P2008-05: Detecting hybrid speciation with low copy nuclear genes and its usefulness in cultivated and invasive plants using taxa of Leontodon /Scorzoneroides (Asteraceae) as model organisms.
Project leader :
Hybrid polyploidization has played a major role in speciation as well as in improvement of crop plants. However relatively little is known about the importance of hybrid speciation at the diploid level.
The primary effect of hybridization is an increase in genetic variation and this often has stimulated formation of ecological races, a critical step in speciation. The genera Leontodon and Scorzoneroides were chosen for the study of low copy nuclear genes because diploid hybridization is reported here.
Scorzoneroides comprises of 29 species where as Leontodon includes around 50 species. Hybrids have been detected in Scorzoneroides, e.g. S. montana x S. pyrenica (S. huguenini Brugger) and S. autumnalis x S. montana (S. lannesii). Moreover, hybrids between members of the genera Scorzoneroides and Leontodon have been postulated based on morphological characters.
Nuclear rDNA (nrDNA) ITS is biparently inherited, however the process of concerted evolution can make isolation of both parental copies difficult, if not impossible. In contrast, low copy nuclear genes are less frequently subjected to concerted evolution, thus making them ideal candidates for identifying parental donors of suspected hybrids or allopolyploids.
Low copy nuclear genes so far have been used only occasionally to identify the origins of hybrid homoploid or allopolyploid in Peonia, Glycine or in Elymus (Sang et al. 1997b, Mason-Gamer 2001, Shannon et al. 2006).
In this study we want to develop most appropriate low copy nuclear genes for these two genera and sequence the suspected diploid hybrids within the genera and test hybrid speciation.
The best markers (two or three) will be used to establish a phylogeney of Leontodon and Scorzoneroides and related genera such as Hypochaeris and Picris.
Project status 01/2010
DNA-Extraction
DNA-Extraction was performed using a modified standard CTAB method by Doyle&Doyle (1987). Up to date, 74 accessions were extracted, out of which were taken 59 samples of 13 taxa in Genus Scorzonerodies, 10 out of 10 taxa in Leontodon, 3 out of 3 taxa in Hypochaeris and 1 out of 1 taxon in Helmintotheca.
Chloroplast genome
In previous projects, investigating regions of the chloroplast genome, in combination with the nuclear rDNA-region ITS, helped to understand the phylogeny of the genus Leontodon, and finally led to a generic status of Leontodon subgenus Oporinia, today known as Scorzoneroides . However, in our studies, cpDNA-Markers are less relevant, due to low informativity on low phylogenetic levels. To confirm previous results, as well as to get a wider basis of information, some chloroplast markers were applied.
For our primer trials, 11 universal markers by Shaw were applied to 17 accessions of our extracted plant material. We were investigating on applicability, ease of aligning and informativity.
The rpl16 Intron best overall scores, with the highest percentage of PIC, good sequences and an easy alignment.
The regions psbJ-petA, psbD-trnT and trnQ-5’rps could also be assembled without to much effort, however, we were not satisfied with the information provided.
Even though ndhF-rpl32 marker showed the second highest number of informative characters, we will not continue using it, due to difficulties with aligning. atpI-atpH showed similar results concerning information content, creating an alignment was of ease.
Bad sequences, mainly caused by monomeric repeats, of the regions trnV-ndhC, trnS-trnfM and petL-psbE led to a poor alignment.
Two loci, trnT-trnD and rpoB-trnC, could not be amplified at all.
As the rpl16 Intron and the atpI-atpH spacer region have proven to be the most promising markers in these primer trials, we will use these loci create a phylogenetic tree of all samples.
Nuclear genome
For better understanding of phylogenetic relationships and to discover possible hybrid taxa we were looking at the nuclear genome in more detail. In contrast to the chloroplast genome which is inherited by the maternal line mostly, the nuclear genome is composed of chromosomes of both parental lineages. The origin of different portions of nuclear DNA and hybrid taxa can be discovered more easily, when looking at these parts.
The first nuclear DNA region we investigated has been already successfully applied in another Asteraceae genus (Vaezi & Brouillet 2009). The chosen gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is composed of exon and intron regions, where we investigated only the stretch spanning from exon 3 to exon 6. Exons are the protein-coding, relatively conserved parts of a gene, introns are more variable spacer regions of a specific function (partly still unknown), but clearly non-coding.
We initially used the primers of the given publication, which has been working only for 1/3 of the Scorzoneroides-Taxa. To investigate also the other samples, we developed new PCR and sequencing primers, which finally gave good sequences for most of them. Possible hybrids and variable taxa had to be cloned.
The main issue of our current work is to analyze the sequence data (editing, aligning, phylogenetic analyses). An additional nuclear marker will be established within the remaining project time.
Project status 02/2011
Seven low-copy nuclear markers to detect speculated hybridization events in Scorzoneroides were tested in our study. Four of these genes namely PgiC, PhyC, GScp, GAPDH, are known in their function. Three markers, A27, A28 and A39, could not be specified, as they stem from a study in conserved orthologous regions from Arabidopsis thaliana. PCR conditions were optimized for all markers. We could not obtain good sequences for PgiC, PhyC, GScp, A27 and A28 markers. Two of the regions, namely GAPDH and A39 could be directly sequenced from PCR reactions. Internal primers were designed to increase PCR reliability among the tested accessions from Hypochaeridinae. For GAPDH, we designed 3 new primers, GAPDHx3FSc, GAPDHx5RSc and GAPDHx6RSc. Two new primers were designed for A39, AT2FSc and AT2Rsc.
Both low-copy nuclear genes showed polymorphic sequences in some accessions, thus cloning was performed for 24 (GAPDH) and 19 (A39) samples, respectively. The total number of colonies obtained by cloning was 622 (367 from GAPDH, 255 from A39), with an average number of 15 colonies per sample. Overall, we used 96 colonies from GAPDH and 84 colonies from A39 to get both copies of each individual sequence that showed polymorphic sites.
The alignment of GAPDH sequences gave information about the intron-exon structure of the amplified fragment in Hypochaeridinae (Figure 3).
Intron and exon lengths were estimated manually by screening the alignment of all available sequence data of this study. We were not able to do the same for the A39 marker (Figure 4).
The resulting phylogenetic trees of both markers did not support ongoing hybridization in the investigated genus. We created a combined tree of both nuclear markers (Figure 1) from this study.
The resolution from the trees, however, is low. This may indicate that the two selected markers are not well-suited for investigations in Hypochaeridinae. Further investigation with other nuclear genes or AFLP data as well as a broader sampling in Scorzoneroides might be needed to answer speculations about hybridization events. A combination of the two nuclear regions GAPDH and A39 with the three plastid markers atpI-atpH, ndhF-rpl32 and rpl16-Intron showed a well-resolved phylogenetic tree (Figure 2).
Foresight:
1.)Chromosome counts and genome size measurements will be done and this will give additional insight to the evolution of the genus Scorzoneroides. Seeds are available for some accessions of 9 species.
2.)Currently, the diploma thesis “Molecular phylogeny and detection of diploid hybrids in Scorzoneroides (Asteraceae)” is in preparation and will be submitted by March, 2011.
Project status 02/2012 - final report
Earlier investigations on the molecular phylogeny of the genus Leontodon using chloroplast regions in combination with the nuclear rDNA region ITS, led to a generic status of Leontodon subgenus Oporinia, today Scorzoneroides. To confirm previous results, as well as to get well-resolved phylogenetic trees, more noncoding regions of the chloroplast genome were sequenced. We were investigating the applicability, ease of alignment and informativity of 11 universal markers. The ndhF-rpl32 intergenic spacer, the rpl16-Intron and atpH-atpI intergenic spacer regions were used to create phylogenetic trees of all samples. Seven low-copy nuclear markers were tested to detect speculated hybridization events in Scorzoneroides. PCR conditions were optimized for all markers. Two of the regions, namely GAPDH and the A39 locus could be directly sequenced from some PCR reactions. Internal primers were designed to increase PCR reliability among the tested accessions from Hypochaeridinae. Both low-copy nuclear genes showed polymorphic sequences in some accessions, which led to cloning. We used 96 colonies from GAPDH and 84 colonies from A39 to get both copies of each individual sequence that showed polymorphic sites. The alignment of GAPDH sequences gave information about the intron-exon structure of the amplified fragment in Hypochaeridinae. Intron and exon lengths were estimated manually by screening the alignment of all available sequence data of this study. We were not able to do the same for the A39 marker. The resulting phylogenetic trees of both markers did not support on-going hybridization in the investigated genus. We created a combined tree of both nuclear markers from this study. The resolution of the markers' phylogenies, however, is low. This indicates that the two selected markers are not optimal for investigations in Hypochaeridinae. A combination of the two nuclear regions GAPDH and A39 locus with the three plastid markers atpH-atpI, ndhF-rpl32 and rpl16-Intron showed a well-resolved phylogenetic tree. In addition to nuclear and plastid DNA markers, karyological methods were used for the accessions where seed material was available. Chromosome counts and genome size measurements from seeds of 9 species of all resulting clades of the phylogenetic analysis were measured. Successful germination was easily obtained for annual species, whereas the germination rate of the included perennial species was very low. Chromosome counts could only be obtained for the annual plants. The basic chromosome number for Scorzoneroides is x=6, two species had a chromosome number of x=5. The genome size of 8 out of 9 species could be measured by flow cytometry. All species investigated possess a small genome size ranging from 1C=1.24pg to 1C=1.87pg.
Publications
Reinhold Stockenhuber: Molecular phylogeny and detection of diploid hybrids in Scorzoneroides (Asteraceae) (POSTER > ABSTRACT)
Stockenhuber, Reinhold M. (2011) Phylogenetic relationship of the genus Scorzoneroides (Compositae) inferred from plastid and low-copy nuclear markers, and karyological data. Diploma Thesis, Institut für Botanik, Universität Wien, Wien.
Lectures
Stockenhuber, Reinhold M.; Barfuss, Michael H.J.; Samuel, Rosabelle (18.01.2010) Molecular phylogeny of Scorzoneroides (Asteraceae). Vortrag: Department Seminar of the Department of Systematic and Evolutionary Botany of the Faculty Center of Biodiversity of the University of Vienna, Vienna/AUSTRIA.
Stockenhuber, Reinhold; Barfuss, Michael H.J.; Cruz-Mazo, Gema; Samuel, Rosabelle (16.09.2010) Molecular phylogeny and detection of diploid hybrids in Scorzoneroides (Asteraceae). Vortrag: 19th International Symposium, “Biodiversity and Evolutionary Biology” of the German Botanical Society (DBG), Vienna/AUSTRIA.
Stockenhuber, Reinhold M. (June 15, 2011) Molecular phylogeny of Scorzoneroides (Asteraceae). Lecture at: Department Seminar (Department of Systematic and Evolutionary Botany of the Faculty Center of Biodiversity), Vienna/AUSTRIA.
