Re-sequencing analysis method for whole genome of brontispa longissima and application of re-sequencing analysis method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a brontispa longissima whole genome re-sequencing analysis method and application thereof. Whole genome sequencing is carried out on the brontispa longissima, and the population difference of the brontispa longissima in different populations is determined. Based on the technical advantages, the invention also provides a method for tracing the brontispa longissima, data comparison analysis is performed on the whole genome DNA of the brontispa longissima to be traced and the purified whole genome DNA of the brontispa longissima, so that the traceability of the brontispa longissima is completed, and accurate tracing and traceability of the brontispa longissima are realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a whole genome resequencing analysis method for a coconut palm leaf beetle and an application thereof. Background Art

[0002] The coconut leaf beetle (Brontispa longissima) is the most important pest of palm plants in tropical my country. It attacks the unexpanded or semi-expanded leaves of the host plant, resulting in a reduction in the fruit yield of the palm plant. In addition, since most palm plants are important greening tree species in southern my country, they can cause serious landscape damage and indirectly affect economic activities such as tourism.

[0003] Existing studies have shown that the genetic diversity of coconut leaf beetles in my country is low to moderate, and its genetic diversity is not related to geographical distance (Chen Zhiming; Wang Guihua; Li Min; Peng Zhengqiang; Ali Habib; Xu Lina et al. (2020): Development of Single Nucleotide Polymorphism (SNP) Markers for Analysis of Population Structure and Invasion Pathway in the Coconut Leaf Beetle Brontispa longissima (Gestro) Using Restriction Site-Associated DNA (RAD) Genotyping in Southern China. In Insects 11 (230): 1-13.), and conventional molecular marker methods are difficult to accurately track and trace the coconut leaf beetles. Therefore, how to accurately track and trace the coconut leaf beetles is a difficult problem that needs to be solved in this field. Summary of the invention

[0004] The object of the present invention is to provide a whole genome resequencing analysis method for coconut palm leaf beetle and its application, by performing whole genome resequencing on coconut palm leaf beetle, it is helpful to accurately track and trace it.

[0005] The present invention provides an analysis method for whole genome resequencing of a coconut palm leaf beetle, comprising the following steps:

[0006] Perform whole genome resequencing on the coconut palm leaf beetle to obtain genomic DNA of the coconut palm leaf beetle;

[0007] The genomic DNA of the tested coconut palm leaf beetle is subjected to maximum eigenvector analysis and / or a population structure phylogenetic tree is constructed for analysis.

[0008] Preferably, the sources of the coconut palm leaf beetles to be tested include: Maldives, Yunnan Province, Guangxi Zhuang Autonomous Region, Hainan Province and Guangzhou City or more.

[0009] Preferably, the coconut leaf beetle includes: a coconut leaf beetle feeding on unexpanded leaves of coconut trees and / or a coconut leaf beetle feeding on expanded leaves of coconut trees.

[0010] The present invention provides application of the analysis method described in the above technical solution in tracing the origin of coconut palm leaf beetles.

[0011] The present invention provides a method for tracing the origin of coconut palm leaf beetles, comprising the following steps:

[0012] The whole genome DNA of the coconut palm leaf beetle to be traced is compared and analyzed with the purified whole genome DNA of the coconut palm leaf beetle, thereby completing the traceability of the coconut palm leaf beetle;

[0013] The reference whole genome DNA of the coconut palm leaf beetle comprises: the genome DNA of the coconut palm leaf beetle purified by feeding with unexpanded leaves of coconut trees.

[0014] Preferably, the purification generation number is ≧10.

[0015] Beneficial effects:

[0016] The present invention clarifies the genetic differences of coconut palm leaf beetles in different geographical populations by sequencing the whole genome of coconut palm leaf beetles, which can be used to analyze the population differences of coconut palm leaf beetles.

[0017] Based on the above technical advantages, the present invention provides a method for tracing the coconut heart leaf beetle, comprising the following steps: performing data comparison and analysis on the whole genome DNA of the coconut heart leaf beetle to be traced and the purified whole genome DNA of the coconut heart leaf beetle, thereby achieving accurate tracking and tracing of the coconut heart leaf beetle. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The whole genome sequencing result of the coconut palm leaf beetle provided by the present invention;

[0020] Figure 2 The predicted gene number of the whole genome of the coconut palm leaf beetle provided by the present invention;

[0021] Figure 3 The number of annotated genes in the whole genome of the coconut palm leaf beetle provided by the present invention;

[0022] Figure 4 The principal component analysis 1 of the population structure of the coconut palm leaf beetle provided by the present invention;

[0023] Figure 5 The principal component analysis 2 of the population structure of the coconut palm leaf beetle provided by the present invention;

[0024] Figure 6 The phylogenetic tree of the population structure of the coconut palm leaf beetle provided by the present invention is constructed;

[0025] Figure 7 The present invention provides a population structure genetic background analysis of the coconut palm leaf beetle 1;

[0026] Figure 8 The present invention provides population structure genetic background analysis of coconut palm leaf beetle 2. DETAILED DESCRIPTION

[0027] Unless otherwise specified, the methods and reagents used in the present invention are conventionally selected.

[0028] Example 1

[0029] Resequencing the whole genome of the coconut palm leaf beetle:

[0030] Using third-generation sequencing technologies such as Illumina, PacBio and Hi-C sequencing, the whole genome of the homozygous coconut palm leaf beetle reared on the unexpanded leaves of coconut trees for more than 10 generations was sequenced and assembled, and the whole genome sequence of the coconut palm leaf beetle was obtained. Figure 1-3 .

[0031] Combination Figure 1-3 The results showed that the genome size of the coconut leaf beetle was 582.19Mb, 372 contigs were assembled, contigN50 was 5.49Kb, and the longest contig was 20.12Kb; the Busco software was used to evaluate the completeness of the genome assembly, and it was found that 98.20% of the sequences were similar to the insect conserved sequence library, and the genome integrity was high; based on the transcriptomes of different instars, full-length transcriptomes and homologous gene sequences, 16,208 protein-coding genes were finally obtained through de novo annotation, homology annotation, and transcriptome annotation methods; based on the comparison of the gene function annotation library, 14,544 genes were found to have similarities with the known function annotation library; RepeatMasker software was used to search for repetitive sequences in the genome, and the coconut leaf beetle repetitive sequence was 261.48Mb.

[0032] Example 2

[0033] Comparison of the genome of the coconut palm leaf beetle:

[0034] A total of 63 samples of coconut beetles were collected from Maldives, Jinghong, Yunnan Province, Xishuangbanna, Yunnan Province, Yuanyang, Yunnan Province, Beihai, Guangxi Zhuang Autonomous Region, Fangchenggang, Guangxi Zhuang Autonomous Region, Hainan Province, and Guangzhou City (see Table 1 for sample information) and whole genome sequencing was performed. A total of 4748.85 million clean reads and 1424.66G clean bases were obtained by Illumina genome sequencing of the 63 samples.

[0035] Table 1 Source information of 63 samples of Brontispa longituba

[0036]

[0037]

[0038] The clean reads of 63 samples were anchored to the reference genome of the coconut palm leaf beetle (i.e., the coconut palm leaf beetle genome purified for more than 10 generations using unexpanded coconut leaves indoors), and the alignment information of the anchored reads was statistically analyzed using Bcftools software. The detected genetic variations were used for downstream analysis. The results showed that a total of 9430926 high-quality SNP variant sites were detected in 63 samples; among the 9430926 SNP variant sites, 41.56% (3919949) were annotated to the mRNA gene region, and 57.30% (5403467) SNPs were annotated to the intergenic region; among the SNPs in the mRNA gene region, 7.34% (692189) were annotated to the CDS region, and 34.12% (3217959) were annotated to the intron region; in the CDS region, 5.25% (494849) were synonymous substitution mutations, and 2.09% (197340) were non-synonymous substitution mutations; 1.14% (107510) were annotated to the promoter region of the gene. The SNP (107510) in the promoter region of the gene and the non-synonymous substitution mutation SNP (197340) in the CDS region may be of great significance in the long-term adaptive evolution of populations of coconut palm beetles in different regions.

[0039] Example 3

[0040] Coconut Heart Leaf Beetle Origin:

[0041] In order to study the population structure of coconut palm leaf beetle populations in different geographical areas, the whole genomes of the 63 samples in Example 2 were subjected to principal component analysis of population structure.

[0042] The largest eigenvector PCA1 and PCA2 values ​​are used to distinguish geographical populations, combined with Figure 4 and Figure 5It can be seen that with PCA1 as the main grouping, the Yunnan Jinghong population (YN-JH) of the coconut palm leaf beetle is clearly separated, and with PCA2 as the main grouping, the Maldives (MEDF) population is separated, that is, it can be divided into three subpopulations. The geographical populations are distinguished by the maximum eigenvector PCA1 and PCA3 values. When PCA3 is used for grouping, the Guangdong Province (GD) population is significantly separated. Based on the PCA1, PCA2 and PCA3 grouping, the 63 samples can be divided into four populations, namely the Maldives (MEDF) population, the Yunnan Jinghong (YN-JH) population, the Guangdong Province (GD) population, and the Guangxi Zhuang Autonomous Region-Hainan Province-Yunnan Province (Xishuangbanna + Yuanyang) (GX-HN-YN_XSBN_YY) population. Among them, the Guangdong (GD) population is more closely related to the Guangxi Zhuang Autonomous Region-Hainan Province-Yunnan Province (Xishuangbanna + Yuanyang) (GX-HN-YN_XSBN_YY) population.

[0043] In order to further explore the relationship between individuals of the coconut palm beetle in different geographical locations, the neighbor joining method was used to construct a phylogenetic tree of individuals in different geographical locations. The results are shown in Figure 6 .

[0044] Combination Figure 6 The results of the phylogenetic tree of different geographical individuals of Brontispa longituba showed that different geographical individuals clustered into different branches and formed different subpopulations based on geographical location information. The Guangdong (GD) population and the Guangxi Zhuang Autonomous Region-Hainan Province-Yunnan Province (Xishuangbanna + Yuanyang) (GX-HN-YN_XSBN_YY) population clustered into one branch, and were more closely related to individuals in Yuanyang, Yunnan and Guangxi Zhuang Autonomous Region; the Maldives (MEDF) population and Jinghong, Yunnan Province (YN-JH) formed a separate branch. Among the Hainan Province-Yunnan Province (Xishuangbanna + Yuanyang) (GX-HN-YN_XSBN_YY) population, the Hainan Province (HN) population was more closely related to the Xishuangbanna, Yunnan Province (YN-XSBN) population than to other populations, and the Guangxi Zhuang Autonomous Region (GX) population was closely related to Yuanyang, Yunnan.

[0045] In order to verify the relationship between individuals of the coconut palm leaf beetle from different geographical locations, the population structure of 63 individuals from different geographical locations was analyzed using admixture software to detect the genetic background of different subpopulations. The results are shown in Figure 7 and Figure 8 .

[0046] Combination Figure 7 and Figure 8It can be seen that when the ancestral population of the coconut palm leaf beetle is 2, that is, K=2, the individuals of the Maldives (MEDF) population and the Jinghong (YN-JH) population in Yunnan are divided into one subgroup with a consistent genetic background, and the other individuals have the same genetic background and are divided into a separate subgroup; when the ancestral population is assumed to be 3, that is, K=3, the 63 coconut palm leaf beetle samples are clearly divided into three subgroups according to their geographical location, namely the Maldives (MEDF) population, the Jinghong (YN-JH) population in Yunnan and other individuals. The Jinghong population in Yunnan and the Maldives population are clearly differentiated, and there is only one phenomenon of gene infiltration between the individuals; among the other individuals, the Guangdong (GD) population has some genetic background of the Jinghong (YN-JH) population in Yunnan. Assuming that the ancestral population is divided into 4, that is, K = 4, the 63 coconut palm leaf beetle samples can be divided into four populations, namely the Maldives (MEDF) population, the Jinghong (YN-JH) population in Yunnan, the Guangdong (GD) population, and the Guangxi Zhuang Autonomous Region-Hainan-Yunnan (Xishuangbanna + Yuanyang) (GX-HN-YN_XSBN_YY) population. At this time, the cross-validation error value is the smallest, which is 0.50982. It is most appropriate to divide the 63 individual samples into 4 subpopulations. Individuals in Yuanyang, Yunnan Province, Hainan Province, and Guangxi Zhuang Autonomous Region all have the genetic background of the Guangdong population, and the genetic background penetration of Guangxi Zhuang Autonomous Region and Yuanyang, Yunnan Province is more obvious. Some individuals in Hainan Province and Yuanyang, Yunnan Province have the genetic background of the Maldives population. Interestingly, the genetic background of individuals in Xishuangbanna, Yunnan Province is single, and the relationship is closer to Hainan (K = 4 or K = 5).

[0047] In summary, Yunnan Province and other places have been invaded by coconut palm leaf beetles from multiple times and different sources. It can be seen that the technical solution provided by the present invention can accurately track and locate coconut palm leaf beetles.

[0048] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for analyzing the whole genome resequencing of Brontispa longituba, characterized in that: The steps include: Perform whole genome resequencing on the coconut palm leaf beetle to obtain genomic DNA of the coconut palm leaf beetle; The genomic DNA of the tested coconut palm leaf beetle is subjected to maximum eigenvector analysis and / or a population structure phylogenetic tree is constructed for analysis.

2. The analysis method according to claim 1, wherein the source of the Brontispa longituba to be detected comprises: One or more of Maldives, Yunnan Province, Guangxi Zhuang Autonomous Region, Hainan Province and Guangzhou City.

3. The analysis method according to claim 1, wherein the coconut palm leaf beetle comprises: Coconut leaf beetles feeding on the unfurled leaves of coconut trees and / or Coconut leaf beetles feeding on the unfolded leaves of coconut trees.

4. Application of the analytical method according to any one of claims 1 to 3 in tracing the origin of coconut palm leaf beetles.

5. A method for tracing the origin of coconut palm leaf beetle, characterized in that: The following steps are involved: The whole genome DNA of the coconut palm leaf beetle to be traced is compared and analyzed with the purified whole genome DNA of the coconut palm leaf beetle, thereby completing the traceability of the coconut palm leaf beetle; The purified whole genome DNA of the coconut palm leaf beetle comprises: the genome DNA of the coconut palm leaf beetle purified by feeding with unexpanded leaves of coconut trees.

6. The method according to claim 5, characterized in that The number of generations of purification is ≧10.

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