Exploiting genome variation to improve next-generation sequencing data analysis and genome editing efficiency in Populus tremulaxalba 717-1B4

Populus species are widely distributed across the Northern Hemisphere. The genetic diversity makes the genus an ideal study system for traits of ecological or agronomic significance. However, sequence variation between the genome-sequenced Populus trichocarpa Nisqually-1 and many other Populus species and hybrids poses significant challenges for research that employs sequence-sensitive approaches, such as next-generation sequencing and sitespecific genome editing. Using the routinely transformed genotype Populus tremulaxalba 717-1B4 as a test case, we utilized established variant-calling pipelines with affordable re-sequencing (similar to 20x) and publicly available transcriptome data to generate a variant-substituted custom genome (sPta717). The sPta717 genome harbors over 10 million SNPs or small indels relative to the P. trichocarpa v3 reference genome. When applied to RNA-Seq analysis, the fraction of uniquely mapped reads increased by 13-28 % relative to that obtained with the P. trichocarpa reference genome, depending on read length and sequence type. The enhanced mapping rates enabled detection of several hundred more expressed genes and improved the differential expression analysis. Similar improvements were observed for DNA-Seq and ChIP-Seq data mapping. The sPta717 genome is also instrumental in guide RNA (gRNA) design for CRISPR-mediated genome editing. We showed that a majority of gRNAs designed from the P. trichocarpa reference genome contain mismatches with the corresponding target sequences of sPta717, likely rendering those gRNAs ineffective in transgenic 717. Awebsite is provided for querying the sPta717 genome by gene model or homology search. The same approach should be applicable to other outcrossing species with a closely related reference genome.