| Summary: | Creating varieties with high nitrogen use efficiency (NUE) is crucial for sustainable agriculture development. In this study, a superior barley doubled haploid line (named DH45) with improved NUE was produced via F<sub>1</sub> microspore embryogenesis with three rounds of screening in different nitrogen levels by hydroponic and field experiments. The molecular mechanisms responsible for the NUE of DH45 surpassing that of its parents were investigated by RNA-seq analysis. A total of 1027 differentially expressed genes (DEGs) were identified that were up- or down-regulated in DH45 under low nitrogen conditions but showed no significant differences in the parents. GO analysis indicated that genes involved in nitrogen compound metabolic processes were significantly enriched in DH45 compared with the parents. KEGG analysis showed the MAPK signaling pathway plant to be highly enriched in DH45 relative to its parents, as well as genes involved in alanine, aspartate and glutamate metabolism, and arginine biosynthesis. In conclusion, our study revealed the potential to fix trait superiority in a line by combining crossing with F<sub>1</sub> microspore culture technologies in future crop breeding and also identified several candidate genes that are expressed in shoots and may enable barley to cope with low-nitrogen stress.
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