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. 2022 Nov 25;12(12):1975.
doi: 10.3390/life12121975.

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

David Vlk  1 , Oldřich Trněný  2 , Jana Řepková  1
Affiliations

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

David Vlk et al. Life (Basel). .

Abstract

Commonly studied in the context of legume-rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant-bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover's root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.

Keywords: differentially expressed gene; gene duplication; nodule-specific cysteine-rich peptide; transcriptome.

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Conflict of interest statement

O.T. is employed by the company Agricultural Research, Ltd., Troubsko, Czech Republic and he declares no conflict of interests. The remaining authors declare no competing interest.

Figures

Figure 1
Figure 1
Intrapopulation distribution of BNF efficiency among accessions evaluated prior to RNA sequencing. Varieties indicated by letter: A–Tatra, K–Kvarta, S–Start, T–Tempus, G–Global. Numerals following the letter indicate number of the parent plant from 2017. Inside the violin plots, median and interquartile ranges indicated by boxplot, minimum and maximum by whiskers, outliers by dots above boxplot. Accessions are ordered by median values of nitrogen fixation. Orange labels–progeny of strong fixators from 2017, green label–progeny of weak fixators from 2017. On the y-axis, measured BNF efficiency is expressed as concentration of ethylene in µmol/mL.
Figure 2
Figure 2
Dependence of measured BNF efficiency expressed as ethylene molar concentration (CE) on plant fresh mass with linear regression lines. Ethylene molar concentration is expressed as ethylene concentration in µmol/mL.
Figure 3
Figure 3
Heatmap of rlog-transformed read counts for 491 differentially expressed genes. Genes are sorted according to hierarchical clustering and the read count values are scaled per row. High-BNF samples are on the right side of the plot (blue line), and low BNF are on the left (red line).
Figure 4
Figure 4
Volcano plot for 491 differentially expressed genes (red dots). The vertical dashed line indicates threshold log2 fold change > 1, the horizontal dashed line indicates threshold padj > 0.05.
Figure 5
Figure 5
Verification of sequencing data using qPCR for 10 genes (subfigures A–J) in T. pratense genotypes with low and high BNF. All samples were analyzed in triplicate and data are presented as means. The x-axis identifies analyzed samples, and the y-axis shows relative expression in the log2 ratio.
Figure 6
Figure 6
Expression divergence between duplicated gene pairs originated from different modes of duplication. Duplicated pairs were divided according to mode of duplication (x-axis) and proportions of genes with conserved versus diverged expression were calculated (y-axis).
Figure 7
Figure 7
WGCNA analysis: Clustering of genes according to their expression profile similarity. First row assigned a module color to each gene. Rows 2 to 12 are red–blue scale heatmaps of Pearson correlation coefficients between traits and expression levels of the genes (red: 1, blue: −1).
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