Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern.Many genetic strategies exist to mitigate potential gene flow;however,the long delay in onset of flow-ering is a severe constraint to research progress.We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes,LEAFY and AGAMOUS.We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T(AtFT)gene under control of either a strong constitutive promoter or a heat-inducible promoter.We successfully obtained flowering in both a male and female clone of poplar,observing a wide range of inflorescence and floral forms among flowers,ramets,and insertion events.Overall,flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes.LFY-targeted events showed small catkins with leaf-like organs,AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers.These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering,which may be of horticultural value.They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

GWAS identifies candidate genes controlling adventitious rooting in Populus trichocarpa

Adventitious rooting(AR)is critical to the propagation,breeding,and genetic engineering of trees.The capacity for plants to undergo this process is highly heritable and of a polygenic nature;however,the basis of its genetic variation is largely uncharacterized.To identify genetic regulators of AR,we performed a genome-wide association study(GWAS)using 1148 genotypes of Populus trichocarpa.GWASs are often limited by the abilities of researchers to collect precise phenotype data on a high-throughput scale;to help overcome this limitation,we developed a computer vision system to measure an array of traits related to adventitious root development in poplar,including temporal measures of lateral and basal root length and area.GWAS was performed using multiple methods and significance thresholds to handle non-normal phenotype statistics and to gain statistical power.These analyses yielded a total of 277 unique associations,suggesting that genes that control rooting include regulators of hormone signaling,cell division and structure,reactive oxygen species signaling,and other processes with known roles in root development.Numerous genes with uncharacterized functions and/or cryptic roles were also identified.These candidates provide targets for functional analysis,including physiological and epistatic analyses,to better characterize the complex polygenic regulation of AR.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants,particularly for wind-pollinated trees such as poplar.We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE(SVL),a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE(SVP),delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars.Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance,and did not cause morphologically obvious or statistically significant effects on leaf characteristics,tree form,or stem volume.Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific.Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.