A 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.

Trifolium pratense L. is a high-yielding and high-quality fodder crop. However, it shows low persistence, which may be overcome by hybridisation with species that produce rhizomes. Hybrids between T. pratense L. and T. medium L. were previously obtained by embryo rescue. The aim of this study was to evaluate 16 characteristics (stem weight, stem length, internode number, length/width ratio of a central leaflet of the trifoliate leaf on the 4 th internode, stem thickness on the 4 th internode, average leaf area of the trifoliate leaf, weight of dry plant, was significantly higher in the hybrids compared to both parental species, which could have a positive impact on the yield. Short rhizomes were observed in the hybrids after the harvest of plants in the second harvest year. The stability of this trait and its impact on the perenniality of the hybrids will be investigated in the next years.Keywords: interspecific hybrids; morphological traits; persistence; red clover; rhizomes; zigzag cloverThe red clover (Trifolium pratense L.) is a widespread forage crop in the Czech Republic as well as in the rest of Europe. It is a high-yielding and high-quality fodder crop from the aspect of both nutrient content and ensilage suitability. Red clover has a high content of proteins, water-soluble carbohydrates, tannins, polyphenol oxidase and polyunsaturated fatty acids.Clover breeding is aimed at the production of fresh and dry matter that is of high quality combined with satisfactory seed yields. Polyploidy induction (tetraploidy 2n = 4x = 28) in red clover is useful for obtaining varieties with higher fodder production compared to diploids because autoploidy increases the cell size, especially in meristematic tissues, and the autoploids usually

Dluhošová J., Řepková J., Jakešová H., Nedělník J. (2016): Impact of interspecific hybridization of T. pratense × T. medium and backcrossing on genetic variability of progeny. Czech J. Genet. Plant Breed., 52: 125-131.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 posthybridization stabilization of hybrid genomes.

Three pairs of generic primers designed for specific reverse transcription polymerase chain reaction amplification of part of the cytorhabdoviruses genomes were successfully tested with a number of red clover samples. Two infected plants showing irregular vein clearing, tissue necrosis and stunted growth symptoms were selected for further studies. Subsequently, two novel plant rhabdoviruses, tentatively named Trifolium pratense virus A (TpVA) and Trifolium pratense virus B (TpVB), were detected in red clover plants. Sequence analyses of nucleotide sequences of their genomic RNAs obtained after high‐throughput sequencing confirmed their affinity to members of the genus Cytorhabdovirus. While TpVA was transmitted by mechanical inoculation to Nicotiana occidentalis 37B and subtransferred to Physalis floridana plants, crude sap transmission of TpVB to a range of herbaceous host plants failed. The bacilliform shape of virions, which has a cytoplasm‐limited distribution, their sizes, and the phylogenetic relationships, support assignment of TpVA and TpVB to two distinct species of the genus Cytorhabdovirus.

A virus detected in symptomatic Trifolium pratense L. plants in the Czech Republic had bacilliform virions which in thin sections occurred solely in phloem tissues and measured 220–500 nm by 30–31.5 nm. The virus was mechanically transmitted to Nicotiana occidentalis Wheeler, accession 37B. The partial nucleotide sequence (540 bp; accession number ) with similarity to open reading frame III of the badnavirus genome was amplified from total genomic DNA, extracted from the plants. The new sequence had 74.4% nucleotide identity to that of Ananas comosus endogenous virus in the polyprotein gene covering reverse transcriptase. The results suggest that the Czech isolate from clover should be regarded as a new member of the genus Badnavirus, for which the name Red clover bacilliform virus is suggested. This is the first report of a tentative member of Badnavirus genus occurring in forage crops.