Spodoptera frugiperda polymorphic microsatellite molecular markers and primers thereof
A technology of Spodoptera frugiperda and molecular markers, applied in the field of DNA molecular markers in molecular biology, can solve the problems of microsatellite marker development difficulties, low abundance of microsatellite sequences, high mutation frequency, etc., and improve local integrated pest management practices Effect
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Embodiment 1
[0048] Example 1 Extraction, quality detection and identification of Spodoptera frugiperda sample DNA
[0049] 1) DNA extraction and quality assessment
[0050] 30 5th instar larvae of Spodoptera frugiperda were removed from their intestinal microbiota to prepare samples. Then, the DNA extraction kit was used to extract the total genomic DNA of Spodoptera frugiperda according to the instructions of the kit. It was used as template DNA for PCR amplification and stored in a refrigerator at 4°C for later use. Among them, 5 sample DNAs were used for preliminary screening of primers for four-base microsatellite candidate sites and optimization of PCR Tm values, and the remaining 25 sample DNAs were used for preliminary verification of polymorphisms at candidate sites. Take 3 μL of sample DNA and use 1% agarose gel electrophoresis to preliminarily detect the quality of its DNA. At the same time, 1.5 μL sample DNA was taken to measure its concentration with an ultraviolet spectrop...
Embodiment 2
[0059] Example 2 Screening of polymorphic four base microsatellite genetic markers
[0060] 1) Prediction of polymorphic microsatellite sites
[0061] CandiSSR software is an efficient polymorphic microsatellite screening software, which can predict a large number of polymorphic microsatellite sites based on multiple assembly sequences. In this study, CandiSSR was used to process the genome data of the above six Spodoptera frugiperda, compared and screened the microsatellite loci in the genome, retrieved the polymorphic microsatellite loci in the genome, and designed a method to amplify the locus at the same time. primers. CandiSSR software parameter setting: the length of the sequence on both sides is 200nt (-1=200), the E value of BLAST is 1e-10 (e=1e-10), the similarity is 95% (-s=95) and the coverage 95% (-c=95).
[0062] The genome sequences of 6 Spodoptera frugiperda were compared and screened using CandiSSR software. A total of 2316 perfect microsatellite sites with...
Embodiment 3
[0078] Example 3 Quality Assessment of 26 Candidate Sites
[0079] 1) The typing effect of the site
[0080] The electropherogram of genotyping shows that the genotyping effect is better, that is, the electropherogram is clean and single, and there are 14 pairs of primers without staggered peaks, miscellaneous peaks, and multiple peaks, representing the following 14 loci: Spf (284, 301, 626, 1018, 1165, 1614, 300, 471, 542, 635, 981, 1167, 1434, 1725). After the second PCR optimization, the typing effect was still unsatisfactory (the peak value was too low, multiple amplification (MA), false amplification (FA)) and there were 12 pairs of primers, representing 12 loci. Therefore, these 12 sites need to be eliminated in subsequent research and analysis, and the specific problems of these 12 sites are shown in Table 4. The large number of multiple amplifications at these loci may be related to the fact that there are significantly more flanking sequence repeats in the genomes o...
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