DDRAD sequencing: An emerging technology added to the biosecurity toolbox for tracing the origin of brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae)

- Principal component analysis (PCA) of the identified SNPs indicated the existence of two main distinct genetic clusters representing individuals sampled from regions where BMSB is native to, China and Japan, respectively, and one broad cluster comprised individuals sampled from countries which have been invaded by BMSB.
- The population genetic structure analysis further discriminated the genetic diversity among the BMSB populations at a higher resolution and distinguished them into five potential genetic clusters..
- Full list of author information is available at the end of the article.
- The BMSB populations from Japan were genetically distant from the other studied populations.
- Similarly, the BMSB populations from China were also genetically differentiated from the Japanese and other populations.
- In the past decades, BMSB has invaded and established in a range of countries irrespective of the environmental conditions .
- As agricultural exports play a significant role in New Zealand’s Gross Domestic Product, the establishment of the pest would be highly detrimental to the country.
- of the genomic regions sampled for sequencing and more reproducible recovery of sequenced regions [31].
- The simulation revealed that more than 100 K fragments produced from most of the RE pairs se- lected except the pairs, MseI-MluCI, MspI-PstI and EcoRI-PstI (Table 1).
- Of the 15 pairs of REs used for the in silico test, nine different pairs of REs were selected for ddRADseq.
- was selected it as the most suitable pair of restriction en- zymes for digesting the BMSB genome via ddRAD se- quencing (Table 1, Additional file 1)..
- Using quality-trimming of the se- quence data, 387,629 SNPs were estimated from 399 BMSB individuals.
- Genetic clusters were observed among the BMSB populations.
- There was a genetic divergence among the BMSB individuals from native regions of China and Japan, while those of invaded countries were more closely related in the net- work.
- One individual from Chile was found in the same clade of the Chinese samples, suggesting that this BMSB Table 1 Summary of the in silico and in vitro tests of RE pairs.
- Note: a Prediction of the DNA segments of 300 – 500 bp against the BMSB genome scaffold.
- The rest of the Chilean samples were dis- tantly related from those in China and Japan but were more closely related to the samples from the European/.
- Moreover, the F ST value between the BMSB populations from the neighbouring countries was very small, for ex- ample, Turkey and Georgia (F ST = 0.0165).
- A Neighbour-net tree constructed using the F ST pairwise values among the individuals from the 12 countries revealed the similar relationships among the BMSB populations from the 12 countries (Fig.
- The tree depicted the overall relationships of the populations and showed that Chinese and Japanese pop- ulations were clustered together, but genetically differ- ent.
- The populations from the invaded countries were genetically linked, but the populations from Romina formed a long branch, indicating the genetic separation from those of the other countries studied (Fig.
- It also demonstrated that the BMSB from the adjoining coun- tries, i.e.
- Five genetic clusters exist in the BMSB populations Furthermore, insights into the BMSB genetic diversity were unravelled by population genetic structure analysis using fastSTRUCTURE.
- 1) and provided more in-depth clus- tering for the BMSB populations from the invaded countries.
- three genetic clusters within the BMSB-invaded coun- tries (Fig.
- The BMSB pop- ulations from China (Cluster 4) and Japan (Cluster 5) were clearly separated from the invasive populations (Fig.
- Fst analysis of the five genetic clusters (Table 3) showed that the genetic distance was moderately higher (Fst >.
- 3 A roadmap of the most likely BMSB invasion pathways constructed based on the results of this study.
- (PCA), phylogenetic analysis and population structure analysis to elucidate the population structure and genetic diversity among the BMSB populations.
- The present study unambiguously proved that the BMSB populations in the two native regions of China and Japan were genet- ically distinct.
- Overall, this study has provided a remarkable resolution in unravelling the Table 2 The group pairwise F ST (Fixation index) between the BMSB populations from 12 countries.
- population structure and estimating the genetic related- ness among the BMSB populations from a set of native and invaded regions of the world..
- Genetics isolation between the BMSB populations from China and Japan.
- The fastSTRUCTURE analysis showed that population structures of the BMSB samples from China and Japan were derived predominantly from.
- These results sug- gested that there is genetic isolation for the BMSB popu- lations of the two countries.
- 2014 [22] also observed genetic divergence between the BMSB popula- tions from China and Japan via haplotype analysis of mitochondrial COII and 12S/CR gene regions.
- We cannot rule out the possibility of the BMSB hitchhiker capabil- ity, however this study suggest that there is limited mi- gration among BMSB populations between the two countries.
- Different colours in a bar represent proportion of the genomic component in a sample derived from different populations.
- Estimation of the BMSB invasion pathway.
- 0.25) between the BMSB populations in Japan and the invaded countries of Chile, Georgia, Italy, Romania, Turkey, and the USA (Table 2).
- Therefore, we can conclude that the BMSB populations in the six in- vaded countries might not have originated from Japanese populations.
- The smallest F ST values of 0.0006 between the BMSB populations from Hungary and Serbia, the neighbouring countries, imply they were possibly from the same origin.
- The Fst analysis also indicated that the BMSB populations from Turkey and Georgia (Table 2) were more likely shared the same source and there were gene flow among them.
- It is also possible for the BMSB populations from Austria and Slovenia (Table 2)..
- The rest of the BMSB samples from Italy were more closely related to the USA and other European populations, suggesting that mul- tiple BMSB invasions have occurred in Italy in the past [16, 40].
- Most of the BMSB specimens from Chile, except one individual, were genetically close to each other (Figs..
- Predication of the BMSB genetic cluster.
- The population structure analysis of BMSB indicated that there were at least five genetic clusters identified among the BMSB populations studied here.
- Besides the genetic clusters of native BMSB populations from China and Japan, there were three genetic clusters in the BMSB-invaded countires.
- The analysis showed that the BMSB populations from the USA, Georgia, Turkey, Italy, Hungary, and Chile were more likely belong to one gen- etic cluster due to their similar genetic structure.
- In saying that, the population genetic structure analysis also showed that the BMSB populations from Slovenia, Austria, and Serbia contained the components of the Europe- established BMSB populations (Fig.
- This analysis indicated that there was inbreeding between the BMSB individuals from the adjoined established European BMSB populations and the Chinese populations (Figs.
- The F ST values be- tween the BMSB samples from Romania and the two Table 4 Analysis of molecular variance (AMOVA) the five genetic clusters derived from the 389 individuals from the 12 countries.
- The results showed that most of the variation in the dataset was found among the individuals within countries.
- Therefore, further study of ddRADseq of BMSB samples from those countries will provide more insights into genetic diversity of the BMSB populations and facilitate in inferring a more in-depth invasion pathway..
- Novel genetic analysis for the BMSB populations from the invade countries.
- This study also provided novel genetic information on the BMSB populations for some of the European coun- tries, such as Austria, Serbia, Slovenia, and Georgia..
- The results showed that the BMSB populations from Turkey were more likely the next generation of the BMSB from the USA whereas those populations from Austria, Serbia and Slovenia were the hybrids of Chinese and USA populations (Fig..
- The results obtained here will be useful for future monitoring of the pest and assessing the potential path- ways of invasion in those countries..
- As an alternative, analysis of genome-wide SNPs via whole genome sequencing can be applied for deeper un- derstanding the BMSB genome and genetic diversity..
- However, it is still more expensive due to the large size of the BMSB genome.
- geographic origin of the BMSB samples, irrespective of their life stages, and sex, that are intercepted at border..
- This study demonstrates that the restriction enzymes pair of EcoRI and MspI is suitable for generating restriction-associated DNA fragments for identifying SNPs within the BMSB genome through ddRAD sequen- cing.
- The selection of restriction enzyme (RE) is the crucial step for experimental ddRADseq analysis and discovery of the SNPs across the genome for genetic diversity study, thus initial in silico tests on the RE used for the li- brary was conducted.
- A Python script (Additional file 6) was devel- oped to calculate the number of the potential sheared segments that could be generated by in silico digestion of the reference genome using different combination of restriction enzymes..
- To further confirm the suitability of the RE pairs, two DNA samples were used for ddRADseq and 18 ddRADSeq libraries (nine pairs of enzymes for each sample) were prepared using Illumina® TruSeq DNA Nano following the manu- facturer’s instructions and ToBo lab ezRAD Protocol- v3.2 [52].
- Then, quality- trimmed sequence datasets, were mapped to the BMSB reference genome, Hhal_2.0, using Borows-Wheeler Aligner (BWA) version in default setting and files with the mapping information (i.e.
- bioinformatics analysis was performed on the high- performance computing platform of the New Zealand eScience Infrastructure (NeSI), Auckland, New Zealand..
- Briefly, genomic DNA was digested at 37 °C for 5 h using 10 Units of the two se- lected restriction enzymes, namely EcoRI and MspI (NEB, MA, USA), and deactivated at 65 °C for 20 mins, then followed by the ligation of Illumina adapter se- quences and unique 8 bp barcodes, which varied by at least three bases.
- To call SNPs from ddRAD- seq data, the raw fastq files were mapped to the scaffold of the reference genome, Hhal_2.0 (RefSeq assembly ac- cession: GCF to produce a mapping file (SAM format) using BWA v .
- Using the SNPs identified from the 389 BMSB individ- uals, we conducted principal component analysis (PCA), neighbour-net tree and population structure analysis using fastSTRUCTURE to elucidate the population structure and genetic diversity among the BMSB popula- tions.
- To further explore the genetic relatedness among the BMSB individuals, a Minimum Spanning Network (MSN) based on the SNP results of each individual was also reconstructed using R package poppr [33]..
- A roadmap of the BMSB invasive pathway was created using Tableau [63] by inputting a excel file with known BMSB background information.
- This work is dedicated to the memory of the late Dr.
- Our special thanks go to the Entomology team members of the PHEL of MPI for supplying the BMSB specimens intercepted at the New Zealand border or from the post-border surveillance and assisting in morphological identification of the specimens.
- Finally, we would like to thank the following researchers for providing the BMSB specimens from a range of different countries:.
- This research was partly supported by the Hungarian Ministry for Innovation and Technology within the framework of the Higher Education Institutional Excellence Program (NKFIH in the scope of plant breeding and.
- This research was also supported by the Ministry for Innovation and Technology within the framework of the Thematic Excellence Programme 2020- Institutional Excellence Subprogram (TKP2020-IKA-12) for research on plant breeding and plant protection..
- GV, PF, JZ and RG collected and provided the BMSB specimens..
- GV, PF and RG also provided substantive revision of the manuscript.
- All authors have read, edited and approved the final version of the manuscript..
- The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in the preparation of the manuscript.
- All BMSB specimens imported into New Zealand were in accordance to the Import Health Standard, Section 22 of the Biosecurity Act 1993..
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