Elucidating crops’ physiological and molecular mechanisms to adapt to low nitrogen environment and promoting nitrogen transfer from senescent leaves to new leaves is crucial in improving Brassica’s nitrogen use efficiency (NUE). Glutamine synthetase gene (GS) plays a vital role in helping plants reassimilate ammonium released from protein degradation in leaves, and it was the focus of our research on this topic. In this study, we identified high (H141) and low (L65) NUE genotypes of Brassica juncea with different responses to low-nitrogen stress. We found that H141 has a lower nitrate content but higher ammonium and free amino acid contents as well as higher nitrate reductase and GS activities in the shoots. These physiological indicators are responsible for the high NUE of H141. Whole-genome resequencing data revealed that 5,880 genes associated with NUE are polymorphic between H141 and L65. These genes participate in various amino acid, carbohydrate, and energy metabolic pathways. Haplotype analysis revealed two haplotypes for BjuB05.GS1.4, Hap1 and Hap2, which have multiple single nucleotide polymorphisms or insertions/deletions in the regulatory regions of the 5´ and 3´ untranslated regions and introns. Furthermore, the shoot NUE of Hap1 is significantly lower than that of Hap2. These two haplotypes of BjuB05.GS1.4 lead to differences in the shoot NUEs of different genetic populations of mustard and are associated with the local soil nitrogen content, suggesting that they might help mustard to adapt to different geographic localities. In conclusion, the results of our study shed light on the physiological and molecular mechanisms underlying different mustard NUE genotypes and demonstrate the enormous potential of NUE breeding in B. juncea.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes. However, few B. juncea (mustard) mitochondrial genomes have been published. We assembled the mitochondrial genomes of three B. juncea subspecies and compared them with previously published genomes. The genomes were phylogenetically classified into A, B, C, and Bna clades. Two variant sites, a transversion (C → A) at nt 79, 573 and a 31-bp copy-number variation between nts 65, 564 and 65, 596, were identified. Based on these variant sites, mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B. juncea accessions. Three mitochondrial genome types (mitotypes MT1–MT3) were identified. In terms of geographical distribution, MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N. Root mustards carried only MT1, leaf and stem mustards carried mainly MT3, and seed mustards carried all three mitotypes, implying that the mitotypes underwent selection during B. juncea domestication. A new form of oil mustard evolved by hybridization between two gene pools in southwest China.