Kirsi Kostamo

Microsatellite marker identification using genome screening and restriction-ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.

Observations on the mode and seasonality of reproduction in Furcellaria lumbricalis (Gigartinales, Rhodophyta) populations in the northern Baltic Sea

Abstract The life cycle of the red alga, Furcellaria lumbricalis, is poorly known in the northern part of the brackish water Baltic Sea, even though it is one of the most common red algal species in the region. To study its life cycle and reproduction, monthly sampling (n=30/sampling site) was carried out for one year on the coast of Finland at four locations spanning 500 km along a salinity gradient (3.6–5.4 psu). In three of the populations studied, only tetrasporangia and spermatangia were produced. In the fourth population, at the lowest (3.6 psu) salinity, all algae examined were vegetative. Winter months were the main season for tetrasporangial production, when sori with masses of sporangia were present. Smaller sori with only a few tetrasporangia were observed during spring at higher salinities, and some sporangia were detected in the population at 5 psu salinity, even during the summer months. The timing and intensity of reproduction was regulated by a combination of factors including seawater salinity, along with seawater transparency, temperature and photoperiod. In conclusion, we hypothesise that three of the populations, living at salinities between 4.9–5.4 psu reproduce by thallus fragmentation and possibly by asexual tetraspore-to-tetraspore cycling without meiosis, and the fourth population, at a salinity of 3.6 psu, survives by thallus fragmentation and reattachment.

New microsatellite markers for Ulva intestinalis (Chlorophyta) and the transferability of markers across species of Ulvaceae

K. Kostamo, J. Blomster, H. Korpelainen, J. Kelly, C.A. Maggs and F. Mineur. 2008. New microsatellite markers for Ulva intestinalis (Chlorophyta) and the transferability of markers across species of Ulvaceae. Phycologia 47: 580–587. DOI: 10.2216/08-16.1 Macroalgal blooms are a growing environmental problem in eutrophicated coastal ecosystems. Members of the green algal genus Ulva are significant contributors to blooms, which are typically dominated by only one of several co-occurring opportunistic species. Our understanding of bloom dynamics, such as the importance of clonality, is limited because previously used genetic markers such as internal transcribed spacer sequences have shown very little resolution. Microsatellites are the marker of choice for such studies, but to date, only five primer pairs have been developed for a single member of this genus, Ulva intestinalis. We have now developed four new microsatellite markers for U. intestinalis using genome screening and restriction–ligation and tested them on individuals from six populations in the Gulf of Finland, Finland. All new markers exhibited polymorphism in U. intestinalis, with the numbers of alleles ranging from 6 to 10. On the basis of assignment tests, FST estimates and analysis of molecular variance, there was genetic differentiation among populations. Where significantly different, expected heterozygosity (HE) was higher than observed heterozygosity (HO), indicating a trend toward heterozygote deficiency. This may indicate that although Ulva spores can disperse relatively efficiently, asexual reproduction can result in genetic differentiation among populations. We also tested the cross-species amplification of our primers and the five primer pairs reported previously on seven species of Ulva, Ulvaria obscura and Umbraulva olivascens (all members of the Ulvaceae). In each species, from five to nine of the loci produced an amplification product, and one to four alleles were discovered at each locus. These markers therefore have great potential for testing hypotheses about the formation and maintenance of multispecies macroalgal blooms.

An improved and cost-effective cDNA-AFLP method to investigate transcription-derived products when high throughput sequencing is not available

Although the cost of high throughput sequencing is decreasing, the cost is still often too high for individual projects targeted at, e.g., genome-wide transcription profiling in non-model organisms. Then, a low-cost alternative is cDNA-AFLP, which we have now considerably modified in order to develop a faster and simpler method to identify and analyze genes involved in specific, possibly adaptive characteristics. Particularly, we wanted to exclude repetitive PCR amplifications, extensive cloning and the presence of overlapping transcripts, which all lower the efficiency of the method.

Genetic Composition of Bryophyte Populations Occupying Habitats Differing in the Level of Human Disturbance

By using microsatellite markers, we investigated the population genetic structure of three mainly clonally propagating bryophyte species—the hepatic Plagiochila asplenioides (L.) Dumort and the mosses Pleurozium schreberi (Willd. ex Brid.) Mitt. and Rhytidiadelphus squarrosus (Hedw.) Warnst—that occupy habitats differing in the level of human disturbance. Sampled populations, located in southern Finland, were classified as natural or disturbed. All species possessed a fair amount of genetic variation both within populations (mean diversities per population of , , and , respectively) and across populations (0.599, 0.618, and 0.400, respectively). On average, natural and disturbed populations possessed similar levels of genetic diversity. Differentiation levels (FST values of 0.356, 0.298, and 0.302, respectively) were significant but showed that there is some gene flow taking place. The Bayesian STRUCTURE analysis provided additional information on the population genetic structures. Although R. squarrosus was the species with the lowest level of genetic variation, its populations inhabiting urban, disturbed habitats tended to contain highly differentiated genotypes of different origins, which suggests that human-mediated dispersal is important in urban habitats. In addition, detailed analyses of the spatial genetic structure of R. squarrosus showed that at short distances (1 to <30 m) individuals are more closely related than expected, while at greater distances (30 to 500 m) kinship decreases with increasing distance. Thus, the rarity of sexual reproduction was visible in the spatial genetic structure within R. squarrosus populations, as aggregations of similar genotypes were found at short distances in populations otherwise showing isolation-by-distance effect.