Received: 5 January 2023 | Accepted: 1 August 2023 DOI: 10.1111/pce.14691 DATA INSIGHT Combined genomics to discover genes associated with tolerance to soil carbonate Silvia Busoms1 | Laura Pérez‐Martín1 | Joana Terés1 | Xin‐Yuan Huang2 | Levi Yant3 | Roser Tolrà1 | David E. Salt4 | Charlotte Poschenrieder1 1 Department of Animal Biology, Plant Biology, and Ecology, Plant Physiology Laboratory, Abstract Bioscience Faculty, Universitat Autònoma de Carbonate‐rich soils limit plant performance and crop production. Previously, local Barcelona, Barcelona, Spain 2 State Key Laboratory of Crop Genetics and adaptation to carbonated soils was detected in wild Arabidopsis thaliana accessions, Germplasm Enhancement, College of allowing the selection of two demes with contrasting phenotypes: A1 (carbonate Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China tolerant, c+) and T6 (carbonate sensitive, c−). Here, A1(c+) and T6(c−) seedlings were 3 Future Food Beacon of Excellence & School grown hydroponically under control (pH 5.9) and bicarbonate conditions (10 mM of Life Sciences, University of Nottingham, NaHCO3, pH 8.3) to obtain ionomic profiles and conduct transcriptomic analysis. In Nottingham, UK 4 parallel, A1(c+) and T6(c−) parental lines and their progeny were cultivated on Future Food Beacon of Excellence & School of Biosciences, University of Nottingham, carbonated soil to evaluate fitness and segregation patterns. To understand the Sutton, UK genetic architecture beyond the contrasted phenotypes, a bulk segregant analysis Correspondence sequencing (BSA‐Seq) was performed. Transcriptomics revealed 208 root and 2503 Charlotte Poschenrieder leaf differentially expressed genes in A1(c+) versus T6(c−) comparison under Email: Charlotte.Poschenrieder@uab.es bicarbonate stress, mainly involved in iron, nitrogen and carbon metabolism, Funding information hormones and glycosylates biosynthesis. Based on A1(c+) and T6(c−) genome SPANISH MINISTRY OF SCIENCE AND contrasts and BSA‐Seq analysis, 69 genes were associated with carbonate tolerance. INNOVATION, Grant/Award Number: PID2019‐1040000RB‐I00 Comparative analysis of genomics and transcriptomics discovered a final set of 18 genes involved in bicarbonate stress responses that may have relevant roles in soil carbonate tolerance. KEYWORDS Arabidopsis, bicarbonate stress, BSA‐Seq: transcriptomics, calcareous soil 1 | INTRODUCTION The main anions present in excess in alkaline soils are HCO3− and CO32− (Al‐Busaidi & Cookson, 2003). Soil carbonates act as pH Soil alkalinity is a highly stressful environmental factor that limits buffers and play an important role in rhizosphere processes, plant growth and crop yield (Filippi et al., 2019; Li et al., 2018). High hampering nutrient availability to plants. The low availability of pH soils are present in 30% of the Earth surface specially located in nitrogen (N), phosphorus (P) and micronutrients such as iron (Fe), zinc areas with arid and semiarid climate. The pH values of most (Zn) and manganese (Mn) produce nutrient deficiencies in many plant calcareous soils are within the range of 7.5–8.5 (Loeppert & Suarez, species cultivated on carbonated soils (Dreyer et al., 2020; Hui et al., 1996) with bicarbonate concentrations between 5 and 35 mmol L−1. 2019; Terés et al., 2019). Moreover, the high pH surrounding plant This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd. Plant Cell Environ. 2023;1–13. wileyonlinelibrary.com/journal/pce | 1 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 | BUSOMS ET AL. roots can alter the membrane potential (Felle, 2001) and inhibit the 2 | M A T E R I A L S AN D M E T H O D S uptake of essential ions, and thus further contribute to nutrient deficiency in sensitive plants. 2.1 | Plant material The uptake of bicarbonate can cause important metabolic disturbances due to both alteration of cell pH homoeostasis and In previous studies, natural variation of A. thaliana populations from dark fixation of inorganic carbon. Bicarbonate can be quickly Catalonia (Busoms et al., 2015) were tested in a multiyear small‐scale incorporated into organic acids, mainly malate, causing inhibition of common garden under carbonated and noncarbonated soils (Terés mineral nutrient transport from roots to shoot, reduction of root et al., 2019). Seeds of six extreme phenotype plants from A1(c+), a soil growth, and oxidative stress in sensitive calcifuges, but not in tolerant carbonate tolerant deme, and six extreme phenotype plants from calcicole species (Lee & Woolhouse, 1969; Sagervanshi et al., 2021). T6(c−), a soil carbonate sensitive deme, were collected from the last Due to these multiple direct and indirect injuries, sensitive species reciprocal transplant experiment performed in 2015 and stored under exhibit a complex syndrome of stress symptoms, including morpho‐ fresh (4°C) and dry conditions until the beginning of hydroponic and anatomical changes in roots, disturbance of water relations, signs of soil cultivation experiments. A1 × T6 and T6 × A1 crosses were iron and/or zinc deficiency, reduction of total photosynthetic generated using the six A1(c+) and six T6(c−) lines (generating 12 pigments and photosynthetic activity; accumulation of osmo‐ crossing lines) and self‐crossed for three generations (F1, F2, F3). Col‐ protectants, soluble sugars, and organic acids; and activation of the 0 seeds and seeds of 18 T‐DNA mutants with Col‐0 background were biosynthesis of antioxidant enzymes (Ahmad et al., 2014; Sarkar et al., purchased from NASC/ABRC (Scholl et al., 2000). Segregating lines 2022; Zou et al., 2021). were genotyped using specific primers and multiplied for T2 A better understanding of plant alkaline tolerance mechanisms generations. The nonexpression of the genes was validated through and cultivation of new varieties of alkali‐tolerant crops is needed to qPCR (Supporting Information: Data set SD1). improve carbonated soils and increase food production (Cao et al., 2022). However, current knowledge of the bicarbonate stress response of plants is limited. Most studies have been conducted on 2.2 | Growth conditions roots of crop species such as Glycine max or Oryza sativa which are species with moderate bicarbonate tolerance (Waters et al., 2018; Selected seeds were surface sterilized by soaking in 70% (v/v) Zhang et al., 2017). Nonetheless, after several decades of effort still ethanol for 1 min, suspended in 30% (v/v) commercial Clorox bleach a proper model is missing to understand the adaptive mechanism to and 1 drop of Tween‐20 for 5 min and rinsed five times in sterile carbonate stress and the tolerance mechanism at the molecular‐ 18 MΩmilli‐Q water. Seeds were kept in water, at 4°C, in the dark genetic level. Early experiments revealed that organic acid accumu- conditions for 48 h to synchronize germination. lation in response to bicarbonate occurred both in sensitive and Hydroponic culture: A. thaliana seeds from six A1(c+) and six T6(c−) tolerant species and it was speculated that differential compart- lines were sown in 0.2 mL tubes containing 0.6% phyto agar (Duchefa) mentation of organic acids may play a role in the tolerance prepared in nutrient solution 1/2 Hoagland solution (HS), pH 5.9 in a (Sagervanshi et al., 2021). In fact, several anion transporters, and growth chamber (12 light/12 dark hours, 150 µmol cm−2·s−1, 40% gene families such as ALMT, NRT/POT and SLAHs were related to humidity and 25°C). The bottom of the tubes containing seedlings was bicarbonate response in G. max (Duan et al., 2018; Waters cut off and the tubes were placed in 150 mL hydroponic container et al., 2018). with aerated nutrient solution 1/2 HS, pH 5.9. After 15 days During recent years, combined omics are applied to success- postgermination, treatment was applied, and seedlings were separated fully characterize plant tolerance responses to complex stress in different sets. The treatments consisted of control (½ HS at pH 5.9), factors (e.g., Mehari et al., 2021). Resequencing‐based bulk high pH (½ HS at pH 8.3) and bicarbonate (½ HS at pH 8.3 and 10 mM segregation analysis (BSA) utilizes a strategy of pooling individuals NaHCO3). Solutions were buffered with different proportions of 2‐(N‐ with extreme phenotypes to conduct rapidly linked marker morpholino)ethanesulfonic acid and BTP (BIS‐TRIS propane) depend- screening (Li & Xu, 2022). Whole‐genome studies are useful to ing on the final pH. Plants of A1(c+), T6(c−) used for transcriptomic detect signature of selection, quantitative trait locus, and single analysis were collected after 48 h under treatment. Plants of A1(c+), nucleotide polymorphism (SNP) markers. However, some important T6(c−), Col‐0 and the 18 T‐DNA knockout mutants were collected after changes affecting the regulation of key genes can remain hidden. 10 days under treatment. Image analysis for root length measurements The combination of genomic and transcriptomic studies allows a were performed with Image J, software16 (Schneider et al., 2012). more accurate screening of the candidate genes involved in specific Greenhouse experiments: four cultivations were conducted physiological processes. Here, we took advantage of the natural consecutively under the same conditions: (1) six A1(c+), six T6(c−), six variation present in two Arabidopsis thaliana populations with A1 × T6 and six T6 × A1 F1 lines; (2) five A1(c+), two T6(c−), 10 A1 × T6 contrasting phenotypes of soil alkalinity tolerance (Terés et al., and four T6 × A1 F2 families; (3) five A1(c+), two T6(c−), 66 A1 × T6 2019) to highlight the loci involved in bicarbonate stress responses and 28 T6 × A1 F3 families; (4) Col‐0 and the 18 T‐DNA mutants of and adaptation to carbonated soils by combining BSA‐Seq and candidate genes. Plants were cultivated in carbonated soil (LP) RNA‐Seq technologies. mixed with perlite (3:1) (Table 1) in 6 × 6 cm square pots under 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 3 T A B L E 1 Physical and chemical Geology Texture O.M. CaCO3 pH properties of the natural carbonated soil Limestones Clay‐loam 47.3 33.25 7.9 excavated from Les Planes d'Hostoles (LP, % Zn Cu Mo P S Mg Fe Mn Na Ca K 42° 03ʹ 45.1″ N; 2° 32ʹ 46.6″ E). Mean (µg/g) 4.9 2.1 0.02 40.3 25.5 33.9 40.8 23.3 39.2 353.8 60 SD 1.9 0.7 0.01 10.5 7.2 9.6 10.7 7 6.1 56.2 20.1 Abbreviations: O.M., organic matter; SD, standard deviation. semicontrolled conditions (12‐h light/12‐h dark photoperiod, tem- Bi‐allelic SNPs were identified using ‘HaplotypeCaller’ and perature between 15°C and 25°C). Five seeds of each line were sown genotyped using ‘GenotypeGVCF’ (both tools in GATK). Data in pots and distributed randomly in the greenhouse. Two weeks after were filtered using GATK SelectVariants using these ‘GATK best germination, seedlings were thinned out so that only one plant per practices’ guidelines: QD < 2.0||MQ < 40.00||FS > 60.0||SOR > 4.0 || pot was left. Irrigation with distilled water was applied twice a week MQRankSum ≤ 8.0 || ReadPosRankSum ≤ 8.0 and a minimum at the bottom of the trays. For the third cultivation, two leaves per coverage of 10 per sample. Genotyping of the pooled sample (19 plant were collected 20 days after germination and stored at −80°C A1 × T6 individuals) was performed with LoFreq, (Wilm et al., 2012) for subsequent DNA extraction and sequencing of the selected as GATK failed with ploidy levels required (ploidy‐38 for 19 diploid plants. Rosette diameter and bolting time were monitored, and silique samples). LoFreq was run with default settings. VCFs were then number was counted at maturity in all the experiments. merged with BCFtools (Li, 2011). The allele frequency (AF) of all variants in the F3‐tolerant pool versus the F0‐sensitive pool was determined using the Multipool programme (Edwards & Gifford, 2.3 | Ionomic analysis 2012). Several intervals with bi‐polar peaks were identified across the genome. Intervals where the AF between the two pools Root ionome was assessed in plants submitted for 10 days to differed considerably and had a positive LOD score were further treatment conditions. Roots were carefully washed with 18 MΩ to explored for candidate genes. remove nutrients from solution. Plant material was dried for 4 days at The whole genome of four A1(c+) and four T6(c−) individuals was 60°C. Approximately 0.1 g was used to perform an open‐air digestion sequenced in a previous study (Busoms et al., 2018) and were merged in Pyrex tubes using 0.7 mL concentrated HNO3 at 110°C for 5 h in a with the two parental samples sequenced for the BSA‐seq experiment. hot‐block digestion system (SC154‐54‐Well Hot Block™, Environ- To obtain a consensus sequence for each deme, private SNPs of T6 mental Express) placed inside a flow cabinet. The concentrations of and A1 (shared for the 6 samples) were selected using GATK and Col‐0 selected elements (Ca, K, Mg, Na, P, S, B, Mo, Cu, Fe, Mn, Zn) were TAIR10 sequence as a reference. Once we obtained the consensus determined by inductively coupled plasma optical emission spectros- sequence, we selected the sites that differ between demes using copy ICP‐OES (Thermo Jarrell‐Ash, model 61E Polyscan). GATK ‘concordance’ command obtaining one vcf file with T16 AF < 0.1 and A1 AF > 0.9 variants and a second vcf file with T16 AF > 0.9 and A1 AF < 0.1 variants. We merged the files using VCFtools (Danecek 2.4 | Genomic analysis et al., 2011) and the amino acid changes between A1 and T6 were obtained and quantified using SNPeff (Cingolani, 2022). Genes with Leaf material from two A1(c+) individuals (F0‐tolerant pool), two T6(c−) 3–10 genetic modifiers were selected as candidates (Supporting individuals (F0‐sensitive pool) and 19 F3 individuals from seven Information: Data set SD3). tolerant families (two T6 × A1 and five A1 × T6) (F3‐tolerant pool) were used for DNA extraction (Supporting Information: Data set SD5). DNA was isolated with a Qiagen DNeasy kit and libraries were prepared 2.5 | RNA isolation and microarray scanning using a TruSeq DNA PCR‐Free Sample Preparation Kit from Illumina. Whole‐genome sequencing was performed on Illumina HiSeq. 2000 by Root tissue from 15‐day‐old plants cultivated under hydroponic Novogene. Read depth was measured after processing and alignment conditions was recollected at 48 h after initiating the treatments. At to the TAIR10 reference (below) at 15× coverage for each individual least 12 plants per line and treatment were pooled to perform three sample and 180× for the pooled sample (19 F3 individuals). biological replicates. Leaves were immersed in liquid nitrogen, homoge- Raw sequence data was processed as follows: (1) read trimming nized to a fine powder, and stored at −80°C. The total RNA of about by default parameters with Sickle (Joshi & Fass, 2011); (2) alignment 100 mg of leaf powder for each biological replicate was extracted using to the TAIR10 reference with bwa mem 0.7.12 (processing with the Maxwell® plant RNA kit (Promega Corporation) following the SAMtools 1.3); (3) removal of duplicate reads using Picard (MarkDu- manufacturer's instructions. To ensure a high quality and quantity RNA plicates); (4) application to read group name correction to the bam material, Experion RNA Analysis Kit (Biorad) was performed. After files using Picard (AddOrReplaceReadGroups); (5) realigning Indels quality control, total RNA was tagged, hybridized and cleaned using using the GATK ‘GenomeAnalysis’ Toolkit (McKenna et al., 2010). GeneChip® 3′ IVT Express Kit. Microarray expression analysis were 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 | BUSOMS ET AL. performed by Affymetrix GeneChip™ Arabidopsis Genome ATH1 Array diversity (Castilla et al., 2020) and they have a great potential for studying Agilent 2100 Bioanalyzer. traits related to adaptation (e.g., Pérez‐Martín et al., 2022) and allelic Differential expression analysis: The function gcrma (package gcrma) variants that specifically interact with the environment (e.g., Busoms et al., was used to adjust background intensities in Affymetrix array data, which 2021). Terés et al. (2019) selected two demes with contrasting include optical noise and nonspecific binding. The R package ‘limma’ was phenotypes in soil carbonate tolerance, A1(c+) and T6(c−), and performed used to test the expression data and search for differentially expressed several studies to characterize the physiological mechanisms activated by genes (DEGs) when pairs of experimental groups were compared. A these demes under carbonate and iron‐deficiency stress. model matrix was defined by specifying the groups that belonged to each sample. Then, limma's functions lmFit and contrasts. fit, together with the model matrix, were used to adjust the expression data to a linear model 3.1 | Root characterization of A. thaliana demes to extract fold changes and confidence statistics associated to the under high pH and bicarbonate stress comparisons of interest. Function eBayes was then used to rank genes in order of evidence for differential expression. The R library ath1121501. The hydroponic cultivation of A1(c+), T6(c−) and Col‐0 plants under db was used to include names and descriptions of the genes associated to control pH (pH 5.9), high pH (pH 8.3), or bicarbonate (bic, 10 mM the microarray's features probed. The resulting p‐values were adjusted NaHCO3, pH 8.3) conditions confirmed the higher tolerance of A1(c+) using the Benjamini and Hochberg's approach (Benjamini & Yekutieli, to both pH 8.3 and bic treatments. Col‐0 exhibited an intermediate 2001) for controlling the false discovery rate. To perform a list of DEGs, response, while T6(c−) was the most sensitive (Figure 1, Pérez‐Martín genes were filtered by adj p‐value (adjusted p‐value) < 0.05 and log fold et al., 2021). Root length was hardly affected by exposure to bic in change (LFC) > 1 and LFC < −1. A1(c+), the deme evolved on the calcareous soil. Contrastingly, root Gene ontology (GO), KEGG pathway and functional protein growth in T6(c−) was strongly inhibited by bic, but less affected by association networks analysis: GO enrichment analysis of DEGs was high pH (Figure 1a). In A. thaliana Col‐0, used as a well‐established implemented by AgriGO V2 (Tian et al., 2017) and FLAME (Thanati reference genotype, root length was more inhibited by bicarbonate et al., 2021). Significant GO terms (p < 0.05) were classified into three than A1(c+), but less than the sensitive deme T6(c−). In contrast to both categories: biological function, molecular process and cellular natural demes, Col‐0 did not show difference in root growth component. Kyoto Encyclopedia of Genes and Genomes (KEGG) inhibition between high pH and the bic treatment (Figure 1a). pathway and STRING version 11.0 were used to understand high‐ The standardized root mineral content revealed clear differences in level function and gene interaction network from differential response to high pH and bic among the three A. thaliana variants expressed genes (Szklarczyk et al., 2019). (Figure 1b). In deme A1(c+) both high pH and bic reduced root Fe, Cu and Zn concentrations in comparison to the control treatment. However, bicarbonate stress enhanced the uptake of macronutrients such as Ca and 2.6 | Statistical analysis of phenomics and ionomics P in A1(c+) and Col‐0 (Supporting Information: Figure S1). In general, high pH and bic reduced Fe contents in both organs in all plants. However, Normal distribution of data was confirmed by Levene's test and A1(c+) distinctively maintained higher Fe levels under bicarbonate nonnormal data were transformed before applying any parametric exposure and the Fe translocation from roots to shoots was clearly tests. Mean‐standardized values (1 < value > 1) of elemental contents enhanced in comparison to the control treatment (Figure 1c). Contrast- of root material were used to represent the radar plots and compare ingly, the sensitive T6(c−) had the lowest leaf Fe content despite having each accession profile. One‐way or multivariate analysis of variance higher root Fe levels (Supporting Information: Figure S1). Because of this, was used to test for significant differences (p < 0.05) between means T6(c−) exhibited a clear inhibition by bicarbonate of Fe translocation from of data with respect to physiological parameters, ionome, fitness and roots to shoots, while high pH strongly enhanced the Fe translocation in gene expression. To test for correlations between two variables, a this sensitive deme (Figure 1c). bivariate fit was applied. To perform multiple comparisons of group means, Tukey's HSD test was conducted. To perform multiple comparisons to a control (wild type [WT]), Dunnett's test was 3.2 | Transcriptional changes in response applied. Statistical analyses and plots were performed using SAS to bicarbonate stress Software JMP v.16.0 and are available at Supporting Information: Data sets SD2 and SD5. Microarray analyses in roots were performed to characterize, at the gene expression level, the mechanisms underlying the differential response to bicarbonate of the two naturally selected accessions. 3 | RES UL TS AND DISC US SION Gene responses were assessed by using samples after 48 h of treatment exposure, when the plants still did not exhibit any foliar Natural variation of wild species provides a relevant complementary symptoms of the stress treatments. We performed pairwise resource to discover novel gene functions. It has been proven that A. comparison to understand the differences between A1(c+)‐tolerant thaliana demes from the north‐east of Spain harbour high genetic and T6(c−) ‐sensitive lines in response to different treatments (pH 8.3 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 5 (a) (c) (b) F I G U R E 1 Root growth and ionome. (a) Root length (cm), (b) root ionome profile (mean‐standardized values of 12 elements) and (c) root Fe concentration (μg/g) and Fe translocation ratio (leaf [Fe]/root [Fe]) of A1(c+), T6(c‐) and Col‐0 Arabidopsis thaliana plants cultivated in hydroponics under control (pH 5.7), high pH (pH 8.3) and bic (10 mM NaHCO3, pH 8.3) conditions for 10 days. Numbers indicate significant differences between treatments per accession and letters indicate significant differences between accessions under the same treatment (t‐test, p < 0.05). [Color figure can be viewed at wileyonlinelibrary.com] vs pH 5.9 and bic vs control pH 5.9) (Supporting Information: Data factors were altered. In T6(c−) only up and down modifications in set SD3). A total of 4595 DEGs were identified, considering accession catalytic activity were found. The analysis of the KEEG pathways and treatment (Figure 2). The bicarbonate treatment caused a higher revealed that the sensitive line activated only two principal pathways number of DEGs than the high pH treatment, indicating that involved in catalytic activity while in A1(c+) more than 20 pathways bicarbonate stress involves more complex processes than simply were up or downregulated (Figure 2d, Supporting Information: Data the specific responses to alkaline pH (Figure 2a). For that, we further set SD4). This analysis indicates that the modifications are being focused our analysis on the pathways found in the plants exposed to produced in the sugar, lipid and protein metabolism of the the bic treatment. tolerant line. Previously, RNA‐Seq of leaves from A1(c+) and T6(c−) individuals In rice, better tolerance to saline‐alkaline conditions was due to revealed that bicarbonate exposure quickly upregulated Fe‐ superior Fe acquisition and higher root‐to‐shoot Fe translocation (Li deficiency related genes in the sensitive T6(c−) but not in the tolerant et al., 2016). This was attributed to enhanced root development and A1(c+) (Pérez‐Martín et al., 2021). In leaves, the highest number of higher expression of genes involved in Fe acquisition and transport in DEGs was observed in T6(c−) but in roots the tolerant A1(c+) exhibited the tolerant variety in comparison to the sensitive one. Contrastingly, a huge response when exposed to bic (Figure 2b). These differences among the DEGs in our tolerant A. thaliana deme, no differential in DEG number may denote contrasted deme strategies toward enhancement of main Fe‐acquisition‐related genes was observed. bicarbonate stress. GO terms indicated that in roots of A1(c+) the Moreover, root expression of bHLH101 was more than 4 times higher main pathways activated after 48 h of exposure were related to in the sensitive T6(c−) than in the tolerant A1(c+) (Supporting activation of biological processes and metabolism located in the Information: Data set S5). bHLH101 is a transcription factor that extracellular region (Figure 2c, Supporting Information: Data set controls plant Fe homoeostasis in a FIT‐independent way (Sivitz et al., SD4). Also, enzymatic regulatory activity, transport and transcription 2012). It is required for proper growth under Fe deficiency 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 | BUSOMS ET AL. (a) (b) (c) (d) (e) F I G U R E 2 Transcriptomics. (a) Total number and heatmap profile of differentially expressed genes (DEGs) from pairwise comparisons bic versus control (dark blue) and high pH versus control (light blue) in A1(c+) and T6(c‐) demes. (b) Total number of pairwise comparisons bic versus control in roots and leaves of A1 (c+) and T6(c‐) demes. Bubble plots indicating (c) significant Gene Ontology (GO) analysis and (d) heatmap of KEGG pathway analysis of A1(c+) and T6(c‐) root DEGs in bic versus control comparison. Arrows indicate up or downregulated genes. (e) Venn diagrams of pairwise comparisons A1(c+) bic versus T6(c‐) bic and A1(c+) control versus T6(c‐) control in roots and leaves. Selected DEGs are highlighted in brown for root and green for leaves. DEGs, GO and KEGG terms were filtered at log fold change (LFC > 1, LFC < −1) and adjusted p < 0.05. [Color figure can be viewed at wileyonlinelibrary.com] 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 7 conditions. These results further confirm that Fe deficiency is earlier not strictly fulfill the Mendelian phenotype ratio (3:1) (Figure 4b,c), perceived by the sensitive T6(c−). suggesting that the ‘soil carbonate tolerance phenotype’ in our demes Focusing on A1(c+) versus T6(c−) comparison under the bic is partial‐dominant or a polygenic trait. Moreover, considering only treatment and excluding the genes also up or downregulated in the plants that were able to flower, seed production was higher in the control conditions, 208 DEGs were identified in roots, 2503 DEGs in A1 × T6 than in the T6 × A1 offsprings (Figure 4c) pointing to a leaves, and 26 DEGs in both tissues (Figure 2d, Supporting potential parental effect. Information: Data set SD3). Two contrasted root strategies imply the possibility of differences in signal perceiving and transduction. To further explore these differential mechanisms, we performed 3.4 | Identification of genes associated with soil protein–protein interaction network functional enrichment analysis carbonate tolerance by BSA‐Seq analysis with the STRING database on the 208 genes (Figure 3, Supporting Information: Data set SD4). These genes can be classified in five main During meiosis, recombination reasserts the complement of alleles keywords according to FLAME: catalytic, binding and ubiquitin segregating in hybrid progeny (Salomé et al., 2012). Thus, the study activity; phosphorus metabolic process and oxidoreductase activity. of F2 populations from contrasted phenotype crosses can provide We found two main cores of genes with high network connectivity: valuable information on genetic mechanism by association (Benowicz one lead by genes involved in nucleus binding (TOPII, CDC20.2, et al., 2020). With the purpose of performing a BSA‐Seq study, plants MAD2, AUR1, FZR3, CYCB1;3) and microtubule movement regula- from 65 families of the A1 × T6 cross and from 28 families of the tion (AT5G60930, ATK5, AT3G20150, AT4G15830) that are T6 × A1 cross were self‐pollinated. F3 progeny plants were cultivated indispensable for normal plant development and fertility but have and screened under the same soil and growth conditions to select the not previously been associated with abiotic stress resistance; and extreme pools (Supporting Information: Data set SD5). We realized another controlled by the NIA1 and Nitrite Reductase 1 (NIR1) genes. that the most sensitive families had very low germination rates, or the NIA1 (or GNR1) encodes the cytosolic minor isoform of nitrate seedlings died after a few days postgermination (NP plants), reductase and NIR1 is involved in the second step of nitrate therefore, we were not able to sequence the sensitive pool. Despite assimilation. Contrastingly, NRT2.3 was less expressed in A1(c+) than this, considering the families with a higher number of flowered in the sensitive T6(c−). NRT2.3 is a high‐affinity nitrate transporter individuals and with an elevated silique production, seven families with still poorly defined functions but linked to the signalling of were selected as the tolerant pool (F3TP) (Figure 4d). several phytohormones (Kiba et al., 2011). The occurrence of The AF comparison of the F3TP with the sensitive parental (T6(c−)) inorganic carbon and nitrogen in karst and carbonated soils affects enabled the identification of the top 0.5% outlier SNPs from the the carbon/nitrogen metabolism of plant species. Under bicarbonate empirical distribution (Figure 4e). The 5617 SNPs of both tails were stress, growth reduction is enhanced ‐due to the inhibition of the associated to 1119 genes, mainly located in chromosome 5. Within photosynthesis and nitrogen metabolism but water use efficiency is this chromosomal group, the higher proportion of SNPs were promoted in tolerant plants (Xia & Wu, 2022). This balance is vital for assigned to the short arm (Figure 4f, Supporting Information: Data plants to adapt to alkaline environments and the regulation of the set SD6). Salomé et al. (2012) found that crossovers and segregation ‘nitrogen hub genes’ found here might be important for C fixation and distortion in F2 populations from different A. thaliana accessions bicarbonate tolerance. were more frequent in chromosomes 1 and 5. Here, 68% of our phenotype‐associated divergent outlier genes belong to chromosome 5, suggesting that this chromosome harbours key polymorphisms that 3.3 | Phenotypic variation for soil carbonate facilitate soil carbonate tolerance. tolerance traits among demes Additionally, we sequenced the whole genome of 6 A1(c+) and 6 T6(c−) individuals collected from their natural habitat. It is well known The natural habitat of A1(c+) and T6(c−) differs mainly in soil pH (A1(c+): that amino acid substitutions in a protein can cause a drastic 7.4; T6(c−): 6.5) and soil carbonate content (A1(c+): 12%; T6(c−): 0.8%) phenotypic effect (Bartlett & Whipple, 2013). The AF comparison (Terés et al., 2019). Natural carbonated soil from a location closer to between A1(c+) and T6(c−) picked out 977 genes with 3–10 A1(c+) deme (pH = 7.9; CaCO3 = 33%) was excavated for studying the nonsynonymous variants with high or moderate predicted effect tolerance capability of each deme and their progeny. F1 progeny of (Supporting Information: Data set SD7). There are many examples of both crosses (A1 × T6 & T6 × A1) exhibited similar growth and fitness genomic variants whose frequencies are correlated with environ- as the tolerant parental A1(c+) (Figure 4a,c), while F2 progeny showed mental variables and temporal changes consistent with natural segregation (Figure 4b,c). In addition to symptoms of iron deficiency selection in A. thaliana (Castilla et al., 2020; Monroe et al., 2022). and reduced growth, T6(c−) grown on alkaline soil suffers from A1(c+) is locally adapted to carbonated soils, thus it was important to delayed flowering and infertility that substantially hamper its fitness consider the genomic differences between A1(c+) and T6(c−) to narrow (Supporting Information: Figure S2, Supporting Information: Data set the BSA results down. The 69 genes in common between these two SD5). Only 35% of F2 progeny plants were able to flower and analyses were selected as the ‘genomic candidate genes list’ reproduce (Figure 4b). In consequence, the fitness of both crosses did (Figure 4g, Supporting Information: Data set SD8). 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 | BUSOMS ET AL. F I G U R E 3 Protein–protein interaction network of bicarbonate stress. Gene protein interaction network of the 208 root differentially expressed genes up or downregulated in the tolerant A1(c+) under the bic treatment. Each sphere corresponds to one gene, nodes represent protein interactions (source: STRING. org) and colours represent Gene Ontology terms. Source: bib. fleming. gr. [Color figure can be viewed at wileyonlinelibrary.com] 3.5 | Association analysis for prediction 2022) water stress responses in wheat (Derakhshani et al., 2020) of candidate genes or chilling stress tolerance in rice (Guo et al., 2020). Here, we performed an association analysis by combining the genomic and It is not feasible to determine the consequences of the missense transcriptomic data described above. The candidate gene lists variants on gene expression, protein structure and function of an obtained from the BSA‐Seq and the parental comparison analysis elevated number of putative candidate genes. Alternatively, (Supporting Information: Data set SD6–8) was compared with the candidate gene lists can be bounded conducting association root and leaf DEGs exclusive of the bic treatment in A1(c+) versus analysis such as the successfully applied to identify genes T6(c−) comparison (Supporting Information: Data set SD3), result- associated with plant architecture in Brassica napus, (Ye et al., ing in 18 matching genes (Figure 5a,b). The expression pattern of 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 9 (a) (b) (c) (d) (e) (f) (g) F I G U R E 4 Arabidopsis thaliana soil carbonate tolerance inheritance and bulk segregant analysis sequencing (BSA‐Seq). (a) Rosette diameter (mm); (b) percentage of flowered plants and plants with more or less than 10 siliques and (c) fitness (silique number) of A1(c+), T6(c‐) parents, F1 and F2 progeny from reciprocal crosses cultivated in natural carbonated soil. (d) Percentage of plants that did not prosper (no plant, purple bars), plants that did not flower (not flower, grey bars) and plants that produce more than 10 siliques (orange bars) from A1 × T6 F3 and T6 × A1 F3 progeny cultivated in natural carbonated soil. Selected families for BSA analysis are indicated in bold. (e) Allele frequency distribution of SNPs detected between the sensitive parent (T6(c‐)) and the F3 tolerant pool. (f) Chromosome distribution of genes associated with the SNPs from BSA left and right tails. (g) Summary table of the SNPs and genes detected and shared between parent's comparison (A1(c+) versus T6(c‐)) and between T6(c‐) and the F3 tolerant pool comparison. Letters indicate significant differences (Tukey's HSD, p < 0.05). [Color figure can be viewed at wileyonlinelibrary.com] these genes confirms that generally they are more expressed in the The SNP changes present in these 18 genes were explored a roots of A1(c+) but also in the leaves of the sensitive T6(c−) deme several missense variants were identified in both demes. The high (Figure 5c,d, Supporting Information: Data set SD9). This suggests number of SNPs present in Allantoate Amido Hydrolase (AHH) and that roots of the tolerant A1(c+) are reacting quickly to the adverse PCH1 of A1(c+), and in INV‐E of T6(c−) stands out (Supporting alkaline condition avoiding secondary effects in the leaves. Information: Data set SD9, Supporting Information: Figure S4). Contrastingly, in the sensitive T6(c−) leaves are promptly stressed, Some of these variants could be responsible for the observed gene especially due to the inhibition of Fe translocation (Figure 1c), and expression differences or be linked to structural changes that leaf gene expression adjusts to these alterations to alleviate affect the regulation of the gene. To evaluate the consequences of further injury. the no‐expression of these genes under carbonate stress, the 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10 | BUSOMS ET AL. (a) (c) (b) (d) F I G U R E 5 Combined analysis for bicarbonate tolerance candidate genes detection. (a) Number and (b) description of putative candidate genes obtained from the genomic and transcriptomic association analysis of A1(c+) and T6(c‐) demes. Relative fold change (bic versus control) of the 18 candidate genes in the (c) leaves and (d) roots of A1(c+) (orange bars) and T6(c‐) (purple bars) plants submitted to bic stress (10 mM NaHCO3, pH 8.3) for 48 h. [Color figure can be viewed at wileyonlinelibrary.com] knock‐out mutants of the 18 genes (T‐DNA lines, Supporting to alteration in sulphate reduction, enhanced glutathione and Information: Data set SD1), together with the wild type of Col‐0 cadmium tolerance (Lee et al., 2020). We have previously shown and A1(c+) and T6(c−), were cultivated on the same carbonated soil that bicarbonate tolerance in A. thaliana was associated with the (LP) and under the same control (pH 5.9) and bicarbonate upregulation of leaf genes related to sulphate acquisition (Pérez‐ hydroponic conditions (10 mM NaHCO3, pH 8.3) used in our Martín et al., 2021). So far FBN6 has mainly been studied in relation previous studies. to chloroplasts, while the role in root development is still unknown. Seeds of m03 (Sulphotransferase12) did not germinate in any Clearly, further characterization of the role of FBN6 in plant condition and, therefore, sot12 could not been tested. Instead, gdh2 development under bicarbonate is required. (m08) germinated normally under carbonated soil and we observed On the other hand, dao1 (m01), aah (m07) and jaz10 (m10) grew an enhanced fitness of this mutant in this condition (Supporting significantly better than the WT under bic stress (Supporting Information: Figure S3A,B). Both mutants csld2 (m15) and fbn6 Information: Figure S3A) but only dao1 exhibited higher fitness (m16) coding, respectively, for Cellulose Synthase Like D2 and when it was cultivated on carbonated soil (Supporting Information: Fibrillin6 died only when treated with bicarbonate stress under Figure S3B). DAO1, coding for a 2‐oxoglutarate and Fe (II)‐dependent hydroponic conditions (Supporting Information: Figure S3A,B), dioxygenase, is a main player in irreversible auxin degradation suggesting hypersensitivity to this stress. CSLD2, is required for (Hayashi et al., 2021) and a higher production of adventitious roots normal root hair development (Bernal et al., 2008). However, the has been shown in the dao1‐1 loss of function mutants (Lakehal et al., inhibition of root hair development alone may not cause plant 2019). Downregulation of DAO1 could enhance root development death. The failure to survive is more likely related to the putative and Fe acquisition, being beneficial in high pH habitats. AHH role of CSLD2 in collaboration with CSLD3 and CSLD5 in catalyzes the degradation of allantoin yielding CO2 and four hemicellulose biosynthesis (Yin et al., 2011) affecting early plant molecules of NH4 (Werner et al., 2008). High levels of allantoin and development. FBN6 is also required for normal plant development downregulation of allantoin degradation has been found to enhance and fbn6 mutants have stunted root growth. This has been related salt stress tolerance in A. thaliana (Lescano et al., 2016). Our findings 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 11 regarding the bicarbonate tolerance of the ahh mutant (Supporting ACKNOWL EDGEM ENTS Information: Figure S3A) and the lower root expression of AHH in Special thanks to Sian Bray for her bioinformatic programming A1(c+) indicate (Figure 5c,d) a role for allantoin also in bicarbonate support. We are grateful to Maria José Almira for her collaboration in tolerance. In A. thaliana, JAZ proteins act as repressors of JA laboratory experiments and graphics; and to Rosa Padilla for signalling and plants overexpressing AtJAZ10 are insensitive to JA processing the ionome data. This research was funded by the (Chung & Howe, 2009; Sun et al., 2017). In our case, jaz10 exhibited a Spanish Ministry of Science & Innovation (PID2019‐104000RB‐I00). better response to bicarbonate stress than the WT and JAZ10 was less expressed in the tolerant A1(c+) deme, suggesting that a ORC I D downregulation of this gene could trigger JA signals important for Roser Tolrà http://orcid.org/0000-0001-8513-3520 tolerant responses. Charlotte Poschenrieder http://orcid.org/0000-0002-3818-0874 Trichome Birefringence Like 19 ‐tbl19 (m11) did not grow or produced more siliques than Col‐0 but was not affected by the RE F ER EN CES carbonate treatment, exhibiting higher tolerance (Supporting Infor- Ahmad, P., Ozturk, M., Sharma, S. & Gucel, S. (2014) Effect of sodium mation: Figure S3A,B). In both demes TBL19 was downregulated in carbonate‐induced salinity‐alkalinity on some key osmoprotectants, protein profile, antioxidant enzymes, and lipid peroxidation in two roots and upregulated in leaves (Figure 5). Under bicarbonate mulberry (Morus alba L.) cultivars. Journal of Plant Interactions, 9, exposure, the knock‐out mutant had high root biomass and the 460–467. Available from: https://doi.org/10.1080/17429145.2013. highest fitness as expressed in number of siliques (Supporting 855271 Information: Figure S3). TBL19, also known as AtXYBAT1 [63], has Al‐Busaidi, A. & Cookson, P. (2003) Salinity—pH relationships in calcareous soils of Oman. Agricultural and Marine Sciences, 8, O‐acetyltransferase activity and is probably involved in the 41–46. Available from: https://journals.squ.edu.om/index.php/ acetylation of cellohexaose, a hemicellulose in A. thaliana cell walls. jams/article/view/615/614 Root apoplastic iron in A. thaliana is mainly stored in the Bartlett, M.E. & Whipple, C.J. (2013) Protein change in plant evolution: operationally defined HC1 fraction of hemicellulose and this tracing one thread connecting molecular and phenotypic diversity. bound‐Fe can be mobilized by coumarin‐type phenolic exudation Frontiers in Plant Science, 4, 382. Available from: https://doi.org/10. 3389/fpls.2013.00382 (Lei et al., 2014). Under bicarbonate exposure, the tolerant A1(c+) Benjamini, Y. & Yekutieli, D. (2001) The control of the false discovery rate has higher exudation of these phenolics than the sensitive T6(c−) in multiple testing under dependency. Annals of Statistics, 2001(29), (Terés et al., 2019). The decrease of acetylation may enhance Fe 1165–1188. Available from: https://doi.org/10.1214/aos/ binding to cell wall hemicellulose and its accessibility to the 1013699998 Benowicz, A., Stoehr, M., Hamann, A. & Yanchuk, A.D. (2020) Estimation coumarin‐type phenolics, so enhancing the root‐to‐shoot Fe of the F2 generation segregation variance and relationships among translocation specifically in A1(c+). growth, frost damage, and bud break in coastal Douglas‐Fir Alkaline/Neutral Invertase (INV‐E) encodes a chloroplast‐ (Pseudotsuga menziesii (Mirb.) Franco) wide‐crosses. Annals of targeted protein, although it is expressed in root tissues in A. thaliana Forest Science, 77, 28. Available from: https://doi.org/10.1007/ s13595-020-0925-9 (TAIR‐BAR). INV‐E catalyzes the irreversible cleavage of sucrose into Bernal, A.J., Yoo, C.‐M., Mutwil, M., Jensen, J., Hou, G., Blaukopf, C. et al. glucose and fructose and some AtINV have been linked to the control (2008) Functional analysis of the cellulose synthase‐like genes of root cell elongation mediated by sugars (Ruan et al., 2010). inv‐e CSLD1, CSLD2, and CSLD4 in tip‐growing Arabidopsis cells. Plant (m17) mutant was clearly affected by the bicarbonate content both in Physiology, 148, 1238–1253. Available from: https://doi.org/10. hydroponics and in soil (Supporting Information: Figure S3A,B). The 1104/pp.108.121939 Busoms, S., Paajanen, P., Marburger, S., Bray, S., Huang, X.Y., SNP changes detected in A1(c+) and T6(c−) were evaluated and we Poschenrieder, C. et al. (2018) Fluctuating selection on migrant found that T6(c−) INV‐E gene contains a large number of variants that adaptive sodium transporter alleles in coastal Arabidopsis thaliana. could affect the expression of the gene (Supporting Information: Proceedings of the National Academy of Sciences, 115, Figure S4, Supporting Information: Data set SD9), supporting the E12443–E12452. Available from: https://doi.org/10.1073/pnas. 1816964115 hypothesis that a proper regulation of INV‐E could play an important Busoms, S., Terés, J., Huang, X.‐Y., Bomblies, K., Danku, J., Douglas, A. role in bicarbonate tolerance. et al. (2015) Salinity is an agent of divergent selection driving local In conclusion, differential bicarbonate tolerance in A. thaliana is adaptation of Arabidopsis to coastal habitats. Plant Physiology, 168, associated with enhanced root‐to‐shoot translocation of iron, proper 915–929. Available from: https://doi.org/10.1104/pp.15.00427 distribution of C/N fixation, and better root development. Associa- Busoms, S., Terés, J., Yant, L., Poschenrieder, C. & Salt, D.E. (2021) Adaptation to coastal soils through pleiotropic boosting of ion and tion analysis of genomics and transcriptomics with natural demes stress hormone concentrations in wild Arabidopsis thaliana. New well‐ or mal‐adapted to soil carbonate allowed the selection of 18 Phytologist, 232, 208–220. Available from: https://doi.org/10.1111/ candidate genes for this differential tolerance. Evaluation of genomic nph.17569 variation and the corresponding knock‐out mutants suggested Cao, Y., Song, H. & Zhang, L. (2022) New insight into plant saline‐alkali tolerance mechanisms and application to breeding. International implications of AHH, CSLD2, DAO1, GDH2, FBN6, INV‐E, JAZ10 Journal of Molecular Sciences, 23, 16048. Available from: https://doi. and TBL19. We propose these genes as candidates for being further org/10.3390/ijms232416048 explored through complementary analysis under bicarbonate stress Castilla, A.R., Méndez‐Vigo, B., Marcer, A., Martínez‐Minaya, J., conditions. Conessa, D., Picó, F.X. et al. (2020) Ecological, genetic and 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12 | BUSOMS ET AL. evolutionary drivers of regional genetic differentiation in Arabidopsis Lee, J.A. & Woolhouse, H.W. (1969) Root growth and dark fixation of thaliana. BMC Evolutionary Biology, 20, 71. Available from: https:// carbon dioxide in calcicoles and calcifuges. New Phytologist, 68, doi.org/10.1186/s12862-020-01635-2 247–255. Available from: https://doi.org/10.1111/j.1469-8137. Chung, H.S. & Howe, G.A. (2009) A critical role for the TIFY motif in 1969.tb06437.x repression of jasmonate signaling by a stabilized splice variant of the Lee, K., Lehmann, M., Paul, M.V., Wang, L., Luckner, M., Wanner, G. et al. JASMONATE ZIM‐domain protein JAZ10 in Arabidopsis. The Plant (2020) Lack of FIBRILLIN6 in Arabidopsis thaliana affects light Cell, 21, 131–145. Available from: https://doi.org/10.1105/tpc.108. acclimation and sulfate metabolism. New Phytologist, 225, 064097 1715–1731. Available from: https://doi.org/10.1111/nph.16246 Cingolani, P. (2022) Variant Annotation and Functional Prediction: Lei, G.J., Zhu, X.F., Wang, Z.W., Dong, F., Dong, N.Y. & Zheng, S.J. (2014) SnpEffIn: Ng, C. & Piscuoglio, S. (Eds.) Variant calling. Methods in Abscisic acid alleviates iron deficiency by promoting root iron molecular biology, 2493. New York, NY: Humana. https://doi.org/10. reutilization and transport from root to shoot in Arabidopsis. Plant, 1007/978-1-0716-2293-3_19 Cell & Environment, 37, 852–863. Available from: https://doi.org/10. Danecek, P., Auton, A., Abecasis, G., Albers, C.A., Banks, E., DePristo, M.A. 1111/pce.12203 et al. (2011) The variant call format and VCFtools. Bioinformatics, 27, Lescano, C.I., Martini, C., González, C.A. & Desimone, M. (2016) Allantoin 2 1 5 6 – 2 1 5 8 . A v a i l a b l e f r o m : h t tp s :// d o i .o r g/1 0 .1 0 9 3 / accumulation mediated by allantoinase downregulation and trans- bioinformatics/btr330 port by Ureide Permease 5 confers salt stress tolerance to Derakhshani, B., Ayalew, H., Mishina, K., Tanaka, T., Kawahara, Y., Arabidopsis plants. Plant Molecular Biology, 91, 581–595. Available Jafary, H. et al. (2020) Comparative analysis of root transcriptome from: https://doi.org/10.1007/s11103-016-0490-7 reveals candidate genes and expression divergence of homoeolo- Li, H. (2011) A statistical framework for SNP calling, mutation discovery, gous genes in response to water stress in wheat. Plants, 9, 9596. association mapping and population genetical parameter estimation Available from: https://doi.org/10.3390/plants9050596 from sequencing data. Bioinformatics, 27, 2987–2993. Available Dreyer, M., Wichmann, M., Rischen, M., Görlach, B.M., Ehmke, A., from: https://doi.org/10.1093/bioinformatics/btr509 Pitann, B. et al. (2020) Ammonium‐driven nitrification plays a key Li, N., Liu, H., Sun, J., Zheng, H., Wang, J., Yang, L. et al. (2018) role in increasing Mn availability in calcareous soils. Journal of Plant Transcriptome analysis of two contrasting rice cultivars during Nutrition and Soil Science, 183, 389–396. Available from: https://doi. alkaline stress. Scientific Reports, 8, 9586. org/10.1002/jpln.201900131 Li, Q., Yang, A. & Zhang, W.‐H. (2016) Efficient acquisition of iron confers Duan, X., Yu, Y., Duanmu, H., Chen, C., Sun, X., Cao, L. et al. (2018) greater tolerance to saline‐alkaline stress in rice (Oryza sativa L.). GsSLAH3, a Glycine soja slow type anion channel homolog, positively Journal of Experimental Botany, 67, 6431–6444. Available from: modulates plant bicarbonate stress tolerance. Physiologia Plantarum, https://doi.org/10.1093/jxb/erw407 164, 145–162. Available from: https://doi.org/10.1111/ppl.12683 Li, Z. & Xu, Y. (2022) Bulk segregation analysis in the NGS era: a review of Edwards, M.D. & Gifford, D.K. (2012) High‐resolution genetic mapping its teenage years. The Plant Journal, 109, 1355–1374. Available with pooled sequencing. BMC Bioinformatics, 13(Suppl 6), S8. from: https://doi.org/10.1111/tpj.15646 Available from: https://doi.org/10.1186/1471-2105-13-S6-S8 Loeppert, R.H. & Suarez, D.L. (1996) Carbonate and gypsum. In: Felle, H.H. (2001) pH: signal and messenger in plant cells. Plant Biology, 3, Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., 577–591. Tabatabai, M.A., Johnston, C.T. & Sumner, M.E. (Eds.) Methods of soil Filippi, P., Jones, E.J., Ginns, B.J., Whelan, B.M., Roth, G.W. & Bishop, T.F.A. analysis. Part 3 chemical methods, 5.3. Madison, USA: Publisher: Soil (2019) Mapping the depth‐to‐soil pH constraint, and the relationship Science Society of America and American Society of Agronomy, chapter with cotton and grain yield at the within‐field scale. Agronomy, 9, 251. 15. pp. 437–474. Available from: https://doi.org/10.3390/agronomy9050251 McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Guo, Z., Cai, L., Chen, Z., Wang, R., Zhang, L., Guan, S. et al. (2020) Kernytsky, A. et al. (2010) The genome analysis toolkit: a Identification of candidate genes controlling chilling tolerance of rice MapReduce framework for analyzing next‐generation DNA in the cold region at the booting stage by BSA‐Seq and RNA‐Seq. sequencing data. Genome Research, 20, 1297–1303. Available from: Royal Society Open Science, 7, 201081. Available from: https://doi. https://doi.org/10.1101/gr.107524.110 org/10.1098/rsos.201081 Mehari, T.G., Xu, Y., Umer, M.J., Shiraku, M.L., Hou, Y., Wang, Y. et al. Hayashi, K., Arai, K., Aoi, Y., Tanaka, Y., Hira, H., Guo, R. et al. (2021) The (2021) Multi‐omics‐based identification and functional characteriza- main oxidative inactivation pathway of the plant hormone auxin. tion of Gh_A06G1257 proves its potential role in drought stress Nature Communications, 12, 6752. Available from: https://doi.org/ tolerance in Gossypium hirsutum. Frontiers in Plant Science, 12, 10.1038/s41467-021-27020-1 746771. Available from: https://doi.org/10.3389/fpls.2021.746771 Hui, X., Luo, L., Wang, S., Cao, H., Huang, M., Shi, M. et al. (2019) Critical Monroe, J.G., Srikant, T., Carbonell‐Bejerano, P., Becker, C., Lensink, M., concentration of available soil phosphorus for grain yield and zinc Exposito‐Alonso, M. et al. (2022) Mutation bias reflects natural nutrition of winter wheat in a zinc‐deficient calcareous soil. Plant and selection in Arabidopsis thaliana. Nature, 602, 101–105. Available Soil, 444, 315–330. Available from: https://doi.org/10.1007/ from: https://doi.org/10.1038/s41586-021-04269-6 s11104-019-04273-w Pérez‐Martín, L., Busoms, S., Almira, M.J., Azagury, N., Terés, J., Tolrà, R. Joshi, N.A. & Fass, J.N. (2011) Sickle: A sliding‐window, adaptive, quality‐ et al. (2022) Evolution of salt tolerance in Arabidopsis thaliana on based trimming tool for FastQ files (Version 1.33) [Software]. siliceous soils does not confer tolerance to saline calcareous soils. Available from: https://github.com/najoshi/sickle Plant and Soil, 476, 455–475. Available from: https://doi.org/10. Kiba, T., Kudo, T., Kojima, M. & Sakakibara, H. (2011) Hormonal control of 1007/s11104-022-05439-9 nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. Pérez‐Martín, L., Busoms, S., Tolrà, R. & Poschenrieder, C. (2021) Journal of Experimental Botany, 62, 1399–1409. Available from: Transcriptomics reveals fast changes in salicylate and jasmonate https://doi.org/10.1093/jxb/erq410 signaling pathways in shoots of carbonate‐tolerant Arabidopsis Lakehal, A., Dob, A., Novák, O. & Bellini, C. (2019) A DAO1‐mediated thaliana under bicarbonate exposure. International Journal of circuit controls auxin and jasmonate crosstalk robustness during Molecular Sciences, 22, 1226. Available from: https://doi.org/10. adventitious root initiation in Arabidopsis. International Journal of 3390/ijms22031226 Molecular Sciences, 20, 4428. Available from: https://doi.org/10. Ruan, Y.L., Jin, Y., Yang, Y.J., Li, G.J. & Boyer, J.S. (2010) Sugar input, 3390/ijms20184428 metabolism, and signaling mediated by invertase: roles in development, 13653040, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.14691 by Readcube (Labtiva Inc.), Wiley Online Library on [24/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License COMBINED GENOMICS TO DISCOVER GENES | 13 yield potential, and response to drought and heat. Molecular Plant, 3, Werner, A.K., Sparkes, I.A., Romeis, T. & Witte, C.P. (2008) Identification, 942–955. Available from: https://doi.org/10.1093/mp/ssq044 biochemical characterization, and subcellular localization of allanto- Sagervanshi, A., Naeem, A., Kaiser, H., Pitann, B. & Mühling, K.H. (2021) Early ate amidohydrolases from Arabidopsis and Soybean. Plant growth reduction in Vicia faba L. under alkali salt stress is mainly caused Physiology, 146, 323–324. Available from: https://doi.org/10. by excess bicarbonate and related to citrate and malate over 1104/pp.107.110809 accumulation. Environmental and Experimental Botany, 192, 104636. Wilm, A., Aw, P.P.K., Bertrand, D., Yeo, G.H.T., Ong, S.H., Wong, C.H. et al. Available from: https://doi.org/10.1016/j.envexpbot.2021.104636 (2012) LoFreq: a sequence‐quality aware, ultra‐sensitive variant Salomé, P.A., Bomblies, K., Fitz, J., Laitinen, R.A.E., Warthmann, N., Yant, L. caller for uncovering cell‐population heterogeneity from high‐ et al. (2012) The recombination landscape in Arabidopsis thaliana F2 throughput sequencing datasets. Nucleic Acids Research, 40, populations. Heredity, 108, 447–455. Available from: https://doi. 11189–11201. Available from: https://doi.org/10.1093/nar/gks918 org/10.1038/hdy.2011.95 Xia, A. & Wu, Y. (2022) Joint interactions of carbon and nitrogen Sarkar, B., Hasanuzzaman, M. & Adak, M.K. (2022) Insights into the role of metabolism dominated by bicarbonate and nitrogen in Orycho- iron supplementation in conferring bicarbonate‐mediated alkaline phragmus violaceus and Brassica napus under simulated karst stress tolerance in maize. Journal of Soil Science and Plant Nutrition, habitats. BMC Plant Biology, 22, 264. Available from: https://doi. 22, 2719–2734. Available from: https://doi.org/10.1007/s42729- org/10.1186/s12870-022-03646-1 022-00839-3 Ye, S., Yan, L., Ma, X., Chen, Y., Wu, L., Ma, T. et al. (2022) Combined BSA‐ Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. (2012) NIH image to Seq based mapping and RNA‐Seq profiling reveal candidate genes ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675. associated with plant architecture in Brassica napus. International Available from: https://doi.org/10.1038/nmeth.2089 Journal of Molecular Sciences, 23, 2472. Available from: https://doi. Scholl, R.L., May, S.T. & Ware, D.H. (2000) Seed and molecular resources org/10.3390/ijms23052472 for Arabidopsis. Plant Physiology, 124, 1477–1480. Available from: Yin, L., Verhertbruggen, Y., Oikawa, A., Manisseri, C., Knierim, B., Prak, L. https://doi.org/10.1104/pp.124.4.1477 et al. (2011) The cooperative activities of CSLD2, CSLD3, and Sivitz, A.B., Hermand, V., Curie, C. & Vert, G. (2012) Arabidopsis bHLH100 CSLD5 are required for normal Arabidopsis development. Molecular and bHLH101 control iron homeostasis via a FIT‐independent Plant, 4, 1024–1037. Available from: https://doi.org/10.1093/mp/ pathway. PLoS One, 7, e44843. Available from: https://doi.org/10. ssr02 1371/journal.pone.0044843 Zhang, H., Liu, X.‐L., Zhang, R.‐X., Yuan, H.‐Y., Wang, M.‐M., Yang, H.‐Y. Sun, Q., Wang, G., Zhang, X., Zhang, X., Qiao, P., Long, L. et al. (2017) Genome‐ et al. (2017) Root damage under alkaline stress is associated with wide identification of the TIFY gene family in three cultivated Gossypium reactive oxygen species accumulation in rice (Oryza sativa L.). species and the expression of JAZ genes. Scientific Reports, 2017(7), 1–9. Frontiers of Plant Science, 8, 1–12. Available from: https://doi.org/ Available from: https://doi.org/10.1038/srep42418 10.3389/fpls.2017.01580 Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta‐Cepas, J. Zou, C.L., Wang, Y.B., Wang, B., Liu, D., Liu, L. & Li, C.F. (2021) Effects of et al. (2019) String v11: protein‐protein association networks with alkali stress on dry matter accumulation, root morphology, ion increased coverage, supporting functional discovery in genome balance, free polyamines, and organic acids of sugar beet. Acta –wide experimental datasets. Nucleic Acids Research, 47, Physiologiae Plantarum, 43, 13. Available from: https://doi.org/10. D607–D613. Available from: https://doi.org/10.1093/nar/gky1131 1007/s11738-020-03194-x Terés, J., Busoms, S., Perez Martín, L., Luís‐Villarroya, A., Flis, P., Álvarez‐ Fernández, A. et al. (2019) Soil carbonate drives local adaptation in Arabidopsis thaliana. Plant, Cell & Environment, 42, 2384–2398. SUPP ORTING INFO RM ATION Available from: https://doi.org/10.1111/pce.13567 Additional supporting information can be found online in the Thanati, F., Karatzas, E., Baltoumas, F., Stravopodis, D.J., Eliopoulos, A. & Supporting Information section at the end of this article. Pavolpoulos, G. (2021) FLAME: a web tool for functional and literature enrichment analysis of multiple gene lists. Biol. (Basel), 10, 665. Available from: https://doi.org/10.3390/biology10070665 Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z. et al. (2017) agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. How to cite this article: Busoms, S., Pérez‐Martín, L., Terés, J., Nucleic Acids Research, 45, W122–W129. Available from: https:// Huang, X.‐Y., Yant, L., Tolrà, R. et al. (2023) Combined doi.org/10.1093/nar/gkx382 genomics to discover genes associated with tolerance to soil Waters, B.M., Amundsen, K. & Graef, G. (2018) Gene expression profiling of carbonate. Plant, Cell & Environment, 1–13. iron deficiency chlorosis sensitive and tolerant soybean indicates key https://doi.org/10.1111/pce.14691 roles for phenylpropanoids under alkalinity stress. Frontiers in Plant Science, 9, 10. Available from: https://doi.org/10.3389/fpls.2018.00010