Marker SSLPs

Microsatellites, or simple sequence length polymorphisms (SSLPs) (Bell and Ecker 1994), can be used as markers in many genomes, including that of Arabidopsis. They are tandemly repeated di- to tetranucleotides flanked by unique sequences. Because they are prone to slippage during DNA replication, the number of repeats can increase or decrease, leading to considerable allelic diversity in natural populations. SSLP marker information is available through the marker site at TAIR (see www.ara-bidopsis.org).

A typical protocol (see, e.g., Lukowitz et al. 2000) comprises a standard PCR, the products of which are analyzed using gel elecrophoresis. Because the differences in lengths between different alleles are often small, they are best assayed on high-percentage agarose gels. Typical reaction conditions are as follows:

0,5 unit Taq polymerase (per 20-jLtl reaction)

template DNA (e.g., 2 jil of miniprep DNA for a 20-|xl réaction)

PCR program:

1 minute at 94°C

cycle 1: 30 seconds at 94°C

30 seconds at 65°C 30 seconds at 72°C cycles 2-10: same as cycle 1, but decrease annealing temperature by 1°C per cycle, i.e., from 65°C in cycle 1 to 56°C in cycle 9. cycles 11-40: 30 seconds at 94°C 30 seconds at 55°C 30 seconds at 72°C

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