Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study

Next-generation (NG) sequencing in a natural population of Populus nigra revealed a mutant with a premature stop codon in the gene encoding hydroxycinnamoyl-CoA : shikimate hydroxycinnamoyl transferase1 (HCT1), an essential enzyme in lignin biosynthesis. The lignin composition of P. nigra trees homozygous for the defective allele was compared with that of heterozygous trees and trees without the defective allele. The lignin was characterized by phenolic profiling, lignin oligomer sequencing, thioacidolysis and NMR. In addition, HCT1 was heterologously expressed for activity assays and crosses were made to introduce the mutation in different genetic backgrounds. HCT1 converts p-coumaroyl-CoA into p-coumaroyl shikimate. The mutant allele, PnHCT-1-Delta 73, encodes a truncated protein, and trees homozygous for this recessive allele have a modified lignin composition characterized by a 17-fold increase in p-hydroxyphenyl units. Using the lignin pathway as proof of concept, we illustrated that the capture of rare defective alleles is a straightforward approach to initiate reverse genetics and accelerate tree breeding. The proposed breeding strategy, called 'breeding with rare defective alleles' (BRDA), should be widely applicable, independent of the target gene or the species.