Hana Jakešová

Identification of barriers to interspecific crosses in the genus Trifolium

SummaryA study of pre- and post-fertilisation barriers after interspecific crosses of diploid and tetraploid Trifolium pratense L. and wild species T. alpestre L., T. medium L. and T. sarosiense Hazsl. was aimed at finding of a promising cross combination for obtaining hybrids. The growth of pollen tubes was arrested in interspecific crosses mainly when T. pratense was at a diploid level. To investigate the post-fertilisation barriers in detail, the hybrid embryo viability was traced by two clearing treatments of immature seeds: (1) using chloral hydrate (which proved to be most appropriate); and (2) a mixture of benzyl benzoate and dibutly phthalate. In interspecific combinations T. pratense (4×) × either T. alpestre or T. sarosiense, enlargement of immature seeds occurred, but no hybrid embryo was traced. Of the wild species used as a male parent for crosses, T. medium was the only exception from the point of view of fertilisation. Globular, heart and the early torpedo stages of hybrid embryos were observed 7 days after pollination (DAP) but only when T. pratense was at a tetraploid level. When T. pratense (2×, 4×) was used as a male parent for interspecific crosses with T. alpestre, T. medium and T. sarosiense, strong defects in various stages of embryogenesis were observed, particularly wrinkled and narrowing embryo sacs caused by an expansion of endothelial cells. We conclude with the following finding: (1) to make crosses only in one direction with T. pratense as a female parent and T. medium as a male; (2) to use tetraploid plants of T. pratense; (3) and to excise hybrid embryos at an early torpedo stage, about 7 DAP.

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.

Impact of Interspecific Hybridization of T. pratense x T. medium and Backcrossing on Genetic Variability of Progeny

Red clover (Trifolium pratense L.) is a high-quality fodder crop which has been hybridized successfully with its wild relative zigzag clover (T. medium L.). The aim of this study was to evaluate the genetic impact of interspecific hybridization and subsequent repeated backcrossing on the variability within hybrid progeny genomes. Nuclear DNA content of 800 and 753 hybrid plants from F-7/F-8 and F-8/F-9 generations, respectively, was measured by flow cytometry. Resulting values were converted to estimated chromosome counts, which were successfully validated on a sample of 28 plants by counting mitotic chromosomes. The two generations showed a similar distribution of various chromosome counts ranging from 22 to 47 chromosomes. In total, 24.0% and 34.3% of plants from the two generations had different numbers of chromosomes from their parental plants. Variability within the hybrid population was assessed by fluorescent in situ hybridization using rDNA probes. Individual plants had a pattern of 5S and 45S rDNA loci rather more similar to that of T. pratense than of T. medium. Numbers of chromosomes with clusters of 5S rDNA ranged from 6 to 14 while those of 45S rDNA varied between 4 and 13. Individual arrangements were almost unique, and some plants possessed also novel formations which were not present in any of the parental species, such as a cluster of 5S rDNA surrounded by 45S rDNA clusters or a 45S rDNA cluster surrounded by 5S rDNA clusters. This suggests complex rearrangements connected with post-hybridization stabilization of hybrid genomes.