucleotide with the specific nucleotide at 84 SNP loci corresponding to the DNA fingerprint spectrum of the fugu obscurus in claim 1, and identifying the fish sample to be detected as the fugu obscurus when the coincidence rate is more than or equal to 95%.
4. The identification method of Fugu obscurus according to claim 3, wherein the fish sample to be tested is the tail fin tissue of fish.
5. The identification method of the fugu obscurus according to claim 3, wherein the identification method specifically 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 spectrum of the fugu obscurus defined in claim 1, and identifying the fish sample to be detected as the fugu obscurus population when the coincidence rate is more than or equal to 95%.
6. The method for identifying Fugu obscurus according to claim 5, wherein the step of identifying specific living areas of Fugu obscurus is further included after the fish sample to be tested is identified as the Fugu obscurus population.
7. The method for identifying the specific living areas of the fugu obscurus according to claim 6, wherein the step of identifying the specific living areas of the fugu obscurus is to calculate genetic distances among samples, and compare and analyze genetic relationship with the samples in the invention, and finally identify the specific living areas of the fugu obscurus as Jiangsu, guangdong or Japan.
8. The identification method of the fugu obscurus according to claim 7, wherein the DNA fingerprint of the fugu obscurus in the living area is:
。
9. the identification method of the fugu obscurus according to claim 7, wherein the DNA fingerprint of the fugu obscurus in the living area of guangdong is:
。
10. the method for identifying Fugu obscurus according to claim 7, wherein the DNA fingerprint of Fugu obscurus in the living area of Japan is:
。
CN202310666880.5A 2023-06-07 2023-06-07 Fugu obscurus DNA finger print and application thereof Pending CN116445632A (en)

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