Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula

Abstract

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

Flavonoid Accumulation Varies in Medicago truncatula in Response to Mercury Stress

“…Under environmental stresses, plant cells initiate gene expression programs at the transcriptional level, which regulate metabolite accumulation to adapt to the new conditions ( Hu et al, 2022 ). In a recent GWAS on mercury tolerance in M. truncatula ( Paape et al, 2022 ), we identified a UGT gene as a candidate gene within the proximity to the top SNP related to Hg tolerance. There were other UGT genes in the same region of chromosome 2, whose substrates are unknown.…”

“…Three Hg-tolerant and one Hg-sensitive cultivar within the HapMap panel were selected according to their seedling relative root growth (RRG), which represents a valid indicator of Hg tolerance in M. truncatula ( García de la Torre et al, 2013 ). Tolerant accessions HM081 (RRG = 99.39), HM080 (RRG = 92.00), and HM175 (RRG = 89.96), and sensitive cultivar HM289 (RRG = 21.74) were selected ( Paape et al, 2022 ). Here we will refer to cultivars HM081 , HM080 , HM175 , and HM289 as LR1, LR2, LR3, and LR4, respectively, with L indicating leaves and R indicating roots.…”

“…In previous studies we were able to identify Hg-tolerant and Hg-sensitive M. truncatula cultivars ( García de la Torre et al, 2013 ; Paape et al, 2022 ) by germplasm screening. As indicated above, the general molecular mechanisms of Hg tolerance and response in plants have been described; however, to our knowledge, the role of flavonoids in the response to Hg stress, has not been investigated.…”

“…All main subclasses of plant flavonoids are present in the genus Medicago ( Gholami et al, 2014 ). Additionally, in a recent genome-wide association study (GWAS) on Hg tolerance in M. truncatula ( Paape et al, 2022 ), we identified a UGT gene as a candidate gene within the proximity to the top SNP related to Hg tolerance. Several additional UGT genes were also present in the same region of chromosome 2, and more than 150 UGTs have been identified in the M. truncatula genome ( Modolo et al, 2007 ).…”

Cadmium toxicity in medicinal plants: An overview of the tolerance strategies, biotechnological and omics approaches to alleviate metal stress

“…Genome-wide association studies (GWAS) have been conducted in Brassica rapa using a 60K Brassica Infinium ® SNP array to understand the mechanisms underlying Cd tolerance ( Chen et al., 2018b ). Another GWAS study in Medicago sativa subjected to cadmium stress reveals that the root and leaf response traits are polygenic with multiple quantitative loci (QTL), and genes such as oxidative stress response genes, P-type transporters genes are associated with Cd tolerance ( Paape et al., 2021 ). Studies have been conducted in the OSNramp5 gene to reduce Cd uptake in rice by CRISPR Cas 9 technology ( Wang et al., 2019 ).…”