A snp molecular marker combination for identifying or distinguishing grouper germplasm resources and application thereof

Abstract

The present application belongs to the technical field of molecular biology, and particularly relates to a SNP molecular marker combination for identifying or distinguishing grouper germplasm resources and application thereof. Genomic DNA of golden money grouper, tiger grouper and golden tiger hybrid grouper is extracted, and 177 common SNP sites are obtained after detection, and specific position information is shown in Table 1 of the specification. The SNP molecular marker combination screened by the present application can be developed into primers, probes or gene chips, and through simple phylogenetic tree analysis, golden money grouper, tiger grouper and golden tiger hybrid grouper can be quickly and accurately distinguished, and unknown detection samples can also be subjected to genetic phylogenetic tree analysis with known golden money grouper, tiger grouper and golden tiger hybrid grouper, so that it can be quickly and accurately identified which kind of grouper the unknown detection sample belongs to, and the present application has the characteristics of simple operation, high efficiency, high accuracy and the like, and has important significance in grouper germplasm resource protection and utilization and grouper genetic breeding work.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a combination of SNP molecular markers for identifying or distinguishing grouper germplasm resources and their applications. Background Technology

[0002] Grouper is a valuable marine fish species with high economic value. The golden grouper and tiger grouper both hold important positions in grouper breeding and aquaculture. The golden grouper has a light grayish-brown body covered with dark brown spots. The tiger grouper has a light yellowish-brown body covered with black spots. The hybrid golden tiger grouper is brown in color, similar in size to the golden grouper, and also has brown spots on its body. Although the three types of grouper differ somewhat in phenotypic characteristics, grouper body color can change with external environmental factors. Furthermore, there are significant differences in morphology and body color between juvenile and adult grouper. These factors can all lead to inaccurate results when identifying species solely based on morphological characteristics.

[0003] Single nucleotide polymorphisms (SNPs) have become major molecular markers due to their wide distribution, large number, genetic stability, and ease of high-throughput automated detection. Although SNP markers are widely used in many animals and plants, their application in the identification and differentiation of grouper germplasm resources is still in its early stages. Therefore, screening for SNP markers that can identify or differentiate between grouper species such as the golden grouper, tiger grouper, and golden-tiger hybrid grouper is of great significance and has broad application prospects for the utilization and conservation of grouper germplasm resources. Summary of the Invention

[0004] The purpose of this invention is to provide a combination of SNP molecular markers for identifying or distinguishing grouper germplasm resources and its application, to quickly distinguish between the golden grouper and the tiger grouper and their hybrid offspring (golden tiger hybrid grouper), to achieve rapid and accurate identification of the three grouper species, and to solve the problem of inaccurate results in grouper species identification.

[0005] This invention provides a combination of SNP molecular markers for identifying or differentiating grouper germplasm resources. The site information of the SNP molecular marker combination is shown in Table 1 of the specification. The grouper includes the golden grouper, tiger grouper, and golden tiger hybrid grouper.

[0006] This invention provides reagents for targeting the SNP molecular marker combinations described in the above-mentioned technical solutions, wherein the reagents include primers and / or probes.

[0007] This invention provides a chip for identifying or differentiating grouper germplasm resources, including the reagents described in the above technical solution, wherein the grouper includes the golden grouper, tiger grouper, and golden tiger hybrid grouper.

[0008] Preferably, the chip is a liquid phase chip.

[0009] This invention provides a kit for identifying or differentiating grouper germplasm resources, including the chip described in the above technical solution, wherein the grouper includes the golden grouper, tiger grouper and golden tiger hybrid grouper.

[0010] This invention provides the application of the reagent, chip, or kit described in the above-described technical solutions in one or more of the following:

[0011] (1) Identification of grouper germplasm resources;

[0012] (2) Differentiate grouper germplasm resources;

[0013] (3) Grouper-assisted breeding;

[0014] The grouper mentioned includes the golden grouper, tiger grouper, and hybrid golden tiger grouper.

[0015] Preferably, the golden tiger hybrid uses tiger stripe as the female parent and golden coin stripe as the male parent.

[0016] This invention provides a method for identifying grouper germplasm resources, comprising the following steps: extracting genomic DNA from the grouper to be identified, extracting site information corresponding to the SNP molecular marker combinations described in the above technical solution, performing phylogenetic tree analysis with known grouper species such as the money grouper, tiger grouper, and golden tiger hybrid grouper, and determining which of the money grouper, tiger grouper, and golden tiger hybrid grouper the grouper to be identified belongs to.

[0017] This invention provides a method for distinguishing three types of grouper germplasm resources, comprising the following steps:

[0018] Genomic DNA was extracted from grouper, and site information corresponding to the SNP molecular marker combinations described in the above technical solution was extracted. A phylogenetic tree was constructed using the maximum likelihood method, and the aggregation results were observed to distinguish three types of grouper. The three types of grouper are the money grouper, the tiger grouper, and the golden tiger hybrid grouper.

[0019] Preferably, the genomic DNA is genomic DNA from the tail fin tissue.

[0020] Beneficial effects:

[0021] This invention extracts genomic DNA from the grouper species *S. coinina*, *S. tigerina*, and *S. spp.* hybrid, and detects 177 shared SNP loci, the specific locations of which are shown in Table 1 of the specification. The SNP molecular marker combinations screened using this invention can be developed into primers, probes, or gene chips. Through simple phylogenetic tree analysis, these three grouper species can be quickly and accurately distinguished. Furthermore, phylogenetic tree analysis can be performed between unknown samples and known *S. coinina*, *S. tigerina*, and *S. spp.* hybrids to quickly and accurately identify which of the three grouper species the unknown sample belongs to. This method is characterized by its simplicity, efficiency, and high accuracy, and is of great significance in the protection and utilization of grouper germplasm resources and in grouper genetic breeding. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 A phylogenetic tree based on 177 SNPs was constructed to screen samples of money-spotted, tiger-spotted, and golden-tiger hybrid spots.

[0024] Figure 2 To validate the phylogenetic tree constructed based on 177 SNPs from samples of money-spotted, tiger-spotted, and golden-tiger hybrids. Detailed Implementation

[0025] This invention provides a combination of SNP molecular markers for identifying or differentiating grouper germplasm resources. The site information of the SNP molecular marker combination is shown in Table 1 of the specification. The grouper includes the golden grouper, tiger grouper, and golden tiger hybrid grouper.

[0026] This invention provides reagents for targeting the SNP molecular marker combinations described in the above-mentioned technical solutions, wherein the reagents include primers and / or probes.

[0027] This invention provides a chip for identifying or differentiating grouper germplasm resources, including the reagents described in the above technical solution, wherein the grouper includes the golden grouper, tiger grouper, and golden tiger hybrid grouper.

[0028] As one implementation method, the chip described in this invention is a liquid phase chip.

[0029] This invention provides a kit for identifying or differentiating grouper germplasm resources, including the chip described in the above technical solution, wherein the grouper includes the golden grouper, tiger grouper and golden tiger hybrid grouper.

[0030] The present invention provides the application of the reagents, chips, or kits described in the above technical solutions in one or more of the following: (1) identification of grouper germplasm resources; (2) differentiation of grouper germplasm resources; (3) grouper-assisted breeding; wherein the grouper includes golden grouper, tiger grouper and golden tiger hybrid grouper.

[0031] In one embodiment, the golden tiger hybrid described in this invention uses tiger stripe as the female parent and coin stripe as the male parent.

[0032] This invention provides a method for identifying grouper germplasm resources, comprising the following steps: extracting genomic DNA from the grouper to be identified, extracting site information corresponding to the SNP molecular marker combinations described in the above technical solution, performing phylogenetic tree analysis with known grouper species such as the money grouper, tiger grouper, and golden tiger hybrid grouper, and determining which of the money grouper, tiger grouper, and golden tiger hybrid grouper the grouper to be identified belongs to.

[0033] This invention provides a method for distinguishing three types of grouper germplasm resources, comprising the following steps:

[0034] Genomic DNA was extracted from grouper, and site information corresponding to the SNP molecular marker combinations described in the above technical solution was extracted. A phylogenetic tree was constructed using the maximum likelihood method, and the aggregation results were observed to distinguish three types of grouper. The three types of grouper are the money grouper, the tiger grouper, and the golden tiger hybrid grouper.

[0035] Preferably, the genomic DNA is tail fin tissue genomic DNA.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a combination of SNP molecular markers for identifying or distinguishing grouper germplasm resources and their applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Screening and validation of specific SNP combination sites

[0039] 1. Caudal fin tissues were collected from 35 golden fin cloven ...

[0040] 2. Based on site-specific probes synthesized in the liquid-phase chip, multiple target sequences located at different genomic positions were captured and enriched using liquid-phase hybridization. Then, libraries were constructed and next-generation sequencing was performed on the captured and enriched target regions. Fastp was used to filter the raw reads to obtain clean reads. The sequences were aligned to the NCBINT database using blastn for contamination assessment. The filtered clean reads were then aligned with the *Pseudomonas australis* genome using BWA alignment software to locate the clean reads on the genome. Based on the alignment results, the genotype of the target sites was determined using the mutation analysis software GATK and the variant detection tool HaplotypeCaller.

[0041] 3. Data were compared and analyzed. Non-shared loci in the three types of grouper—golden grouper, tiger grouper, and golden tiger hybrid grouper—were discarded and not included in subsequent analyses. A total of 177 SNPs were identified and located in the golden grouper genome in NCBI (GenBank: GCA_021130775.1), as detailed in Table 1 below.

[0042] Table 1. Location information of 177 SNPs

[0043]

[0044]

[0045]

[0046]

[0047] 4. Based on the genotypic data of the 177 SNPs, a phylogenetic tree was constructed using the maximum likelihood method with IQ-TREE version 2.3.6 software, and the results were visualized using the online tool tvBOT. The results are as follows: Figure 1 As shown, tiger stripes (green), coin stripes (purple), and golden tiger hybrid stripes (red) each cluster into a large branch. Therefore, based on these 177 SNP loci, the three species can be well distinguished.

[0048] 5. Caudal fin tissues were collected from 101 golden fin, 52 tiger fin, and 44 golden tiger hybrid fins, and DNA was extracted. Genetic microarray analysis was used to analyze the samples, and 177 SNPs corresponding to Table 1 were extracted. Based on the locus information, a phylogenetic tree was constructed using the maximum likelihood method with IQ-TREE version 2.3.6 software. The results are as follows: Figure 2As shown, the tiger stripe (green), coin stripe (purple), and golden tiger hybrid (red) clades each cluster to form large branches, providing excellent differentiation. This verifies that these 177 SNPs can effectively distinguish between the three species: tiger stripe, coin stripe, and golden tiger hybrid.

[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of reagents, chips, or kits targeting SNP molecular marker combinations in one or more of the following: (1) Identification of grouper germplasm resources; (2) Differentiate grouper germplasm resources; (3) Grouper-assisted breeding; The site information of the SNP molecular marker combination is as follows, and the grouper is selected from the golden grouper, tiger grouper and golden tiger hybrid grouper; 2. The application according to claim 1, characterized in that, The reagents, kits, or chips include primers and / or probes.

3. The application according to claim 1, characterized in that, The chip is a liquid phase chip.

4. The application according to claim 1, characterized in that, The aforementioned golden tiger hybrid uses tiger stripe as the female parent and coin stripe as the male parent.

5. A method for identifying grouper germplasm resources, characterized in that, The process includes the following steps: extracting the genomic DNA of the grouper to be identified, extracting the site information corresponding to the SNP molecular marker combination described in claim 1, performing phylogenetic tree analysis with known grouper species such as the money grouper, tiger grouper, and golden tiger hybrid grouper, and determining which of the money grouper, tiger grouper, and golden tiger hybrid grouper the grouper to be identified belongs to.

6. A method for distinguishing three types of grouper germplasm resources, characterized in that, Includes the following steps: Genomic DNA was extracted from grouper, and site information corresponding to the SNP molecular marker combinations described in claim 1 was extracted. A phylogenetic tree was constructed using the maximum likelihood method, and the aggregation results were observed to distinguish three types of grouper. The three types of grouper are golden grouper, tiger grouper, and golden tiger hybrid grouper.

7. The method according to claim 5 or 6, characterized in that, The genomic DNA in question is genomic DNA from the caudal fin tissue.

Images