Genome-wide association studies of root system architecture traits in a broad collection of Brassica genotypes

Abstract

The root systems of Brassica species are complex. Eight root system architecture (RSA) traits, including total root length, total root surface area, root average diameter, number of tips, total primary root length, total lateral root length, total tertiary root length, and basal link length, were phenotyped across 379 accessions representing six Brassica species (B. napus, B. juncea, B. carinata, B. oleracea, B. nigra, and B. rapa) using a semi-hydroponic system and image analysis software. The results suggest that, among the assessed species, B. napus and B. oleracea had the most intricate and largest root systems, while B. nigra exhibited the smallest roots. The two species B. juncea and B. carinata shared comparable root system complexity and had root systems with larger root diameters. In addition, 313 of the Brassica accessions were genotyped using a 19K Brassica single nucleotide polymorphism (SNP) array. After filtering by TASSEL 5.0, 6,213 SNP markers, comprising 5,103 markers on the A-genome (covering 302,504 kb) and 1,110 markers on the C-genome (covering 452,764 kb), were selected for genome-wide association studies (GWAS). Two general linear models were tested to identify the genomic regions and SNPs associated with the RSA traits. GWAS identified 79 significant SNP markers associated with the eight RSA traits investigated. These markers were distributed across the 18 chromosomes of B. napus, except for chromosome C06. Sixty-five markers were located on the A-genome, and 14 on the C-genome. Furthermore, the major marker-trait associations (MTAs)/quantitative trait loci (QTLs) associated with root traits were located on chromosomes A02, A03, and A06. Brassica accessions with distinct RSA traits were identified, which could hold functional, adaptive, evolutionary, environmental, pathological, and breeding significance.

Keywords: Brassica; GWAS; marker-root trait associations; plant breeding; root system architecture.

Figure 1

Correlation analysis between eight root system architecture traits as determined by a Spearman rank-based variable correlation test. Traits include total root length (TRL/cm), total surface area of roots (TRSA/cm²), root average diameter (RAD/cm), number of tips (NTP), total primary root length (TPRL/cm), total lateral root length (TLRL/cm), total tertiary root length (TTRL/cm), and basal link length (BLL/cm). ‘Corr. Coef.’ denotes the coefficient values of the correlation, with the strength of the correlation indicated in different colors. 0.0, no significant correlation (p-value > 0.05).

Figure 2

Principal components analysis (PCA) biplot among six Brassica species including B. napus, B. oleracea, B. rapa, B. nigra, B. carinata, and B. juncea. Traits include total root length (TRL/cm), total surface area of roots (TRSA/cm²), root average diameter (RAD/cm), number of tips (NTP), total primary root length (TPRL/cm), total lateral root length (TLRL/cm), total tertiary root length (TTRL/cm), and basal link length (BLL/cm). The different species are indicated by different colors. With the increase of PC1, most of the values of the root system architecture (RSA) traits increased, except for RAD. With the increase of PC2, RAD, NTP, and TRSA increased, while values of other traits decreased. Most of the B. juncea, B. nigra, B. rapa, and B. carinata genotypes are located in a cloud on the left side of the biplot, while most B. napus and B. oleracea genotypes are located on the right side of the biplot with relatively larger values for the root traits. This suggests that B. napus and B. oleracea have relatively larger root systems.

Figure 3

Bar plot of eight root system architecture traits among six Brassica species. B. napus, and B. oleracea had a relatively greater total root length (TRL/cm), total surface area of roots (TRSA/cm²), root average diameter (RAD/cm), number of tips (NTP), total primary root length (TPRL/cm), total lateral root length (TLRL/cm), and total tertiary root length (TTRL/cm), and basal link length (BLL/cm). In general, values for six of the eight traits (except for NTP and BLL) were lowest in B. nigra. Different letters indicate significant differences.

Figure 4

Percentage of genotypes with small, medium, or large sized root systems in six Brassica species – (A) B. napus, (B) B. juncea, (C) B. rapa, (D) B. nigra, (E) B. oleracea, and (F) B. carinata. Most B. napus and B. oleracea genotypes had larger-sized root systems, while most B. juncea, B. rapa, B. nigra, and B. carinata genotypes had smaller-sized root systems. Genotypes with medium-sized root system represented the smallest percentage for all the species. .

Figure 5

Bayesian cluster analysis of 313 Brassica accessions representing five species, including B. napus, B. oleracea, B. rapa, B. carinata, and B. juncea, estimated with STRUCTURE based on 6,213 SNP markers using 50,000 burn-in iterations and Markov Chain Monte Carlo (MCMC) lengths. The value of K determined following Evanno et al. (2005), and the population structure determined with the Puechmaille (2016) and Li and Liu (2018) alternative, indicated three clusters for all runs (A, B). Detailed Bayesian clustering of the 313 accessions is shown in (C), with each colour represents one ancestry component. The simplified view suggests three ancestral populations. The clusters were obtained based on the standard 70% threshold of similarity with STRUCTURE software (e.g., Yu et al., 2021). Genotypes not meeting this threshold were classified as admixtures or not belonging to any of the groups (Yu et al., 2021).

Figure 6

Manhattan plots of the PCA + K MLM models for identifying root system architecture traits loci in 313 Brassica accessions representing five species, including B. napus, B. oleracea, B. rapa, B. carinata, and B. juncea. Traits include (A) total root length (TRL/cm), (B) total surface area of roots (TRSA/cm²), (C) root average diameter (RAD/cm), (D) number of tips (NTP), (E) total primary root length (TPRL/cm), (F) total lateral root length (TLRL/cm), (G) total tertiary root length (TTRL/cm), and (H) basal link length (BLL/cm). The dashed horizontal lines indicate the Bonferroni-adjusted significance threshold (“logarithm-of-odds” (LOD) score). The dots above the significance threshold indicate single nucleotide polymorphisms (SNPs) associated with each trait.