The starting mechanism for paramutation: cytoplasm as a factor --Zavalishina, AN, Tyrnov, VS The phenomenon of paramutation in maize has been investigated for a long time and on a large scale, on both the genetic and the molecular levels. However, to this day the reasons and mechanisms of the origin of paramutation have not been ascertained.

Study of progeny of androgenesis in vivo demonstrate that in lines with substituted cytoplasm, paramutations arise as a regular phenomenon. By transference of the genome, including dominant genes B1 and Pl1, onto different cytoplasms, in some generations these genes transferred from a state of active expression to reduction of expression, from the state B1 and Pl1 to the state of paramutagenic genes B` and Pl` (MNL 69:120-121, 1995; MNL 72:74-75, 1998). It happened, more often, after 2-3 generations, rarely 4-5, since genome and different cytoplasm were joined in the initial androgenic plant. Such regularity in the transformation of the genome was observed by transference of the genome by the method of androgenesis both on sterile and on normal cytoplasms. Sometimes genes B1 and Pl1 became paramutagenic in the first generation by the union of different cytoplasms and genomes and manifested in the phenotype of the initial androgenic plant. It is possible this happens because of a significant difference in the substituted cytoplasm from the initial one. For example, paramutagenic genes B`or Pl` or both, manifested relatively often in androgenic haploids produced on S-type cytoplasm. An exclusive case was observed when, in progeny of androgenic plants with normal cytoplasm, paramutagenic genes were not discovered in 15 generations. Probably in this case, both substituted and initial cytoplasms did not differ from one another. In the initial maize lines, which were donors of the genomes for the androgenic plants, paramutagenic genes were not discovered in more than 20 generations.

Confirmation that the cytoplasm, but not androgenesis, influences the origin of paramutagenic genes, comes from results obtained by crossing of lines with subsequent backcrossing or selfing. In our experiments with the transference of genomes, including the dominant genes A1, B1, Pl1, R1 or a1, B1, Pl1, R1, in a line with CMS, paramutagenic genes B`, Pl1`, R` also manifested in backcrossed progeny. It must be noted that, as well as by androgenesis, paramutagenic genes more often manifested in some years after the beginning of backcrossing and their expression decreased to a state of B`, Pl`, R`. Analogous results were also obtained by self-pollination. From the hybrid, the maternal parent of which was line W23 with normal cytoplasm, and the male parent was a line with the genes a1, B1, Pl1, R1, a line was picked out with these genes in a homozygous condition. This line preserves active expression of color genes in three generations. In subsequent generations, expression of these genes first decreases in solitary individuals, and then in all plants up to full suppression of expression of B`, Pl`.

Once more, confirmation of cytoplasm participation in the origin of paramutations was obtained in an experiment in which reversion of paramutagenic genes to a state of complete expression on the initial cytoplasm was observed. For that purpose, a special experiment was organized in which two lines were used — the initial Brown marker line with the genes B1 and Pl1 in a state of complete expression and its line-analogue, produced by the method of androgenesis in vivo on cytoplasm of line W23, in which paramutagenic genes B` and Pl` then manifested. We carried out a series of direct and reverse saturating crosses between these two lines. In progenies, both in direct and in reverse crosses, only plants with paramutagenic genes B` and Pl` first appeared. Partial reversion to a state of active expression of paramutagenic genes B` and Pl’ was discovered after the third backcross, and complete reversion after the fourth backcross, on the initial cytoplasm of the Brown marker line. In spite of the same heterozygosity (normal and paramutagenic genes) on cytoplasm W23, reversion was not discovered.

Additional arguments, confirming indirectly, that namely the initial cytoplasm, but not heterozygosity, influences reversion of paramutagenic genes, follow our experiments. By both backcrossing and androgenesis in vivo we produced analogous lines with sterile cytoplasms. Initially, genomes of these analogues included all or part of genes A1, B1, Pl1, R1. The fact is well-known, that for production of progeny from analogous lines with CMS they must be pollinated by pollen of the initial lines. This procedure was used every year. As a result, in some generations in all analogous lines on sterile cytoplasms, irrespective of its production by androgenesis or by backcrossing, paramutagenic genes B’, Pl’, R’ were manifested. The more generations were produced in analogous lines, the more strongly suppressed was expression of genes B1, Pl1, R1, which came with pollen. They also became paramutagenic. In all these numerous experiments, which continued sometimes more than dozens of years, we not once discovered reversion of paramutagenic genes to a state of complete expression, in spite of constant heterozygosity in result of pollination.

Obviously, starting or switching of expression of nuclear genes is in some manner associated with cytoplasm. This connection was not discovered till now, probably, for several reasons. First of all, changes of genome arise, as a rule, in some generations after the union of the genome with a different cytoplasm, and often these changes first manifest not in complete expression, but as a small decrease of expression of some color genes. Secondly, after this process has happened and genes become paramutagenic, they that was for some generations, irrespective of the cytoplasmic background.

The importance of research into the role of the cytoplasm is also that substituted cytoplasm influences a change in quantitative signs. In our experiments with change of color genes' expression, a change in quantitative signs influencing crops was observed (MNL 75:57-58, 2001). It is possible that by transference of the genome onto different cytoplasms, one may obtain not only changes of selectively significant signs, but a fixation of these changes.

Probably, cytoplasmic factors play a more important role, than has been considered till now, in the instability of the genome and in the formative process, having evolutionary and selective value. We are ready to cooperate for discussion of this problem and joint research.
 
 


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