Four large indels detected by cpTILLING in barley chloroplast mutator seedlings

In a previous work, a polymorphism detection strategy based on mismatch digestion was applied to the chloroplast genome of barley seedlings that carried the chloroplast mutator (cpm) genotype through many generations. Sixty-two different one- or two-nucleotide-polymorphisms were detected along with four large indels: an insertion of 15 bp in the intergenic region between tRNAHis and rps19 genes, a deletion of 620 bp in the psbA gene, a deletion of 79 bp in the intergenic region between rpl33 and rps18 genes and a deletion of 45 bp in the rps3 gene. In the present investigation, we analyzed direct repeats located at the borders of those four large indels. Furthermore, we investigated the consequences of protein expression of large indels located in coding regions. The deletion of 620 bp in the psbA gene was lethal at the second leaf stage when homoplastomic. The deletion of 45 bp in the rps3 gene, which eliminates 15 amino acids, did not affect the viability of the seedlings in homoplastomy. Interestingly, the deleted segment is also lacking in the wild type version of the rps3 gene of maize and sorghum. The presence of direct repeats at the borders of the four large indels suggests that they could have originated by illegitimate recombination. This would be in agreement with a previous hypothesis that the Cpm gene product would correspond to a mismatch repair (MMR) protein devoted to maintain plastome stability by playing fundamental roles in mismatch repair during replication and avoiding illegitimate recombination.