Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

Transposable elements in a marginal plant population

Background
How new forms arise in nature has engaged evolutionary biologists since Darwin's seminal treatise on the origin of species. Transposable elements (TEs) may be among the most important internal sources for intraspecific variability. Thus, we aimed to explore the temporal dynamics of several TEs in individual genotypes from a small, marginal population of Aegilops speltoides. A diploid cross-pollinated grass species, it is a wild relative of the various wheat species known for their large genome sizes contributed by an extraordinary number of TEs, particularly long terminal repeat (LTR) retrotransposons. The population is characterized by high heteromorphy and possesses a wide spectrum of chromosomal abnormalities including supernumerary chromosomes, heterozygosity for translocations, and variability in the chromosomal position or number of 45S and 5S ribosomal DNA (rDNA) sites. We propose that variability on the morphological and chromosomal levels may be linked to variability at the molecular level and particularly in TE proliferation.

Results
Significant temporal fluctuation in the copy number of TEs was detected when processes that take place in small, marginal populations were simulated. It is known that under critical external conditions, outcrossing plants very often transit to self-pollination. Thus, three morphologically different genotypes with chromosomal aberrations were taken from a wild population of Ae. speltoides, and the dynamics of the TE complex traced through three rounds of selfing. It was discovered that: (i) various families of TEs vary tremendously in copy number between individuals from the same population and the selfed progenies; (ii) the fluctuations in copy number are TE-family specific; (iii) there is a great difference in TE copy number expansion or contraction between gametophytes and sporophytes; and (iv) a small percentage of TEs that increase in copy number can actually insert at novel locations and could serve as a bona fide mutagen.

Conclusions
We hypothesize that TE dynamics could promote or intensify morphological and karyotypical changes, some of which may be potentially important for the process of microevolution, and allow species with plastic genomes to survive as new forms or even species in times of rapid climatic change.

Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

The Transcriptional Regulation of Retrotransposon BARE

The purpose of this research project was to understand the steps of the retrotransposon BARE (BArley REtrotransposon) life cycle, from regulation of transcription to Virus-Like Particle (VLP) formation and ultimate integration back into the genome. Our study concentrates mainly on BARE1 transcriptional regulation because transcription is the crucial first step in the retrotransposon life cycle. The BARE element is a Class I LTR (Long Terminal Repeat) retrotransposon belonging to the Copia superfamily and was originally isolated in our research group. The LTR retrotransposons are transcribed from promoters in the LTRs and encode proteins for packaging of their transcripts, the reverse transcription of the transcripts into cDNA, and integration of the cDNA back into the genome. BARE1 is translated as a single polyprotein and cleaved into the capsid protein (GAG), integrase (IN), and reverse transcriptase-RNaseH (RT-RH) by the integral aspartic proteinase (AP). The BARE retrotransposon family comprises more than 104 copies in the barley (Hordeum vulgare) genome. The element is bound by long terminal repeats (LTRs, 1829 bp) containing promoters required for replication, signals for RNA processing, and motifs necessary for the integration of the cDNA. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Several basic questions concerning transcription are explored in the thesis: BARE1 transcription control, promoter choice in different barley tissues, start and termination sites for BARE transcripts, and BARE1 transcript polyadenylation (I). Polyadenylation is an important step during mRNA maturation, and determines its stability and translatability among other characteristics. Our work has found a novel way used by BARE1 to make extra GAG protein, which is critical for VLP formation. The discovery that BARE1 uses one RNA population for protein synthesis and another RNA population for making cDNA has established the most important step of the BARE1 life cycle (III). The relationship between BARE1 and BARE2 has been investigated. Besides BARE, we have examined the retrotransposon Cassandra (II), which uses a very different transcriptional mechanism and a fully parasitic life cycle. In general, this work is focused on BARE1 promoter activity, transcriptional regulation including differential promoter usage and RNA pools, extra GAG protein production and VLP formation. The results of this study give new insights into transcription regulation of LTR retrotransposons.