P2010-02: Tracing post-polyploidization genome restructuring in the Eastern Alpine autopolyploid complex of Senecio carniolicus (Asteraceae)
Project leader :
Polyploidy is recognized as major force driving plant evolution, diversification, and speciation. An indirect effect of polyploidization is that it often triggers genomic rearrangements as well as genic, genomic or epigenetic changes imposed by merging of more than two chromosome complements in one nucleus, contributing to functional genome diploidization. Type and extent of such changes vary among different plant groups and may involve homeologous recombination, changes in epigenetic regulation, or changes in the dynamics of repetitive DNA, most notably transposable elements. These changes have so far mostly been studied in allopolyploids (i.e., polyploids formed by merging and duplication of genomes of different species), but little is known about such genome dynamics in newly formed autopolyploids (i.e., polyploids originating by duplication of homologous chromosome sets within a taxon).
Recent methodological advances in next-generation sequencing, allow for the first time a comprehensive investigation of repetitive DNA, the major component of such genomic changes, in organisms with uncharacterized or only poorly characterized genomes. The Quaternary autopolyploid complex of Senecio carniolicus (Asteraceae) is a frequent and abundant element of alpine and subnival vegetation on siliceous bedrock in the Eastern Alps and comprises three main cytotypes (diploid, tetraploid and hexaploid), which show a highly complex distribution pattern and a high incidence of cytotype mixture.
We will use novel next-generation sequencing techniques combined with molecular cytogenetic analyses (FISH) to assess the dynamics of selected repetitive DNA families in all cytotypes and to evaluate their contribution to evolution of these genomes and to the diploidisation of polyploids. The incidence and directionality of such changes data will be interpreted in an existing phylogenetic framework, and their putative role as isolating mechanism between cytotypes will be addressed.
Project status 02/2011
The project, which started in 2010, aims to analyze in a comparative context the genome evolution in the autopolyploid complex of Senecio carniolicus. We have isolated genomic DNAs of multiple genotypes and cytotypes of Senecio (2x, 4x, and 6x), one of which (basic diploid) was selected for 454 next generation sequencing. The sequencing has been performed and the sequences (500 000 reads) were analyzed. Numerous clusters of repetitive DNA types were recovered and are now being analyzed in all cytotypes using experimental approaches. Briefly, primers were designed to all clusters that show similarity to tandemly repeated DNA types, and used for PCR amplification, cloning and sequencing of multiple clones. At the same time, sequence clusters and contigs are characterized further using bioinformatic tools.
The analyses of selected tandem and dispersed repeats in three main cytotypes of Senecio carniolicus will continue until cloned and well characterized sequences are available. These will in turn be used for mapping in chromosomes via molecular cytogenetic techniques. Comparative analyses of the localization of various repeats in chromosomes of various cytotypes should provide information about the dynamics of repetitive DNA in the genomes following polyploidization and reproductive isolation. In parallel, the same repeat types will be analyzed on larger scale in multiple accessions of the cytotypes using Southern blotting.
Project status 02/2012
The project aims to analyse the genome evolution in the autopolyploid complex of Senecio carniolicus (family Asteraceae). In 2010 we have isolated genomic DNAs of multiple genotypes and cytotypes of Senecio carniolicus (2x, 4x, and 6x), and one of these (diploid) was selected for 454 next generation sequencing (NGS). All sequences (0.5 mln reads) continue to be analysed and numerous clusters of repetitive DNA types were already recovered. Primers designed to four most prominent clusters showing similarity to tandem repeats were used to amplify, clone and sequence these repeats. Necessary optimization of the amplification protocols was successful. Two of the repeats (clusters 104 and 105) to encompass short tandem repeats (below 100bp), while the clusters 88 and 52 are composed of repeats significantly longer than average tandemly repeated DNAs (longer than 800 bps). First DNA sequence data for these four clusters indicate some heterogeneity of the repeats within each of the clusters and among cytotypes. Amplification of the same satellite DNA types from multiple cytotypes (diploids, tetraploids and hexaploids) allows direct comparison of their repeat similarity.
Clones containing single inserts of the two shorter DNA repeats (cluster 104 and 105) are now prepared for fluorescent labelling. Actively dividing meristematic tissues have recently been fixed and will be used as a source of chromosomes for in situ localization (FISH) of the isolated satellite DNAs in the genomes of all ploidy levels. Preliminary cytogenetic analyses allowed optimization of FISH protocol. The comparative detailed analyses of the four isolated tandem repeats in Senecio carniolicus will also include Southern blotting quantification of the repeat amount in various cytotypes (using same cloned inserts as for FISH). Several families of retrotransposons (both copia and gypsy type) has also been identified in NGS data. Primers for these families are being designed, and will be used for amplification and characterization of these DNA sequences and furthermore for comparative analyses of different ploidy levels. Combined analyses of all types of data will allow insight into the dynamics of repetitive DNA in the genomes following polyploidization and apparent reproductive isolation of different ploidy levels.