Crop breeding for high nitrogen use efficiency (NUE) or tolerance to low nitrogen fertilization is thought to be an ideal solution to reduce the cost, carbon footprint, and other environmental problems caused by the excess use of nitrogen fertilizers. As a model plant for cereal crops, barley has many advantages, including good adaptability, a short growth period, and high natural stress resistance or tolerance. Therefore, research on improving NUE in barley is not only beneficial for nitrogen-efficient barley breeding but will also inform NUE improvement in other cereal crops. In this review, recent progress in understanding barley’s response to nitrogen nutrition, evaluation of NUE or low-nitrogen tolerance, quantitative trait loci (QTL) mapping and gene cloning associated with improving NUE, and breeding of nitrogen-efficient barley is summarized. Furthermore, several biotechnological tools that could be used for revealing the molecular mechanisms of NUE or breeding for improving NUE in barley are introduced, including GWAS, omics, and gene editing. The latest research ideas in unraveling the molecular mechanisms of improving NUE in other crops are also discussed. Thus, this review provides a better understanding of improving the NUE of barley and some directions for future research in this area.

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Although nitrogen (N) deficiency greatly affects N absorption and metabolism in barley, the global transcriptomic changes in morphological and physiological adaptation to altered N availability remains largely unclear. We conducted a comparative transcriptome analysis of roots in A9-29 (low N tolerant line of barley) and Hua 30 (low N-sensitive variety of barley) under low N conditions to elucidate the responses and the underlying molecular mechanism. The results demonstrated that the root architecture was strongly influenced and that the root morphological indexes (total root length, total root area surface, and root volume) were remarkably promoted in A9-29 compared to Hua30 under low N stress. The transcriptome analysis of roots identified 1779 upregulated differentially expressed genes (DEGs) and 1487 downregulated DEGs specifically expressed in A9-29 under low N stress. Specific DEGs in A9-29 were largely enriched in energy metabolism, lipid metabolism, and the metabolism of other amino acids. In addition, transcription factor genes ERFs and IAA-related genes were specifically expressed in A9-29. To conclude, this study could provide a foundation for improving low N tolerance in barley.