An epigenetic marker associated with the variation of black spots in body color during overwintering of red tilapia and its application

Abstract

This invention discloses an epigenetic marker related to the variation of body color black spots in red tilapia during overwintering, and further utilizes this epigenetic marker to develop detection primers and a method for determining the variation of body color black spots in red tilapia during overwintering. This method can be applied to the screening of red tilapia exhibiting body color black spot variation during overwintering, selecting genetically stable red tilapia as broodstock for epigenetic breeding. Therefore, by utilizing an epigenetic marker related to the variation of body color black spots in red tilapia during overwintering, a new approach and method are provided for its epigenetic breeding, offering a new scheme for improving the variation of body color black spots in red tilapia.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to an epigenetic marker related to the variation of body color black spots during the overwintering period of red tilapia and its application. Background Technology

[0002] Red tilapia (Oreochromis spp.) belongs to the order Perciformes, family Cichlidae, and genus Tilapia. It is a superior mutant species resulting from the hybridization of Nile tilapia and Mozambique tilapia and multiple generations of selective breeding. Red tilapia boasts a rich variety of body colors, including red, orange, gold, pink, and tangerine. Furthermore, red tilapia is characterized by rapid growth, short reproductive cycle, wide salinity tolerance, strong adaptability to complex aquaculture systems, high economic value, and vibrant body color with no black membrane in the abdominal cavity. These characteristics have led to its widespread cultivation in Southeast Asia and my country. Body color is a significant economic value of fish, and the vibrant coloration of red tilapia makes it widely popular among consumers.

[0003] In the actual industrialized farming of red tilapia, some red tilapia exhibit a mutation in body coloration characterized by black spots during the overwintering period. Initially, these spots appear as diffuse black spots concentrated on the lower abdomen, caudal fin, and anal fin. As the overwintering period progresses, these black spots gradually become denser and spread throughout the body, forming clusters and deepening in color. After the overwintering period, the black spots gradually diminish but do not disappear. Therefore, this mutation in body coloration of red tilapia significantly impacts its economic value and restricts its large-scale industrial farming. However, the environmental and genetic factors contributing to this mutation are still unclear. Furthermore, the complexity of the red tilapia's genetic background and its self-incompatibility greatly limit the genetic breeding of superior strains and the selection of new varieties.

[0004] Therefore, effectively screening for red tilapia with stable and non-mutant black spot coloration during overwintering is crucial for solving the current problems faced by red tilapia factory farming and improving its economic value. Existing technologies for screening red tilapia body color mainly focus on the application of molecular markers, such as the Chinese invention patent "Detection Primers for Microsatellites Related to Black Spot Traits in Overwintering Red Tilapia and Their Application," which provides two pairs of microsatellite detection primers related to the black spot trait in overwintering red tilapia; and the Chinese invention patent "A Method for Judging Black Spot Traits in Overwintering Red Tilapia," which uses QTL mapping of the black spot trait and finds a SNP mutation site at the LG3_11647206 that is significantly associated with the black spot trait across the entire genome. However, current screening methods for red tilapia body color breeding still focus on traditional breeding techniques.

[0005] Epigenetics is the science that studies how environmental factors alter gene expression without changing the DNA sequence, thus affecting gene expression. These alterations include DNA methylation, histone modifications, and RNA-related mechanisms. DNA methylation is the most widely and thoroughly studied biochemical modification of DNA bases within epigenetics. It is an exceptionally conserved epigenetic mechanism that can alter the expression levels of the transcriptome within cells and cause phenotypic differences without changing the genomic base sequence. Furthermore, DNA methylation exhibits tissue specificity, stable and variable sequences, and the ability to be inherited across generations. Epigenetic breeding is a breeding method that has emerged in recent years. It incorporates epigenetic information into traditional genetic breeding. Therefore, it can accelerate the breeding process and obtain superior traits more quickly. Simultaneously, because epigenetic markers reflect the influence of the environment on gene expression, it allows for more accurate selection of breeding targets. It also increases genetic variability, helping to overcome limitations in the breeding process, and improves trait stability by selecting traits that are stable under environmental changes through epigenetic marker selection.

[0006] Epigenetic breeding is currently mainly applied to the improvement of crops and livestock. In crop breeding, the selection of epigenetic markers can improve crop yield, stress resistance, and other aspects. In livestock improvement, selecting individuals with favorable epigenetic markers can improve meat quality, milk quality, and reproductive capacity. Epigenetic breeding is still in the research and development stage, but with the rapid development of high-throughput sequencing technology, epigenomics, and bioinformatics, its application prospects are extremely broad. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an epigenetic marker related to the variation of body color black spots in red tilapia during overwintering and its application.

[0008] Epigenetics is the science that studies how environmental factors alter gene expression without changing the DNA sequence, thus transmitting information. Based on this science, a new breeding method that has emerged in recent years is epigenetic breeding, which incorporates the utilization of epigenetic information into traditional genetic breeding. This invention uses DNA methylation modification markers associated with black spot trait variations during overwintering to screen for red tilapia with stable body color variations, thus avoiding the impact of black spot trait on the economic benefits of red tilapia, and also to screen for red tilapia broodstock with stable body color traits during overwintering.

[0009] The first objective of this invention is to provide an epigenetic marker related to the variation of body color black spots in red tilapia during overwintering.

[0010] A second objective of this invention is to provide the application of the aforementioned epigenetic markers and / or their detection reagents in detecting the variation of body color black spots in overwintering red tilapia.

[0011] A third objective of this invention is to provide the application of the aforementioned epigenetic marker detection reagent in the preparation of a kit for detecting the variation of body color black spots in overwintering red tilapia.

[0012] The fourth objective of this invention is to provide a method for detecting the variation of body color black spots in red tilapia during overwintering.

[0013] The fifth objective of this invention is to provide a kit for detecting the variation of black spots in red tilapia during overwintering.

[0014] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0015] This invention claims protection for an epigenetic marker related to the variation of body color black spots during the overwintering period of red tilapia, the epigenetic marker being located in LG3:11575201~11575800, with a reference genome of GCA_001858045.3.

[0016] Preferably, the epigenetic markers are CpG1 to CpG13, wherein CpG1 is located at ChrLG3: 11575499, CpG2 at ChrLG3: 11575510, CpG3 at ChrLG3: 11575531, CpG4 at ChrLG3: 11575533, CpG5 at ChrLG3: 11575552, and CpG6 at ChrLG3: 115755. 89. CpG7 is located in ChrLG3: 11575599, CpG8: 11575617, CpG9 is located in ChrLG3: 11575630, CpG10 is located in ChrLG3: 11575667, CpG11 is located in ChrLG3: 11575683, CpG12 is located in ChrLG3: 11575722, and CpG13 is located in ChrLG3: 11575732.

[0017] It also claims protection for the use of the epigenetic markers and / or their detection reagents in detecting the variation of body color black spots in overwintering red tilapia.

[0018] And the application of the epigenetic marker detection reagent in the preparation of a kit for detecting the variation of body color black spots in overwintering red tilapia.

[0019] Preferably, the reagent is a primer.

[0020] More preferably, the nucleotide sequences of the primers are shown in SEQ ID NO 1 and 2.

[0021] SEQ ID NO 1: 5'-TAGGTGTGAATGTGAGTGTGAATGG-3';

[0022] SEQ ID NO 2: 5'-CAATCAAAATTAACACCAACCCCCT-3'.

[0023] This invention also claims a method for detecting the variation of body color black spots in red tilapia during overwintering, wherein the DNA of the sample to be tested is treated with bisulfite, and then the epigenetic markers are detected, and the variation of black spots during overwintering is determined based on their methylation level.

[0024] Preferably, the epigenetic markers are CpG1 to CpG13, wherein CpG1 is located at ChrLG3: 11575499, CpG2 at ChrLG3: 11575510, CpG3 at ChrLG3: 11575531, CpG4 at ChrLG3: 11575533, CpG5 at ChrLG3: 11575552, and CpG6 at ChrLG3: 115755. 89. CpG7 is located in ChrLG3: 11575599, CpG8: 11575617, CpG9 is located in ChrLG3: 11575630, CpG10 is located in ChrLG3: 11575667, CpG11 is located in ChrLG3: 11575683, CpG12 is located in ChrLG3: 11575722, and CpG13 is located in ChrLG3: 11575732.

[0025] Among the 13 CpG sites, individuals with a methylation level higher than 50% at each CpG site were red tilapia with stable body color and no black spots during overwintering, while individuals with a methylation level lower than 25% at each CpG site were red tilapia with black spot mutations in body color during overwintering.

[0026] Preferably, the primers shown in SEQ ID NO 1 and 2 are used for detection.

[0027] SEQ ID NO 1: 5'-TAGGTGTGAATGTGAGTGTGAATGG-3';

[0028] SEQ ID NO 2: 5'-CAATCAAAATTAACACCAACCCCCT-3'.

[0029] This invention also claims a kit for detecting the variation of black spots in red tilapia during overwintering, containing the epigenetic marker detection reagents described above.

[0030] Preferably, it also contains DNA bisulfite treatment reagent and / or PCR amplification reagent.

[0031] Preferably, the reagent detects the methylation level of the epigenetic marker.

[0032] Preferably, the primers shown in SEQ ID NO 1 and 2 are used for detection.

[0033] SEQ ID NO 1: 5'-TAGGTGTGAATGTGAGTGTGAATGG-3';

[0034] SEQ ID NO 2: 5'-CAATCAAAATTAACACCAACCCCCT-3'.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention provides an epigenetic marker related to the variation in body color black spots during the overwintering period of red tilapia, and further utilizes this epigenetic marker to develop detection primers and a method for determining the variation in body color black spots during the overwintering period of red tilapia. This method can be applied to the screening of red tilapia exhibiting body color black spot variation during the overwintering period, selecting genetically stable red tilapia as broodstock, and applying it to the epigenetic breeding of red tilapia.

[0037] Therefore, by utilizing epigenetic markers related to the variation of body color and black spots in red tilapia during overwintering, a new approach and method have been provided for its epigenetic breeding, offering a new solution for improving the variation of body color and black spots in red tilapia. Attached Figure Description

[0038] Figure 1 The black spot variation on the body surface of red tilapia during the overwintering period is shown in Figure 1. A and B are normal red tilapia with a red body color. C and D are red tilapia with black spots and red body color, with the black spots mainly concentrated on the lower abdomen, which is also the experimental sampling location.

[0039] Figure 2 This image shows the stained structure of paraffin sections of the skin on the ventral side of a group of red tilapia. HE staining and L-dopa staining were performed on the skin tissue sections. HE staining was used to observe the skin tissue structure, dividing it into EP (epidermis), DE (dermis), PL (pigment cell layer), P (peritoneum), and M (melanin band). L-dopa staining was used to verify that the pigment cells in the epidermis are melanocytes, thus correlating the black spot trait of the red tilapia group with melanocytes. "Black" refers to red tilapia exhibiting the black spot trait; "Red" refers to red tilapia without body color variation.

[0040] Figure 3This image shows a genome-wide DNA methylation map of skin tissue constructed using whole-genome bisulfite sequencing (WGBS). At a significance correction threshold of q < 0.001 (marked in red), significantly differentially methylated regions are located on chromosomes LG2, LG3, LG7, LG10, and LG14. The x-axis represents chromosomes, and the y-axis represents the p-values ​​of different DMRs located on each chromosome.

[0041] Figure 4 The epigenetic marker dmr_2598 (LG3: 11575201~11575800) was identified on ChrLG3 using the WGBS method and was significantly associated with the black spot trait of body color during the overwintering period of red tilapia. The significance value of this DMR was 5.86E-19.

[0042] Figure 5 This is a schematic diagram of the original sequence of dmr_2598 (LG3: 11575201~11575800), and the specific locations of 13 CpG sites within the interval. The methylation level mainly occurs on the negative strand sequence.

[0043] Figure 6 To verify the differential methylation levels of the epigenetic marker dmr_2598 (LG3: 11575201~11575800) related to the black spot trait in overwintering red tilapia, the methylation levels of red tilapia with black spot variation during the wintering period were compared. Statistical comparison of methylation levels in this region showed that the methylation level of red tilapia with black spot variation was significantly reduced, with an overall methylation level of 1.9%; the overall methylation level of red tilapia without body color variation was 63.5%. Black refers to red tilapia with black spot variation, and Red refers to red tilapia without body color variation.

[0044] Figure 7 The proportions of 13 differentially expressed CpG sites within the dmr_2598 (LG3: 11575201~11575800) region were statistically analyzed and calculated. In the Black group, almost all CpG sites had a methylation percentage of 0%, with only the 10th CpG site at 25%. In the Red group, almost all CpG sites had a methylation percentage of over 50%, with the 4th and 7th CpG sites at 37.5%.

[0045] Figure 8 This diagram illustrates the color variation of red tilapia under low temperature (16℃) conditions in an in vivo laboratory experiment. The results show that low temperature causes black coloration to appear on the body surface of red tilapia.

[0046] Figure 9To verify the differential methylation levels of the epigenetic marker dmr_2598 (LG3: 11575201~11575800) related to the black spot trait in overwintering red tilapia during in vivo experiments, the methylation level of red tilapia with black spot variation was significantly reduced, with an overall methylation level of 5.7%; while the overall methylation level of normal red tilapia was 91.3%.

[0047] Figure 10 The results represent the differential methylation rates of 13 CpG sites of the epigenetic marker dmr_2598 (LG3: 11575201~11575800) in red tilapia under simulated low-temperature conditions in the laboratory. In the Black group, the percentage of methylation at all 13 CpG sites was below 12.5%, while in the Red group it was above 75%. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1

[0050] I. Experimental Methods

[0051] Red tilapia and black tilapia exhibiting the black spot trait, used for sequencing library construction, were derived from a full-sib family. 150 Red and 150 Black tilapia were selected from each family, and DNA was extracted from the ventral skin tissue of 48 individuals from each family based on the degree of color variation. The DNA concentration of each individual's skin tissue sample was determined using a Qubit 3.0 fluorescence spectrophotometer, and the samples were mixed in equal volumes according to the sequencing company's requirements. Sequencing was then performed on an Illumina HiSeq™ 250 high-throughput sequencing platform, and the acquired sequencing data were subsequently analyzed. HE staining and L-dopa staining were used to observe the morphological changes in tissues exhibiting the black spot trait.

[0052] 1. Collection and preservation of skin tissue samples from red tilapia

[0053] Fifty red tilapia from the same family that exhibited black spot mutation during hibernation and 150 red tilapia without black spot mutation were selected as the experimental (Black) group and the control (Red) group. Within the experimental and control groups, individuals with extreme color differences were selected: 48 red tilapia with the most pronounced black spot mutation (Black group) and 48 red tilapia with the most vibrant color (Red group). Skin tissue samples were taken from the lower ventral side of each individual using scissors and tweezers, and then preserved in 96-well plates filled with alcohol.

[0054] 2. Observation of skin tissue structure and identification of melanocytes

[0055] HE staining was used to observe the structure of the ventral skin tissue of Red and Black tilapia. The results showed that the black bands were mainly concentrated in the epidermis. Meanwhile, L-dopa staining is a specific staining method targeting melanocytes, which are dendritic cells that secrete melanin. Melanocytes contain tyrosinase (DOPA oxidase), which converts tyrosine into melanin. Therefore, by using DOPA as a substrate and targeting the tyrosinase within the melanocytes, melanin is generated, eliminating the need for further staining to reveal a brownish-black color.

[0056] 3. DNA extraction from skin tissue samples and construction of mixed-pool sequencing libraries

[0057] Genomic DNA was extracted from 48 individuals in each of the Black and Red groups (a total of 96 individuals) using a genomic DNA extraction kit. The DNA quality of all individuals was assessed using agarose gel electrophoresis, and DNA concentration was measured using Qubit 3.0 to ensure that the DNA quality and concentration used for sequencing library construction met the standards. Simultaneously, the DNA concentration of the 48 individuals in each group was diluted to 20 ng / μL according to sequencing requirements, and then mixed in equal volumes.

[0058] 4. High-throughput sequencing of mixed-sample DNA libraries

[0059] High-throughput sequencing was performed using an Illumina HiSeq™ 2500. The raw sequencing data was filtered to remove low-quality reads, adapters, and reads with excessive N, thus obtaining clean data.

[0060] 5. Data analysis and DMR identification

[0061] (I) Data Comparison

[0062] The obtained clean data were compared with the Nile tilapia reference genome using Bismark-0.23.1 software. The tilapia reference genome version was GCA_001858045.3 (https: / / ftp.ensembl.org / pub / release-111 / fasta / oreochromis_niloticus / dna / Oreochromis_niloticus.O_niloticus_UMD_NMBU.dna.toplevel.fa.gz). The successfully aligned sequences were used for further analysis.

[0063] (II) Identification of DMR (Differential Methylation Regions)

[0064] DMR identification includes calculating the DNA methylation level of the whole genome, the methylation level of each base site, and analyzing DNA methylation differences, including identifying differentially methylated regions and differentially methylated sites.

[0065] Specifically, BatMeth2 software was used to calculate methylation levels, with parameters set to a methylation window of 600 bp and a step rate of 200 bp to calculate differentially methylated regions (DMRs) and visualize them at the chromosome level. This yielded a genome-wide methylation map, and data on differential methylation levels, locations, methylation states, and significance levels were collected. Subsequently, a significantly differentially methylated map was constructed from the genome-wide methylation map based on a significance threshold q < 0.001 and a differential methylation level meth.dif ≥ 25. The next step was to annotate the differentially methylated map to obtain significantly differentially methylated regions (DMRs) and their corresponding gene information.

[0066] II. Experimental Results

[0067] Histomorphological observation of the skin tissue on the lower abdomen of red tilapia with black spot mutation revealed that the body color black spot mutation is mainly closely related to the large-scale aggregation of melanocytes.

[0068] Whole-genome bisulfite sequencing was used to obtain 31.64 GB of data from the Red group and 31.08 GB of data from the Black group of skin tissue in the lower ventral region, for a total of 62.72 GB of raw data. Through quality control and DMR analysis, all DMRs were mapped at the whole-genome level, and a whole-genome DNA methylation map was constructed. Figure 3A total of 3943 DMRs were identified based on the thresholds q < 0.001 and meth.diff ≥ 25. Among them, DMRs with similar positions were found enriched in the ChrLG3 chromosome region of ChrLG3: 11574600–11577200. Furthermore, a highly significant DMR, dmr_2598 (ChrLG3: 11575201–11575800), was found within this region, with a significance level of 5.86E-19 (e.g., dmr_2598). Figure 4 Furthermore, 13 significantly different CpG sites were located in the dmr_2598 (ChrLG3: 11575201~11575800) region, such as... Figure 5 Among them, CpG1 is located at ChrLG3: 11575499, CpG2 is located at ChrLG3: 11575510, CpG3 is located at ChrLG3: 11575531, CpG4 is located at ChrLG3: 11575533, CpG5 is located at ChrLG3: 11575552, CpG6 is located at ChrLG3: 11575589, and CpG7 is located at C hrLG3: 11575599, CpG8: 11575617, CpG9 is located in ChrLG3: 11575630, CpG10 is located in ChrLG3: 11575667, CpG11 is located in ChrLG3: 11575683, CpG12 is located in ChrLG3: 11575722 and CpG13 is located in ChrLG3: 11575732.

[0069] Example 2: Epigenetic DMR identification of black spot variation trait in red tilapia during overwintering.

[0070] I. Experimental Methods

[0071] According to the whole-genome bisulfite sequencing analysis in Example 1, a significantly differentially expressed epigenetic DMR, dmr_2598 (LG3: 11575201~11575800), was identified in red tilapia during the overwintering period. This DMR contained 13 CpG loci (CpG1 at ChrLG3: 11575499, CpG2 at ChrLG3: 11575510, CpG3 at ChrLG3: 11575531, CpG4 at ChrLG3: 11575533, and CpG5 at ChrLG3: 11575533). The epigenetic modification markers (CpG10, CpG11, CpG12, and CpG13) are located in ChrLG3 (11575552, ...

[0072] 1. Experimental subjects

[0073] Similar to the Red and Black groups in Example 1 (48 tilapia individuals in each group), a total of 96 tilapia individuals were collected. Skin tissue samples from the lower abdomen were cut and placed in 96-well plates filled with alcohol for preservation, following the method in Example 1.

[0074] 2. Extract DNA from the sample

[0075] Genomic DNA was extracted from the lower abdominal skin tissue of each individual according to the method in Example 1. The DNA concentration of 48 individuals in each group was diluted to 20 ng / μL and mixed in equal amounts as template DNA.

[0076] 3. Design primers

[0077] Design primers for the dmr_2598 (LG3: 11575201~11575800) epigenetic marker. The designed primers are as follows:

[0078] SEQ ID NO 1: 5'-TAGGTGTGAATGTGAGTGTGAATGG-3';

[0079] SEQ ID NO 2: 5'-CAATCAAAATTAACACCAACCCCCT-3'.

[0080] 4. Bisulfite sequencing (Bs-seq)

[0081] Template DNA bisulfite treatment protocol based on EZ DNA Methylation-Gold TM The kit is used; the genomic DNA template from skin tissue undergoes bisulfite modification, resulting in the conversion of DNA bases. After conversion, unmethylated C bases in the template DNA are converted to U, while methylated C bases remain unchanged. Subsequent first-generation sequencing and statistical analysis allow for the calculation and comparison of methylation levels within this region.

[0082] 5. Methylation-specific primer PCR reaction

[0083] Epigenetic marker fragments were amplified using Sangon Taq PCR Mix premix.

[0084] The PCR reaction system consisted of 2 μL of bisulfite-treated genomic DNA as a template, 1 μL of forward and reverse primers, 25 μL of 2×Taq PCR Mix premix, and water added to a total volume of 50 μL.

[0085] The reaction procedure was as follows: pre-denaturation at 94℃ for 7 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 45 s, with a cycle number of 35, and a final extension at 72℃ for 10 min.

[0086] 6. PCR product purification, ligation, transformation, and bacterial pickling

[0087] The PCR products were purified using a gel extraction kit. An overnight ligation reaction was performed under the following conditions: 1 μL T4 DNA ligase, 0.5 μL pGEM-T easy plasmid, 5 μL 2× rapid ligation buffer, 1.5 μL purified PCR product, and finally water to bring the total volume to 10 μL.

[0088] The ligation product was then transformed into Sangon Biotech Trans5α Chemically Competent Cells; finally, after overnight culture in plates, single clones were selected for expansion culture.

[0089] 7. Single-clonal first-generation Sanger sequencing and detection of epigenetic marker methylation levels

[0090] The bacterial culture after monoclonal expansion, along with the primers shown in SEQ ID NO: 1 and 2, was sent to the company for first-generation Sanger sequencing. The sequenced files were first aligned using SnapGene software, and then BiQ_Analyzer software was used to assess the methylation status of the sequences and calculate the methylation level of CpG sites.

[0091] II. Experimental Results

[0092] The sequencing results were first aligned with the tilapia sequences in LG3: 11575201–11575800 (GCA_001858045.3) using SnapGene software. Then, BiQ_Analyzer software was used to assess the methylation status of the sequences and calculate the methylation level of CpG sites. For each single-clone sequencing result, the sequence was aligned with dmr_2598 (LG3: 11575201–11575800) to statistically analyze the methylation modifications at these 13 CpG sites.

[0093] In the pooled samples from the Red and Black groups, eight single-clone sequences were sequenced from each group. The methylation frequency of the 13 CpG sites in Example 1 was calculated, and the results are as follows: Figure 6 The results showed that the methylation level of dmr_2598 in red tilapia exhibiting the black spot variation was 1.9% (2 / 104), which was significantly lower than the methylation level of 63.5% (66 / 104) in red tilapia without the black spot variation. In the Red group, the methylation rate at almost all 13 CpG sites was above 50%, while in the Black group, only the CpG10 site showed methylation at 25%, while the other CpG sites remained unmethylated. Figure 7 .

[0094] Example 3: In vivo experiment to identify epigenetic DMR related to the black spot variation trait in overwintering red tilapia.

[0095] I. Experimental Methods

[0096] 1. Experimental subjects

[0097] In a laboratory environment simulating the low temperature of overwintering, 13 red tilapia of the same lineage without body color variation (Red group) and 13 red tilapia with black spot variation (Black group) were used. Skin tissue samples were cut from the lower abdomen and placed in 96-well plates filled with alcohol for preservation.

[0098] The method to simulate the low-temperature environment during the overwintering period is to use a chiller to cool the aquaculture circulating water to 16℃.

[0099] Genomic DNA was extracted from the lower abdominal skin tissue of each individual according to the method in Example 1. The DNA concentration of 13 individuals in each group was diluted to 20 ng / μL and mixed in equal amounts as template DNA.

[0100] The methylation of 13 CpG sites in dmr_2598 was evaluated according to the method in Example 2.

[0101] II. Experimental Results

[0102] In in vivo experiments simulating the effects of low-temperature environments on the body color changes of red tilapia, black spot mutations appeared in the red tilapia under low-temperature conditions, such as... Figure 8 Sequencing results are as follows: Figure 7 As shown, the SnapGene software was first used to align the tilapia sequence at positions LG3: 11575201–11575800, and then the BiQ_Analyzer software was used to evaluate the methylation site status of the sequence and calculate the methylation level of the CpG site.

[0103] In the pooled samples from the Red and Black groups, eight single-clonal sequences were sequenced from each group, and the overall methylation frequency was calculated as follows: Figure 9 The methylation level of dmr_2598 in red tilapia exhibiting the black spot trait was 5.7% (9 / 104), which was significantly lower than the methylation level of 91.3% (95 / 104) in red tilapia without the black spot trait. Specifically, in the Red group, the methylation rate at all 13 CpG sites was above 75%, while in the Black group, the methylation rate at all 13 CpG sites was below 12.5%. Figure 10 .

[0104] Therefore, an epigenetic marker highly associated with the black spot variation trait exists in dmr_2598 (LG3:11575201~11575800). This marker can be used to determine whether red tilapia will experience body color variations due to environmental changes during overwintering, which is beneficial for targeted breeding and improving trait stability. Example 4: A method for detecting epigenetic markers related to the black spot variation trait in red tilapia during overwintering.

[0105] 1. Extraction and processing of genomic DNA from samples

[0106] Genomic DNA was extracted from individual samples using a genomic DNA extraction kit. Template DNA bisulfite treatment was performed according to the EZ DNA Methylation-Gold protocol. TM The kit was used.

[0107] 2. Methylation-specific primer PCR reaction

[0108] The primers for the PCR reaction are:

[0109] SEQ ID NO: 1: 5'-TAGGTGTGAATGTGAGTGTGAATGG-3';

[0110] SEQ ID NO: 2: 5'-CAATCAAAATTAACACCAACCCCCT-3'.

[0111] The PCR reaction system consisted of 2 μL of bisulfite-treated genomic DNA as a template, 1 μL of forward and reverse primers, 25 μL of 2×Taq PCR Mix premix, and water added to a total volume of 50 μL.

[0112] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 7 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0113] 3. PCR product purification, ligation, transformation, and bacterial pickling

[0114] The PCR product from the previous step was purified by gel extraction; then an overnight ligation reaction was performed under the following conditions: 1 μL T4 DNA ligase, 0.5 μL pGEM-T easy plasmid, 5 μL 2× rapid ligation buffer, 1.5 μL of purified PCR product, and finally water to a final volume of 10 μL. The ligation product was transformed into competent cells; after overnight plating and culture, single colonies were picked for further expansion.

[0115] 4. Single-clonal first-generation Sanger sequencing and verification of epigenetic marker methylation levels

[0116] Each single-clone culture, along with the primers shown in SEQ ID NO1 and 2, was sent to the company for first-generation Sanger sequencing. The sequenced files were first aligned using SnapGene software, and then BiQ_Analyzer software was used to assess the methylation status of the sequences and calculate the methylation levels of the 13 CpG sites.

[0117] 5. Result Interpretation

[0118] Single-clone sequencing results were aligned using SnapGene software to check the consistency between the sequencing sequences and dmr_2598 (LG3: 11575201~11575800). Subsequently, the sequences of each single clone were input into BiQ_Analyzer software for alignment and evaluation against the reference sequences ChrLG3:11575201~11575800. Analysis was performed on the 13 CpG sites in each single-clone sequence (CpG1 at ChrLG3:11575499, CpG2 at ChrLG3:11575510, CpG3 at ChrLG3:11575531, CpG4 at...). The methylation status and methylation level of CpG5 (CrLG3: 11575533), CpG6 (CrLG3: 11575552), CpG7 (CrLG3: 11575589), CpG8 (CrLG3: 11575599), CpG9 (CrLG3: 11575617), CpG10 (CrLG3: 11575667), CpG11 (CrLG3: 11575683), CpG12 (CrLG3: 11575722), and CpG13 (CrLG3: 11575732).

[0119] Individuals with methylation levels above 50% at 13 CpG sites in the dmr_2598 differential methylation region were red tilapia with stable body color and no black spots during overwintering, while individuals with methylation levels below 25% at the same 13 CpG sites were red tilapia with black spot mutations during overwintering. Therefore, by comparing the methylation levels of epigenetic markers related to the black spot mutation trait in red tilapia during overwintering, it can be determined whether red tilapia will develop the black spot trait due to changes in low environmental temperatures.

[0120] Example 5: A kit for detecting the variation of black spots in red tilapia during overwintering.

[0121] I. Composition

[0122] Primers with nucleotide sequences as shown in SEQ ID NO: 1 and 2, DNA bisulfite treatment reagent, and PCR amplification reagent, wherein the PCR amplification reagent is 2×Taq PCR Mix premix and ddH2O, and the DNA bisulfite treatment reagent is EZ DNAMethylation-Gold. TM Kit.

[0123] II. Instructions for Use

[0124] Same as Example 4.

Claims

1. The application of a reagent for detecting the methylation level of an epigenetic marker related to the variation of body color black spots in overwintering red tilapia in the preparation of a kit for detecting the variation of body color black spots in overwintering red tilapia, characterized in that, The epigenetic markers are located in LG3: 11575201 to 11575800, with the reference genome being GCA_001858045.

3. The epigenetic markers are 13 CpG sites located at CpG1 to CpG13 in the nucleotide sequence shown in SEQ ID NO:

3. CpG1 is located at position 299 of the nucleotide sequence shown in SEQ ID NO:

3. CpG2 is located at position 310 of the nucleotide sequence shown in SEQ ID NO:

3. CpG3 is located at position 331 of the nucleotide sequence shown in SEQ ID NO:

3. CpG4 is located at position 333 of the nucleotide sequence shown in SEQ ID NO:

3. CpG5 is located at position 352 of the nucleotide sequence shown in SEQ ID NO:

3. CpG6 is located at position 389 of the nucleotide sequence shown in SEQ ID NO:

3. CpG7 is located at position 399 of the nucleotide sequence shown in SEQ ID NO:

3. CpG8 is located at position 417 of the nucleotide sequence shown in SEQ ID NO:

3. CpG9 is located at position 430 of the nucleotide sequence shown in SEQ ID NO:

3. CpG10 is located at position 467 of the nucleotide sequence shown in SEQ ID NO:

3. CpG11 is located at position 483 of the nucleotide sequence shown in SEQ ID NO:

3. CpG12 is located at position 522 of the nucleotide sequence shown in SEQ ID NO:

3. CpG13 is located at position 532 of the nucleotide sequence shown in SEQ ID NO:3; Individuals with methylation levels above 50% at all 13 CpG sites are red tilapia with stable body color and no black spots during overwintering, while individuals with methylation levels below 25% at all 13 CpG sites are red tilapia with black spot mutations during overwintering.

2. The application according to claim 1, characterized in that, The detection reagent is a primer.

3. The application according to claim 2, characterized in that, The kit also contains DNA bisulfite treatment reagents and / or PCR amplification reagents.

4. A method for detecting the variation of body color black spots in red tilapia during overwintering, characterized in that, The DNA of the test samples was treated with bisulfite, and its epigenetic markers were detected. The methylation level was used to determine the variation of black spots during winter. The epigenetic markers are located in LG3: 11575201 to 11575800, with the reference genome being GCA_001858045.

3. The epigenetic markers are 13 CpG sites located at CpG1 to CpG13 in the nucleotide sequence shown in SEQ ID NO:

3. CpG1 is located at position 299 of the nucleotide sequence shown in SEQ ID NO:

3. CpG2 is located at position 310 of the nucleotide sequence shown in SEQ ID NO:

3. CpG3 is located at position 331 of the nucleotide sequence shown in SEQ ID NO:

3. CpG4 is located at position 333 of the nucleotide sequence shown in SEQ ID NO:

3. CpG5 is located at position 352 of the nucleotide sequence shown in SEQ ID NO:

3. CpG6 is located at position 389 of the nucleotide sequence shown in SEQ ID NO:

3. CpG7 is located at position 399 of the nucleotide sequence shown in SEQ ID NO:

3. CpG8 is located at position 417 of the nucleotide sequence shown in SEQ ID NO:

3. CpG9 is located at position 430 of the nucleotide sequence shown in SEQ ID NO:

3. CpG10 is located at position 467 of the nucleotide sequence shown in SEQ ID NO:

3. CpG11 is located at position 483 of the nucleotide sequence shown in SEQ ID NO:

3. CpG12 is located at position 522 of the nucleotide sequence shown in SEQ ID NO:

3. CpG13 is located at position 532 of the nucleotide sequence shown in SEQ ID NO:3; Individuals with methylation levels above 50% at all 13 CpG sites are red tilapia with stable body color and no black spots during overwintering, while individuals with methylation levels below 25% at all 13 CpG sites are red tilapia with black spot mutations during overwintering.

Images