Pair of PCR (Polymerase Chain Reaction) primers and application thereof in identifying crassostrea gigas, Fujian oyster and hybrid thereof

Abstract

The invention relates to a pair of PCR (polymerase chain reaction) primers and application thereof in identification of crassostrea gigas, Fujian oysters and hybrids thereof, and belongs to the field of molecular biology, sequences of the primers are shown as SEQ ID NO.1-2, and the invention also provides application of the primers in identification of crassostrea gigas, Fujian oysters and hybrids thereof. According to the method, the crassostrea gigas, the Fujian oyster and the hybrid thereof can be identified, and the method is simple and convenient.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a pair of PCR primers and application thereof in identifying Crassostrea gigas, Crassostrea fujianensis and hybrids thereof. Background Art

[0002] China's diverse oyster species and wide morphological variation make accurate phenotypic identification difficult. Furthermore, incomplete reproductive isolation between oyster species often results in hybridization. The shellfish industry, in particular, frequently employs hybridization to cultivate superior varieties, significantly impacting the conservation, discovery, and utilization of shellfish germplasm. The long oyster and the Fujian oyster are closely related species, allowing for smooth forward and reverse hybridization, with the offspring exhibiting a degree of hybrid vigor. This has led to a large number of hybrid seedlings appearing on the market. Given the limited research foundation on biological traits such as genetic stability and environmental adaptability of hybrid seedlings, and the difficulty of distinguishing oyster species based solely on external morphological characteristics, there is an urgent need for testing methods to accurately identify hybrid individuals and guide relevant companies in making accurate production decisions. Furthermore, the indiscriminate use of hybrids can easily deplete local germplasm resources, severely impacting the full utilization and conservation of local biological resources. Against this backdrop, there is an urgent need to develop accurate and rapid identification technologies to meet these needs.

[0003] In recent years, scientists have developed many new technologies for identifying shellfish species using genetic material. These include barcode genes such as COI, 16S rDNA, 18S rDNA, and ITS. Although mitochondrial barcode genes can accurately identify long oysters and Fujian oysters, because oyster mitochondria are maternally inherited, the identification of hybrids requires reliance on nuclear genes. However, commonly used barcode genes in the cell nucleus cannot accurately distinguish between the two species. Currently, most approaches for identifying shellfish hybrids are based on the detection of single nucleotide polymorphisms (SNPs) and microsatellite (SSR) loci in the nuclear genome. Methods based on SNP detection identify single nucleotide variants that differ significantly between species (CN104046683B). This method requires a sufficient sample size for statistical validation, and the SNP detection method is relatively cumbersome. In addition, there are detection methods based on microsatellite variant sites (CN105624307B, CN108220456B). This method requires designing primers targeting interspecies conserved sequences on both sides of the microsatellite site. Because microsatellite sites often exhibit length variation between individuals and the sequence length difference between the two species is small, it is easy to cause misjudgment. Therefore, existing technologies cannot simultaneously distinguish between the long oyster, the Fujian oyster, and their hybrids. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a pair of PCR primers and their use in identifying the long oyster, the Fujian oyster, and their hybrids. Based on the structural variation (SV) in the genomes of these species, a species identification marker is developed, providing a simple, efficient, and accurate method for distinguishing the long oyster, the Fujian oyster, and their hybrids. This method solves the technical problem of the difficulty in identifying the long oyster, the Fujian oyster, and their hybrids.

[0005] The present invention is achieved through the following technical solutions:

[0006] A pair of PCR primers, wherein the primers are: F: 5'-TCAATGTGACCTCTGATGGCT-3' (SEQ ID NO. 1), R: 5'-GCCTTCATCGTGAATGCTGAC-3' (SEQ ID NO. 2).

[0007] The present invention also provides an application of the primer pair in identifying Crassostrea gigas, Crassostrea fujianensis, and their hybrids, wherein the application method is as follows:

[0008] Step 1: extract genomic DNA from oyster samples, use it as a template, and perform PCR amplification using the primers.

[0009] Step 2: The amplified product is subjected to agarose gel electrophoresis, and typing identification can be performed based on the difference in band positions displayed by gel imaging: if the band position shows a sequence size of 266 bp, the oyster sample is a long oyster; if the band position shows a sequence size of 537 bp, the oyster sample is a Fujian oyster; if there are bands in both the 266 bp and 537 bp regions, the oyster sample is a hybrid of a long oyster and a Fujian oyster.

[0010] Furthermore, the PCR reaction system and reaction procedure are as follows:

[0011] The 25 μL PCR amplification system is as follows: 12.5 μL of 2× RapidTaq MasterMix, 1 μL of each 10 μM upstream and downstream primers, 50-500 ng of DNA template, and ddH2O is used to make up to 25 μL.

[0012] The amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 15 s. The three steps of denaturation and extension were repeated 30 times, and extension was performed at 72°C for 5 min.

[0013] The beneficial effects of the present invention compared with the prior art are as follows: the method of the present invention can distinguish between Crassostrea gigas, Crassostrea fujianensis and their hybrids, and the method is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the electrophoresis result of PCR amplification of DNA mixture of long oyster, Fujian oyster, and long oyster and Fujian oyster using specific primers: gel well 1 is DL2000, gel wells 2 and 3 are long oyster, gel wells 4 and 5 are Fujian oyster, gel wells 6 and 7 are the amplified products of the mixed DNA of long oyster and Fujian oyster.

[0015] Figure 2 The electrophoresis results of PCR amplification of DNA from Crassostrea gigas, Crassostrea fujianensis, a mixture of Crassostrea gigas and Crassostrea fujianensis, and a hybrid of Crassostrea gigas and Crassostrea fujianensis using specific primers: gel well 1 is DL2000, gel well 2 is Crassostrea gigas, gel well 3 is Crassostrea fujianensis, gel well 4 is the amplified product of the mixed DNA of Crassostrea gigas and Crassostrea fujianensis, and gel wells 5, 6, and 7 are the hybrid of Crassostrea gigas and Crassostrea fujianensis. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below with reference to specific examples and accompanying drawings. Unless otherwise specified, the materials, reagents, and molecular marker probes used in the following examples can all be purchased from the company through commercial channels.

[0017] The following description is intended to further illustrate the present invention, rather than to limit it:

[0018] Example 1 Primer design and screening process

[0019] Bioinformatics analysis based on the third-generation whole-genome resequencing data of Fujian oysters showed that the resequenced genome of Fujian oysters had 178,471 structural variations compared with the reference genome of long oysters. Among them, 36,057 overlapped with genes in the long oyster genome annotation file, of which 15,340 were located in the exon region, which may directly affect the function of the gene. The insertion and deletion sites in genes such as RPLs11, RPS35, RPS5, and RPLP0 were screened as candidates. Through multiple experimental verifications of primers designed on both sides of the insertion and deletion, the RPLP0 primer pair was finally selected. In this region, the long oyster has a stable 271bp structural deletion compared with the Fujian oyster, and the electrophoresis bands are obviously different and easy to distinguish.

[0020] Example 2

[0021] Differentiation and identification of Crassostrea gigas and Crassostrea fujianensis using primers screened in Example 1

[0022] 1) Using the DNE29 universal genomic DNA extraction kit (Nobel Biotech), genomic DNA of Crassostrea gigas and Crassostrea fujianensis was extracted according to the instructions:

[0023] Take 5-30 mg of fresh oyster tissue and place it in a 1.5 mL centrifuge tube. Add 200 μL of Buffer SL and 20 μL of Proteinase K (20 mg / ml). Digest at 56°C for 3 h, vortexing for 15 min / time. Add 4 μL of RNase A (25 mg / ml) and let it stand at room temperature for 5 min. Add 220 μL of Buffer VL, vortex and treat at 70°C for 10 min. Centrifuge at 13,400 × g for 2 min and transfer the supernatant to a new centrifuge tube to remove impurities and prevent clogging of the column. Add 220 μL of anhydrous ethanol to the supernatant, vortex and transfer to an adsorption column loaded in a collection tube. Centrifuge at 13,400 × g for 1 min and discard the waste liquid. Add 500 μL of Centrifuge Buffer WB1 at 13,400×g for 1 min and discard the waste liquid; add 600μL of Buffer WB2 with anhydrous ethanol and centrifuge at 13,400×g for 1 min and discard the waste liquid; repeat the addition of 600μL of Buffer WB2 with anhydrous ethanol and centrifuge at 13,400×g for 1 min and discard the waste liquid; place the adsorption column in an empty collection tube and centrifuge at 13,400×g for 2 min; take only the adsorption column and place it in a new collection tube, open the lid for 3 minutes to evaporate the ethanol; drop 50μL of Buffer EB onto the central adsorption membrane and let it stand for 5 minutes, then centrifuge at 13,400×g for 1 min to obtain the DNA solution.

[0024] 2) The concentration and purity of the DNA solution were measured using a Nanodrop micro-spectrophotometer. PCR amplification was performed using the DNA of Crassostrea gigas, the DNA of Fujian oysters, and a mixture of the DNA of Crassostrea gigas and Fujian oysters (1:1) as templates, referring to the instructions for use of the 2× Rapid Taq MasterMix product from Novozymes.

[0025] The 25 μL PCR amplification system was as follows: 2× Rapid Taq Master Mix 12.5 μL, Primer 1 (10 μM) 1 μL, Primer 1 (10 μM) 1 μL, DNA template 50-500 ng, and made up to 25 μL with ddH2O.

[0026] The specific primer sequences are as follows:

[0027] Upstream primer F: 5'-TCAATGTGACCTCTGATGGCT-3'

[0028] Downstream primer R: 5'-GCCTTCATCGTGAATGCTGAC-3'

[0029] The amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 15 s. The three steps of denaturation and extension were repeated 30 times, and extension was performed at 72°C for 5 min.

[0030] 3) Take 5 μL of the PCR amplification product and run it on 1.2% agarose gel electrophoresis. The gel imaging results are as follows: Figure 1 As shown, gel well 1 is DL2000, gel wells 2 and 3 are long oysters, and the band position shows a sequence size of 266bp; gel wells 4 and 5 are Fujian oysters, and the band position shows a sequence size of 537bp; gel wells 6 and 7 are mixed DNA amplification products of long oysters and Fujian oysters, with bands in the 266bp and 537bp regions.

[0031] Example 2

[0032] Referring to the operation method of Example 1, genomic DNA of long oyster, Fujian oyster, and hybrid of long oyster and Fujian oyster was extracted. After the concentration and purity were determined by Nanodrop, PCR amplification was performed on the long oyster DNA, Fujian oyster DNA, a mixture of long oyster and Fujian oyster DNA (1:1), and hybrid DNA of long oyster and Fujian oyster using the primers. The electrophoresis results are as follows: Figure 2 As shown, gel well 1 is DL2000, gel well 2 is long oyster, and the band position shows that the sequence size is 266bp; gel well 3 is Fujian oyster, and the band position shows that the sequence size is 537bp; gel well 4 is the mixed DNA amplification product of long oyster and Fujian oyster, with bands in the 266bp and 537bp regions; gel wells 5, 6, and 7 are hybrids of long oyster and Fujian oyster, with bands in the 266bp and 537bp regions.

Claims

1. A pair of PCR primers, characterized in that: The primers are: F: 5'-TCAATGTGACCTCTGATGGCT-3', R: 5'-GCCTTCATCGTGAATGCTGAC-3'.

2. Use of the primers according to claim 1 in identifying Crassostrea gigas, Crassostrea fujianensis and their hybrids, characterized in that: The application method is: Step 1, extracting genomic DNA from oyster samples, using it as a template, and performing PCR amplification using the primers; Step 2: The amplified product is subjected to agarose gel electrophoresis, and typing identification can be performed based on the difference in band positions displayed by gel imaging: if the band position shows a sequence size of 266 bp, the oyster sample is a long oyster; if the band position shows a sequence size of 537 bp, the oyster sample is a Fujian oyster; if there are bands in both the 266 bp and 537 bp regions, the oyster sample is a hybrid of a long oyster and a Fujian oyster.

3. The use according to claim 2, characterized in that The PCR reaction system and reaction procedure are as follows: 25 μL of PCR amplification system is as follows: 12.5 μL of 2×RapidTaq Master Mix, 1 μL of each of 10 μM upstream and downstream primers, 50-500 ng of DNA template, and ddH2O is used to make up to 25 μL.

4. The use according to claim 2, characterized in that The amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 15 s. The three steps of denaturation and extension were repeated 30 times, and extension was performed at 72°C for 5 min.

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