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Plant-rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study's main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover ( Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation ( EFD ) and molybdate transporter 1 ( MOT1 ). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15 N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.

The genus clover ( Trifolium sp.) is one of the most economically important genera in the Fabaceae family. More than 10 species are grown as manure plants or forage legumes. Red clover's ( T. pratense ) genome size is one of the smallest in the Trifolium genus, while many clovers with potential breeding value have much larger genomes. Zigzag clover ( T. medium ) is closely related to the sequenced red clover; however, its genome is approximately 7.5x larger. Currently, almost nothing is known about the architecture of this large genome and differences between these two clover species. We sequenced the T. medium genome (2n = 8x = 64) with ∼23× coverage and managed to partially assemble 492.7 Mbp of its genomic sequence. A thorough comparison between red clover and zigzag clover sequencing reads resulted in the successful validation of 7 T. pratense - and 45 T. medium -specific repetitive elements. The newly discovered repeats led to the set-up of the first partial T. medium karyotype. Newly discovered red clover and zigzag clover tandem repeats were summarized. The structure of centromere-specific satellite repeat resembling that of T . repens was inferred in T. pratense . Two repeats, TrM300 and TrM378, showed a specific localization into centromeres of a half of all zigzag clover chromosomes; TrM300 on eight chromosomes and TrM378 on 24 chromosomes. A comparison with the red clover draft sequence was also used to mine more than 105,000 simple sequence repeats (SSRs) and 1,170,000 single nucleotide variants (SNVs). The presented data obtained from the sequencing of zigzag clover represent the first glimpse on the genomic sequence of this species. Centromeric repeats indicated its allopolyploid origin and naturally occurring homogenization of the centromeric repeat motif was somehow prevented. Using various repeats, highly uniform 64 chromosomes were separated into eight types of chromosomes. Zigzag clover genome underwent substantial chromosome rearrangements and cannot be counted as a true octoploid. The resulting data, especially the large number of predicted SSRs and SNVs, may have great potential for further research of the legume family and for rapid advancements in clover breeding.

Red clover ( Trifolium pratense ) is an important forage plant worldwide. This study was directed to broadening current knowledge of red clover's coding regions and enhancing its utilization in practice by specific reanalysis of previously published assembly. A total of 42,996 genes were characterized using Illumina paired-end sequencing after manual revision of Blast2GO annotation. Genes were classified into metabolic and biosynthetic pathways in response to biological processes, with 7,517 genes being assigned to specific pathways. Moreover, 17,727 enzymatic nodes in all pathways were described. We identified 6,749 potential microsatellite loci in red clover coding sequences, and we characterized 4,005 potential simple sequence repeat (SSR) markers as generating polymerase chain reaction products preferentially within 100-350 bp. Marker density of 1 SSR marker per 12.39 kbp was achieved. Aligning reads against predicted coding sequences resulted in the identification of 343,027 single nucleotide polymorphism (SNP) markers, providing marker density of one SNP marker per 144.6 bp. Altogether, 95 SSRs in coding sequences were analyzed for 50 red clover varieties and a collection of 22 highly polymorphic SSRs with pooled polymorphism information content >0.9 was generated, thus obtaining primer pairs for application to diversity studies in T. pratense . A set of 8,623 genome-wide distributed SNPs was developed and used for polymorphism evaluation in individual plants. The polymorphic information content ranged from 0 to 0.375. Temperature switch PCR was successfully used in single-marker SNP genotyping for targeted coding sequences and for heterozygosity or homozygosity confirmation in validated five loci. Predicted large sets of SSRs and SNPs throughout the genome are key to rapidly implementing genome-based breeding approaches, for identifying genes underlying key traits, and for genome-wide association studies. Detailed knowledge of genetic relationships among breeding material can also be useful for breeders in planning crosses or for plant variety protection. Single-marker assays are useful for diagnostic applications.

The issue of moulds and, thus, contamination with mycotoxins is very topical, particularly in connexion with forages from grass stands used at the end of the growing season. Deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM) and aflatoxins (AFL) are among the most common mycotoxins. The aim of the paper was to determine concentrations of mycotoxins in selected grasses (Lolium perenne, Festulolium pabulare, Festulolium braunii) and their mixtures with Festuca rubra an/or Poa pratensis during the growing season as a marker of grass safety, which was assessed according to content of the aforementioned mycotoxins. During the growing season grass forage was contaminated with mycotoxins, most of all by DON and ZEA. The contents of AFL and FUM were zero or below the limit of quantification. Moreover, the level of the occurrence of mould was quantified as ergosterol content, which was higher at the specific date of cut. All results were statistically processed and significant changes were discussed.