Ploidy determination, genotype identification and genome size estimation
To create a plant that was suitable for genome sequencing, and also was more juvenile to promote transformability and regenerability when making transgenic plants, we regenerated plantlets from anther callus ofP. tomentosa (using a male elite clone LM50, that otherwise shows low transformation efficiency). Though from anther culture, the conservation of ploidy level of the regenerated plantlet (GM15) (Fig. 1a) was determined by a number of approaches. This included flow cytometry (Fig. 1b), which showed that both genotypes were diploids. It was further confirmed by chromosome counts (Fig. 1c), and the use of allele-specific primers located on each of the 19 chromosomes (they had been previously developed for genotype identification: Table S1); the electrophoretic images of all amplified alleles of clone LM50 and its anther derived plant GM15 appeared identical (Fig. 1d). Finally, we estimated the genome sizes of both GM15 and LM50 by K-mer sequence analysis. It suggested they are almost the same, approximately 800 Mb as would be expected for a diploid poplar (Fig. 1e). We therefore conclude that the anther regenerated clone GM15 used for seqeuencing developed from somatic cells in the anther, not from gametes; it is thus a legitimate representative of its parent P. tomentosa genotype, LM50.