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.