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Reading Nuclear and Chloroplast DNA from the Same Sequencing Reads

Phylogenetic analysis is a widely used research method for understanding the evolutionary history and classification of plants. To date, the chloroplast (cp) genome has been commonly used in plant phylogenetic analyses. The chloroplast genome has the advantage of being small and easy to analyze, but because it is maternally inherited, it can follow a different evolutionary trajectory than nuclear DNA, which limits its use as an information source.

To obtain more comprehensive phylogenetic information, nuclear DNA information, which is inherited from both parents, is also important. Nuclear DNA provides a more holistic genetic perspective than cpDNA. However, acquiring full nuclear DNA information was previously associated with high costs. In recent years, highly cost-effective methods for obtaining nuclear DNA data have emerged (e.g., target capture, RNA sequencing), but it has generally been necessary to prepare chloroplast and nuclear DNA data separately.

In this study, to address this challenge, we focused on the development and validation of a new method that obtains nuclear DNA phylogenetic information from the “exact same” raw (unprocessed) read sequence data used for chloroplast genome analysis.

We utilized a new computational tool called Read2Tree. Read2Tree enables the direct and efficient extraction of conserved nuclear gene sequences from raw read sequence data.

Main Features and Advantages of Read2Tree

• Cost-effective: Using the same raw data as chloroplast analysis reduces data acquisition costs.
• Comprehensive nuclear data retrieval: Reference genome-guided approach allows extraction of a large number of conserved nuclear genes.
• Streamlined analysis: Functions as an integrated pipeline from gene identification through alignment generation to phylogenetic tree construction.
• No custom preparation required: Uses existing reference datasets, eliminating the need to design and validate species-specific probe sets or databases.
• Useful for discordance analysis: Outputs individual gene alignment data, which can be used to investigate causes of phylogenetic discordance (such as discrepancies between gene trees and species trees).

Method Validation: An Example Using Aurantioideae Plants

To validate the effectiveness and usefulness of this new method (nuclear DNA phylogenetic analysis using Read2Tree), we targeted the plant group Aurantioideae. Aurantioideae, which includes citrus and its close relatives, is an important group with complex phylogenetic relationships.

We used Read2Tree to build a nuclear DNA dataset for approximately 40 Aurantioideae species. The method was then validated using the following approaches:

• Comparison with existing nuclear DNA methods: The nuclear DNA phylogenetic tree obtained by Read2Tree was compared with the tree generated by RAD-Seq, another nuclear DNA method used in previous studies. This comparison evaluated the reliability of Read2Tree.
• Integration with chloroplast phylogenetic analysis: Phylogenetic trees of chloroplast genomes were constructed from the same raw data and compared with the nuclear DNA trees produced by Read2Tree. This approach investigated cytonuclear discordance, the mismatch between chloroplast and nuclear phylogenies.
• Analysis of discordance causes: Using individual gene alignment data output by Read2Tree and combining it with other methods (D-statistic , DFOIL , QuIBL , etc.), we analyzed whether observed phylogenetic discordance was caused by complex evolutionary processes such as incomplete lineage sorting (ILS), introgression, or ancient introgression.

Results (Method Effectiveness)

The results of this study revealed the following points, demonstrating the effectiveness of the method using Read2Tree:

• Concordance of nuclear DNA phylogenies: The nuclear DNA phylogenetic tree constructed with Read2Tree strongly matched the results obtained by RAD-Seq in previous studies. This finding indicates that Read2Tree can generate reliable nuclear DNA data for plant phylogenetic analyses.
• Proof of cost efficiency: It was reconfirmed that using the same raw data for chloroplast and nuclear DNA analyses with this method is highly cost-effective.
• Utility for discordance cause analysis: Utilizing Read2Tree output data revealed that discordance between nuclear DNA and chloroplast phylogenies observed in Aurantioideae, as well as discrepancies among gene trees, was primarily caused by incomplete lineage sorting (ILS), and that introgression and ancient introgression also contributed in specific lineages. This finding demonstrates that Read2Tree is useful not only for tree construction but also for elucidating complex evolutionary processes.

Conclusion

This study demonstrated that the computational tool Read2Tree is a highly effective and versatile method for plant phylogenetic analyses. In particular, its ability to cost-effectively obtain nuclear DNA data from the same raw reads used for chloroplast genome analysis , to construct accurate phylogenetic trees, and to analyze the causes of phylogenetic discordance resulting from complex evolutionary mechanisms like incomplete lineage sorting and introgression are significant strengths. Aurantioideae plants served as an excellent example to validate the effectiveness of this new method.

This method has the potential to contribute to more detailed and reliable reconstructions of evolutionary histories in future plant phylogenetic research.

Potential Applications in Food Research

The phylogenetic analysis method developed and validated in this study using Read2Tree is a powerful tool for elucidating the evolutionary history and genetic relationships of plants in detail. This insight not only advances evolutionary research but also holds great potential for research on plants that serve as our everyday food resources.

For example, groups like the Aurantioideae used in this study include plant species that are important for food and medicinal uses worldwide. Understanding their precise phylogenetic relationships and the complex evolutionary history of hybridization is crucial for crop improvement in agriculture and for the conservation and utilization of valuable genetic resources (such as wild relatives) that may become future food sources.

This new method using Read2Tree enables efficient retrieval of information from both chloroplast and nuclear DNA using the same dataset, allowing more comprehensive and detailed phylogenetic analyses of many food crop species that were previously difficult to analyze.

Moreover, the ability to analyze complex genetic processes such as incomplete lineage sorting (ILS) and introgression provides the foundational understanding at the genetic level of how diverse traits of food plants—such as high nutritional value, specific flavors, and resistance to pests and diseases—have been acquired and maintained throughout evolutionary history.

In this way, this method can be said to offer an important foundation for deepening our understanding of the diversity of plants used as food and for driving research toward the development of improved varieties and sustainable resource utilization.