Breeding for an elite malting barley cultivar with acid soil tolerance

Barley is widely cultivated for food, malting, and feed, but its productivity is limited on acid soils (30–40% of arable land) due to sensitivity to aluminum toxicity, which rapidly inhibits root growth and nutrient/water uptake. With global warming accelerating soil acidification, improving barley tolerance to acid soils is urgent for food security. Natural variation in Al tolerance exists in barley, largely controlled by HvAACT1, a root-expressed citrate transporter that detoxifies Al via citrate exudation from root tips. A 1-kb CACTA-like transposon insertion upstream of HvAACT1 enhances its expression and citrate secretion, conferring tolerance; European accessions exhibit an alternative regulatory mechanism via a multiretrotransposon-like insertion and DNA methylation changes. Two-rowed hulled malting barley, widely used for brewing, typically lacks the 1-kb insertion, underscoring the need to introgress this allele into elite malting backgrounds to enhance acid soil tolerance. This study aimed to breed an elite malting cultivar with improved acid soil tolerance by introgressing the 1-kb insertion controlling HvAACT1 expression and to evaluate its agronomic performance on acidic soils.

Prior work established broad natural variation in barley Al tolerance and identified HvAACT1 as a key determinant mediating Al-activated citrate efflux from root tips. Enhanced HvAACT1 expression due to a 1-kb CACTA-like transposon upstream of the gene confers higher tolerance and is associated with barley expansion into acidic Far-Eastern regions. In European barley, a multiretrotransposon-like insertion and its methylation state regulate HvAACT1 expression, indicating convergent strategies for acid soil adaptation. Transgenic approaches have improved Al tolerance in barley, including overexpression of wheat TaALMT1 (increasing malate efflux) and HvAACT1 (under a maize ubiquitin promoter), and a higher Al-tolerant variety (Litmus) was developed from an acid-tolerant line; however, deployment has been constrained by public acceptance issues and grain color (aleurone) traits affecting quality. Two-rowed hulled malting cultivars generally lack the beneficial 1-kb insertion, motivating marker-assisted introgression as a non-transgenic route for malting barley improvement.

Breeding and genotyping: The 1-kb insertion allele upstream of HvAACT1 was introgressed from the Al-tolerant Japanese landrace Murasakimochi into the elite two-rowed malting cultivar Haruna Nijo via multiple backcrossing. Haruna Nijo (female) × Murasakimochi F1 progeny were repeatedly backcrossed to Haruna Nijo to generate BC1F1, BC2F1, and BC3F1. After two selfing cycles, 83 BC3F3 recombinant chromosome substitution lines were genotyped with 1,536 EST-derived SNPs using the Illumina GoldenGate platform; 691 polymorphic markers were used. Markers were positioned using consensus maps to identify those linked to HvAACT1. An isogenic line carrying the Murasakimochi allele at the HvAACT1 locus in the Haruna Nijo background was selected and further backcrossed to Haruna Nijo to BC4, then selfed to BC4F10 (designated Haruna Nijo-AT) for analyses. Acid soil seedling test: Parents and BC4F3 were grown 10 days on acidic Andosol (pH ~4.8) to assess root growth inhibition. Molecular assays: Presence of the 1-kb insertion was confirmed by PCR on genomic DNA (primers: 5′-GGTCCAACACTCTACCCTTCCTT-3′ and 5′-GGTGCGAGTTGCCCCTAGCTATTACAGA-3′), products resolved on 1.0% agarose gel. HvAACT1 expression was quantified by RT-qPCR on root RNA (RNeasy extraction; cDNA by ReverTra Ace; primers 5′-TTCGTCGCTGAAGAAGATGC-3′ and 5′-GCACATTGCAGGCATCTGAA-3′); Actin served as internal control (primers 5′-TCGCTCCACCTGAGAGGAAG-3′ and 5′-GCTAGGATGGACCCTCCGAT-3′); relative expression analyzed by ΔΔCt on Bio-Rad CFX96 with four biological replicates. Physiological assays: Seedlings were germinated and grown hydroponically (0.5 mM CaCl2, then one-fifth Hoagland, pH 6.0; 22–25 °C; aerated; solution changed every 2 days). For citrate exudation, 24-day-old seedlings were exposed to 10 µM Al in 1.0 mM CaCl2 (pH 5.0) for 6 h; exudates were purified via cation- and anion-exchange resin columns (Amberlite IR-120B H+; AG 1×8 formate), eluted with 2 N HCl, dried, reconstituted, and citrate quantified enzymatically. For Al accumulation, 8-day-old seedlings were exposed to 10 µM Al in 1.0 mM CaCl2 (pH 5.0) for 24 h; 0–1 cm root tips were excised, extracted with 1 N HCl, and Al measured by ICP-MS (Agilent 7700X). Al localization was visualized by 0.1% eriochrome cyanine staining. Field trials: Conducted at Okayama University over two seasons. 2022–2023: Acidic soil (pH 4.9) and neutral soil (pH 6.5); three plots per line; each plot 140 × 90 cm with 6 plants; fertilizer 15–15–15 (N–P2O5–K2O) at 4 g per plot; sown mid-November 2022, harvested end of May 2023; measured plant height, spike number, seed yield, and straw weight per plant; photographed. 2023–2024: Acidic soil repeated at slightly higher pH (5.0); plots 160 × 90 cm with 8 plants; same agronomic measurements. Grain mineral analysis: Dried grains digested in 61% HNO3 up to 140 °C; mineral elements quantified by ICP-MS (Agilent 7700X/8900). Statistics: One-way ANOVA with Tukey’s test for field traits (Fig. 4); Student’s t-test for molecular and physiological assays (Figs. 2, 3; Supplementary Fig. 5). Biological replicates: minimum n=3; detailed n specified in figure legends.

• Introgression of the 1-kb upstream insertion at HvAACT1 from Murasakimochi into Haruna Nijo produced a near-isogenic malting line (Haruna Nijo-AT) with enhanced acid soil tolerance while retaining Haruna Nijo agronomic and grain appearance traits.
• HvAACT1 expression in roots was ~30-fold higher in Haruna Nijo-AT than in Haruna Nijo (RT-qPCR, n=4, P<0.01).
• Root citrate secretion under Al exposure was ~7-fold higher in Haruna Nijo-AT than in Haruna Nijo (6 h at 10 µM Al; enzymatic assay; n=3; significant by t-test), consistent with enhanced Al detoxification.
• Al accumulation in 0–1 cm root tips after 24 h at 10 µM Al was significantly lower in Haruna Nijo-AT than in Haruna Nijo (ICP-MS; n=4; P<0.05–0.01), corroborated by weaker eriochrome cyanine staining in Haruna Nijo-AT root tips.
• Field trial 2022–2023: Under neutral soil (pH 6.5), no significant differences in plant height, spike number, seed yield, or straw biomass between Haruna Nijo-AT and Haruna Nijo. Under acidic soil (pH 4.9), Haruna Nijo-AT had greater plant height and spike number, and produced 3.3× higher seed yield and >3× higher straw biomass than Haruna Nijo (ANOVA/Tukey, P<0.05).
• Field trial 2023–2024 (acidic soil pH 5.0): Haruna Nijo-AT produced ~2× higher seed yield than Haruna Nijo; plant height, spike number, and straw weight were also higher.
• Grain mineral element profiles did not differ between Haruna Nijo-AT and Haruna Nijo within a given soil type; grains from acidic soil showed higher Fe, Cu, Mn, and Zn than those from neutral soil, likely reflecting greater micronutrient bioavailability in acidic conditions.

The study demonstrates that targeted introgression of a 1-kb regulatory insertion upstream of HvAACT1 into an elite malting barley background is sufficient to substantially enhance tolerance to acid soils. Elevated HvAACT1 expression in Haruna Nijo-AT increases Al-activated citrate efflux, reducing Al binding in root tips and mitigating Al toxicity, thereby maintaining root function. This physiological improvement translated into large agronomic gains on acidic soils across two field seasons (2–3.3× yield increases and higher biomass), while performance under neutral conditions remained unchanged—indicating the trait specifically benefits acid soil environments without penalizing yield potential elsewhere. Compared with agronomic liming, which is costly and often ineffective for subsoil Al, and with transgenic approaches that face public acceptance and grain quality issues, marker-assisted introgression provides a practical, non-transgenic route compatible with malting quality requirements. The results support deploying this allele broadly in malting germplasm to overcome the current sensitivity of two-rowed hulled malting barley to acidic soils.

By introgressing a 1-kb upstream transposon that enhances HvAACT1 expression, the authors developed Haruna Nijo-AT, an elite malting barley line with markedly improved acid soil tolerance. The line exhibits substantially higher HvAACT1 expression, greater citrate secretion, reduced Al accumulation in roots, and 2–3.3-fold yield gains on acidic soils, without compromising performance on neutral soils or altering grain appearance or mineral composition. This marker-assisted, non-transgenic strategy offers a sustainable and economical approach to improving malting barley productivity on acid soils. Future work could extend introgression to additional elite malting backgrounds, test performance across diverse acidic environments and seasons, and evaluate comprehensive malting quality parameters to ensure industry compatibility.

• Field validation was conducted at a single location across two years; broader multi-location trials across diverse acid soils would strengthen generalizability.
• Year-to-year differences in yield gains likely reflect climatic variation and slight pH differences (pH 4.9 vs 5.0), indicating environmental sensitivity of effect size.
• Detailed malting quality assessments beyond grain appearance and mineral composition were not reported; potential subtle quality impacts were not evaluated.
• Development of near-isogenic lines via backcrossing and selfing requires multiple years, which may limit rapid deployment, though marker-assisted selection mitigates this.