A short communication: Genome editing and mutation detection of triploid tree induced by CRISPR/Cas9

Although the CRISPR/Cas9 system has been successfully used for diploid trees breeding, its application remains limited in polyploid trees. The detection of CRISPR/Cas9-induced mutations in polyploid trees remains challenging due to the genomic complexity caused by multiple sets of chromosomes in a single cell. To explore methods for genome editing and mutation detection in polyploid trees, this study used diploid poplar Populus alba × P. glandulosa (poplar 84 K) and triploid poplar Populus × aldatomentosa CL. 741 (poplar 741) as the research materials. CRISPR/Cas9 constructs were delivered via Agrobacterium -mediated transformation, enabling targeted mutagenesis of PagSPL26 , PagSPL35, and PalMYB3R-1 genes. We used a method based on a two-round PCR strategy coupled with Hi-TOM platform, allowing effective identification of biallelic and triallelic knockout mutations in both diploid and triploid genetic backgrounds. Sequencing analysis revealed that 100% of edited diploid plants carried homozygous mutations, whereas 57.14% of edited triploids exhibited homozygous mutations. Unlike conventional T-cloning followed by Sanger sequencing, this study uses chromosome-specific primers to amplify target regions covering CRISPR-induced edits sites across chromosomes. Combined with Hi-TOM platform, this study enables accurate quantification of mutation types and allele frequencies, significantly enhancing the precision of selecting homozygous edits in polyploids. These results establish the successful application of CRISPR/Cas9 for generating mutations in polyploid trees and enable efficient screening of homozygous mutations, thereby broadening the scope of precision genome editing in polyploid species and accelerating both functional genomics and trait improvement in perennial forest trees. • CRISPR/Cas9 successfully applied to triploid poplar. • Two-round PCR + Hi-TOM enables precise mutation detection. • Chromosome-specific primers allow accurate allele quantification. • 57.14% of edited triploids showed homozygous mutations. • Provides efficient screening for polyploid genome editing.