Abstract

Simple and economical microsatellite marker detection is important for successful and widespread adoption of marker technology in various breeding programs. A non-automated but fast, reliable and cost effective protocol for microsatellite marker detection is described that requires only standard molecular laboratory equipment. The protocol consists of a leaf sampling method for 96-well plates, tissue grinding using a bead-based grinder, DNA extraction using a modified heating method, and marker detection using polyacrilamid MegaGel dual vertical electrophoresis. At least 200 data points can be generated by one person in a single day from sample preparation to DNA extraction, PCR run, electrophoresis, and data recording. The cost of consumable chemicals for marker analyses was about U.S. $0.25 per data point. The protocol is adaptable for use in field settings to support marker-assisted selection and breeding line development, as well as for genotyping and mapping purposes. This relatively simple and inexpensive marker detection could expand the participation of small laboratories.  Wider participation from various breeding programs in both developed and developing countries will benefit many crop improvement programs.

Key words: High throughput, low cost, microsatellite marker, polyacrylamid gel electrophoresis, rice (Oryza sativa), marker-assisted selection, breeding line.