RESUMEN
BACKGROUND: The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). RESULTS: Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. CONCLUSIONS: Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.
RESUMEN
The sweet cherry is an important fruit species that is widespread globally. In addition to the well-known traditional and modern varieties, a myriad of landraces is present in Europe, as well as in southern Italy. This study aims to evaluate the population structure, genetic relationships, and cases of duplicate samples in a collection of 143 accessions using GBS-derived SNP markers. The genetic material under investigation includes modern commercial varieties, ancient European and American varieties, landraces, and individuals retrieved from small orchards. Some of the known varieties were genetically analyzed here for the first time. In addition, several genotypes were collected from the Basilicata region (southern Italy), an area largely unexplored for sweet cherry genetic resources. The relationships among genotypes were assessed using four different methods: allele frequency and ancestry estimation, principal component analysis, Neighbor-Joining tree, and identity-by-state estimation. The analyses returned quite congruent results and highlighted the presence of four main genetic groups, namely: (i) American varieties, (ii) the 'Germersdorfer-Ferrovia' cluster, (iii) the 'Burlat' group, and (iv) the group of Italian landraces. The main drivers of clustering were ancestry, geographical distribution, and some important traits such as self-compatibility. The sweet cherries from Basilicata, herewith examined for the first time, were mostly distributed within the group of Italian landraces, being particularly linked to the autochthonous varieties of the Campania region. However, some genotypes were outside this group, thus suggesting the introduction of genetic material from other Italian regions or from European countries. The considerable amount of American and European modern varieties analyzed are genetically very closely related, suggesting a reduced genetic basis. In addition, we highlighted the discriminating ability of SNP markers to distinguish between an original variety and its mutant. Overall, our results may be useful in defining conservation strategies for sweet cherry germplasm and developing future breeding programs to enlarge the genetic basis of commercial varieties.