Introduction

Genetic fingerprinting has been utilized in the identification of individuals, breeds, cultivars and species, in genetic mapping in connection with animal and plant breeding or medical applications, and in a range of population genetic and ecological applications (1,2,3,4,5,6). Among DNA fingerprinting methods, speciesspecific microsatellite analyses are of major importance. Microsatellites are highly abundant within eukaryotic genomes (6,7). The markers consist of repeated 1- to 6-bp-long DNA motifs. Different forms (alleles) vary in the number of units of the repeat motifs. The genomic areas containing microsatellites are known to be highly variable, which makes their analyses useful for many applications (3,6). Even in species in which the level of genetic variation detected by other methods may be low, a considerable level of variation is often detected using microsatellites (8,9,10). A disadvantage is that the present methodology used to identify microsatellite markers is quite time-consuming and expensive (11), although a number of attempts to identify improved techniques have been carried out, and a number of different protocols are available (3,12,13).

The aim of the present study was to develop a new methodology for a faster and easier way to identify microsatellite markers when compared with traditional methods. The approach includes genome screening techniques with inter-simple sequence repeats (ISSR) primers (14) in order to find microsatellite regions and to obtain sequence information flanking one side of the microsatellites, as well as a restrictionligation technique with a specific adaptor to allow sequence walking in order to obtain sequence information flanking the other side of the microsatellites. Each ISSR primer contains a repeat sequence flanked by one or a few different nucleotides acting as an anchor. To allow wide testing of the applicability of the identified method, we used the method on a range of plant material and also one animal species.

Materials and Methods

The process for microsatellite identification was initiated with the screening of genomic DNA, extracted using DNeasy® Plant Mini kit (Qiagen, Valencia, CA, USA) for the plant species in question, and using standard procedures for animal tissues (15) for the raccoon dog material, with a set of 12 ISSR primers. Different primers or primer combinations were used until a desired number of microsatellite regions (i.e., well-amplified ISSR products) were detected. The PCRs involved the use of two different primers (the possible 6 × 6 combinations shown in Table 1) when following procedure A and the use of single primers when following procedure B (any primer of the 12 primers listed in Table 1). There are many other possible primer sequences besides those listed in Table 1 and used in the present study. The selection of primers was based on knowledge of the occurrence of different repeat sequences in genomes (3). Microsatellite primer identification was tested on plant material, representing 21 species of bryophytes and three algal species collected from different parts of the Northern Hemisphere, mainly from Northern Europe, and on one animal species, the raccoon dog.

Table 1.  ISSR Primer Sequences Used in the Genome Screening (Click to enlarge)

Procedure A

Cross-amplification including the three following types of PCRs was conducted: (i) amplification with only one ISSR primer; (ii) amplification with another ISSR primer; and (iii) amplification with both ISSR primers at the same time. Amplification reactions were conducted in a volume of 20 µL. The reaction mixture contained about 10–20 ng genomic DNA 1.2 U DyNAzyme™ II DNA polymerase (Finnzymes, Espoo, Finland), 1× PCR buffer, 0.4 µL 10 mM dNTP mixture, and 1 µL of a single 5 µM primer or 1 µL each of two 5 µM primers. The PTC-200 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) was programmed for 4 min denaturation at 94°C, followed by 45 cycles of denaturation at 94°C for 45 s, annealing at 50°−52°C for 45 s, and elongation at 72°C for 90 s. An additional 8-min elongation followed the last cycle. All amplification products were electrophoretically separated on 1.4% agarose gels. Only clear fragments, mostly in size range of 300–900 bp, resulting from two-primer amplifications and therefore containing different microsatellite repeats at the ends of the sequence were excised and purified with QIAquick® Gel Extraction kit (Qiagen) or a comparable product and sequenced with one of the two primers. The use of two different primers and the presence of different microsatellite repeats at the ends of the sequence are necessary for a satisfactory sequencing result. Single-primer control PCRs are conducted to distinguish the two-primer amplification products from the singleprimer amplification products. Based on the sequencing result, the first specific microsatellite primer was identified for the microsatellite at one end or both ends of the sequenced fragment.