Estimation of stoichiometry of marker sequences of the sugar beet (Beta vulgaris) mitochondrial genome by real-time PCR

Abstract

47 Plant mitochondrial genomes exhibit some features distinguishing them from the mitochondrial genomes of animals and fungi, the main of which are a large size (with the coding capacity being similar) and a complex organization. It was shown that the mitochondrial DNA fraction (mtDNA) contains concatemers, circular molecules with linear fragments, and subgenomic circular and linear variants whose stoichiometry may vary [1‐5]. The traditional representation of full-length sequences of plant mitochondrial genomes in the form of the “master” chromosome apparently does not reflect their state in vivo , because it does not take into account the lowcopy-number and recombinant sequences located in untypical environment. The trait of cytoplasmic male sterility (CMS) is related to the presence in mitochondria of a special genome type. In the case of Owen’s type of CMS in sugar beet ( Beta vulgaris ), this is the Svulg genome. This trait is expressed only in a special nuclear environment and consists in the inability of the plant to produce fertile pollen. CMS is usually caused by the formation of chimerical sequences, with this process being facilitated by a high recombination activity of plant mitochondrial genome. To date, two variants of the main chromosome of B. vulgaris mitochondrial genome have been sequenced: the N type, which ensures the formation of fertile pollen, and the Svulg -type [6, 7]. A large number of regions unique for each genome type does not make it possible to assign CMS to a loss or acquisition of a certain sequence in sugar beet. Additionally, the analysis of translation patterns of N- type and Svulg type mitochondria did not allow the CMS determinant to be determined at the protein level [8]. The relationship between CMS and selective amplification of certain subgenomic variants has been demonstrated for the haricot ( Phasoleus vulgaris ) [9]. Probably, the expression of CMS in B. vulgaris is also ensured not only by the presence of the genome (the “master” chromosome) but also by the composition and stoichiometry of subgenomic variants. In this case, the number of copies of subgenomic variants of mtDNA in the cytoplasm of sterile and fertile plants should be determined to find the relationship between CMS and the mitochondrial genome structure. In this study, we performed a comparative analysis of the number of copies of marker sequences in N -type and Svulg -type mitochondrial genomes. For this purpose, we for the first time used the real-time PCR—one of the most sensitive and precise methods of determination of the amount of certain sequences in DNA samples [10].