Mitochondrial genome sequencing and analysis of the invasive Microstegium vimineum: a resource for systematics, invasion history, and management.

Premise of the Research: Plants remain underrepresented among species with sequenced mitochondrial genomes (mitogenomes), due to the difficulty in assembly with short-read technology. Invasive species lag behind crops and other economically important species in this respect, representing a lack of tools for management and land conservation efforts. Methodology: The mitogenome of Microstegium vimineum, one of the most damaging invasive plant species in North America, was sequenced and analyzed using long-read data, providing a resource for biologists and managers. We conducted analyses of genome content, phylogenomic analyses among grasses and relatives based on mitochondrial coding regions, and an analysis of mitochondrial single nucleotide polymorphism in this invasive grass species. Pivotal Results: The assembly is 478,010 bp in length and characterized by two large, inverted repeats, and a large, direct repeat. However, the genome could not be circularized, arguing against a "master circle" structure. Long-read assemblies with data subsets revealed several alternative genomic conformations, predominantly associated with large repeats. Plastid-like sequences comprise 2.4% of the genome, with further evidence of Class I and Class II transposable element-like sequences. Phylogenetic analysis placed M. vimineum with other Microstegium species, excluding M. nudum, but with weak support. Analysis of polymorphic sites across 112 accessions of M. vimineum from the native and invasive ranges revealed a complex invasion history. Conclusions: We present an in-depth analysis of mitogenome structure, content, phylogenetic relationships, and range-wide genomic variation in M. vimineum's invasive US range. The mitogenome of M. vimineum is typical of other andropogonoid grasses, yet mitochondrial sequence variation across the invasive and native ranges is extensive. Our findings suggest multiple introductions to the US over the last century, with subsequent spread, secondary contact, long-distance dispersal, and possibly post-invasion selection on awn phenotypes. Efforts to produce genomic resources for invasive species, including sequenced mitochondrial genomes, will continue to provide tools for their effective management, and to help predict and prevent future invasions.

Chromosome Level Genome Assembly and Annotation of Highly Invasive Japanese Stiltgrass (Microstegium vimineum).

The invasive Japanese stiltgrass (Microstegium vimineum) affects a wide range of ecosystems and threatens biodiversity across the eastern USA. However, the mechanisms underlying rapid adaptation, plasticity, and epigenetics in the invasive range are largely unknown. We present a chromosome-level assembly for M. vimineum to investigate genome dynamics, evolution, adaptation, and the genomics of phenotypic plasticity. We generated a 1.12-Gb genome with scaffold N50 length of 53.44 Mb respectively, taking a de novo assembly approach that combined PacBio and Dovetail Genomics Omni-C sequencing. The assembly contains 23 pseudochromosomes, representing 99.96% of the genome. BUSCO assessment indicated that 80.3% of Poales gene groups are present in the assembly. The genome is predicted to contain 39,604 protein-coding genes, of which 26,288 are functionally annotated. Furthermore, 66.68% of the genome is repetitive, of which unclassified (35.63%) and long-terminal repeat (LTR) retrotransposons (26.90%) are predominant. Similar to other grasses, Gypsy (41.07%) and Copia (32%) are the most abundant LTR-retrotransposon families. The majority of LTR-retrotransposons are derived from a significant expansion in the past 1-2 Myr, suggesting the presence of relatively young LTR-retrotransposon lineages. We find corroborating evidence from Ks plots for a stiltgrass-specific duplication event, distinct from the more ancient grass-specific duplication event. The assembly and annotation of M. vimineum will serve as an essential genomic resource facilitating studies of the invasion process, the history and consequences of polyploidy in grasses, and provides a crucial tool for natural resource managers.

Evolutionary Dynamics of Transposable Elements Following a Shared Polyploidization Event in the Tribe Andropogoneae.

Both polyploidization and transposable element (TE) activity are known to be major drivers of plant genome evolution. Here, we utilize the Zea-Tripsacum clade to investigate TE activity and accumulation after a shared polyploidization event. Comparisons of TE evolutionary dynamics in various Zea and Tripsacum species, in addition to two closely related diploid species, Urelytrum digitatum and Sorghum bicolor, revealed variation in repeat content among all taxa included in the study. The repeat composition of Urelytrum is more similar to that of Zea and Tripsacum compared to Sorghum, despite the similarity in genome size with the latter. Although LTR-retrotransposons were abundant in all species, we observed an expansion of the copia superfamily, specifically in Z. mays and T. dactyloides, species that have adapted to more temperate environments. Additional analyses of the genomic distribution of these retroelements provided evidence of biased insertions near genes involved in various biological processes including plant development, defense, and macromolecule biosynthesis. Specifically, copia insertions in Zea and T. dactyloides were significantly enriched near genes involved in abiotic stress response, suggesting independent evolution post Zea-Tripsacum divergence. The lack of copia insertions near the orthologous genes in S. bicolor suggests that duplicate gene copies generated during polyploidization may offer novel neutral sites for TEs to insert, thereby providing an avenue for subfunctionalization via TE insertional mutagenesis.

Methods for accurate quantification of LTR-retrotransposon copy number using short-read sequence data: a case study in Sorghum.

Transposable elements (TEs) are ubiquitous in eukaryotic genomes and their mobility impacts genome structure and function in myriad ways. Because of their abundance, activity, and repetitive nature, the characterization and analysis of TEs remain challenging, particularly from short-read sequencing projects. To overcome this difficulty, we have developed a method that estimates TE copy number from short-read sequences. To test the accuracy of our method, we first performed an in silico analysis of the reference Sorghum bicolor genome, using both reference-based and de novo approaches. The resulting TE copy number estimates were strikingly similar to the annotated numbers. We then tested our method on real short-read data by estimating TE copy numbers in several accessions of S. bicolor and its close relative S. propinquum. Both methods effectively identify and rank similar TE families from highest to lowest abundance. We found that de novo characterization was effective at capturing qualitative variation, but underestimated the abundance of some TE families, specifically families of more ancient origin. Also, interspecific reference-based mapping of S. propinquum reads to the S. bicolor database failed to fully describe TE content in S. propinquum, indicative of recent TE activity leading to changes in the respective repetitive landscapes over very short evolutionary timescales. We conclude that reference-based analyses are best suited for within-species comparisons, while de novo approaches are more reliable for evolutionarily distant comparisons.

Phylogenetic reconstruction using four low-copy nuclear loci strongly supports a polyphyletic origin of the genus Sorghum.

BACKGROUND AND AIMS: Sorghum is an essential grain crop whose evolutionary placement within the Andropogoneae has been the subject of scrutiny for decades. Early studies using cytogenetic and morphological data point to a poly- or paraphyletic origin of the genus; however, acceptance of poly- or paraphyly has been met with resistance. This study aimed to address the species relationships within Sorghum, in addition to the placement of Sorghum within the tribe, using a phylogenetic approach and employing broad taxon sampling. METHODS: From 16 diverse Sorghum species, eight low-copy nuclear loci were sequenced that are known to play a role in morphological diversity and have been previously used to study evolutionary relationships in grasses. Further, the data for four of these loci were combined with those from 57 members of the Andropogoneae in order to determine the placement of Sorghum within the tribe. Both maximum likelihood and Bayesian analyses were performed on multilocus concatenated data matrices. KEY RESULTS: The Sorghum-specific topology provides strong support for two major lineages, in alignment with earlier studies employing chloroplast and internal transcribed spacer (ITS) markers. Clade I is composed of the Eu-, Chaeto- and Heterosorghum, while clade II contains the Stipo- and Parasorghum. When combined with data from the Andropogoneae, Clade II resolves as sister to a clade containing Miscanthus and Saccharum with high posterior probability and bootstrap support, and to the exclusion of Clade I. CONCLUSIONS: The results provide compelling evidence for a two-lineage polyphyletic ancestry of Sorghum within the larger Andropogoneae, i.e. the derivation of the two major Sorghum clades from a unique common ancestor. Rejection of monophyly in previous molecular studies is probably due to limited taxon sampling outside of the genus. The clade consisting of Para- and Stiposorghum resolves as sister to Miscanthus and Saccharum with strong node support.