Comparison of six genomic DNA extraction methods for molecular downstream applications of apple tree (Malus X domestica)

Abstract

Extraction of high quality DNA is crucial for any molecular genetic analysis. However, it is difficult to be obtained from problematic plant tissue, high in phenolic compounds, such as apple leaves. Despite the variety of commercially available kits for DNA isolation, no study has been done so far evaluating their potential for apple tree. We have tested six different kits and compared their performance on five to ten samples of apple tree (Malus X domestica) leaves. Genomic DNA was extracted following manufacturers’ protocols and amplified by touchdown PCR using 12 different SSR markers. The quality of DNA and PCR products was proven on agarose gel; additionally, DNA concentrations were measured using fluorimeter. Results showed high level of variation for concentrations and DNA purities; the highest yield (more than 512 ng/µL) was obtained with E.Z.N.A. SP Plant DNA Kit (Omega bio-tek), although DNA was not absolutely pure. The highest DNA sample purity was obtained using the DNeasy Plant Pro Kit (Qiagen); however, it resulted in the lowest DNA concentration (13 ng/µL). Despite big differences in DNA yields, all kits performed well for further PCR amplification. We conclude that choosing suitable method for DNA extraction of the particular sample plays a big role for the quality of DNA and its downstream applications. Extraction with DNeasy Plant Pro Kit (Qiagen) was the most efficient, as it resulted in the purest DNA. Despite its relatively low DNA yield, concentrations were still high enough for further PCR amplification. Obtained results indicate the optimal DNA extraction method used for problematic plant species in molecular studies.

Keywords

• malus x domestica
• dna extraction
• pcr amplification
• simple sequence repeat primers
• problematic plant tissues