A SNP molecular marker associated with the body length of Hexagrammos ogakii and its application

Abstract

This invention belongs to the field of molecular genetic breeding technology, and relates to a SNP molecular marker related to the body length of *Hexagrammos oysteri* and its application. This SNP molecular marker is located at positions 26369518, 25096948, and 24794721 on chromosome 14. Based on this SNP molecular marker, this invention designs amplification primers. Using these primers, it is possible to identify or assist in identifying the body length trait of *Hexagrammos oysteri*, assist in screening or breeding *Hexagrammos oysteri* with superior body length traits, and not be limited by the growth stage. Screening can be carried out in the early stages of fry development, greatly improving the breeding efficiency of *Hexagrammos oysteri*.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetic breeding technology and relates to an SNP molecular marker related to the body length of the Ōtaki Hexagrammos and its application. Background Technology

[0002] Otaki Hexagram ( Hexagrammosotakii ) belongs to the order Scorpaeniformes ( Scorpaeniformes ), Hexagramidae ( Hexagrammidate ), Hexagram Hexagrammos The rockfish (Hexagrammos octopus) is an important marine economic fish. Due to its tender flesh, delicious taste, and high nutritional value, it is widely loved by consumers and has significant economic value. With the strong market demand for rockfish, the catch volume is gradually increasing, while the catch amount and the number of individuals caught are decreasing year by year, leading to increasing pressure on its resources and a more serious trend of resource decline.

[0003] Currently, breeding efforts for *Hexagrammos ogakii* rely on traditional methods, which are inefficient, depend on phenotypic measurements, cannot achieve early selection, prolong the breeding cycle, and are susceptible to environmental interference, leading to significant biases in heritability estimation and affecting the accuracy of breeding. Research on functional genes related to growth traits is weak, and there is a lack of efficient molecular markers, making it difficult to meet the needs of precision breeding. High-throughput genotyping is costly, and traditional GWAS requires large samples and has a high false-positive rate, hindering the widespread application of these technologies and limiting the development of molecular breeding for *Hexagrammos ogakii*.

[0004] Molecular markers are genetic markers that reflect genomic differences between individuals or populations, possessing heritable and detectable characteristics. Among them, single nucleotide polymorphisms (SNPs) refer to variations in a single base in the genome (such as transitions, transversions, insertions, or deletions), with a variation frequency greater than 1%, characterized by high-density distribution and diaellic properties. As a third-generation molecular marker, SNPs have become a core tool in modern molecular breeding due to their high density, high detection efficiency, and functional relevance. Simplified genome sequencing (GBS) technology offers advantages such as high throughput and low cost, enabling efficient screening of SNP loci across the entire genome. However, currently, there is a lack of molecular markers for marker-assisted breeding of Hexagrammos oytaki for body length traits, which restricts the development of the Hexagrammos oytaki industry. Summary of the Invention

[0005] This invention provides SNP molecular markers and their amplification primers for the rapid detection of body length advantage in Hexagrammos otakii, and establishes a genotyping method based on these SNP sites. This allows for early selection during the fry stage, choosing individuals with growth potential for aquaculture, greatly reducing breeding workload and significantly shortening breeding time. It enables early and accurate identification of the growth traits of this species, and can then be used to rapidly screen parent individuals with excellent growth potential, accelerating the breeding and genetic improvement process of Hexagrammos otakii, and improving breeding efficiency and aquaculture profits.

[0006] The technical solution provided by this invention is as follows: a SNP molecular marker related to the body length of the large-scaled six-lined fish, wherein the SNP molecular marker is any one of the following:

[0007] SNP1: Located at position 26369518 on chromosome 14, with a polymorphic base sequence of C / T;

[0008] SNP2: Located at position 25096948 on chromosome 14, with a polymorphic base sequence of T / G;

[0009] SNP3: Located at position 24794721 on chromosome 14, with a polymorphic base combination of A / C.

[0010] Amplification primers for detecting the SNP molecular markers include:

[0011] The primers used to amplify the SNP1 site have nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5 of the sequence listing;

[0012] The primers used to amplify the SNP2 site have nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7 of the sequence listing;

[0013] The primers used to amplify the SNP3 site have nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9 of the sequence listing.

[0014] The SNP molecular marker or the amplification primers described herein may be used in any of the following ways:

[0015] (1) Used for identification or auxiliary identification of the body length characteristics of the Ōtaki Hexagrammos;

[0016] (2) Used to assist in the screening or breeding of large-scaled six-lined fish with excellent body length traits;

[0017] (3) Prepare products for identification or auxiliary identification of the body length trait of Hexagrammos otakii, and for auxiliary screening or breeding of Hexagrammos otakii with excellent body length trait.

[0018] Products used for identifying or assisting in the identification of body length traits in Hexagrammos otakii, and for assisting in the screening or breeding of Hexagrammos otakii with superior body length traits, include amplification primers for detecting the SNP molecular markers; the products are reagents or kits.

[0019] A method for identifying or assisting in the identification of body length traits in *Hexagrammos oysteri*, comprising using the aforementioned amplification primers to detect the genotypes of alleles shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in a *Hexagrammos oysteri* sample, and determining the body length trait of the sample based on the detected genotypes; wherein, in individuals carrying the SNP1 locus, the genotype for long body length is TT, and the genotype for short body length is CC; in individuals carrying the SNP2 locus, the genotype for long body length is TT, and the genotype for short body length is GG; and in individuals carrying the SNP3 locus, the genotype for long body length is AA, and the genotype for short body length is CC.

[0020] A method for assisting in the screening or breeding of Hexagrammos otakii with superior body length traits is disclosed. This method utilizes the amplification primers described herein to detect the genotypes of alleles shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the Hexagrammos otakii sample to be tested. Samples with superior body length traits are then screened and bred based on the detected genotypes. Specifically, individuals carrying the SNP1 locus have the genotype TT with superior body length traits; individuals carrying the SNP2 locus have the genotype TT with superior body length traits; and individuals carrying the SNP3 locus have the genotype AA with superior body length traits.

[0021] Compared with the prior art, the beneficial effects of the present invention are: the SNP molecular markers and amplification primers provided by the present invention can quickly detect or identify individuals with excellent body length traits, and can be selected at any stage of the growth of Hexagrammos otakii, without being limited by the growth stage. It can be used for trait identification and screening in the early stage of fish fry, which greatly improves the breeding efficiency of Hexagrammos otakii. Attached Figure Description

[0022] Figure 1 This is a diagram showing the LOD (logarithmic dominance) distribution of SNP molecular markers for the body length trait of *Hexagrammos octopus* in this embodiment of the invention.

[0023] Figure 2 This is an electrophoresis image of DNA extraction;

[0024] Figure 3 The three SNP molecular markers in this embodiment of the invention were verified by Sanger sequencing; where A is SNP1; B is SNP2; and C is SNP3. Detailed Implementation

[0025] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0026] (I) Screening of SNP molecular markers related to body length of Hexagrammos otakii

[0027] 229 healthy Hexagrammos oysters from the same breeding batch were randomly selected for sampling. Each fish was numbered, and the body length of each fish was measured and recorded. The dorsal muscle was taken into a 5ml cryovial, immediately placed in liquid nitrogen, and then stored in a -80℃ freezer for subsequent genomic DNA extraction. Molecular markers at the whole genome level were developed using the GBS method.

[0028] 1. Genomic DNA extraction and detection

[0029] DNA was extracted using a standard procedure with a DNA extraction kit. The detection of DNA samples in this invention mainly includes three methods: (1) agarose gel electrophoresis to analyze the purity and integrity of the DNA; (2) Nanodrop detection of DNA purity (OD). 260 / OD 280 (3) Qubit accurately quantifies DNA concentration.

[0030] DNA samples that pass inspection are digested with enzymes to form small fragments. These fragments then undergo end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification to prepare high-throughput sequencing libraries. Following a pre-defined protocol, the libraries are purified by electrophoresis and gel extraction to select libraries with the desired insertion length for subsequent sequencing.

[0031] 2. Library construction and high-throughput sequencing

[0032] The genome was digested with enzymes to construct a library. To ensure the quantity and quality of SNP markers, the optimal digestion scheme was selected. The Ecori+NIAIII (Hin1II) digestion scheme was adopted, and the digestion fragment size was about 400~500bp. After the library passed the quality inspection after fragment length screening, it was put into high-throughput sequencing. The sequencing platform was Illumina Nova6000 and the sequencing mode was PE150. The raw data generated by sequencing was preprocessed by quality filtering to obtain CleanData. The specific processing steps are as follows: (1) Remove reads with adapters; (2) Remove reads containing more than 5% of N (N means that the base information cannot be determined); (3) Remove low-quality reads (the number of bases with quality value Q≤10 accounts for more than 20% of the entire read); (4) Calculate the raw sequencing volume, effective sequencing volume, Q20, Q30, and GC content.

[0033] 3. Bioinformatics Analysis

[0034] After sequencing data is processed, quality control is performed to remove low-quality sequences and adapter sequences, resulting in CleanData. The CleanData is aligned to a reference genome, and SNPs (single nucleotide polymorphisms) and InDels (insertions and deletions) are detected using GATK software. Detected variant sites are then filtered for quality. After obtaining polymorphic SNP markers, genotyping and encoding are performed, and a genetic map is constructed using led-map3, followed by map evaluation. QTL association analysis is conducted using QTL mapping software to identify regions significantly associated with the trait. Based on LOD values, the number and genetic locations of SNP markers significantly associated with body length are determined.

[0035] 4. Results Analysis

[0036] The body length LOD threshold was determined to be 1.5 using a whole-genome scan combined with a substitution test (1,000 replicates). The distribution of body length-related SNP molecular marker LOD values ​​on chromosomes is as follows: Figure 1 As shown, based on the threshold screening, three SNP molecular markers with a high correlation to body length were identified, all located on chromosome 14, namely:

[0037] SNP1: chr14_26369518 is located at position 26369518 on chromosome 14, with a polymorphic base sequence of C / T;

[0038] SNP2: chr14_25096948 is located at position 25096948 on chromosome 14, with a polymorphic base sequence of T / G;

[0039] SNP3: chr14_24794721 is located at position 24794721 on chromosome 14, with a polymorphic base combination of A / C.

[0040] Correlation analysis was performed on the body length distribution of 229 individuals carrying different genotypes of *Hexagrammos otakii* using Welch ANOVA in SPSS software for three SNP loci. Statistical analysis revealed that individuals carrying SNP1 had the genotype TT for longer body length and CC for shorter body length; individuals carrying SNP2 had the genotype TT for longer body length and GG for shorter body length; and individuals carrying SNP3 had the genotype AA for longer body length and CC for shorter body length. The results showed that the pairwise differences in body length between the three genotypes corresponding to the three SNP loci were significant (p < 0.05). Specific results are shown in Tables 1-3.

[0041] Table 1. Correlation analysis between SNP1 locus and body length in Hexagrammos ogakii (mean ± standard deviation)

[0042] ;

[0043] Note: Letters in the same row indicate specific significant differences. p <0.05).

[0044] Table 2. Correlation analysis between SNP2 loci and body length in Hexagrammos otakii (mean ± standard deviation)

[0045] ;

[0046] Note: Letters in the same row indicate specific significant differences. p <0.05).

[0047] Table 3. Correlation analysis between SNP3 loci and body length in Hexagrammos ogakii (mean ± standard deviation)

[0048] ;

[0049] Note: Letters in the same row indicate specific significant differences. p <0.05).

[0050] As shown in Tables 1-3, at SNP1, individuals with the TT genotype were significantly longer than those with the CT and CC genotypes. Similarly, at SNP2, individuals with the TT genotype were significantly longer than those with the TG and GG genotypes. At SNP3, individuals with the AA genotype were also significantly longer than those with the AC and CC genotypes.

[0051] (II) Verification of SNP molecular markers related to body length of Hexagrammos otakii

[0052] To verify the accuracy of the SNP, the validation experiment used reconstructed, synchronously bred *Hexagrammos oysteri* individuals. Artificial breeding began in November 2024, and after five months of rearing, 89 *Hexagrammos oysteri* individuals were randomly selected in April 2025. The body length of each fish was measured and recorded using calipers with an accuracy of 0.01 cm. Simultaneously, muscle tissue was collected for DNA extraction and genotyping.

[0053] 1. DNA extraction and quality testing of sample tissues

[0054] The extraction procedure was performed strictly according to the standard procedure in the instructions of the Sangon Biotech Ezup column-based animal tissue genomic DNA extraction kit. The extracted genomic DNA was examined using 1% agarose gel electrophoresis to observe band integrity, and its purity was assessed by detecting the OD260 / 280 value (1.7-2.0) using a spectrophotometer, meeting the requirements for standard PCR.

[0055] 2. Primer design

[0056] The three SNP molecular markers screened in (I) were sequenced and verified (sequence information: SEQ ID NO.1-3). Based on the sequence information of the three SNP sites, three pairs of primers were designed using Primer Premier 5 software for sequencing and verification. The primer sequences are shown in Table 4.

[0057] Table 4. Primer sequence information for the three SNP sequences

[0058] .

[0059] SNP1 corresponds to position 202 in SEQ ID NO.1, where m is either t or c.

[0060] SNP2 corresponds to position 202 in SEQ ID NO.2, where m is either t or g.

[0061] SNP3 corresponds to position 202 in SEQ ID NO.3, where m is either a or c.

[0062] 3. PCR amplification

[0063] The extracted genomic DNA and designed primers were used for PCR amplification. The PCR reaction system and reaction conditions are shown in Tables 5 and 6.

[0064] Table 5 PCR reaction system

[0065] ;

[0066] Table 6 PCR Reaction Conditions

[0067] .

[0068] 4. Sequencing

[0069] Two μL of the reaction product was used for 2% agarose gel electrophoresis. Qualified samples were then sequenced to determine the genotype of each sample at three SNP molecular markers. Electrophoresis images of some samples are shown below. Figure 2 .

[0070] Genomic DNA samples from 89 large-scaled six-lined rockfish were sequenced using the Sanger method to determine the genotypes of three SNP molecular marker mutation sites, and the body length data of individuals with different genotypes were collected. Figure 3 This presentation showcases the Sanger sequencing validation results of genotypes at three SNP loci in 89 randomly selected Hexagrammos oysterfish. Figure 3 As shown in A, there are significant differences in body length among individuals with different SNP1 genotypes in Hexagrammos otakii; among them, individuals carrying the TT genotype have significantly longer body lengths than individuals carrying the CT and CC genotypes. Figure 3 The results from the B study showed that individuals carrying the TT genotype at the SNP2 locus had significantly longer body lengths than individuals carrying the TG and GG genotypes. Figure 3 The results showed that individuals carrying the AA genotype at the SNP3 locus had significantly longer body lengths than individuals carrying the AC and CC genotypes.

[0071] In summary, the three SNP loci provided by this invention are significantly associated with the body length of *Hexagrammos oysteri*. The results of body length identification using genotyping via sequencing are consistent with those obtained by directly measuring body length. Genotype can be determined using the primers in Table 4, a simple and reliable procedure. This demonstrates that these three SNP markers can provide a reliable basis for identifying the growth status of *Hexagrammos oysteri*, enabling rapid and accurate screening for fish with superior traits. In production, by testing parent *Hexagrammos oysteri* and selecting individuals with the dominant genotypes at these three SNP loci, faster-growing offspring can be obtained, accelerating the production and breeding process of *Hexagrammos oysteri*.

Claims

1. A SNP molecular marker related to the body length of Hexagrammos otakii, characterized in that, The nucleotide sequence of the SNP molecular marker is any one of the following: SNP1: the nucleotide sequence is shown in SEQ ID NO. 1 of the sequence listing; the polymorphic base at position 201 of m is C / T; SNP2: the nucleotide sequence is shown in SEQ ID NO. 2 of the sequence listing; the polymorphic base at position 201 of m is T / G; SNP3: the nucleotide sequence is shown in SEQ ID NO. 3 of the sequence listing; the polymorphic base at position 201 of m is A / C.

2. The SNP molecular marker of claim 1 is used in any one of the following: (1) for identifying or assisting in identifying the body length trait of O. Dabryi; (2) for assisting in screening or breeding O. Dabryi with excellent body length trait; (3) for preparing products for identifying or assisting in identifying the body length trait of O. Dabryi, and for assisting in screening or breeding O. Dabryi with excellent body length trait; wherein The genotype of an individual carrying the SNP1 site with excellent body length trait is TT; the genotype of an individual carrying the SNP2 site with excellent body length trait is TT; the genotype of an individual carrying the SNP3 site with excellent body length trait is AA.

3. A method for identifying or assisting in identifying the body length trait of O. Dabryi, characterized by: detecting the genotype of the SNP molecular marker of claim 1 in the sample of the O. Dabryi to be tested, and determining the body length trait of the sample to be tested according to the detected genotype; wherein the genotype of an individual carrying the SNP1 site with long body length trait is TT, and the genotype of an individual carrying the SNP1 site with short body length trait is CC; the genotype of an individual carrying the SNP2 site with long body length trait is TT, and the genotype of an individual carrying the SNP2 site with short body length trait is GG; the genotype of an individual carrying the SNP3 site with long body length trait is AA, and the genotype of an individual carrying the SNP3 site with short body length trait is CC.

4. A method for assisting in screening or breeding of Hexagrammos otakii having excellent body length traits, characterized by: detecting the genotype of the SNP molecular marker of claim 1 in the sample of the O. Dabryi to be tested, and screening the sample with excellent body length trait for breeding according to the detected genotype; wherein the genotype of an individual carrying the SNP1 site with excellent body length trait is TT; the genotype of an individual carrying the SNP2 site with excellent body length trait is TT; the genotype of an individual carrying the SNP3 site with excellent body length trait is AA.

Images