Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.).

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 15N 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.

Comparison of Biogenic Amines and Mycotoxins in Alfalfa and Red Clover Fodder Depending on Additives.

In the production of fermented feed, each crop can be contaminated with a variety of microorganisms that may produce natural pollutants. Biogenic amines, mycotoxins, and undesirable organic acids can decrease health feed safety. The aim of this study was to compare the counts of microorganisms, levels of biogenic amines, and the mycotoxins in forage legumes, and also to compare the occurrence of microorganisms and levels of mycotoxins in green fodder and subsequently produced silage and the influence of additives on the content of natural harmful substances in silage. The experimental plot was located in Troubsko and Vatín, in the Czech Republic. Two varieties of Medicago sativa and one variety of Trifolium pratense were compared. Green fodder and subsequently produced silage reaching up to 23% of dry matter were evaluated and prepared using a bio-enzymatic additive and a chemical additive. Green fodder of Medicago sativa was more contaminated by Enterococci than Trifolium pratense fodder. The obvious difference was determined by the quality of silage leachate. The silage prepared from Medicago sativa fodder was more contaminated with butyric acid. Fungi were present in higher counts in the anaerobic environment of green fodder and contaminated it with zearalenone and deoxynivalenol. Lower counts of fungi were found in silage, although the zearalenone content did not change. Lower content of deoxynivalenol was detected in silage, compared with green fodder. Silages treated with a chemical additive were found not to contain butyric acid. Lower ethanol content was determined, and the tendency to reduce the risk of biogenic amines occurrence was evident. The additives proved to have no influence on the content of mycotoxins.

Determination of Phytoestrogen Content in Fresh-Cut Legume Forage.

The aim of the study was to determine phytoestrogen content in fresh-cut legume forage. This issue has been much discussed in recent years in connection with the health and safety of feedstuffs and thus livestock health. The experiments were carried out on two experimental plots at Troubsko and Vatín, Czech Republic during June and July in 2015. Samples were collected of the four forage legume species perennial red clover (variety "Amos"), alfalfa (variety "Holyne"), and annuals Persian clover and Alexandrian clover. Forage was sampled twice at regular three to four day intervals leading up to harvest and a third time on the day of harvest. Fresh and wilted material was analyzed using liquid chromatography-mass spectrometry (LC-MS). Higher levels ( p < 0.05) of isoflavones biochanin A (3.697 mg·g (-1) of dry weight) and formononetin (4.315 mg·g (-1) of dry weight) were found in red clover than in other species. The highest isoflavone content was detected in red clover, reaching 1.001% of dry matter ( p < 0.05), representing a risk for occurrence of reproduction problems and inhibited secretion of animal estrogen. The phytoestrogen content was particularly increased in wilted forage. Significant isoflavone reduction was observed over three to four day intervals leading up to harvest.