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

Inactive Publication Date: 2020-07-17
SICHUAN UNIV
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Problems solved by technology

However, studies have shown that the development of microsatellite markers in Lepidoptera insects is relatively difficult, and the proportion of usable microsatellites obtained under the same library construction method is less than that of other species (Meglécz & Solignac, 1998), which is consistent with the characteristics of Lepidoptera insect genomes Related, the abundance of microsatellite sequences in Lepidoptera insects is relatively low (Giajie and Zhang Dexi

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  • Spodoptera frugiperda polymorphic microsatellite molecular markers and primers thereof
  • Spodoptera frugiperda polymorphic microsatellite molecular markers and primers thereof
  • Spodoptera frugiperda polymorphic microsatellite molecular markers and primers thereof

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses spodoptera frugiperda polymorphic microsatellite molecular markers and primers thereof. Nucleotide sequences of the microsatellite molecular markers are correspondingly shown as SEQ ID NO.1-12, and primer pairs of the microsatellite molecular marker are shown as SEQ ID NO.13-36. The invention further discloses a preparation method of the polymorphic microsatellite molecularmarker. According to the invention, polymorphism experimental verification is carried out on 33 sites by predicting four-base microsatellite sites with polymorphism in the genome of spodoptera frugiperda, and twelve four-base microsatellite markers with good polymorphism are obtained through screening for the first time, wherein the four-base microsatellite markers comprise four moderate polymorphism markers and eight high polymorphism markers, so that molecular technical support is provided for genetic research on invasion of spodoptera frugiperda, and molecular ecological research on the highly-multiphagous species, namely the grassland spodoptera litura, is facilitated.

Description

technical field [0001] The invention belongs to the technical field of DNA molecular markers of molecular biology, in particular to a polymorphic microsatellite molecular marker of Spodoptera frugiperda and a primer thereof. Background technique [0002] Spodoptera frugiperda, also known as fall armyworm, belongs to the genus Spodoptera of the family Noctuida (Lepidoptera) and is native to tropical and subtropical regions of America (Sparks, 1979). As a polyphagous pest, this insect has a wide range of hosts and can reproduce rapidly and migrate long distances. It has now developed into a major agricultural pest worldwide (Ge Shishuai et al., 2019). The insect invaded Africa in early 2016 and has great potential for further spread and economic damage. It has now spread to 44 countries in Africa, and the occurrence area has exceeded 2.2 million km 2 , has caused serious damage to African agricultural ecosystems (Goergen et al., 2016). Without control measures, fall armyworm ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 岳碧松张雪莲王磊
Owner SICHUAN UNIV
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