Selecting plants with increased total polyphenol oxidases in the genus Trifolium

One of the aims in red clover (Trifolium pratense) breeding is to increase the polyphenol oxidase (PPO) activity, which may effectively reduce protein breakdown in silage and when cattle are fed by fresh clover. We analysed total PPO activity spectrophotometrically and on the level of gene expression using real-time quantitative PCR in single plants derived from an interspecific T. pratense × T. medium hybrid. Experiments were performed for two years and evaluated according to the general linear model with three factors (family, year, and cut). The analysis revealed considerable variability in total PPO activity between individuals and between families. Four families and two individuals with significantly higher PPO activity were selected. Their PPO activity ranged from 3.411 to 3.547 mkatal/min/g and from 4.041 to 5.731 mkatal/min/g, respectively, in comparison with the control variety Amos (2.370 mkatal/min/g). The majority of PPO transcripts were expressed by the two genes PPO1/5 and PPO2. In some genotypes, the PPO5 gene was expressed. Quantitative PCR confirmed the highest activity of PPO genes in seven hybrid plants with higher DNA contents corresponding to 30 chromosomes with 815 013 copies per plant. Our results indicate the suitability of combining two methods for improved selection: initial expression analysis to assess the PPO transcript level indicating gene activity and subsequent enzymatic assay.