Koi-TAT gene related to golden and silver scale character of koi, SNP molecular marker, primer pair and application

Abstract

The invention relates to the technical field of animal molecular genetics, molecular marker breeding and aquatic genetic breeding, in particular to a Koi-TAT gene related to golden and silver scale traits of fancy carp, an SNP molecular marker, a primer pair and application. According to the invention, a functional gene highly associated with the Koi gold and silver scale character is found, a specific molecular marker and a detection method are developed on the basis of the gene, and by utilizing the molecular marker and the detection method disclosed by the invention, an individual with excellent gold and silver scale character can be efficiently identified, and the molecular marker is suitable for molecular-assisted selective breeding and early-stage seed selection. The invention provides an important molecular tool for phenotype genetic improvement of aquarium fish, also provides a basis for molecular mechanism research of gold and silver scale formation, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of animal molecular genetics, molecular marker breeding, and aquatic genetic breeding, and particularly to the Koi-TAT gene, SNP molecular markers, primer pairs, and applications related to the gold and silver scales of koi carp. Background Technology

[0002] Koi ( Cyprinus carpio var. Koi The golden-silver scale trait is an important ornamental fish with numerous strains. Its body color and scale luster are key factors in its aesthetic and commercial value. Among them, the golden-silver scale trait, characterized by scales with a silvery-white or golden-yellow metallic sheen, has attracted significant attention from breeders and consumers. Traditional breeding relies on phenotypic selection, which suffers from long cycles and low accuracy, especially in the early stages where it is difficult to accurately identify individuals with the target trait.

[0003] Existing research indicates that the gold and silver scales of koi carp are controlled by genetic factors, but the core functional genes have not yet been identified or effective molecular markers have been developed.

[0004] Therefore, there is an urgent need to screen out functional genes that are highly associated with the traits of gold and silver scales, as well as specific molecular markers and detection methods. Summary of the Invention

[0005] To address the aforementioned technical challenges, in a first aspect, the present invention provides the Koi-TAT (Tyrosine Aminotransferase) gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0006] This invention discovered that the Koi-TAT gene, with a length of 6277 bp and containing 12 exons, is associated with iris cells in the skin tissue of gold and silver scale koi and the formation of gold and silver scale traits. Real-time quantitative PCR showed that this gene was highly expressed in the dorsal scale tissue of gold and silver scale koi, with an expression level more than 13 times that of non-gold and silver scale koi (P<0.01).

[0007] Furthermore, this invention provides a protein encoded by the Koi-TAT gene.

[0008] Furthermore, this invention provides the application of the Koi-TAT gene in identifying the gold and silver scales trait in koi or in molecular-assisted breeding of the gold and silver scales trait in koi.

[0009] In a specific implementation scheme, the expression level of the Koi-TAT gene in the dorsal scale tissue of gold and silver scale koi is higher than that in non-gold and silver scale koi.

[0010] Preferably, the expression level of the Koi-TAT gene in the dorsal scale tissue of gold and silver scale koi is more than 13 times that in non-gold and silver scale koi.

[0011] Secondly, the present invention provides an SNP molecular marker related to the gold and silver scale trait of koi carp. The SNP molecular marker is located at the 2504th base of the sequence shown in SEQ ID NO.1 and is an A / T mutation, wherein the allele for gold and silver scale koi carp is A and the allele for non-gold and silver scale koi carp is T.

[0012] In the fourth intron of the Koi-TAT gene, this invention identified a SNP locus, designated SNP-TAT-2504A / T. Mutations at this locus lead to significant changes in the abundance of Koi-TAT gene transcripts, thereby significantly affecting the silver metallic sheen of koi scales. Gold and silver scale-type individuals exhibit the "A" allele at this locus, while non-gold and silver scale-type individuals exhibit the "T" allele.

[0013] Thirdly, the present invention provides primer pairs for detecting the SNP molecular marker, the primer pairs being shown in SEQ ID NO.2 and SEQ ID NO.3.

[0014] In some implementations, the primer pairs can be used with Sanger sequencing or high-throughput sequencing platforms.

[0015] The product length after PCR amplification using this primer pair is 314 bp, which can be directly used for Sanger sequencing or SNP chip typing, thereby enabling rapid determination of an individual's genotype.

[0016] Fourthly, the present invention provides the application of the SNP molecular markers or primer pairs in identifying the gold and silver scales trait of koi or in molecular-assisted breeding of the gold and silver scales trait of koi.

[0017] Preferably, the present invention provides the SNP molecular marker or the primer pair for early identification. Application of distinguishing gold and silver scales in koi. That is, determining whether a fish carries the dominant gold and silver scale allele or its gold and silver scale trait by using its genotype during the juvenile stage.

[0018] Fifthly, the present invention provides a method for identifying the gold and silver scale trait of koi carp, comprising: detecting the genotype of the sample to be tested; if the 2504th base of the sequence shown in SEQ ID NO.1 is A, then the sample to be tested is a gold and silver scale koi carp; if the 2504th base of the sequence shown in SEQ ID NO.1 is T, then the sample to be tested is a non-gold and silver scale koi carp.

[0019] Preferably, the primer pair is used to amplify the sample by PCR, and then the genotype of the sample to be tested is detected.

[0020] Sixthly, the present invention provides a molecular-assisted breeding method for koi with the gold and silver scale trait, comprising: using the SNP molecular marker to screen koi individuals carrying the dominant allele A, in order to increase the expression frequency of the gold and silver scale trait in offspring.

[0021] By using the aforementioned SNP molecular markers for parental screening, the proportion and stability of gold and silver scales in offspring can be improved.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention identifies a functional gene highly associated with the golden and silver scale trait in koi carp and develops specific molecular markers and detection methods based on this gene. These methods can efficiently identify individuals with superior golden and silver scale traits and are suitable for molecular-assisted selection breeding and early selection. This invention provides an important molecular tool for phenotypic genetic improvement of ornamental fish and lays the foundation for research on the molecular mechanisms of golden and silver scale formation, demonstrating broad application prospects. Attached Figure Description

[0023] Figure 1 This is the result of real-time quantitative PCR for the Koi-TAT gene.

[0024] Figure 2 This is a schematic diagram of the Koi-TAT gene structure. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0026] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing.

[0027] Example 1: Analysis of Koi-TAT gene expression levels Total RNA was extracted from the dorsal scale tissues of both golden and non-golden silver scale koi. cDNA was obtained using a reverse transcription kit and amplified by real-time quantitative PCR using Koi-TAT specific primers (SEQ ID NO.4 and SEQ ID NO.5). The PCR amplification program was as follows: 95℃ for 15 min; 95℃ for 10 s, 60℃ for 20 s, 72℃ for 32 s (collecting fluorescence signal), for 40 cycles. The expression levels in golden and non-golden silver scale koi were analyzed, and the results are as follows: Figure 1 As shown, the results indicated that the abundance of the koi-TAT transcript in gold and silver scale koi was significantly upregulated, and the expression level of the Koi-TAT gene in the dorsal scale tissue of gold and silver scale koi was more than 13 times that of non-gold and silver scale koi.

[0028] Example 2: SNP molecular markers related to the gold and silver scales of koi carp A schematic diagram of the Koi-TAT gene structure is shown below. Figure 2 As shown, based on the Koi-TAT gene, this embodiment provides an SNP molecular marker related to the gold and silver scale trait of koi carp, which is located at the 2504th base of the sequence shown in SEQ ID NO.1 and shows an A / T mutation, in which the allele of gold and silver scale koi carp is A and the allele of non-gold and silver scale koi carp is T.

[0029] Furthermore, this embodiment provides a method for identifying the gold and silver scale characteristics of koi carp, the steps of which are as follows: Ten gold-silver scale koi and ten non-gold-silver scale koi were selected, and genomic DNA was extracted. PCR amplification was performed using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3. The PCR amplification program was as follows: 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 5 min. The 314 bp PCR amplification product was subjected to Sanger sequencing to analyze the genotypes at the SNP molecular markers, and then the consistency with the phenotype was verified.

[0030] The test results showed that 8 out of 10 gold and silver scale koi had the allele A, and 9 out of 10 non-gold and silver scale koi had the allele T, indicating that the SNP molecular markers of this invention have high accuracy in identifying the gold and silver scale trait of koi.

[0031] Example 3: Molecular-assisted breeding using SNP molecular markers This embodiment provides a molecular-assisted breeding method for koi with the gold and silver scale trait. Using the SNP molecular markers from Example 2, koi individuals carrying the dominant allele A are screened as parents to increase the frequency of the gold and silver scale trait in offspring. The specific steps are as follows: Step 1: Genotyping of the parent population. From the candidate female and male koi parent populations, 3mm × 3mm caudal fin tissue was cut from each fish, and each fish was uniquely numbered. Detailed pedigrees and phenotypic records of the golden-silver scale trait were established. High-quality genomic DNA was extracted from the collected samples using a commercially available animal genomic DNA extraction kit. After quality control, all sample DNA was diluted to 50 ng / μL. The TaqMan probe method was used to detect the dominant SNP sites closely linked to the golden-silver scale trait identified in Example 2. Based on the fluorescence signal type, the genotype of each candidate parent at this SNP site was determined as follows: AA: Carrying two dominant alleles A, the best parent for breeding the golden-silver scale trait; AT: Carrying one dominant allele A and one inferior allele T, can be used as a parent, but the offspring will show phenotypic segregation; TT: Not carrying a dominant allele, the offspring are extremely unlikely to exhibit the ideal golden-silver scale trait, and is eliminated from the core breeding population.

[0032] Step Two: Formulating the Parental Mating Plan. Based on the genotyping results from Step One, and considering other important economic traits such as body size, coloration, and health status of the parents, the optimal mating plan is formulated. The recommended mating combination is AA (female) × AA (male). Theoretically, 100% of their offspring will have the AA genotype, ensuring stable inheritance of the golden and silver scales trait, and exhibiting a uniform phenotype.

[0033] Step 3: Artificial Insemination and Seedling Cultivation. During the koi breeding season, according to the established mating plan, the selected parent fish are induced to spawn, eggs are collected, and semen is extracted for artificial insemination. The fertilized eggs are placed in a sterilized hatching pond for incubation. After the fry swim to a horizontal position, they are transferred to outdoor earthen ponds with pre-prepared natural food for rearing, and then managed according to standardized seedling cultivation procedures.

[0034] Step 4: Offspring Verification and Reselection. When the offspring fry reach a body length of 3-5 cm, 100 individuals are randomly selected, and genotyping is performed again using the aforementioned SNP molecular marker. This verifies whether all offspring have the AA genotype. The gold and silver scale phenotypes of the offspring are recorded, and association analysis is performed with the genotype results to further confirm the predictive accuracy of the SNP marker in this breeding population.

[0035] Step 5: Establishment of the core breeding population. Verified offspring individuals with the AA genotype and superior phenotype are incorporated into the core breeding population as parental sources for the next generation. By repeating these steps, the golden and silver scale traits can be rapidly fixed and enhanced within the population, significantly accelerating the selection and breeding process for superior strains.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The Koi-TAT gene, characterized by, Its nucleotide sequence is shown in SEQ ID No.

1.

2. The protein encoded by the Koi-TAT gene as described in claim 1.

3. The application of the Koi-TAT gene as described in claim 1 in identifying the gold and silver scales of koi or in molecular-assisted breeding of the gold and silver scales of koi.

4. The application according to claim 3, characterized in that, The expression level of the Koi-TAT gene in the dorsal scale tissue of gold and silver scale koi is higher than that in non-gold and silver scale koi. Preferably, the expression level of the Koi-TAT gene in the dorsal scale tissue of gold and silver scale koi is more than 13 times that in non-gold and silver scale koi.

5. SNP molecular markers associated with the gold and silver scales of koi carp, characterized in that, The SNP molecular marker is located at the 2504th base of the sequence shown in SEQ ID NO.1, and exhibits an A / T mutation, with the allele of A for gold and silver scale koi and the allele of T for non-gold and silver scale koi.

6. The primer pair for detecting the SNP molecular marker of claim 5, characterized in that, The primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.

3.

7. The application of the SNP molecular marker of claim 5 or the primer pair of claim 6 in identifying the gold and silver scale trait of koi or in molecular-assisted breeding of the gold and silver scale trait of koi.

8. A method for identifying the gold and silver scales of koi carp, characterized in that, include: The genotype of the sample to be tested is determined. If the 2504th base of the sequence shown in SEQ ID NO.1 is A, then the sample to be tested is a gold-silver scale koi; if the 2504th base of the sequence shown in SEQ ID NO.1 is T, then the sample to be tested is a non-gold-silver scale koi.

9. The method according to claim 8, characterized in that, The genotype of the sample to be tested is then detected by PCR amplification using the primer pair described in claim 6.

10. A molecular-assisted breeding method for koi carp with gold and silver scale traits, characterized in that, include: Using the SNP molecular markers described in claim 5, koi individuals carrying the dominant allele A were screened to increase the frequency of the gold and silver scale trait in offspring.

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