Format: ORAL

Zhi-Qiong Mo1,2,3, Chao-Nan Fu1, AlexTwyford 3, Pete M. Hollingsworth4, Jun-Bo Yang2, De-Zhu Li2,6, Lian-Ming Gao1,6*

1 CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 2 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JH, UK 5 Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK 6 Lijiang Forest Biodiversity National Observation and Research Station, Kunming Institute of Botany, Chinese Academy of Sciences, Lijiang 674100, Yunnan, China

Using the deep genome skimming (DGS) data to assembly target nuclear orthologous genes for phylogenomics has recently become attractive, but little practical exploration has been done for herbarium specimens. Sequences flanking target genes are useful for phylogenetic inference of shallow taxonomic levels, while it is underutilized in studies possibly due to its finite recovery. We evaluated the optimal sequencing depth and performance for recovering target nuclear gene from specimen accessions, by subsampling genome sequencing data (10, 15 and 20 sequencing coverage) with Rhododendron as example. To explore whether the sequence recovery of target exons and their flanking regions improved or not, we utilized exons only or allowed intron-containing sequences (supercontigs) as reference for read sorting in assembly process using HybPiper, respectively. Additionally, phylogenetic analyses were conducted to test the phylogenetic utilities of plastomes and the captured nuclear exons and their flanking sequences. Our results showed that DGS can be well applied for herbarium specimens to recover large number of nuclear genes, and the optimal sequencing depth for high-quality gene assembly from specimens was ~15 coverage. Further analyses suggested that the adoption of supercontigs as reference can capture more higher-integrity target exons and flanking regions, and can be used as an alternative to increasing sequencing depth to improve sequence recovery in the case of limited sequencing data. The high-integrity nuclear sequences recovered here and plastome sequences recovered robust phylogenetic relationships in Rhododendron. It is also noteworthy that the recovered non-targeted exon-flanking regions showed great promise for shallow-scale phylogenetic relationships. The exons and flanking regions captured by adding introns into references for assembly shed new light on resolving interspecies relationships by using DGS data which recover high-integrity target nuclear genes and their flanking regions for phylogenomics of an expanding sampling covering both the extant and the natural history collections.