CN-122024830-A - Method for accurately evaluating germplasm purity of crassostrea gigas

Abstract

The invention discloses a method for accurately evaluating the germplasm purity of crassostrea gigas, which relates to the technical field of aquatic genetic breeding biology and has the core that indexes are built based on NCBI crassostrea gigas genome sequences by means of a full genome resequencing technology, 150 crassostrea gigas and Fujian oysters are collected as natural populations, outer membrane DNA sequencing is extracted, 635 specific SNP loci are screened out, pure characteristic sequences are built, 2 detection sequences of 635 loci of a sample to be tested are compared with the characteristic sequences after sequencing, and a consistency average value is calculated according to the same base number/sequence length multiplied by 100%, so that the germplasm purity value is obtained. The method fills the blank that the prior art cannot quantitatively evaluate, has high accuracy and high reliability, and can provide key technical support for crassostrea gigas breeding improvement, germplasm protection, industry standardization and the like.

Inventors

• QI HAIGANG
• CONG RIHAO
• ZHANG TAIPING
• Pang Meiqian
• WANG CHAOGANG
• LIU MINGKUN
• LI LI
• ZHANG GUOFAN

Assignees

• 中国科学院海洋研究所

Dates

Publication Date

20260512

Application Date

20260202

Claims (7)

1. 1. A method for accurately evaluating the purity of crassostrea gigas germplasm, which is characterized in that: Firstly, constructing indexes by respectively adopting bwa program, samtools program and gatk program of bioinformatics software based on a crassostrea gigas genome sequence GCA_025765685.3 in an NCBI database; Collecting crassostrea gigas of natural population and crassostrea gigas of natural population respectively, extracting genome DNA from mantle tissue of oyster individual, and finally carrying out whole genome resequencing; Step three, adopting fastp program for filtering the sequencing data; Comparing the re-sequencing sequences of the crassostrea gigas and the crassostrea gigas by adopting bwa program to obtain crassostrea gigas genome C.gigas, and constructing an index for the bam file by adopting samtools program; Generating a genome variation vcf file by adopting a gatk program based on the bam file; step six, counting the allele frequency of the mononucleotide variation site by adopting a vcftools program, and then identifying specific alleles in the crassostrea gigas; step seven, 635 crassostrea gigas single nucleotide polymorphism specific alleles are obtained; step eight, extracting genome DNA from mantle tissue of oyster individuals to be detected, and carrying out whole genome re-sequencing; and step nine, comparing the sequencing sequence of the oyster to be detected with the crassostrea gigas pure-breed characteristic sequence, then respectively calculating the consistency values of the 2 detection sequences and the crassostrea gigas pure-breed characteristic sequence according to a preset formula, and finally taking the average value of the two consistency values as the crassostrea gigas germplasm purity value of the sample to be detected.
2. 2. A method for accurately assessing the purity of crassostrea gigas germplasm according to claim 1, characterized in that the amount of sequencing data of each oyster individual is not less than 12G.
3. 3. A method for accurately assessing the purity of crassostrea gigas germplasm according to claim 1, characterized in that said screening criteria for specific alleles is that the frequency of such alleles in crassostrea gigas populations is greater than 96%.
4. 4. A method for accurately assessing the purity of crassostrea gigas germplasm according to claim 1, characterized in that said screening criteria for specific alleles is that the frequency of such alleles in the Fujian oyster population is less than 5%.
5. 5. A method for precisely assessing the purity of a crassostrea gigas germplasm according to claim 1 wherein genomic single nucleotide polymorphic sites are identified according to steps three-five above and then 2 alleles of 635 sites in step 5 in each sample are organized into 2 test sequences.
6. 6. The method for precisely assessing the purity of crassostrea gigas according to claim 1, wherein the sample sizes of crassostrea gigas of the natural population and crassostrea gigas of the natural population are 150.
7. 7. A method for precisely assessing the purity of a crassostrea gigas germplasm according to claim 1 wherein the formula is sequence identity = number of identical bases/(sequence length x 100%).

Description

Method for accurately evaluating germplasm purity of crassostrea gigas Technical Field The invention relates to the technical field of aquatic genetic breeding biology, in particular to a method for accurately evaluating the germplasm purity of crassostrea gigas. Background The crassostrea gigas is used as shellfish cultivated on a global scale, and is more a main cultivated oyster species in northern coastal areas of China, and is naturally distributed in the pacific ocean areas of northwest, including coastal areas of northern China, korean, japanese and the like. The crassostrea gigas is rich in nutrition and high in economic value, and is a high-quality seafood which can be directly eaten. As one of oyster species with the largest cultivation standard in China, the current crassostrea gigas cultivation almost completely depends on the supply of artificially bred offspring. In order to enhance the adaptability of crassostrea gigas fries to high temperature, means such as hybridization with Fujian oysters in the south of China, backcrossing and the like are often adopted to improve the properties of crassostrea gigas fries in the artificial breeding process. Along with the continuous deep progress of crassostrea gigas genetic breeding work and the increase of diversified quality cultivation requirements, the precise control of genetic background in the breeding process becomes more important. In order to ensure accurate and controllable genetic breeding process, and ensure stable characters and breeding suitability of the cultured strain, it is highly desirable to establish a crassostrea gigas germplasm purity assessment method to promote crassostrea gigas genetic breeding, offspring seed breeding and high quality development of related industries. On the other hand, in view of the fact that the artificial breeding area of crassostrea gigas is adjacent to the naturally distributed sea area, a crassostrea gigas germplasm fine evaluation technology is also urgently needed to monitor the influence of exogenous gene flows on local natural populations, so that technical support is provided for evaluating and protecting genetic diversity of crassostrea gigas natural populations. At present, no technical method for accurately evaluating the purity of crassostrea gigas germplasm exists, and only oyster species identification technology based on oyster genetic or genetic information exists, which mainly comprises the following steps: According to the sequence difference of the mitochondrial COX1 genes, the oysters of different species such as crassostrea gigas, fujian oysters, crassostrea gigas, hong Kong oysters and the like are distinguished, and the oysters of different species are essentially identified, and the germplasm purity or blood purity evaluation of the oysters of a single species is not involved. Part of the technology can realize the identification of the first-generation oysters of the hybrid between the crassostrea gigas and other oyster species (such as Fujian oysters) by means of a small amount of molecular markers, and does not involve the fine quantitative evaluation of the germplasm purity of the crassostrea gigas. Based on the above, the invention screens a large number of alleles of high-reliability specific single nucleotide polymorphic sites within the whole genome range of the crassostrea gigas by means of genome high coverage resequencing, counts the number of the alleles of the specific single nucleotide polymorphic sites in crassostrea gigas individuals, and realizes high-efficiency and accurate quantitative evaluation of crassostrea gigas germplasm purity by calculating the proportion of alleles of pure ancestral sites in the crassostrea gigas genome. Disclosure of Invention Aiming at the problems, the invention provides a method for accurately evaluating the purity of crassostrea gigas germplasm, which has the core thought that by means of a genome high coverage resequencing technology, a large number of high-reliability alleles of specific single nucleotide polymorphic sites are screened in the whole genome range of crassostrea gigas, and the blood lineage proportion of crassostrea gigas specific alleles is calculated by counting the number of crassostrea gigas pure specific SNP site alleles in an individual to be detected, so that the accurate quantitative evaluation of the purity of crassostrea gigas germplasm is realized. The single nucleotide polymorphism refers to DNA sequence polymorphism caused by single nucleotide variation on genome level, has the characteristics of large quantity, wide distribution, high stability, easy detection and the like, is the most commonly used molecular marker in the current genetic breeding and germplasm identification, and screens out specific SNP sites which only occur at high frequency in crassostrea gigas and at low frequency or not occur in crassostrea gigas by comparing the whole genome sequences of crassostrea gigas and related species (F