Rapid identification method of leiocassis longirostris polymorphism EST-SSRs based on mixed tissue transcriptome and verification method of EST-SSRs

Abstract

The invention discloses a rapid identification method of leiocassis longirostris polymorphism EST-SSRs based on a mixed tissue transcriptome and a verification method of the EST-SSRs, and relates to the field of biological breeding, and the method specifically comprises the following steps: obtaining leiocassis longirostris transcriptome data, screening a plurality of leiocassis longirostris transcriptome EST-SSRs from the transcriptome data, namely EST-SSRs, and verifying the leiocassis longirostris transcriptome EST-SSRs. And carrying out genetic diversity analysis on different geographical culture groups of the leiocassis longirostris by using the EST-SSRs. The invention provides a low-cost and rapid leiocassis longirostris identification method.

Description

Technical Field

[0001] The invention relates to the field of biological breeding, and in particular to a rapid identification method of polymorphic EST-SSRs of long-snout catfish based on mixed tissue transcriptome and a verification method of EST-SSRs. Background Art

[0002] The long-snouted catfish (Leiocassis longirostris) belongs to the order Silurifomes, the family Bagrid, the genus Catfish, also known as catfish, commonly known as Jiangtuan and Feituo. It is mainly distributed in the Yangtze River Basin, the Pearl River Basin, the Huaihe River Basin, the Liaohe River Basin and the rivers and lakes connected to it. Because its snout is longer than that of other catfish, it is called the long-snouted catfish. The meat is delicious and tender, rich in nutrition, and is favored by consumers. Historically, the natural fishery resources of the long-snouted catfish in the Yangtze River Basin were extremely rich. However, in recent years, due to overfishing, water pollution and water conservancy project construction, its natural germplasm resources have been increasingly depleted, and the breeding population is also extremely scarce. It is urgent to carry out research on breeding of good varieties. To carry out breeding research, a large number of molecular markers are urgently needed. However, compared with other farmed economic fish, the molecular markers of the long-snouted catfish are relatively scarce, and it is urgent to develop a large number of molecular markers for its germplasm resource evaluation and genetic breeding research.

[0003] At present, the research on long-snouted catfish mainly focuses on reproductive biology, breeding mode, stress resistance and nutritional components, while the research on genetic background and current status of germplasm resources is relatively weak. If traditional breeding methods are used to carry out improved breed selection, it is relatively time-consuming and labor-intensive. Especially for aquatic animals with a relatively long breeding cycle such as long-snouted catfish, molecular marker-assisted breeding technology is urgently needed to accelerate the breeding process of improved breeds. With the rapid development of molecular marker technology, molecular marker technologies such as Random Amplification Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Inter Simple Sequence Repeat (ISSR), Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) have successively blossomed in the research of genetic breeding of aquatic animals. Microsatellites, also known as simple sequence repeats, are tandem repeat sequences in the genome composed of 1 to 6 nucleotides as basic repeating units. With their good repeatability, high polymorphism, genetic stability, and co-dominant inheritance, they are widely used in aquatic animal population genetic variation analysis, parentage testing, growth trait association analysis, genetic map construction, molecular marker-assisted breeding and other related research. Compared with traditional SSR development technology, microsatellites developed based on high-throughput transcriptome sequencing technology, also known as expressed sequence tag SSR (EST-SSR), have relatively low polymorphism, but due to their large number, high throughput, short cycle, low cost, and direct association with certain functional genes, they are widely used in large-scale development and utilization research of aquatic animal molecular markers. In recent years, the emergence of massive high-throughput sequencing data has also provided convenient conditions for the development of microsatellite markers for aquatic animals. At the same time, a large number of bioinformatics software for high-throughput transcriptome data mining EST-SSRs, such as MISA software (MIcroSAtellite identification tool) and CandiSSR software, SSRMMD (Simple SequenceRepeat Molecular Marker Developer), etc., have been widely used in the screening of polymorphic EST-SSR sites, effectively accelerating the development of molecular markers for the assessment of aquatic animal germplasm resources.

[0004] Based on high-throughput transcriptome sequencing technology, the transcriptome data of mixed tissue samples (liver, muscle and brain) of female and male long-snouted catfish were obtained. After assembly and de-redundancy, the transcriptome Unigenes of male and female long-snouted catfish were obtained. Then, MISA software and SSRMMD software were used to mine EST-SSRs in transcriptome Unigenes and identify polymorphic EST-SSRs, respectively. Finally, the 20 EST-SSRs with high polymorphism obtained were used to evaluate the genetic diversity of different geographical populations of farmed long-snouted catfish, in order to lay a theoretical foundation for the analysis of genetic diversity of farmed long-snouted catfish populations, the protection and utilization of germplasm resources, the construction of high-density genetic linkage maps, and molecular marker-assisted breeding of improved varieties.

[0005] However, there are no reports on the rapid identification of polymorphic EST-SSRs based on the transcriptome of mixed tissues (liver, muscle and brain) of long-snout catfish.

[0006] Therefore, technicians in this field are committed to developing a low-cost and rapid method for identifying long-snout catfish based on EST-SSRs. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a low-cost and rapid method for identifying long-snout catfish.

[0008] To achieve the above object, the present invention provides a rapid identification method of polymorphic EST-SSRs of long-snout catfish based on mixed tissue transcriptome, characterized in that it comprises the following steps:

[0009] Step 1, obtaining the transcriptome data of long-snout catfish;

[0010] Step 2, screening a number of long-snout catfish transcriptome EST-SSR sequences from the transcriptome data obtained in step 1, namely EST-SSRs;

[0011] Step 3: Use EST-SSRs to analyze the genetic diversity of different geographical breeding populations of longnose catfish.

[0012] In a preferred embodiment of the present invention, the step 1 specifically includes:

[0013] Eight male and female longnose catfish aged 17 months were used as research materials. The liver, muscle and brain tissues were cut and sequenced using the Illumina Truseq sequencing platform based on the Illumina Novaseq 6000 platform. TMThe RNA sample preparation kit method was used to construct transcriptome sequencing libraries of male and female long-snouted catfish. After the sequencing data were assembled and de-redundant using Trinity software, the female long-snouted catfish transcriptome Unigenes data, namely LLF.fa, and the male long-snouted catfish transcriptome Unigenes data, namely LLM.fa, were obtained.

[0014] In another preferred embodiment of the present invention, the step 2 specifically includes:

[0015] Using MISA software, multiple EST-SSR sequences were obtained from the LLF.fa and LLM.fa databases of long-snouted catfish, with the minimum repeat numbers of mono-, di-, tri-, tetra-, penta- and hexanucleotide SSRs set to 10, 6, 5, 5, 5 and 5, respectively, and the minimum interval of composite SSR set to 100.

[0016] In another preferred embodiment of the present invention, the step 3 specifically comprises:

[0017] Step 3.1, randomly select several sequences from the screened polymorphic EST-SSRs, and design specific PCR amplification primers, i.e., EST-SSRs primers, based on the sequence characteristics of the long-snouted catfish reference genome in GenBank and the conserved flanking sequences on both sides of the polymorphic EST-SSRs;

[0018] Step 3.2, select fin ray samples from different geographical populations of long-snout catfish, randomly select a number of equal samples from each population, and extract genomic DNA from all individuals as template DNA;

[0019] Step 3.3, use EST-SSRs primers to perform PCR on the template DNA, estimate the concentration of the PCR product based on the agarose gel electrophoresis results, and dilute the product 10 times, mix it with the LIZ500 internal standard, and place it on the sample rack of the ABI 3730XL sequencer for capillary electrophoresis;

[0020] Step 3.4: Data processing to obtain analysis results.

[0021] In another preferred embodiment of the present invention, the step 3.4 specifically includes:

[0022] Step 3.4.1, use GeneMarker 2.2 software to read the sequencing results of ABI 3730XL gene sequencer, obtain the gene-related information of each sample, and then perform statistics on the size of microsatellite, i.e., EST-SSR fragments;

[0023] Step 3.4.2, using GenAlEx 6.0 software to calculate and analyze the genetic diversity parameters of different populations of long-snouted catfish, and perform principal coordinate analysis (PCoA) and Hardy-Weinberg equilibrium (HWE) test, and calculate the polymorphic information content (PIC) of different microsatellite loci according to the following calculation formula;

[0024]

[0025] Where P i , P j are the frequencies of the i-th and j-th alleles in the population, respectively, and n is the number of alleles;

[0026] Step 3.4.3: Use MEGA software to construct and analyze the UPGMA cluster tree of different groups of long-snouted catfish;

[0027] Step 3.4.4: Use Structure software to map the genetic structure, Structure Harverter software to determine the optimal K value, CLUMPP software to merge the K value matrix, Distruct software to draw the Bayesian genotype cluster diagram, and analyze the results.

[0028] In another preferred embodiment of the present invention, the gene-related information obtained in step 3.4.1 is not limited to: allele number, peak map and genotype information.

[0029] In another preferred embodiment of the present invention, the genetic diversity parameters calculated and analyzed in step 3.4.2 include but are not limited to: the number of alleles, i.e., Na, the effective number of alleles, i.e., Ne, the observed heterozygosity, i.e., Ho, the expected heterozygosity, i.e., He, the Nei's genetic distance and genetic similarity between populations, and the genetic differentiation index between populations.

[0030] In another preferred embodiment of the present invention, in the Hardy-Weinberg equilibrium test in step 3.4.2, a balance test is performed on the microsatellite loci of several EST-SSRs selected in step 3.1 corresponding to each different geographical population of the selected long-snouted catfish. If P < 0.05, the locus of the corresponding population deviates significantly from the Hardy-Weinberg equilibrium.

[0031] In another preferred embodiment of the present invention, in step 3.4.2, when analyzing the genetic differentiation index among populations, an ANOVA molecular variance analysis among different populations is also performed.

[0032] A method for verifying polymorphic EST-SSRs of long-snout catfish based on mixed tissue transcriptomes is characterized by comprising the following steps:

[0033] Step a, randomly selecting a number of sequences from the screened polymorphic EST-SSRs, combining the long-snout catfish reference genome sequence characteristics in GenBank and the conserved flanking sequences on both sides of the polymorphic EST-SSRs, designing specific PCR amplification primers, i.e., EST-SSRs primers;

[0034] Step b, extracting genomic DNA from fin samples taken from different geographical groups of cultured long snout catfish, randomly selecting a number of DNA samples and mixing them in equal amounts, and using them as templates for subsequent PCR amplification, performing PCR, and after the PCR products are detected by electrophoresis, using silver staining to develop and fix the images, and taking pictures with a digital camera after the bands are clearly colored, and determining the optimal annealing temperature, effectiveness and polymorphism of each primer;

[0035] At the same time, several equal samples are randomly selected from the fin tissue samples of different geographical groups of long-snouted catfish corresponding to step b, and DNA is extracted respectively for primer universality detection and genetic diversity analysis of different breeding groups. If the primer amplification obtains a PCR product of the expected size and has more than one band, it proves that the primer is polymorphic.

[0036] Technical Effects

[0037] 1. The present invention is based on the transcriptome data of mixed tissues (liver, muscle and brain) of long-snouted catfish in the Yangtze River Delta region, and develops polymorphic EST-SSRs suitable for long-snouted catfish in the Yangtze River Delta region on a large scale. The high-throughput transcriptome sequencing data of male and female individuals of long-snouted catfish in the Yangtze River Delta region are assembled separately to obtain corresponding Unigenes data, and then the SSRMMD software is used to identify the different EST-SSRs, thereby facilitating the development of polymorphic EST-SSRs for the germplasm resource assessment of long-snouted catfish in the Yangtze River Delta region, effectively improving the detection efficiency of polymorphic EST-SSR sites, with a polymorphic site detection rate of up to 90%, saving a lot of manpower, material resources and financial resources.

[0038] 2. The present invention develops corresponding polymorphic EST-SSR markers based on the long-snouted catfish in the Yangtze River Delta region, effectively analyzes the genetic differences of different groups of long-snouted catfish in the Yangtze River Delta region, and calculates the genetic diversity parameters of different long-snouted catfish groups based on the genotype differences of different long-snouted catfish individuals at different mutation sites using relevant bioinformatics software, thereby effectively evaluating the current status of germplasm resources of different groups of long-snouted catfish in the Yangtze River Delta region, and effectively analyzing the current status of germplasm resources of long-snouted catfish in the Yangtze River Delta region, which has important guiding significance for the later genetic breeding research of long-snouted catfish in the Yangtze River Delta region.

[0039] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the result of UPGMA cluster analysis based on Nei's genetic distance in a preferred embodiment of the present invention;

[0041] Figure 2 It is a principal coordinate analysis (PCoA) result based on Nei's genetic distance between different individuals of long-snout catfish in a preferred embodiment of the present invention;

[0042] Figure 3 It is a δK variation curve diagram of analyzing the genetic structure of different groups of long-snout catfish in a preferred embodiment of the present invention;

[0043] Figure 4 A cluster diagram for analyzing the genetic structure of different populations of long-snout catfish according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0045] Example:

[0046] 1. Source of transcriptome data of long-snout catfish

[0047] The transcriptome data were based on the Illumina Novasek 6000 sequencing platform and the Illumina Truseq sequencing technology. TM The RNA sample prep kit method was used to construct transcriptome sequencing libraries of male and female long-snouted catfish. After the sequencing data were assembled and de-redundant using Trinity software, the female long-snouted catfish transcriptome Unigenes data (hereinafter referred to as LLF.fa) and male long-snouted catfish transcriptome Unigenes data (hereinafter referred to as LLM.fa) were obtained.

[0048] 2. Distribution characteristics of EST-SSR sites and quantitative characteristics of polymorphic EST-SSRs in the transcriptome of long-snouted catfish Using MISA software, 105,961 and 97,640 EST-SSR sequences were obtained from the LLF.fa and LLM.fa databases of long-snouted catfish, respectively. Among them, dinucleotide repeat types were the most, accounting for 57.00% and 57.55%, respectively; mononucleotide repeats were second, accounting for 30.92% and 30.74%, respectively; the number of other base repeat types was relatively small, and decreased with the increase of the number of base repeat types. At the same time, the search analysis results based on SSRMMD software showed that the number of polymorphic EST-SSRs also decreased gradually with the increase of the number of base repeat types; it can be seen that the types of EST-SSRs in the transcriptome of long-snouted catfish are extremely rich, ranging from mononucleotides to hexanucleotides, but mainly concentrated in mononucleotides, dinucleotides and trinucleotides (see Table 1).

[0049] Table 1. Transcriptome and polymorphic EST-SSRs information of male and female longnose catfish

[0050]

[0051] 3. Validity and polymorphism verification of polymorphic EST-SSRs in long-snouted catfish

[0052] Thirty sequences were randomly selected from the screened polymorphic EST-SSRs. Combined with the sequence characteristics of the long-snouted catfish reference genome (GenBank accession number: GCA_041146805.1) and the conserved flanking sequences on both sides of the polymorphic EST-SSRs, specific PCR amplification primers were designed using Primer Premier 5.0 software for marker validity and polymorphism detection.

[0053] Fin samples from different geographical populations of farmed longnose catfish (BS, CZ, MS, SD and XT) were used to extract genomic DNA using the Tiangen Marine Animal Tissue Genomic DNA Extraction Kit (DP324-03). 25 DNA samples were randomly selected and mixed in equal amounts as templates for subsequent PCR amplification. The optimal annealing temperature of each primer was screened on an Eppendorf Mastercycler X50s gradient PCR instrument. The PCR reaction system was 20 μl, including: 2 μl template DNA, 0.5 μl of each forward and reverse primer (10 μM), 10 μl of 2×Taq PCR MasterMix (Tiangen Biochemical, KT211), and then ddH2O was used to make up the total volume to 20 μl; the reaction procedure was 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, annealing (48~70℃) for 30 s, 72℃ extension for 25 s, 72℃ extension for 8 min after 35 cycles, and 4℃∞ storage. After the PCR products were detected by 8% non-denaturing polyacrylamide gel electrophoresis, they were fixed by silver staining. After the bands were clearly colored, they were photographed and saved with a digital camera to determine the optimal annealing temperature, effectiveness and polymorphism of each primer. After obtaining the optimal annealing temperature of each primer, 30 tails were randomly selected from the fin tissue samples of different geographical groups of long-snouted catfish (sampled in Baoshan District, Shanghai (hereinafter referred to as BS), Changzhou City, Jiangsu Province (hereinafter referred to as CZ), Meishan City, Sichuan Province (hereinafter referred to as MS), Shunde City, Guangdong Province (hereinafter referred to as SD) and Xiantao City, Hubei Province (hereinafter referred to as XT)) stored in the Fengxian Research Base in the early stage, totaling 150 samples, and DNA was extracted respectively according to the instructions of Tiangen Biochemical Marine Animal Tissue Genomic DNA Extraction Kit (DP324-03), which was used for primer universality detection and genetic diversity analysis of different breeding groups.

[0054] The results showed that among the 30 pairs of EST-SSR primers, 3 pairs of amplified products had only a single band, and the remaining 27 pairs of EST-SSR primers amplified PCR products of the expected size and had more than one polymorphic band, with a polymorphism rate of 90%; non-denaturing polyacrylamide gel electrophoresis, as a classic method for SSR marker detection, showed that 20 pairs of primers had relatively high polymorphism and could be used for genetic diversity analysis of different geographical populations of long-snout catfish. Information on the 20 pairs of polymorphic EST-SSR primers is shown in Table 2.

[0055] 4. Genetic diversity analysis of different geographical culture populations of longnose catfish

[0056] The samples used for the analysis of genetic diversity of long-snouted catfish in different geographical locations were taken from fin ray samples of different geographical populations (BS, CZ, MS, SD and XT) stored at the Fengxian Research Base of our unit in 2023. 30 samples were randomly selected from each population for subsequent extraction of genomic DNA.

[0057] The genomic DNA of individuals from different geographical populations of long-snouted catfish was extracted using the Tiangen Biochemical Marine Animal Genomic DNA Extraction Kit. The quality of the sample DNA was tested by 1.5% agarose gel electrophoresis, and the concentration and purity of the sample DNA were tested by NanoVue Plus spectrophotometer. 260 / A 280 DNA samples with a ratio of about 1.8 were diluted to 50 ng / μl and stored in a -20°C freezer.

[0058] A total of 150 samples from five geographical populations of long-snouted catfish were amplified by PCR using 20 pairs of polymorphic EST-SSR primers on an Eppendorf Mastercycler X50s gradient PCR instrument. The PCR reaction system was 20 μl, including: 2 μl template DNA, 0.5 μl forward and reverse primers (10 μM), 10 μl 2× Taq PCR Master Mix (Tiangen Biochemical, KT211), and then the total volume was supplemented to 20 μl with ddH2O; the reaction program was 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, annealing (optimal annealing temperature in Table 2) for 30 s, 72℃ extension for 25 s, 72℃ extension for 8 min after 35 cycles, and 4℃∞ storage. The concentration of PCR products was estimated according to the results of agarose gel electrophoresis, and the products were diluted 10 times, mixed with LIZ500 internal standards (sizes were 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 base, respectively), and placed on the sample rack of the ABI 3730XL sequencer for capillary electrophoresis detection.

[0059] Table 2. Information of 20 pairs of polymorphic EST-SSR primers

[0060]

[0061] Data processing: GeneMarker 2.2 software was used to read the sequencing results of the ABI 3730XL gene sequencer. After obtaining the number, peak map, genotype and other information of each sample allele, the size of the microsatellite fragment was counted and the results were entered into Excel for future use. The number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Nei's genetic distance and genetic similarity between populations, and genetic differentiation index between populations were calculated using GenAlEx 6.0 software, and principal coordinates analysis (PCoA) and Hardy-Weinberg equilibrium (HWE) test were performed. The polymorphism information content (PIC) of different microsatellite loci was calculated according to the calculation formula of Bostein et al. The UPGMA (Unweighted pair-group method with arithmetic means) clustering tree of different populations of long-snouted catfish was constructed using MEGA software. The genetic structure was mapped using Structure (version 2.3.4) software. Harverter software determined the optimal K value, CLUMPP software merged the K value matrix, and Distruct software drew the Bayesian genotype clustering diagram. The main analysis results are shown below.

[0062] (1) The results of genetic diversity analysis of different geographical populations of long-snouted catfish showed that 20 pairs of EST-SSR primers detected a total of 219 polymorphic sites, with an average of 10.95 alleles detected per primer pair. The average number of effective alleles in the Changzhou population was the highest (4.13), and the lowest in the Baoshan population (3.03). The average observed heterozygosity of the five geographical populations of long-snouted catfish ranged from 0.7083 (BS) to 0.8133 (SD), while the average expected heterozygosity ranged from 0.6343 (BS) to 0.7276 (CZ). The average polymorphic information content was the highest in the Changzhou population (0.6865), followed by the Meizhou population (0.6847) and the Xiantao population (0.6733), and the lowest in the Baoshan population (0.5740) (see Table 3). The average Ho values ​​of the five long-snouted catfish populations were all greater than their He values, indicating that these five long-snouted catfish populations had varying degrees of excess heterozygosity. Most of the genetic parameters of the Changzhou population of long-snouted catfish are higher than those of other geographical populations, and are expected to be used in subsequent research on the breeding of long-snouted catfish.

[0063] Table 3. Genetic diversity parameters of different geographical populations of longnose catfish

[0064]

[0065] (2) The results of Hardy-Weinberg equilibrium test of 20 microsatellite loci (see Table 4) showed that the microsatellite locus LL14 deviated significantly from Hardy-Weinberg equilibrium in all five long-snouted catfish populations (P<0.05). The BS population had the most microsatellite loci that deviated significantly from Hardy-Weinberg equilibrium, with 12 microsatellite loci that deviated significantly from Hardy-Weinberg equilibrium (P<0.05). The CZ population and SD population had 9 microsatellite loci that deviated significantly from Hardy-Weinberg equilibrium (P<0.05). The MS population had 8 microsatellite loci that deviated significantly from Hardy-Weinberg equilibrium (P<0.05). The XT population had the least microsatellite loci that deviated significantly from Hardy-Weinberg equilibrium, with only 7 loci that deviated significantly from Hardy-Weinberg equilibrium (P<0.05). Among the 100 population combinations (5 populations × 20 loci), 55 population loci were in accordance with Hardy-Weinberg equilibrium; the remaining 45 population loci deviated significantly from Hardy-Weinberg equilibrium (P<0.05).

[0066] Table 4 Genetic diversity parameters of 20 microsatellite loci of longnose catfish

[0067]

[0068]

[0069] (3) The genetic distances between different geographical populations of long-snouted catfish ranged from 0.0334 to 0.2266, with an average genetic distance of 0.1291 between each population. The genetic distance between the Baoshan population and other geographical populations was relatively large (0.1594-0.2266), with an average genetic distance of 0.2060 between each population. The genetic distances between other two geographical populations ranged from 0.0334 to 0.0971, with an average genetic distance of 0.0778. The results of genetic similarity showed that the Changzhou population and the Xiantao population of long-snouted catfish had the highest genetic similarity (0.9671), while the Baoshan population and the Xiantao population had the lowest genetic similarity (0.7972) (see Table 5). The results of UPGMA cluster analysis based on Nei's genetic distance ( Figure 1 ) showed that the Changzhou population first clustered with the Xiantao population, then with the Meishan population, then with the Shunde population, and finally with the Baoshan population, indicating that the Baoshan population was relatively distantly genetically related to other geographical populations.

[0070] Table 5 Genetic similarity coefficient (lower left corner) and genetic distance (upper right corner) of different geographical populations of long-snouted catfish

[0071]

[0072] (4) The genetic differentiation index among different populations of long-snouted catfish ranged from 0.0138 to 0.0558 (Table 6). The results showed that only 5.24% of the genetic variation among different populations of long-snouted catfish came from between populations, and most of the genetic variation came from individuals within the population (94.76%). The Baoshan population (BS) and Changzhou population (CZ), Shunde population (SD) and Xiantao population (XT) of long-snouted catfish had a moderate degree of genetic differentiation (0.0536-0.0558), while the genetic differentiation between other two populations was weak (0.0138-0.0438). In addition, the average genetic differentiation index and gene flow among different populations of long-snouted catfish were 0.0524 and 6.9604, respectively. The results of molecular variance analysis (ANOVA) showed that among the total genetic variation, only 4.72% of the genetic variation came from between populations, and 95.28% of the genetic variation came from genetic differences between individuals within a population (Table 7).

[0073] Table 6 Genetic differentiation index (F) between two populations of longnose catfish ST )

[0074]

[0075]

[0076] Table 7 AMOVA molecular variance analysis among different populations of longnose catfish

[0077]

[0078] (5) In animal population genetics, principal coordinate analysis (PCoA) can visualize the similarities and differences between different populations. The results of principal coordinate analysis (PCoA) based on Nei's genetic distance between different individuals of long-nosed catfish show (see Figure 2 ), the individuals in the BS group had obvious differences from those in other groups, among which the genetic differences between individuals in the CZ and XT groups were the smallest, and the genetic differences between individuals in the BS and XT groups were the largest.

[0079] (6) The genetic structure of different populations of long-snout catfish was analyzed. By assuming that the K value was 1-10, a δK variation curve was drawn. When δK was the largest, the optimal K value was 2 (see Figure 3 ), indicating that the 150 long-nosed catfish individuals can be best divided into two groups. Different colors in the cluster diagram represent individuals of different long-nosed catfish groups (see Figure 4 ), the results showed that the genetic structures of different individuals in the five long-snouted catfish populations were mixed to varying degrees, and the genetic composition of different populations was more diverse.

[0080] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A rapid identification method for polymorphic EST-SSRs of long-snout catfish based on mixed tissue transcriptome, characterized in that: The steps include: Step 1, obtaining the transcriptome data of long-snout catfish; Step 2, screening a number of long-snout catfish transcriptome EST-SSR sequences from the transcriptome data obtained in step 1, namely EST-SSRs; Step 3: Use EST-SSRs to analyze the genetic diversity of different geographical breeding populations of longnose catfish.

2. The method according to claim 1, characterized in that The step 1 specifically includes: Eight male and eight female longnose catfish aged 17 months were used as research materials. The liver, muscle and brain tissues were cut and sequenced using the Illumina Novaseq 6000 sequencing platform. TM The RNA sample preparation kit method was used to construct transcriptome sequencing libraries of male and female long-snouted catfish. After the sequencing data were assembled and de-redundant using Trinity software, the female long-snouted catfish transcriptome Unigenes data, namely LLF.fa, and the male long-snouted catfish transcriptome Unigenes data, namely LLM.fa, were obtained.

3. The method according to claim 2, characterized in that The step 2 specifically includes: Using MISA software, multiple EST-SSR sequences were obtained from the LLF.fa and LLM.fa databases of long-snouted catfish, with the minimum repeat numbers of mono-, di-, tri-, tetra-, penta- and hexanucleotide SSRs set to 10, 6, 5, 5, 5 and 5, respectively, and the minimum interval of composite SSR set to 100.

4. The method according to claim 1, characterized in that The step 3 specifically includes: Step 3.1, randomly select several sequences from the screened polymorphic EST-SSRs, and design specific PCR amplification primers, i.e., EST-SSRs primers, based on the sequence characteristics of the long-snouted catfish reference genome in GenBank and the conserved flanking sequences on both sides of the polymorphic EST-SSRs; Step 3.2, select fin ray samples from different geographical populations of long-snout catfish, randomly select a number of equal samples from each population, and extract genomic DNA from all individuals as template DNA; Step 3.3, use EST-SSRs primers to perform PCR on the template DNA, estimate the concentration of the PCR product based on the agarose gel electrophoresis results, and dilute the product 10 times, mix it with the LIZ500 internal standard, and place it on the sample rack of the ABI 3730XL sequencer for capillary electrophoresis; Step 3.4: Data processing to obtain analysis results.

5. The method according to claim 4, characterized in that The step 3.4 specifically includes: Step 3.4.1, use GeneMarker 2.2 software to read the sequencing results of ABI 3730XL gene sequencer, obtain the gene-related information of each sample, and then perform statistics on the size of microsatellite, i.e., EST-SSR fragments; Step 3.4.2, using GenAlEx 6.0 software to calculate and analyze the genetic diversity parameters of different populations of long-snouted catfish, and perform principal coordinate analysis (PCoA) and Hardy-Weinberg equilibrium (HWE) test, and calculate the polymorphic information content (PIC) of different microsatellite loci according to the following calculation formula; Where P i , P j are the frequencies of the i-th and j-th alleles in the population, respectively, and n is the number of alleles; Step 3.4.3: Use MEGA software to construct and analyze the UPGMA cluster tree of different groups of long-snouted catfish; Step 3.4.4: Use Structure software to map the genetic structure, Structure Harverter software to determine the optimal K value, CLUMPP software to merge the K value matrix, Distruct software to draw the Bayesian genotype cluster diagram, and analyze the results.

6. The method according to claim 5, characterized in that The gene-related information obtained in step 3.4.1 is not limited to: allele number, peak graph and genotype information.

7. The method according to claim 5, characterized in that The genetic diversity parameters calculated and analyzed in step 3.4.2 include, but are not limited to: the number of alleles, i.e., Na, the effective number of alleles, i.e., Ne, the observed heterozygosity, i.e., Ho, the expected heterozygosity, i.e., He, the Nei's genetic distance and genetic similarity between populations, and the genetic differentiation index between populations.

8. The method according to claim 5, characterized in that In the Hardy-Weinberg equilibrium test in step 3.4.2, a balance test is performed on the microsatellite loci of several EST-SSRs selected in step 3.1 corresponding to each different geographical population of the selected long-snouted catfish. If P < 0.05, the locus of the corresponding population deviates significantly from the Hardy-Weinberg equilibrium.

9. The method according to claim 7, characterized in that In step 3.4.2, when analyzing the genetic differentiation index among groups, ANOVA molecular variance analysis among different groups is also performed.

10. A method for verifying the polymorphic EST-SSRs of long-snout catfish based on mixed tissue transcriptome according to any one of claims 1 to 3, characterized in that: The steps include: Step a, randomly selecting a number of sequences from the screened polymorphic EST-SSRs, combining the long-snout catfish reference genome sequence characteristics in GenBank and the conserved flanking sequences on both sides of the polymorphic EST-SSRs, designing specific PCR amplification primers, i.e., EST-SSRs primers; Step b, extracting genomic DNA from fin samples taken from different geographical groups of cultured long snout catfish, randomly selecting a number of DNA samples and mixing them in equal amounts, and using them as templates for subsequent PCR amplification, performing PCR, and after the PCR products are detected by electrophoresis, using silver staining to develop and fix the images, and taking pictures with a digital camera after the bands are clearly colored, and determining the optimal annealing temperature, effectiveness and polymorphism of each primer; At the same time, several equal samples are randomly selected from the fin tissue samples of different geographical groups of long-snouted catfish corresponding to step b, and DNA is extracted respectively for primer universality detection and genetic diversity analysis of different breeding groups. If the primer amplification obtains a PCR product of the expected size and has more than one band, it proves that the primer is polymorphic.

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