Indel marker for identifying gender of different habitats of large yellow croaker and application thereof

Abstract

This invention discloses an indel marker for identifying the sex of large yellow croaker from different habitats and its application. The indel marker is a 12 bp insertion / deletion nucleotide sequence located at position 47617128 of chromosome 3 of the large yellow croaker, as shown in SEQ ID NO.1. Female individuals have the sequence shown in SEQ ID NO.1, while male individuals lack the sequence shown in SEQ ID NO.1. The invention also discloses primers and kits for amplifying the marker, a method for identifying the sex of large yellow croaker from different habitats using the primers or kits, and the application of the aforementioned primers, kits, or methods in identifying the sex of large yellow croaker from different habitats. This invention features universality, high resolution and specificity, low cost and ease of operation, and also has industrialization potential.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to an indel marker for identifying the sex of large yellow croaker in different habitats and its application. Background Technology

[0002] Most fish lack heteromorphic chromosomes, making their sex-determining regions difficult to identify directly at the morphological and molecular levels. This characteristic makes accurate sex identification of live large yellow croakers impossible using traditional morphological methods, especially during the juvenile stage (undifferentiated gonads). Furthermore, significant sexual dimorphism exists in large yellow croaker aquaculture, making asexual reproduction a highly valuable industrial practice. Developing sex-specific molecular markers provides a technological foundation for achieving asexual breeding of large yellow croakers and analyzing the evolutionary and domestication selection processes of sex-determining genes.

[0003] A study identified a sex-specific deletion marker between the dmrt1 and cfap157 genes in the Daiqu population. This marker was validated with 100% accuracy in 276 Daiqu large yellow croakers, but the accuracy in the Min-Yue Dong population was only 79.6%, indicating a divergence in the sex-determining region between the two groups. Another study screened a perfectly sex-linked SNP using a whole-genome scan of the Min-Yue Dong large yellow croaker. Based on this SNP, an allele-specific PCR (AS-PCR) detection system was developed (using two universal primers and one Y-chromosome-specific primer, amplifying only a 348 bp band in females, while generating an additional 194 bp Y-chromosome-specific band in males). This marker was validated with 100% accuracy in nearly 2200 individuals from both the Min-Yue Dong and Daiqu populations. Further localization analysis showed that the sex-determining region in the Min-Yue Dong population is located on chromosome 22, in stark contrast to the location on chromosome 3 in the Daiqu population, revealing genetic differentiation in the sex-determining mechanism between populations. It is worth noting that although the marker is also effective in the Daiqu tribe, the design of deletion markers specific to the Daiqu tribe can more accurately adapt to the genetic background of this population. Research on the sex determination mechanism of the Naozhou tribe large yellow croaker is relatively lagging. Due to its long-term wild existence and lack of domestication, basic biological research on the Naozhou tribe started relatively late, and targeted sex-linked marker studies have not yet been reported.

[0004] In addition to geographical population differences, genetic and morphological differentiation between wild and farmed populations (different ecological environments, referred to as habitats) also influences the application of sex markers. There are differences in genetic diversity between wild and farmed populations. Studies based on mitochondrial D-loop region analysis and microsatellite markers have shown that the haplotype diversity index, observed heterozygosity, and Shannon index of wild populations are higher than those of farmed populations. Other studies have shown differences in sex dimorphism expression; in wild populations, female body length and male body height have the greatest direct effect on body weight; while in farmed populations, the total length of both male and female fish has the most significant effect on body weight. Therefore, it is necessary to develop an indel marker for sex in large yellow croaker from different habitats. Summary of the Invention

[0005] The purpose of this invention is to provide an indel marker for identifying the sex of large yellow croaker from different habitats.

[0006] The present invention also aims to provide a primer for amplifying the indel marker and a kit including the primer, as well as a method for identifying the sex of large yellow croaker from different habitats using the primer or kit.

[0007] The final object of the present invention is to provide the application of the above-described primers, kits or methods in identifying the sex of large yellow croaker in different habitats.

[0008] The first objective of the present invention can be achieved by the following technical solution: an indel marker for identifying the sex of large yellow croaker from different habitats, wherein the indel marker is a 12bp insertion / deletion nucleotide sequence at position 47617128 of chromosome 3 of large yellow croaker, the sequence being as shown in SEQ ID NO.1, with female individuals having the sequence shown in SEQ ID NO.1 and male individuals lacking the sequence shown in SEQ ID NO.1.

[0009] Based on high-throughput resequencing data, this invention screens sex-associated SNPs and InDel molecular markers from wild and farmed populations of large yellow croaker, as well as from the Naozhou and Eastern Fujian-Guangdong populations. Combining population genetics analysis, GWAS, and FST methods, stable sex-associated loci are identified, and specific primers are designed to achieve efficient sex identification. Furthermore, a universal sex molecular marker for large yellow croaker applicable to wild populations, farmed populations, and different geographical subpopulations is developed.

[0010] The development process of sex indel markers for large yellow croaker in different habitats in this invention includes: sample collection → morphological feature analysis → resequencing and SNP / Indel mining → GWAS analysis → sex-associated indel marker screening → primer design and validation → molecular marker application.

[0011] in:

[0012] Preferably, sample collection includes:

[0013] A total of 146 samples (58 females and 88 males) of different large yellow croaker groups were collected. Among them, 20 wild large yellow croaker (NZ) samples (10 females and 10 males) were collected in the waters near Naozhou Island, Zhanjiang City, Guangdong Province (longitude: 110.51639°; latitude: 20.75070°); 68 wild large yellow croaker samples (22 females and 46 males) were collected in the waters off Liu'ao Town, Zhangpu City, Fujian Province (longitude: 117.70330°; latitude: 23.89065°); and 58 domesticated and farmed large yellow croaker samples (26 females and 32 males) were collected from Sandu Marine Food Co., Ltd., Ningde City, Fujian Province (longitude: 119.57384°; latitude: 26.60169°).

[0014] Preferably, the morphological characteristics analysis of male and female large yellow croaker includes:

[0015] Images were acquired from well-preserved large yellow croaker specimens. The specimens were fixed to foam boards, and the fins were spread out using pins to ensure natural extension without abnormal bending. Each specimen was then photographed individually. During photography, the camera was fixed so that the lens was completely perpendicular to the specimen's shooting surface, maintaining consistent specimen placement and focus. Distinctive and easily identifiable points on the large yellow croaker were selected as landmarks. Geometric morphology methods were used to perform Procrustes superposition and average morphological deformation analysis on both sexes. TPS interpolation was used to transform the difference between the two average shapes into a smooth, continuous deformation field, and the spatial distribution pattern of this difference across the entire morphology was visually visualized using a deformation regular grid.

[0016] Preferably, resequencing and SNP / Indel mining include DNA extraction and library preparation, high-throughput sequencing, quality control, alignment and variant detection, and PCA analysis.

[0017] Preferably, DNA extraction and library construction includes: extracting high-quality genomic DNA using the CTAB method and constructing an Illumina sequencing library.

[0018] Preferably, high-throughput sequencing includes 150bp paired-end sequencing on the Illumina NovaSeq platform.

[0019] Preferably, quality control includes: using FASTP to remove adapters and low-quality sequences from the raw sequencing data.

[0020] Preferably, the alignment and variant detection include: aligning clean reads to a reference genome (reference genome version GCF_000972845.2) using BWA-MEM, followed by SNP and Indel detection using GATK.

[0021] Preferably, the PCA analysis includes:

[0022] Use PLINK to filter variability data (e.g., MAF > 0.05, missing rate < 10%).

[0023] Principal component analysis was performed using PCA.

[0024] Plot a principal component diagram in R, using colors to distinguish between males and females.

[0025] Preferably, gender association analysis (GWAS) includes:

[0026] Using PLINK for association analysis based on gender phenotype:

[0027] plink--bfile filtered_data--assoc--pheno sex.txt--allow-no-sex

[0028] Use Bonferroni to correct the significance threshold.

[0029] Generate QQ plots and Manhattan plots (using the qqman or CMplot R packages).

[0030] Preferably, the FST analysis process includes:

[0031] Significance threshold: The significance threshold of FST (FST>0.2) was determined by permutation test (1000 times).

[0032] Preferably, Indel marker development includes: screening Indel sites with a length of ≥3bp within the sex-associated interval, and with clear male-female differentiation.

[0033] Preferably, the marker type includes: Indel-based co-dominant molecular markers, suitable for conventional PCR and electrophoresis platforms. Detection efficiency: Sex typing can be achieved with a single pair of primers, with an accuracy of ≥98%.

[0034] The final selected indel marker is located at ri-3-47617128 (reference genome version GCF_000972845.2), which contains a 12 bp insertion / deletion nucleotide sequence, as shown in SEQ ID NO.1.

[0035] Specifically: 5'-TGCAGGCCGATG-3' (as shown in SEQ ID NO.1).

[0036] The second objective of the present invention can be achieved by the following technical solution: a primer for amplifying the indel label, the primer comprising an upstream primer and a downstream primer, the sequence of the upstream primer being shown in SEQ ID NO.2, and the sequence of the downstream primer being shown in SEQ ID NO.3.

[0037] Specifically:

[0038] Forward: 5'-CCACCACTTCATTCCTGGACA-3' (as shown in SEQ ID NO.2);

[0039] Reverse: 5'-CTCATTCCTCTGCTCCCTACAA-3' (as shown in SEQ ID NO.3).

[0040] The present invention also provides a kit for identifying the sex of large yellow croaker from different habitats, the kit comprising the primers described above.

[0041] This invention also provides a method for identifying the sex of large yellow croakers from different habitats, comprising the following steps:

[0042] (1) Extract genomic DNA from the sample to be tested;

[0043] (2) Using the genomic DNA of the sample to be tested as a template, perform PCR amplification using the primers or the kit described above;

[0044] (3) The sex of the large yellow croaker was determined based on the electrophoresis image. Individuals with only the 131bp fragment were female large yellow croakers, while individuals with both the 131bp and 119bp fragments were male large yellow croakers.

[0045] In this method for identifying the sex of large yellow croaker from different habitats:

[0046] Preferably, in step (2), the PCR reaction system used for PCR amplification is: 10 μL of 2×Taq MasterMix, 0.5 μL each of upstream and downstream primers with a concentration of 10 μM, 50 ng of genomic DNA, and ddH2O to 20 μL.

[0047] Preferably, the PCR reaction program used in step (2) during PCR amplification is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 9 s, 35 cycles, 72℃ extension for 5 min.

[0048] Preferably, in step (3), females have a single band (131bp) and males have two bands (131bp and 119bp).

[0049] The last objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned primers, kits, or methods in identifying the sex of large yellow croakers from different habitats.

[0050] The present invention has the following advantages:

[0051] (1) Universality: Applicable to sex identification of large yellow croaker from Naozhou and eastern Fujian and Guangdong, and also applicable to sex identification of wild and farmed large yellow croaker;

[0052] (2) High resolution and specificity: Combining GWAS and FST analysis, potential sex-determining regions can be identified, and Indel sites can be accurately located;

[0053] (3) Low cost and easy operation: No complicated equipment is required. Ordinary laboratories can complete the test through routine PCR, and the cost per sample is ≤5 yuan.

[0054] (4) Industrialization potential: The developed molecular markers can be converted into reagent kits to promote the asexual farming and genetic breeding of large yellow croaker. Attached Figure Description

[0055] Figure 1 This is an analysis of the morphological differences of large yellow croaker in Example 1. Figure A shows the geometric morphological markers of the large yellow croaker body. Figure B shows the shape deformation mesh diagram of female and male large yellow croaker constructed based on the geometric morphological markers. The left image in Figure B is the average morphological mesh of females, the right image in Figure B is the average morphological mesh of males, and Figure C uses the average shape of males as a reference to visualize the relative deformation of females.

[0056] Figure 2 The images shown are typical examples of anatomical and histological identification of large yellow croaker used for resequencing and molecular marker verification in Examples 2 and 5. Image A is an external image of the ovary of a female large yellow croaker, Image B is an external image of the testis of a male large yellow croaker, Image C is a histological section of the ovary, with oocytes visible under microscopic structure, and Image D is a histological section of the testis, with spermatocytes and spermatids visible under microscopic structure.

[0057] Figure 3 This is an example of the population structure and sex-related analysis of male and female large yellow croaker in Example 2. Figure A shows the principal component analysis (PC1 vs PC2) based on whole-genome SNPs, with the ellipse representing the 95% confidence interval. Figure B compares the distribution of observed values ​​and theoretical expected values ​​in the sex association analysis. Figure C is a whole-genome Manhattan plot of sex-related SNP loci, with the horizontal axis representing chromosome location and the vertical axis representing -log. 10(P), the red line represents the Bonferroni-corrected significance threshold (P = 5 × 10⁻⁶). -8 The blue line represents the suggested threshold (P = 1 × 10). -5 The significant peak on chromosome 3. Figure D is a Manhattan plot of sex-associated SNPs on chromosome 3 of large yellow croaker. Each point represents a SNP locus, the horizontal axis is its physical location on chromosome 3 (unit: Mb), and the vertical axis is the significance of the association after logarithmic transformation (-log). 10 (p-value)), the red dashed line represents the significance threshold after Bonferroni correction, and the E-plot is a local Manhattan plot of the 45-50Mb region of chromosome 3;

[0058] Figure 4 For the FST analysis of male and female SNP data of large yellow croaker in Example 3, Figure A is the genome-wide FST distribution map of male and female large yellow croaker. Each point in the figure represents the FST value of a SNP locus. The horizontal axis is its physical location in the whole genome, and the vertical axis is the FST value. Different chromosomes are distinguished by different colors. Figure B is the FST distribution map of SNPs on chromosome 3. Each point represents the FST value of a single SNP on chromosome 3. The horizontal axis is the physical location (unit: Mb), and the vertical axis is the FST value. Figure C is the sliding plot of the average FST of chromosome 3 within a 1Mb window.

[0059] Figure 5 For the population structure and sex correlation analysis of male and female large yellow croaker in Example 4, Figure A is a whole-genome Manhattan diagram of sex-related Indel loci, with the horizontal axis representing chromosome position and the vertical axis representing -log 10 (P), the red line represents the Bonferroni-corrected significance threshold (P = 5 × 10⁻⁶). -8 The blue line represents the suggested threshold (P = 1 × 10). -5 Figure B is a Manhattan plot of sex association on chromosome 3 of large yellow croaker. Each point represents an INDEL locus, with the horizontal axis representing its physical location on chromosome 3 (unit: Mb), and the vertical axis representing the significance of the association after logarithmic transformation (-log). 10 (p-value)), the red dashed line represents the significance threshold after Bonferroni correction, and Figure C is a local Manhattan plot of the 45–50Mb region of chromosome 3, magnified to show the sex association signal in the 45–50Mb region of Chr3.

[0060] Figure 6 This is a typical electrophoresis result from Example 5. Detailed Implementation

[0061] Unless otherwise specified, all reagents and materials used in the following examples are commercially available. Furthermore, unless otherwise specified, all laboratory instruments used are standard laboratory equipment.

[0062] Example 1: Morphological characteristics analysis of male and female large yellow croaker

[0063] Landmarks were selected from points on the large yellow croaker that are easily identifiable and have distinct characteristics. These were categorized into three types: Type I (intersections between different tissues), Type II (depressions or protrusions within the structure), and Type III (extreme points within the structure), as detailed below. Figure 1 As shown in Figure A, Procrustes superposition and average morphological deformation analysis were performed on both sexes using geometric morphology methods. The morphological deformation mesh results show that males and females are highly consistent in key morphological parameters such as body length ratio, dorsal and ventral contour, and head structure, with no obvious morphological deviations observed. Figure 1 (Figures B-C)

[0064] Specifically, the analysis of morphological differences in large yellow croaker is as follows: Figure 1 As shown, Figure A represents the geometric morphological markers of the large yellow croaker. Figure B shows the body deformation mesh diagrams of female and male large yellow croakers constructed based on the geometric morphological markers. The left image in Figure B is the average morphological mesh of the female, and the right image is the average morphological mesh of the male. The mesh deformation represents the morphological differences between the two sexes in the overall body shape. Stretched or contracted areas indicate morphological changes in the corresponding parts. The mesh shapes of the male and female are basically the same, and no obvious stretching or compression is shown, indicating that the male and female large yellow croakers do not show significant differences in overall body structure. Figure C visualizes the relative deformation of the female using the average shape of the male as a reference. The deformation diagram is constructed based on the superposition of Procrustes and morphological averaging. The deformation diagram shows that the arrows of most Landmarks are short and the changes are slight, indicating that there is no systematic shift in the local structure of the male and female body shapes, supporting the conclusion that the overall morphology of the two sexes is consistent.

[0065] Conclusion: Large yellow croaker did not exhibit morphological sex dimorphism between male and female individuals. Their body shape differences may be mainly driven by individual developmental stage or environmental factors, rather than sex itself.

[0066] Example 2: GWAS analysis based on SNP resequencing of male and female large yellow croaker

[0067] Anatomical sex confirmation

[0068] Target: All large yellow croaker individuals used for resequencing;

[0069] Operating steps:

[0070] Vivo-anesthesia (MS-222, 100 mg / L, immersion for 5 min);

[0071] Gonadal tissue was removed via ventral dissection;

[0072] Histological sections of gonads:

[0073] Fixation: Bouin's solution for 24 hours;

[0074] Sectioning: Paraffin sections were 5 μm thick;

[0075] Staining: HE staining;

[0076] The interpretation criteria are shown in Table 1 below:

[0077] Table 1 Gender Determination Criteria

[0078] gender Microscopic features Illustrated Example female oocyte Figure 2 Figures A and C male Fine lobule structure Figure 2 Figures B and D

[0079] PCA analysis results ( Figure 3 (Figure A in the middle):

[0080] Principal component analysis results showed that male and female individuals exhibited significant separation along the PC1 and PC2 dimensions. Figure 3 Figure A shows that there are genetic differences between sexes. This provides a basis for subsequent detection of sex-related variations.

[0081] QQ image analysis ( Figure 3 (Figure B in the middle):

[0082] The QQ plot illustrates the distribution relationship between the observed p-values ​​and the expected p-values. The results show ( Figure 3 (Figure B) The vast majority of loci are distributed along the main diagonal, but deviations occur in extremely significant regions, suggesting the presence of variant loci highly associated with sex.

[0083] Manhattan diagram analysis ( Figure 3 (Figure C in the middle):

[0084] Manhattan plots reveal the presence of sex-related SNP sites on chromosome Chr3. Figure 3 (Figure C in the middle)

[0085] Partial Manhattan diagram ( Figure 3 (Diagrams D-E):

[0086] A significant correlation peak was observed at 46.5–49.5 Mb on chromosome 3. Figure 3 (See Figure D). A magnified view of the 45–50 Mb region of chromosome 3, showing the strongest sex-related correlation at 47.3–48.0 Mb. Figure 3 (China E diagram).

[0087] Specifically, the population structure and sex correlation analysis of large yellow croaker are as follows: Figure 3As shown, Figure A is the principal component analysis (PC1 vs PC2) based on genome-wide SNPs. Female (red) and male (blue) individuals show significant segregation on PC1, indicating sex-related genetic differentiation. The ellipse represents the 95% confidence interval. Figure B compares the distribution of observed values ​​with the theoretical expected values ​​in the sex association analysis. The tails slightly deviate from the diagonal, suggesting the presence of a true association signal. Figure C is the genome-wide Manhattan plot of sex-related SNP loci, with the horizontal axis representing chromosome location and the vertical axis representing -log. 10 (P), the red line represents the Bonferroni-corrected significance threshold (P = 5 × 10⁻⁶). -8 The blue line represents the suggested threshold (P = 1 × 10). -5 The significant peak on chromosome 3. Figure D is a Manhattan plot of sex-associated SNPs on chromosome 3 of large yellow croaker. Each point represents a SNP locus, the horizontal axis is its physical location on chromosome 3 (unit: Mb), and the vertical axis is the significance of the association after logarithmic transformation (-log). 10 (p-value)), the red dashed line represents the significance threshold after Bonferroni correction. Significantly associated sites are highly concentrated in the approximately 46.5-49.5 Mb interval of Chr3. Figure E is a local Manhattan plot of the 45-50 Mb interval of chromosome 3. The magnified view shows the sex-associated signal in the 45-50 Mb interval of Chr3. Some variant snps in this region show high linkage disequilibrium (LD), which may constitute the structural or regulatory regions related to sex determination.

[0088] Information on SNPs selected based on GWAS and passing the Bonferroni correction (BONF) significance threshold is presented in Table 2.

[0089] Table 2 shows the SNPs selected based on GWAS and passing the Bonferroni-corrected (BONF) significance threshold (P<0.05).

[0090] CHR SNP UNADJ GC BONF HOLM SIDAK_SS SIDAK_SD FDR_BH FDR_BY 3 rs:3:47459569 1.485e-10 6.223e-09 0.001083 0.001083 0.001082 0.001082 0.0007442 0.01219 3 rs:3:47458183 2.041e-10 8.095e-09 0.001488 0.001488 0.001487 0.001487 0.0007442 0.01219 3 rs:3:47555780 1.462e-09 4.122e-08 0.01066 0.01066 0.01061 0.01061 0.003554 0.05822 3 rs:3:47823050 2.964e-09 7.394e-08 0.02162 0.02162 0.02139 0.02139 0.005405 0.08853 3 rs:3:47498003 5.721e-09 1.274e-07 0.04172 0.04172 0.04086 0.04086 0.006246 0.1023 3 rs:3:47506146 6.498e-09 1.415e-07 0.04739 0.04739 0.04628 0.04628 0.006246 0.1023 3 rs:3:47638843 6.5e-09 1.415e-07 0.04741 0.04741 0.0463 0.0463 0.006246 0.1023 3 rs:3:47638636 6.851e-09 1.478e-07 0.04997 0.04997 0.04874 0.04874 0.006246 0.1023

[0091] Note: CHR: Chromosome; SNP: SNP Identifier; UNADJ: Unadjusted P-value; GC: Genomic Control Adjusted P-value; BONF: Bonferroni Correction; HOLM: Holm-Bonferroni Correction; SIDAK_SS: Sidák Single Step Correction; SIDAK_SD: Sidák Step-Down Correction; FDR_BH: Benjamini-Hochberg FDR; FDR_BY: Benjamini-Yekutieli FDR.

[0092] Example 3: Results of whole-genome FST analysis

[0093] Results of whole-genome FST analysis ( Figure 4 (Figure A in the middle):

[0094] Across the entire genome, most SNPs had FST values ​​between 0 and 0.1, indicating a low level of overall genomic differentiation between males and females. A high FST peak was observed on chromosome 3, suggesting that this chromosome may carry important genetic variations related to sex determination.

[0095] FST analysis results of chromosome 3 ( Figure 4 (Figures B-C):

[0096] On Chr3, particularly in the 46.5–49.5 Mb region, the FST value is significantly elevated. This extreme differentiation usually indicates strong sex selection pressure or sex-determining loci. A 1 Mb sliding window analysis of the average FST on chromosome 3 helps reduce the impact of random fluctuations and identify genomic regions with persistently high differentiation. The results show that the average FST in the 46–50 Mb segment of chromosome 3 is much higher than in other regions, further validating this as a hotspot of significant male-female differentiation. This region is consistent with the aforementioned GWAS and Manhattan plot analyses, providing cross-validation support.

[0097] Specifically, the Fst analysis of male and female SNP data of large yellow croaker is as follows: Figure 4As shown in Figure A, the genome-wide FST distribution of large yellow croaker (SNP) loci in males and females is illustrated. Each point represents the FST value of a single SNP locus. The horizontal axis represents its physical location in the genome, and the vertical axis represents the FST value. Different chromosomes are distinguished by different colors. Most SNP loci have low FST values, suggesting a low overall level of genetic differentiation between males and females. However, an area with abnormally high FST values ​​was observed on chromosome 3, suggesting that this area may be related to sex determination. Figure B shows the FST distribution of SNPs on chromosome 3. Each point represents the FST value of a single SNP on chromosome 3. FST values, with the horizontal axis representing physical location (unit: Mb) and the vertical axis representing FST values, show multiple sites with significantly high FST values ​​in the approximately 46–49 Mb range, indicating significant male-female differentiation in this region, which may be a candidate region for sex determination or regulation. Figure C is a sliding plot of the average FST value in a 1 Mb window on chromosome 3. This figure shows the distribution of the average FST value calculated by a 1 Mb sliding window on chromosome 3. The horizontal axis represents the starting position of the sliding window, and the vertical axis represents the average FST of SNPs within that window. A significantly higher average FST peak than the background was observed in the approximately 46–50 Mb range.

[0098] Example 4: GWAS analysis based on Indel male and female large yellow croaker resequencing

[0099] Manhattan diagram analysis ( Figure 5 (Figure A in the middle):

[0100] Manhattan plot reveals the presence of a sex-linked Indel locus on chromosome Chr3. Figure 5 (Figure A in the middle)

[0101] Partial Manhattan diagram ( Figure 5 (Figures B-C):

[0102] A significant correlation peak was observed at 46.5-49.5 Mb on chromosome 3. Figure 5 (See Figure B). A magnified view of the 45-50 Mb region of chromosome 3, showing the strongest sex-related correlation at 47.4-48.0 Mb. Figure 5 (Figure C in the middle)

[0103] Information on Indels selected based on GWAS and passing the Bonferroni correction (BONF) significance threshold is presented in Table 3.

[0104] Table 3 shows the Indels (P<0.05) selected based on GWAS and corrected for Bonferroni significance threshold (BONF).

[0105] CHR SNP A1 F_A F_U A2 CHISQ P OR 3 ri-3-47617128 C 0.512 0.1509 CTGCAGGCCGATG 36.14 1.84E-09 5.903 3 ri-3-47823327 A 0.3415 0.7019 AACTAAGTCAGTC 33.12 8.65E-09 0.2202 3 ri-3-47458918 CTGT 0.358 0.717 C 33.02 9.10E-09 0.2201 3 ri-3-47460264 CGT 0.5904 0.2453 C 31.06 2.51E-08 4.434

[0106] Note: CHR: Chromosome; SNP: SNP Identifier; A1: Allele 1, effect allele (target allele), the direction of the effect detected in association analysis (usually a minor allele or a target phenotype associated allele); F_A: Frequency in Cases, the frequency of A1 in the case group (e.g., males), the frequency of the A1 allele in the male population; F_U: Frequency in Controls, the frequency of A1 in the control group (e.g., females); A2: Allele 2, reference allele (baseline allele); CHISQ: Chi-square Statistic, a chi-square test statistic that measures the difference in genotype frequencies between male and female populations (the larger the value, the more significant); P: P-value, the uncorrected association significance P-value; OR: Odds Ratio, the odds ratio (effect strength), OR = (F_A / (1-F_A)) / (F_U / (1-F_U)) (OR>1: A1 increases the probability of males).

[0107] Example 5 Molecular marker verification

[0108] 1. DNA extraction (fin tissue)

[0109] Reagents and equipment:

[0110] Reagent kit: TIANGEN Marine Animal Genomic DNA Extraction Kit (DP324);

[0111] Tissue homogenizer: Tissuelyser LT (QIAGEN).

[0112] step:

[0113] Sample processing:

[0114] Take ≤20mg of fin tissue (fixed with ethanol), chop it with a sterile blade, add 180μL GA buffer + 20μL Proteinase K for lysis, and digest in a 56℃ water bath with shaking for 3 hours (twice the time of ordinary animal tissue);

[0115] DNA purification:

[0116] Add 200 μL of GB buffer, incubate at 70 °C for 10 min, add 200 μL of anhydrous ethanol, transfer to the adsorption column, and centrifuge at 12,000 rpm for 30 s (repeat twice).

[0117] Washout:

[0118] Elute with 60 μL of preheated (65°C) ultrapure water;

[0119] Output: Concentration ≥25ng / μL, OD260 / 280=1.82±0.05.

[0120] 2. Primer design and synthesis

[0121] Tagging information:

[0122] Name: ri-3-47617128;

[0123] Location: Chr3: 47617128-47617139;

[0124] Variance: 12bp deletion (male-specific).

[0125] Primer design:

[0126] Forward primer: 5'-CCACCACTTCATTCCTGGACA-3'

[0127] Reverse primer: 5'-CTCATTCCTCTGCTCCCTACAA-3'

[0128] Product length:

[0129] -Wild type: 131bp;

[0130] - Deletion type: 131bp - 12bp = 119bp.

[0131] Synthesis Specifications:

[0132] Synthetic Company: Sangon Biotech

[0133] Purity: PAGE purification

[0134] Reconstitution concentration: 100 μM (stock solution), dilute working solution to 10 μM;

[0135] The PCR reaction system used was as follows: 10 μL of 2×Taq MasterMix, 0.5 μL each of 10 μM upstream and downstream primers, 50 ng (2 μL) of genomic DNA, and 20 μL of ddH2O (6.4 μL).

[0136] The PCR reaction program used was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 9 s, 35 cycles, 72℃ extension for 5 min.

[0137] 3. Electrophoresis detection:

[0138] Gel concentration: 3% agarose gel (containing 0.5×GelRed);

[0139] Electrophoresis conditions: 100V × 1h;

[0140] Loading volume: 5 μL PCR product;

[0141] The results are shown in Table 4:

[0142] Table 4. Judgment of Electrophoresis Detection Results

[0143] gender Number of stripes Fragment size female 1 item 131bp male 2 131bp + 119bp

[0144] Note: Ladder H2 DNA Marker was used (bands: 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1031 bp).

[0145] 4. Sample Validation Design

[0146] The sex of the samples was determined by anatomical and histological identification (see Example 2 for specific methods), as shown in Table 5 below.

[0147] Table 5 shows the sex of the samples obtained by anatomical and histological identification.

[0148]

[0149] 5. Classification Results

[0150] Typical electrophoresis results for some samples are as follows: Figure 6 It shows that, from Figure 6 As can be seen, phenotypically females have only a 131bp fragment, while phenotypically males have both 131bp and 119bp fragments. That is: females: a single band (131bp); males: two bands (131bp and 119bp).

[0151] 6. The accuracy statistics are shown in Table 6 below:

[0152] Table 6. Accuracy Statistics of the Method of the Invention

[0153]

[0154] Conclusion: Primers designed using the ri-3-47617128 marker can achieve 100% accuracy in sex identification. They are applicable to wild Naozhou tribes, eastern Fujian and Guangdong tribes, and domesticated eastern Fujian and Guangdong tribes. The entire test can be completed within 4 hours, with a cost of less than 3 yuan per sample, making it suitable for large-scale screening.

[0155] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A primer for amplifying an indel marker to identify the sex of large yellow croaker from different habitats, characterized in that, The primers include an upstream primer and a downstream primer. The sequence of the upstream primer is shown in SEQ ID NO.2, and the sequence of the downstream primer is shown in SEQ ID NO.

3. The indel marker is a 12 bp insertion / deletion nucleotide sequence at position 47617128 of chromosome 3 of the large yellow croaker, as shown in SEQ ID NO.

1. Female individuals have the sequence shown in SEQ ID NO.1, while male individuals lack the sequence shown in SEQ ID NO.

1.

2. A kit for identifying the sex of large yellow croaker from different habitats, characterized in that: The kit includes the primers described in claim 1.

3. A method for identifying the sex of large yellow croaker from different habitats, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA of the sample to be tested as a template, perform PCR amplification using the primers described in claim 1 or the kit described in claim 2; (3) Based on the electrophoresis image, the sex of the large yellow croaker is determined. Individuals with only the 131bp fragment are female large yellow croakers, while individuals with both the 131bp and 119bp fragments are male large yellow croakers.

4. The method according to claim 3, characterized in that, In step (2), the PCR amplification process used the following PCR reaction system: 10 μL of 2× Taq MasterMix, 0.5 μL each of upstream and downstream primers at a concentration of 10 μM, 50 ng of genomic DNA, and ddH2O to 20 μL.

5. The method according to claim 3, characterized in that, In step (2), the PCR amplification process used was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 9 s, 35 cycles, and 72℃ extension for 5 min.

6. The application of the primers of claim 1 or the kit of claim 2 in identifying the sex of large yellow croaker in different habitats.

7. The application of the method according to any one of claims 3-5 in identifying the sex of large yellow croaker from different habitats.

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