During summer and fall of 2010, I completed an extensive collection of reed canarygrass (Phalaris arundinacea) along the six major rivers in the Czech Republic. This material will be assessed for genetic variation along with commercial cultivars. The specific research questions being asked are:
A) Does genetic variation differ along the CR rivers (Labe, Orlice, Vltava, Berounka, Lužnice, Dyje)?
B) Does genetic variation within populations differ along the river banks vs. inland from the river edges?
All of these rivers, with the exception of the Dyje, feed into the North Sea basin. The Dyje River runs into the Black Sea basin. There may be differences between these rivers that feed into the basins, although we do not have enough Black Sea basin rivers sampled to make this determination. Genetic variation assessment consisted of phenotypic observations of plants and populations and the use of inter-simple sequence repeats (ISSRs) to assess nuclear DNA genetic (amino acid sequence) variation (being done now).
Who knows the parasitic species growing in the trees (right-hand side of the following picture)?
And who can identify the red-leafed plant growing along with the reed canarygrass??
Initial results from our N. American reed canarygrass ISSR analysis, particularly that most of the genetic variation is within rather than among N. American populations, are being contrasted with Czech populations. Higher levels of variation within N. American populations caused us to expand from our initial proposal of collecting only 10 plants/population and 5 populations for each of the 5 major rivers. In a meeting with my hosts in July 2010 after my arrival, we expanded this to 6 rivers and sampling every 30 km along each river within the country, including above and below confluences. This was due to a new paper that had just been published that noted a distance of ~30 km separated river populations for different species.
At each sampling site, we collected from at least 5 locations along the river edges each of which were 100 m apart. At each site, as many as 15-18 samples were collected along the rivers (Table 1). In addition, we also included perpendicular transects within each site to determine whether the genetic variation along the rivers was the same or different from the populations extending back from the water's edge. At each site, if plants existed away from the river edges, we collected 1-3 transects with as many as 4-39 individuals for analyses (Table 1). Our total collections from the rivers, encompassed by ~13 000 km of driving around the country on multiple trips (July - November, 2010), totaled 30 sites within the six rivers and 155 locations at these sites for a total of 465 genotypes collected along all rivers (Table 1). At these locations, a total of 38 transects perpendicular to the rivers' edges were run and a total of 370 genotypes were collected (Table 1). Thus, our grand total of genotypes for Czech rivers is 835 genotypes--a very large sampling which will make our research papers and inferences very powerful.
In addition to these wild population collections in the Czech Republic, we collected large population transects in the wet meadows (two perpendicular and intersecting transects through the middle of each population) near some of the major historic fish ponds in the Trebon basin (Table 2). We also sampled along the Golden Canal (Zlata Stoka), using the same collection technique for the rivers (Table 1). The Golden Canal (Sites No. 12-13) feeds fresh water from the Lužnice River into all of these ponds. We hypothesized that the Golden Canal could be a major vector to spread reed canarygrass propagules (vegetative) through the fish pond system in Trebon. At Site No. 11, Rzmberk Pond, we collected two sets of perpendicular transects, one set each in the unmowed and mowed sections of Mokre Louky wet meadow. The unmowed wet meadow is a historic section of reed canarygrass that has been extensively studied by two of my hosts, Drs. Jan Květ and Keith Edwards; it is know to have a decreasing nutrient gradient across the site. The mowed wet meadow is harvested several times/year and used for biofuel production by Lane Aurora spa nearby. We theorized that the mowed and unmowed sections might be genetically divergent due to mowing. Genetic variation here may be important first steps in determining if there are differences between the mowed (cultivated) and unmowed (wild) populations and specific genotypes and whether the latter could be bred and selected for non-invasive reed canary grass cultivars for biofuel production. In 2011, I anticipate visiting the biofuel plants for Lazne Aurora as well as discussing future breeding directions with the Faculty of Agriculture, in particular Prof. Ing. Stanislav Kužel CSc.
Other collection sites for wild populations surfaced as we traveled around the country, including Site No. 10 (Rzmberk Pond; Table 2), which is where our University of Minnesota graduate student, Gina Quiram, sampled purple loosestrife populations, Lythrum salicaria, in 2008. It is also the site where Dr. Jan Květ has conducted numerous ecological studies for many decades. The historic salt marsh, the Nesyt Fish Pond, in South Moravia (near Lednice) has a unique population of reed canarygrass that are partially salt-tolerant and also grow in partial shade. Whether or not genetic changes have occurred in the population due to salt and shade conditions will be determined. The Nesyt Fish Pond has been studied extensively since 1968 by Drs. Jan Květ and Štěpán Husák (Institute of Botany). I visited this site with both of them in October 2010 so we could collect reed canarygrass samples (Site No. 32, Table 2). One biofuel cultivar, 'Chrastava', which was developed by the Czech Republic was also sourced from the breeders by Dr. Vladislav Čurn (Table 2). I also found 3 ornamental cultivars and have 4 genotypes of these 3 cultivars (Table 2). The genetic variation within cultivars will be compared with the wild Czech populations, as well as the N. American types (several of which were brought to the Czech Republic in July 2010 as tissue cultured specimens). Total germplasm collected or sourced were 4 cultivars, 4 canal or wet meadow sites, 7 transects (304 genotypes) within wet meadows, and 10 sets (30 genotypes) along the Zlata Stoka Table 2).
Genotypes were assessed for GPS coordinates (latitude, longitude, altitude) and the dominant plant community species were noted. Of particular interest was the unique correlation of reed canarygrass growing with nettles (Urtica) in all locations except one. What is unique about this finding is we have never observed this association in N. American populations. We also noted any morphological (phenotypic) abnormalities: tip burn, chlorosis, necrosis, insect damage (leaf borers vs. chewing insects), pathogens (Puccinia, Ustilago), potential vector(s) (recent flooding, Trumpeter swans) or other unusual characteristics. These were noted for each sample or population; should these be correlated or linked with any of the DNA markers in the future this information could be used to create linkage maps.
Leaf samples were collected (1 recently expanded leaf/plant), marked with site, location and plant-specific codes, kept on ice during collection and transport, and brought into the lab for descriptions. Leaf tips were cut into small (3-4 mm) pieces, placed in eppendorf tubes, labeled and frozen (-20•C) until extraction. The remainder of each leaf were placed in individual, labeled bags and frozen as back up samples in case they were needed. It took ~1-2 hours to cut, sample and freeze a single population of samples (15-40 genotypes). DNA extractions take 5 hours per 30 samples, so I have spent the bulk of time during November-December (when not teaching or preparing for lectures) doing extractions. We have now completed DNA extractions of all 465 samples collected along the rivers; the 370 perpendicular river transect samples are nearly completely extracted as well. I anticipate finishing extractions within the next week.
We ordered and have now received 3-5 ISSR primers from the University of British Columbia (UBC) that were used to score the N. American populations: UBC 810, 828, 853, 881, and 890. Once all of the DNA extractions are completed I will commence polymerase chain reactions (PCRs) and run gels to score the primers for differences between samples. Data analyses will follow. This will take several months to accomplish but I anticipate having this done by spring 2011.
At a later date, provided time allows, we will conduct similar genetic analysis of reed canarygrass from the European germplasm banks. I have obtained seed from the germplasm bank curators of ~150 sample populations from across the European Union. Seeds of related Phalaris species have also been sent and are being kept in the Ecosystems Biology seed vault. Also, if time allows, I will sample Phalaris herbarium specimens from the National Museum in Prague Herbarium. Dr. Otakar Sida is assembling all of the collections of reed canarygrass for this purpose during 2011.
We will be able to publish at least one if not 2-3 peer-reviewed publications from this research. It will add to the published body of knowledge on reed canarygrass and, more importantly, add new information about vegetative and seed spread of the species, whether rivers differ above and below confluences and if transects within populations differ from those by the water's edge. All of this will greatly influence future decision-making for use of Phalaris as a biofuel crop and risk assessment for potential future spread of this species within the Czech Republic.