# Genome-Wide Association Study Elucidates the Genetic Architecture of Manganese Tolerance in Brassica napus

Harsh Raman, Zetao Bai, Brett Mcvittie, Sourav Mukherjee, Hugh D. Goold, Yuanyuan Zhang, Nay Chi Khin, Yu Qiu, Nawar Shamaya, Shengyi Liu, et al.

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Harsh Raman, Zetao Bai, Brett Mcvittie, Sourav Mukherjee, Hugh D. Goold, et al.. Genome-Wide Association Study Elucidates the Genetic Architecture of Manganese Tolerance in Brassica napus. Plant, Cell and Environment, 2025, 10.1111/pce.15433. hal-04984122

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ORIGINAL ARTICLE

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# Genome-Wide Association Study Elucidates the Genetic Architecture of Manganese Tolerance in *Brassica napus*

Harsh Raman<fnref n="1" /> ID | Zetao Bai² ID | Brett McVittie<fnref n="1" /> ID | Sourav Mukherjee³ ID | Hugh D. Goold⁴,⁵ ID | Yuanyuan Zhang² ID | Nay Chi Khin<fnref n="1" />,⁶ ID | Yu Qiu<fnref n="1" /> ID | Nawar Shamaya<fnref n="1" /> ID | Shengyi Liu² ID | Regine Delourme⁷ ID | Barry J. Pogson⁶ ID | Sureshkumar Balasubramanian³ ID | Rosy Raman<fnref n="1" /> ID

**Correspondence:** Harsh Raman (harsh.raman@dpi.nsw.gov.au)

**Received:** 14 July 2024 | **Revised:** 2 February 2025 | **Accepted:** 4 February 2025

**Funding:** This research was supported by NSW DPIRD and GRDC, partly under the National Brassica Germplasm Improvement Programs (DAN00208), and DPI2204-021RTX led by H.R. N.K., H.R. and B.J.P. acknowledge the support of the ARC Training Centre for Future Crops Development (IC210100047).

**Keywords:** canola | genetic architecture | GWAS | manganese tolerance | soil acidity | yeast and ectopic expression

## ABSTRACT

*Brassica napus* (canola) is a significant contributor to the world's oil production and is cultivated across continents, yet acidic soils with aluminium (Al³⁺) and manganese (Mn²⁺) toxicities limit its production. The genetic determinants underlying natural variation for acidic soil tolerance in canola are unknown and need to be determined. Through genome-wide association analysis of 326 canola accessions, we identified three QTLs for tolerance to Mn²⁺ toxicity on chromosomes A09, C03, and C09. Allelism tests between four tolerance sources confirmed that at least one locus on A09 controls Mn²⁺ tolerance in canola. Integrated analyses of genomic and expression QTL and Mn²⁺ tolerance data revealed that *BnMTP8.A09*, possibly in conjunction with *BnMATE.C03*, *BnMTP8.C04* and *BnMTP8.C08*, play a central role in conferring Mn²⁺ tolerance in canola. Gene expression analysis showed that variation in *BnMTP8.A09* expression could account for upto 74% of the variation in Mn²⁺ tolerance between individuals with extreme phenotypes. Yeast complementation assays and ectopic expression in Arabidopsis show that *BnMTP8.A09* can complement manganese-hypersensitive yeast mutant strain *PMR1Δ* and Arabidopsis *atmtp8* mutant background, respectively and restore Mn²⁺ tolerance to wild-type levels. Our multi-omics research approach unveils the genetic architecture of Mn²⁺ tolerance and identifies *BnMTP8.A09* as a causal gene imparting tolerance to Mn²⁺ toxicity in canola.

## 1 | Introduction

Soil acidity affects approximately 50% of the world's arable land and limits the production of crops, especially in tropical and subtropical regions (Kochian 1995). The projected effects of global climate change are likely to exacerbate manganese

(Mn²⁺) toxicity over the coming decades (Fernando and Lynch 2015). At low pH (< 5.5), exchangeable Al³⁺, Mn²⁺, and H⁺ ions get solubilized into a solution form, which causes toxicities to plants. With growing global food demands, increasing productivity from the marginal and problematic soil is essential. The relative importance of each ion toxicity varies

<footnote n="1">¹New South Wales Department of Primary Industries and Regional Development, Wagga Wagga Agricultural Institute, Wagga Wagga, Australia | ²The Key Laboratory of Biology and Genetic Improvement of Oil Crops, the Ministry of Agriculture of PRC, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, Hubei, China | ³Monash University, Clayton, Australia | ⁴New South Wales Department of Primary Industries and Regional Development, Elizabeth Macarthur Agricultural Institute, Menangle, Australia | ⁵School of Natural Sciences, Macquarie University, Sydney, Australia | ⁶ARC Training Centre for Future Crops Development, Research School of Biology, Australian National University, Canberra, Australia | ⁷INRA, Agrocampus Ouest, Université de Rennes 1, UMR1349 Institut de Génétique, Environnement et de Protection des Plantes, Le Rheu, France</footnote>

<footnote>Harsh Raman and Zetao Bai contributed equally.</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly
cited.</footnote>

<footnote>© 2025 The Author(s). *Plant, Cell & Environment* published by John Wiley & Sons Ltd.</footnote>
across soils with different chemistries: Al³⁺ toxicity primarily inhibits root growth; while, Mn²⁺ toxicity causes interveinal and leaf margin chlorosis, brown and necrotic lesions, leaf cupping, and crinkling; with both resulting in reduced crop yield (Bergmann 1992; Foy 1983; Marschner 1995). Mn²⁺ toxicity can also result in the inhibition of net photosynthesis assimilation, accumulation of reactive oxygen species, disruption of the activity of critical enzymes, and impairment of absorption, translocation, and utilisation of essential nutrients for plant growth, including Ca²⁺, Fe³⁺, Zn²⁺, and Mg²⁺ (Bloom and Lancaster 2018; Horst 1988; Li 2021).

Surface soil acidity can be ameliorated by applying lime. However, it is challenging to incorporate lime in deeper layers to correct the subsoil acidity, and it takes several years before these soils become productive for commercial cropping. In nature, to cope with toxic levels of Mn²⁺ ions, plants have evolved several strategies, such as sequestration into subcellular compartments, activation of the antioxidant system, and regulation of the uptake, translocation, and distribution of Mn (Fecht-Christoffers et al. 2006; Li et al. 2019; Peiter et al. 2007; Pittman 2005). Several proteins play a role in the homoeostasis and detoxification of Mn²⁺. These include various transporters such as Natural Resistance Associated Macrophage Protein (NRAMP1, NRAMP3, NRAMP4, NRAMP5), Iron Transporter (IRT1), ATP-binding cassette (ABC, multidrug resistance-associated proteins, MRP), iron-regulated transporters (IREG) and Cation/H⁺ Exchanger (CAX, CAX2, ECA1), Cation diffusion Facilitator (CDF, or metal tolerance protein, MTP), and P-type ATPase (ZIP) (Castaings et al. 2021; Li et al. 2019).

Natural variation for tolerance to Mn²⁺ toxicity is described in several plant species, including *Brassica napus* (Basu et al. 2001; Delhaize et al. 2003; Foy 1984; Horiguchi 1988; Kassem et al. 2004; Khan and McNeilly 1998; Mizuno et al. 2008; Moroni et al. 2003; Peiter et al. 2007; Pradeep et al. 2020; Raman et al. 2017; Schaaf et al. 2002; Wratten and Scott 1979). Studies have shown that *AtMTP11*, *BnMTP8.C04*, *BnMTP9.A07*, *ShMTP1*, and *NRAMP5* confer tolerance to Mn²⁺ in plants (Delhaize et al. 2007; Delhaize et al. 2003; Gu et al. 2022; Noor et al. 2023). However, the genetic basis of natural variation in tolerance to Mn²⁺ toxicity was not described in diverse *B. napus* germplasm.

*B. napus* (canola/rapeseed, $2n = 4x = 38$, genome $A^nA^nC^nC^n$) is a widely grown critical crop of importance to agriculture and is sensitive to Mn²⁺ toxicity. It contributes approximately 12% of the global edible vegetable oil supply (FAO STAT, https://www.fao.org/faostat/) and protein for feedstock. In addition, canola oil accounts for 80%-85% of the renewable sources for biodiesel production (Tursi 2019). To develop Mn²⁺ tolerant canola cultivars suitable for cultivation on acid soils, we have previously mapped a major locus, *BnMTP8.A09* on chromosome A09, for tolerance to Mn²⁺ toxicity in a biparental doubled haploid population derived from a cross between Darmor-*bzh* (French cultivar) and Yudal (Korean cultivar) using QTL mapping approach (Raman et al. 2017) near the chromosome A09 orthologues of the *AtMTP8* transporter gene of *A. thaliana* (Delhaize et al. 2003). QTL mapping studies often capture only a slice of the genetic architecture of a trait because only alleles that differ between parental lines segregate (Holland 2007).

Understanding the genetic architecture of Mn²⁺ tolerance genes in genetically diverse germplasm provides insights into crop adaptation and assists the development of canola varieties tolerant to Mn²⁺ toxicity.

Herein, we present the genetic architecture of Mn²⁺ tolerance in diverse canola accessions, which shows multiple loci contribute tolerance to Mn²⁺ toxicity. In addition, we identify *BnMTP8.A09* variants underlie a major QTL for tolerance to Mn²⁺ toxicity in canola using genome-wide association studies (GWAS) and allelic complementation tests. The *BnMTP8.A09* gene is a causal candidate that acts through transcript level regulation, explains a substantial proportion of variation in Mn²⁺ toxicity and is associated with tolerance to Mn²⁺ toxicity under laboratory, glasshouse, and field conditions.

## 2 | Materials and Methods

### 2.1 | Plant Materials

To investigate the genetic architecture of loci controlling tolerance to Mn²⁺ toxicity, we carried out six experiments in laboratory, glasshouse and field conditions (Methods S1). The GWAS panel consisted of 415 *B. napus* spring, semi-winter and winter accessions representing Australian, Asian, North American and European breeding programmes (Experiment 1).

To test the genetic inheritance, chromosomal locations and relevance of GWAS associations in the *B. napus* breeding programmes, we generated four F₂ intercross populations derived from P3083 (Chinese cultivar, tolerant to Mn²⁺) × ZY003 (Chinese cultivar, sensitive to Mn²⁺), Mutu (Japanese cultivar, tolerant to Mn²⁺) × RSO-96 (sensitive to Mn²⁺), Darmor-*bzh* (French cultivar, tolerant to Mn²⁺) × Mutu, and Darmor-*bzh* × Jet Neuf (French cultivar) (Experiment 2–3; Table S1). The relationship between Mn²⁺ tolerance and *BnMTP8.A09* expression was established using a subset of 20 GWAS accessions (Experiment 4). For validation of Mn²⁺ tolerance under field conditions, parental lines and 175 doubled haploid (DH) lines derived from Darmor-*bzh*/ Yudal (Korean cultivar) population (Pilet et al. 1998; Raman et al. 2017) plus 15 controls complemented with a glasshouse bioassay (Experiment 5, Table S1).

### 2.2 | Phenotypic Evaluation for Tolerance to Mn²⁺ Toxicity

Natural variation of *B. napus* accessions for tolerance to Mn²⁺ toxicity was assessed in different environments (Table S1). Response of *B. napus* GWAS panel and F₂ lines to Mn²⁺ tolerance was assessed in a nutrient solution supplemented with 125 µM of manganese tetrahydrate (MnCl₂·4H₂O) as described previously (Raman et al. 2017). Seedlings were grown in a laboratory maintained at 20 ± 1°C and 200 µmolm⁻²s⁻¹ light intensity under 16 h of light and 8 h of dark regime. Statistical valid experiment designs were followed and detailed (Methods S2). After 96 h of Mn²⁺ treatment, the critical symptoms of Mn²⁺ toxicity as the extent of chlorosis on
cotyledonary lobes were scored quantitatively as “1”–“5” as described earlier (Raman et al. 2017). After scoring each F₂ population and the parental lines for Mn²⁺ tolerance, 2–3-week-old seedlings from each population were transplanted in plastic pots to raise F₂:₃ progenies. Plants were selfed to ensure purity and avoid cross-pollination for different phenotyping and genotyping experiments. Each F₂:₃ line was assessed for Mn²⁺ tolerance as described above. To determine differences in Mn²⁺ accumulation among extreme accessions, we collected shoot tissue from the young seedlings grown in a nutrient solution (Raman et al. 2017). Pooled tissue samples across replicates were washed thrice with deionised water and then dried at 80°C in an oven for 72 h. In addition to Mn, we also determined the concentration of Ca and Fe, as excess Mn restricts the uptake and translocation of these essential elements, using inductively coupled plasma mass spectrometry (ICP-MS) as described previously (Delhaize et al. 2007).

## 2.3 | Evaluation of Lines for Field and Glasshouse Performance

A total of 192 lines, including 175 from DH population derived from Darmor-*bzh* and Yudal, two parental lines (Darmor-*bzh* and Yudal) and 15 controls were evaluated to test their performance on acid soil at Mangoplah, NSW, Australia (35.3532054° S, 147.2901734° E, Experiment 5). Symptoms of Mn²⁺ toxicity were visually scored as tolerant (0) and sensitive (1). Lines segregation for Mn²⁺ tolerance (in control accessions) were scored as intermediate (0.5). Experiment 5 also involved the evaluation of 20 DH lines from the Darmor-*bzh*/Yudal population with different *BnMTP8* alleles for tolerance and sensitivity to Mn²⁺ toxicity. Acid soil was collected from the top 30 cm layers from the Mangoplah site and tested for pH. The soil had a pH of 4.3 in CaCl₂, and Mn content was 198 µM. The soil suitability for phenotyping to Mn²⁺ tolerance was validated using a bioassay conducted in an environment-controlled growth chamber (Percival, USA) with a 250 µmol M⁻² S¹ photon flux density, 50% humidity and a 22°C/20°C (16/8 h) day/night temperature regime.

## 2.4 | DNA Isolation

Genomic DNA was isolated following a standard phenol/chloroform extraction method for whole-genome resequencing and targeted gene sequencing of the *BnMTP8.A09* gene using the Sanger-sequencing approach at the Australian Genomic Research Facility (http://www.agrf.com). Low-density DArTseq genotyping of F₂ lines was performed based on the genotyping-by-sequencing method (Raman et al. 2016).

## 2.5 | Whole Genome Resequencing and SNP Identification

We resequenced 415 accessions using the WGR approach at the commercial Novogene and Illumina HiSeqXTen services (BGI-Shenzhen, China). Clean paired-end reads were mapped to the *B. napus* reference genome sequence, version 4.1 of the Darmor-*bzh*,

downloaded from the Genoscope website (https://www.genoscope.cns.fr/brassicanapus, Chalhoub et al. 2014) using BWA-MEM with default parameters (Li and Durbin 2009). The SNPs for each line were identified using the pipeline of Sentieon DNAseq (v201711.05, https://www.sentieon.com). The filtering was accomplished with function, variant filtration in GATK (v3.4-46-gbc02625, https://soCDFware.broadinstitute.org/gatk/) using the parameters of QUAL < 30, MQ < 50 and QD < 2. The extent of heterozygosity and the minor allele frequency (MAF) were calculated using VCFtools (v0.1.13). We used only 326 accessions that had high-quality DNA resequencing data. High-quality SNPs with MAF > 0.05 and missing rate < 0.1 were used for GWAS analysis.

## 2.6 | Population Structure, GWAS and Candidate Gene Identification

The tagSNPs, extracted using plink (v1.9) (Purcell et al. 2007), were used as input files for phylip software (v3.697) (https://evolution.genetics.washington.edu/phylip.html) to construct the phylogenetic tree. The tree was visualised using the FigTree package (v1.4.4, http://tree.bio.ed.ac.uk/soCDFware/figtree/). The population structure was constructed using admixture software (v1.3.0, www.genetics.ucla.edu/software/admixture). We estimated LD in VCFtools, using high-quality SNPs. The LD between marker pairs was estimated using the correlation coefficient of the allelic frequencies (*r*²), considering all the possible allele combinations. The physical distance when the decay of linkage disequilibrium (LD) reached the half maximum (1/2 LD distance) in a GWAS population was calculated using VCFtools (v0.1.13, http://vcftools.github.io). We accounted for the population structure and the kinship matrix for GWAS. The latter was calculated using the Efficient Mixed-Model Association eXpedited (EMMAX)-kin method. GWAS was conducted using the EMMAX software with a linear mixed model (Kang et al. 2010), which corrects spurious associations due to population structure. The LD heatmap, Manhattan and Quantile-Quantile (Q-Q) plots were drawn in the R program (v4.0.5, https://www.r-project.org/). The candidate genes for GWAS loci were extracted based on physical positions of highly significant associated SNP ± ½ LD distance (kb). Haplotype analysis was performed by extracting the SNP data of candidate genes including gene body and flanking region (2 kb upstream/downstream regions) and calculating the phenotypic differentiation between haplotypes.

## 2.7 | Selective Sweep Analyses

The SNP data sets with missing rates < 0.1 were used for selective sweep analysis. The fixation index (*F*<sub>ST</sub>) and the nucleotide diversity (π) were analysed using the VCFtools package. We analysed a cohort of 50 extreme accessions (25 tolerant accessions with a mean score of < 2 and 25 sensitive accessions with a mean score of ≥ 4) from the GWAS panel consisting of 415 accessions. The SNP data for both cohorts were extracted from filtered SNP data of the GWAS panel. In total, 2,226,172 SNPs for each cohort were used for selective sweep analysis. The *t*-test of significance was used to determine
the differences between tolerant and sensitive cohorts. The threshold of $F_{ST} > 0.19$, which corresponded to the top 5% of sites, was used to detect the selective sweep.

## 2.8 | BnMTP8.A09 Allele Mining in B. napus
Global Germplasm

Allelic variation, as SNPs and InDELs, in the *BnMTP8.A09* gene was investigated in the published data set (https://bnaomics. ocri-genomics.net/) of resequenced 2311 *B. napus* accessions, representing 1259 accessions from Asia, 929 accessions from Europe, 60 from North America, two from South America, 38 from Oceania and four from Africa. (Lu et al. 2019; Song et al. 2020; Tang et al. 2021; Wu et al. 2019). These accessions included three ecotypes, spring (354 accessions), winter (756 accessions) and semi-winter (1122 accessions).

## 2.9 | Genetic Architecture of Mn²⁺ Tolerance

To describe the genetic architecture underlying Mn²⁺ tolerance, the reported QTL region related to the Mn²⁺ tolerance of *B. napus* was integrated with significant SNP associations identified in the GWAS panel. Their physical positions or QTL intervals were aligned to the Darmor-*bzh* reference genome sequence of *B. napus* v4.1 and were drawn using the MapChart programme.

## 2.10 | BnMTP8.A09 Gene Expression and
Structural Variation Analyses

We investigated gene expression using tails, that is, extreme 20 phenotypes (Mn²⁺ tolerant with 1–2 score and Mn²⁺ sensitive with 4–5 scores) of the DH population derived from the Darmor-*bzh*/Yudal and GWAS panel (Table S1). Twelve biological replicates (four plants/replicate × three replications) from each treatment were taken within 1 h during the light cycle at 10 days in hydroponic. Cotyledonary leaves showing tolerant and sensitive phenotypes were pooled from four plants per replicate and flash-frozen in liquid nitrogen. cDNA synthesis and the relative expression of the *BnMTP8.A09* gene were calculated by the relative quantification method using UBC9 as the reference gene, as outlined previously (Raman et al. 2019). Gene-specific primers for the *BnMTP8.A09* used for the expression analysis are given in Table S2. We also obtained sequence information for *MTP8* paralogs from whole-genome resequencing data of the 326 canola accessions. Variation across the *MTP8* paralogs was extracted using the gene model information or manually identifying gene regions based on BLAT homology. The physical positions of different *MTP8* paralogs (NCBI GenBank accession; AT3G58060.1, MTP8 cation efflux family protein) were confirmed with those of the sequenced *MTP8* genes on the ‘Darmor-*bzh*’ assembly v4.1. For each accession, the *BnMTP8* nucleotide sequences were aligned using MUSCLE as implemented in the software package Genious (https://www.geneious.com). Structural variation, the number of polymorphic sites within the gene and the promoter region were identified. The selection pressure ($K_a/K_s$ values for

paralogous genes in *Arabidopsis*) of target genes was calculated using KaKs_Calculator v3.0 software (Zhang 2022). The functional domains were verified using information from the NCBI conserved domain database. Protein-protein-interaction network using the STRING (search tool for recurring instances of neighbouring genes) database (Version 11.0, http://string-db.org/).

## 2.11 | Arabidopsis Transformation and
BnMTP8.A09 Expression Analysis

A gene fragment of *BnMTP8.09* from *B. napus* cv. Darmor-*bzh* (Mn²⁺ tolerant) flanked by *AttR* restriction sites was synthesised (Genscript). To insert the gene fragment into the destination vector, pMDC32, Gateway LR reaction was performed (Method S2). Plasmid DNA from a single colony of transformed *E. coli* cells was isolated and confirmed for cloned *BnMTP8.A09* gene fragment in the plasmid via Nanopore sequencing (Warren Bach, Oxford Nanopore Technologies Ltd). Mn²⁺ sensitive *atmtp8* lines were grown in pots under controlled environment conditions (16 h photoperiod at 22°C–25°C, humidity of 55% and a light intensity of about 100–150 µE in growth cabinet, Percival USA) at the Wagga Wagga Agricultural Institute and transformed using the floral dipping method (Clough and Bent 1998; Zhang et al. 2022) using *A. tumefaciens* GV3101 strain harbouring *pro35S-BnMTP8.A09-nosT*. A rapid screening method developed by Harrison et al. (2006) was used for the isolation of successful transgenic plants. The plants were then confirmed for the presence of hygromycin phosphotransferase gene (HptII) and *BnMTP8.A09* gene in the genome through PCR amplification (Phire Plant Direct PCR Kit, Thermo Fisher) and determination of *BnMTP8.A09* gene expression levels (Power SYBR Green RNA-to-CT 1-Step Kit, Applied Biosystems). To determine the expression of the *BnMTP8.A09* gene, total RNA from tissue samples were extracted with GeneJET Plant RNA Purification Kit (Thermo Fisher). The RT-qPCR was carried out using Power SYBR Green RNA-to-CT in QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). PROTEIN PHOSPHATASE 2A SUBUNIT A3 (PP2AA3) gene was used as the housekeeping gene.

## 2.12 | eQTL Mapping on BnMTP8.A09

To analyse the expression regulation and potential interaction of *BnMTP8.A09*, we collected the RNAseq data and genotyping data of *B. napus* from the databases, including BnIR (http://yanglab.hzau.edu.cn/BnIR) and National Genomics Data Center (GSA Bioprojects: PRJCA002835, PRJCA002836 and PRJCA013095). The gene expression level was determined by TPM (Transcripts Per Million) and then used for eQTL mapping on target genes. The normalised gene expression values were used as the phenotype for eQTL analysis. GWAS-SNPs with MAF > 0.01 were utilised to perform eQTL mapping using Genome-wide Efficient Mixed Model Association (Zhou and Stephens 2012) to detect associations of SNP-gene pairs. The threshold value for determining significant associations was set as −log₁₀(1/n), where ‘n’ represents the total number of SNPs in the *B. napus* population. Based on the distance between eQTL and target genes, we
subdivided an eQTL into cis-eQTL if its lead eSNP was found
within 1 Mb from the transcription start site or transcription end
site of the target gene; otherwise, including located on different
chromosomes, it was assumed as trans-eQTL.

2.13 | Functional Complementation in Yeast

We used the hypersensitive yeast strain *pmr1Δ* for functional
complementation assay (Experiment 6). The coding sequence of
the *BnMTP8.A09* gene (*BnaA09g37250D*) was translated to a
peptide sequence and then backtranslated using the EMBOSS
backtranslate tool with *Saccharomyces cerevisiae* codon prefer-
ences. A gene cassette was designed *in-silico* by incorporating
the *S. cerevisiae* Tef2 promoter (700 bp) and *S. cerevisiae* TDH1
terminator (224 bp) flanking the *S. cerevisiae* codon-optimised
coding sequence. The construct was synthesised (Azenta) with a
*Not*I site 5' of the promoter sequence and *BamHI* site 3' of the
terminator sequence and was cloned into a polylinker of the
low-copy yeast expression vector PRS-413 (Euroscarf). The
plasmid was transformed into yeast knockout collection strain
BY4741 Δ*PMR1* by incubating exponential phase cells at 42°C in
360 µL of 50% PEG 3500, 100 mM Lithium Acetate, containing
10 µL herring sperm (Sigma). Transformed cells were plated on
*Saccharomyces cerevisiae* medium with histidine dropout
(Sigma). Vector-only controls were transformed into BY4741
Δ*PMR1* and By4741. Cells were normalised to an optical density
of 1, and a serial dilution was plated on yeast minimal media
without histidine and with various concentrations of MnCl₂.
Spot assays were photographed after 48 h.

3 | Results

**3.1 | Genotypic Variation for Mn²⁺ Tolerance**

For elucidation of the genetic architecture underlying tolerance
to Mn²⁺ toxicity, we carried out six experiments (Methods S1). To
screen for Mn²⁺ tolerance we first scored symptoms of Mn²⁺
toxicity after 5 days of Mn²⁺ treatment in 415 accessions of *B. napus* (Table S1). Mn²⁺ toxicity was typically characterised by
extensive chlorosis on the cotyledonary lobes (Figure 1A). Several
accessions showed interveinal and marginal leaf chlorosis with
some displaying cupping and necrotic spots on the leaf, sugges-
tive of Mn²⁺ sensitivity. However, there were Mn²⁺ tolerant ac-
cessions which revealed no such visible symptoms or had limited
chlorosis (Figure 1B–F). We scored each accession for their Mn²⁺
tolerance, visibly on a scale of 1 (Mn²⁺ tolerant) to 5 (Mn²⁺
sensitive). While a majority of accessions (72.29%) were generally
sensitive to Mn²⁺ with scores above 3, and 26.51% were inter-
mediate (Mn²⁺ tolerance scale ≥ 2−≤ 33) we observed significant
variation among diverse accessions (Table S1, Figure 1G).

**3.2 | Genome-Wide Association Analysis (GWAS)**
**Reveals Genetic Architecture for Mn<sup>2+</sup> Tolerance**

To ascertain the genetic relatedness of the accessions, we car-
ried out whole genome resequencing (WGR) at a moderate
depth (4.69×-99.59× with an average of 13.03×, Table S2). WGR

provided 8 789 769 high-quality single nucleotide polymor-
phisms (SNPs) mapped to the reference genome of *B. napus* cv.
Darmor-*bzh* v.4.1. Filtering-out variants with MAF < 0.05 and
missing rate > 0.1 resulted in a total of 2,226,172 high-quality
SNPs used for GWAS analysis, which averages to roughly one
SNP marker per 20 Kb of the canola genome. Population
structure analysis revealed four genetically distinct clades: I, II,
III, and IV in the GWAS population, consistent with their
geographic origins (Figure 2A–C, Table S3). We calculated
pairwise SNP linkage disequilibrium (LD) using 1,981,597 tag-
ged SNP derived from haploblocks. The LD patterns were var-
iable across the whole (*A*<sup>n</sup>*C*<sup>n</sup>) genome and within the *A*<sup>n</sup> and
the *C*<sup>n</sup> subgenomes. Consistent with previous studies
(Chalhoub et al. 2014), the LD decays faster in the *A*<sup>n</sup> sub-
genome than in the *C*<sup>n</sup> subgenome (Figure 2D). Using Mn<sup>2+</sup>
toxicity phenotypes of the cotyledons and accounting for pop-
ulation structure and kinship coefficients, we identified 34
significant SNP associations (binned into three loci) for Mn<sup>2+</sup>
tolerance on chromosomes A09, C03, and C09 (Figure 3A,
Figure S1B, Table S4). There was a good fit between observed
and expected SNP associations (Figure 3B). Twenty-nine SNP
associations (of 34 SNPs) were identified within the 300 kb
genomic region on A09 (Table S4). Our results indicate that
multiple loci contribute to tolerance to Mn<sup>2+</sup> toxicity.

3.3 | Validation and Fine Mapping of the GWAS Loci Using Bi-Parental Populations

To verify the linkage between Mn²⁺ tolerance and SNP markers,
we performed selective sweep analysis using *F*<sub>ST</sub> and ratio test,
utilising a cohort of 50 extreme accessions from the GWAS panel
(Table S5). Although current breeding programmes are not
intentionally selecting for Mn²⁺ tolerance, our results showed
that the A09 genomic region is subjected to passive selection
under acid soil conditions (Figure S1A). Haplotype association
analysis revealed that A09 haplotypes showed a statistically sig-
nificant difference in Mn²⁺ tolerance (Figure 3C–G).

To validate the genetic linkage between Mn²⁺ tolerance and A09 genomic region, we evaluated two F₂ populations derived from P3083 (China, tolerant to Mn²⁺) × ZY003 (China, sensitive to Mn²⁺), and Mutu (Japan, tolerant to Mn²⁺) × RSO 96 (sensitive to Mn²⁺), in hydroponic culture. Both populations showed approximately monogenic segregation for Mn²⁺ tolerance in a dominant manner (Table S6, Figure S2). QTL analysis revealed a single genomic region for Mn²⁺ tolerance on chromosome A09 (Figure S3). We compared the physical positions of SNPs associated with Mn²⁺ tolerance with those identified in the earlier study from Darmor-*bzh*/Yudal (Raman et al. 2017). We found that one genomic region on chromosome A09 (26.36 Mb to 27.16 Mb) is shared across the GWAS, F₂, and DH (Darmor-*bzh*/Yudal) panels (Table S5), suggesting common allelic variation at this locus for Mn²⁺ tolerance in *B. napus*. To test whether Mutu and Darmor-*bzh*, earlier described sources of Mn²⁺ tolerance (Moroni et al. 2003, Raman et al. 2017) also harbour the same gene(s), we tested the crosses made between three sources of tolerance. There was no segregation among F₂ progenies derived from Darmor-*bzh* (tolerant to Mn²⁺) × Mutu (tolerant to Mn²⁺) and Darmor-*bzh* (tolerant to Mn²⁺) × Jet Neuf (tolerant to Mn²⁺) Table S6, Figure S2). These results
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** | Critical symptoms and natural variation in tolerance to manganese (Mn²⁺) toxicity in *B. napus* accessions as evident in cotyledon and fully expanded leaves. (A) Mn²⁺-sensitive accessions show extensive chlorosis on cotyledons of young seedlings grown on 125 µM MnCl₂·4H₂O on Day 4 from germination, while Mn²⁺ tolerant accessions did not show such symptoms, as observed in Mn²⁺ sensitive accessions. (B) Mn²⁺ sensitive accessions show extensive chlorosis, curling and necrosis on mature leaves of seedlings (21 days after germination), while Mn²⁺ tolerant accessions show no such symptoms observed in Mn²⁺ sensitive accessions (marked with arrow). Stereomicroscope images showing normal leaf development in Mn²⁺ tolerant accession (C) and a range of toxicity symptoms (D) chlorosis; E: leaf curing and chlorosis (F) dark brown leaf speckles on 3-4-week-old plants) in Mn²⁺ sensitive accessions. (G) Frequency distributions of Mn²⁺tolerance scores in the 415 *B. napus* accessions of GWAS panel. The tolerance scores are based on the extent of leaf chlorosis. Each line had four plants/replicate and replicated thrice (12 biological replicates). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

suggest that parental lines Darmor-*bzh*, Jet-Neuf, and Mutu have the same or similar alleles that control Mn²⁺ tolerance. This result also corroborates that Darmor-*bzh* and Jet-Neuf share a common ancestry.

**3.4 | Candidate Genes Underlying Significant Loci Contributing to Mn²⁺ Tolerance**

We searched candidate genes based on LD with significantly (*P* > 3.0) associated SNPs (Table S5) and found 19 genes that

were located within the 7.8 kb regions of the GWAS-SNPs on A09, C03, and C09 chromosomes (Figure S1B-D, Table S7), including Metal Tolerance Protein 8 (MTP8, designated as *BnMTP8.A09* in *B. napus*), which has previously been shown to confer natural variation in Mn²⁺ tolerance in Arabidopsis (Delhaize et al. 2003) making it an obvious candidate for further investigation. Protein-protein-interaction network also revealed that candidate genes prioritised in this study are related to cation transport and intercellular homoeostasis (Figure S1E-G). Candidate genes include several Zn transporters (ZAT, AT2G04620, AT3G12100, *MTPB1*, AT1G51610, *MTPA2*),
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** | Genetic diversity, population structure and linkage disequilibrium in the AHGDS panel of *B. napus*. (A) Circular phylogenetic tree showing grouping (I–IV) of 326 accessions based on the neighbour-joining method. (B) population structure of 326 accessions revealed by the Bayesian method, STRUCTURE. (C) Principal component plots show three predominant clades of 326 accessions: Australia (I), Europe (II), Asia (III), and others (IV). (D) Genome and subgenome-wide LD decay plots. The horizontal black lines are the standard critical $R^2$ value, and the vertical red, black, and blue lines represent the AC, A and C subgenomes of *B. napus*. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

high-affinity Mn²⁺ transporter involved in Fe, Cd and Co acquisition (NRAMP1), vacuolar transporter involved in intercellular metal (Fe, Mn, Cd and Co) homoeostasis, IREG2, (IRON REGULATED 2) encoding FPN2, a tonoplast localised Ni transport protein network with MTP8, and TMN1 (Transmembrane 9) gene which interacts with proteins involved in Golgi transport complex-related vascular transport, intercellular protein transport, and transfer from ER via Golgi.

We performed functional domain analysis and sequenced the full length of *BnMTP8.A09* coding alleles (1,966 bp) from the parental lines of the Darmor-*bzh*/Yudal DH population using A09 sequence-specific primers, which revealed 12 polymorphic SNPs and InDELs (Table S8, Figure 4A) for Mn²⁺ tolerance. In addition, we characterised sequence variants in *BnMTP8* from a

data set of 2,289 *B. napus* sequenced accessions (http://yanglab.hzau.edu.cn/BnIR). These data revealed that the *BnMTP8.A09* downstream sequence had the most sequence variants (55.6%) consistent with its association, suggestive of that being the candidate gene (Figure 4B–C).

## 3.5 | Gene Expression Variation in *BnMTP8.A09* Explains 74% of Natural Variation in Mn²⁺ Tolerance

To assess whether *BnMTP8.A09* is the major locus associated with Mn²⁺ tolerance in *B. napus*, we performed a selective DNA genotyping of 20 DH lines of Darmor-*bzh*/Yudal segregating for Mn²⁺ tolerance by Sanger sequencing. We found almost
<figure><img src="image_4.png" /><figcaption>**FIGURE 3** | Natural variation in Mn²⁺ tolerance loci in *B. napus*. (A) Manhattan plots showing SNP associations for Mn²⁺ tolerance in a GWAS panel of *B. napus*. Three genomic regions (A09, C03 and C09) were associated with Mn²⁺ tolerance. Only QTL above the threshold–log(p-value) > 2.5E⁻⁸ were included in this figure. The closest candidate gene (with suffix *Bna*) that maps near the highly significant SNP is also shown. (B) QQ plot showing a relationship between observed (y axis) and expected (x axis) LOD scores and a line of fit (red dashed line). Local linkage disequilibrium (LD) heatmap of the genomic region containing the most significant SNP associated with Mn²⁺ tolerance on chromosomes A09 (C), C03 (D), and C09 (E). The genomic region is based on the physical map position of the most significant SNP on the Darmor-*bzh* reference assembly ±10 kb. LD estimated as r² is shown in colour keys. Haplotype showing association with Mn²⁺ tolerance on chromosome A09 (F) and C09 (G). Box plots for Mn²⁺ tolerance grouped by alleles of the top SNP markers (maximum LOD scores) on A09, and C09 chromosomes. The central bold line within the box represents the median; box edges indicate the upper and lower quartiles; whiskers show the 50% interquartile range, and points indicate outliers. Two-tailed two-sample Wilcoxon tests determined *p* values. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**FIGURE 4** | Structural variants and their distribution in *BnMTP8.A09* (*BnaA09g37250D*) gene. (A) Schematic representation of the *BnaA09g37250D* (*BnMTP8.A09*, 1966 bp) gene encoding a cation diffusion facilitator protein showing seven exons and six introns in black and grey colour, respectively. The inverted triangle symbols indicate natural population SNPs/InDels located on *BnaA09g37250D*. The colours of inverted triangle symbols indicate different variations of functional types. (See detailed information in Table S3B). (B) Annotation and the proportion of SNPs and InDels of *BnaA09g37250D* in 2289 accessions of *B. napus*. (C) Linkage disequilibrium heatmap of population SNPs/InDels on *BnaA09g37250D*. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

complete linkage between *BnMTP8.A09* alleles and Mn²⁺ tolerance (Figure S4A). To assess whether the expression level of the *BnMTP8* gene correlates with phenotypic difference in Mn²⁺ toxicity, we compared the expression levels of *BnMTP8.A09* in cotyledonary leaves from extreme 20 accessions that were categorised as sensitive (S) or tolerant (T) to Mn²⁺ toxicity (Table S9). There are six copies of the *BnMTP8*, located on the homoeologous regions on the *A*ⁿ and *C*ⁿ subgenomes (A04/C04, A07/C06 and A09/C08) differ only by a few SNPs (Figure S4B-C, Table S10). We designed multiple primers with a 3' mismatch to specifically amplify and quantify the *MTP8* sequence on chromosome A09 (gene ID: BnaA09g37250D in Darmor-bzh v4.1 reference, Table S2). The amplified products were sequence verified to ensure that PCR amplification is specific to only *BnMTP8.A09*. Our analysis revealed a striking correlation between sensitive and tolerant lines, with the expression levels being visibly high in tolerant lines compared to sensitive ones (Figure 5A, Figure S5).

Quantitative reverse-transcription PCR analysis revealed that expression levels of *BnMTP8.A09* could explain up to 74% variance in the Mn²⁺ tolerance phenotype (R² = 0.74, Nominal Logistic regression with expression level as a factor and phenotype (sensitive/tolerant) as a response, *p* < 0.0001). In addition, among the tolerant varieties, there was a marginal yet significant increase in *BnMTP8.A09* expression upon Mn²⁺ treatment (Figure 5B). Based on this striking correlation, we conclude that Mn²⁺ dependent transcriptional control of *BnMTP8.A09* expression levels accounts for most of the variation in Mn²⁺ toxicity in *B. napus* accessions. We further raised the same 20 diverse accessions differing in *BnMTP8.A09*

expression (Figure 5C) with and without Mn²⁺ treatments in controlled environment conditions (22°C, 50% humidity, 16/8 h). As expected, tolerant lines showing high *BnMTP8.A09* expression lines did not show critical symptoms of Mn²⁺ toxicity and accumulated 5.04× higher biomass than Mn²⁺ sensitive and low *BnMTP8.A09* expression lines. Low *BnMTP8.A09* expression lines carrying sensitive alleles for Mn²⁺ tolerance showed reduced SPAD values, a proxy for chlorophyll content of the expanded leaf, and shoot biomass in high Mn²⁺ concentration (125 µm MnCl₂) compared to tolerant lines with high *BnMTP8.A09* expression (Figure 5C,D). Mn²⁺ tolerance positively correlated with fresh and dry shoot weights (Figure 5E,F). The chlorophyll (SPAD values) had a negative relationship with Mn²⁺ tolerance; scored as 1 being highly tolerant and 5 being highly sensitive (Figure 5E). Chlorophyll is essential for net photosynthesis assimilation, which could affect biomass production. In previous studies, high biomass was positively related to seed yield in *B. napus* (Raman et al. 2020; Raman et al. 2017; Raman et al. 2016). Preliminary ICP-MS analysis revealed that *B. napus* lines sensitive to Mn²⁺ toxicity accumulated more manganese than Mn²⁺ tolerant lines; shoot Mn content and Mn²⁺ tolerance score were positively correlated (*r* = 0.75, Figure 5F). Furthermore, Mn concentration negatively correlated (*r* = 0.5) with Fe and positively correlated with Ca (*r* = 0.8) accumulation (Figure S6). Interestingly, in one accession, Primor accumulated ~13× more Mn than other tolerant lines (without showing critical symptoms of Mn²⁺ toxicity, Table S11), suggesting that it can sequestrate excessive Mn²⁺ into the vacuole. Further work is required to understand such interactions and implications on plant growth and development in acidic soils.
<figure><img src="image_6.png" /><figcaption>**FIGURE 5** | Legend on next page.</figcaption></figure>
## 3.6 | Expression QTL Analysis Revealed the Central Role of *BnMTP8.A09* in the Genetic Architecture of Mn²⁺ Tolerance

To investigate what regulates the expression of *BnMTP8.A09*, which explained close to 74% of the variation in Mn²⁺ tolerance, we carried out the expression quantitative trait loci (eQTL) mapping of *BnMTP8.A09* in the *B. napus* population using RNA sequencing data from seedling leaves of 154 accessions (Figure S7A). Four eQTL of *BnMTP8.A09* were detected on chromosomes A09, C03, C04 and C08 (Figure 6A) and three of them colocalized with Mn²⁺ tolerance-related loci (Figure 3A, 6A). Based on the physical distance between eQTL and the target gene, we found the eQTL on A09 is cis-eQTL (<1 Mb) of *BnMTP8.A09*. *BnMTP8.A09* showed a higher expression among different homologues (Figure S7B) and is cis-regulated by SNPs of its flanking sequence. These findings strongly support our hypothesis that *BnMTP8.A09* is the target causal gene in the QTL for Mn²⁺ tolerance. Interestingly, we found three *trans*-eQTL of the *BnMTP8* homologues and two of them were on C04 and C08 (Figure 6B); although, there was no significant GWAS SNP within each of the two loci probably due to their expression dosage compensation effect on *BnMTP8.A09*. For the colocalized eQTL and QTL on C03, we found a candidate gene, Fe homoeostasis-related FERRIC REDUCTASE DEFECTIVE3 (*BnaC03g49020D*, *BnFRD3.C03/BnMATE.C03*), a member of the MATE gene family that is also involved in cross-talk with Zn tolerance (Pineau et al. 2012; Rogers and Guerinot 2002), contribute to Mn²⁺ tolerance and also could have genetic interaction with *MTP8* involved in Mn²⁺ tolerance network. The *BnMTP8.A09* also showed epistatic effects with the QTL on C03 and C09 (Table S12). Nevertheless, these results indicated that *BnMTP8.A09* plays a central role in Mn²⁺ tolerance in *B. napus*. Next, we asked which of the specific polymorphism(s) could explain most of the variation in *BnMTP8.A09* of *B. napus* 2280 accessions. We found that only one InDel(Ref/Alt = AT/A), in nine Asian and European accessions (Figure S7C), showed a significant association with *BnMTP8.A09* (*BnaA09g37250D*) expression in 289 *B. napus* accessions (Figure S7D). Interestingly, the same InDel was previously found to be associated with seed oil content (Figure S7E), suggestive of pleiotropy (Yang et al. 2023).

## 3.7 | *BnMTP8.A09* Alleles Confer Tolerance to Mn²⁺ Toxicity Under Field Conditions and Are Under Purifying Selection

To assess whether allelic variation in *BnMTP8.A09* indeed makes a difference to plants, we tested 175 doubled haploid (DH) lines from the Darmor-*bz*h/Yudal (DY) population with natural alleles that segregate for Mn²⁺ tolerance (Raman et al. 2017), along with parental lines of DY population and 15 controls in acid soil conditions at Mangoplah, NSW, Australia (2022, soil pH: 4.5, Mn = 198 µM, Figure S8A). We observed critical symptoms for Mn²⁺ toxicity, manifested as leaf chlorosis, at physiological maturity (BBCH GS89) when plots were waterlogged due to excessive rainfall and subjected to high temperatures (Figure S8B), though conditions (warm and dry winter and early spring) at earlier stages were not favourable to assess Mn²⁺ toxicity in the cotyledon/seedling stage. However, symptoms of Mn²⁺ toxicity were apparent in reshoots and plants regenerated from preharvest shattered seeds in the plots (Figure S9). We took an opportunistic approach and, scored symptoms of Mn²⁺ toxicity (leaf chlorosis) on re-shooted plants, and related them with Mn²⁺ tolerance scores from the nutrient screening experiment. Interestingly, cotyledon chlorosis scores of DH lines assessed in nutrient culture (Raman et al. 2017) were positively correlated with leaf chlorosis in the field (*r* = 0.45, Figure S6A). To verify the results, we selected 20 lines from the DYDH population that had consistent scores for Mn²⁺ tolerance in nutrient solution and field conditions and had contrasting marker alleles associated with Mn²⁺ tolerance at *BnMn²⁺*.A09 locus (Raman et al. 2017). These lines were evaluated in acidic soil having high Mn²⁺ (pH 4.5), collected from Mangoplah under glasshouse conditions at Wagga Wagga. Test lines showed differences in tolerance to Mn²⁺ toxicity and a positive correlation (*r* = 0.9) with cotyledon scores in nutrient culture. These findings conclusively demonstrated that *BnMTP8.A09* indeed confers tolerance to Mn²⁺ toxicity not only in lab conditions but also in field conditions. To assess whether *BnMTP8.A09* alleles may have undergone selection during *B. napus* domestication, we determined nonsynonymous sites (*Ka*)/synonymous sites (*Ks*) of *BnMTP8.A09*. Our results suggested that *BnMTP8.A09* is under purifying selection (*p* = 0) irrespective of *B. napus* ecotypes (Figure S10). These findings suggest conservation of gene function across spring, winter and semi-winter types due to adaptation to environments with Mn²⁺ toxicity.

**FIGURE 5** | MTP8 functions in Mn²⁺ tolerance in *B. napus*. (A) Relative expression levels of *MTP8* in Mn²⁺ tolerant (T, *n* = 10) and sensitive (S) lines (*n* = 10) and (B) of 20 diverse accessions of *B. napus* after 6 days of stress in the 125 µM MnCl₂·4H₂O and the corresponding control (9 µM of MnCl₂·4H₂O, -ve Mn²⁺ treatment). Relative expression levels calculated with *UBC9* as an internal control are shown. Statistical significance was tested by one-way ANOVA, which showed the effect of the genotypic groups (A) or manganese treatment (B) is significant (*p* = 0.05; **** = *p* < 0.0001). (C) Genetic variation for Mn²⁺ tolerance among 20 diverse accessions of *B. napus*. Chlorosis was measured as 1 (tolerant) to 5 (sensitive) scale. (D) Per cent reduction in chlorophyll content and shoot weights of 20 accessions that show the extreme phenotypes for Mn²⁺ tolerance and sensitivity measured after 3-4 weeks in control nutrient solution (-ve Mn²⁺ treatment) and with 125 µM of MnCl₂·4H₂O (C). Chlorophyll content was measured with SPAD. Above-ground shoot biomass was measured on a fresh and dry weight basis. (D) Per cent reduction to control treatment was calculated as the Trait value of Mn²⁺ plus treatment minus trait value of the control treatment)/trait value to control treatment × 100. The means of 12 biological replicates were plotted in the R package. (E) Phenotypic correlation between Mn²⁺ tolerance, chlorophyll content, and fresh and dry biomass of 20 accessions. (F) Correlation between Mn²⁺ tolerance scores and shoot Mn²⁺ content. [Color figure can be viewed at wileyonlinelibrary.com]
<figure><img src="image_7.png" /></figure>

**FIGURE 6** | Expression QTL (eQTL) analysis revealing the genetic architecture of Mn²⁺ tolerance in *B. napus*. (A) Manhattan plot of eQTL for *BnMTP8.A09* in 326 *B. napus* accessions. Each point represents an SNP/InDel in the *B. napus* GWAS population. The candidate-expressed genes (*BnMATE.C03*, *BnMTP8.C04* and *BnMTP8.C08*) in *trans*-eQTL which could influence the expression of *BnMTP8.A09* were marked. (B) The regulatory network of *BnMTP8.A09* mediated the response to Mn²⁺ tolerance in *B. napus*. One cis-eQTL and three trans-eQTL colocalized with Mn²⁺ tolerance QTL on Chromosomes A09, C03, C04 and C08 indicated that *BnMTP8.A09* play an important role in Mn²⁺ tolerance in *B. napus*. The epistatic effects of the QTL between A09 and C03/C04/C08/C09 suggest the complex regulatory mechanism of Mn²⁺ tolerance in *B. napus*. The green line (eQTL) next to the short blue line (candidate gene) indicates the corresponding candidate gene located in the eQTL region. [Color figure can be viewed at wileyonlinelibrary.com]

## 3.8 | *BnMTP8.A09* Is a Bonafide Mn²⁺ Transporter

To verify the contribution of the *BnMTP8.A09* gene in natural variation in Mn²⁺ tolerance, we performed a complementation assay using the Mn-hypersensitive yeast strain *pmr1Δ*. *Pmr1* is a P₂-type Ca-ATPase responsible for transporting both Ca²⁺ and Mn²⁺ into the Golgi apparatus, a major pathway for the cellular detoxification of manganese (Antebi and Fink 1992). Yeast assays revealed that the *BnMTP8.A09* gene imparts the Mn²⁺ tolerance in the yeast strain *pmr1Δ*, which tolerated elevated Mn²⁺ levels (50 mM, i.e., 400× dose), that enabled discrimination of Mn²⁺ tolerant lines from sensitive ones in nutrient solution (Figure 7).

To investigate whether the *BnMTP8.A09* enhances the level of Mn²⁺ tolerance in planta, we employed heterologous expression of *BnMTP8.A09* in Arabidopsis Col-0 ecotype and the Mn²⁺ sensitive T-DNA mutant, *atmtp8*, using the floral dip method for transgenesis. The transformants were confirmed through hygromycin resistance, *BnMTP8.A09*-specific PCR markers (Figure S11) and *in vitro* Mn²⁺ tolerance screening. Overexpression of *BnMTP8.A09* did not improve Mn²⁺ tolerance in the Col-0 background (Figure S12). However, 35S:*BnMTP8.A09:Col-0* transgenic lines exhibited significant *BnMTP8.A09* expression (2,373–100,085 folds) and increased plant biomass compared to both Col-0, and

the Mn²⁺-sensitive T-DNA insertion mutant lines, *atmtp8.1* and *atmtp8.2* under 1 mM MnCl₂ treatment (Figure S12B, C). Furthermore, ectopic expression of *BnMTP8.A09* restored Mn²⁺ tolerance in *atmtp8* (SALK_140266C) at 500 µM and 1 mM MnCl₂ (Figure 8A). The *atmtp8* mutant showed increased cotyledon lobe chlorosis as compared to 35S:*BnMTP8.A09:atmtp8* transgenic lines and Col-1. Additionally, 35S:*BnMTP8.A09:atmtp8* lines exhibited *BnMTP8.A09* expression levels between 66 and 487 fold higher and increased plant biomass (approximately fivefold) under 1 mM MnCl₂ treatment compared to mutant *atmtp8* line, attributed to the presence of *BnMTP8.A09* transgene. (Figure 8B,C). Moreover, the root length of WT and 35S:*BnMTP8.A09:atmp8* lines were longer (1.4–1.8 fold) compared to *atmtp8* mutant lines under Mn²⁺ treatment (Figure 8D). These findings suggest that *BnMTP8.A09* functions as a bonafide Mn²⁺ transporter.

## 4 | Discussion

### 4.1 | Natural Variation in Mn²⁺ Tolerance in *B. napus*

*B. napus* was domesticated and selected for consumer preferences in Southern Europe, where carbonate-rich soils are highly prevalent (Gómez-Campo and Prakash 1999; Prakash et al. 2011).
<figure><img src="image_8.png" /><figcaption>**FIGURE 7** | Effect of the *B. napus* MTP8 expression on tolerance to Mn²⁺ toxicity. Yeast cells (Mn²⁺ hypersensitive yeast mutant *pmr1Δ*) carrying empty PRS vector, *BnMTP8* gene (BnA09g37250D in PRS vector) were spotted on the yeast medium (pH 4.4) without MnCl₂ (control) and with MnCl₂ concentrations (6.25, 12.5, 25 and 50 mM). The plates were incubated for 48 h and photographed.</figcaption></figure>

In this study, we assessed a diversity panel representing spring, semi-winter and winter ecotypes and identified only five (1.2%) highly Mn²⁺ tolerant *B. napus* accessions (which had the least chlorosis scores of ≤ 1) of spring-type from Asia (Pakistan, Japan), and winter-type from Europe (France, Germany, Table S1). These results suggest that populations of *B. napus* had developed adaptation mechanisms to minimise the harmful effects of toxic ions in acid soils by natural selection and/or by passive selection by breeders.

## 4.2 | BnMTP8.A09 Controls Natural Variation for Mn²⁺ Tolerance in B. napus

Through GWAS, selective sweep and eQTL analyses, we uncovered 3 to 4 genomic regions for Mn²⁺ tolerance (Figures 3,6, S1), suggesting that genome-wide association approaches are suitable for revealing the architecture of Mn²⁺ tolerance. One of the highly significant association peaks was located close to the *BnMn*²⁺*A09* locus, previously identified by QTL analysis in the *B. napus* DH population (Raman et al. 2017). However, gene expression level and gene function were not investigated to Mn²⁺ tolerance. In addition, QTL/eQTL for Mn²⁺ tolerance on C03 and C09 chromosomes were not identified in earlier studies. The *trans*-eQTL associated with Mn²⁺ tolerance on chromosomes A09 and C03 (Figure 7) were mapped within the LD of GWAS-SNPs, suggesting that both genes (*BnMTP8.A09* and *BnMATE.C03*) are likely correlated. Future studies need to validate the role of new

genomic regions (e.g. *BnMATE.C03*) for their contribution in Mn²⁺ tolerance.

We identified several highly significant SNP associations that were located near the transporter genes (Table S7), implicated in metal ion transport in different plant species (Chu et al. 2017; De Caroli et al. 2020; Leung et al. 2019; Tsunemitsu et al. 2018). For example, MTP8 is a tonoplast localised member of the CDF and functions in roots as an Mn²⁺ transporter (Eroglu et al. 2016). MATE transporters are implicated in turgor-regulating chloride channels and xenobiotic detoxification by transmembrane export across the plasma membrane; this gene confers Al³⁺ tolerance by mediating citrate efflux from root cells in wheat, barley, sorghum and Arabidopsis to chelate Al³⁺ (Ryan et al. 2009; Upadhyay et al. 2019; Zhang et al. 2017). The ABCC5 (C09) encodes a High-Affinity Inositol Hexakisphosphate Transporter. It plays a role in the signalling of guard cells, phytate storage, cellular potassium ion homoeostasis, and response to salt stress (Andolfo et al. 2015; Lemtiri-Chlieh et al. 2000). ABC transporters are responsible for detoxifying many compounds from the cytoplasm (Klein et al. 2006).

## 4.3 | BnMTP8.A09 Imparts Mn²⁺ Tolerance in Canola and Yeast

In this study, we show for the first time the functionality of *BnMTP8.A09* that underlies natural variation in Mn²⁺ tolerance in *B. napus* using yeast complementation assay in a strain
<figure><img src="image_9.png" /><figcaption>**FIGURE 8** | Phenotypic and *BnMTP8.A09* gene expression analyses of Arabidopsis. (A) Plant growth of 14-day old wild type Col-0, *atmp8* and two 35S::*BnMTP8.A09*-lines grown on 1/4 MS, pH 4.8 supplemented either 0 µM (control), 500 µM and 1 mM of MnCl₂. (B) *BnMTP8.A09* expression quantification of Col-0, *atmp8* and three *BnMTP8.A09*-overexpressing lines, normalised to the lowest basal expression level of *atmp8*. *, **, *** denote significance of *p* < 0.05, < 0.01 and < 0.001, respectively. (C) Fresh plant biomass and (D) root length of Col-0, ectopic expression lines of *BnMTP8.A09* in *atmp8*, and mutant *atmp8* in response to 500 µM and 1 mM of MnCl₂ treatments. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

lacking the Golgi-mediated cytoplasmic efflux carrier PMR1 (Dürr et al. 1998), and ecotypic and phenotypic expression using natural alleles under controlled environment and field conditions. *MTP8* homologues have been shown to enhance tolerance to Mn²⁺ toxicity, Mn sequestration and plant growth in different species (Chen et al. 2013; Delhaize et al. 2003; Eroglu et al. 2016; Mills et al. 2008). *AtMTP8* is also reported to alleviate the antagonistic interference of Mn²⁺ with Fe²⁺ by loading Mn²⁺ into the root vacuole at high pH conditions in Arabidopsis (Eroglu et al. 2016; Farthing et al. 2023). In *B. napus*, three homologues; *BnMTP3*, *BnMTP8* (BnaCnng31720D on

chromosome C04/*BnMTP8.C04*, AT3G58060D) and *BnMTP9.A07* (BnaA07g34970D, AT1G79520D) are shown to load Mn²⁺ into vesicles for subsequent delivery to the vacuole or secretion into extracellular spaces and maintain Mn²⁺ homoeostasis in the roots and shoots (Gu et al. 2021; Gu et al. 2022). However, the role(s) of *BnMTP3*, *BnMTP8* and *BnMTP9* in natural variation for Mn²⁺ tolerance in canola was not shown in these studies. In this study, we illustrate the extent of natural variation and genetic architecture for Mn²⁺ tolerance in canola using a GWAS panel of 415 accessions, and identification of candidate genes for tolerance to Mn²⁺ toxicity
(BnMTP8.A09, BnMATE.C03, BnMTP8.C04, BnMTP8.C08 and BnABCC.C09). Our phenotypic on ecotypically BnMTP8 expressing lines showed that Mn²⁺ toxicity affects both shoot and root characteristics as reported earlier (Gu et al. 2022).

## 4.4 Validation of High Throughput Method for Screening Germplasm for Mn²⁺ Tolerance

Evaluating natural or transgenic *B. napus* lines under field conditions is challenging, as the phenotypic expression of Mn²⁺ toxicity depends on soil pH and weather conditions. Sparrow and Uren (1987) reported that waterlogging with low redox potential, water deficit, and heat episodes can lead to excessive Mn²⁺ absorption and toxicity, affecting plant physiology and development across different soil types. Our results are consistent with literature that suggests warm and waterlogged conditions favour the expression of Mn²⁺ toxicity genes in acidic soil containing high exchangeable Mn²⁺. In addition, the critical symptoms of tolerance for Mn²⁺ toxicity and symptoms were variable across the field plots. It is emphasised that Mn²⁺ toxicity is not only a limitation in the acidic soil; the availability and/or unavailability of other ions, such as Al³⁺, H⁺, Fe²⁺, and Ca²⁺, could compromise the expression of Mn²⁺ tolerance under field conditions. We rated the plots that showed no symptoms of Mn²⁺ toxicity as tolerant, while plots showing > 10% of plants were scored as Mn²⁺ sensitive. Therefore, this criterion was subjected to ascertainment bias. Our data showed that hydroponic-based screening is more robust and reliable than field and glasshouse screening of large breeding germplasm that requires a large volume of soil with desired characteristics. Many plants can be screened in less than 10 days in a hydroponic system, providing highly reliable phenotyping for Mn²⁺ tolerance. This work laid a good foundation for canola breeding for Mn²⁺ tolerant cultivars. High throughput phenotyping, in conjunction with molecular markers, could provide an efficient pipeline for tracking Mn²⁺ tolerance alleles in the *B. napus* breeding programmes.

### Acknowledgements

The authors thank Mr. Kevin McCrea for making the Mangoplah field available for germplasm evaluation, Ms. Helen Burn for organising the field site; Ms. Hannah Roe for help in phenotyping; Mr. Warren Bartlett for a sowing field trial and Novogene Corporation Inc., China, for re-sequencing accessions. This research was supported by NSW DPIRD and GRDC, partly under the National Brassica Germplasm Improvement Programs (DAN00208), and DPI2204-021RTX led by H.R. N.K., H.R. and B.J.P. acknowledge the support of the ARC Training Centre for Future Crops Development (IC210100047). Quantitative gene expression work was supported by a Denis and Maisie Carr Award (SM) and funds from Monash University (SB).

### Data Availability Statement

The data supporting the findings and supplementary data are available within the paper. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA:CRA0014759) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section.