SNP molecular marker related to body length of hexagrammos otakii and application of SNP molecular marker

Abstract

The invention belongs to the technical field of molecular genetic breeding, and relates to an SNP molecular marker related to the body length of hexagrammos otakii and application of the SNP molecular marker. The SNP molecular marker is respectively located at the 26369518th site, the 25096948th site and the 24794721th site of the 14 # chromosome. An amplification primer is designed on the basis of the SNP molecular marker. The primer can be used for identifying or assisting in identifying the body length character of the hexagrammos otakii and assisting in screening or breeding the hexagrammos otakii with the excellent body length character, is not limited by the growth stage, can be used for screening at the early stage of fry, and greatly improves the breeding efficiency of the hexagrammos otakii.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular genetic breeding, and relates to a SNP molecular marker related to the body length of Hexagrammos otakii and application thereof. BACKGROUND

[0002] Hexagrammos otakii belongs to the order Scorpaeniformes, the family Hexagrammidae, and the genus Hexagrammos, and is an important marine economic fish. Hexagrammos otakii Due to its tender meat, delicious taste and high nutritional value, Hexagrammos otakii is deeply loved by consumers and has high economic value. Scorpaeniformes With the increasing demand for Hexagrammos otakii, the amount of fishing is gradually increasing, while the catch and fishing individuals are decreasing year by year, resulting in increasing pressure on the resources and a serious trend of resource decline. Hexagrammos otakii Hexagrammos otakii Currently, the breeding of Hexagrammos otakii is limited to traditional breeding, and the traditional breeding method is low in efficiency, depends on phenotype measurement, cannot realize early selection, prolongs the breeding cycle, and is easily affected by environmental factors, resulting in large deviation of genetic force estimation and affecting the accuracy of breeding.

[0003] At present, the research on functional genes related to growth traits is weak, efficient molecular markers are lacking, and it is difficult to meet the demand for precision breeding.

[0004] Molecular markers are genetic markers that can reflect the differences in genomes between individuals or populations, and have the characteristics of heredity and detectability. SUMMARY

[0005] ​The application provides a SNP molecular marker for quickly detecting Oxyeleotris marmoratus with body length advantage and an amplification primer thereof, establishes a genotyping method based on the SNP site, and can perform early selection in fry stage, selects individuals with growth potential for breeding, greatly reduces breeding workload, significantly shortens breeding time, realizes early and accurate identification of growth traits of the species, and is further used for quickly screening parent individuals with excellent growth potential, speeds up Oxyeleotris marmoratus breeding and genetic improvement process, and improves breeding efficiency and breeding income.

[0006] The technical scheme provided by the application is as follows: a SNP molecular marker related to Oxyeleotris marmoratus body length, wherein the SNP molecular marker is any one of the following: SNP1: located at position 26369518 of chromosome 14, and the polymorphic base is C / T; SNP2: located at position 25096948 of chromosome 14, and the polymorphic base is T / G; SNP3: located at position 24794721 of chromosome 14, and the polymorphic base is A / C.

[0007] An amplification primer for detecting the SNP molecular marker, comprising: a primer for amplifying the SNP1 site, and the nucleotide sequence is shown in SEQ ID NO. 4 and SEQ ID NO. 5 of the sequence listing; a primer for amplifying the SNP2 site, and the nucleotide sequence is shown in SEQ ID NO. 6 and SEQ ID NO. 7 of the sequence listing; a primer for amplifying the SNP3 site, and the nucleotide sequence is shown in SEQ ID NO. 8 and SEQ ID NO. 9 of the sequence listing.

[0008] The SNP molecular marker or the amplification primer is applied to any one of the following: (1) used for identifying or assisting in identifying the body length trait of Oxyeleotris marmoratus; (2) used for assisting in screening or breeding Oxyeleotris marmoratus with excellent body length traits; (3) used for preparing a product for identifying or assisting in identifying the body length trait of Oxyeleotris marmoratus, assisting in screening or breeding Oxyeleotris marmoratus with excellent body length traits.

[0009] The product for identifying or assisting in identifying the body length trait of Oxyeleotris marmoratus, assisting in screening or breeding Oxyeleotris marmoratus with excellent body length traits comprises an amplification primer for detecting the SNP molecular marker, and the product is a reagent or a kit.

[0010] A method for identifying or assisting in identifying the body length trait of Hexagrammos otakii, which detects the genotype of the allele represented by any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 in the sample of the Hexagrammos otakii to be tested by using the amplification primer, and determines the body length trait of the sample to be tested according to the detected genotype; wherein the genotype of the individual carrying the SNP1 site is TT for the long body length trait and CC for the short body length trait; the genotype of the individual carrying the SNP2 site is TT for the long body length trait and GG for the short body length trait; and the genotype of the individual carrying the SNP3 site is AA for the long body length trait and CC for the short body length trait.

[0011] A method for assisting in screening or breeding Hexagrammos otakii with excellent body length traits, which detects the genotype of the allele represented by any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 in the sample of the Hexagrammos otakii to be tested by using the amplification primer, and breeds the sample with excellent body length traits according to the detected genotype; wherein the genotype of the individual carrying the SNP1 site is TT for the excellent body length trait; the genotype of the individual carrying the SNP2 site is TT for the excellent body length trait; and the genotype of the individual carrying the SNP3 site is AA for the excellent body length trait.

[0012] Compared with the prior art, the beneficial effects of the present application are that the SNP molecular marker and the amplification primer provided by the present application can quickly detect or identify individuals with excellent body length traits, can be selected at any stage of the growth of Hexagrammos otakii, is not limited by the growth stage, can identify and screen traits at an early stage of the fry, and greatly improves the breeding efficiency of Hexagrammos otakii. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is an LOD (log of odds ratio) distribution diagram of the body length trait SNP molecular marker of Hexagrammos otakii in the embodiments of the present application. Figure 2 It is a DNA extraction electropherogram. Figure 3 It is sanger sequencing verification of the three SNP molecular markers in the embodiments of the present application; wherein A is SNP1, B is SNP2, and C is SNP3. DETAILED DESCRIPTION

[0014] The specific embodiments of the present application are described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0015] (I) Screening of SNP molecular markers related to the body length of Hexagrammos otakii Randomly selected the same batch of healthy breeding good health of 229 Takifushi six-line fish to sample, each fish was numbered, measured and recorded the body length data, take the back muscle in 5 ml cryogenic tube, immediately into liquid nitrogen, and then into the-80℃ refrigerator, send for subsequent genomic DNA extraction, using GBS method to develop whole genome level molecular markers.

[0016] 1. Genomic DNA extraction and detection Using DNA extraction kit using standard process to extract DNA, the detection of DNA samples in the present application mainly includes three methods: (1) agarose gel electrophoresis analysis of DNA purity and integrity; (2) Nanodrop detection of DNA purity (OD 260 / OD 280 ratio); (3) Qubit accurate quantification of DNA concentration.

[0017] The DNA samples that pass the inspection are treated by enzyme digestion to become small fragments, and the fragments are subjected to end repair, A tailing, sequencing adapter addition, purification, PCR amplification and other processes to complete the preparation of high-throughput sequencing library. According to the preset scheme, the library is electrophoresed and gel purified to select the library with the target interval fragment length for subsequent sequencing.

[0018] 2. Library construction and high-throughput sequencing The genome is digested for library construction. To ensure the number and quality of SNP markers, the most suitable enzyme digestion scheme is selected, and the enzyme digestion scheme of Ecori+NIAIII (Hin1II) is used, and the enzyme digestion fragment size is about 400-500 bp. After the library quality inspection of the fragment length screening is qualified, high-throughput sequencing is carried out, the sequencing platform is Illumina Nova6000, and the sequencing mode is PE150. The raw data (Raw data) generated by sequencing is pretreated by quality filtering to obtain CleanData. The specific processing steps are as follows: (1) remove reads with adaptor; (2) remove reads with N (N represents undetermined base information) content greater than 5%; (3) remove low-quality reads (the number of bases with quality value Q≤10 accounts for more than 20% of the entire read); (4) count the original sequencing amount, effective sequencing amount, Q20, Q30, and GC content.

[0019] 3. Bioinformatics analysis After sequencing data is obtained, the obtained data is quality controlled to remove low-quality sequences and adapter sequences to obtain CleanData. The CleanData is aligned to a reference genome, and GATK software is used for SNP (single nucleotide polymorphism) and InDel (insertion and deletion) detection, and quality filtering is performed on the detected variation sites. After obtaining the polymorphic SNP markers, the markers are genotyped and encoded, and a genetic map is constructed using led-map3, and the map is evaluated. QTL correlation analysis is performed using QTL positioning software to obtain regions significantly associated with traits. According to the LOD value, the number of SNP molecular markers and genetic positions significantly related to body length traits are screened.

[0020] 4. Result analysis The body length LOD threshold is determined to be 1.5 by whole genome scanning combined with substitution test (1,000 times of repetition), and the distribution of body length-related SNP molecular markers on the chromosome is as shown in Figure 1 According to the threshold, 3 SNP molecular markers with high association intensity with body length are screened, all of which are located on chromosome 14, and are respectively: SNP1: chr14_26369518 is located at position 26369518 of chromosome 14, and the polymorphic base is C / T; SNP2: chr14_25096948 is located at position 25096948 of chromosome 14, and the polymorphic base is T / G; SNP3: chr14_24794721 is located at position 24794721 of chromosome 14, and the polymorphic base is A / C.

[0021] For the 3 SNP sites, Welch ANOVA test in SPSS software is used to analyze the correlation of body length data distribution of 229 individuals of Daikoku Hexagram carrying different genotypes. Statistical analysis found that the genotype with longer body length in individuals carrying SNP1 site is TT, and the genotype with shorter body length is CC; the genotype with longer body length in individuals carrying SNP2 site is TT, and the genotype with shorter body length is GG; the genotype with longer body length in individuals carrying SNP3 site is AA, and the genotype with shorter body length is CC. The results show that the three SNP sites correspond to three genotypes with significant differences in body length (p<0.05). The specific results are shown in Tables 1-3.

[0022] Table 1 Correlation analysis of SNP1 site of Daikoku Hexagram and body length (average ± standard deviation) ; Note: The same row of letters represents a specific significant difference p <0.05).

[0023] Table 2 Correlation analysis between SNP2 locus of Onigiri goby and body length (mean ± standard deviation) ; Note: The same row of letters represents a specific significant difference p <0.05).

[0024] Table 3 Correlation analysis between SNP3 locus of Onigiri goby and body length (mean ± standard deviation) ; Note: The same row of letters represents a specific significant difference p <0.05).

[0025] As can be seen from Tables 1-3, at the SNP1 locus, the body length of individuals with TT genotype was significantly higher than that of individuals with CT and CC genotypes. At the SNP2 locus, the body length of individuals with TT genotype was also significantly higher than that of individuals with TG and GG genotypes. At the SNP3 locus, the body length of individuals with AA genotype was also significantly higher than that of individuals with AC and CC genotypes.

[0026] (II) Verification of SNP molecular markers related to the body length of Onigiri goby In order to verify the accuracy of the SNP, the verification test used the same period of reproduction of Onigiri goby individuals, which were artificially bred in November 2024. After 5 months of cultivation, 89 individuals of Onigiri goby were randomly selected in April 2025, and the body length data of each fish was measured and recorded using a vernier caliper with an accuracy of 0.01 cm. At the same time, muscle tissue was taken for DNA extraction and genotyping.

[0027] 1. DNA extraction from sample tissue and quality detection The extraction steps were carried out according to the standard procedures of the Ezup column animal tissue genomic DNA extraction kit of Shengong Biology, strictly following the instructions. The extracted genomic DNA was observed for band integrity using 1% agarose gel electrophoresis, and the OD260 / 280 value (1.7-2.0) was detected by spectrophotometer to evaluate the purity, which met the requirements of ordinary PCR.

[0028] 2. Primer design The three SNP molecular markers selected in (I) were sequenced and verified (sequence information: SEQ ID NO. 1-3). According to the sequence information of the three SNP loci, three pairs of primers were designed for sequencing verification using Primer Premier 5 software, and the primer sequences are shown in Table 4.

[0029] Table 4 Primer sequence information of 3 SNP sequences .

[0030] SNP1 corresponds to the 202nd position in SEQ ID NO. 1, and m at this site is t or c.

[0031] SNP2 corresponds to the 202nd position in SEQ ID NO. 2, and m at this site is t or g.

[0032] SNP3 corresponds to the 202nd position in SEQ ID NO. 3, and m at this site is a or c.

[0033] 3. PCR amplification PCR amplification was performed using the extracted genomic DNA and the designed primers, and the PCR reaction system and reaction conditions are shown in Table 5 and Table 6.

[0034] Table 5 PCR reaction system ; Table 6 PCR reaction conditions .

[0035] 4. Sequencing 2 μL of the reaction product was taken for 2% agarose gel electrophoresis detection, and qualified samples were used for sequencing to determine the genotype of each sample at the 3 SNP molecular markers. Part of the electropherogram is shown in Figure 2 .

[0036] The genomic DNA samples of 89 D. lacustris were sequenced using the Sanger method to determine the genotype of the 3 SNP molecular markers mutation sites, and the body length data of individuals with different genotypes were counted. Figure 3 The Sanger sequencing verification results of the genotypes of 3 SNP sites of 89 randomly selected D. lacustris are shown in Figure 3 It can be seen from Fig. A that there is a significant difference in body length between individuals with different genotypes of SNP1 of D. lacustris; the body length of individuals carrying TT genotype is significantly higher than that of individuals carrying CT and CC genotypes; Figure 3 Fig. B shows that the body length of individuals carrying TT genotype at SNP2 site is significantly higher than that of individuals carrying TG and GG genotypes; Figure 3 Fig. C shows that the body length of individuals carrying AA genotype at SNP3 site is significantly higher than that of individuals carrying AC and CC genotypes.

[0037] In conclusion, the three SNP sites provided by the application are significantly associated with the body length of O. latipes, the identification result of the body length of O. latipes by the sequencing-based genotyping method is consistent with the identification result by directly measuring the body length, the genotype can be determined by the primers in table 4, the operation is simple and reliable, and it is indicated that the three SNP analysis markers can provide a reliable basis for identifying the growth of O. latipes and are used for quickly and accurately screening O. latipes with excellent traits. In production, by detecting the parents of O. latipes, selecting individuals with the three SNP site advantage genotypes as parents, and obtaining the offspring of O. latipes with faster growth, the production and breeding process of O. latipes can be accelerated.

Claims

1. A SNP molecular marker related to the body length of Hexagrammos otakii, characterized in that, The SNP molecular marker is any one of the following: SNP1: located at position 26369518 on chromosome 14, the polymorphic base is C / T; SNP2: located at position 25096948 on chromosome 14, the polymorphic base is T / G; SNP3: located at position 24794721 on chromosome 14, the polymorphic base is A / C.

2. An amplification primer for detecting the SNP molecular marker of claim 1, characterized in that, It comprises: a primer for amplifying the SNP1 site, the nucleotide sequence of which is shown in SEQ ID NO. 4, SEQ ID NO. 5 of the sequence listing; a primer for amplifying the SNP2 site, the nucleotide sequence of which is shown in SEQ ID NO. 6, SEQ ID NO. 7 of the sequence listing; a primer for amplifying the SNP3 site, the nucleotide sequence of which is shown in SEQ ID NO. 8, SEQ ID NO. 9 of the sequence listing.

3. The SNP molecular marker of claim 1 or the amplification primer of claim 2 is used in any one of the following: (1) for identifying or assisting in identifying the body length trait of Oryzias latipes; (2) for assisting in screening or breeding Oryzias latipes with excellent body length traits; (3) for preparing a product for identifying or assisting in identifying the body length trait of Oryzias latipes, assisting in screening or breeding Oryzias latipes with excellent body length traits.

4. A product for identifying or assisting in identifying the body length trait of Oryzias latipes, assisting in screening or breeding Oryzias latipes having an excellent body length trait, characterized by comprising: The product comprises amplification primers for detecting the SNP molecular marker of claim 1; and the product is a reagent or a kit.

5. The product of claim 4, wherein: The amplification primer is the primer of claim 2.

6. A method for identifying or assisting in the identification of the body length trait of the large-scaled rockfish, characterized in that: The amplification primer of claim 2 is used to detect the genotype of any one of the alleles shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 in the sample of Oryzias latipes to be tested, and the body length trait of the sample to be tested is determined according to the detected genotype; wherein the genotype of the individual carrying the SNP1 site with long body length trait is TT, and the genotype of the individual carrying the SNP1 site with short body length trait is CC; the genotype of the individual carrying the SNP2 site with long body length trait is TT, and the genotype of the individual carrying the SNP2 site with short body length trait is GG; the genotype of the individual carrying the SNP3 site with long body length trait is AA, and the genotype of the individual carrying the SNP3 site with short body length trait is CC.

7. A method for assisting in the screening or breeding of *Hexagrammos otakii* with superior body length traits, characterized in that: The amplification primer of claim 2 is used to detect the genotype of any one of the alleles shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 in the sample of Oryzias latipes to be tested, and the sample with excellent body length trait is screened for breeding according to the detected genotype; wherein the genotype of the individual carrying the SNP1 site with excellent body length trait is TT; the genotype of the individual carrying the SNP2 site with excellent body length trait is TT; and the genotype of the individual carrying the SNP3 site with excellent body length trait is AA.

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