Tsimafei Padvitski

The goal of any plant breeding program is to improve quality of a target crop. Crop quality is a comprehensive feature largely determined by biological background. To improve the quality parameters of crops grown for the production of fiber, a functional approach was used to search for genes suitable for the effective manipulation of technical fiber quality. A key step was to identify genes with tissue and stage-specific pattern of expression in the developing fibers. In the current study, we investigated the relationship between gene expression evaluated in bast fibers of developing flax plants and the quality parameters of technical fibers measured after plant harvesting. Based on previously published transcriptomic data, two sets of genes that are upregulated in fibers during intrusive growth and tertiary cell wall deposition were selected. The expression level of the selected genes and fiber quality parameters were measured in fiber flax, linseed (oil flax) cultivars, and wild species that differ in type of yield and fiber quality parameters. Based on gene expression data, linear regression models for technical stem length, fiber tensile strength, and fiber flexibility were constructed, resulting in the identification of genes that have high potential for manipulating fiber quality. Chromosomal localization and single nucleotide polymorphism distribution in the selected genes were characterized for the efficacy of their use in conventional breeding and genome editing programs. Transcriptome-based selection is a highly targeted functional approach that could be used during the development of new cultivars of various crops.

Fiber flax is an important source of natural fiber and a comprehensive model for the plant fiber biogenesis studies. Cellulose-synthase (CesA) and cytoskeletal genes are known to be important for the cell wall biogenesis in general and for the biogenesis of flax fibers in particular. Currently, knowledge about activity of these genes during the plant growth is limited. In this study, we have investigated flax fiber biogenesis by measuring expression of CesA and cytoskeletal genes at two stages of the flax development (seedlings and stems at the rapid growth stage) in several flax subspecies (elongatum, mediterraneum, crepitans). RT-qPCR has been used to quantify the expression of LusСesA1, LusСesA4, LusСesA7, LusСesA6, Actin, and α-Tubulin genes in plant samples. We report that CesA genes responsible for the secondary cell wall synthesis (LusCesA4, LusCesA7) have different expression pattern compared with CesA genes responsible for the primary cell wall synthesis (LusCesA1, LusCesA6): an average expression of LusCesA4 and LusCesA7 genes is relatively high in seedlings and further increases in stems at the rapid growth stage, whereas an average expression of LusCesA1 and LusCesA6 genes decreases. Interestingly, LusCesA1 is the only studied gene with different expression dynamics between the flax subspecies: its expression decreases by 5.2-10.7 folds in elongatum and mediterraneum but does not change in crepitans subspecies when the rapid growth stage and seedlings are compared. The expression of cytoskeleton genes (coding actin and α-tubulin) is relatively stable and significantly higher than the expression of cellulose-synthase genes in all the studied samples.

In silico search for the nucleotide sequences encoding cellulose synthases in the flax genome was carried out, together with the comparison of the identified sequences with the orthologous genes in dicotyledonous plants. The analysis resulted in the identification of 32 flax candidate genes, 16 of which encoded cellulose synthases with high possibility rate, while the remaining 16 encoded cellulose synthase-like proteins (Csl). The phylogenetic analysis of the protein products of the cellulose synthase genes allowed dividing them into six groups comprising cellulose synthases of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7, and CesA8. Paralogous sequences belonging to the classes CesA1/10 and CesA5/6/2/9, associated with the primary cell wall formation, were characterized by the higher intra-class similarity rate, than the orthologous sequences. At the same time, the genes that control cellulose biosynthesis for the secondary cell wall formation constituted distinct clades, CesA4, CesA7, and CesA8. The analysis of the 16 selected flax candidate cellulose synthase genes demonstrated that the flax genome contains at least 12 different variants of cellulose synthase genes, which belong to all six cellulose synthase clades. In such a way, at this stage, the cellulose synthase genes from all ten known CesA classes have been identified in the flax genome; however, their correct attribution to each of these classes requires some additional study.