Genetic improvement of nitrogen use efficiency in barley

Grain yield is the key target in barley production. A powerful tool to increase yield has been the use of fertilizers of which nitrogen (N) is the main nutrient for the productivity and grain quality. Yet excessive application leads to environmental pollution and high production costs. Therefore, improvement of nitrogen use efficiency (NUE) is fundamental for sustainable agriculture. Only limited research has been conducted on barley NUE. A few QTLs controlling NUE have been identified, albeit they are not stable across experiments due to low marker density, limited genetic diversity and small population size. Thus, the aims of this project are to investigate the barley tolerance to low-N, propose a candidate gene pool related to NUE and to develop advanced barley materials with improved NUE through CRISPR/Cas9 genome editing. Hydroponic screening of barley was conducted under low-N for 7 NUE related traits to identify low to moderate NUE commercial cultivars such as Compass with potential for further NUE improvement. 66 significant marker trait associations (MTAs) associated with NUE were identified through a genome wide association study (GWAS) using 282 barley accessions which is a subset of a larger worldwide barley panel. Key genes retrieved from the GWAS mainly belonged to nitrate transporters, asparagine synthetase gene family, several transcription factor families and protein kinases. High-affinity nitrate transporter 2.7 (HvNRT2.7) and a chloroplast envelope membrane protein (HvARE1) on chromosome 7H, protein NRT1/PTR FAMILY 8.3 (HvNRT1) gene on 4H and asparagine synthetase 2 (glutamine hydrolyzing) (HvASN2) on 1H were identified as promising loci for NUE improvement under low-N. In addition, a part of this research proposes the use of chlorate ions as an analogue to nitrate for rapid and large scale NUE screening of barley using a multiparent advanced generation inter-cross (MAGIC) population developed from four commercial barley cultivars Compass, GrangeR, LaTrobe and Lockyer. An independent GWAS identified 9 MTAs and a candidate gene pool with D2H71173319_GA, D2H655480684_ CT and D3H37942201_CT repeatedly identified in both experiments under low-N. MTA4H546806926_TC which exhibits concordance with high NUE phenotype can further be explored under different genetic backgrounds and successfully applied in marker-assisted selection (MAS). CRISPR/Cas9 gene editing of the HvARE1 gene successfully generated 22 potential mutant lines from barley cv. Golden Promise. Transformation efficiency was 87% based on the Cas9 and CaMV35S promoter specific PCR detection. 18% of T1 and 24% of T2 individuals were mutants based on PCR-RE assay. Missense and frameshift mutations were identified by Sanger sequencing from both T1 and T2 generations. Interestingly, the are1 mutants had a 1.5 to 2.8-fold increase of the chlorophyl content at the grain filling stage and a delayed leaf senescence than that of the wild type. Overall, the are1 mutants had better performance in terms of agronomic and physiological traits related to NUE under both low and optimal N. The results of this research can be successfully adopted to improve commercial barley cultivars for high NUE and yield through CRISPR/Cas9 gene editing