CN111455088B - Fusarium layering SSR molecular marker and application thereof - Google Patents

Abstract

Fusarium SSR molecular markers and application thereof belong to the technical field of DNA molecular markers. The fusarium SSR molecular marker is obtained by detecting the distribution of the fusarium SSR according to genome data of the fusarium, designing primers, and carrying out primary screening and secondary screening to obtain 12 SSR molecular markers with abundant polymorphism and different simple sequence repeating units. The invention has the beneficial effects that: based on genome sequence information of the layered fusarium, 12 SSR molecular markers which are different from the former and have high polymorphism and good repeatability are obtained, so that the molecular markers are very effective, and the genetic diversity of the layered fusarium and the population genetic structure of the layered fusarium can be systematically revealed.

Description

Fusarium layering SSR molecular marker and application thereof

Technical Field

The invention belongs to the technical field of DNA molecular markers, and particularly relates to a Fusarium layering SSR molecular marker and application thereof.

Background

Fusarium layeringFusarium proliferatum) Is a pathogenic bacteria of plant diseases worldwide, has wide host range, and can cause rice ear rot, corn ear rot, soybean root rot, mango malformation, eggplant flower rot, tomato leaf blight and the like. The pathogenic bacteria not only can reduce crop yield, but also can produce various mycotoxins (such as fumonisin, fusarin, fusaric acid and the like), has strong pathogenicity, teratogenicity and carcinogenicity, and can cause serious threat to the life health of human and animal. At present, research on Fusarium layering at home and abroad is focused on aspects of pathogen identification classification, biological characteristics, toxin formation and the like, and little is known about genetic diversity and population genetic characteristics of the Fusarium layering. The microbial systems in all the places are affected by climate, soil conditions and the like, so that fusarium groups in all the strata are not completely identical. Therefore, it is necessary to develop suitable molecular markers, systematically study genetic structure and genetic differentiation of Fusarium layering, understand the relationship between Fusarium layering genetic variation and factors such as geography, climate, variety and the like and related evolution rules, and provide theoretical basis for formulating scientific and reasonable disease control measures.

Simple repeated sequences (SSR, simple sequence repeats), also known as microsatellite loci, are DNA sequences consisting of 1-6 base sequences in tandem and are commonly distributed in the genome of eukaryotes. SSR markers are commonly used molecular biological markers, and the principle is as follows: because of different repetition numbers due to different alleles, specific primers can be designed according to conserved sequences at two sides of the repeated sequences to carry out PCR amplification. The SSR molecular marker has the advantages of good repeatability, high polymorphism, co-dominant inheritance and the like, and is widely applied to research fields of species evolution, genetic map construction, gene positioning, genetic diversity, molecular auxiliary breeding and the like. However, traditional microsatellite development approaches are time consuming and inefficient, mainly by constructing genomic libraries. With the development of sequencing technology, the repeated sequence fragment information is obtained by using a high-throughput sequencing technology, so that the efficiency of SSR marker development is remarkably improved. However, until now, development and utilization of SSR molecular markers layered in fusarium genome have been little studied, and only Monnief developed a report of 6 pairs of polymorphic SSR primers from the simple sequence repeat spacer (ISSR) (Monnief et al, development of Simple Sequence Repeat (SSR) markers for discrimination among isolates ofFusarium proliferatumJournal of Microbiological Methods, 2016, 126:12-17), which is far from sufficient for the stratification of genetic variation of Fusarium. Since the research of the genome sequencing of the fusarium is started later, no report of developing SSR markers by using the genome sequence of the fusarium is found. Therefore, the invention develops the SSR molecular marker with abundant fusarium polymorphism according to the fusarium complete genome sequence information.

Disclosure of Invention

Aiming at the current situation that the fusarium microsatellite marker is not developed by using the genome sequence of fusarium, the invention aims to design and provide the SSR molecular marker of fusarium and the application thereof. The invention develops the molecular marker of the fusarium oxysporum microsatellite with polymorphism by applying a bioinformatics method, establishes a technical system of the fusarium oxysporum microsatellite, and provides a powerful research tool for genetic diversity and population genetic research of the fusarium oxysporum.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the fusarium SSR molecular marker is characterized by comprising 12 SSR molecular markers which are rich in polymorphism and contain different simple sequence repeating units, and specifically comprises FP03, wherein the nucleotide sequence is SEQ ID NO.1; FP08, nucleotide sequence of SEQ ID NO.2; FP13 with the nucleotide sequence of SEQ ID NO.3; FP21 with the nucleotide sequence of SEQ ID NO.4; FP24 with the nucleotide sequence of SEQ ID NO.5; FP26 with the nucleotide sequence of SEQ ID NO.6; FP31 with the nucleotide sequence of SEQ ID NO.7; FP32 with the nucleotide sequence of SEQ ID NO.8; FP35 with the nucleotide sequence of SEQ ID NO.9; FP43 with the nucleotide sequence of SEQ ID NO.10; FP48 with the nucleotide sequence of SEQ ID NO.11; FP51 has the nucleotide sequence of SEQ ID NO.12.

The fusarium layered SSR molecular marker is characterized in that 12 pairs of primers for amplifying the 12 SSR molecular markers containing different simple sequence repeating units comprise: the nucleotide sequences of the primers of the amplified FP03 are SEQ ID NO.13 and SEQ ID NO.14, the nucleotide sequences of the primers of the amplified FP08 are SEQ ID NO.15 and SEQ ID NO.16, the nucleotide sequences of the primers of the amplified FP13 are SEQ ID NO.17 and SEQ ID NO.18, the nucleotide sequences of the primers of the amplified FP21 are SEQ ID NO.19 and SEQ ID NO.20, the nucleotide sequences of the primers of the amplified FP24 are SEQ ID NO.21 and SEQ ID NO.22, the nucleotide sequences of the primers of the amplified FP26 are SEQ ID NO.23 and SEQ ID NO.24, the nucleotide sequences of the primers of the amplified FP31 are SEQ ID NO.25 and SEQ ID NO.26, the nucleotide sequences of the primers of the amplified FP32 are SEQ ID NO.27 and SEQ ID NO.28, the nucleotide sequences of the primers of the amplified FP35 are SEQ ID NO.29 and SEQ ID NO.30, the nucleotide sequences of the primers of the amplified FP43 are SEQ ID NO.31 and SEQ ID NO.32, the nucleotide sequences of the primers of the amplified FP48 are SEQ ID NO.33 and SEQ ID NO.34, and the nucleotide sequences of the primers of the amplified FP51 are SEQ ID NO.35 and SEQ ID NO.36.

The application of the SSR molecular marker in the genetic diversity analysis of fusarium layering.

The application of the SSR molecular marker in the genetic diversity analysis of fusarium, comprises the following steps:

(1) Collecting, separating and identifying fusarium;

(2) Extracting the genome DNA of fusarium strains from the layer;

(3) Performing PCR amplification on the genome DNA obtained in the step (2) to obtain a PCR amplification product;

(4) SSR genotyping: diluting the PCR amplification product obtained in the step (3) by 30 times, then carrying out denaturation, loading the sample to a full-automatic gene sequencer for fragment analysis, reading data by using software GeneMapper v3.25, and determining the genotype according to the size of the separated fragment;

(5) Analysis of genetic diversity.

The application of the SSR molecular marker in the genetic diversity analysis of fusarium, which is characterized in that the PCR reaction system in the step (3) is as follows: 50 ng/. Mu.L of template DNA 1. Mu.L, mgCl 2.5. Mu.L of 25 mM, 2.5 mM dNTPs 1. Mu.L, 10. Mu.M forward and reverse primers each 1. Mu.L, 5U/. Mu.L of DNA Taq enzyme 0.2. Mu.L, 10 XPCR buffer 2.5. Mu.L, deionized water to 25. Mu.L, the forward 5' end of the primers carrying a blue 6-FAM or green HEX specific fluorescent label for amplifying the size of the band of interest, the PCR amplification procedure being: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, annealing at 55℃for 20s, extension at 72℃for 30s, total of 35 cycles, extension at 72℃for 10min.

The application of the SSR molecular marker in the genetic diversity analysis of fusarium, which is characterized in that the genetic diversity analysis method in the step (5) comprises the following steps: the method comprises the steps of calculating and observing an allelic factor Na, an effective allelic factor Ne, shannon's diversity index I, an observed heterozygosity Ho, an expected heterozygosity He, an inter-population Nei's genetic distance D and a genetic similarity I by using POPGENE version1.32, calculating the percentage of total variation occupied by different variation levels among individuals in the population, the population and the individuals by using Alequin3.1 software, calculating the statistical variance component and the contribution rate, clearly causing the main source of the total variation of the population, calculating the genetic differentiation coefficient FST among the populations, estimating a gene flow Nm according to FST=1/(4Nm+1), constructing a non-weighted group average method UPGMA cluster map of the population based on the Nei's genetic distance, carrying out inter-population genetic Structure analysis by using a Bayesian cluster method of Structure V2.3.4 software, and drawing a generated population genetic Structure diagram by using DISTRUCT software.

The invention has the beneficial effects that: based on genome sequence information of the layered fusarium, 12 SSR molecular markers which are different from the former and have high polymorphism and good repeatability are obtained, so that the molecular markers are very effective, and the genetic diversity of the layered fusarium and the population genetic structure of the layered fusarium can be systematically revealed.

Drawings

FIG. 1 is SSR genotype of Fusarium layering FP 03;

FIG. 2 is SSR genotype of Fusarium layering FP 08;

FIG. 3 is SSR genotype of Fusarium layering FP 13;

FIG. 4 is SSR genotype of Fusarium layering FP 21;

FIG. 5 is SSR genotype of Fusarium layering FP 24;

FIG. 6 is SSR genotype of Fusarium layering FP 26;

FIG. 7 is SSR genotype of Fusarium layering FP 31;

FIG. 8 is SSR genotype of Fusarium layering FP 32;

FIG. 9 is SSR genotype of Fusarium layering FP 35;

FIG. 10 is SSR genotype of Fusarium layering FP 43;

FIG. 11 is SSR genotype of Fusarium layering FP 48;

FIG. 12 is SSR genotype of Fusarium layering FP 51;

FIG. 13 is a UPGMA cluster map of Fusarium layering populations based on Nei's genetic distance;

FIG. 14 is a genetic block diagram of Fusarium layering populations based on a STRUCTURE analysis.

Detailed Description

For the purpose of illustrating the technical content and structural features of the fusarium SSR molecular marker in detail, the following description is made in conjunction with the embodiments and the accompanying drawings, and it is to be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: fusarium layering whole genome SSR scanning and primer design

In view of availability, representativeness and importance of the data, the whole genome sequence of Fusarium ET1 strain (https:// www.ncbi.nlm.nih.gov/genome/2434) is downloaded from NCBI database, the SSR sequence is searched by using SSR Hunter software, 2-6 nucleotides are selected as repeated motifs, and the number of repetitions is at least 6. SSR Primer design was performed with Primer 5 software using the detected SSR and 150bp flanking sequences upstream and downstream thereof. The general principle of primer design is: the primer length is 19-25 bp; GC content is 40% -60%; the Tm value of the annealing temperature is 52-58 ℃, and the difference between the Tm values of the upstream primer and the downstream primer is not more than 5 ℃; the PCR amplification length is 100-300 bp; primer dimers, hairpin structures, mismatches, etc. are avoided as much as possible. The synthetic primer carries a specific fluorescent label of 6-FAM (blue) or HEX (green) at the 5' end of the forward primer for amplifying the size of the target band, and the fluorescent primer is synthesized by biological engineering (Shanghai) Co., ltd.

Example 2: screening of SSR molecular markers

In the results of the above genome SSR scan and primer design, 52 pairs of SSR primers were then picked and their specificity in the genome was detected by local Blast. After pre-experiments, the fragment length of the amplified product is 100-300 bp, and the amplified product has 12 microsatellite loci with diversity and contains different simple sequence repeating units (Table 1). SSR genotypes of fusarium falcatum FP03, FP08, FP13, FP21, FP24, FP26, FP31, FP32, FP35, FP43, FP48 and FP51 are shown in fig. 1, fig. 2, fig. 3, fig. 4, fig. 5, fig. 6, fig. 7, fig. 8, fig. 9, fig. 10, fig. 11 and fig. 12, respectively. The primers of the 12 SSR sites all show polymorphism, and provide markers for analysis of genetic diversity of fusarium.

Table 1 shows 12 SSR molecular markers of Fusarium

Example 3: application of SSR molecular markers in genetic diversity of fusarium layering

Step 1: fusarium layering collection, separation and identification

The present example uses Fusarium layering as samples collected from 3 populations (FY 03, FY08, FY 13) in Hangzhou, zhejiang and 2 populations (NN 05, NN 12) in Guangxi nan. Fusarium is separated from the infected sample of rice spike rot. Specific primers, EF1 (5'-ATGGGTAAGGAGGACAAGAC-3') and EF2 (5'-GGAAGTACCAGTGATCATGTT-3'), were designed based on the conserved sequence of the protein translational Elongation Factor (EF) gene, and PCR amplification was performed on the strain. Sequencing the PCR amplified product, submitting the sequence in Fusarium TEF sequence database (FUSARIUM-ID v 1.0) for alignment, and finally selecting the Fusarium through sequence alignment.

Step 2: extracting layer to obtain genome DNA of Fusarium strain

Extracting the layer by adopting a modified CTAB method to obtain fusarium genome DNA.

(1) 0.2g of fusarium mycelium sample is taken, added with liquid nitrogen, fully ground into powder, and filled into a centrifuge tube.

(2) 600 μLCTAB extract (100 mM Tris-HCl,700 mMNaCl,10 mMEDTA,1% CTAB,1% beta-mercaptoethanol, pH=8) was added, mixed upside down, and placed in a 65℃water bath for 40 min, during which the centrifuge tube was turned 1 time every 10min.

(3) An equal volume (600 μl) of chloroform was added: isoamyl alcohol (24:1), and is evenly mixed by turning over, and centrifuged at 12000 rpm and 4 ℃ for 10min.

(4) The upper aqueous phase was collected and transferred to a new centrifuge tube.

(5) An equal volume of chloroform was added: isoamyl alcohol (24:1), and is evenly mixed by turning over, and centrifuged at 12000 rpm and 4 ℃ for 10min.

(6) The upper aqueous phase was collected and transferred to a new centrifuge tube.

(7) 2.5 volumes of pre-chilled isopropanol were added and centrifuged at 12000 rpm at 4℃for 10min.

(8) The supernatant was carefully decanted, 1 mL of 75% ethanol was added, and the mixture was centrifuged for 5 min and repeated twice.

(9) And (5) drying at room temperature.

(10) Adding 30 μl of sterile water, dissolving at room temperature, and preserving at-20deg.C.

Step 3: PCR amplification

The PCR reaction system is as follows: 50 ng/. Mu.L of template DNA 1. Mu.L of MgCl 25 mM ₂ 2.5 mu.L, 2.5 mM dNTPs 1. Mu.L, 10. Mu.M forward and reverse primers 1. Mu.L, 5. 5U/. Mu.L DNA, respectivelyTaqEnzyme 0.2. Mu.L, 10 XPCR buffer 2.5. Mu.L, deionized water was added to 25. Mu.L. The forward 5' end of the primer carries a 6-FAM (blue) or HEX (green) specific fluorescent label for amplifying the size of the target band. The PCR amplification procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 55℃for 20s, extension at 72℃for 30s for 35 cycles; extending at 72℃for 10min.

Step 4: SSR genotyping

The PCR amplified product was denatured after 30-fold dilution, loaded onto ABI3730XL full-automatic gene sequencer for fragment analysis, data read using software GeneMapper v3.25, and genotype was determined according to the size of the isolated fragment.

Step 5: analysis of genetic diversity

(1) Genetic diversity of SSR sites

Calculation of observed allele factors for each SSR site using POPGENE version1.32 [ ]Na) Effective allelic factors [ ]Ne) Shannon's diversity indexI) Observing heterozygosityHo) Desired heterozygosity [ ]He）。

As a result, it was found that the allele factor of 12 SSR sites [ ]Na) 4-12; effective allelic factors [ (]Ne) Between 1.484 and 3.865 (Table 2). Wherein, the allelic factors of the FP03 and FP21 loci are at most [ ]Na=12), the effective allele factor of the FP21 locus is the highest [ ]Ne=3.865). Shannon information index [ ]I) Observing the heterozygosity between 0.693 and 1.587Ho) between 0.333 and 0.836, the desired heterozygosity [ ]He) Between 0.389 and 0.857.

TABLE 2 genetic diversity of SSR loci in Fusarium on layer populations

(2) Fusarium layering population genetic diversity level

Observations were calculated for each population using POPGENE version1.32Allelic factors [ ]Na) Effective allelic factors [ ]Ne) Shannon's diversity indexI) Observing heterozygosityHo) Desired heterozygosity [ ]He). Average allelic factors of 5 layers of Fusarium populationNa) Effective allelic factors [ ]Ne) And Shannon's information index [ ]I) Observing heterozygosityHo) and the desired degree of heterozygosityHe) 5.07, 2.414, 1.014, 0.51 and 0.518, respectively (table 3). Wherein, NN05 population genetic diversity is most abundantNa=5.93，Ne=2.83，I= 1.21), the minimum FY03 population%Na=4.62，Ne=2.08，I=0.87）。

TABLE 3 estimation of genetic diversity of Fusarium populations

(3) Genetic differentiation and gene communication between Fusarium layering populations

Calculating genetic differentiation coefficient between groups by Arlequin 3.1 softwareF _ST ). According toF _ST =1/（4Nm+1) estimation of Gene flowNm). Genetic differentiation coefficient between two populationsF _ST ) The variation range is 0.015-0.237, and the gene flow is @Nm) 0.80 to 16.42 (Table 4). The gene flow between groups of different regions from the same rich region (FY) is high (FY:Nm=16.42）。

TABLE 4 genetic differentiation coefficients between Fusarium populations @F _ST Under diagonal) and gene flowNmOn the diagonal line)

(4) Molecular analysis of variance of Fusarium layering populations

Calculation of inter-population, inter-population individuals and individuals using molecular variance analysis of variance (AMOVA) with aleucin 3.1 softwareThe different levels of variation between the bodies are percentages of the total variation. AMOVA analysis shows that obvious genetic variation exists in the groups of 5 layers of FusariumP< 0.001) (table 5). The genetic variation among the populations accounts for 12.85% of the total variation, and the individuals among the populations account for 87.15%, which means that the genetic variation is mainly distributed among fusarium individuals.

TABLE 5 molecular variance analysis of Fusarium populations

(5) Genetic distance and cluster analysis between Fusarium layering populations

Between 5 populationsNei’sGenetic distance [ ]D) 0.018-0.396, and genetic similarity is calculated as #I) Between 0.673 and 0.982 (Table 6). Wherein, the genetic relationship between FY13 and NN05 groups is the farthest, and the genetic distance is the largestD=0.396), the lowest genetic similarityI=0.673); and the genetic distance between FY03 and FY08 groups is minimizedD=0.018), the highest genetic similarityI=0.982), indicating that the relationship between populations is closest.

Based on 5 populationsNei’sGenetic distance, UPGMA cluster tree is constructed for the Fusarium layering group (figure 13), hangzhou groups FY03 and FY08 are clustered preferentially, and the Fusarium layering group and Hangzhou group FY13 are clustered together, and then the Fusarium layering group and the Hangzhou group are clustered with Guangxi nan Ning NN12 and NN05 groups sequentially. It can be seen that the difference between the populations in the same region is small, and the regions have a certain genetic difference.

TABLE 6 Fusarium species groupNei’sGenetic distance [ ]DUnder diagonal) and genetic similarityIOn the diagonal line)

(6) Genetic structural analysis of Fusarium layering populations

Inter-population genetic Structure analysis was performed using bayesian clustering with Structure V2.3.4 software, with burn-in 100 000 times and run-length 1 000 000 times of iterations, and the generated population genetic Structure was drawn with DISTRUCT software (fig. 14). Inter-individual genetic shuffling was evident from different populations, where the fusarium layering populations (NN 05, NN 12) from nanning were significantly different from the Hangzhou populations (FY 03, FY08 and FY 13), consistent with UPGMA analysis results.

The invention develops 12 SSR markers based on genome sequence information of fusarium, has the characteristics of high polymorphism and good repeatability, is a reliable and effective molecular marker, and can study genetic diversity of fusarium and genetic structure of fusarium population.

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications could be made by those skilled in the art without departing from the principles of the invention, which modifications would also be considered to be within the scope of the invention.

Sequence listing

SEQ ID NO.1

attgtctcgctcgtcgtcaatgcaacctagatacttatggctgagctcacaagtctgaagtgacgatgaatcttatctaaagatagagtcagtgctatctttgcctttcattcacgcaacattcaacgaaaatttagtaagacagacgctTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCtttcattcatagataataaacgtaaaaatcggcctataaggagatagccgcgagttcgtcttgggagattgagacgacttgtaaggagtatatcaccagacactcaacaagcagcacaacttgttggcataattggtttctgttattaga

SEQ ID NO.2

tccctcgttgtaatcaacgctaccttccaccacgtatccgccttggagatacttcggagatagctctgctcgaagcgcagatctggcaaaggaggtaggtgggaatgcctgggcgccggtcggagaacgctgtaggggatgtcctcttcaTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGtcaattccaccatcgtgctcgtgcttgacgtgctctagggtcacaggctgtcttctctcggctgctgtgggcgggttgatatcaagagcaggatccggatggcgttcgatgctggcagcctggatagttgcggcaagctctgtgggagct

SEQ ID NO.3

tgtctccaggaaccatctatgccaactgatccagcgccactactgtacgtgcccagaagaggcgcttcacatgccgtcctgccttgatactctctaaagagtagaggaggcgacgcctgatgacgatgatggaatcgaccgaatggcaatTCATCATCATCATCATCATCATCATCATCAtcgagaggtggttttgcggcgtaggcatgtatgatatacgcaagggactcgatgttggccagccttaccataaccagctgcagcagtacgtgttgatgtctctttccacacccggaacaactccgcatagcacaaaattgcacacctcac

SEQ ID NO.4

cagatgcatagtgatcaatatcgatgtcactggcaaatacaataagtcttgataccaagaccggagacatcttgttgccgccgactccgggtctaagttaggtatttctacccctgctttcaatgccaagactcgccatctctttcagccCTTCTTCTTCTTCTTCTTCTTCTTcatagtaagaagaagaatgcccagtatctgagtatgtcactcataaggtccctgtcattcagcgagcccgtctccgtggccgttggccgcttactcccctgtcatcaatcgtcctgcgccttcggggatttctgtattaagcatattcgt

SEQ ID NO.5

tctcgagctactgggccaatgggcacagctgtaatgggaacatcaagttccgagaatggcgtaccatggcggaagctgttcctaggtgttatctccagatcaggagatcgagggctggtcttctccatctggtaagggacaaaggcgggcTGCTGTTGCTGTTGCTGTTGCTGTTGCTGTTGCTGTTGCTGTTGCTGTtgcaattgctgttgatgttgggcaaatgactgcgtgggcttcttctgagggggaggcaccgaagaagcaccaagacgggacgggggaagctggggcttcccaggaatggacagtttagggagtcggcgcagaccttggtgaggaagctgg

SEQ ID NO.6

ggtcaatactcacataataagcccatttcccagtacgagaagaaaatcaacgataaacatgacgatgagaagaattatatcaaagtgatcaaggatcgcgtctcgcagcacaagccaactcttcataatgagaagccggatcatcatcacCATCATCATCATCATCATCATCATgacaaggataatgagcatgctaagagacccaagcctcatcataaccatcaagaggctgagcaccatggggactctaagaaatcagaggactactacccttacaacaaaccgaaacaccaattcgaggagcaatctaagcacaatcacgac

SEQ ID NO.7

ctgtttgaaagaccaaagtttgtttgtcgtcacgggcaaaaaggccacgagtacaaggtctcggccgattacagtcaaatagagtaatttcaatcgatgttgaagagttggatagtcgcaatgaatacgttacaatctggaagaggttaaGGAATTGGAATTGGAATTGGAATTGGAATTGGAATTgatgcgatgacggagatataagagaatagagttatcgcgtagtgtcctactttaaatctaacgtacctatcggcagacaaaagggttaaagcacctggataagcagaacacagccgctaactccactcacctgcttcagaaactggggga

SEQ ID NO.8

gatatccaggcggcggtcaacgcgaccagtacgatgatggttatggccatcaacagggaggcaaccagcaacaacacggtggaaacactgactcctactaccaagacgatcagtactacgatcaaggctacgacaaccgtggcccaaatcACAACAACAACAACAACAACAACAaccatgatggttactacgacgaatcgtgagcatcatgtccctcctctcatagtcctttcactaacacactagtagtggttactacaacgccgaccccaataacccctaccagcaggatggaggctattacgacggccacgatcaatacca

SEQ ID NO.9

caagatttcgagaaagagatggaggatatatataatgaagagaatggtggtcctgccgattcctcaaagctttcaccgaaacgccccgatcacctacgcattccaagtattaaccatgatctagatgctgctgttgaggttgaagacgatAGCAGCAGCAGCAGCAGCAGCagtgacagtaacgcagaagtggatagtactattatcaatcaatcggaggataaggtgaaacatgagctggacgtgaccggttatggtcctgaccgttggaagactgccgagaaatccggccttgatgccggccgcgattctgttatcatc

SEQ ID NO.10

ctaggtgcggtcgagttgccaaggccttttggtgtagcggagttagttgtattgttggagccgagttctattgtctttcgagtcgagtcaggtatgatatcgtttcgatcgttctagtcgtgtctcgtcgcaatcgggtgcaaggtgtgtGCGTGAGCGTGAGCGTGAGCGTGAGCGTGAGCGTGAGCGTGAgcgtatgtgtttgtattataatatggaggtgatgatcgatgctgtaacgtaatgcaatgtgatgtgatgtgacgcttcttgggggtccgagtttctggccagatgatgataatttgtaatgctgagggctcagccggcagttccctttag

SEQ ID NO.11

tcttttcaagttttctgaagcgaacacaaagtctgttgagatgacgagtgaatacgacgcattggacaacatcatccaggatgccgttcctgggctaggatctcgaaagcaagacagaaaagggcggaacgtccaggaaggaggaagaagAGGAGCAGGAGCAGGAGCAGGAGCAGGAGCAGGAGCAGGAGCaggaagtgatggcccgtttggaaacagtcatcaaacaacaggctcgtacaaatctctggtggaacctagtgttttcaatatgagcttgttactaccgccaacgttggtcttcctgcagcgtctcaagaacatcgtccctcccggctcaga

SEQ ID NO.12

cacagcggcgttggtattgctgctttgcgactggagagcgactttgaagaggttgggtcgagatgctccatgtatactttgtgatgtggtagatgagaccatagtctacctatgtcagtgatcaagccatggcggagcggtcgcagcaaaACACACACACACACACACACACACACctttgtcttgctgtagagcttagagccgtcgccaagggcatcattctcatcgttgacggacatgcgctcgaagcgcgcctcaattgcacgggtagccattattgggagcaagggcgggatggactgttaatgttagtgaaagacgaacga

SEQ ID NO.13

TCATTCACGCAACATTC

SEQ ID NO.14

ATCTCCCAAGACGAACT

SEQ ID NO.15

TCTGGCAAAGGAGGTAGG

SEQ ID NO.16

AAGACAGCCTGTGACCCTA

SEQ ID NO.17

AGAAGAGGCGCTTCACA

SEQ ID NO.18

AACATCGAGTCCCTTGC

SEQ ID NO.19

TGCCAAGACTCGCCATCT

SEQ ID NO.20

ACGGGCTCGCTGAATGA

SEQ ID NO.21

CCATCTGGTAAGGGACAA

SEQ ID NO.22

AAGAAGCCCACGCAGTC

SEQ ID NO.23

ATAAGCCCATTTCCCAGTA

SEQ ID NO.24

ATGGTGCTCAGCCTCTT

SEQ ID NO.25

GTTGGATAGTCGCAATG

SEQ ID NO.26

GGTGCTTTAACCCTTTT

SEQ ID NO.27

CGGTGGAAACACTGACT

SEQ ID NO.28

TAATAGCCTCCATCCTG

SEQ ID NO.29

GGTCCTGCCGATTCCTC

SEQ ID NO.30

CCTCCGATTGATTGATA

SEQ ID NO.31

GGAGCCGAGTTCTATTG

SEQ ID NO.32

CGATCATCACCTCCATAT

SEQ ID NO.33

GACGCATTGGACAACAT

SEQ ID NO.34

ATCACTTCCTGCTCCTG

SEQ ID NO.35

CGTTGGTATTGCTGCTT

SEQ ID NO.36

CCGTCAACGATGAGAAT

Sequence listing

<110> China institute of Rice

<120> Fusarium layering SSR molecular marker and application thereof

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 333

<212> DNA

<213> FP03(FP03)

<400> 1

attgtctcgc tcgtcgtcaa tgcaacctag atacttatgg ctgagctcac aagtctgaag 60

tgacgatgaa tcttatctaa agatagagtc agtgctatct ttgcctttca ttcacgcaac 120

attcaacgaa aatttagtaa gacagacgct ttcttcttct tcttcttctt cttcttcttc 180

ttctttcatt catagataat aaacgtaaaa atcggcctat aaggagatag ccgcgagttc 240

gtcttgggag attgagacga cttgtaagga gtatatcacc agacactcaa caagcagcac 300

aacttgttgg cataattggt ttctgttatt aga 333

<210> 2

<211> 333

<212> DNA

<213> FP08(FP08)

<400> 2

tccctcgttg taatcaacgc taccttccac cacgtatccg ccttggagat acttcggaga 60

tagctctgct cgaagcgcag atctggcaaa ggaggtaggt gggaatgcct gggcgccggt 120

cggagaacgc tgtaggggat gtcctcttca tcgtcgtcgt cgtcgtcgtc gtcgtcgtcg 180

tcgtcaattc caccatcgtg ctcgtgcttg acgtgctcta gggtcacagg ctgtcttctc 240

tcggctgctg tgggcgggtt gatatcaaga gcaggatccg gatggcgttc gatgctggca 300

gcctggatag ttgcggcaag ctctgtggga gct 333

<210> 3

<211> 330

<212> DNA

<213> FP13(FP13)

<400> 3

tgtctccagg aaccatctat gccaactgat ccagcgccac tactgtacgt gcccagaaga 60

ggcgcttcac atgccgtcct gccttgatac tctctaaaga gtagaggagg cgacgcctga 120

tgacgatgat ggaatcgacc gaatggcaat tcatcatcat catcatcatc atcatcatca 180

tcgagaggtg gttttgcggc gtaggcatgt atgatatacg caagggactc gatgttggcc 240

agccttacca taaccagctg cagcagtacg tgttgatgtc tctttccaca cccggaacaa 300

ctccgcatag cacaaaattg cacacctcac 330

<210> 4

<211> 324

<212> DNA

<213> FP21(FP21)

<400> 4

cagatgcata gtgatcaata tcgatgtcac tggcaaatac aataagtctt gataccaaga 60

ccggagacat cttgttgccg ccgactccgg gtctaagtta ggtatttcta cccctgcttt 120

caatgccaag actcgccatc tctttcagcc cttcttcttc ttcttcttct tcttcatagt 180

aagaagaaga atgcccagta tctgagtatg tcactcataa ggtccctgtc attcagcgag 240

cccgtctccg tggccgttgg ccgcttactc ccctgtcatc aatcgtcctg cgccttcggg 300

gatttctgta ttaagcatat tcgt 324

<210> 5

<211> 348

<212> DNA

<213> FP24(FP24)

<400> 5

tctcgagcta ctgggccaat gggcacagct gtaatgggaa catcaagttc cgagaatggc 60

gtaccatggc ggaagctgtt cctaggtgtt atctccagat caggagatcg agggctggtc 120

ttctccatct ggtaagggac aaaggcgggc tgctgttgct gttgctgttg ctgttgctgt 180

tgctgttgct gttgctgttg caattgctgt tgatgttggg caaatgactg cgtgggcttc 240

ttctgagggg gaggcaccga agaagcacca agacgggacg ggggaagctg gggcttccca 300

ggaatggaca gtttagggag tcggcgcaga ccttggtgag gaagctgg 348

<210> 6

<211> 324

<212> DNA

<213> FP26(FP26)

<400> 6

ggtcaatact cacataataa gcccatttcc cagtacgaga agaaaatcaa cgataaacat 60

gacgatgaga agaattatat caaagtgatc aaggatcgcg tctcgcagca caagccaact 120

cttcataatg agaagccgga tcatcatcac catcatcatc atcatcatca tcatgacaag 180

gataatgagc atgctaagag acccaagcct catcataacc atcaagaggc tgagcaccat 240

ggggactcta agaaatcaga ggactactac ccttacaaca aaccgaaaca ccaattcgag 300

gagcaatcta agcacaatca cgac 324

<210> 7

<211> 336

<212> DNA

<213> FP31(FP31)

<400> 7

ctgtttgaaa gaccaaagtt tgtttgtcgt cacgggcaaa aaggccacga gtacaaggtc 60

tcggccgatt acagtcaaat agagtaattt caatcgatgt tgaagagttg gatagtcgca 120

atgaatacgt tacaatctgg aagaggttaa ggaattggaa ttggaattgg aattggaatt 180

ggaattgatg cgatgacgga gatataagag aatagagtta tcgcgtagtg tcctacttta 240

aatctaacgt acctatcggc agacaaaagg gttaaagcac ctggataagc agaacacagc 300

cgctaactcc actcacctgc ttcagaaact ggggga 336

<210> 8

<211> 324

<212> DNA

<213> FP32(FP32)

<400> 8

gatatccagg cggcggtcaa cgcgaccagt acgatgatgg ttatggccat caacagggag 60

gcaaccagca acaacacggt ggaaacactg actcctacta ccaagacgat cagtactacg 120

atcaaggcta cgacaaccgt ggcccaaatc acaacaacaa caacaacaac aacaaccatg 180

atggttacta cgacgaatcg tgagcatcat gtccctcctc tcatagtcct ttcactaaca 240

cactagtagt ggttactaca acgccgaccc caataacccc taccagcagg atggaggcta 300

ttacgacggc cacgatcaat acca 324

<210> 9

<211> 321

<212> DNA

<213> FP35(FP35)

<400> 9

caagatttcg agaaagagat ggaggatata tataatgaag agaatggtgg tcctgccgat 60

tcctcaaagc tttcaccgaa acgccccgat cacctacgca ttccaagtat taaccatgat 120

ctagatgctg ctgttgaggt tgaagacgat agcagcagca gcagcagcag cagtgacagt 180

aacgcagaag tggatagtac tattatcaat caatcggagg ataaggtgaa acatgagctg 240

gacgtgaccg gttatggtcc tgaccgttgg aagactgccg agaaatccgg ccttgatgcc 300

ggccgcgatt ctgttatcat c 321

<210> 10

<211> 342

<212> DNA

<213> FP43(FP43)

<400> 10

ctaggtgcgg tcgagttgcc aaggcctttt ggtgtagcgg agttagttgt attgttggag 60

ccgagttcta ttgtctttcg agtcgagtca ggtatgatat cgtttcgatc gttctagtcg 120

tgtctcgtcg caatcgggtg caaggtgtgt gcgtgagcgt gagcgtgagc gtgagcgtga 180

gcgtgagcgt gagcgtatgt gtttgtatta taatatggag gtgatgatcg atgctgtaac 240

gtaatgcaat gtgatgtgat gtgacgcttc ttgggggtcc gagtttctgg ccagatgatg 300

ataatttgta atgctgaggg ctcagccggc agttcccttt ag 342

<210> 11

<211> 342

<212> DNA

<213> FP48(FP48)

<400> 11

tcttttcaag ttttctgaag cgaacacaaa gtctgttgag atgacgagtg aatacgacgc 60

attggacaac atcatccagg atgccgttcc tgggctagga tctcgaaagc aagacagaaa 120

agggcggaac gtccaggaag gaggaagaag aggagcagga gcaggagcag gagcaggagc 180

aggagcagga gcaggaagtg atggcccgtt tggaaacagt catcaaacaa caggctcgta 240

caaatctctg gtggaaccta gtgttttcaa tatgagcttg ttactaccgc caacgttggt 300

cttcctgcag cgtctcaaga acatcgtccc tcccggctca ga 342

<210> 12

<211> 326

<212> DNA

<213> FP51(FP51)

<400> 12

cacagcggcg ttggtattgc tgctttgcga ctggagagcg actttgaaga ggttgggtcg 60

agatgctcca tgtatacttt gtgatgtggt agatgagacc atagtctacc tatgtcagtg 120

atcaagccat ggcggagcgg tcgcagcaaa acacacacac acacacacac acacaccttt 180

gtcttgctgt agagcttaga gccgtcgcca agggcatcat tctcatcgtt gacggacatg 240

cgctcgaagc gcgcctcaat tgcacgggta gccattattg ggagcaaggg cgggatggac 300

tgttaatgtt agtgaaagac gaacga 326

<210> 13

<211> 17

<212> DNA

<213> FP03 Forward primer (FP 03 forward primer)

<400> 13

tcattcacgc aacattc 17

<210> 14

<211> 17

<212> DNA

<213> FP03 reverse primer (FP 03 reverse primer)

<400> 14

atctcccaag acgaact 17

<210> 15

<211> 18

<212> DNA

<213> FP08 forward primer (FP 08 forward primer)

<400> 15

tctggcaaag gaggtagg 18

<210> 16

<211> 19

<212> DNA

<213> FP08 reverse primer (FP 08 reverse primer)

<400> 16

aagacagcct gtgacccta 19

<210> 17

<211> 17

<212> DNA

<213> FP13 forward primer (FP 13 forward primer)

<400> 17

agaagaggcg cttcaca 17

<210> 18

<211> 17

<212> DNA

<213> FP13 reverse primer (FP 13 reverse primer)

<400> 18

aacatcgagt cccttgc 17

<210> 19

<211> 18

<212> DNA

<213> FP21 forward primer (FP 21 forward primer)

<400> 19

tgccaagact cgccatct 18

<210> 20

<211> 17

<212> DNA

<213> FP21 reverse primer (FP 21 reverse primer)

<400> 20

acgggctcgc tgaatga 17

<210> 21

<211> 18

<212> DNA

<213> FP24 forward primer (FP 24 forward primer)

<400> 21

ccatctggta agggacaa 18

<210> 22

<211> 17

<212> DNA

<213> FP24 reverse primer (FP 24 reverse primer)

<400> 22

aagaagccca cgcagtc 17

<210> 23

<211> 19

<212> DNA

<213> FP26 forward primer (FP 26 forward primer)

<400> 23

ataagcccat ttcccagta 19

<210> 24

<211> 17

<212> DNA

<213> FP26 reverse primer (FP 26 reverse primer)

<400> 24

atggtgctca gcctctt 17

<210> 25

<211> 17

<212> DNA

<213> FP31 forward primer (FP 31 forward primer)

<400> 25

gttggatagt cgcaatg 17

<210> 26

<211> 17

<212> DNA

<213> FP31 reverse primer (FP 31 reverse primer)

<400> 26

ggtgctttaa ccctttt 17

<210> 27

<211> 17

<212> DNA

<213> FP32 forward primer (FP 32 forward primer)

<400> 27

cggtggaaac actgact 17

<210> 28

<211> 17

<212> DNA

<213> FP32 reverse primer (FP 32 reverse primer)

<400> 28

taatagcctc catcctg 17

<210> 29

<211> 17

<212> DNA

<213> FP35 forward primer (FP 35 forward primer)

<400> 29

ggtcctgccg attcctc 17

<210> 30

<211> 17

<212> DNA

<213> FP35 reverse primer (FP 35 reverse primer)

<400> 30

cctccgattg attgata 17

<210> 31

<211> 17

<212> DNA

<213> FP43 forward primer (FP 43 forward primer)

<400> 31

ggagccgagt tctattg 17

<210> 32

<211> 18

<212> DNA

<213> FP43 reverse primer (FP 43 reverse primer)

<400> 32

cgatcatcac ctccatat 18

<210> 33

<211> 17

<212> DNA

<213> FP48 forward primer (FP 48 forward primer)

<400> 33

gacgcattgg acaacat 17

<210> 34

<211> 17

<212> DNA

<213> FP48 reverse primer (FP 48 reverse primer)

<400> 34

atcacttcct gctcctg 17

<210> 35

<211> 17

<212> DNA

<213> FP51 forward primer (FP 51 forward primer)

<400> 35

cgttggtatt gctgctt 17

<210> 36

<211> 17

<212> DNA

<213> FP51 reverse primer (FP 51 reverse primer)

<400> 36

ccgtcaacga tgagaat 17

Claims (6)

1. The fusarium SSR molecular marker is characterized by comprising 12 SSR molecular markers which are rich in polymorphism and contain different simple sequence repeating units, and specifically comprises FP03, wherein the nucleotide sequence of the SSR molecular marker is SEQ ID NO.1; FP08, nucleotide sequence of SEQ ID NO.2; FP13 with the nucleotide sequence of SEQ ID NO.3; FP21 with the nucleotide sequence of SEQ ID NO.4; FP24 with the nucleotide sequence of SEQ ID NO.5; FP26 with the nucleotide sequence of SEQ ID NO.6; FP31 with the nucleotide sequence of SEQ ID NO.7; FP32 with the nucleotide sequence of SEQ ID NO.8; FP35 with the nucleotide sequence of SEQ ID NO.9; FP43 with the nucleotide sequence of SEQ ID NO.10; FP48 with the nucleotide sequence of SEQ ID NO.11; FP51 has the nucleotide sequence of SEQ ID NO.12.

2. The fusarium layered SSR molecular marker of claim 1, wherein the 12 pairs of primers for amplifying the 12 SSR molecular markers comprising different simple sequence repeat units comprise: the nucleotide sequences of the primers of the amplified FP03 are SEQ ID NO.13 and SEQ ID NO.14, the nucleotide sequences of the primers of the amplified FP08 are SEQ ID NO.15 and SEQ ID NO.16, the nucleotide sequences of the primers of the amplified FP13 are SEQ ID NO.17 and SEQ ID NO.18, the nucleotide sequences of the primers of the amplified FP21 are SEQ ID NO.19 and SEQ ID NO.20, the nucleotide sequences of the primers of the amplified FP24 are SEQ ID NO.21 and SEQ ID NO.22, the nucleotide sequences of the primers of the amplified FP26 are SEQ ID NO.23 and SEQ ID NO.24, the nucleotide sequences of the primers of the amplified FP31 are SEQ ID NO.25 and SEQ ID NO.26, the nucleotide sequences of the primers of the amplified FP32 are SEQ ID NO.27 and SEQ ID NO.28, the nucleotide sequences of the primers of the amplified FP35 are SEQ ID NO.29 and SEQ ID NO.30, the nucleotide sequences of the primers of the amplified FP43 are SEQ ID NO.31 and SEQ ID NO.32, the nucleotide sequences of the primers of the amplified FP48 are SEQ ID NO.33 and SEQ ID NO.34, and the nucleotide sequences of the primers of the amplified FP51 are SEQ ID NO.35 and SEQ ID NO.36.

3. Use of an SSR molecular marker according to claim 1 or 2 in analysis of genetic diversity of fusarium layering.

4. Use of an SSR molecular marker according to claim 3 in the analysis of genetic diversity of fusarium layering, comprising the steps of:

(1) Collecting, separating and identifying fusarium;

(2) Extracting the genome DNA of fusarium strains from the layer;

(3) Performing PCR amplification on the genome DNA obtained in the step (2) to obtain a PCR amplification product;

(4) SSR genotyping: diluting the PCR amplification product obtained in the step (3) by 30 times, then carrying out denaturation, loading the sample to a full-automatic gene sequencer for fragment analysis, reading data by using software GeneMapper v3.25, and determining the genotype according to the size of the separated fragment;

(5) Analysis of genetic diversity.

5. The use of SSR molecular markers according to claim 4 in genetic diversity analysis of fusarium layering, wherein the PCR reaction system in step (3) is: 50 ng/. Mu.L of template DNA 1. Mu.L, mgCl 2.5. Mu.L of 25 mM, 2.5 mM dNTPs 1. Mu.L, 10. Mu.M forward and reverse primers each 1. Mu.L, 5U/. Mu.L of DNA Taq enzyme 0.2. Mu.L, 10 XPCR buffer 2.5. Mu.L, deionized water to 25. Mu.L, the forward 5' end of the primers carrying a blue 6-FAM or green HEX specific fluorescent label for amplifying the size of the band of interest, the PCR amplification procedure being: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, annealing at 55℃for 20s, extension at 72℃for 30s, total of 35 cycles, extension at 72℃for 10min.

6. The use of SSR molecular markers according to claim 4 for genetic diversity analysis of fusarium, wherein the method of genetic diversity analysis in step (5) comprises the steps of: the method comprises the steps of calculating and observing an allelic factor Na, an effective allelic factor Ne, shannon's diversity index I, an observed heterozygosity Ho, an expected heterozygosity He, an inter-population Nei's genetic distance D and a genetic similarity I by using POPGENE version1.32, calculating the percentage of total variation occupied by different variation levels among individuals in the population, the population and the individuals by using Alequin3.1 software, calculating the statistical variance component and the contribution rate, clearly causing the main source of the total variation of the population, calculating the genetic differentiation coefficient FST among the populations, estimating a gene flow Nm according to FST=1/(4Nm+1), constructing a non-weighted group average method UPGMA cluster map of the population based on the Nei's genetic distance, carrying out inter-population genetic Structure analysis by using a Bayesian cluster method of Structure V2.3.4 software, and drawing a generated population genetic Structure diagram by using DISTRUCT software.

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