Molecular Markers Associated with Body Fat Traits of Grass Carp and Their Applications

Abstract

This application relates to the technical field of grass carp, and specifically relates to molecular markers associated with the body fat traits of grass carp and their applications. This application discloses SNPs molecular markers associated with the body fat traits of the grass carp FABP3 gene, which are located at the base positions of 29945677, 29948389, and 29948502 on chromosome 19 of the grass carp reference genome GCF_019924925.1. The above three SNPs molecular markers are closely associated with body fat traits such as the abdominal fat index, liver fat content, and muscle fat content of grass carp. The above molecular markers can be applied to molecular assisted selection breeding of low-fat grass carp strains.

Description

Technical Field

[0001] This application relates to the technical field of grass carp, specifically to molecular markers associated with grass carp body fat traits and their applications. Background Art

[0002] The grass carp (Ctenopharyngodon idellus) is the largest freshwater economic fish in China, with a farming output of 5.94 million tons in 2023. With the development of intensive farming models, artificial compound feeds have been widely used. However, in order to reduce feed cost inputs, producers often supplement excessive amounts of cheap energy feeds to save the use of protein feeds, resulting in excessive deposition of grass carp body fat, which in turn brings problems such as frequent occurrence of hepatobiliary syndrome and decline in fish meat quality.

[0003] Fatty acid binding protein 3 (FABP3) is a member of the intracellular lipid-binding protein superfamily, which has a strong affinity for long-chain fatty acids. Its main function is to promote the uptake of fatty acids by cells from the external environment and transport them to intracellular mitochondria for β-oxidation to meet the energy metabolism requirements of cells. FABP3 is widely distributed in tissues with high demand for fatty acids such as the myocardium, skeletal muscle, and adipocytes. Given the important role of FABP3 in fatty acid uptake, intracellular transport, and utilization, the association between FABP3 gene polymorphisms and grass carp body fat traits was explored and analyzed in order to provide technical support for the breeding of low-fat grass carp strains. Summary of the Invention

[0004] This application discloses SNPs molecular markers associated with the grass carp FABP3 gene and body fat traits, which are located at the base positions of 29945677, 29948389, and 29948502 on chromosome 19 of the grass carp reference genome GCF_019924925.1. The above three SNPs molecular markers are closely associated with body fat traits such as the grass carp abdominal fat index, liver fat content, and muscle fat content. The above molecular markers can be applied to molecular-assisted selection breeding of low-fat grass carp strains. For this reason, at least the following technical solutions are disclosed in the embodiments of this application:

[0005] In the first aspect, the embodiment discloses molecular markers associated with grass carp body fat traits, including the nucleotide sequence formed by the single nucleotide G>A mutation at position 29945677 on chromosome 19 of the grass carp reference genome GCF_019924925.1; the nucleotide sequence formed by the single nucleotide G>T mutation at position 29948389 on chromosome 19 of the grass carp reference genome GCF_019924925.1; and at least one of the nucleotide sequences formed by the single nucleotide C>T mutation at position 29948502 on chromosome 19 of the grass carp reference genome GCF_019924925.1.

[0006] Second aspect, the embodiments disclose a primer set. The primer set includes: DNA molecules shown in SEQ ID NO: 1 and 2; and / or DNA molecules shown in SEQ ID NO: 3 and 4. The primer set is used for amplifying a nucleotide sequence containing single nucleotide mutation sites at three positions in the molecular marker described in the first aspect. In some embodiments, the DNA molecules shown in SEQ ID NO: 1 and 2 are used to amplify a nucleotide sequence formed by a single nucleotide G>A mutation at position 29945677 on chromosome 19 of the grass carp reference genome GCF_019924925.1. In some embodiments, the DNA molecules shown in SEQ ID NO: 3 and 4 are used to amplify a nucleotide sequence formed by a single nucleotide G>T mutation at position 29948389 on chromosome 19 of the grass carp reference genome GCF_019924925.1 and a nucleotide sequence formed by a single nucleotide C>T mutation at position 29948502 on chromosome 19 of the grass carp reference genome GCF_019924925.1.

[0007] Third aspect, the embodiments disclose a kit. The kit includes the primer set described in the second aspect. The kit is a PCR amplification kit, including the primer set described in the second aspect and other reagents for PCR amplification.

[0008] Fourth aspect, the embodiments disclose a method for screening excellent individuals with grass carp body fat traits using a molecular marker. The method includes: extracting genomic DNA from the fin rays of the grass carp to be selected; performing PCR amplification on the genomic DNA using the primer set described in the second aspect; sequencing the PCR amplification product; determining the genotypes of the grass carp at position 29945677 on chromosome 19, position 29948389 on chromosome 19, and / or position 29948502 on chromosome 19 of the reference genome GCF_019924925.1 according to the sequencing base peak map results; judging the breeding value of the grass carp to be selected in terms of body fat traits according to the genotypes, and screening out grass carp individuals containing low-fat dominant genotypes.

[0009] In some embodiments, the body fat traits include at least one of body weight, body length, condition factor, viscerosomatic index, intraperitoneal fat index, liver fat content, and muscle fat content.

[0010] In some embodiments, the genotypes at position 29,945,677 on chromosome 19 of the reference genome GCF_019924925.1 include GG, GA, and AA. Among them, the body weights of individuals with GG, GA, and AA genotypes increase in sequence. The body lengths of individuals with GG, GA, and AA genotypes increase in sequence. The condition factor of individuals with GG, GA, and AA genotypes decreases in sequence. The viscerosomatic index of individuals with GG, GA, and AA genotypes decreases in sequence. The abdominal fat index of individuals with GG, GA, and AA genotypes decreases in sequence. The liver fat content of individuals with AA genotype is lower than that of GG and GA genotypes. The muscle fat content of individuals with AA genotype is lower than that of GG and GA genotypes. In summary, the AA genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0011] In some embodiments, the genotypes at position 29,948,389 on chromosome 19 of the reference genome GCF_019924925.1 include GG, GT, and TT. Among them, the viscerosomatic index of individuals with GG genotype is lower than that of GT and TT genotypes. The abdominal fat index of individuals with GG genotype is lower than that of GT and TT genotypes. The liver fat contents of individuals with GG, GT, and TT genotypes increase in sequence. The muscle fat contents of individuals with GG, GT, and TT genotypes increase in sequence. In summary, the GG genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0012] In some embodiments, the genotypes at position 29,948,502 on chromosome 19 of the reference genome GCF_019924925.1 include CC, CT, and TT. Among them, the body weights of individuals with TT, CT, and CC genotypes increase in sequence. The body lengths of individuals with TT, CT, and CC genotypes increase in sequence. The condition factor of individuals with CC genotype is lower than that of CT and TT genotypes. The viscerosomatic indices of individuals with TT, CT, and CC genotypes decrease in sequence. The abdominal fat indices of individuals with TT, CT, and CC genotypes decrease in sequence. The liver fat contents of individuals with TT, CT, and CC genotypes decrease in sequence. The muscle fat contents of individuals with TT, CT, and CC genotypes decrease in sequence. In summary, the CC genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0013] In the fifth aspect, the embodiments disclose the application 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 at least one of screening excellent individuals with grass carp body fat traits and molecular assisted selection breeding of low-fat grass carp strains. Description of the Drawings

[0014] Figure 1Agarose gel electrophoresis map of the PCR amplification product of the grass carp FABP3 gene fragment provided for the example. Samples 1-3 are the 476bp target fragments amplified by the forward primer of SEQ ID NO:1 and the reverse primer of SEQ ID NO:2; Samples 4-6 are the 635bp target fragments amplified by the forward primer of SEQ ID NO:3 and the reverse primer of SEQ ID NO:4.

[0015] Figure 2 Sequencing peak map of grass carp SNP chr19:29945677 typing provided for the example.

[0016] Figure 3 Sequencing peak map of grass carp SNP chr19:29948389 typing provided for the example.

[0017] Figure 4 Sequencing peak map of grass carp SNP chr19:29948502 typing provided for the example. Detailed implementation manners

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer and more understandable, the present application will be further described in detail below in conjunction with examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application. The reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.

[0019] Association Analysis of Single Nucleotide Polymorphisms of Grass Carp FABP3 Gene with Body Fat Traits

[0020] 1. Experimental materials

[0021] The grass carp used in the experiment was taken from the Fangcun breeding base of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, and was the F3 generation selection line of the same batch of breeding and the same pond culture. Randomly select 294 individuals for trait association analysis, accurately measure the body fat trait indicators, and at the same time cut fin samples and store them in anhydrous ethanol at low temperature.

[0022] 2. Genomic DNA extraction

[0023] Cut a small amount of grass carp fin samples, chop them up and digest and lyse them with proteinase K, centrifuge to take the supernatant; use the magnetic bead separation technology and use an automatic nucleic acid extractor to extract genomic DNA.

[0024] 3. PCR and SNPs typing

[0025] PCR amplifies the target fragment of the grass carp FABP3 gene, and the primer sequences are shown in Table 1:

[0026] Table 1 Primer sequence information for PCR amplification of grass carp FABP3 gene fragment

[0027]

[0028] The PCR reaction system was as follows: 1.0 μL of grass carp genomic DNA template, 1.0 μL of each forward and reverse primer, 10 μL of 2× premixed Taq enzyme, and ddH2O was added to make up to 20 μL. The PCR reaction program was: pre-denaturation at 94°C for 60 s; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, for 32 cycles. After sequencing the PCR amplification products, the genotypes of the grass carp samples to be tested were determined according to the base peak maps at the mutation sites.

[0029] 4. Genetic parameter analysis

[0030] The effective number of alleles (Ne), expected heterozygosity (He), and observed heterozygosity (Ho) of the SNPs loci in the experimental population were calculated using the genetic software PopGene, and the deviation of the population from Hardy-Weinberg equilibrium was estimated by Chi-square test; the polymorphic information content (PIC) was calculated using Picalc software. The results are shown in Table 2. All three SNPs loci were moderately polymorphic loci (0.25 < PIC < 0.50), and the loci chr19:29945677 and chr19:29948502 were in Hardy-Weinberg equilibrium (P > 0.05).

[0031] Table 2 Genetic parameter information of three SNPs in the grass carp FABP3 gene

[0032] SNP Locus Effective Allele Number Expected Heterozygosity Observed Heterozygosity Polymorphism Information Content Hardy-Weinberg Equilibrium chr19:29945677 1.6217 0.3840 0.3673 0.3099 0.4556 chr19:29948389 1.1685 0.1445 0.1224 0.1338 0.0083 chr19:29948502 1.9908 0.4985 0.5102 0.3738 0.6876

[0033] 5. Trait association analysis

[0034] Using the SPSS 19.0 statistical software, the multivariate analysis of variance module of the general linear model (GLM) was selected to perform a significance test on the genotypes of different SNPs loci and body fat traits.

[0035] Table 3 Association analysis of SNP locus chr19:29945677 and body fat traits

[0036] Genotype GG GA AA Number of Individuals 164 108 22 Body Weight / g 1600.27±158.21 1620.87±159.86 1636.09±216.83 Body Length / cm <![CDATA[46.26±1.80 b > <![CDATA[46.75±2.03 ab > <![CDATA[47.40±2.27 a > Condition Factor <![CDATA[1.62±0.10 a > <![CDATA[1.59±0.11 a > <![CDATA[1.53±0.08 b > Viscerosomatic Index 7.87±0.76 7.79±0.83 7.76±0.93 Abdominal Fat Index 2.21±0.43 2.13±0.49 2.05±0.48 Liver Fat Content 7.73±3.62 8.03±3.71 7.50±4.13 Muscle Fat Content <![CDATA[0.20±0.08 ab > <![CDATA[0.22±0.08 a > <![CDATA[0.18±0.07 b >

[0037] As shown in Table 3, the dominant genotype of the body fat trait at the SNP locus chr19:29945677 in grass carp was AA. Compared with GG and GA, individuals with the AA genotype not only had good growth performance and slender body characteristics, but also had lower body fat trait indicators such as abdominal fat index, liver fat content, and muscle fat content, and could be selected as grass carp individuals with low-fat dominant genotypes.

[0038] Association analysis of SNP locus chr19:29948389 with body fat traits

[0039]

[0040]

[0041] As shown in Table 4, the dominant genotype of body fat traits at SNP locus chr19:29948389 is GG. Compared with GT and TT, individuals with the GG genotype have significantly lower body fat trait indexes such as viscerosomatic index, abdominal fat index, liver fat content, and muscle fat content, and can be selected as grass carp individuals with low-fat dominant genotypes.

[0042] Table 5 Association analysis of SNP locus chr19:29948502 with body fat traits

[0043] Genotype CC CT TT Number of Individuals 82 150 62 Body Weight / g <![CDATA[1639.11±173.26 a > <![CDATA[1613.23±161.69 ab > <![CDATA[1566.15±147.54 b > Body Length / cm <![CDATA[47.08±2.11 a > <![CDATA[46.40±1.82 b > <![CDATA[46.11±1.90 b > Condition Factor <![CDATA[1.57±0.10 b > <![CDATA[1.61±0.11 a > <![CDATA[1.60±0.10 ab > Viscerosomatic Index <![CDATA[7.54±0.86 b > <![CDATA[7.92±0.76 a > <![CDATA[8.00±0.69 a > Abdominal Fat Index <![CDATA[1.96±0.45 b > <![CDATA[2.22±0.46 a > <![CDATA[2.33±0.36 a > Liver Fat Content <![CDATA[7.27±3.34 c > <![CDATA[7.88±3.64 b > <![CDATA[8.41±4.15 a > Muscle Fat Content <![CDATA[0.19±0.07 b > <![CDATA[0.21±0.08 ab > <![CDATA[0.23±0.09 a >

[0044] As shown in Table 5, the dominant genotype of body fat traits at SNP locus chr19:29948502 is CC. Compared with CT and TT, individuals with the CC genotype not only have good growth performance and slender body shape characteristics, but also have lower body fat trait indexes such as abdominal fat index, liver fat content, and muscle fat content, and can be selected as grass carp individuals with low-fat dominant genotypes.

[0045] 6. Application of SNP locus

[0046] Based on the above research, the embodiment also discloses a primer set. The primer set includes: DNA molecules shown in SEQ ID NO:1 and 2; and / or DNA molecules shown in SEQ ID NO:3 and 4. The primer set is used to amplify the nucleotide sequence containing 3 single nucleotide mutation sites in the molecular marker described in the first aspect.

[0047] In some embodiments, the DNA molecules shown in SEQ ID NO:1 and 2 are used to amplify the nucleotide sequence formed by a single nucleotide G>A mutation at position 29945677 on chromosome 19 of the grass carp reference genome GCF_019924925.1. In some embodiments, the length of the amplification product is 476bp, and the single nucleotide G>A mutation at position 29945677 on chromosome 19 of GCF_019924925.1 is located at position 301bp of the amplification product with a length of 476bp.

[0048] In some embodiments, DNA molecules such as those shown in SEQ ID NO: 3 and 4 are used to amplify a nucleotide sequence formed by a single nucleotide G>T mutation at position 29948389 on chromosome 19 of the grass carp reference genome GCF_019924925.1 and a nucleotide sequence formed by a single nucleotide C>T mutation at position 29948502 on chromosome 19 of the grass carp reference genome GCF_019924925.1. In some embodiments, the length of the amplification product is 635 bp, and the single nucleotide G>A mutation at position 29948389 on chromosome 19 of GCF_019924925.1 is located at 384 bp of the amplification product with a length of 635 bp. In some embodiments, the length of the amplification product is 635 bp, and the single nucleotide C>T mutation at position 29948502 on chromosome 19 of GCF_019924925.1 is located at 497 bp of the amplification product with a length of 635 bp.

[0049] The embodiment discloses a kit. The kit includes the primer set described above. The kit is a PCR amplification kit, including the primer set and other reagents for PCR amplification.

[0050] The embodiment discloses a method for screening excellent individuals with grass carp body fat traits using molecular markers. The method includes: extracting genomic DNA from grass carp fin rays; performing PCR amplification on the genomic DNA using the primer set described above; sequencing the PCR amplification product; determining the genotypes of the grass carp at positions 29945677, 29948389, and / or 29948502 on chromosome 19 of the reference genome GCF_019924925.1 according to the sequencing results; judging the breeding value of the candidate grass carp in terms of body fat traits according to the genotypes, and screening out grass carp individuals containing low-fat dominant genotypes.

[0051] In some embodiments, the body fat traits include at least one of body weight, body length, condition factor, viscerosomatic index, abdominal fat index, liver fat content, and muscle fat content.

[0052] In some embodiments, the genotypes at position 29,945,677 on chromosome 19 of the reference genome GCF_019924925.1 include GG, GA, and AA. Among them, the body weights of individuals with GG, GA, and AA genotypes increase in sequence. The body lengths of individuals with GG, GA, and AA genotypes increase in sequence. The condition factors of individuals with GG, GA, and AA genotypes decrease in sequence. The viscerosomatic indices of individuals with GG, GA, and AA genotypes decrease in sequence. The abdominal fat indices of individuals with GG, GA, and AA genotypes decrease in sequence. The liver lipid content of individuals with AA genotype is lower than that of GG and GA genotypes. The muscle lipid content of individuals with AA genotype is lower than that of GG and GA genotypes. In summary, the AA genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0053] In some embodiments, the genotypes at position 29,948,389 on chromosome 19 of the reference genome GCF_019924925.1 include GG, GT, and TT. Among them, the viscerosomatic index of individuals with GG genotype is lower than that of GT and TT genotypes. The abdominal fat index of individuals with GG genotype is lower than that of GT and TT genotypes. The liver lipid contents of individuals with GG, GT, and TT genotypes increase in sequence. The muscle lipid contents of individuals with GG, GT, and TT genotypes increase in sequence. In summary, the GG genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0054] In some embodiments, the genotypes at position 29,948,502 on chromosome 19 of the reference genome GCF_019924925.1 include CC, CT, and TT. Among them, the body weights of individuals with TT, CT, and CC genotypes increase in sequence. The body lengths of individuals with TT, CT, and CC genotypes increase in sequence. The condition factor of individuals with CC genotype is lower than that of CT and TT genotypes. The viscerosomatic indices of individuals with TT, CT, and CC genotypes decrease in sequence. The abdominal fat indices of individuals with TT, CT, and CC genotypes decrease in sequence. The liver lipid contents of individuals with TT, CT, and CC genotypes decrease in sequence. The muscle lipid contents of individuals with TT, CT, and CC genotypes decrease in sequence. In summary, the CC genotype is the dominant genotype for the body fat traits of grass carp and can be used for screening low-fat grass carp.

[0055] In some embodiments, the PCR reaction system is 20 μL in total and includes: 1.0 μL of grass carp genomic DNA template, 1.0 μL each of forward and reverse primers, 10 μL of 2× premixed Taq enzyme, and the balance of ddH2O. The PCR reaction program is: pre-denaturation at 94 °C for 60 s; denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 40 s, for 32 cycles.

[0056] In some embodiments, after the PCR product is amplified and sequenced, the genotype is determined according to the corresponding base in the peak map at the SNPs locus. Among them, when at least one of the chr19:29945677 locus is the AA genotype, the chr19:29948389 locus is the GG genotype, and the chr19:29948502 locus is the CC genotype, the obtained grass carp individuals are considered to be excellent grass carp strains with low body fat content.

[0057] The embodiment also discloses the application of the molecular marker, the primer set or the kit described in the above embodiment in at least one of screening excellent individuals with grass carp body fat traits and molecular assisted selection breeding of low-fat grass carp strains.

[0058] As mentioned above, the above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application.

Claims

1. A method for screening grass carp individuals with excellent body fat traits using molecular markers, comprising: Extracting genomic DNA from the fin rays of the selected grass carp; Performing PCR amplification on the genomic DNA using a primer set; Sequencing the PCR amplification product; Determine the genotype of the grass carp at position 29945677 of chromosome 19, position 29948389 of chromosome 19 and / or position 29948502 of chromosome 19 of the reference genome GCF_019924925.1 according to the sequencing base peak graph result; Determine the breeding value of the selected grass carp in terms of body fat traits according to the genotype, and screen out grass carp individuals with low-fat advantage genotypes; Wherein, the primer set includes a DNA molecule as shown in SEQ ID NOs: 1 and 2 for amplifying a nucleotide sequence formed by a single nucleotide G>A mutation at position 29945677 of chromosome 19 of the grass carp reference genome GCF_019924925.1, and a DNA molecule as shown in SEQ ID NOs: 3 and 4 for amplifying a nucleotide sequence formed by a single nucleotide G>T mutation at position 29948389 of chromosome 19 of the grass carp reference genome GCF_019924925.1 and a nucleotide sequence formed by a single nucleotide C>T mutation at position 29948502 of chromosome 19 of the grass carp reference genome GCF_019924925.1; Among them, the genotypes at position 29945677 of chromosome 19 of the reference genome GCF_019924925.1 include GG, GA and AA, and the AA genotype is a dominant genotype of the body fat trait of grass carp, and is used for screening low-fat grass carp; Among them, the genotypes at position 29948389 of chromosome 19 of the reference genome GCF_019924925.1 include GG, GT and TT, and the GG genotype is the dominant genotype of the body fat trait of grass carp, which is used for screening low-fat grass carp; Among them, the genotypes at position 29948502 of chromosome 19 of the reference genome GCF_019924925.1 include CC, CT and TT, and the CC genotype is a dominant genotype of the body fat trait of grass carp, and is used for screening low-fat grass carp; The body fat traits include: at least one of body mass, body length, fatness, visceral body index, abdominal fat index, liver fat content and muscle fat content; Among them, at chromosome 19, No. 29945677 of the reference genome GCF_019924925.1, the body weight of individuals with GG, GA, and AA genotypes increases successively, the body length of individuals with GG, GA, and AA genotypes increases successively, the fatness of individuals with GG, GA, and AA genotypes decreases successively, the visceral body index of individuals with GG, GA, and AA genotypes decreases successively, the abdominal fat index of individuals with GG, GA, and AA genotypes decreases successively, the liver fat content of individuals with AA genotype is lower than that of GG and GA genotypes, and the muscle fat content of individuals with AA genotype is lower than that of GG and GA genotypes; Among them, at chromosome 19, position 29948389 of the reference genome GCF_019924925.1, the visceral index of individuals with GG genotype was lower than that of GT and TT genotypes, the abdominal fat index of individuals with GG genotype was lower than that of GT and TT genotypes, the liver fat content of individuals with GG, GT and TT genotypes increased in sequence, and the muscle fat content of individuals with GG, GT and TT genotypes increased in sequence; Among them, at chromosome 19, No. 29948502 of the reference genome GCF_019924925.1, the body weight of individuals with TT, CT and CC genotypes increases successively, the body length of individuals with TT, CT and CC genotypes increases successively, the fatness of individuals with CC genotypes is lower than that of CT and TT genotypes, the visceral body index of individuals with TT, CT and CC genotypes decreases successively, the abdominal fat index of individuals with TT, CT and CC genotypes decreases successively, the liver fat content of individuals with TT, CT and CC genotypes decreases successively, and the muscle fat content of individuals with TT, CT and CC genotypes decreases successively.

Images