CRISPR/Cas9 gene editing and natural variation analysis demonstrate the potential for HvARE1 in improvement of nitrogen use efficiency in barley.

Nitrogen is a major determinant of grain yield and quality. As excessive use of nitrogen fertilizer leads to environmental pollution and high production costs, improving nitrogen use efficiency (NUE) is fundamental for a sustainable agriculture. Here, we dissected the role of the barley abnormal cytokinin response1 repressor 1 (HvARE1) gene, a candidate for involvement in NUE previously identified in a genome-wide association study, through natural variation analysis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. HvARE1 was predominantly expressed in leaves and shoots, with very low expression in roots under low nitrogen conditions. Agrobacterium-mediated genetic transformation of immature embryos (cv. Golden Promise) with single guide RNAs targeting HvARE1 generated 22 T0 plants, from which four T1 lines harbored missense and/or frameshift mutations based on genotyping. Mutant are1 lines exhibited an increase in plant height, tiller number, grain protein content, and yield. Moreover, we observed a 1.5- to 2.8-fold increase in total chlorophyll content in the flag leaf at the grain filling stage. Delayed senescence by 10-14 d was also observed in mutant lines. Barley are1 mutants had high nitrogen content in shoots under low nitrogen conditions. These findings demonstrate the potential of ARE1 in NUE improvement in barley.

Genomic structural equation modelling provides a whole-system approach for the future crop breeding.

KEY MESSAGE: Using genomic structural equation modelling, this research demonstrates an efficient way to identify genetically correlating traits and provides an effective proxy for multi-trait selection to consider the joint genetic architecture of multiple interacting traits in crop breeding. Breeding crop cultivars with optimal value across multiple traits has been a challenge, as traits may negatively correlate due to pleiotropy or genetic linkage. For example, grain yield and grain protein content correlate negatively with each other in cereal crops. Future crop breeding needs to be based on practical yet accurate evaluation and effective selection of beneficial trait to retain genes with the best agronomic score for multiple traits. Here, we test the framework of whole-system-based approach using structural equation modelling (SEM) to investigate how one trait affects others to guide the optimal selection of a combination of agronomically important traits. Using ten traits and genome-wide SNP profiles from a worldwide barley panel and SEM analysis, we revealed a network of interacting traits, in which tiller number contributes positively to both grain yield and protein content; we further identified common genetic factors affecting multiple traits in the network of interaction. Our method demonstrates an efficient way to identify genetically correlating traits and underlying pleiotropic genetic factors and provides an effective proxy for multi-trait selection within a whole-system framework that considers the joint genetic architecture of multiple interacting traits in crop breeding. Our findings suggest the promise of a whole-system approach to overcome challenges such as the negative correlation of grain yield and protein content to facilitating quantitative and objective breeding decisions in future crop breeding.

Using chlorate as an analogue to nitrate to identify candidate genes for nitrogen use efficiency in barley.

Nitrogen (N) is one of the most important macronutrients for crop growth and development. Large amounts of N fertilizers are applied exogenously to improve grain yield and quality, which has led to environmental pollution and high cost of production. Therefore, improvement of N use efficiency (NUE) is a very important aspect for sustainable agriculture. Here, a pilot experiment was firstly conducted with a set of barley genotypes with confirmed NUE to validate the fast NUE screening, using chlorate as an analogue to nitrate. High NUE genotypes were susceptible to chlorate-induced toxicity whereas the low NUE genotypes were tolerant. A total of 180 barley RILs derived from four parents (Compass, GrangeR, Lockyer and La Trobe) were further screened for NUE. Leaf chlorosis induced by chlorate toxicity was the key parameter observed which was later related to low-N tolerance of the RILs. There was a distinct variation in chlorate susceptibility of the RILs with leaf chlorosis in the oldest leaf ranging from 10 to 80%. A genome-wide association study (GWAS) identified 9 significant marker-trait associations (MTAs) conferring high chlorate sensitivity on chromosomes 2H (2), 3H (1), 4H (4), 5H (1) and Un (1). Genes flanking with these markers were retrieved as potential targets for genetic improvement of NUE. Genes encoding Ferredoxin 3, leucine-rich receptor-like protein kinase family protein and receptor kinase are responsive to N stress. MTA4H5468 which exhibits concordance with high NUE phenotype can further be explored under different genetic backgrounds and successfully applied in marker-assisted selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01239-8.

Genome-Wide Association Study and Identification of Candidate Genes for Nitrogen Use Efficiency in Barley (Hordeum vulgare L.).

Nitrogen (N) fertilizer is largely responsible for barley grain yield potential and quality, yet excessive application leads to environmental pollution and high production costs. Therefore, efficient use of N is fundamental for sustainable agriculture. In the present study, we investigated the performance of 282 barley accessions through hydroponic screening using optimal and low NH4NO3 treatments. Low-N treatment led to an average shoot dry weight reduction of 50%, but there were significant genotypic differences among the accessions. Approximately 20% of the genotypes showed high (>75%) relative shoot dry weight under low-N treatment and were classified as low-N tolerant, whereas 20% were low-N sensitive (≤55%). Low-N tolerant accessions exhibited well-developed root systems with an average increase of 60% in relative root dry weight to facilitate more N absorption. A genome-wide association study (GWAS) identified 66 significant marker trait associations (MTAs) conferring high nitrogen use efficiency, four of which were stable across experiments. These four MTAs were located on chromosomes 1H(1), 3H(1), and 7H(2) and were associated with relative shoot length, relative shoot and root dry weight. Genes corresponding to the significant MTAs were retrieved as candidate genes, including members of the asparagine synthetase gene family, several transcription factor families, protein kinases, and nitrate transporters. Most importantly, the high-affinity nitrate transporter 2.7 (HvNRT2.7) was identified as a promising candidate on 7H for root and shoot dry weight. The identified candidate genes provide new insights into our understanding of the molecular mechanisms driving nitrogen use efficiency in barley and represent potential targets for genetic improvement.