A microsatellite molecular marker related to growth traits of Pelteobagrus fulvidraco and its application

Abstract

The present invention discloses a microsatellite molecular marker related to the growth traits of yellow catfish and its application, belonging to the technical field of molecular marker genetic breeding. The repeat sequence of the microsatellite fragment is (TTCTC) n , the value range of n is any integer between 6 and 13; the growth traits include total length, body length, body weight, body height, head length, tail peduncle length and tail peduncle height. The present invention also provides a primer pair for amplifying the above-mentioned microsatellite fragment, the primer pair comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.18. By amplifying the sample using the above-mentioned primer pair, microsatellite fragments can be specifically amplified, and these microsatellite fragments are closely related to the growth traits of yellow catfish. The use of this primer pair can screen yellow catfish parents with better growth potential, and can provide effective molecular markers for molecular breeding of yellow catfish.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker genetic breeding, in particular to a microsatellite molecular marker related to growth traits of yellow catfish and an application thereof. Background Art

[0002] In the selection of aquatic breeding targets, the selection of growth rate as the target trait accounts for 47.85%. Animal growth is a complex biological metabolic process. The GH / IGF1 axis is the main regulatory part of the hypothalamus-pituitary-liver axis, which regulates the growth and development of vertebrates through the secretion of various growth factors and hormones. The GH / IGF1 axis is mainly composed of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor 1 / 2 (IGF1 / IGF2), insulin-like growth factor receptor (IGF1R / IGF2R) and insulin-like growth factor binding protein (IGFBPs). These genes have been successfully applied to improve the growth-related traits of fish. For example, the IGF-like gene is the main effect gene affecting muscle growth rate. Its effect in the selection of channel catfish (Ictalurus punctatus) has been verified. For example, the mRNA expression level in the muscle of the fast-growing strain is more than twice that in the muscle of the slow-growing strain. At present, the main breeding directions of yellow catfish are to improve its stress resistance through hybridization ("Huangyou No. 1") and to increase its growth rate through sex control ("Quanxiong No. 1"). However, there are few reports on the application of molecular marker-assisted breeding methods to the breeding process of yellow catfish growth traits.

[0003] The genetic information carried by microsatellites can be used to improve growth traits during breeding. Microsatellites are short repetitive sequences in the genome, consisting of 1 to 6 bases. Microsatellites are widely distributed in the genomes of eukaryotic organisms, with one microsatellite approximately every 10 to 550 kb. Microsatellites have become one of the most effective molecular genetic markers used in molecular breeding due to their specific PCR amplification, high polymorphic information capacity (PIC), good primer versatility, high mutation rate, codominance, low DNA usage, fast reaction speed, simple operation, and good result reproducibility. Microsatellites can reveal genetic diversity better than other molecular markers such as RFLP, RAPD, and AFLP. Pelteobagrus pelteobagrus is an important freshwater economic fish in my country, and it is farmed in 27 provinces across the country. In recent years, the production has grown rapidly, from 54,800 tons in 2003 to 587,800 tons in 2021, an increase of nearly 11 times. The production of freshwater fish ranks 10th, second only to bulk freshwater fish, tilapia and sea bass. At present, the research on microsatellites in yellow catfish mainly focuses on the following four aspects: first, the analysis of economic trait association, such as the microsatellite screening of GH gene in yellow catfish; second, the use of microsatellites to evaluate the population genetic structure and genetic diversity of yellow catfish, such as the use of 8 microsatellites to analyze the slight differences between the populations of yellow catfish in the Yangtze River and Baiyangdian Lake; third, the construction of genetic maps and QTL analysis, using 100 F1 individuals of wild (♂) and artificially cultivated (♀) yellow catfish as the mapping population, integrating 13 SSR markers, 89 SRAP markers, and 26 TRAP markers to construct the genetic linkage map of yellow catfish; fourth, the screening of microsatellites in some important trait-related genes from the transcriptome, such as a large number of microsatellites involved in sex determination were screened from the gonad and brain transcriptome of yellow catfish. There are few studies on microsatellite markers for growth traits in yellow catfish, only the screening of GH gene microsatellites and the positioning of QTLs for growth traits.

[0004] The yellow catfish insulin-like growth factor 1a receptor (igf1ra) gene is the core gene of the GH / IGF1 axis and the downstream receptor of insulin-like growth factor (IGF1). It is 96549bp in length and is located on chromosome 13. The development of microsatellite molecular markers related to growth traits based on the yellow catfish insulin-like growth factor 1a receptor (igf1ra) gene is of great significance for the selection of growth traits of yellow catfish using molecular marker-assisted breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a microsatellite molecular marker related to the growth traits of yellow catfish and its application, so as to solve the problems existing in the above-mentioned prior art. The amplification primers developed by the present invention are used to detect the genotype of the microsatellite locus related to the growth traits of yellow catfish, so as to realize the selection of the growth traits of yellow catfish by molecular marker-assisted breeding.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a microsatellite fragment related to the growth traits of yellow catfish, wherein the repetitive sequence of the microsatellite fragment is (TTCTC) n , the value range of n is any integer between 6 and 13;

[0008] The microsatellite fragments include the alleles shown in the following table:

[0009]

[0010] The growth traits include total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

[0011] The present invention also provides a primer pair for amplifying the microsatellite fragment, the primer pair comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.18.

[0012] The invention also provides a kit containing the primer pair.

[0013] The invention also provides application of the primer pair or the kit in detecting growth traits of yellow catfish.

[0014] The present invention also provides a method for detecting the growth traits of yellow catfish, comprising the following steps:

[0015] Extracting DNA from yellow catfish samples to be tested;

[0016] Using the sample DNA of yellow catfish to be tested as a template, performing PCR amplification using the primer pair, performing genotyping on the amplified product, and determining the growth traits of the yellow catfish to be tested according to the genotyping results;

[0017] The genotyping results showed that the growth traits of the CF genotype yellow catfish were significantly higher than those of the EF genotype yellow catfish; the body length was significantly higher than those of the FG genotype yellow catfish and the FF genotype yellow catfish; the total length was significantly higher than those of the BH genotype yellow catfish and the FF genotype yellow catfish;

[0018] The genotyping results showed that the caudal peduncle height of yellow catfish with CF genotype, DF genotype and FG genotype was significantly higher than that of yellow catfish with FF genotype.

[0019] The growth traits include total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

[0020] Furthermore, the reaction system of the PCR amplification was 1 μL of DNA, 10 μL of 2×Taq PCR Mix (Beijing Tiangen Biochemical Co., Ltd.), 0.5 μL of each of the upstream and downstream primers, and ddH2O was added to make up to 20 μL;

[0021] The reaction conditions of the PCR amplification were pre-denaturation at 94° C. for 4 min, denaturation at 94° C. for 30 s, annealing for 30 s, extension at 72° C. for 30 s, 35 cycles, and extension at 72° C. for 5 min.

[0022] The invention also provides application of the primer pair or the kit in screening yellow catfish with excellent growth traits.

[0023] The present invention also provides a method for screening yellow catfish with excellent growth traits, comprising the following steps:

[0024] Extracting DNA from yellow catfish samples to be tested;

[0025] Using the yellow catfish sample DNA to be tested as a template, performing PCR amplification using the primer pair, and performing genotyping on the amplified product;

[0026] If the genotyping result is CF genotype, the yellow catfish to be tested is an individual with excellent growth traits;

[0027] The growth traits include total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

[0028] Furthermore, the reaction system of the PCR amplification was 1 μL of DNA, 10 μL of 2×Taq PCR Mix (Beijing Tiangen Biochemical Co., Ltd.), 0.5 μL of each of the upstream and downstream primers, and ddH2O was added to make up to 20 μL;

[0029] The reaction conditions of the PCR amplification were as follows: pre-denaturation at 94° C. for 4 min, denaturation at 94° C. for 30 s, annealing for 30 s, extension at 72° C. for 30 s, 35 cycles, and extension at 72° C. for 5 min.

[0030] The invention also provides the application of the primer pair or the kit in molecular marker-assisted breeding of growth traits of yellow catfish.

[0031] The present invention discloses the following technical effects:

[0032] The present invention finds a microsatellite fragment on the yellow catfish insulin-like growth factor 1a receptor (igf1ra) gene, and the repeating sequence of the microsatellite fragment is (TTCTC) n, the value range of n is any integer between 6 and 13. At the same time, a pair of primers for amplifying the above-mentioned microsatellite fragments were screened out, and the correlation between the genetic diversity and main growth traits of the candidate yellow catfish population was analyzed to find the dominant genotype that is beneficial to the growth traits.

[0033] The present invention uses the above-screened primer pair to amplify samples and can specifically amplify microsatellite fragments. These microsatellite fragments are closely related to the growth traits of yellow catfish. The use of this primer pair can screen yellow catfish parents with better growth potential and provide effective molecular markers for molecular breeding of yellow catfish.

[0034] By using the primer pair developed by the invention to detect the genotype of microsatellite loci related to the growth traits of yellow catfish, molecular marker-assisted breeding of the growth traits of yellow catfish can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the statistical result of the microsatellite loci of the igf1ra gene, where Di: dibase; Tetra: tribase; Tri: tetrabase; Penta: pentabase; Hexa: hexabase;

[0037] Figure 2 This is the gel electrophoresis diagram of the primers for the microsatellite locus of the igf1ra gene, where the fragment sizes of maker from top to bottom are: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 600bp, 250bp, 100bp; the 10 primers were amplified using the same DNA sample (sample numbers: 001, 002, 002, 005, 007, 008, 009, 009, 001, 001, 002, 003, 004, 005, 006, 007, 008, 009, 009, 001 ... 11); The corresponding relationship between the red numbers and primers is: 0-IGF1A2, 1-IGF1A3, 2-IGF1A4, 3-IGF1A5, 4-IGF1A6, 5-IGF1A7, 6-IGF1A8, 7-IGF1A9, 8-IGF1A10, 9-IGF1A1; The last red frame is the single sample test result of primers IGF1A1-IGF1A10, and the DNA sample No. 018 was selected;

[0038] Figure 3 The microsatellite locus of igf1ra gene (TTCTC) nThe ABI typing diagram corresponds to the BG genotype (183bp / 208bp), DF genotype (193bp / 203bp), CE genotype (188bp / 198bp) and AH genotype (178bp / 213bp) from top to bottom. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1 Screening of microsatellite loci and primer design for the igf1ra gene of yellow catfish

[0045] The yellow catfish samples used for microsatellite marker screening in this embodiment were from Brothers Family Farm in Dongpo District, Meishan, Sichuan. The experimental fish were purebred yellow catfish breeding population F2, with 20 males and 20 females of the purebred yellow catfish breeding system as parent F1, and self-breeding was carried out in the same spawning pond. After hatching, the fertilized eggs were transferred to the same pond for breeding, which was the F2 population, and the samples came from the same batch of F2 population fry. 200 12-month-old yellow catfish were collected in May 2022. About 1g of yellow catfish caudal fin tissue was collected, immersed in a centrifuge tube containing 75% (volume percentage) alcohol, and stored at -20°C. At the same time, the growth-related traits of the samples such as total length, body length, weight, body height, head length, tail peduncle length and tail peduncle height were recorded.

[0046] About 0.5 g of yellow catfish tail fin tissue was cut from each sample, and the original sample was stored in a -20°C refrigerator for later use. The tissue was washed with ultrapure water, and DNA was extracted from the tail fin tissue according to the steps using the QIAamp DNAMini kit (Tiangen). The extracted DNA concentration and purity were measured and recorded by NanoDrop2000, and DNA was re-extracted for samples that did not reach 100 ng / μL. 10 μL of the qualified DNA sample was taken and diluted to 50 ng / μL for subsequent experiments, and the remaining DNA sample was stored at -20°C for later use.

[0047] The yellow catfish igf1ra gene (GeneID: 113652381) was downloaded from the NCBI database. The full length is 96549 bp and is located on chromosome 13. The microsatellite loci in the igf1ra gene were searched using Krait software to determine the type, number of repeats, and location of the microsatellite loci. The igf1ra gene contains 79 microsatellite loci, including 48 dibasic (Di), 5 tribasic (Tetra), 13 tetrabasic (Tri), 3 pentabasic (Penta), and 1 hexabasic (Hexa). Figure 1 ). Primers were designed according to the following principles: there should be no undetermined sequence at the position of the primer; the primer should be neither too close nor too far from the core sequence, about 10 to 60 bp; the primer sequence should consist of 18 to 27 nucleotides; the product should be between 100 and 300 bp; the primer sequence should be rich in G / C, about 20 to 80%, preferably about 50%, with regular distribution of G / C; special attention should be paid to avoid more than 5 consecutive A / T nucleotides; the Tm value of the primer should be controlled between 57 and 68 °C; the primers should have low complementarity or no complementarity; the length and Tm value of the two primers should not differ too much; and dimers should be avoided as much as possible between the primers. A total of 10 pairs of primers were designed, and the primer information is shown in Table 1. The primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and diluted to 10 μmol / L for use.

[0048] Table 1 Primer information on the microsatellite locus of the igf1ra gene

[0049]

[0050]

[0051] PCR amplification was performed using the primers in Table 1. The total reaction system was 20 μL: 1 μL DNA, 10 μL 2×Taq PCR Mix (Beijing Tiangen Biochemical Co., Ltd.), 0.5 μL each of upstream and downstream primers, and ddH2O was used to make up to 20 μL. Reaction conditions: 94°C pre-denaturation for 4 min, 94°C denaturation for 30 s, annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C extension for 5 min. 5 μL of the amplified product was taken for 2.0% agarose gel electrophoresis detection. Among the 10 pairs of primers, the amplified bands of IGF1A2, IGF1A3, IGF1A4, IGF1A8 and IGF1A10 were weak, the amplified band of IGF1A5 had a stray band, and the target fragment bands amplified by the four pairs of primers IGF1A1, IGF1A6, IGF1A7 and IGF1A9 were clear without stray bands, indicating that the primers were of good quality ( Figure 2 ).

[0052] Example 2 Yellow catfish igf1ra gene microsatellite locus (TTCTC) n Genotyping and polymorphism analysis

[0053] The polymorphism detection of microsatellites is semi-automatically detected by collecting fluorescent signals, so the 5' or 3' end of the common primer needs to be labeled with a fluorescent modification group. In this example, two commonly used fluorescent modification groups, FAM (green) and TAMRA (orange), were selected. Four pairs of primers, IGF1A1, IGF1A6, IGF1A7 and IGF1A9, with clearer bands, were selected to design fluorescent signal primers for subsequent experiments.

[0054] The PCR products obtained by fluorescent primer amplification were detected by electrophoresis on a 96-well capillary 3730xl DNA analyzer, and capillary electrophoresis genotyping scanning was performed using an ABI 3730XL instrument, and the scanning results were analyzed by GeneMaper software. Gene frequencies and genotype frequencies were statistically analyzed based on the typing results. Cervus 3.0 software was used to calculate alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC). Genepop 3.4 software was used to perform Hardy-Weinberg equilibrium test.

[0055] The results showed that 10 samples were selected for preliminary genotyping experiments with each primer. The genotyping results of IGF1A1, IGF1A6 and IGF1A7 primers were poor, so only IGF1A9 primers were retained for subsequent trait association analysis. The core sequence of the microsatellite locus corresponding to the IGF1A9 primer is TTCTC, which belongs to a 5-base microsatellite and is denoted as (TTCTC) n .

[0056] Igf1ra gene microsatellite locus (TTCTC) n Typing results were obtained from 195 yellow catfish samples, and a total of 8 alleles were detected. The lengths of the alleles were 178bp, 183bp, 188bp, 193bp, 198bp, 203bp, 208bp and 213bp, respectively. The size of each fragment differed by n 5bp (core sequence TTCTC), and the value of n ranged from 6 to 13. Therefore, the igf1ra gene microsatellite locus (TTCTC) n The Ne is 8, Ho is 0.77, He is 0.81, PIC is 0.78, PIC>0.5, and some genotyping results are as follows Figure 3 , microsatellite locus (TTCTC) n The allele distribution is shown in Table 2. Among them, the E gene has the highest frequency, accounting for 0.33, and the proportion of heterozygotes is much higher than that of homozygotes. The Hardy-Weinberg (HW) equilibrium test was performed using Genepop 3.4 software, and the results showed that (TTCTC) n The HW balance P value of the site was 0.60, which was consistent with HW balance.

[0057] Table 2 Microsatellite loci (TTCTC) n Allele distribution and frequency

[0058]

[0059] Example 3 Yellow catfish igf1ra gene microsatellite locus (TTCTC) n Gene polymorphism and trait association analysis

[0060] SPSS26.0 software general linear model was used to analyze the microsatellite loci of igf1ra gene (TTCTC) n The association between gene polymorphism and the main growth traits of Pelteobagrus fulvidraco, such as total length, body length, weight, body height, head length, caudal peduncle length and caudal peduncle height, was shown in Table 3. The results were expressed as mean ± standard deviation. LSD and Duncan methods were used for multiple comparisons, with the threshold of significant difference set at P < 0.05 and the threshold of extremely significant difference set at P < 0.01.

[0061] Table 3 (TTCTC)n Mean values ​​and multiple comparisons of growth traits of different genotypes at each locus

[0062]

[0063]

[0064] Note: The mean is followed by the standard error. Different lowercase letters in the same row indicate significant differences (P < 0.05). Different uppercase letters in the same row indicate extremely significant differences (P < 0.01). Genotype AB is represented by the length of the allele amplification product, which is 178 bp / 183 bp. The same is true for other genotypes.

[0065] From Table 3, we can see that (TTCTC) n The growth traits of the CF genotype (188bp / 203bp) at the locus were significantly higher than those of the FF genotype (198bp / 203bp). The body length of the CF genotype was significantly higher than that of the FG genotype (203bp / 208bp) and FF genotype (203bp / 203bp). The total length of the CF genotype was significantly higher than that of the BH genotype (183bp / 213bp) and FF genotype. The caudal peduncle height of the CF genotype, DF genotype (193bp / 203bp) and FG genotype (203bp / 208bp) genotype was significantly higher than that of the FF genotype. Therefore, (TTCTC) n The CF genotype of the locus is a dominant genotype and can be used as a candidate genotype for growth traits. Therefore, PCR amplification of the yellow catfish sample was performed with the IGF1A9 primer. If the amplification product contained fragments of 188 bp and 203 bp in length at the same time, the yellow catfish to be tested was an individual with a dominant growth trait.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer pair for amplifying a microsatellite fragment related to the growth traits of Pelteobagrus fulvidraco, characterized in that: The primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.17 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.18; The growth traits are total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

2. A kit comprising the primer pair according to claim 1.

3. Use of the primer pair described in claim 1 or the kit described in claim 2 in detecting growth traits of Pelteobagrus fulvidraco, wherein the growth traits are total length, body length, weight, body height, head length, caudal peduncle length and caudal peduncle height.

4. Use of the primer pair according to claim 1 or the kit according to claim 2 in screening yellow catfish with excellent growth traits, wherein the growth traits are total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

5. A method for screening yellow catfish with excellent growth traits, characterized in that: The following steps are involved: Extracting DNA from yellow catfish samples to be tested; Using the yellow catfish sample DNA to be tested as a template, performing PCR amplification using the primer pair described in claim 1, and performing genotyping on the amplified product; The primer pairs described are capable of amplifying the following alleles: ； The allele includes a repeating sequence (TTCTC) n, wherein the value of n is any integer between 6 and 13, and when the alleles are A, B, C, D, E, F and H, the values ​​of n are 6, 7, 8, 9, 10, 11, 12 and 13, respectively; If the genotyping result is CF genotype, the yellow catfish to be tested is an individual with excellent growth traits; The growth traits are total length, body length, body weight, body height, head length, caudal peduncle length and caudal peduncle height.

6. The method according to claim 5, characterized in that The reaction system of the PCR amplification is 1 μL DNA, 10 μL 2×Taq PCR Mix, 0.5 μL each of upstream and downstream primers, and ddH2O is added to 20 μL; The reaction conditions of the PCR amplification were pre-denaturation at 94° C. for 4 min, denaturation at 94° C. for 30 s, annealing for 30 s, extension at 72° C. for 30 s, 35 cycles, and extension at 72° C. for 5 min.

7. Use of the primer pair according to claim 1 or the kit according to claim 2 in molecular marker-assisted breeding of growth traits of Pelteobagrus fulvidraco, wherein the growth traits are total length, body length, weight, body height, head length, caudal peduncle length and caudal peduncle height.

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