Complete plastome sequencing of Allium paradoxum reveals unusual rearrangements and the loss of the ndh genes as compared to Allium ursinum and other onions.

In this work the complete chloroplast DNAs of Allium paradoxum and Allium ursinum, two edible species of Allium subg. Amerallium (the first lineage), were sequenced, assembled, annotated, and compared with complete Allium plastomes of the second and third evolutionary lines from GenBank database. The A. ursinum plastome contains 90 predicted genes (81 unique) including 5 pseudogenes, while A. paradoxum has 88 predicted genes (79 unique) including 19 pseudogenes. The comparative analysis has revealed that the A. paradoxum plastome differs markedly from those of other species. Due to many deletions, the A. paradoxum plastome is the shortest of known for Allium species, being only 145,819 bp long. The most prominent distinctions are (1) a 4825 bp long local inversion that spans from the ndhE to the rpl32 gene in the small single copy region and (2) pseudogenization, or the loss of all NADH-genes. In contrast, the plastome of A. ursinum - a species from the first evolutionary line (as well as A. paradoxum) - resembles the Allium species of the second and third evolutionary lines, showing no large rearrangements or discrepancies in gene content. It is unclear yet whether only A. paradoxum was affected by some evolutionary events or its close relatives from both sect. Briseis and other sections of Amerallium were altered as well. We speculate the sunlight-intolerant, shade-loving nature of A. paradoxum and the impairment of the ndh genes in its plastome could be interrelated phenomena.

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products.

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum.

BACKGROUND: The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. RESULTS: KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. CONCLUSIONS: The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time.