Application of lateolabrax japonicus DNA fingerprint spectrum in lateolabrax japonicus identification

Abstract

The invention discloses application of a lateolabrax japonicus DNA fingerprint spectrum in lateolabrax japonicus identification. The lateolabrax japonicus DNA fingerprint spectrum comprises 546 specific SNP (Single Nucleotide Polymorphism) sites. The DNA fingerprint spectrum provided by the invention is obtained based on simplified genome sequencing, and when the DNA fingerprint spectrum is used for identifying lateolabrax japonicus, compared with a traditional method, a more objective, more accurate and more reliable identification result can be provided.

Description

Technical Field

[0001] The invention relates to identification of Lateolabrax japonicus, and in particular to identification of Lateolabrax japonicus by utilizing Lateolabrax japonicus DNA fingerprint spectrum. Background Art

[0002] Early variety identification methods mainly relied on morphological identification, which is time-consuming and easily affected by the environment. With the development of molecular biology, the emergence of molecular marker technology has provided a new means for variety identification. Molecular marker technology has the advantages of short cycle, no influence of environmental conditions, and high-throughput testing and analysis. It has been widely used in variety identification, seed purity identification, etc. With the advancement of biotechnology, molecular markers such as RFLP, AFLP, SSR, and SNP have been used to construct DNA fingerprint maps. SNP molecular markers are one of the recommended markers for constructing DNA fingerprint databases in the BMT molecular testing guidelines of the International Union for the Protection of New Varieties of Plants (UPOV). Compared with traditional markers, this technology has co-dominance, and the detection content is the difference in DNA sequence information, mainly di-allelic variation, easy to realize data integration and sharing, the site distribution density on the genome is higher and more uniform, SNP sites are distributed more in the intragenic region, increasing the probability of being associated with functional genes, easy to realize high-throughput detection, and low cost per data point. According to the typing results, SNP sites that are evenly distributed on chromosomes and have high polymorphism information are selected as core markers, and the core SNP markers are used to construct the variety DNA fingerprint map, providing an important basis for the authenticity and specificity identification of the variety. Summary of the invention

[0003] The invention provides application of a DNA fingerprint of Lateolabrax japonicus in identification of Lateolabrax japonicus.

[0004] The present invention constructs a DNA fingerprint of Lateolabrax japonicus, including 546 specific SNP sites. The SNP sites and the specific nucleotides therein are shown in Table 1.

[0005] Table 1:

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[0013] Advantages of the present invention:

[0014] The DNA fingerprint of Lateolabrax japonicus provided by the present invention includes 546 specific SNP sites. The DNA fingerprint provided by the present invention is obtained based on simplified genome sequencing, and when used for Lateolabrax japonicus identification, it can provide a more objective, more accurate and more reliable identification result compared with the traditional method. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a graph of the number of chromosome SNP markers.

[0016] Figure 2 It is the chromosome distribution map of the locus.

[0017] Figure 3 It's a DNA fingerprint.

[0018] Figure 4 It is a genetic similarity distribution map.

[0019] Figure 5-1 , 5-2 ,5-3 is the NJ clustering diagram of Lateolabrax japonicus. Specific embodiments

[0020] 1. Construction of DNA fingerprint of Lateolabrax japonicus

[0021] The present invention selects three different geographical groups of japonica seabass, including Zhejiang, Annex and Qingdao, 30 fish from each group, takes the tail fin, and soaks in alcohol to preserve the samples at room temperature. The samples are sent to Shijiazhuang Boredy Biotechnology Co., Ltd. for DNA extraction and sequencing, and the DNA fingerprint of japonica seabass is analyzed and constructed.

[0022] 1. DNA Sample Extraction

[0023] use The DNA extraction kit (magnetic bead method) is used to extract sample DNA. This kit uses the specific recognition and efficient binding of magnetic beads to DNA to separate and purify high-quality genomic DNA from the sample. The principle is that the magnetic beads embedded with special technology have a strong adsorption effect on nucleic acids under specific conditions. By changing the conditions, the magnetic beads can release the adsorbed nucleic acids, thereby achieving the purpose of separating and purifying nucleic acids. The entire operation process is safe and convenient, and can be operated manually or applied to high-throughput automated extraction platforms.

[0024] 2. GenoBaits Experiment

[0025] The technology uses biotin-labeled probes to hybridize with the target region of the genome in a liquid system to form a double strand. The molecules carrying biotin are adsorbed by streptavidin-coated magnetic beads to capture the target hybridized with the probe. The captured target library can be used or The sequencer was used for sequencing analysis.

[0026] DNA extraction

[0027] use DNA extraction kit (magnetic bead method) was used to extract sample DNA.

[0028] The extracted DNA samples were subjected to two tests: (1) 1% agarose gel electrophoresis was used to analyze the purity and integrity of the DNA; (2) Qubit was used to accurately quantify the DNA concentration. DNA that passed the quantitative quality test entered the next step of the experiment.

[0029] Library construction

[0030] DNALibrary Prep Kit uses enzyme digestion to construct DNA library. The library constructed by this kit can be directly used for high-throughput sequencing and can also be used for hybridization capture of target segments. End Repair Enzyme is used for fragmentation, and the broken DNA is repaired at the end and then connected to the A tail. Ultra DNA Ligase connects the DNA fragments with the sequencing adapter, and then uses carboxyl-modified magnetic beads ( The ligation product is purified by DNA Clean Beads. The ligation product is added with sequencing primers with barcodes and a high-fidelity PCR reaction system for PCR amplification. Different barcodes are used to distinguish different samples. The amplified product purified by carboxyl magnetic beads can be used for probe hybridization experiments.

[0031] Take 500ng of the constructed DNA library and add Panel, Block I and The Block II mixture was concentrated to dryness and added 2X Hyb Buffer and Hyb Buffer Enhancer, incubate at 65℃ for 2 hours to complete the hybridization reaction. DNA ProbeBeads, elution removes unbound DNA, retaining only The target DNA fragments of the panel hybridization are enriched and purified by PCR amplification to complete the construction of the hybridization capture library.

[0032] After the library is constructed, Qubit2.0 is used for preliminary quantification, and the effective concentration of the library is accurately quantified using the qPCR method to ensure the quality of the library. After the library is qualified, it enters the sequencing stage.

[0033] The sequencing data was filtered, and the clean reads after quality control were compared with the reference genome sequence using the software BWA (mem comparison mode). The clean reads were located on the reference genome by comparison. The genotype information on the core SNP sites was extracted, and the sample deletion and mutation statistics were performed based on the 546 SNP markers after filtering. The distribution of the 546 SNP markers shown in Table 1 on the chromosome after filtering was statistically analyzed.

[0034] The characteristic DNA fingerprints of 90 materials were constructed using the 546 SNP marker combinations shown in Table 1 after filtering. Figure 3 shown.

[0035] Based on 546 SNP markers, the genetic similarity of 90 materials was compared pairwise. The genetic similarity calculation formula is: GS = nij / Nij*100%, where nij is the number of SNP marker sites detected in both materials but with no genotype difference, and Nij is the number of SNP marker sites detected in both materials.

[0036] The distribution statistics of pairwise genetic similarity are shown in Figure 4 shown.

[0037] 2. Application of DNA fingerprint of Lateolabrax japonicus

[0038] Take fish samples and use a high-throughput DNA extraction kit - DNA extraction kit (magnetic bead method) was used to extract sample DNA.

[0039] Specific amplification primers for the 546 specific SNP sites in Table 1 were used for PCR reaction, sequencing, and Figure 3 The DNA fingerprint of the spotted sea bass shown in the figure is compared with the core SNP sites to determine whether the sample is spotted sea bass. Figure 5-1 , Figure 5-2 , Figure 5-3 The cluster diagrams were compared to determine the relationship between the samples and the Fujian (F1-F30), Qingdao (Q1-Q30) and Zhejiang (Z1-Z30) populations.

Claims

1. The application of DNA fingerprint of Lateolabrax japonicus in the identification of Lateolabrax japonicus is characterized by: The DNA fingerprint of L. japonicus includes 546 SNP sites, and the SNP sites and the specific nucleotides therein are as follows:

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