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Medicago truncatulais a model legume that has been extensively investigated in diverse subdisciplines of plant science.Medicago littoraliscan interbreed withM. truncatulaandM. italica; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated thatM. truncatulaaccessions are heterogeneous but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverseM. truncatulaaccessions within the genus, we assembled 54 plastid genomes (plastomes) using publicly available next-generation sequencing data and conducted phylogenetic analyses using maximum likelihood. Five accessions showed high levels of plastid DNA polymorphism. Three of these highly polymorphic accessions contained sequences from bothM. truncatulaandM. littoralis.Phylogenetic analyses of sequences placed some accessions closer to distantly related species suggesting misidentification of source material.Most accessions were placed within the TLI clade and maximally supported the interrelationships of three subclades. TwoMedicagoaccessions were placed within aM. italicasubclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within theM. littoralissubclade. Our results suggest that theM. truncatulaaccession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.

 

Comprising 501 genera and around 14,000 species, Papilionoideae is not only the largest subfamily of Fabaceae (Leguminosae; legumes), but also one of the most extraordinarily diverse clades among angiosperms. Papilionoids are a major source of food and forage, are ecologically successful in all major biomes, and display dramatic variation in both floral architecture and plastid genome (plastome) structure. Plastid DNA-based phylogenetic analyses have greatly improved our understanding of relationships among the major groups of Papilionoideae, yet the backbone of the subfamily phylogeny remains unresolved. In this study, we sequenced and assembled 39 new plastomes that are covering key genera representing the morphological diversity in the subfamily. From 244 total taxa, we produced eight datasets for maximum likelihood (ML) analyses based on entire plastomes and/or concatenated sequences of 77 protein-coding sequences (CDS) and two datasets for multispecies coalescent (MSC) analyses based on individual gene trees. We additionally produced a combined nucleotide dataset comprising CDS plus matK gene sequences only, in which most papilionoid genera were sampled. A ML tree based on the entire plastome maximally supported all of the deep and most recent divergences of papilionoids (223 out of 236 nodes). The Swartzieae, ADA (Angylocalyceae, Dipterygeae, and Amburaneae), Cladrastis, Andira, and Exostyleae clades formed a grade to the remainder of the Papilionoideae, concordant with nine ML and two MSC trees. Phylogenetic relationships among the remaining five papilionoid lineages (Vataireoid, Dermatophyllum , Genistoid s.l., Dalbergioid s.l., and Baphieae + Non-Protein Amino Acid Accumulating or NPAAA clade) remained uncertain, because of insufficient support and/or conflicting relationships among trees. Our study fully resolved most of the deep nodes of Papilionoideae, however, some relationships require further exploration. More genome-scale data and rigorous analyses are needed to disentangle phylogenetic relationships among the five remaining lineages. 

The genus Trifolium is the largest of the tribe Trifolieae in the subfamily Papilionoideae (Fabaceae). The paucity of mitochondrial genome (mitogenome) sequences has hindered comparative analyses among the three genomic compartments of the plant cell (nucleus, mitochondrion and plastid). We assembled four mitogenomes from the two subgenera (Chronosemium and Trifolium) of the genus. The four Trifolium mitogenomes were compact (294,911–348,724 bp in length) and contained limited repetitive (6.6–8.6%) DNA. Comparison of organelle repeat content highlighted the distinct evolutionary trajectory of plastid genomes in a subset of Trifolium species. Intracellular gene transfer (IGT) was analyzed among the three genomic compartments revealing functional transfer of mitochondrial rps1 to nuclear genome along with other IGT events. Phylogenetic analysis based on mitochondrial and nuclear rps1 sequences revealed that the functional transfer in Trifolieae was independent from the event that occurred in robinioid clade that includes genus Lotus. A novel, independent fission event of ccmFn in Trifolium was identified, caused by a 59 bp deletion. Fissions of this gene reported previously in land plants were reassessed and compared with Trifolium. 

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single‐copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short‐read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single‐molecule, long‐read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long‐read data. We generated a pipeline,plastidGenomeAssemblyUsingLong‐read data (ptGAUL), to address the problem of plastome assembly using long‐read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long‐read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four newJuncus(Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements inJuncusplastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub:https://github.com/Bean061/ptgaul.

 

Plant nuclear genomes harbor sequence elements derived from the organelles (mitochondrion and plastid) through intracellular gene transfer (IGT). Nuclear genomes also show a dramatic range of repeat content, suggesting that any sequence can be readily amplified. These two aspects of plant nuclear genomes are well recognized but have rarely been linked. Through investigation of 31Medicagotaxa we detected exceptionally high post‐IGT amplification of mitochondrial (mt) DNA sequences containingrps10in the nuclear genome ofMedicago polymorphaand closely related species. The amplified sequences were characterized as tandem arrays of five distinct repeat motifs (2157, 1064, 987, 971, and 587 bp) that have diverged from the mt genome (mitogenome) in theM. polymorphanuclear genome. The mtrps10‐like arrays were identified in seven loci (six intergenic and one telomeric) of the nuclear chromosome assemblies and were the most abundant tandem repeat family, representing 1.6–3.0% of total genomic DNA, a value approximately three‐fold greater than the entire mitogenome inM. polymorpha. Compared to a typical mt gene, the mtrps10‐like sequence coverage level was 691.5–7198‐fold higher inM. polymorphaand closely related species. In addition to the post‐IGT amplification, our analysis identified the canonical telomeric repeat and the species‐specific satellite arrays that are likely attributable to an ancestral chromosomal fusion inM. polymorpha. A possible relationship between chromosomal instability and the mtrps10‐like tandem repeat family in theM. polymorphaclade is discussed.

 

The plastid genome (plastome), while surprisingly constant in gene order and content across most photosynthetic angiosperms, exhibits variability in several unrelated lineages. During the diversification history of the legume family Fabaceae, plastomes have undergone many rearrangements, including inversions, expansion, contraction and loss of the typical inverted repeat (IR), gene loss and repeat accumulation in both shared and independent events. While legume plastomes have been the subject of study for some time, most work has focused on agricultural species in the IR‐lacking clade (IRLC) and the plant modelMedicago truncatula. The subfamily Papilionoideae, which contains virtually all of the agricultural legume species, also comprises most of the plastome variation detected thus far in the family. In this study three non‐papilioniods were included among 34 newly sequenced legume plastomes, along with 33 publicly available sequences, to assess plastome structural evolution in the subfamily. In an effort to examine plastome variation across the subfamily, approximately 20% of the sampling represents the IRLC with the remainder selected to represent the early‐branching papilionoid clades. A number of IR‐related and repeat‐mediated changes were identified and examined in a phylogenetic context. Recombination between direct repeats associated withycf2resulted in intraindividual plastome heteroplasmy. Although loss of the IR has not been reported in legumes outside of the IRLC, one genistoid taxon was found to completely lack the typical plastome IR. The role of the IR and non‐IR repeats in the progression of plastome change is discussed.