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Fusarium proliferatum SSR molecular marker and application thereof

A technology for the generation of Fusarium spp. and molecular markers, which is applied in the determination/inspection of DNA/RNA fragments, instruments, microorganisms, etc. Insufficient genetic variation of bacteria, etc., to achieve the effect of good repeatability and high polymorphism

Pending Publication Date: 2020-07-28
CHINA NAT RICE RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there have been few studies on the development and utilization of SSR molecular markers in the Fusarium sp. genome, and only Moncrief has developed 6 pairs of polymorphic SSR primers from the simple intersequence repeat region (ISSR) (Moncrief et al., Development of simple sequence repeat (SSR) markers for discrimination among isolates of Fusarium proliferatum . Journal of Microbiological Methods, 2016,126: 12-17), which is far from enough to emerge genetic variation of Fusarium
Due to the late start of the research on the genome sequencing of Fusarium exocytoma, there is no report on the development of SSR markers using the genome sequence of Fusarium extrinsicum

Method used

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  • Fusarium proliferatum SSR molecular marker and application thereof
  • Fusarium proliferatum SSR molecular marker and application thereof
  • Fusarium proliferatum SSR molecular marker and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Genome-wide SSR scanning and primer design of Fusarium sp.

[0034] Considering the availability, representativeness and importance of the data, the whole genome sequence of the emerging Fusarium sp. ET1 strain (https: / / www.ncbi.nlm.nih.gov / genome / 2434) was downloaded from the NCBI database, using The SSR Hunter software searches the SSR sequence among them, selects 2-6 nucleotides as repeat motifs, and repeats at least 6 times. Using the detected SSR and its upstream and downstream 150bp flanking sequences, Primer 5 software was used to design SSR primers. The general principles of primer design are: primer length 19~25bp; GC content 40%~60%; annealing temperature Tm value 52~58°C, and the Tm value difference between upstream and downstream primers is not more than 5°C; PCR amplification length 100~300bp ; Try to avoid primer dimers, hairpin structures, mismatches, etc. The synthetic primers have specific fluorescent labels of 6-FAM (blue) or HEX (green) a...

Embodiment 2

[0035] Example 2: Screening of SSR molecular markers

[0036] From the results of the above genome SSR scanning and primer design, 52 pairs of SSR primers were selected at random, and the specificity of these primers in the genome was detected by local Blast. After the pre-experiment, 12 microsatellite loci with diversity in the fragment length of the amplified product ranging from 100 to 300 bp were selected, and contained different simple sequence repeating units (Table 1). figure 1 , figure 2 , image 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 and Figure 12 The SSR genotypes of Fusarium sp. FP03, FP08, FP13, FP21, FP24, FP26, FP31, FP32, FP35, FP43, FP48 and FP51 are shown, respectively. The primers of the 12 SSR loci showed polymorphism, which provided markers for the analysis of the genetic diversity of Fusarium sp.

[0037] Table 1. 12 SSR molecular markers of Fusarium

[0038]

Embodiment 3

[0039] Example 3: Application of SSR Molecular Markers in Genetic Diversity of Fusarium sp.

[0040] Step 1: Collection, isolation and identification of layered Fusarium

[0041] In this example, Fusarium sp. collected from 3 populations (FY03, FY08, FY13) in Hangzhou, Zhejiang and 2 populations (NN05, NN12) in Nanning, Guangxi were used as samples. Fusarium was isolated from a susceptible sample of rice ear rot. Specific primers were designed according to the conserved sequence of the protein translation elongation factor (EF) gene, EF1 (5'-ATGGGTAAGGAGGACAAGAC-3') and EF2 (5'-GGAAGTACCAGTGATCATGTT-3'), and the strain was amplified by PCR. After the PCR amplification products were sequenced, the sequences were submitted to the Fusarium TEF sequence database (FUSARIUM-ID v 1.0) for comparison, and finally Fusarium sp. was selected by sequence comparison.

[0042] Step 2: Extraction of Genomic DNA from the Outer Fusarium Strains

[0043] The Fusarium genomic DNA was extracte...

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Abstract

The invention discloses a fusarium proliferatum SSR molecular marker and an application thereof, and belongs to the technical field of DNA molecular markers. According to the fusarium proliferatum SSRmolecular marker, SSR distribution of the fusarium proliferatum is carried out according to the genomic data of the fusarium proliferatum, a primer is designed and primary screening and secondary screening are performed to obtain 12 SSR molecular markers which are rich in polymorphism and contain different simple sequence repeat units. The makers have the beneficial effects that based on genome sequence information of the fusarium proliferatum, 12 SSR molecular markers which are different from previous ones and have high polymorphism and good repeatability are obtained, the markers are very effective molecular markers, and the genetic diversity and the population genetic structure of the fusarium proliferatum can be systematically revealed.

Description

technical field [0001] The invention belongs to the technical field of DNA molecular markers, and in particular relates to the SSR molecular markers of Fusarium exfoliates and the application thereof. Background technique [0002] Layered Fusarium ( Fusarium proliferatum ) is a worldwide pathogen of plant diseases and has a wide host range, causing rice ear rot, corn ear rot, soybean root rot, mango malformation, eggplant flower rot, tomato leaf blight, etc. . The pathogen not only reduces crop yield, but also produces a variety of mycotoxins (such as fumonisins, fusaricin and fusaric acid, etc.), which are highly pathogenic, teratogenic and carcinogenic, and pose a serious threat to human and animal life and health. At present, domestic and foreign researches on Fusarium epiphyllum mainly focus on identification and classification of pathogenic bacteria, biological characteristics, toxin formation, etc. Little is known about its genetic diversity and population genetic ch...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11G16B40/00
CPCC12Q1/6895G16B40/00C12Q2600/156
Inventor 王玲黄世文刘连盟
Owner CHINA NAT RICE RES INST
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