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.

Agricultural Research Ltd Zahradní 1 664 41 Troubsko Czech Republic

Department of Experimental Biology Masaryk University 625 00 Brno Czech Republic

Department of Zoology Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Institute of Microbiology of the Academy of Sciences of the Czech Republic 142 20 Prague Czech Republic

Red Clover and Grass Breeding 724 47 Hladké Životice Czech Republic

Research Institute for Fodder Crops Ltd 664 41 Troubsko Czech Republic

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Genes Associated with Biological Nitrogen Fixation Efficiency Identified Using RNA Sequencing in Red Clover (Trifolium pratense L.)

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