Biomics

A multiplex in silico RAPD-analysis of their complete genomes, which differ markedly in size, was performed for several plant species. From Crucifera family three lines of Arabidopsis thaliana with a genome size of about 135 million bp were investigated. The potato Solanum tuberosum and tomato S. lycopersicum from the Solanaceae family with genomes 840 and 828 million bp respectively were analyzed. Two subspecies of rice Oryza sativa indica and O. sativa japonica (500 million bp each), diploid wheat Triticum urartu (5 billion bp) and Aegilops tauschii (4.3 billion bp), as well as tetraploid wheat T. turgidum and T. dicoccoides (both species have genomes of about 12 billion bp) and hexaploid bread wheat T. aestivum (17 billion bp) were analyzed in silico for the presence of annealing sites in these genomes of a set of decamer primers was performed using the ABCDNA_GS program that we created earlier. A feature of primers containing 40% G and C bases is their trinucleotide composition with a complete absence of thymines, which helps to exclude the formation of homo- and heterodimers of such primers. It is shown that for plants with small genome sizes (for example, the Crucifera family), a satisfactory level of DNA polymorphism is achieved when a set of 12 primers is used in multiplex analysis, whereas 6 such primers are quite sufficient for large genomes. It is proposed to modify the RAPD-analysis in such a way that only short amplicons that can be separated by capillary gel electrophoresis and their lengths measured with accuracy to the nucleotide will be taken into account. The selected range of amplicons from 51 to 500 nucleotides accommodates 450 imaginary DNA cells, which are designated as a DNA[+]-cell if it contains a fragment(s) of DNA and DNA[-]-cell in their absence, which in binary is displayed as "1" and " 0 " respectively. The calculations of the number of possible combinations with different filling of such imaginary DNA cells, exceeding in some cases a googol (10¹⁰⁰), are given. Based on the results obtained in silico, genetic barcodes are constructed that allow visual observation of differences between the analyzed plant species. Minor differences found in Arabidopsis lines and rice subspecies allow us to predict the applicability of this approach to unambiguous DNA-cataloguing of plant cultivars. Moreover, this approach can be quite applicable to animal breeds, fungal races, and even microbial strains, requiring only an adjustment of the number of multiplex primers depending on the size of the genomes of the studied species. In addition to RAPD analysis, other methods of detecting DNA polymorphism can be used for the purpose of DNA-cataloguing, which provide accurate determination of the size of the resulting amplicons. Bioinformatic analysis of the complete genomes of the studied species using the ABCDNA_GS program can also serve in these cases to predict the results and pre-select sets of multiplex primers. It is noted that such DNA certification is extremely important for breeding and agricultural production.

DNA polymorphism, genome, multiplex PCR, primers, RAPD analysis, bioinformatic analysis, in silico, virtual amplicons, genetic barcoding, DNA-cataloguing, cultivar, selection

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