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ssr primers and applications for genetic diversity and kinship analysis of Curvularia lunata

A technique of genetic diversity and kinship, which is applied in the field of SSR primers and applications for the analysis of genetic diversity and kinship of Curvularia lunata, to achieve the effects of shortening the analysis cycle, being convenient to use, and improving research efficiency

Active Publication Date: 2021-06-01
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the study of fungal genetic diversity and genetic structure, a large number of SSR molecular markers have been developed and applied. Amplified polymorphic DNA (random amplified polymorphic DNA, RAPD) and simple repeat sequence interval (inter-simple sequence repeats, ISSR) two molecular markers for genetic diversity analysis

Method used

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  • ssr primers and applications for genetic diversity and kinship analysis of Curvularia lunata
  • ssr primers and applications for genetic diversity and kinship analysis of Curvularia lunata
  • ssr primers and applications for genetic diversity and kinship analysis of Curvularia lunata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 SSR primer development

[0070] (1) Analysis of genomic SSR loci and primer design

[0071] Download the whole genome sequence of Curvularia lunata from the NCBI (National Center for Biotechnology Information) website, use MISA software to search for SSR sites of 2 to 6 nucleotides in the genome sequence, and the search condition is 2 nucleotides The number of repetitions must be ≥6, and the number of repetitions of 3 to 6 nucleotides must be ≥5. Then extract the 200 bp sequence upstream and downstream of the SSR site, and use Primer 5.0 software to design SSR primers, setting parameters: primer length 20-24 bp, annealing temperature 50-65 °C, GC content 40-60%, PCR product length 150-350 bp. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0072] (2) DNA extraction and PCR

[0073] 6 strains of Curvularia lunata with different geographical distribution (CL17-27, CL15-27, CL17-30, CL17-35, CL17-64 and CL15-16, see Table 3 for deta...

Embodiment 2

[0088] Example 2 Using SSR primers to analyze the genetic diversity of different populations of Curvularia lunene

[0089] (1) Tested strains and medium

[0090] 24 strains of Curvularia crescens were isolated from diseased plants in the cornfields of Shangqiu City, Zhoukou City and Zhumadian City in Henan Province by tissue separation method (Table 3), then purified by single spore isolation, and transferred to a PDA slope at 4 °C Save it. The medium used was potato dextrose agar (PDA) medium: 200 g of potatoes, 20 g of glucose, 15 g of agar, and 1 L of distilled water.

[0091] Table 3 Information of 24 strains of Curvularia lunene

[0092]

[0093]

[0094] (2) DNA extraction and PCR

[0095] The specific operation is the same as in Example 1, and the amplification primers are CL-SSR2, CL-SSR6, CL-SSR7, CL-SSR19, CL-SSR24, CL-SSR29, CL-SSR35, CL-SSR37, CL-SSR42 and CL-SSR45 ;

[0096] (3) Genetic diversity analysis of different populations of Curvularia lunene

...

Embodiment 3

[0101] Example 3 Utilizes SSR primers to analyze the phylogenetic relationship between Curvularia lunata strains

[0102] (1) test bacterial strain and culture medium are with embodiment 2;

[0103] (2) The specific operation of DNA extraction and PCR is the same as in Example 1, and the amplification primers are CL-SSR2, CL-SSR6, CL-SSR7, CL-SSR19, CL-SSR24, CL-SSR29, CL-SSR35, CL-SSR37 , CL-SSR42 and CL-SSR45;

[0104] (3) Genetic relationship analysis between Curvularia lunata strains

[0105] According to 10 pairs of SSR primers (CL-SSR2, CL-SSR6, CL-SSR7, CL-SSR19, CL-SSR24, CL-SSR29, CL-SSR35, CL-SSR37, CL-SSR42 and CL-SSR45), 24 new The results of PCR amplification of Curvularia lunata were calculated using NTSYSpc2.10 software to calculate the genetic similarity coefficient (Table 5) between the strains, and the UPGMA method was used to cluster and build a phylogenetic tree ( image 3 ). According to the genetic similarity coefficient between the strains or the phylo...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to an SSR primer for analyzing the genetic diversity and kinship of Curvularia lunene and its application. The nucleotide sequences of the SSR primers used for the analysis of the genetic diversity and kinship of Curvularia lunata are shown in SEQ ID NO.1-40. The present invention also provides a kit for analyzing the genetic diversity and kinship of Curvularia lunata, and the development of the primers and the kit provides a new technique for studying the genetic diversity and kinship analysis of Curvularia lunene ways and means.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an SSR primer for analyzing the genetic diversity and kinship of Curvularia lunene and its application. Background technique [0002] Curvularia lunata is a very important plant pathogenic fungus. Its host range is very wide, and most plants in the family Fabaceae, Cucurbitaceae, Compositae, Solanaceae, Malvaceae and Gramineae can be infected by Curvularia crescens. Among the plant diseases caused by the pathogen, Curvularia maize leaf spot is one of the most serious plant diseases. This disease is a devastating leaf disease on maize. It can occur throughout the growth period of maize. The peak of the disease is from tasseling to filling stage. The typical symptom is a round disease with gray center and yellowish brown edge on the leaves. spots with a yellowish halo around them. Since the 1990s, the disease has seriously occurred in the corn planting areas of 11 provin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 胡艳红崔林开郭金利李梦琪和志华
Owner HENAN UNIV OF SCI & TECH
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