A molecular marker of a body color related gene site of a large yellow croaker, a detection primer pair and a probe, a kit and a use thereof

Abstract

This invention discloses a molecular marker, detection primer pair, and probe for a gene locus related to body color in large yellow croaker, a kit, and its uses. The molecular marker is shown in SEQ ID NO: 1, located at positions 1307-1316 of the CDS region of the bco1l gene; when this molecular marker is present at positions 1307-1316 of the bco1l gene CDS region, the large yellow croaker... bco1l The gene can degrade carotenoids, resulting in the transparent body color of the large yellow croaker; when this molecular marker is missing, the encoded amino acid terminates prematurely, altering the gene function, and the large yellow croaker... bco1l The gene cannot degrade carotenoids, resulting in the yellow body color of the large yellow croaker. This molecular marker can easily, quickly, and stably identify the genotype of the gene locus related to body color in different individuals of the large yellow croaker, which helps to identify the parents for breeding new strains of large yellow croaker that cannot degrade carotenoids, and can effectively improve breeding efficiency and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of molecular markers, and more particularly to a molecular marker, detection primer pair and probe for a gene locus related to body color in large yellow croaker, a kit and its uses. Background Technology

[0002] Among vertebrates, aquatic animals exhibit remarkable diversity in body color, with yellow and red hues being among the most economically valuable traits. These colors significantly enhance their ornamental and commercial value. Fish possess the most diverse types of pigment cells. Carotenoids are the core pigments for coloring yellow and red pigment cells, playing a crucial role in the formation of yellow and red body color. However, since vertebrates cannot synthesize carotenoids de novo, their body color depends on dietary intake, and more importantly, on the metabolism and transport of these pigments within the body. Key processes such as cleavage, isomerization, and deposition are regulated by specific enzymes and transport proteins. Carotenoid oxygenases are key regulators of carotenoid processing in animals; mutations and disruptions of carotenoid oxygenases can lead to changes in the carotenoid state and color of animal tissues. For example, bco2 A genetic mutation caused the Darwin's finches' beaks to change from pink to yellow; in Norwegian sheep and cattle, bco2 Gene mutations can also cause white fat to turn yellow; in salmon, bco1l and bco2l Gene expression levels are related to flesh color in scallops. bco11 Downregulation of the gene resulted in the adductor muscle turning orange and accompanied by carotenoid deposition. Therefore, the regulation of carotenoid metabolism and pigment cell development has always been a key research focus in aquaculture species breeding programs.

[0003] Large yellow croaker ( Larimichthys crocea Large yellow croaker (Croatia spp.) is one of China's most representative marine aquaculture species, often hailed as the "national fish" of marine aquaculture. Its tender flesh and unique golden-yellow body color are highly favored by consumers, giving it significant market value and economic importance. According to the *China Fisheries Statistical Yearbook* (2025), the yield of large yellow croaker reached 297,683 tons in 2024. However, long-term closed breeding systems and intensive artificial selection have led to the gradual degradation of germplasm resources in aquaculture populations, with changes in body color being one of the most significant impacts. Wild large yellow croaker typically exhibit a bright golden-yellow color, which holds cultural value and visual appeal in China, allowing it to command a premium in the market. In contrast, fish raised in net cages for extended periods often exhibit fading or grayish-white discoloration upon exposure to light, significantly reducing their aesthetic appeal and commercial value, while also increasing harvesting costs. Therefore, selecting for yellow-colored large yellow croaker is an important direction in large yellow croaker breeding.

[0004] Marker-assisted selection (MAG) is an effective way to improve the breeding efficiency of large yellow croaker. Yellow body color is usually closely related to the metabolic regulation of carotenoids; therefore, developing new large yellow croaker body color-related genes provides both a theoretical basis for the body color formation mechanism and technical support for the breeding of yellow-bodied large yellow croaker varieties. To date, no molecular markers have been developed for large yellow croaker body color-related gene loci. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for a gene locus related to body color in large yellow croaker.

[0006] To achieve the above objectives, the present invention provides a molecular marker for a gene locus related to body color in large yellow croaker, characterized in that the molecular marker is... bco1l The SEQ ID NO: 1 is located at positions 1307-1316 of the gene's CDS region; when bco1l When the molecular marker is present at positions 1307-1316 of the gene's CDS region, the large yellow croaker... bco1l The gene can degrade carotenoids, making the large yellow croaker transparent; when bco1l When this molecular marker is deleted from positions 1307-1316 of the gene's CDS region, the encoded amino acid terminates prematurely, altering the gene's function. This affects the large yellow croaker. bco1l The genes cannot degrade carotenoids, which is why the large yellow croaker is yellow in color.

[0007] The present invention also provides a primer pair and a probe for detecting the molecular marker, characterized in that the sequence of the primer pair is shown in SEQ ID NO: 4-5, and the sequence of the probe is shown in SEQ ID NO: 6.

[0008] The present invention also provides a detection kit for identifying molecular markers related to the body color of large yellow croaker, characterized in that the kit includes the primer pair and the probe.

[0009] The present invention also provides the primer pairs and probes and the kit thereof for use in detecting and identifying the body color of large yellow croaker.

[0010] The present invention also provides a method for identifying molecular marker genotypes of gene loci related to body color in large yellow croaker, characterized in that the molecular markers are detected in the large yellow croaker to be tested using the primer pair and probe or the kit.

[0011] The present invention also provides a method for identifying the body color of large yellow croaker, characterized in that the primer pair and probe or the kit is used to detect the molecular marker in the large yellow croaker to be tested, and the body color of the large yellow croaker is determined according to whether the molecular marker is missing or not.

[0012] Furthermore, the following steps are included:

[0013] Genomic DNA was extracted from the large yellow croaker to be tested as a sample;

[0014] The samples were subjected to TaqMan amplification using the primer pairs and probes or the kit described above, and the amplification curves and fluorescence intensity were detected.

[0015] Based on the color and fluorescence intensity of the amplification curve, it is determined whether the large yellow croaker to be tested carries a deletion variant;

[0016] When the amplification curve is blue, the genotype of the tested individual is GGTCAAACTT / GGTCAAACTT, the body color-related gene in the tested individual functions normally, and the large yellow croaker has a transparent body color; when the amplification curve is red, the genotype of the tested individual is a deletion heterozygous GGTCAAACTT / -, and the large yellow croaker has a transparent body color; if the amplification curve is green, the genotype of the tested individual is a deletion homozygous - / -, which indicates that the body color-related gene cannot degrade carotenoids, and the large yellow croaker has a yellow body color.

[0017] Furthermore, the Taqman amplification system, calculated in 20 μL, includes 10 μL of Pro Taq HS premixed probe qPCR kit, 1 μL each of 10 μM primer pair and probe, 1 μL of 30 ng / μL DNA template, and the remainder is ddH2O.

[0018] Furthermore, the reaction program for the Taqman amplification is: 95℃ for 30 s, 62℃ for 20 s, for 40 cycles; the fluorescence channel is set to FAM, and a melting curve is not required.

[0019] This invention, through resequencing and GWAS analysis of transparent and yellow-bodied large yellow croaker, identified INDEL:Chr24_17700698, a key marker significantly associated with yellow body color, located in... bco1l A 10bp deletion in the coding region of the gene: GGTCAAACTT. In individuals with a transparent body phenotype, the genotype shows homozygous deletion (GGTCAAACTT / GGTCAAACTT) and heterozygous deletion (GGTCAAACTT / -), while in individuals with a yellow body phenotype, the genotype is homozygous deletion (- / -). Furthermore, this INDEL is located in exon 10 of the bco1l gene; after the deletion mutation, a stop codon appears prematurely in exon 11, altering both the type and number of encoded amino acids.

[0020] Further verification of protein expression revealed that this deficiency led to... bco1l-Δ10The mutation cannot function properly to degrade carotenoids. To further verify the relationship between this deletion mutation and carotenoid metabolism, the applicant constructed mutant and non-mutated bco1l protein expression vectors and transformed them into E. coli strains capable of synthesizing β-carotene. The results showed that E. coli expressing bco1l normally could decompose the β-carotene produced, resulting in a white product. However, the product of E. coli expressing bco1l-Δ10 was yellow, indicating that β-carotene was not decomposed. Further HPLC analysis of the products showed that the β-carotene content in the product of E. coli expressing bco1l was extremely low, while the product of E. coli expressing bco1l-Δ10 contained a large amount of residual β-carotene.

[0021] Using this deletion-designed probe, the Taqman method can be used to determine the sample genotype through the amplification curve of the specific probe: the fluorescence intensity peak of the deletion heterozygous (GGTCAAACTT / -) sample is only half that of the non-deletion homozygous (GGTCAAACTT / GGTCAAACTT), and there is no signal in the bco1l-Δ10 deletion homozygous (- / -). The reaction was carried out using a CFX96 fluorescence quantitative thermal cycler and the amplification curve was observed.

[0022] Through application and validation in different populations, it was demonstrated that the developed molecular markers have good stability and high identification accuracy. They can conveniently and accurately identify the genotypes of gene loci related to body color in large yellow croaker individuals. They have important application value in basic theoretical research related to the formation mechanism of yellow body color in large yellow croaker and in guiding the breeding of large yellow croaker varieties.

[0023] The beneficial effects of this invention are: it can easily, quickly and stably identify the genotypes of body color-related gene loci in different individuals of large yellow croaker, which helps to identify the parents for the breeding of new strains of large yellow croaker that cannot degrade carotenoids, and can effectively improve breeding efficiency and economic benefits.

[0024] The molecular markers developed in this invention will also benefit basic research on the mechanism of yellow body color formation in large yellow croaker. It also provides a basis for selective breeding of large yellow croaker with yellow body color based on the molecular markers of this invention, and provides a technical roadmap (such as...). Figure 12 ). Attached Figure Description

[0025] Figure 1 These are comparison photos of large yellow croakers with transparent and yellow bodies.

[0026] Figure 2 The GWAS analysis results of 18 transparent-colored and 20 yellow-colored juvenile large yellow croakers are shown, along with the location of the significantly correlated locus Chr24_17700698.

[0027] Figure 3 This diagram shows a comparison of the bco1l gene sequences with and without deletion mutations (non-deletion homozygous) and with deletion mutations (deletion heterozygous, deletion homozygous).

[0028] Figure 4 This diagram shows a comparison of the amino acid sequences encoded by the bco1l gene with and without deletion mutations (non-deletion homozygous) and with deletion mutations (deletion heterozygous, deletion homozygous).

[0029] Figure 5 This diagram shows a comparison of the effects of bco1l-encoded proteins with and without deletion mutations (non-deletion homozygous) on the degradation of β-carotene.

[0030] Figure 6 This is a diagram showing the relationship between genotype and fish body color.

[0031] Figure 7 This is an electrophoresis result of detecting large yellow croaker using primer pairs (SEQ ID NO: 2-3).

[0032] Figure 8 This diagram shows the locations of Taqman-specific primers and probes. The yellow portion represents the CDS sequence, the red boxes represent SEQ ID NO:4 and SEQ ID NO:5 respectively, and the green box represents SEQ ID NO:6.

[0033] Figure 9 The figure shows the results of Taqman specific probe detection of the bco1l genotype of wild large yellow croaker from nine different geographical populations using two primers and one probe.

[0034] Figure 10 The results of the experiment to detect the frequency of homozygous missing alleles are shown in the figure.

[0035] Figure 11 The results of population detection in Examples 2, 3, 4, and 5 are shown.

[0036] Figure 12 Two technical roadmaps for selective breeding based on the molecular markers of this invention are shown. Detailed Implementation

[0037] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0038] Example 1: Acquisition and verification of molecular markers for body color-related gene loci in large yellow croaker

[0039] The applicant observed that a small number of juvenile large yellow croakers in the nursery population exhibited a distinct golden-yellow body color, which is significantly different from the common transparent phenotype (see...). Figure 1 Where A represents the transparent phenotype of large yellow croaker, B is a magnified image of the tail end of the transparent phenotype of large yellow croaker in A, C represents the yellow phenotype of large yellow croaker, and D is a magnified image of the tail end of the yellow phenotype of large yellow croaker in A), the applicant collected DNA from 38 juvenile large yellow croakers of different body colors, including 20 transparent croakers and 18 yellow croakers, and performed whole-genome resequencing. Subsequently, based on genome-wide association analysis (GWAS), two SNPs and one INDEL were detected on chromosome 24 that were significantly associated with the golden body color phenotype, but neither SNP caused any amino acid changes. At position 17,700,698 on chromosome 24, -log10(P) = 16.3279 (see...). Figure 2 Figure A shows the GWAS analysis results of 18 transparent and 20 yellow-bodied juvenile large yellow croakers, and Figure B shows the location of the significantly associated locus Chr24_17700698. This deletion, totaling 10 bp, was located at... bco1l Nucleotides 1307-1316 of the gene are located in exon 10 (see...) Figure 3 The genotypes of the 18 transparent individuals used for testing included 14 homozygous GGTCAAACTT / GGTCAAACTT and 4 heterozygous GGTCAAACTT / -. The genotypes of the 20 golden-yellow individuals all showed homozygous deletion mutations, i.e., - / -.

[0040] Continuous tracking of the golden-colored juvenile fish revealed no individuals that maintained their golden color during the day after adulthood, and no heterozygous mutant genotypes were detected. Further analysis of the genotypes of another group of 2072 large yellow croakers showed that only one large yellow croaker exhibited a homozygous mutation (- / -), indicating that the frequency of homozygous mutant genotypes is low and difficult to detect under natural conditions, while the frequency of heterozygous mutant genotypes is high, allowing for targeted breeding through parent selection.

[0041] When no deletion mutation occurs at position Chr24_17700698 bco1l The gene is 1,566 bp in length and encodes 522 amino acids; when a 10 bp deletion mutation occurs at this position, bco1l-Δ10 The entire sequence is 1,556 bp, with the stop codon TGA located at positions 1417-1419, encoding 472 amino acids (see...). Figure 4 ).

[0042] To further verify the association between this deletion mutation and carotenoid metabolism, pET-32a(+)-bco1l and pET-32a(+)-bco1l-Δ10 protein expression vectors were constructed and transformed into *E. coli* strains capable of synthesizing β-carotene. The results showed that *E. coli* expressing bco1l normally could decompose the produced β-carotene, resulting in a white product. However, *E. coli* expressing bco1l-Δ10 produced a yellow product, indicating that β-carotene was not decomposed (see [link to relevant documentation]). Figure 5 Further high-performance liquid chromatography (HPLC) analysis of the above products showed that the β-carotene content in the *E. coli* products expressing *bco1l* was extremely low, while the *E. coli* products expressing *bco1l-Δ10* contained a large amount of β-carotene residue (see...). Figure 5 The relationship between genotype and fish body color is shown in [reference needed]. Figure 6 As shown.

[0043] The above results indicate that the deletion of the bco1l gene GGTCAAACTT (SEQ ID NO:1) is closely related to carotene metabolism in large yellow croaker.

[0044] To better detect the deletion of GGTCAAACTT (SEQ ID NO:1), specific primers were designed to detect the genotype of the body color-related gene locus in the large yellow croaker individuals to be tested. The sequences are as follows:

[0045] Primer Δ10-F: 5'-TCGTGTCATCGTATTTTCTGGAGTA-3'SEQ ID NO:2;

[0046] Primer Δ10-R: 5'-TCCATCCAACAGATCGCCCT-3'SEQ ID NO: 3.

[0047] If the body color is yellow, a 500bp band should appear; if the body color is transparent, no band should appear.

[0048] Test results as follows Figure 7As shown. Y1-Y18 represent the PCR amplification results of 18 yellow-bodied samples, and WT1-WT22 represent the PCR amplification results of 22 clear-bodied samples. From... Figure 7 It can be seen that the body color of Y10-Y11 and Y14-16 is yellow, but no 500bp band appears; while the body color of WT4, WT7, and WT8 is transparent, but weak bands appear. Therefore, this primer pair cannot accurately detect the genotype of large yellow croaker body color-related genes with 100% accuracy.

[0049] To more accurately determine genotype, this invention designs novel primer pairs and probes based on quantitative fluorescence methods to detect the genotype of body color-related gene loci in individual large yellow croakers. (See below) Figure 8 The sequence is as follows:

[0050] Q-bco1l-96 bp-F: 5'-GACACCTGATCTCTGCTTGAAT-3'SEQ ID NO:4;

[0051] Q-bco1l-96 bp-R: 5'-ACAAACACCGGCTCTGAAG-3'SEQ ID NO:5;

[0052] ProbeQ-bco1l-WT: 5'-CCAAGTTTGACCTCGTCACAAGGA-3'SEQ ID NO:6.

[0053] The two primers and one probe were simultaneously placed into the Taqman reaction system and amplified using a CDX96 real-time quantitative thermal cycler.

[0054] The Taqman reaction system is 20 μL, including: 10 μL of 2X Pro Taq HS Probe Premix from the Pro Taq HS Premixed Probe qPCR Kit, 1 μL each of two primers and probes at a concentration of 10 μM, 1 μL of DNA template at 30 ng / μL, and ddH2O added to make up to 20 μL.

[0055] The reaction program for the CDX96 fluorescence quantitative thermal cycler is: (95℃ 30s; 62℃, 30s), 40 ×. The fluorescence channel is set to FAM, and a melting curve is not required.

[0056] After verifying the genotype through sequencing, the accuracy of the amplification curve is verified, enabling accurate determination of the genotype of the individual being tested.

[0057] 1) Large yellow croaker bco1 genotype detection

[0058] The samples were taken from a population of 38 resequencing tails, including 2 tails with no deletion homozygous DNA, 2 tails with deletion heterozygous DNA, and 2 tails with deletion homozygous DNA. A negative control group was designed to ensure the reliability of the experimental environment.

[0059] The detection method is described above, and the results are shown in Table 1. The results show that the Ct value of the homozygous WT fragment without deletion is the smallest, followed by the Ct value of the heterozygous fragment with deletion. The homozygous Δ10 fragment with deletion and the negative control have no Ct value, indicating that the target fragment was not detected.

[0060] Table 1. WT probes for different genotypes bco1 Quantitative expression scale

[0061]

[0062] 2) Detection of bco1 genotype in large yellow croaker from different population groups.

[0063] The samples were 401 large yellow croakers from 9 different geographical groups (including 21 from Nantong, 30 from Zhoushan, 9 from Ningbo, 41 from Wenzhou, 273 from Ningde, 13 from Fuzhou, 2 from Xiamen, 6 from Zhanjiang, and 6 from Haikou) whose DNA was used for testing. All of them were transparent.

[0064] The detection method is described above, and the results are shown above. Figure 9 None of the individuals exhibited homozygous mutations with - / - deletion.

[0065] Figure 9 This figure shows the results of TaqMan specific probe detection of the bco1l genotype in nine different geographic populations of wild large yellow croaker using two primers and one probe. Figure A shows the amplification curves of the TaqMan specific probe detection of the bco1l genotype in nine different geographic populations of wild large yellow croaker using two primers and one probe. When the amplification curve is blue, the genotype of the tested individual is GGTCAAACTT / GGTCAAACTT, which is homozygous without deletion; when the amplification curve is red, the genotype of the tested individual is heterozygous with deletion GGTCAAACTT / -, which is heterozygous with deletion; when the amplification curve is green, the genotype of the tested individual is homozygous with deletion - / -, which is homozygous with deletion. As can be seen from the figure, the most common type of homozygous individuals are those without deletion (blue line), the fewest heterozygous individuals are those with deletion (red line), and no homozygous individuals with deletion were found (green line).

[0066] Figure B shows the results of TaqMan specific probe detection of the bco1l genotype in nine different geographical populations of wild large yellow croaker using two primers and one probe, and the fluorescence intensity results of samples with different genotypes are statistically analyzed. + / + indicates homozygous individuals without deletion, + / - indicates heterozygous individuals with deletion, and - / - indicates homozygous individuals with deletion. As can be seen from the figure, homozygous individuals without deletion were the most numerous, heterozygous individuals with deletion were fewer, and no homozygous individuals with deletion were found.

[0067] The test results were completely consistent with the actual body color of the large yellow croaker.

[0068] 3) To detect the frequency of missing homozygous alleles, the inventors used BWA to align the resequencing data of 2072 large yellow croakers to the genome, and used GATK to screen for variants and count the genotypes of these individuals.

[0069] See results Figure 10 The results showed that among 2072 individuals, 2056 were homozygous without deletion, 15 were heterozygous without deletion, and 1 was homozygous without deletion. The allele frequency was 0.0041, indicating that this variation is rare in the large yellow croaker population. It is difficult to obtain a yellow body color strain under natural or artificial selection.

[0070] Example 2: Samples of 5-month-old golden-yellow croaker juveniles during group rearing in the same batch. bco1l Genotyping

[0071] Sample: 14 five-month-old golden-yellow croaker juveniles (14 of which were transparent and 0 were yellow) from the same batch of 38 GWAS analysis samples during the group rearing process.

[0072] Method: Same as in Example 1.

[0073] See results Figure 11 A.

[0074] Figure 11 This demonstrates the application of three primers to different stages of the same batch of golden-bodied large yellow croaker juveniles during group rearing. bco1l Genotyping results are shown in the image. + / + indicates homozygous deletion, + / - indicates heterozygous deletion, and - / - indicates homozygous deletion. Figure A shows the fluorescence intensity statistics of 14 5-month-old samples. The image shows that 10 samples were + / +, 4 were + / -, all transparent (14 in total), and 0 were yellow. These results perfectly match the actual body color of the large yellow croaker.

[0075] Example 3: Samples of 16-month-old golden-yellow croaker juveniles during group rearing in the same batch. bco1l Genotyping

[0076] Sample: 152 juvenile golden-yellow croakers (152 of which were transparent and 0 were yellow) of 16 months of age from the same batch of GWAS analysis samples.

[0077] See results Figure 11 B.

[0078] Figure 11This demonstrates the application of three primers to different stages of the same batch of golden-bodied large yellow croaker juveniles during group rearing. bco1l Genotyping results. + / + indicates homozygous deletion, + / - indicates heterozygous deletion, and - / - indicates homozygous deletion. Figure B shows the fluorescence intensity statistics of 152 16-month-old samples. The figure shows: 146 samples were + / +, 6 were + / -, all transparent, totaling 152; 0 samples were yellow. The test results are completely consistent with the actual body color of the large yellow croaker.

[0079] Example 4: 18-month-old samples of golden-yellow croaker juveniles during group rearing in the same batch. bco1l Genotyping

[0080] Sample: 80 golden-yellow croaker juveniles (80 transparent and 0 yellow) of 18 months age from the same batch of GWAS analysis samples during the group rearing process.

[0081] See results Figure 11 C.

[0082] Figure 11 This demonstrates the application of three primers to different stages of the same batch of golden-bodied large yellow croaker juveniles during group rearing. bco1l Genotyping results. + / + indicates homozygous deletion, + / - indicates heterozygous deletion, and - / - indicates homozygous deletion. Figure C shows the fluorescence intensity statistics of 80 18-month-old samples. The figure shows that 75 samples were + / +, 5 were + / -, all transparent, totaling 80; 0 samples were yellow. The test results are completely consistent with the actual body color of the large yellow croaker.

[0083] Example 5: Samples of 24-month-old golden-yellow croaker juveniles during group rearing in the same batch. bco1l Genotyping

[0084] Sample: 300 golden-yellow croaker juveniles aged 24 months from the same batch of group breeding (300 of which were transparent and 0 were yellow).

[0085] See results Figure 11 D.

[0086] Figure 11 This demonstrates the application of three primers to different stages of the same batch of golden-bodied large yellow croaker juveniles during group rearing. bco1lGenotyping results. + / + indicates homozygous deletion, + / - indicates heterozygous deletion, and - / - indicates homozygous deletion. D shows the fluorescence intensity statistics of 300 24-month-old samples. The figure shows: 274 samples were + / +, 26 were + / -, all transparent (300 in total); 0 samples were yellow. The test results are completely consistent with the actual body color of the large yellow croaker.

[0087] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A molecular marker for identifying the body color of Larimichthys crocea, characterized in that, The molecular marker is a polynucleotide, and its nucleotide sequence is shown in SEQ ID NO:

1. When the molecular marker is present in the genome of the large yellow croaker, the body color of the large yellow croaker is transparent; when the molecular marker is missing from the genome of the large yellow croaker, the body color of the large yellow croaker is yellow.

2. A primer pair and a probe for detecting the molecular marker of claim 1, wherein the primer pair comprises a forward primer and a reverse primer, and the probe comprises a forward probe and a reverse probe. The sequences of the primer pairs are shown in SEQ ID NO: 4-5, and the sequences of the probes are shown in SEQ ID NO:

6.

3. A detection kit for identifying the body color of Pseudosciaena crocea, characterized in that, The kit includes the primer pair and probe as described in claim 2.

4. The use of the primer pair and probe of claim 2 or the kit of claim 3 for identifying the body color of large yellow croaker.

5. A method for identifying the body color of large yellow croaker, characterized in that, Using the primer pair and probe described in claim 2 or the kit described in claim 3, the large yellow croaker to be tested is detected, and the body color of the large yellow croaker is determined based on the presence of the fragment shown in SEQ ID NO:

1.

6. The method as described in claim 5, characterized in that, Includes the following steps: Genomic DNA was extracted from the large yellow croaker to be tested as a sample; The samples were amplified by qPCR using the primer pair and probe described in claim 2 or the kit described in claim 3, and the amplification curve and fluorescence intensity were detected. Based on the amplification curve and fluorescence intensity, it is determined whether the large yellow croaker to be tested carries a deletion variant; The fluorescence intensity peak of the heterozygous deletion sample was only half that of the homozygous non-deleted sample, the homozygous deletion sample had no signal, the large yellow croakers with both homozygous non-deleted and heterozygous deletion samples were transparent, and the large yellow croakers with homozygous deletion samples were yellow.

7. The method as described in claim 6, characterized in that, The qPCR amplification system, in 20 μL volumes, includes 10 μL of 2X Pro Taq HS Probe Premix from the Pro Taq HS premixed probe qPCR kit, 1 μL each of 10 μM primer pair and probe, 1 μL of 30 ng / μL DNA template, and the remainder is ddH2O.

8. The method as described in claim 6, characterized in that, The reaction program for qPCR amplification is as follows: 95℃ for 30 s, 62℃ for 20 s, 40 cycles; the fluorescence channel is set to FAM, and a melting curve is not required.

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