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Primer combination and application of est-ssr molecular marker of cereal cyst nematode
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A technique of combining cereal cyst nematodes and primers, which is applied in the field of molecular biology and can solve problems such as no relevant reports on cereal cyst nematodes
Active Publication Date: 2019-12-17
NANJING AGRICULTURAL UNIVERSITY
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[0005] At present, SSR molecular markers have been successfully developed in the research fields of soybean, potato and other plant parasitic nematodes and applied to related genetic diversity research [8-9] , but there is no related report in cereal cyst nematode
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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 source geographic location JSTZ Taixing Jichuan Street, Taizhou City, Jiangsu Province N 32.18398, E 120.03166 JSNT Yazhou Town, Haian County, Nantong City, Jiangsu Province N 32.39689, E 120.33246 JSYC Louwang Town, Yandu District, Yancheng, Jiangsu Province N 33.30354, E 119.81794 AHSZ Baitu Town, Xiao County, Suzhou City, Anhui Province N 34.13760, E...
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...
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Abstract
The invention discloses a primer combination with heterodera avenae EST-SSR (expressed sequence-simple sequence repeat) molecular markers and application of the primer combination. Primer sequences of the primer combination with the heterodera avenae EST-SSR molecular markers are shown as SEQ ID NO:1-16. EST data information is analyzed by the aid of MISA software according to transcriptome sequence information of heterodera avenae, large quantities of SSR loci are searched, and EST-SSR primers are designed according to SSR flanking sequences. The primer combination and the application have the advantages that the 8 EST-SST molecular markers which are high in polymorphism are screened by means of STR (short tandem repeat) typing detection after PCR (polymerase chain reaction) amplification is carried out, and the genetic diversity of 17 geographic populations of heterodera avenae can be effectively analyzed; the primer combination can be applied to research on population genetic structures, the genetic variation level and the like of the heterodera avenae in China.
Description
technical field [0001] The invention belongs to the technical field of molecular biology, and relates to the primer combination and application of the cereal cyst nematode EST-SSR molecular marker. Background technique [0002] Cereal cyst nematode (Heterodera avenae) was first discovered in 1989 in the wheat fields of Tianmen County, Hubei Province, my country. [1] , So far, the hazards have been distributed in 16 provinces (cities) including Henan, Hebei, Shandong, Jiangsu, Anhui, etc. [2] , has brought a great threat to my country's wheat production and food security. [0003] Since the first discovery of cereal cyst nematode in my country for more than 20 years, a large number of researches on the morphology, biology, selection of disease-resistant varieties and disease control technology of this pathogenic nematode have been carried out. Research on population genetic diversity and population genetic structure is relatively lagging behind. Pathotype determination resu...
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