Genetic and agronomic approaches to improve waterlogging stress tolerance in barley (Hordeum vulgare L)

Waterlogging remains a significant constraint to barley production across the globe in high rainfall areas. Breeding stress-resilient cultivars is arguably the most economical way of tackling the problem. However, under severe waterlogging combined genetic and agronomic solutions might be more effective. Waterlogging tolerance is conferred by several complimentary mechanisms with root cortical aerenchyma (RCA) formation being one of the major ones. The aims of this project were to: 1) evaluate the contribution of RCA to waterlogging tolerance using near-isogenic lines (NILs) which differ in a quantitative trait locus (QTL) for RCA formation (RCA-QTL); 2) identify marker trait associations (MTA) for RCA and adventitious root (AR) survival under waterlogging conditions; 3) fine map the RCA-QTL; and 4) evaluate the impact of nitrogen fertilizer application to mitigate waterlogging induced penalties imposed on plants. A wide range of agronomic and engineering solutions are currently being used by grain growers to reduce crop losses from waterlogging. The effects of waterlogging on plant growth, and advantages and disadvantages of various agronomic and engineering solutions which are used to mitigate waterlogging damage were reviewed. It is concluded that further research should be focused on: cost/benefit analyses of different drainage strategies; understanding the mechanisms of nutrient loss during waterlogging and quantifying the benefits of nutrient application; increasing soil profile de-watering through soil improvement and agronomic strategies; revealing the specificity of the interaction between different management practices and environment as well as among management practices; and more importantly, combined genetic, agronomic and engineering strategies for varying environments. Waterlogging stress tolerance is conferred by several complimentary mechanisms, with RCA formation being one of the major ones. In this study, three pairs of NILs which have a large proportion of the genetic background of commercial barley varieties but differ in the RCA-QTL were used to investigate the impact of RCA on overall waterlogging tolerance. Eight other genotypes contrasting in waterlogging tolerance were used as controls. Field trials were conducted in 2019 and 2020. The waterlogging treatment began at the two-three leaf stage and continued for two months. Water was subsequently drained, and crops were allowed to recover for final grain yield. Under waterlogging conditions, genotypes with RCA-QTL had a significantly higher proportion of aerenchyma, more white adventitious roots, higher leaf normalised difference vegetation index (NDVI), more shoot biomass, and a higher tiller number than genotypes without RCA-QTL. The average increase in yield due to the introgression of the RCA-QTL was 1.8 t/ha (about 20%) under waterlogging conditions. Importantly, the addition of the RCA-QTL to three commercial varieties showed no significant negative effects or penalty on plant growth, yield, and grain quality attributes under control conditions. It is concluded that the introgression of an RCA-QTL into other varieties is a promising breeding target for mitigating losses caused by waterlogging. The genotype YF225 showed a degree of waterlogging tolerance but had no RCA-QTL, indicating the existence of alternate waterlogging tolerance mechanisms. A genome-wide association (GWAS) approach using 18,132 single nucleotide polymorphisms (SNPs) in a panel of 697 barley genotypes, was conducted to identify marker trait associations (MTA) conferring the RCA and AR formation under waterlogging conditions. Experiments were conducted in 2020 and 2021 in tanks filled with soil and then validated in field experiments. GWAS analysis was conducted using general linear models (GLM), mixed linear models (MLM) and Fixed and random model Circulating Probability Unification models (FarmCPU model), with the FarmCPU showing to be the best suited model. Six and five significant (approximately -log10 (p) ‚Äöv¢‚Ä¢ 5.5) MTA were identified for AR and RCA formation under waterlogged conditions, respectively. The highest -log10 (p) MTA for aerenchyma and adventitious root development were approximately 8 and 9 on chromosome 4H and 2H, respectively. The combination of different MTA showed to be more effective in forming RCA and producing more AR under waterlogging stress. Genes from an ethylene responsive factor (ERF) family and a potassium transporter family for RCA formation; and genes from the major facilitator superfamily (MFS) transporter and a leucine-rich repeat (LRR) family for AR formation were the potential candidate genes involved under waterlogging conditions. Several genotypes which performed consistently well under different conditions can be used in breeding programs to develop waterlogging tolerant varieties. All above experiments have confirmed that RCA enhanced waterlogging tolerance and the introgression of RCA-QTL into commercial variety could significantly increase grain yield under waterlogging conditions. The addition of the RCA-QTL to the commercial varieties showed no significant grain yield penalty. For effective use of this QTL by barley breeders, it is crucial to fine map the QTL and identify better molecular markers or even gene markers. Over 2000 lines from different generations of backcross between Planet (no RCA-QTL) and Tam407227 (with RCA-QTL wild type barley genotype) were used to fine mapping the RCA-QTL on 4H chromosome. The experiments were conducted during 2018-2021. The 2017 reference genome of cv. Morex was used as a road map to rapidly narrow physical interval around the RCA-QTL. Diversity Arrays Technology (DArT) and simple sequence repeat (SSR) marker were used for gene fine mapping analysis. The markers developed in this study can effectively distinguish between tolerant and sensitive progenies. The fine mapping analysis finally narrowed down to a physical interval of less than 20 Mb. Further research on predicted candidate gene isolation and functional characterization is underway. The results will pave the way for breeders to develop more waterlogging tolerant barley cultivars to mitigate waterlogging stress. Waterlogging decreases the availability of nitrogen (N) in the soil. The application of nitrogen fertilizers is one of the important agronomic management strategies that can alleviates waterlogging damage. In this study, two pairs of near-isogenic lines (NIL) Planet+/Planet and Macquarie+/Macquarie were used to investigate the effect of nitrogen fertilizer application to mitigate waterlogging-induced penalties-imposed on plants. Planet+ and Macquarie+ genotype have Planet and Macquarie background, respectively, with root cortical aerenchyma-quantitative trait locus (RCA-QTL) being introgressed. Experiment was conducted in 2021 in large tanks filled with soil. Waterlogging treatment started at two and half leaf stage and last for two months. There were five different combinations of nitrogen (urea) application given at sowing, during waterlogging (foliar spray), and after waterlogging (at recovery) stages. Normalized Difference Vegetation Index (NDVI) was used to evaluate the growth performance under waterlogged conditions. Tolerant genotypes (Planet+ and Macquarie+) demonstrated higher biomass production than sensitive ones (Planet and Macquarie) under waterlogging conditions. Foliar urea spray during waterlogging period significantly improved plants‚ÄövÑv¥ waterlogging tolerance with the average increase in biomass production being 23%. The double dose (30 N kg/ha) foliar application was more effective than a single dose (15 N kg/ha). The double dose foliar application enhanced final biomass production by about 12% for tolerant genotypes and 14% for sensitive genotypes. Based on cost benefit analysis, double dose foliar application is more profitable than single dose for farmers. The application of nitrogen at the recovery stage was less effective for the final biomass production but slightly delayed the maturity time. It can be concluded that application of the nitrogen fertilizer is an effective approach in alleviating yield losses in barley crop caused by waterlogging.