2 2 5The Brassica genus contains a diverse range of oilseed and vegetable crops important for human nutrition 1 . Crops of particular agricultural importance include three diploid species, Brassica rapa (AA), Brassica nigra (BB) and Brassica oleracea (CC), and three allopolyploid species, B. napus (AACC), B. juncea (AABB) and Brassica carinata (BBCC). The evolutionary relationships among these Brassica species are described by what is called the 'triangle of U' model 2 , which proposes how the genomes of the three ancestral Brassica species, B. rapa, B. nigra and Brassica oleracae, combined to give rise to the allopolyploid species of this genus. B. juncea formed by hybridization between the diploid ancestors of B. rapa and B. nigra, followed by spontaneous chromosome doubling. Subsequent diversifying selection then gave rise to the vegetable-and oil-use subvarieties of B. juncea. These subvarieties include vegetable and oilseed mustard in China, oilseed crops in India, canola crops in Canada and Australia, and condiment crops in Europe and other regions 3 . Cultivation of B. juncea began in China about 6,000 to 7,000 years ago 4 , and flourished in India from 2,300 BC onward 5 .The genomes of B. rapa, B. oleracea and their allopolyploid offspring B. napus have been published recently [6][7][8] , and are often used to explain genome evolution in angiosperms [6][7][8] . The genomes of all Brassica species underwent a lineage-specific whole-genome triplication 6,7,9 , followed by diploidization that involved substantial genome reshuffling and gene losses 6,10-13 . In general, plant genomes are typically repetitive, polyploid and heterozygous, which complicates genome assembly 14 . The short read lengths of next-generation sequencing hinder assembly through complex regions, and fragmented draft and reference genomes usually lack skewed (G+C)-content sequences and repetitive intergenic sequences. Furthermore, in allopolyploid species, homoeolog expression dominance or bias, and specifically differential homoelog gene expression, has often been detected, for instance in Gossypium [15][16][17] Triticum 18,19 and Arabidopsis 20,21 , but the role of this phenomenon in selection for phenotypic traits remains mechanistically mysterious 22 .We reported here the draft genomes of an allopolyploid, B. juncea var. tumida, constructed by de novo assembly using shotgun reads, single-molecule long reads (PacBio sequencing), genomic (optical) mapping (BioNano sequencing) and genetic mapping, serving to resolve complicated allopolyploid genomes. The multiuse allopolyploid B. juncea genome offers a distinctive model to study the underlying genomic basis for selection in breeding improvement. These findings place this work into the broader context of plant breeding, highlighting The Brassica genus encompasses three diploid and three allopolyploid genomes, but a clear understanding of the evolution of agriculturally important traits via polyploidy is lacking. We assembled an allopolyploid Brassica juncea genome by shotgun and single-m...

For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle.

BackgroundThe mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution.ResultsWe obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes.ConclusionsExtensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.