Barley (*Hordeum vulgare* L.) is a globally significant cereal crop, utilized for human consumption, malting, and animal feed. However, its productivity is severely hampered by acid soils, which account for 30-40% of arable land. This limitation stems from barley's high sensitivity to aluminum (Al) toxicity. Al ions inhibit root elongation, impacting water and nutrient uptake, and consequently reducing crop yields. The increasing soil acidification due to global warming necessitates urgent improvements in barley's acid soil tolerance for food security. Previous research identified *HvAACT1* (Al-activated citrate transporter 1) as a major gene governing Al tolerance in barley. *HvAACT1*, primarily expressed in roots, encodes a citrate transporter. Upon Al exposure, it releases citrate, which detoxifies Al by forming harmless complexes. Natural variations in Al tolerance are linked to a 1-kb CACTA-like transposon insertion upstream of *HvAACT1*, acting as a promoter to enhance its expression and citrate secretion. This insertion is prevalent in some six-rowed barley cultivars but absent in two-rowed hulled barley (typically used for malting). The importance of developing acid-tolerant malting barley is underscored by the significant portion (approximately 30%) of barley production used for brewing and distilling. This study aimed to address this need by breeding a malting barley cultivar with enhanced acid soil tolerance through the introgression of the beneficial 1-kb transposon.

Extensive research has explored the genetic basis of aluminum tolerance in barley. A key finding was the identification of *HvAACT1*, a gene encoding a citrate transporter crucial for Al detoxification. The natural variation in Al tolerance within barley populations has been linked to a 1-kb transposon insertion upstream of *HvAACT1*, which enhances its expression and subsequently citrate secretion. Different mechanisms of *HvAACT1* regulation have been observed in East Asian and European barley accessions, highlighting independent evolutionary adaptations to Al toxicity. Previous attempts to breed Al-tolerant barley using transgenic approaches and conventional breeding have shown promise but faced challenges, such as public acceptance of transgenic crops and issues with grain quality. This study builds on these prior efforts by employing a conventional breeding approach focusing on the introgression of the functionally validated 1-kb transposon.

To breed an Al-tolerant malting barley cultivar, the researchers employed a backcrossing strategy. The 1-kb transposon insertion from the Al-tolerant cultivar Murasakimochi (a six-rowed barley) was introgressed into the elite malting cultivar Haruna Nijo (a two-rowed hulled barley). Multiple backcrosses with Haruna Nijo were performed, and marker-assisted selection using 1536 SNP markers (691 polymorphic between the parent lines) was utilized to track the introgression of the target 1-kb insertion linked to the *HvAACT1* locus. One isogenic line carrying the Murasakimochi allele at the *HvAACT1* locus in the Haruna Nijo genetic background was selected (designated as Haruna Nijo-AT) and further self-pollinated to BC4F10. The acid soil tolerance of Haruna Nijo-AT was evaluated through hydroponic experiments and field tests conducted over two years in both acidic (pH 4.9-5.0) and neutral (pH 6.5) soils. Hydroponic experiments measured root growth, citrate secretion (enzymatic method), and Al accumulation in root tips (ICP-MS). Field tests assessed plant height, spike number, seed yield, and straw weight. The presence of the 1-kb insertion in Haruna Nijo-AT was confirmed via PCR. Expression analysis of *HvAACT1* was performed using RT-PCR, and Al staining of root tips with Eriochrome cyanine was conducted. Mineral element profiles of the grains were also analyzed using ICP-MS.

The introgression of the 1-kb transposon from Murasakimochi into Haruna Nijo significantly enhanced acid soil tolerance. Haruna Nijo-AT displayed a substantially increased expression of *HvAACT1* (30 times higher than Haruna Nijo), significantly higher citrate secretion from roots (seven times higher), and substantially reduced Al accumulation in root tips. Field tests demonstrated that Haruna Nijo-AT produced 2-3.3 times more grain yield than Haruna Nijo under acidic soil conditions while maintaining similar grain quality (size and color) and a similar mineral profile. The increase in yield was consistent across two years of field trials despite slight variations in soil pH and climatic conditions. No significant difference in yield or other traits was observed between the two cultivars under neutral soil conditions.

The results confirm that the introgression of the 1-kb transposon, leading to increased *HvAACT1* expression and citrate secretion, is a highly effective strategy for enhancing acid soil tolerance in malting barley. The significant yield increase observed under acidic conditions, without compromising grain quality or mineral composition, highlights the practical significance of this breeding approach. This strategy offers a sustainable and economical alternative to liming, which is often an infeasible solution for improving barley productivity in acid soil regions. This study provides a successful example of utilizing natural genetic variation to enhance crop resilience to abiotic stress, a critical approach in the context of climate change and sustainable agriculture. The successful development of Haruna Nijo-AT demonstrates the potential of marker-assisted selection in expediting conventional breeding programs.

This study successfully bred a new malting barley cultivar, Haruna Nijo-AT, with significantly improved acid soil tolerance through the introgression of a 1-kb transposon regulating *HvAACT1* expression. Haruna Nijo-AT exhibits enhanced Al tolerance, increased yield under acidic conditions, and maintains desirable grain quality. This approach represents a valuable tool for developing other acid-tolerant barley cultivars and enhancing food security in areas with acid soils. Future research could investigate the application of this strategy to other barley cultivars and explore the potential for further yield improvements.

While the study demonstrates a significant yield increase in field trials, the specific environmental conditions (soil type, pH, climate) of the testing location may limit the generalizability of the findings to other regions. Furthermore, larger-scale field trials across diverse geographic locations would be beneficial to assess the robustness and adaptability of Haruna Nijo-AT under variable environmental conditions. Long-term studies are also needed to evaluate the sustained performance of this cultivar over multiple growing seasons.