Species-Wide Variation in Shoot Nitrate Concentration, and Genetic Loci Controlling Nitrate, Phosphorus and Potassium Accumulation in Brassica napus L

Abstract

Large nitrogen, phosphorus and potassium fertilizer inputs are used in many crop systems. Identifying genetic loci controlling nutrient accumulation may be useful in crop breeding strategies to increase fertilizer use efficiency and reduce financial and environmental costs. Here, variation in leaf nitrate concentration across a diversity population of 383 genotypes of Brassica napus was characterized. Genetic loci controlling variation in leaf nitrate, phosphorus and potassium concentration were then identified through Associative Transcriptomics using single nucleotide polymorphism (SNP) markers and gene expression markers (GEMs). Leaf nitrate concentration varied over 8-fold across the diversity population. A total of 455 SNP markers were associated with leaf nitrate concentration after false-discovery-rate (FDR) correction. In linkage disequilibrium of highly associated markers are a number of known nitrate transporters and sensors, including a gene thought to mediate expression of the major nitrate transporter NRT1.1. Several genes influencing root and root-hair development co-localize with chromosomal regions associated with leaf P concentration. Orthologs of three ABC-transporters involved in suberin synthesis in roots also co-localize with association peaks for both leaf nitrate and phosphorus. Allelic variation at nearby, highly associated SNPs confers large variation in leaf nitrate and phosphorus concentration. A total of five GEMs associated with leaf K concentration after FDR correction including a GEM that corresponds to an auxin-response family protein. Candidate loci, genes and favorable alleles identified here may prove useful in marker-assisted selection strategies to improve fertilizer use efficiency in B. napus.

Keywords: GWAS; associative transcriptomics; casparian strip; diversity population; fertilizer; nitrogen; nutrient use efficiency; suberin.

Figure 1

Leaf nitrate concentrations of Brassica napus plants grown in compost. Data are means of 339 genotypes for which crop growth type had been assigned including 169 winter-, 11 semiwinter-, and 123 spring-oilseed rape (OSR), 27 swede, six fodder and three kale types. Boxes represent the mid two quartiles with the median drawn; whiskers above and below the boxes indicate the 90 and 10th percentiles with any outliers shown. Boxes organized by mean leaf nitrate concentration between crop growth types from highest (left) to lowest (right).

Figure 2

–log₁₀p-values of SNPs and GEMs associated with leaf nitrate concentration (A,B, respectively), leaf phosphorus concentration (C,D, respectively) and leaf potassium concentration (E,F, respectively) in order of markers within the Brassica napus pan-transcriptome. Upper, gold, dashed line represents Bonferroni corrected significance threshold; lower, yellow, dashed line represents FDR corrected significance threshold (p = 0.05).

Figure 3

Effects of reference allele and most frequent allelic variant at lead-marker loci on leaf nitrate concentration. Each panel refers to a specific association peak; lead-markers at each peak are listed in table 1. Boxes represent the mid two quartiles with the median drawn; whiskers above and below the boxes indicate the 90 and 10th percentiles with any outliers shown. A, T, G, and C alleles represent adenine, thymine, guanine and cytosine nucleotide calls, respectively. S, R, and K alleles represent Strong (C or G), purine (A or G) or Keto (C or T) nucleotide calls, respectively, thus called due to unresolvable variation in closely related genes. Differences in leaf nitrate concentration between alleles at each loci significant at p < 0.001.

Figure 4

Effects of reference allele and most frequent allelic variant at lead-marker loci on leaf phosphorus concentration. Each panel refers to a specific association peak; lead-markers at each peak are listed in table 1. Boxes represent the mid two quartiles with the median drawn; whiskers above and below the boxes indicate the 90 and 10th percentiles with any outliers shown. A, T, G, and C alleles represent adenine, thymine, guanine and cytosine nucleotide calls, respectively. Differences in leaf phosphorus concentration between alleles at each loci significant at p < 0.001 except locus on chromosome A6; p = 0.010.

Figure 5

Effect of leaf transcript abundance of genes underlying candidate GEMs on leaf potassium concentration. Transcript abundance was quantified and normalized as reads per kb per million aligned reads (RPKM) for each genotype. r² values and respective significance values calculated in GenStat are shown.