Screening and application of specific primers for scot-PCR of Ustilago cane whip

A sugarcane whip smut, specific technology, applied in the sugarcane whip smut SCoT-PCR specific primer screening and application field, can solve the problems that plant pathogenic fungi have not been reported, and achieve high polymorphism detection efficiency Effect

Active Publication Date: 2017-09-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of SCoT molecular markers to smut and even plant pathogenic fungi has not been reported so far.

Method used

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  • Screening and application of specific primers for scot-PCR of Ustilago cane whip
  • Screening and application of specific primers for scot-PCR of Ustilago cane whip
  • Screening and application of specific primers for scot-PCR of Ustilago cane whip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The optimization of embodiment 1 SCoT-PCR reaction system

[0035] (1) Test materials

[0036] 1. Test strains

[0037]

[0038] 2. Primers to be tested

[0039] The present invention designs 30 primers in total and entrusts Shanghai Sangon Bioengineering Co., Ltd. to synthesize them.

[0040]

[0041] (2) Experimental method

[0042] 1. DNA extraction, amplification procedures and product detection

[0043] Genomic DNA of the mating-type strain of Ustilago cane whip was extracted by CTAB method. For details, refer to "Optimization of ISSR-PCR reaction system for Ustilago cane whip", and the quality of the extracted DNA was preliminarily detected by 1% agarose gel electrophoresis , and then use a UV spectrophotometer to measure the concentration and purity of the DNA sample, dilute the DNA to 50 ng / μL, and store it at -20°C for later use. The PCR amplification program was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 50°C for...

Embodiment 2

[0059] Example 2 Verification and primer screening of SCoT-PCR optimization system

[0060] According to embodiment 1 optimization test gained reaction system, with primer Ss -SCoT2, Ss -SCoT10, Ss -SCoT12 amplifies the 10 tested strains to verify the stability and reliability of the reaction system. Depend on Figure 7 It can be seen that the amplified bands of different strains are clearly identifiable, the main band is stable, and the polymorphism is rich, indicating that this system can be used for the SCoT-PCR amplification reaction of Ustilago sagoides.

[0061] Using this system, using the DNA of No. 10 strain as a template, 10 primers that can produce clear amplification products and have rich polymorphisms were selected from the 30 primers tested, namely: Ss -SCoT2, Ss -SCoT10, Ss -SCoT12, Ss -SCoT13, Ss -SCoT14, Ss -SCoT15, Ss -SCoT19, Ss -SCoT20, Ss -SCoT28, Ss -SCoT29. The statistical results of polymorphism of each primer are shown in Ta...

Embodiment 3

[0063] Example 3 Application of SCoT-PCR in Genetic Diversity Analysis

[0064] The electrophoresis results of the SCoT amplification products were manually compared and corrected, and the presence of a band was recorded as 1, and the absence of a band was recorded as 0, forming a 0, 1 matrix. Use POPGENE 1.32 software to calculate the effective number of alleles (Ne), Nei's gene diversity index (H), Shannon's information index (I); use NTSYS-pc 2.1 software to conduct similarity analysis to form a similarity coefficient matrix, according to UPGMA method Cluster analysis.

[0065] The results of genetic similarity analysis showed that the similarity coefficients of the 10 strains ranged from 0.6228 to 0.8953, with an average of 0.7578, among which the No. 7 strain and the No. 8 strain from ROC16 in Ganzhou, Jiangxi had the highest similarity coefficient ( 0.8953); the similarity coefficient between No. 5 strain from Guangdong Wengyuan ROC10 and No. 7 strain from Jiangxi Ganzh...

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Abstract

The invention belongs to the field of biotechnology, and specifically discloses a sugarcane smut SCoT-PCR (polymerase chain reaction) specific primer screening and an application. The primer sequence is shown as SEQ ID NO:1 to 10. Ten primers are used for amplifying ten sugarcane smut matching strains from different areas and hosts, totally, 86 bands are amplified, averagely, each primer amplifies 8.6 bands, the polymorphic rate is 59.3%. The screened specific primer has relatively high polymorphic detection efficiency, and can be used for genetic diversity analysis on sugarcane smut; and theoretical direction for selection of disease-resistant varieties of sugarcane is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically discloses the screening and application of SCoT-PCR specific primers for Ustilago cane whip. Background technique [0002] sugar cane( Saccharum officinarum L.) is the most important sugar crop in the world and a renewable biomass energy crop with more development potential. by Ustilago cane whip ( Sporisorium scitamineum ) caused by sugarcane smut is an important sugarcane disease worldwide. At present, the main sugarcane varieties in my country are generally infected with smut, and the incidence rate in the field is high, and the economic loss is very serious. Breeding for disease resistance is the most effective and economical method to prevent and control sugarcane smut. However, the physiological races of S. smut in sugarcane areas in my country are not clear so far. There is a large blindness in breeding for resistance to smut in sugarcane. Breeding for resistance to s...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2600/156
Inventor 沈万宽徐刚红罗明珠陈双吴夏明
Owner SOUTH CHINA AGRI UNIV
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