In a groundbreaking study, scientists have successfully assembled the chromosome-level genome of the Japanese sawyer beetle, Monochamus alternatus. This achievement was made possible through a combination of advanced sequencing technologies, including Nanopore, Illumina short-read sequencing, and chromosome conformation capture (Hi-C). The successful assembly of the beetle’s genome provides valuable genomic resources for future research on topics such as ecology, genetics, evolution, and the interaction between the pinewood nematode and its insect vector.
The research team obtained samples of M. alternatus from a laboratory strain reared at the Key Laboratory of Forest Protection of National Forestry and Grassland Administration in Beijing, China. These samples were sourced from a strain that had been reared in the laboratory for approximately 30 generations. The scientists utilized a single female adult beetle to construct libraries for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing, and Hi-C. The adults were starved for 24 hours, and their internal guts were removed to minimize contamination from gut microbes. Additionally, larvae, pupae, and additional adult beetles were collected for transcriptome sequencing. All collected samples were stored at ultra-low temperatures until further usage.
For short-read sequencing, the researchers extracted genomic DNA using a reliable extraction kit and prepared a pair-end library with an insert size of approximately 300 base pairs. This library was then sequenced using the Illumina NovaSeq 6000 platform, resulting in a staggering 42.5 gigabases of Illumina short reads.
Long-read sequencing, on the other hand, involved isolating high molecular weight genomic DNA and selecting long DNA fragments using the PippinHT system. The researchers used the ONT library preparation protocol, which included an A-ligation reaction and ligation with adapters. Subsequently, the constructed DNA library was sequenced using the Nanopore PromethION sequencer instrument, generating an impressive 142.7 gigabases of long reads.
To perform Hi-C sequencing, the scientists employed a modified protocol where an adult M. alternatus beetle was cross-linked using a formaldehyde solution. After cross-linking, the sample was processed to separate the nuclei, which were then subjected to further treatment and enzymatic digestion. The resulting Hi-C library was constructed and sequenced using the Illumina HiSeq platform. Ultimately, a total of 81.7 gigabases of clean Hi-C data was generated.
For transcriptome sequencing, RNA was extracted separately from the larvae, pupae, and adult beetles of M. alternatus. The extracted RNA was used to construct libraries, which were then sequenced using the Illumina NovaSeq 6000 platform. In total, 18.9 gigabases of transcriptome data was obtained.
Using the data obtained from the various sequencing methods, the scientists were able to estimate the genome size, heterozygosity rate, and duplication rate of M. alternatus. The estimated genome size was found to be approximately 667 megabases, with a heterozygosity rate of 1.31% and a duplication rate of 1.55%.
The researchers then embarked on the assembly of the M. alternatus genome, first generating a draft genome at the contig level using NextDenovo software. The contig assembly was further refined using a tool called Purge_dups to remove redundant fragments. Next, the team performed Hi-C analysis to anchor the contigs to chromosome-scale linkage groups, offering a more complete and comprehensive view of the genome. After two rounds of polishing using ONT and Illumina reads, the scientists successfully obtained a chromosome-level genome for M. alternatus, which had a size of 767.12 megabases, an N50 of 82.0 megabases, a maximum length of 149.24 megabases, and a GC content of 32.35%.
The study also included the prediction and annotation of protein-coding genes in the M. alternatus genome. The researchers utilized three approaches, including RNA-based, ab initio, and homology-based methods, to predict the genes. A non-redundant consensus official gene set (OGS) was generated by combining the evidence from these methods. 16,284 protein-coding genes were identified, with 11,244 genes functionally annotated.
Additionally, the study investigated the presence of transposable elements (TEs) and non-coding RNA in the M. alternatus genome. The researchers used different methods to detect TEs, ultimately identifying a total of 576,182 TEs, including retroelements and DNA transposons. The presence of transposable elements plays a crucial role in shaping the genome and influencing genetic variability within a species. Moreover, the study annotated transfer RNA (tRNA) and ribosome RNA (rRNA) genes, identifying 498 tRNA genes and 107 rRNA genes.
This chromosome-level assembly of the Japanese sawyer beetle genome provides scientists with valuable tools and resources for future research on this species. With the availability of this high-quality genome assembly, researchers can investigate various aspects of M. alternatus, including its ecology, genetics, evolution, and the intricate relationship between the insect vector and the pinewood nematode. The data obtained from this study will undoubtedly facilitate a better understanding of the biology and intricate interactions of this economically important beetle.
Overall, the successful assembly of the chromosome-level genome of the Japanese sawyer beetle marks a significant milestone in the field of genomics and opens up new avenues of research for scientists studying this insect species. The insights gained from this study may have far-reaching implications for pest management strategies, forest protection efforts, and our understanding of the intricate ecological dynamics at play within forest ecosystems.
