Primer combination with heterodera avenae EST-SSR (expressed sequence-simple sequence repeat) molecular markers and application of primer combination
A kind of technology of grain cyst nematode and primer combination, which is applied in the field of molecular biology and can solve the problem of no relevant reports on the grain cyst nematode and the like
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0019] Embodiment 1: cyst separation
[0020] All the CCN soil samples collected in the field were naturally air-dried, crushed and mixed evenly, and 200mL of soil was taken, rinsed with strong water and stirred continuously, and left to stand for 1-2 minutes, and the suspension was filtered through 40-mesh and 100-mesh mesh screens in sequence Repeat the above steps 3 times. The residue on the 100-mesh sieve was gently rinsed under a stream of water, rinsed into a funnel with filter paper and filtered. After filtering, pick the full cysts under a stereoscope and refrigerate them for later use.
Embodiment 2
[0021] Embodiment 2: the extraction of cyst DNA
[0022] Pick one mature and plump cyst of each cyst nematode population (Table 2) to be tested, put it on a glass slide that has been dripped with 20 μL lysis buffer, and grind it thoroughly, and recover it into a 200 μL PCR tube; freeze it in liquid nitrogen 1min, 65°C water bath for 2min, repeat 3 times; finally incubate at 65°C for 1.5h, treat at 95°C for 10min, centrifuge at 14000r / min for 1min, take the supernatant and store it at -20°C for later use.
[0023] Table 2 Sources of cereal cyst nematode populations
[0024] group
[0025] Primer pair TW81(5`-GTT TCC GTA GGT GAA CCT GC-3`) and AB28(5`-ATA TGC TTAAGT TCA GCG GGT-3`) [10] The ITS region of the extracted nematode DNA was amplified, and the amplified product was photographed and recorded under a gel imaging system to confirm that the nematode DNA was successfully extracted.
Embodiment 3
[0026] Embodiment 3: Design EST-SSR primers
[0027] Search and download the EST sequences of cereal cyst nematodes in NCBI, use EST-trimmer (http: / / www.pgrc.ipk-gatersleben.de / misa / download / est trimmer.pl) software to screen effective EST sequences, and delete sequences After the EST sequence with a length of less than 100bp or greater than 700bp, remove the "cap" and "tail" structures of the mRNA (that is, remove 5 or more consecutive A and T within 50bp at both ends of the EST sequence). MISA software was used to search for EST sequences containing SSR sequences and both flanking sequences of SSRs were greater than 40 bp. According to the target EST sequences obtained by screening, Primer Premier 5.0 software was used to design SSR marker primers. The design principles of the primers follow three points: 1) The length of the primer is between 18-25 bp, and it is located in the flanking region of the SSR; 2) The GC content of the primer is between 40% and 60%, and the anneal...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More