Molecular markers of MTTP gene related to growth and body fat traits of grass carp and its application

Abstract

The present application relates to the technical field of grass carp, and specifically to MTTP gene molecular markers related to grass carp growth and body fat traits and their applications. The molecular marker is located at 13970224bp of chromosome 1 of the grass carp reference genome GCF_019924925.1, and the mutation type is G / A. The molecular marker is closely related to economic traits such as grass carp body weight, body length, fatness, visceral body index, abdominal fat index and liver fat content, among which the GG genotype is the dominant genotype for growth and body fat traits. The molecular marker can be applied to molecular assisted selection breeding of low-fat fast-growing grass carp strains.

Description

Technical Field

[0001] The present application relates to the technical field of grass carp, and in particular to MTTP gene molecular markers related to growth and body fat traits of grass carp and applications thereof. Background Art

[0002] Microsomal triglyceride transfer protein (MTTP) is a unique lipid transport protein that was originally isolated from bovine liver cell microsomes and is also highly expressed in intestinal epithelial cells. Its main function is to participate in intracellular lipid transport and the assembly and secretion of very low-density lipoprotein (VLDL) in the endoplasmic reticulum. Studies have shown that MTTP plays a key role in the transfer of neutral lipids to apolipoprotein B in the early stages of lipoprotein assembly. Studies on zebrafish have found that the function of the MTTP gene in fish is similar to that of mammals, and the expression of this gene is associated with fat accumulation in intestinal epithelial cells.

[0003] Grass carp (Ctenopharyngodon idella) is a major freshwater economic farmed fish in China. In 2023, the farmed output exceeded 5.9 million tons, the highest among all freshwater fish. However, with the development of intensive models, farmed grass carp usually have problems with excessive body fat deposition, such as fatty liver and intra-abdominal fat accumulation. Given that MTTP is crucial in maintaining normal lipid metabolism and transport, cholesterol homeostasis, lipoprotein transport, and low-density lipoprotein particle remodeling in the body, and that MTTP gene mutations may lead to reduced or absent function of microsomal triglyceride transfer protein, the association between MTTP gene polymorphism and economic traits such as grass carp body fat was explored and analyzed, in order to provide technical support for the selection and breeding of low-fat and high-quality grass carp strains. Summary of the invention

[0004] The present application discloses a molecular marker associated with growth and body fat traits in the MTTP gene of grass carp (Gene ID: 127519104), which is located at 13970224bp (Chr01: 13970224) of chromosome 1 of the grass carp reference genome (NCBI number: GCF_019924925.1). The molecular marker is closely associated with economic traits such as grass carp body weight, body length, fatness, visceral body index, abdominal fat index and liver fat content. The above molecular marker can be applied to molecular assisted selection breeding of low-fat fast-growing grass carp strains. To this end, the present application embodiment discloses at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose molecular markers associated with growth and body fat traits of grass carp, including a nucleotide sequence formed by a base G>A mutation at 13970224bp on chromosome 1 of the grass carp reference genome GCF_019924925.1.

[0006] In the second aspect, the embodiment discloses a primer set. The primer set includes: DNA molecules shown in SEQ ID NOs: 1, 2 and 3. SEQ ID NOs: 1 and 2 in the primer set are used to amplify the target fragment of the MTTP gene containing the molecular marker described in the first aspect; SEQ ID NO: 3 in the primer set is used to further detect the single nucleotide variation of the molecular marker.

[0007] In a third aspect, an embodiment discloses a nucleic acid comprising a nucleotide sequence formed by a base G>A mutation at 13970224bp of chromosome 1 of the grass carp reference genome GCF_019924925.1.

[0008] In a fourth aspect, the embodiment discloses a kit. The kit includes the primer set described in the second aspect. The kit is a SNaPshot kit, including the primer set described in the second aspect and other reagents for SNaPshot typing detection.

[0009] In the fifth aspect, the embodiment discloses a method for screening grass carp individuals with excellent growth and body fat traits using molecular markers. The method comprises: extracting fin genomic DNA of the selected grass carp; performing PCR amplification on the genomic DNA using DNA molecules as shown in SEQ ID NO: 1 and 2; performing a single base extension reaction on the product of the PCR amplification using a DNA molecule as shown in SEQ ID NO: 3; sequencing the product of the single base extension reaction; determining the genotype of the grass carp at 13970224bp of chromosome 1 of the reference genome GCF_019924925.1 according to the sequencing result; and screening out grass carp individuals with a low-fat and fast-growing advantage genotype according to the genotype.

[0010] In some embodiments, the PCR reaction system includes, in 15 μL, 1.0 μL of grass carp genomic DNA template, 0.3 μL of the DNA molecule shown in SEQ ID NO: 1, 0.3 μL of the DNA molecule shown in SEQ ID NO: 2, 1.5 μL of 10× buffer, 1.5 μL of MgCl2, 0.3 μL of DNTP, 0.3 μL of Taq enzyme and the remainder of ddH2O.

[0011] In some embodiments, the single base extension reaction system includes 2 μL of PCR product, 1 μL of SNaPshotMix reagent, 0.1 μL of single base extension primer, and the balance of ddH2O in a volume of 6.0 μL.

[0012] In some embodiments, the growth trait includes at least one of body mass and body length; the body fat trait includes at least one of fatness, visceral body index, abdominal fat index and liver fat content.

[0013] In some embodiments, the genotype at 13970224bp of chromosome 1 of the grass carp reference genome GCF_019924925.1 includes GG, GA and AA. Among them, the body weight of individuals with AA, GA and GG genotypes increases successively. The body length of individuals with AA, GA and GG genotypes increases successively. The fatness of individuals with AA, GA and GG genotypes decreases successively. The visceral body index of individuals with AA, GA and GG genotypes decreases successively. The abdominal fat index of individuals with AA, GA and GG genotypes decreases successively. The liver fat content of individuals with AA, GA and GG genotypes decreases successively. In summary, the GG genotype is a dominant genotype for growth and body fat traits of grass carp, and can be used for the screening of low-fat fast-growing grass carp.

[0014] In the sixth aspect, the embodiments disclose the use of the molecular marker described in the first aspect, the primer set described in the second aspect, or the kit described in the third aspect in screening individuals with excellent growth and body fat traits of grass carp, and molecular assisted selection breeding of low-fat and fast-growing grass carp strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The embodiment provides a peak graph of SNaPshot typing sequencing of grass carp Chr01:13970224 site, wherein the peak graph of genotype GG individual is a blue single peak; the peak graph of genotype GA individual is a blue and green double peak; the peak graph of genotype AA individual is a green single peak. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0017] Identification of molecular markers associated with growth and body fat traits in grass carp

[0018] 1. Experimental Materials

[0019] The grass carp used in the experiment was obtained from the breeding base of the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences. 296 healthy individuals were randomly selected from the F3 breeding population of grass carp bred in the same batch and raised in the same pond for growth and body fat trait index measurement for trait association analysis. Fin ray samples were cut from each fish and stored in anhydrous ethanol at -20℃ for later use.

[0020] 2. Genomic DNA Extraction

[0021] A small amount of grass carp fin ray samples were cut and chopped, digested and cleaved with proteinase K, and centrifuged to obtain the supernatant; genomic DNA was extracted using a kit (Tiangen Biochemical Technology Co., Ltd.), dissolved in double distilled water, and its purity and concentration were detected by ultraviolet spectrophotometer, and its integrity was detected by 1.2% agarose gel electrophoresis.

[0022] 3. SNaPshot classification

[0023] The forward and reverse amplification primers shown in Table 1 were designed. First, PCR amplification was performed to obtain a nucleotide sequence containing the base at 13970224 bp of chromosome 1 of the grass carp reference genome GCF_019924925.1, which is a fragment of the MTTP gene with a size of 132 bp.

[0024] Table 1 Sequence information of SNaPshot typing primers at SNP sites of grass carp MTTP gene

[0025]

[0026] The PCR reaction system was: 1.0 μL of grass carp genomic DNA template, 0.3 μL each of forward amplification primer (SEQ ID NO: 1) and reverse amplification primer (SEQ ID NO: 2), 1.5 μL of 10× buffer, 1.5 μL of MgCl2, 0.3 μL of DNTP, 0.3 μL of Taq enzyme, and ddH2O to 15 μL. The PCR reaction program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C for 3 min.

[0027] After PCR amplification, 3 μL of PCR product was purified with ExoI and FastAP. ExoI was used to remove the remaining primers in the reaction product, and FastAP was used to remove the remaining DNTP in the reaction. The purification reaction system was: 3 μL of PCR product, 0.2 μL of ExoI, 0.8 μL of FastAP, 0.7 μL of ExoI buffer, and ddH2O was added to 7.0 μL. The purification reaction procedure was: 37°C for 15 min, 80°C for 15 min.

[0028] After purification, the extension primer was used for single base extension reaction. The extension reaction system was: 2 μL of the purified PCR product, 1 μL of SNaPshot Mix reagent, 0.1 μL of single base extension primer (SEQ ID NO: 3), and ddH2O was added to 6.0 μL. The extension reaction program was: 96°C for 1 min; 96°C for 10 s, 52°C for 5 s, 60°C for 30 s, and 30 cycles.

[0029] Take 1.0 μL of the above single-base extension product, add 10.0 μL of loading buffer, denature at 95 °C for 3 min, immediately place in an ice-water bath, transfer to a 3730XL sequencer for genotyping sequencing, and determine the genotype of the grass carp sample to be tested according to the base peak map read by the software at the mutation site.

[0030] 4. Genetic parameter analysis

[0031] Use the genetic software PopGene to calculate the effective number of alleles (Ne), expected heterozygosity (He), and observed heterozygosity (Ho) of the SNP locus in the experimental population, and estimate the deviation from Hardy-Weinberg equilibrium of the population by Chi-square test; use Picalc software to calculate the polymorphism information content (PIC). The results are shown in Table 2. This SNP locus belongs to a moderately polymorphic locus (0.25 < PIC < 0.50), and the locus Chr01:13970224 is in Hardy-Weinberg equilibrium (P > 0.05).

[0032] Table 2 Genetic parameter information of SNPs in the MTTP gene of grass carp

[0033]

[0034] 5. Trait association analysis

[0035] Use the SPSS 19.0 statistical software and select the one-way ANOVA module of the general linear model for the significance test of SNP genotypes and economic traits.

[0036] Table 3 Association analysis of the SNP locus Chr01:13970224 in grass carp with growth and body fat traits

[0037] genotype AA GA GG Number of individuals 123 122 51 Body mass, g <![CDATA[1577.20±158.82 b ]]> <![CDATA[1618.43±168.40 b ]]> <![CDATA[1674.37±140.95 a ]]> Body length, cm <![CDATA[45.99±1.92 c ]]> <![CDATA[46.59±1.80 b ]]> <![CDATA[47.75±1.79 a ]]> <![CDATA[Condition factor, g / cm 3 > <![CDATA[1.62±0.10 a ]]> <![CDATA[1.60±0.11 a ]]> <![CDATA[1.54±0.10 b ]]> Visceral index, % <![CDATA[8.00±0.77 a ]]> <![CDATA[7.88±0.74 a ]]> <![CDATA[7.31±0.81 b ]]> Abdominal fat index, % <![CDATA[2.32±0.44 a ]]> <![CDATA[2.16±0.43 b ]]> <![CDATA[1.82±0.39 c ]]> Liver fat content, % <![CDATA[8.21±3.94 a ]]> <![CDATA[7.85±3.71 ab ]]> <![CDATA[6.88±2.79 b ]]>

[0038] Note: Shoulder marks with the same letter in the same row indicate no significant difference (P > 0.05), and different letters indicate significant difference (P < 0.05).

[0039] The results are shown in Table 3. The dominant genotype for growth and body fat traits of the SNP locus Chr01:13970224 in grass carp is GG. Compared with the AA and GA genotypes, the GG genotype population not only has excellent growth performance and slender body shape characteristics, but also has significantly lower body fat trait indicators such as abdominal fat index and liver fat content, and can be selected as the low-fat and fast-growing dominant genotype grass carp.

[0040] Application of molecular markers

[0041] Based on the above research, the embodiment also discloses a primer set. The primer set includes DNA molecules shown in SEQ ID NOs: 1, 2 and 3. SEQ ID NOs: 1 and 2 in the primer set are used to amplify the target fragment of the MTTP gene containing the molecular marker described in the first aspect; SEQ ID NO: 3 in the primer set is used to further detect the single nucleotide variation of the molecular marker.

[0042] In some embodiments, a nucleotide sequence formed by a base G>A mutation at 13970224 bp of chromosome 1 of the grass carp reference genome GCF_019924925.1 is amplified using a DNA molecule as shown in SEQ ID NO: 1 and 2. In some embodiments, the length of the amplified product is 132 bp, and the base G>A mutation at 13970224 bp of chromosome 1 of GCF_019924925.1 is located at 92 bp of the amplified product having a length of 132 bp.

[0043] The embodiment discloses a kit. The kit includes the primer set. The kit is a SNaPshot kit, including the primer set and other reagents for SNaPshot typing detection.

[0044] The embodiment discloses a method for screening grass carp individuals with excellent growth and body fat traits using molecular markers. The method comprises: extracting fin genomic DNA of the selected grass carp; performing PCR amplification on the genomic DNA using DNA molecules as shown in SEQ ID NO: 1 and 2; performing a single base extension reaction on the product of the PCR amplification using a DNA molecule as shown in SEQ ID NO: 3; sequencing the product of the single base extension reaction; determining the genotype of the grass carp at 13970224bp of chromosome 1 of the reference genome GCF_019924925.1 according to the sequencing result; and screening grass carp individuals with a low-fat and fast-growing advantage genotype according to the genotype.

[0045] In some embodiments, the growth trait includes at least one of body mass and body length; the body fat trait includes at least one of fatness, visceral body index, abdominal fat index and liver fat content.

[0046] In some embodiments, the genotype at 13970224bp of chromosome 1 of the grass carp reference genome GCF_019924925.1 includes GG, GA and AA. Among them, the body weight of individuals with AA, GA and GG genotypes increases successively. The body length of individuals with AA, GA and GG genotypes increases successively. The fatness of individuals with AA, GA and GG genotypes decreases successively. The visceral body index of individuals with AA, GA and GG genotypes decreases successively. The abdominal fat index of individuals with AA, GA and GG genotypes decreases successively. The liver fat content of individuals with AA, GA and GG genotypes decreases successively. In summary, the GG genotype is a dominant genotype for growth and body fat traits of grass carp, and can be used for the screening of low-fat fast-growing grass carp.

[0047] In some embodiments, the PCR reaction system includes 15 μL of grass carp genomic DNA template 1.0 μL, 0.3 μL of the DNA molecule shown in SEQ ID NO: 1, 0.3 μL of the DNA molecule shown in SEQ ID NO: 2, 1.5 μL 10× buffer, 1.5 μL MgCl2, 0.3 μL DNTP, 0.3 μL Taq enzyme and the balance ddH2O. The PCR reaction program is: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C for 3 min.

[0048] In some embodiments, the method further comprises the step of purifying the PCR amplification product. The purification system comprises 7.0 μL: 3 μL PCR amplification product, 0.2 μL ExoI, 0.8 μL FastAP, 0.7 μL ExoI buffer, and the balance ddH2O. The purification treatment procedure is: 37°C for 15 min, 80°C for 15 min.

[0049] In some embodiments, the single base extension reaction system includes 6.0 μL of the purified PCR amplification product, 1 μL of SNaPshot Mix reagent, 0.1 μL of the DNA molecule shown in SEQ ID NO: 3, and the remainder of ddH2O. The program of the single base extension reaction is: 96°C for 1 min; 96°C for 10 s, 52°C for 5 s, 60°C for 30 s, and 30 cycles.

[0050] In some embodiments, when the Chr01:13970224 locus is a GG genotype, the resulting grass carp is considered to be an excellent individual with a faster growth rate and a lower body fat content.

[0051] The embodiments also disclose the use of the molecular markers described in the above embodiments, the primer sets described in the above embodiments, or the kits described in the above embodiments in at least one of screening individuals with excellent growth and body fat traits of grass carp and molecular-assisted selection breeding of low-fat and fast-growing grass carp strains.

[0052] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A primer set, comprising a DNA molecule as shown in SEQ ID NO:1, a DNA molecule as shown in SEQ ID NO:2 and a DNA molecule as shown in SEQ ID NO:3, wherein the DNA molecule as shown in SEQ ID NO:1 and the DNA molecule as shown in SEQ ID NO:2 are used for PCR amplification of grass carp genomic DNA, and the DNA molecule as shown in SEQ ID NO:3 is used for single base extension reaction of the PCR amplification product.

2. A kit comprising the primer set as claimed in claim 1 and other reagents for Snapshot typing detection.

3. Methods for screening grass carp individuals with excellent growth and body fat traits using molecular markers, including: Extracting genomic DNA from the fin rays of the selected grass carp; Performing PCR amplification on the genomic DNA using the DNA molecules shown in SEQ ID NO:1 and SEQ ID NO:2; Using the DNA molecule shown in SEQ ID NO: 3, the product amplified by PCR is subjected to a single base extension reaction; sequencing the product of the single base extension reaction; According to the sequencing result, the genotype of the grass carp at 13970224bp of chromosome 1 of the reference genome GCF_019924925.1 is determined, and the genotype at 13970224bp of chromosome 1 of the grass carp reference genome GCF_019924925.1 includes GG, GA and AA, wherein the body weight of individuals with AA, GA and GG genotypes increases successively, the body length of individuals with AA, GA and GG genotypes increases successively, the fatness of individuals with AA, GA and GG genotypes decreases successively, the visceral body index of individuals with AA, GA and GG genotypes decreases successively, the abdominal fat index of individuals with AA, GA and GG genotypes decreases successively, and the liver fat content of individuals with AA, GA and GG genotypes decreases successively; According to the genotype, grass carp individuals with dominant genotypes in body weight, body length, fatness, visceral body index, abdominal fat index and liver fat content are screened out, and the GG genotype is the dominant genotype for growth and body fat traits of grass carp.

4. The method according to claim 3, wherein the PCR reaction system comprises, in 15 μL: 1.0 μL grass carp genomic DNA template, 0.3 μL of the DNA molecule shown in SEQ ID NO: 1, 0.3 μL of the DNA molecule shown in SEQ ID NO: 2, 1.5 μL 10× buffer, 1.5 μL MgCl2, 0.3 μL DNTP, 0.3 μL Taq enzyme and the balance ddH2O.

5. The method according to claim 3, wherein the single base extension reaction system comprises, in 6.0 μL: 2.0 μL of the product of the PCR amplification reaction, 1.0 μL of Snapshot Mix reagent, 0.1 μL of the DNA molecule shown in SEQ ID NO: 3 and the balance ddH2O.

6. Use of the primer set described in claim 1 or the kit described in claim 2 in screening individuals with excellent growth traits and body fat traits of grass carp, wherein the growth trait includes at least one of body weight and body length; the body fat trait includes at least one of fatness, visceral body index, abdominal fat index and liver fat content.

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