Molecular markers, kits and genotyping methods for predicting high content of highly unsaturated fatty acids in mirror carp muscle

Abstract

The application discloses a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acids in mirror carp muscle, relates to the technical field of biomolecular detection, and specifically relates to a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acids in mirror carp muscle. The molecular marker for predicting high content of highly unsaturated fatty acids in mirror carp muscle is shown as SEQ ID NO. 1. The primer pair for predicting high content of highly unsaturated fatty acids in mirror carp muscle is hufaf1 and hufar1. The genotype detection method comprises the following steps: one, extracting DNA; two, PCR amplification; and three, Sanger sequencing, and the mirror carp with a C single peak at the 75th bp is selected. The application can identify individuals with potential high content of highly unsaturated fatty acids in muscle based on living body non-damage and rapidness through the above-mentioned molecular marker, the kit and the genotype detection method, so that precise and efficient breeding of the high content of highly unsaturated fatty acids in mirror carp is realized.

Description

Technical Field

[0001] This invention relates to the field of biomolecular detection technology, specifically to molecular markers, kits, and genotype detection methods for predicting high levels of highly unsaturated fatty acids in the muscle of mirror carp. Background Technology

[0002] With the improvement of people's living standards, the focus is no longer solely on the quantity of aquatic products supplied, but increasingly on the nutrients that significantly impact human health, such as highly unsaturated fatty acids (HUFAs), mainly including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA). HUFAs are difficult for the human body to synthesize, but are indispensable for promoting the intellectual and visual development of infants and young children. Furthermore, they play important physiological functions in protecting the cardiovascular system, anti-cancer, anti-Alzheimer's disease, anti-inflammatory, and immune regulation. Considering the huge production and consumer market of carp, if the HUFA content trait in muscle can be genetically improved, it is expected to significantly enhance the market competitiveness of carp-related products and promote the healthy development of the carp industry.

[0003] However, the HUFA content trait can only be measured after the carp has reached marketable size and been slaughtered, by taking a portion of the muscle sample. This makes it impossible to directly select for this trait using traditional breeding methods. Therefore, there is an urgent need for a method that can rapidly screen carp individuals with high HUFA content potential based on live, non-invasive methods, in order to achieve precise and efficient breeding of this trait. Summary of the Invention

[0004] To address the problems existing in the background art, the present invention provides a molecular marker, reagent kit, and genotype detection method for rapidly screening potential individuals with high HUFA (highly unsaturated fatty acids) content in the muscle of mirror carp based on in vivo non-destructive methods.

[0005] This invention provides a molecular marker for predicting high levels of highly unsaturated fatty acids (HUFA) in the muscle of mirror carp. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; SEQ ID NO.1 contains a C / T mutation at position 360 bp from the 5' end.

[0006] The present invention provides primer pairs for predicting high content of highly unsaturated fatty acids in the muscle of mirror carp. The primer pairs are hufaf1 and hufar1. The sequence of primer hufaf1 is 5'-ACTTACAGGACTTAAAGGGATAG-3' (as shown in SEQ ID NO.2), and the sequence of primer hufar1 is 5'-CAGGCATAACATAATGACTACC-3' (as shown in SEQ ID NO.3).

[0007] This invention relates to a kit for predicting high levels of highly unsaturated fatty acids in the muscle of mirror carp, the kit comprising primers hufaf1 and hufar1.

[0008] Furthermore, the kit also includes 10×Taq Buffer, MgCl2, dNTPs, and Taq DNA polymerase.

[0009] This invention provides a method for predicting the genotype of high-unsaturated fatty acid content in the muscle of mirror carp. The method comprises the following steps:

[0010] 1. Extract genomic DNA from the fin rays of the carp to be tested;

[0011] 2. Using the extracted DNA as a template, PCR amplification was performed using primer pairs hufaf1 and hufar1 to obtain PCR products.

[0012] 3. Sanger sequencing was performed on the PCR products, and the mirror carp with a single C peak at 75bp were selected as the target mirror carp, which are the mirror carp with the potential to obtain high muscle content of highly unsaturated fatty acids.

[0013] The sequence of primer hufaf1 is 5'-ACTTACAGGACTTAAAGGGATAG-3', and the sequence of primer hufar1 is 5'-CAGGCATAACATAATGACTACC-3'.

[0014] Furthermore, the PCR product in step two is 284 bp.

[0015] Furthermore, the PCR reaction system in step two is as follows: 13.7 μL deionized water, 2 μL 10×Taq Buffer, 1.6 μL MgCl2, 0.4 μL 10 mM dNTPs, 0.6 μL 10 μM primer hufaf1, 0.6 μL 10 μM primer hufar1, 0.1 μL 5 U / μL Taq DNA polymerase, and 1 μL 100 ng / μL genomic DNA;

[0016] PCR amplification conditions: denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 59℃ for 30 s, extension at 72℃ for 20 s, for a total of 30 cycles; extension at 72℃ for 2 min; incubation at 4℃.

[0017] Beneficial effects of this invention:

[0018] This invention enables the rapid, non-invasive screening of potential individuals with high HUFA content in the muscle of mirror carp using the aforementioned molecular markers, kits, and genotype detection methods, thereby facilitating precise and efficient breeding of this trait.

[0019] This invention eliminates the need for carp to reach marketable size, enabling early selection; it eliminates the need to slaughter the individuals to be tested, requiring only live samples of regenerable fins to complete genotyping, ensuring the survival of superior parent stock and subsequent breeding; the method is highly accurate, simple to operate, and quick. Attached Figure Description

[0020] Figure 1 This is the Sanger sequencing diagram of the individual (CC type) that shows a single C peak at the 75th base in the PCR product Sanger sequencing of Example 1.

[0021] Figure 2 This is the Sanger sequencing diagram of the individual (AA type) that shows a single peak of A at the 75th base in the PCR product Sanger sequencing of Example 1;

[0022] Figure 3 This is the Sanger sequencing diagram of the individual (CA type) that shows a C and A peak at the 75th base in the PCR product Sanger sequencing of Example 1.

[0023] Figure 4 This refers to the genotype data detected in Example 1 and their corresponding HUFA content. Figure 4 One star represents P < 0.05, and three stars represent P < 0.001. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0026] Example 1

[0027] This embodiment involves sampling and phenotypic determination of mirror carp hatched at the same time and raised in the same pond under the same conditions until they reach marketable size (~1500g) at two years of age. 300 individuals were randomly selected and transferred to the workshop. After anesthesia, fin tissue (~100mg) was collected and placed in a 2ml EP tube containing 75% ethanol. Simultaneously, muscle tissue from both sides, including the back and abdomen, was collected. Each sample was minced, mixed thoroughly, and 100g of each was placed in a sealed bag. Both fin and muscle samples were stored at -20℃ for later use.

[0028] Genomic DNA was extracted from fin rays using a DNA extraction kit, and fatty acids in muscle samples were determined by gas chromatography.

[0029] Using extracted genomic DNA as a template, PCR amplification was performed using primer pairs hufaf1 and hufar1 to obtain PCR products. The results were detected by 1% agarose gel electrophoresis, showing a 284 bp band. The sequence of primer hufaf1 was 5'-ACTTACAGGACTTAAAGGGATAG-3', and the sequence of primer hufar1 was 5'-CAGGCATAACATAATGACTACC-3'. The PCR reaction system is shown in Table 1; PCR amplification conditions: 94℃ denaturation for 5 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, for a total of 30 cycles; 72℃ final extension for 2 min; 4℃ incubation.

[0030] Table 1

[0031] Deionized water 13.7ul 10×Taq Buffer 2ul <![CDATA[MgCl2]]> 1.6ul 10mM dNTPs 0.4ul 10uM primer hufaf1 0.6ul, 10uM primer hufar1 0.6ul 5 U / ul Taq DNA polymerase 0.1 ul 100 ng / ul genomic DNA 1ul

[0032] The PCR product (as shown in SEQ ID NO.4, a total of 284 bp) was subjected to Sanger sequencing. Individuals showing a single C peak at the 75th base were named CC type. Figure 1 As shown; individuals exhibiting a single peak (A) are named type AA, such as... Figure 2 As shown; individuals exhibiting both C and A peaks are named CA type, such as... Figure 3 As shown. The PCR product is not the complete molecular marker for predicting high HUFA content in mirror carp muscle (but a part of SEQ ID NO.1), and the 75th base of the PCR product corresponds to the 360th bp of SEQ ID NO.1.

[0033] The detected genotype data were compared with the corresponding HUFA (HUFA+DHA+EPA+ARA) content data, and the results are shown in Table 2 and... Figure 4 As shown.

[0034] Table 2

[0035]

[0036] Note: Different letters in the same row of the table indicate significant differences (P<0.05).

[0037] The mirror carp tested (CC type) exhibiting a single C peak at position 75 bp of the PCR product (position 360 bp of SEQ ID NO.1) had significantly higher muscle HUFA content than the CA (P<0.001) and AA types (P<0.001). This demonstrates that the method of the present invention yields accurate and reliable results and can be used for early selection of mirror carp based on HUFA content.

Claims

1. A molecular marker for predicting the content of highly unsaturated fatty acids in the muscle of mirror carp, characterized in that, The molecular marker nucleotide sequence is shown in SEQ ID NO.1; there is a C / T mutation at 360 bp from the 5' end of SEQ ID NO.1, and the content of highly unsaturated fatty acids in the muscles of genotype CC at 360 bp of SEQ ID NO.1 is higher than that in the muscles of genotypes CA and AA.

2. A method for predicting the high content of highly unsaturated fatty acids in the muscles of mirror carp, characterized in that, This method is performed in the following steps: Step 1: Extract genomic DNA from the fin rays of the carp to be tested; Step 2: Using the extracted DNA as a template, perform PCR amplification with primer pair hufaf1 and hufar1 to obtain PCR products; Step 3: Perform Sanger sequencing on the PCR products and select mirror carp that show a single C peak at the 75th bp of the PCR product sequence as shown in SEQ ID NO.4, which are mirror carp with the potential to obtain high muscle content of highly unsaturated fatty acids. The sequence of primer hufaf1 is 5'-ACTTACAGGACTTAAAGGGATAG-3', and the sequence of primer hufar1 is 5'-CAGGCATAACATAATGACTACC-3'.

3. The method for predicting high levels of highly unsaturated fatty acids in the muscle of mirror carp according to claim 2, characterized in that, The PCR product is 284 bp.

4. The method for predicting high levels of highly unsaturated fatty acids in the muscle of mirror carp according to claim 2 or 3, characterized in that, The PCR reaction system in step two is as follows: 13.7 μL deionized water, 2 μL 10×Taq Buffer, 1.6 μL MgCl2, 0.4 μL 10 mM dNTPs, 0.6 μL 10 μM primer hufaf1, 0.6 μL 10 μM primer hufar1, 0.1 μL 5 U / μL Taq DNA polymerase, and 1 μL 100 ng / μL genomic DNA; PCR amplification conditions: denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 59℃ for 30 s, extension at 72℃ for 20 s, for a total of 30 cycles; extension at 72℃ for 2 min; incubation at 4℃.

Images