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

Oldřich Trněný,Jan Hofbauer,Daniela Knotová,Jana Řepková,Michaela Matoušková,David Vlk,Jan Jansa,Hana Jakešová,Jan Nedělník,Eliška Macková,Lubomír Piálek,Karel Vejražka

doi:10.3390/ijms20215470

Abstract

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.

Highlights

The family Fabaceae, consisting of more than 750 genera and 19,000 species, is the third largest family of flowering plants and, in terms of agricultural importance, the second most important family, after Poaceae
Rather than finding new genes in the biological (atmospheric) nitrogen fixation (BNF) process, our study focused on highlighting loci in the red clover genome that are potentially beneficial for BNF, and which should be selected as fixed in starting plant material for breeding new high-BNF rate varieties
We identified polymorphisms in key genes strongly associated with BNF rate: EFD, which negatively regulates the nodulation process and positively influences cell differentiation in the late stages of nodulation, and molybdate transporter type 1 (MOT1), which is responsible for molybdate intake of nodule cells

Summary

Introduction

The family Fabaceae, consisting of more than 750 genera and 19,000 species, is the third largest family of flowering plants and, in terms of agricultural importance, the second most important family, after Poaceae. This initiates a complex process of biological (atmospheric) nitrogen fixation (BNF). In this relationship, the plant provides the bacteria a source of carbon and energy, in addition to phosphorus and other mineral nutrients and anoxic shelter, and the bacteria supply the plant with nitrogen acquired from the atmosphere, converted into organic compounds utilizable in plant metabolism [5]. Soil N enrichment due to effective BNF is environmentally more sustainable than application of synthetic N fertilizers depending on utilization of nonrenewable sources of energy

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