InDel labeled primer of crassostrea gigas Haida No.3 and application of InDel labeled primer

Abstract

The invention relates to an InDel marker primer for identifying crassostrea gigas' Haida No.3 'and application of the InDel marker primer, and belongs to the field of molecular biology. The invention provides a method for identifying the specific InDel marker of the Haida No.3 group by using the primer, DNA of a crassostrea gigas sample is extracted by adopting an SDS method, the marker primer is used for carrying out PCR amplification on the sample to be detected, and the specific InDel marker site of the Haida No.3 group is detected; the determination standard is as follows: the amplified fragment length of the 'Haida No.3' crassostrea gigas is 236bp, and the amplified fragment length of the crassostrea gigas of other strains and wild populations is 274bp. Technical support is provided for paternity identification and germplasm resource protection of crassostrea gigas' Haida No.3 '.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and particularly relates to an InDel marker primer of Crassostrea gigas "Haida No.3" and application thereof. BACKGROUND

[0002] Oyster is the most widely cultured and highest yield marine shellfish in the world, and plays a very important role in China's mariculture. In 2019, the new oyster variety Crassostrea gigas "Haida No.3" (variety registration number: GS-01-007-2018) was approved by the National Aquatic Germplasm and Breeding Committee. Crassostrea gigas "Haida No.3" takes shell black and growth rate as target traits, adopts hybrid breeding technology combining family selection and population selection, and is bred for 6 generations. It is suitable for cultivation in northern coastal areas such as Shandong and Liaoning. Under the same cultivation conditions, the average shell height of 10-month-old oysters increases by 32.9%, and the average soft body weight increases by 64.5%. The left and right shells and mantle are black, which has significant economic benefits. However, the inter-strain hybridization method can breed Crassostrea gigas individuals similar to "Haida No.3" in appearance, which will affect the purity of "Haida No.3" and cause degeneration of germplasm. In addition, the surface attachments of oysters also affect the identification of germplasm. Therefore, it is of great scientific significance and application value to develop a molecular marker that can be used to quickly identify Crassostrea gigas "Haida No.3". It not only can standardize the market and protect the rights and interests of seedling enterprises, but also has important significance for germplasm resource protection and management.

[0003] InDel molecular fingerprint markers have significant advantages over other molecular markers in the identification of variety resources due to their high polymorphism, convenient detection, intuitive results, and good stability. Although InDel markers have been widely used in other species, there is no InDel marker that can be used to quickly and accurately identify Crassostrea gigas "Haida No.3". SUMMARY

[0004] The technical problem to be solved by the present application is to provide an InDel marker and corresponding primer for identifying Crassostrea gigas "Haida No.3". The application of the marker and corresponding primer can successfully identify Crassostrea gigas "Haida No.3".

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] An InDel marker and corresponding primer for identifying Crassostrea gigas "Haida No.3", the marker and corresponding primer are shown in Table 1:

[0007] Table 1 InDel marker and corresponding primer for identifying Crassostrea gigas "Haida No.3"

[0008]

[0009] The application provides a method for identifying Crassostrea gigas "Haida No.3" using the above-mentioned primer, and the method specifically comprises the following steps:

[0010] DNA of the Crassostrea gigas sample is extracted by the SDS method, and the DNA to be tested is subjected to PCR amplification using the marker primer. The PCR reaction system is 10 μL, which comprises 0.5 μL of each of the outer primers SBF and SBR, 1 μL of DNA template, 5 μL of 2x PCR master mix, and ddH2O is supplemented to 10 μL. The amplification condition is pre-denaturation: 95℃, 2 min; denaturation: 95℃, 15 s; annealing: 58℃, 20 s; extension: 72℃, 15 s; final extension: 72℃, 4 min; cycle number 35; storage: 4℃, ∞. The PCR product is detected by 1.8% agarose gel electrophoresis at 100 V for 45 min to detect the fragment size. According to the fragment size, it is judged whether it is the Crassostrea gigas "Haida No.3" population; the determination standard is that if the amplification fragment size is 236 bp, it is the Crassostrea gigas "Haida No.3" population; and if the amplification fragment size is 274 bp, it is other strains or wild Crassostrea gigas population.

[0011] Compared with the prior art, the application has the following beneficial effects:

[0012] The application mines the specific InDel site of the Crassostrea gigas "Haida No.3" by using the resequencing data of seven representative Crassostrea gigas breeding strains and wild populations, and designs a primer based on the InDel sequence, which can be used for quickly identifying the Crassostrea gigas "Haida No.3". Compared with the traditional molecular marker identification method, the primer does not need to be subjected to Sanger sequencing or fluorescence detection of the amplification fragment, and can be directly and intuitively identified by electrophoretogram, thereby providing technical support for parentage identification and germplasm resource protection of the Crassostrea gigas "Haida No.3". BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The application method is applied to identification of 160 Crassostrea gigas from different sources. DETAILED DESCRIPTION

[0014] The technical solutions of the application are further explained by the following examples, but the protection scope of the application is not limited in any form by the examples.

[0015] Example 1

[0016] 1. Specific InDel marker of Crassostrea gigas "Haida No.3"

[0017] 1.1 Sample selection

[0018] Collect 30 individuals of each of "Haida No. 1", "Haida No. 2", "Haida No. 3", Japanese strain, Korean strain, shell orange strain, shell white strain and wild population, a total of 240 individuals.

[0019] 1.2 Genomic DNA extraction

[0020] Collect the adductor muscle and freeze it using liquid nitrogen, then store it at -80℃. The adductor muscle tissue of each individual is ground at 4℃ using a high-speed cryogenic grinder, then digested overnight using PK enzyme, and then high-quality DNA of each individual is extracted using the standard phenol chloroform method. The concentration is determined by Qubit 4.0 fluorescence quantifier, the quality is identified by agarose electrophoresis, and the absorbance value is determined by NanoDrop 2000, to ensure that the DNA concentration is greater than 50 ng / μL, the total amount is greater than 3 μg, and OD260 / OD280 is between 1.6 and 1.8.

[0021] 1.3 Library construction and high-throughput sequencing

[0022] The qualified DNA sample is randomly broken into fragments with a length of 300bp-500bp using transposase and the sequencing adapter is connected to the enzyme digestion product, then purified by magnetic beads, PCR amplified and library size sorted to complete the construction of the sequencing library. The constructed library is preliminarily quantified by Qubit 4.0 fluorescence quantifier, then the library fragment size is detected by Agilent 2100 bioanalyzer, and after the fragment size meets the expectation, the library is quantified by qPCR. The "pair-end 150" sequencing strategy is adopted, and the whole genome resequencing is performed on the Illumina high-throughput sequencing platform. The average insert length of the library is 350bp, and 150bp double-end sequencing is performed, and 6Gb of sequencing data is generated for each individual.

[0023] 1.4 Sequencing data quality control and alignment

[0024] The raw sequencing data is quality controlled using fastp 0.20.1 tool, and bases with a quality value less than 30 are considered to be low-quality bases, and reads with 40% of the bases having a quality value less than 30 are filtered out, the purpose being to filter low-quality reads; sliding trimming is performed with 4 bases as a sliding window, and windows with an average quality less than 20 are removed, the purpose being to filter low-quality bases; if reads 1 and reads 2 have overlapping regions and one of them has a higher quality, then the read with higher quality is used to correct the other. In addition, fastp runs the adapter removal program by default to trim the sequencing adapter sequence.

[0025] 1.5 InDel identification

[0026] InDel typing was performed using GATK 4.1.9.0, generating GVCF files for each individual by the HaplotypeCaller module, and then jointly analyzing these GVCFs using the GenotypeGVCFs module to improve the accuracy of typing. In order to minimize the error rate, the InDel was hard filtered using the VariantFiltration protocol, and the parameters used were as follows: QD>2.0, FS<200.0, SOR<10.0, MQRankSum>-12.5, ReadPosRankSum>-8.0, minimumdepth>3. The obtained InDel genotypes were then filtered using the VCFtools 0.1.13 program according to the standard of minor allele frequency greater than 0.05 and deletion rate less than 10%.

[0027] 1.6 Screening of "Haida No. 3" specific InDel markers

[0028] Based on the filtered InDel dataset, the global Fst value of "Haida No. 3" and other populations was calculated using VCFtools, with 0.9 as the threshold value of Fst. In order to visually read the amplified fragments in polyacrylamide and agarose gels, InDels with a length greater than 20 bp were further screened as candidate sites, and finally one strain-specific InDel molecular marker site was selected, located at chromosome NC047567.1 18,524,258bp to 18,524,296bp of NCBI genome version GCF_902806645.1, with a length of 38 bp, and the sequence information is CAGAGGTTTACCTTCTTTAAGGTACACCTAAGACCCTA.

[0029] 1.7 Primer design

[0030] The sequence of 88 bp upstream and 148 bp downstream of the INDEL site was extracted from the reference genome as a template. The primers were designed using the NCBI website (https: / / www.ncbi.nlm.nih.gov) according to the following standards: primer length, 18-24 bp; GC content, 40-60%; theoretical melting temperature (Tm), 58-62℃. The final primer sequence is: upstream primer GAGAGCATCGGTGCACACT, downstream primer ACAGAACCCTTAATACATGGC.

[0031] 2 Verification of "Haida No. 3" specific InDel markers in Crassostrea gigas

[0032] 2.1 Selection of verification population

[0033] Select and mark the non-full sibling and non-half sibling of "Haida No. 1", "Haida No. 2", "Haida No. 3" and Japanese strain, Korean strain, shell orange strain, shell white strain, wild population, 20 individuals in each group, a total of 160 individuals.

[0034] 2.2 InDel PCR amplification detection

[0035] High-quality DNA of 160 individuals was extracted using the standard phenol chloroform method, and the DNA of the samples to be tested was amplified by PCR using the marker primer. The PCR amplification procedure was as follows: the PCR reaction system was 10 μL, including 0.5 μL of each of the outer primers SBF and SBR, 1 μL of DNA template, 5 μL of 2x PCR master mix, and ddH2O was added to 10 μL. The amplification conditions were pre-denaturation: 95℃, 2min; denaturation: 95℃, 15s; annealing: 58℃, 20s; extension: 72℃, 15s; final extension: 72℃, 4min; cycle number 35; storage: 4℃, ∞. The PCR product was detected by 1.8% agarose gel electrophoresis at 100V for 45min, and 20bp DNA ladder was used as a fragment size indicator marker. After electrophoresis, the gel was placed in a full-automatic gel imaging analysis system for photographing and preservation.

[0036] 2.3 Identification standard and application of "Haida No. 3" Pinctada martensii

[0037] The determination standard is: the PCR amplification fragment size is 236bp, which is "Haida No. 3" Pinctada martensii population; if the amplification fragment size is 274bp, it is other strain or wild Pinctada martensii population.

[0038] Actual application: PCR detection was performed on 160 individuals of the verification population (as shown in Figure 1 The results showed that the amplification fragment length of "Haida No. 3" Pinctada martensii population was around 236bp, and the amplification fragment of the other 7 representative strains of Pinctada martensii and wild population was around 274bp. The amplification fragment length of "Haida No. 3" Pinctada martensii population was significantly smaller than that of other populations, and the detection success rate was 100%.

Claims

1. An InDel marker for identifying the long oyster "Haida No. 3", characterized in that The InDel marker is a deletion sequence specific to the long oyster "Haida No. 3", located from 18,524,258bp to 18,524,296bp from the 5' end of the long oyster international genome version GCF_902806645.1 chromosome number NC 047567.1, with a length of 38bp and a sequence of CAGAGGTTTACCTTCTTTAAGGTACACCTAAGACCCTA.

2. A primer for detecting the InDel marker according to claim 1, characterized in that: The upstream primer nucleotide sequence of the primer is GAGAGCATCGGTGCACACT, and the downstream primer nucleotide sequence is ACAGAACCCTTAATACATGGC.

3. According to claim 2, the method for identifying the "Haida No. 3" long oyster population using InDel-labeled primers is characterized by: The DNA of the long oyster sample was extracted by SDS method, and the labeled primers were used to perform PCR amplification on the test sample. The fragment size of the amplified product was detected by 1.8% agarose gel electrophoresis to determine whether it was the "Haida No. 3" long oyster population.

4. The method according to claim 3, characterized in that If the amplified fragment size is 236bp, it is the "Haida No. 3" long oyster population; if the amplified fragment size is 274bp, it is other strains or wild long oyster populations.

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