Tilling: the modern technology in «reverse» genetic of plants (review)

There are two basic approaches in genetic studies called «forward» and «reverse» genetics. While the «forward» genetics studies the inheritance of traits (phenotype) in living organisms and identifies genetic factors that influence the expression of these traits (working on the principle of «from phenotype to genotype»), the «reverse» genetics reveals the function of gene by changing its structure or activity with subsequent analysis of the associated changes in phenotype (the «from genotype to phenotype» principle). With the development of large-scale genomic sequencing technologies the «reverse» genetics received substantial support, taking the leading position both in scientific and applied fields. Basic principles underlying the novel method of «reverse» genetics called TILLING (for Targeting Induced Local Lesions IN Genomes) are reviewed in this paper. TILLING combines the classic mutation analysis with modern approaches to detecting the nucleotide substitutions in a target locus in genome. The method is highly effective and is applicable to a wide range of biological objects, thereby winning a world-wide recognition. The key stages of preparation for TILLING analysis are described: obtaining of mutagenized population and development of TILLING platform, which includes the organized collection of mutants and database with information about the collection. The main approaches to the detection of point mutations currently used worldwide, including new approaches based on NGS methods (Next Generation Sequencing), are presented. The technical requirements crucial for the successful conducting of TILLING-analysis, as well as variations and modifications of existing techniques designed to solve various scientific tasks, are highlighted. The results obtained using the TILLING method by the group of authors in their research of specificity of partners recognition during the development of mutualistic symbiosis between the garden pea ( Pisum sativum L.) and nodule bacteria Rhizobium leguminosarum bv. viciae are also presented. Authors were able to identify a number of mutants in pea receptor kinase gene LykX, which is the most likely candidate for the determinant of plant’s increased selectivity toward bacterial microsymbionts. Further study of obtained mutants will help to reveal the function of LykX and its role in symbiosis between pea and nodule bacteria.