Efficient low-cost DNA extraction and multiplex fluorescent PCR method for marker-assisted selection in breeding

Marker-assisted selection (MAS) is an increasingly important tool in current breeding efforts for improved crop plants and animal breeds. It enables detection of favourable alleles in early developmental stages and thus may result in substantial cost savings. Until now, however, the high costs of the required chemicals and materials, together with the still very labour-intensive methods, have been an obstacle to widespread application of MAS. A new multiplex-polymerase chain reaction (PCR)-based method has been developed for reliable low-cost, high-throughput screening. By its use 3366 apple seedlings were screened with an average hands-on time from DNA extraction to data ready for analysis of < 4 h per 96 plants, and at a cost below US$ 0.5 per marker per plant. Factors that have a strong effect on segregation ratios such as elevated levels of outcrossing are easily detected, as a significant correlation was observed between deviation from expected segregation ratios in some affected markers and the level of outcrossing in a cross. The new method is suitable for many crop species and, provided that suitable buffers are used for DNA extraction, for animals too.