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

Abstract

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 tremula×alba 717-1B4 as a test case, we utilized established variant-calling pipelines with affordable re-sequencing (~20×) 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. A website 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.

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

“…Approximately 12–32 million (M) paired-end 75-bp reads were obtained per sample. To identify T-DNA insertion sites, individual reads were mapped to the vector as well as the Populus tremula x alba custom genome v1.1 45 using BLAT. Custom Perl scripts were used to filter for paired reads flanking an insertion site, e .…”

Salicylic acid activates poplar defense against the biotrophic rust fungusMelampsora larici‐populinavia increased biosynthesis of catechin and proanthocyanidins

“…Primer sequences for all genes used in this study are given in Supporting Information Table S1. WT from 35°C, SRP146326 and SRP146339 for transgenic line F10 from 27°C, and SRP146340, SRP146325, and SRP146322 for F10 from 35°C. Sequence reads were quality checked and mapped onto a variant-substituted P. tremula 9 alba genome (sP-ta717) v1.1 as described (Xue et al, 2015) using Tophat2 v2.0.12 (Kim et al, 2013). Transcript abundance was estimated by HTSeq (Anders et al, 2015) and differential expression performed using DESeq2 (Love et al, 2014).…”

Separate Pathways Contribute to the Herbivore-Induced Formation of 2-Phenylethanol in Poplar

“…Gray poplar is a hybrid species derived from a cross between silver poplar (Populus alba) and quaking aspen (Populus tremula) and BLAST searches with the different P. trichocarpa AAAD genes as query and the genomes of P. 3 canescens, P. alba, and P. tremula (http://aspendb. uga.edu/index.php/databases/spta-717-genome; Xue et al, 2015;Zhou et al, 2015) as templates revealed that all three species possess putative AADC1, AADC2, and AAS1 orthologs (Supplemental Fig. S8).…”