SSR (Simple Sequence Repeat) molecular marker primers for monochamus alternatus and application of SSR molecular marker primers

Abstract

The invention provides an SSR (Simple Sequence Repeat) molecular marker primer group for genetic diversity analysis of monochamus alternatus and an application method of the SSR molecular marker primer group. And the limitation of population analysis only depending on morphological characteristics in the prior art is overcome. The invention provides a group of polymorphic SSR primer groups with serial numbers of Mona1 to Mona14, and the polymorphic SSR primer groups are obtained by screening on the basis of simplified genomes of monochamus alternatus. The primer groups can remarkably improve the resolution of genetic diversity analysis, and a powerful tool is provided for researching the population genetic structure, evolutionary relationship and ecological adaptability of monochamus alternatus. The invention also comprises sequences of the SSR primer groups and a method for genetic analysis by using the primer groups, and the method can be used for clustering analysis and population genetic analysis of monochamus alternatus. An efficient, environment-friendly and high-specificity means is provided for population genetic research and scientific control of monochamus alternatus, and the important forestry pest can be better understood and managed.

Description

Technical Field

[0001] The invention relates to the technical field of insect genetic engineering, in particular to a group of SSR molecular marker primers of Monochamus alternatus and an application method thereof. Background Art

[0002] The pine beetle is a highly destructive pest in the pine forest ecosystem, posing a serious threat to the survival of coniferous tree species such as Masson pine. Its larvae bore into the trees, causing tunnels to form in the trunks, which seriously affects the growth of the trees and the quality of the wood. What is more serious is that the pine beetle is also the main vector of pine wood nematodes, aggravating the death of pine trees and the loss of forest resources. In recent years, with the intensification of global warming and the rapid expansion of artificial forest areas, the scope of damage caused by the pine beetle has also been expanding. Especially in some areas where coniferous tree species such as Masson pine are densely distributed, the explosive damage caused by the pine beetle has become a prominent problem in forestry production. This has not only led to serious losses of forest resources, but also had an impact on the ecological environment that cannot be ignored.

[0003] In recent years, the scope and degree of pine wood nematode disease in my country have continued to expand, causing serious damage to my country's pine forest resources, natural landscapes and ecological environment. Faced with such a severe situation, it is urgent to find an efficient, environmentally friendly and highly specific prevention and control method. As the main vector of pine wood nematodes, the prevention and control of pine beetles is particularly important. By developing microsatellite loci for pine beetles, we can further explore the population genetic mechanism of pine beetles, achieve accurate identification and monitoring of pine beetle populations, and then understand their distribution patterns, migration paths and reproductive habits, providing strong support for the formulation of scientific and effective prevention and control strategies. Summary of the invention

[0004] The purpose of the present invention is to provide a set of SSR molecular marker primer sets for Monochamus alternatus, and a method for using the primer sets to perform genetic diversity analysis of Monochamus alternatus, so as to overcome the limitations of the prior art that population analysis relies solely on morphological characteristics. The SSR molecular marker primer sets of the present invention can significantly improve the resolution of genetic diversity analysis, and provide a powerful tool for in-depth research on the population genetic structure, evolutionary relationship and ecological adaptability of Monochamus alternatus.

[0005] The present invention achieves the above-mentioned purpose through the following technical means:

[0006] The polymorphic SSR primer sets included in the present invention are obtained based on the simplified genome screening of Monochamus alternatus, and are numbered: Mona1, Mona2, Mona3, Mona4, Mona5, Mona6, Mona7, Mona8, Mona9, Mona10, Mona11, Mona12, Mona13, and Mona14.

[0007] The sequences of the above-mentioned SSR primer set also included in the present invention are shown in Table 3.

[0008] The present invention also includes an application of the SSR molecular marker primer set for genetic analysis, and the application can be used for cluster analysis and population genetic analysis of Monochamus alternatus. DETAILED DESCRIPTION

[0009] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0010] Embodiment 1:

[0011] The Monochamus alternatus used in this experiment was collected from the Monochamus alternatus plant source traps hanging in the pine forests of Xingning District, Nanning City, Guangxi, Chengzhong District, Liuzhou City, Babu District, Hezhou City, Cangwu County, Wuzhou City, and Jingxi County, Baise City from May to July 2022. After the Monochamus alternatus was removed, it was placed in a 2mL cryopreservation tube, placed in anhydrous ethanol and stored at -20°C. The chest muscle was taken and the whole genome DNA was extracted using the animal tissue genomic DNA extraction kit provided by Beijing Quanshijin Biotechnology Co., Ltd. according to the instructions. After passing the quality and concentration tests, it was stored at -20°C for use.

[0012] In this experiment, three pine beetles were selected from Xingning District of Nanning City, Cangwu County of Wuzhou City, and Jingxi County of Baise City, and sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for simplified genome sequencing.

[0013] Embodiment 2:

[0014] FastQC (version 0.11.7) was used to control the data quality, and the sliding window method of Fastp (v0.20.0) was used to filter the raw data to obtain high-quality sequencing data. After the data of the three samples were integrated, the sequences were stacked into one high-quality sequence using FLASH (version 1.2.11).

[0015] As shown in Table 1, after simplified genome sequencing, the three samples were filtered and obtained 6.32 million to 6.87 million high-quality sequences, with an average base content of 900 million to 980 million; the average proportion of high-quality sequences was 96.33%, and the average proportion of high-quality bases was 95.28%. Among them, the average Q20 was 97.4%, the average Q30 was 97.71%, and the average GC content was 38.95%, indicating that the sequencing results were good.

[0016] Table 1 Simplified genome sequencing results of three samples of Monochamus alternatus

[0017]

[0018] Embodiment 3:

[0019] Microsatellite identification tool (MISA) was used to search for SSR loci in all simplified genomic sequences. The search parameters were as follows: mono-10, di-6, tri-5, Tetra-5, penta-5, and hexa-5. The maximum interval allowed between two different SSRs in the composite sequence was set to 100 bp, and reverse complementary and shifted sequences were considered as one type of SSR.

[0020] A total of 95,612 SSR loci were found in 2,577,819 sequences. Among all the sequences, 83,097 sequences contained SSR loci, and 18,986 sequences had SSR flanking sequences longer than 20 bp. A total of 12,115 sequences contained more than one SSR locus, and a total of 12,325 SSR loci existed in a composite form. Among the SSR loci searched, the SSR motif types of mononucleotide repeats, dinucleotide repeats, and trinucleotide repeats appeared more frequently, accounting for 51.43%, 28.79%, and 16.74%, respectively. The frequencies of tetranucleotide repeats, pentanucleotide repeats, and hexanucleotide repeats were relatively low, accounting for 1.50%, 0.45%, and 1.06%, respectively. Among the SSR models with a higher frequency, there were 2, 4, and 10 SSR models of mononucleotide repeats, dinucleotide repeats, and trinucleotide repeats, respectively. Among the SSR models of mononucleotide repeats, the A / T model has the largest frequency, accounting for 98.9% of the total; the C / G model has a lower frequency, accounting for 1.1% of the total. Among the SSR models of dinucleotide repeats, the AT / AT model has the largest frequency, accounting for 87.37% of the total; the AG / CT model and the AC / GT model have a frequency of 6.09% and 5.72% respectively, accounting for a larger proportion than CG / CG. Among the SSR models of trinucleotide repeats, the AAT / AAT model has the largest frequency, accounting for 77.34% of the total, and the frequencies of the remaining 9 models are all less than 5% (Table 2).

[0021] Table 2 SSR motif results statistics

[0022]

[0023] Embodiment 4:

[0024] Primers were designed for both ends of the site using primer3 v2.3.6 for the SSR sequences in the clusters with polymorphisms > 2. The length of the target fragment was controlled at 100-400 bp, and the position of the target fragment was from 1 base before the repeat sequence to 5 bases after the repeat sequence. Other parameters were set as default. All designed primers were screened, and the screening criteria were as follows: the SSRs type of the designed primers was non-mononucleotide repeat motifs and non-complex repeat motifs; only SSRs with one SSR on each read were selected to design primers to avoid the amplification of the second SSR during the amplification process to affect the polymorphism; the designed primers must be in a certain Clusters, and the length polymorphism of the SSRs in the Clusters must be ≥2; each designed primer must be supported by two primers in the Clusters, and the primer results that are exactly the same must be removed.

[0025] After the 5' end of the upstream primer was modified with fluorescent groups 5'-FAM, 5'-HEX and 5'-ROX, PCR amplification was performed on 15 samples from five populations in Xingning District, Nanning City, Chengzhong District, Liuzhou City, Babu District, Hezhou City, Cangwu County, Wuzhou City and Jingxi County, Baise City, Guangxi. The reaction system (25μL) included F primer (1μL), R primer (1μL), genomic DNA (1μL), and 1.1×T3 Super PCR Mix (22μL) (Beijing Qingke Biotechnology Co., Ltd.). The reaction procedure was as follows: pre-denaturation at 98℃, 3min; denaturation at 98℃, 15s; annealing at 53-55℃, 15s; extension at 72℃, 15s; final extension at 72℃, 2min. After obvious bands were detected by 1% agarose gel electrophoresis, capillary electrophoresis genotyping was performed, and 14 polymorphic primers were finally selected for data analysis.

[0026] The whole genome of Monochamus alternatus from 5 sampling points was amplified by PCR using 192 pairs of randomly selected SSR primers. A total of 98 pairs of primers failed to amplify partially, and 94 pairs of primers amplified successfully. Among the 94 pairs of sequences that were successfully amplified, 57 pairs of primers had good peak quality. After excluding sequences with a small number of alleles, 14 polymorphic primers were finally obtained (Table 3). Among them, there were 5 dinucleotide repeat SSR sites, 8 trinucleotide repeat SSR sites, and 1 tetranucleotide repeat SSR site.

[0027] Table 3 SSR primer information

[0028]

[0029] Embodiment 5:

[0030] The genotyping data were sorted into Excel according to the order of loci, and Popgene1.32 was used to calculate the number of alleles, effective number of alleles, Shannon index, observed heterozygosity and expected heterozygosity of each locus. MicrosatelliteToolkit v3.1.1 was used to calculate the polymorphic information content of each locus.

[0031] The results are shown in Table 4. The number of alleles at each site ranged from 3 to 10, and the number of effective alleles ranged from 1.515 to 7.266. The Shannon index of all sites was 0.533 to 2.062, with an average of 1.279; the observed heterozygosity was 0.067 to 0.867, with an average of 0.452; the expected heterozygosity was 0.287 to 0.862, with an average of 0.622; the genetic diversity was 0.644 to 0.931, with an average of 0.811; and the polymorphic information content was 0.261 to 0.847, with an average of 0.582.

[0032] Table 4 Site polymorphism information

[0033]

[0034] Embodiment 6:

[0035] Based on the site polymorphism information of 14 pairs of primers for Monochamus alternatus, the genetic distance coefficient was calculated by ntsys-pc software, and the UPMGA cluster diagram of 15 Monochamus alternatus from 5 regions was constructed ( Figure 1 The results showed that these 14 pairs of SSR primers could distinguish Monochamus alternatus from different regions, indicating that this set of primers is suitable for cluster analysis and population genetic analysis of Monochamus alternatus.

[0036] Figure 1 UPMGA cluster diagram of 15 Monochamus alternatus from 5 regions

[0037] It is particularly reminded that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for providing a set of SSR molecular marker primers for Monochamus alternatus and using the primers to analyze the genetic diversity of Monochamus alternatus. The method comprises: The polymorphic SSR primer sets were obtained based on the simplified genome screening of Monochamus alternatus and were numbered: Mona1, Mona2, Mona3, Mona4, Mona5, Mona6, Mona7, Mona8, Mona9, Mona10, Mona11, Mona12, Mona13, and Mona14.

2. The sequences of the above SSR primer sets are shown in Table 1. Table 1 SSR primer information 3. The present invention also includes an application of the SSR molecular marker primer set for genetic analysis, which can be used for cluster analysis and population genetic analysis of Monochamus alternatus.

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