Department of Plant Agriculture, Biotechnology Division, University of Guelph, Guelph, Canada

Microsatellites, or simple sequence repeats (SSR), can be defined as tandem repeats of 2 to 6 bp motifs (Hearne et al. 1992). These highly polymorphic repetitive DNA elements have the potential to be utilized as molecular markers in plant genetic research and for breeding purposes. The polymorphic nature of SSR can be evaluated by performing PCR amplification of microsatellites (using primers designed from the DNA sequences that flank the SSR of interest) and electrophoretic separation of the resulting amplification products. As an extension of previous work in this laboratory (William et al. 1997), analysis of microsatellite polymorphism across a group of eleven barley (Hordeum vulgare L.) cultivars was performed. The purpose of this research was to evaluate the extent of polymorphism and potential usefulness of large-motif microsatellites.

Primers for PCR analysis were selected from Becker and Heun 1995; Liu et al. 1996; J. Russell (pers. comm.) and from Genbank database sequences. PCR reactions were performed using PCR profile #3, (Liu et al. (1996)), modified to include 35 instead of 30 repeats of the reaction cycle, in a PTC-100 programmable thermal controller (MJ research).

A set of 28 dimeric-containing SSR and 13 other microsatellites that contained larger motif regions were assessed. Eight of the large motif and 17 of the dimeric SSR were polymorphic (Table 1). It was possible to uniquely identify the eleven cultivars using data from four of the large motif SSR (HVM5, NIRE, MYB2, HVSS1); three of the dimeric SSR (HVM60, WAXY, RCAB); or from two of the complete set of SSR (HVM5, HVM68), thereby illustrating that SSR containing all possible motif sizes have potential as molecular markers. Diversity index values (DI = 1-S(allele frequency)_i²) for the large motif microsatellites ranged from 0.17 to 0.74, with two to five alleles found per polymorphic locus. These values compared favourably to those for the set of dimeric SSR also analysed across the same cultivar set.

References

Becker, J., M. Heun, 1995. Barley microsatellites: allele variation and mapping. Plant Mol. Biol. 27:835-845.

Hearne, C.M., S. Ghosh, J.A. Todd, 1992. Microsatellites for linkage analysis of genetic traits. Trends Genet. 8:288-294.

Liu Z-W, R.M. Biyashev, M.A. Saghai Maroof, 1996. Development of simple sequence repeat DNA markers and their integration into a barley linkage map. Theor. Appl. Genet. 93:869-96

William M, I. Dorocicz, K.J. Kasha, 1997. Use of microsatellite DNA to distinguish malting and nonmalting cultivars. J. Amer. Soc. Brew. Chem. 55:107-110

Table 1: Polymorphism for 25 microsatellites across a representative set of 11 cultivars

Notes:

Cultivars: 1 Brier 7 TR306

2 Condor 8 Winchester

3 Phoenix 9 Jackson

4 Rodeo 10 Leger

5 Steptoe 11 McGregor

6 Harrington

PCR product sizes are in bp

0 = null allele

DI = diversity index