Multiplex Nuclear SSR Amplification in Scots Pine (Pinus sylvestris L.)

Abstract

Studies in population genetics usually involves large numbers of samples and loci. Using multiplex PCR, amplifying simultaneously more than one locus in the same reaction, becomes a convenient, rapid, and easy tool when working with these large sample sizes. Many researchers have discussed the conditions that influence the PCR reaction and the difficulties of multiplexing. The development of SSR is difficult due to the cost and labor involved. Scots pine has a very large genome. Because the genome is not sequenced the SSR markers available are limited, but the SSR markers can be transferred from species in the same genus. Using primers developed on Pinus taeda and Pinus pinaster we developed an optimized protocol for SSR multiplex applications in Pinus sylvestris. A number of 300 primers were tested. From these tests 4 multiplex mixes emerged each containing 3 primers. The system was tested on 15 samples from forests in Romania. The conditions were optimized using the Type-it kit from Qiagen. This protocol can be used for population genetics studies that involve a large number of samples and loci, reducing the cost. It is an alternative to the florescent pre-labeled primers technique that can be pool mixed or multiplex, but with less cost.