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Primer set of puccinia recondita tritici EST-SSR molecular marker, detection method and application thereof

A technology of wheat leaf rust and molecular markers, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of cumbersome steps, strong randomness, and difficult conditions to control, and achieve simple operation , convenient detection, and stable PCR amplification results

Inactive Publication Date: 2017-08-18
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems that the monitoring of wheat leaf rust is highly random, the conditions are not easy to control, and the steps are relatively cumbersome, the present invention provides a primer set for wheat leaf rust EST-SSR molecular markers and its detection method and application

Method used

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  • Primer set of puccinia recondita tritici EST-SSR molecular marker, detection method and application thereof
  • Primer set of puccinia recondita tritici EST-SSR molecular marker, detection method and application thereof
  • Primer set of puccinia recondita tritici EST-SSR molecular marker, detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Acquisition of RNA-seq data of wheat leaf rust and synthesis of EST-SSR primers

[0044] The RNA of wheat leaf rust was extracted, and the samples were submitted to Huada (Shenzhen) Company for RNA-Seq sequencing.

[0045] Through sequencing and assembly on the Illumina Hiseq2000 platform, 46,008 Unigenes were obtained from Pythora tritici, and the N50 reached 1,064 nt; using MISA software to search for SSR sites from the 46,008 Unigene sequences, a total of 3,729 SSR sites were obtained, distributed in 3,085 In Unigenes; Primer3 software was used to design primers, and 37 pairs of primer sequences were randomly selected and synthesized by Shanghai Sangon Bioengineering Co., Ltd. Primer sequences are shown in Table 3.

[0046] Table 3 Sequence information of 37 pairs of primers

[0047]

[0048]

Embodiment 2

[0050] Extraction of whole genome DNA of wheat leaf rust

[0051] 1) Weigh about 200 μg of ureterospores of wheat leaf rust, put them in 2mL centrifuge tubes, put them into a liquid nitrogen pot for pre-cooling, and grind them fully with a tissue grinder;

[0052] 2) Quickly add 800 μL of 65°C preheated CTAB solution (add 2% mercaptoethanol before use) to the centrifuge tube, put the centrifuge tube in a 65°C water bath for 1 hour, and mix the sample by inverting every 5 minutes during this period;

[0053] 3) After cooling to room temperature, add 800 μL of phenol: chloroform: isoamyl alcohol (25:24:1), mix well for 10 minutes, and then centrifuge at 12000 rpm for 15 minutes;

[0054] 4) Aspirate the supernatant, put it into a new centrifuge tube, add 800 μL of chloroform:isoamyl alcohol (24:1), mix for 10 minutes, and then centrifuge at 12000 rpm for 10 minutes;

[0055] 5) Transfer the supernatant to a new tube, add 1 mL of -20°C pre-cooled anhydrous ethanol, mix well, and p...

Embodiment 3

[0060] The amplification system is: 2 μL of template DNA, 1 U of Taq DNA polymerase, 1.0 mmol / L of dNTPs, 1.5 μL of upstream and downstream primers, 1×PCR Buffer in a 25 μL total system, and the rest is supplemented with sterile distilled water to 25.0 μL.

[0061] The reaction program was: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 55-59°C for 1 min, extension at 72°C for 1 min, and 35 cycles, and finally extension at 72°C for 2 min, and storage at 10°C.

[0062] The detection method is as follows: take 5 μL of the amplified product, add 0.5 μL of denatured sample loading indicator, and separate it by electrophoresis in a polyacrylamide gel with a concentration of 10%. Silver staining.

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Abstract

Relating to the field of plant protection, the invention specifically discloses a primer set of a puccinia recondita tritici EST-SSR molecular marker, a detection method and application thereof. The primer set of the Puccinia recondita tritici EST-SSR molecular marker includes primers shown as nucleotide sequences SEQ ID No:1-37. The detection method for puccinia recondita tritici genetic diversity employs the primer set to analyze the puccinia recondita tritici diversity and the population genetic structure, has the characteristics of simple operation, convenient detection, stable PCR amplification result, clear electrophoretic band and rich polymorphism, plays an important role in puccinia recondita tritici toxicity monitoring and genetic diversity analysis.

Description

technical field [0001] The invention belongs to the field of plant protection, and in particular relates to a primer set for EST-SSR molecular markers of wheat leaf rust fungus and a detection method and application thereof. Background technique [0002] Wheat leaf rust is one of the most serious diseases in wheat production. Using disease-resistant varieties is the safest, economical and effective control method. However, due to the continuous mutation of wheat leaf rust fungus, the frequency of toxicity changes every year, resulting in the continuous "loss" of resistance genes in wheat, resulting in a shortened lifespan of resistant varieties. Therefore, understanding the genetic diversity and Changes in group dynamics are significant. [0003] The genetic variation of wheat leaf rust is fast, and the effective monitoring of its genetic change trend can effectively control the occurrence of the disease. At present, the molecular markers used for the monitoring of wheat l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 杨文香王志张娜刘大群
Owner HEBEI AGRICULTURAL UNIV.
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