SSR and SNP molecular marker primer combination for genetic identification of southern catfish family and application thereof

Abstract

The application discloses a kind of SSR and SNP molecular marker primer combination for genetic identification of southern catfish family and application thereof, belong to molecular biology field, the SSR molecular marker primer has 9 pairs, nucleotide sequence as shown in SEQ ID No.1-SEQ ID No.18;The SNP molecular marker primer has 6 pairs, nucleotide sequence as shown in SEQ ID No.19-SEQ ID No.30.The application uses 9 microsatellite sites and 6 southern catfish gene SNP mutations as molecular markers, which can distinguish different families of southern catfish, and construct the genetic pedigree of the breeding southern catfish family.The technology can lay a foundation for improving the germplasm purity of southern catfish breeding population and scientific breeding.The method of the application has the characteristics of simple operation and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to a primer combination of SSR and SNP molecular markers for genetic identification of a family of Silurus meriaionalis and application thereof. BACKGROUND

[0002] Clear genetic pedigree information is crucial for genetic breeding, family selection and parent management of farmed fish. Due to the fact that parent individuals, offspring individuals or even second-generation offspring individuals are often selected as breeding parents in fish farms, the pedigree of farmed fish is usually complex, which can easily cause inbreeding depression. Before the pairing plan of parent fish is carried out, the genetic relationship between individuals in the breeding population is analyzed, and a scientific pairing strategy is designed according to the genetic background. The individuals with the farthest genetic relationship are used as the parents of the next generation, which can effectively avoid inbreeding and loss of population genetic diversity. Maintaining the minimization of the average genetic relationship between parent fish is the best way to maintain high population genetic diversity and avoid the reduction of product quality, and is also an important basis for implementing the parent fish pairing scheme.

[0003] Silurus meriaionalis (Chen), also known as southern catfish or large-mouth catfish, belongs to the order Siluriformes, the family Siluridae and the genus Silurus, has the characteristics of large individual, fast growth, strong disease resistance and easy artificial breeding, and is an important economic freshwater fish in China. However, the genetic information of the parent fish of commodity fish in the farm is usually unclear, and molecular marker assisted analysis is often needed when studying the genetic pedigree of different families.

[0004] Microsatellite markers (SSR), also known as short tandem repeats or simple sequence length polymorphism, are a kind of DNA sequences composed of several base motifs in tandem repeats, which are widely distributed in different positions of fish genomes. The variability of the number of repeats in different genetic materials leads to the high variability of the length of microsatellite markers. Due to the great variation in the length and number of repeats, microsatellite markers have become a highly efficient method for detecting population genetic polymorphism, which can quickly identify homozygotes and heterozygotes, accurately determine the genetic relationship between individuals, and the results are reliable, simple and time-saving. Single nucleotide polymorphism markers (SNP) are also known as the third generation of DNA molecular markers, which are polymorphisms of DNA sequences at the chromosomal genome level caused by single nucleotide variation. This variation includes single base deletion and insertion, single nucleotide substitution, etc. SNPs are widely distributed in fish genomes, almost throughout the entire genome. In the population, SNP markers are di-allelic, so the allele frequency of any population can be estimated, and the genetic stability is high. Some SNPs located in genes may directly affect the structure of proteins or gene expression levels, and thus affect the phenotype of fish.

[0005] There are some reports on the screening of SSR markers and SNP sites of P. sinensis. Quan et al. (2006) and Quan et al. (2007) developed 31 microsatellite markers for P. sinensis by magnetic bead enrichment method, and found that some of the microsatellite markers could be cross-species amplified in other Pseudobrama fish such as Pseudobrama simus. Xu Dandan (2013) used "454" sequencing technology to obtain a large number of P. sinensis microsatellite sequences, and screened out 48 highly polymorphic microsatellite markers from them. Xie Mimim (2016) used high-throughput sequencing technology to screen out 77,634 SNP sites from the RAD database of P. sinensis, and used 26,714 SNP markers to construct a high-density genetic linkage map of P. sinensis. This map laid a foundation for the research of quantitative trait mapping, related genetic mechanism of economic traits, and molecular marker-assisted selection of P. sinensis. These technologies only screened SSR markers or SNP markers of P. sinensis, and did not apply them or effectively identify their diversity. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a combination of SSR and SNP molecular marker primers for genetic identification of P. sinensis family and application thereof, which can accurately distinguish different P. sinensis breeding families from the genetic point of view.

[0007] The technical solution of the present application is: a combination of SSR and SNP molecular marker primers for genetic identification of P. sinensis family, the SSR molecular marker primers have 9 pairs, and the nucleotide sequences are shown in SEQ ID No. 1-SEQ ID No. 18; the SNP molecular marker primers have 6 pairs, and the nucleotide sequences are shown in SEQ ID No. 19-SEQ ID No. 30.

[0008] A kit for genetic identification of P. sinensis family, the kit comprises the combination of SSR and SNP molecular marker primers.

[0009] The combination of SSR and SNP molecular marker primers or the kit is applied in the genetic identification of P. sinensis family.

[0010] Microsatellite marker data analysis: for the results of microsatellite marker STR typing, Cervus 2.0 software is used for parentage analysis of genotype data, and the main analysis contents include allele number (A), observed heterozygosity (Ho), expected heterozygosity (He) polymorphic information content (PIC), and invalid allele frequency, and finally STRUCTURE software and POLYSAT are used for family identification.

[0011] SNP data analysis: all obtained sequences were aligned using ClustalX1.83 software, analyzed using MEGA5.05 software, and the proportion of variation sites (V), the proportion of conserved sites (C), single nucleotide mutation sites (S) and the proportion of parsimony information sites (Pi) were calculated; genetic distance and genetic differentiation index (Fst) were analyzed using ClustalX1.83 software; haplotype diversity (h), nucleotide diversity (pi) and haplotype number (H) were calculated using Dnasp4.50 software; allele frequency, inbreeding coefficient (Fis) and other indicators including effective allele number (Ne), allele number (Na), Shannon index (I), observed heterozygosity (Obs Het), observed homozygosity (ObsHom), expected homozygosity (Exp Hom), expected heterozygosity (Exp Het), Nei's expected heterozygosity (Nei), Nei's genetic distance and average heterozygosity (Ave Het) were analyzed using PopGen3.2 software.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] 1、The present application uses 9 microsatellite sites and 6 SNP mutations of pangasian catfish genes as molecular markers, which can distinguish different families of pangasian catfish, and construct a genetic pedigree of the breeding pangasian catfish family. The technology can lay a foundation for improving the germplasm purity of pangasian catfish breeding population and scientific breeding.

[0014] 2、The method of the present application has the characteristics of simple operation and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The agarose gel electrophoresis diagram of 9 microsatellite markers used in the present application;

[0016] Figure 2 Structure genetic structure analysis diagram of different pangasian catfish populations;

[0017] Figure 3 PCA scatter plot of genetic distance of pangasian catfish individuals based on Bruvo distance;

[0018] Figure 4 Agarose gel electrophoresis diagram of 6 genes of pangasian catfish;

[0019] Figure 5 Agarose gel electrophoresis diagram of 6 genes of pangasian catfish;

[0020] Figure 6 Agarose gel electrophoresis diagram of 6 genes of pangasian catfish. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0022] I. Experimental materials:

[0023] Two southern catfish families from two different farms A and B were used, each family including 30 parent individuals and 100 offspring individuals. The parent population from the two farms was abbreviated as AP and BP, and the offspring population from the two farms was abbreviated as AF and BF.

[0024] II. DNA extraction

[0025] 1. Collecting the parent population and offspring population of different breeding families of southern catfish: all samples were collected by non-invasive method to collect part of the tail fin tissue and stored in 95% ethanol in the laboratory refrigerator at -20°C.

[0026] 2. Extracting southern catfish genomic DNA: about 30 mg of tail fin tissue was cut from all collected southern catfish and placed in a centrifuge tube containing 200 μL of GA buffer, vortexed for 15 sec. 20 μL of Proteinase K solution was added, and the tissue was placed at 56°C until it was dissolved. Then 200 μL of buffer GB was added, and the solution was placed at 70°C for 10 min, and 200 μL of anhydrous ethanol was added, and mixed well. The solution was added to the adsorption column CB3, and after centrifugation and washing several times, the waste liquid was completely removed. After drying at room temperature, the adsorption column CB3 was transferred to a new centrifuge tube, 50 μL of ddH2O was added, and the DNA solution was collected by centrifugation. To improve the purity and concentration, the step was repeated. After detecting the DNA by agarose gel electrophoresis, it was stored at -20°C for subsequent experiments.

[0027] III. Southern catfish SSR primer design and PCR amplification

[0028] The microsatellite primers of southern catfish (Table 1) were grouped and labeled with three fluorescent labels FAM, STAM, and DCM, and the extracted southern catfish genomic DNA was amplified by PCR using fluorescent primers.

[0029] Table 1 Southern catfish microsatellite primers used in the present application

[0030]

[0031] The reaction system includes 20 μL of 2x Taq-PCR master mix, including rapid DNA Polymerase, Mg 2+PCR conditions were as follows: pre-denaturation at 98℃ for 30 s; denaturation at 94℃ for 10 s; annealing at appropriate annealing temperature for 15 s; extension at 72℃ for 6 s, 34 cycles; final extension at 72℃ for 1 min, and storage at 4℃.

[0032] The PCR products were verified by 1.5% agarose gel electrophoresis, and the PCR products of bright and clear target bands were sent to Yee Microgene (Beijing) Co., Ltd. for capillary electrophoresis to complete STR typing.

[0033] Agarose gel electrophoresis Figure 1 ) analysis showed that 9 pairs of microsatellite markers obtained single and clear bands, the sizes of the amplification products of these markers were consistent with the expected, the amplification results were good, and were suitable for STR typing.

[0034] Four, genetic diversity and genetic structure analysis of SSR markers

[0035] (1) SSR diversity information of different families of southern catfish

[0036] The SSR diversity analysis results (Table 2) showed that the number of alleles of 9 microsatellite markers used in the application was between 4 and 12, and the diversity was in a relatively high range. The genetic diversity of the parent populations AP (Ho=0.859, He=0.759, PIC=0.733) and BP (Ho=0.822, He=0.739, PIC=0.701) in the application was obviously higher than that of the offspring populations AF (Ho=0.808, He=0.688, PIC=0.643) and BF (Ho=0.781, He=0.691, PIC=0.646). This is consistent with the actual situation.

[0037] Table 2 Genetic diversity parameters of southern catfish populations from different families at 9 microsatellite loci

[0038]

[0039]

[0040] Note: Number of individuals (N), number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC), frequency of null alleles (Null), inbreeding coefficient (Fis)

[0041] (2) Population genetic structure analysis based on SSR

[0042] STRUCTURE analysis of population genetic structure of 260 P. ovatus from two families Figure 2 ) showed that there was a clear peak when ΔK = 2, indicating that there were two genetic structures in the parent and offspring populations of P. ovatus in the present application, which was highly consistent with the actual situation that all samples came from two families. When only discussing family A or family B, genetic structure analysis showed that there were 6 and 5 genetic structures in the two families, respectively, indicating that the content of the two families was complex.

[0043] (3) Individual genetic distance analysis based on SSR

[0044] The genetic distance between individuals of all families was statistically analyzed, and PCA analysis was performed according to Bruvo's distance, and the results Figure 3 ) showed that family A and family B were clearly distinguished, and the parent population AP and the offspring population AF in family A were not obviously distinguished, but were mixed together, and the parent population BP and the offspring population BF in family B were also mixed together, and were not obviously distinguished. It is shown that the individual genetic distance analysis based on SSR can clearly distinguish two different families.

[0045] V. Design of P. ovatus SNP marker primers and PCR amplification

[0046] According to the gene sequences of the pomc, cart, gh genes of Pimelodus and the Y chromosome marker genes ym1, ym2 and ym3 published in the GenBank database, the CDS regions of these genes were PCR amplified. The PCR amplification method was to use the DNA sample of P. ovatus extracted in step two as the template, and use the SNP marker primers in Table 3 for PCR amplification.

[0047] Table 3: Amplification primers of the CDS regions of related genes of P. ovatus in the present technology

[0048]

[0049]

[0050] The reaction system includes 20 μL 2 × Taq-PCR master mix, including fast DNA polymerase, Mg2+, dNTPs, PCR stabilizer and enhancer 10 μL and 0.5 μL each of forward and reverse primers (Table 2), 0.5 μL DNA template and 8.5 μL ddH2O. The PCR conditions are pre-denaturation at 98℃ for 30 s; denaturation at 94℃ for 10 s; annealing at the respective annealing temperature for 15 s; extension at 72℃ for 6 s, 34 cycles; extension termination at 72℃ for 1 min, and then storage at 4℃.

[0051] PCR products were verified by 1.5% agarose gel electrophoresis. Agarose gel electrophoresis Figure 4 ) shows that the agarose gel electrophoresis results of the pomc, cart and gh genes amplified in the present application show that the obtained target bands are about 1000 bp in size, which is consistent with the expected size of the target gene fragments. The sizes of ym1, ym2 and ym3 of the amplified male southern catfish individuals are all less than 500 bp, which is consistent with the expected size of the three genes, and all female individuals do not amplify ym1, ym2 and ym3, which is consistent with the characteristics of Y chromosome specific genes. The agarose electrophoresis bands are clear and bright, showing that they have a high concentration.

[0052] Six, genetic diversity and genetic structure analysis of SNP markers

[0053] (1) Population genetic diversity of different genes of southern catfish

[0054] The genetic diversity parameters based on nucleotide sequences (Table 4) show that the genetic diversity of the parent populations AP (π = 0.1272-0.1572, Hd = 0.8942-0.9688) and BP (π = 0.1170-0.1375, Hd = 0.8667-0.9342) in the present application is significantly higher than that of the offspring populations AF (π = 0.0804-0.0973, Hd = 0.2615-0.3127) and BF (π = 0.0265-0.0832, Hd = 0.2325-0.3075). This is consistent with the actual situation.

[0055] Table 4 Genetic diversity parameters of different genes of southern catfish

[0056]

[0057]

[0058] Note: Number of individuals (N), nucleotide diversity (π), number of haplotypes (h), haplotype diversity (Hd)

[0059] (2) Allele frequency and inbreeding coefficient analysis of southern catfish SNP

[0060] Integrating the sequences of six genes of southern catfish, a sequence with a length of 3522 bp was obtained, from which 15 SNP loci (loc1-loc15) were detected. The alleles of SNP are generated due to the variation of ATCG bases, and the base variation of the 15 SNP loci in the present application is shown in Table 5.

[0061] Table 5 Base variation of SNP loci in the present application

[0062]

[0063] (3) SNP-based population polymorphism analysis

[0064] The heterozygosity analysis results (Table 6) using the obtained 15 SNP sites show that the SNP polymorphisms of the parent populations AP (I = 0.9541, Nei = 0.1964, Ave Het = 0.1978) and BP (I = 0.9472, Nei = 0.1908, Ave Het = 0.1972) in the present application are obviously higher than those of the offspring populations AF (I = 0.2649, Nei = 0.1003, Ave Het = 0.1025) and BF (I = 0.2597, Nei = 0.1032, Ave Het = 0.1110). This shows that the gene heterozygosity level of the parent populations of P. sinensis is higher, which is highly consistent with the actual situation and the results of the SSR application.

[0065] Table 6 SNP polymorphism information of different P. sinensis populations

[0066]

[0067]

[0068] Note: Na: number of alleles; Ne: number of effective alleles; I: Shannon index; Obs_Hom: observed homozygosity; Obs_Het: observed heterozygosity; Exp_Hom: expected homozygosity; Exp_Het: expected heterozygosity; Nei: Nei's expected heterozygosity; Ave Het: average heterozygosity

[0069] (4) Population genetic distance identification analysis

[0070] The genetic distances of the four populations were calculated according to the nucleotide sequences and SNP differences of P. sinensis populations, and a distance tree was constructed according to the genetic distances (Fig. 2). Figure 5 It can be seen that AP and AF from family A are clustered into one branch, while BP and BF from family B are clustered into a second branch, and the two families are clearly distinguished, which is consistent with the conclusion of the SSR analysis.

[0071] (5) Individual genetic distance identification analysis of different families of P. sinensis

[0072] The genetic distances between individuals were calculated based on the multi-gene sequences and SNP differences of 260 P. sinensis from two families, and an individual clustering tree was constructed (Fig. 3). Figure 6). The results show that these individuals of P. sinensis are divided into two branches, the first branch contains 116 individuals, 113 individuals from family A, accounting for 86.92% of all individuals of family A, and 3 individuals from family B, in which a large number of individuals from AP are gathered with AF, which is consistent with their close genetic relationship. The second branch of the clustering tree contains a total of 144 individuals, of which 127 individuals are from family B, accounting for 97.69% of all individuals of family B, and the clustering of the parent population and the offspring population is consistent with that of family A.

[0073] The present application identifies two families from 260 individuals of P. sinensis based on SSR markers, identifies the genetic diversity characteristics of the parent population and the offspring population, verifies them by SNP markers, constructs a clustering tree divided into two branches with obvious differences, verifies the conclusion of the SSR research, and at the same time, based on the genetic variation parameter analysis of nucleotide sequences and SNP mutations, also verifies the genetic diversity characteristics of the parent population and the offspring population in the SSR markers, so that the conclusion of the present application has higher scientificity and credibility. In summary, the present application effectively distinguishes two families of P. sinensis by combining 9 microsatellite sites and 6 SNP mutations of P. sinensis genes as molecular markers, and this technology can lay a foundation for improving the germplasm purity of P. sinensis breeding population and scientific breeding.

Claims

1. A combination of SSR and SNP molecular marker primers for genetic identification of P. ovatus family, characterized by, The SSR molecular marker primers are 9 pairs, and the nucleotide sequences are shown as SEQ ID No. 1-SEQ ID No. 18; the SNP molecular marker primers are 6 pairs, and the nucleotide sequences are shown as SEQ ID No. 19-SEQ ID No.

30.

2. A kit for genetic identification of P. ovatus family, characterized by, The kit comprises the combination of the SSR and SNP molecular marker primers according to claim 1.

3. The combination of the SSR and SNP molecular marker primers according to claim 1 or the kit according to claim 2 is applied to genetic identification of a family of Pelteobagrus vachelli.

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