IRLC Research Articles

Atypical rhizobia trigger nodulation and pathogenesis on the same legume hosts

The emergence of commensalism and mutualism often derives from ancestral parasitism. However, in the case of rhizobium-legume interactions, bacterial strains displaying both pathogenic and nodulation features on a single host have not been described yet. Here, we isolated such a bacterium from Medicago nodules. On the same plant genotypes, the T4 strain can induce ineffective nodules in a highly competitive way and behave as a harsh parasite triggering plant death. The T4 strain presents this dual ability on multiple legume species of the Inverted Repeat-Lacking Clade, the output of the interaction relying on the developmental stage of the plant. Genomic and phenotypic clustering analysis show that T4 belongs to the nonsymbiotic Ensifer adhaerens group and clusters together with T173, another strain harboring this dual ability. In this work, we identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host.

Novel Perspectives on Chloroplast tRNA Genomic and Structural Variations Imply the Evolution of Papilionoideae (Fabaceae)

Papilionoideae is the most species-rich subfamily of the third largest angiosperm family Fabaceae. One constituent large group, the inverted-repeat-lacking clade (IRLC), is well-known for the broad loss of one IR copy. Accumulating observations of massive plastomic disparities have made IRLC a well-suited model for exploring plastome evolution. However, there is still a large amount left to explore. The present study focused on the plastid tRNA (pttRNA) evolution within Papilionoideae, employing the currently densest sampling strategies for both the IRLC (156) and non-IRLC (109) lineages. Strikingly, our results revealed abundant inter-lineage variabilities in both tRNA sequences and structures, including a 3 nt difference in the average size of trnS-UGA, the consensus sequence disparities across 29 tRNAs, the distinct 3 nt indels in trnA-UGC, and an impressive 248 nt intron loss of IRLC trnI-GAU (potential markers). Additionally, there was unequal stability of the atypical secondary structures in trnS-GGA and trnS-UGA, as well as significantly diverse compositions of substitution events in all compared tRNAs (p < 0.05). Ultimately, these findings not only demonstrate the significant differences and unique markers of IRLC pttRNAs compared to other non-IRLC Papilionoideae, but also draw an important conclusion that the large losses of one IR potentially promote highly diverse evolutionary patterns of IRLC, which could partly compensate for the potential IR-lacking impacts.

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Chloroplast genome analyses of Caragana arborescens and Caragana opulens

BackgroundNumerous species within the genus Caragana have high ecological and medicinal value. However, species identification based on morphological characteristics is quite complicated in the genus. To address this issue, we analyzed complete plastid genome data for the genus.ResultsWe obtained chloroplast genomes of two species, Caragana arborescens and Caragana opulens, using Illumina sequencing technology, with lengths of 129,473 bp and 132,815 bp, respectively. The absence of inverted repeat sequences in the two species indicated that they could be assigned to the inverted repeat-lacking clade (IRLC). The genomes included 111 distinct genes (4 rRNA genes, 31 tRNA genes, and 76 protein-coding genes). In addition, 16 genes containing introns were identified in the two genomes, the majority of which contained a single intron. Repeat analyses revealed 129 and 229 repeats in C. arborescens and C. opulens, respectively. C. arborescens and C. opulens genomes contained 277 and 265 simple sequence repeats, respectively. The two Caragana species exhibited similar codon usage patterns. rpl20-clpP, rps19-rpl2, and rpl23-ycf2 showed the highest nucleotide diversity (pi). In an analysis of sequence divergence, certain intergenic regions (matK-rbcL, psbM-petN, atpA-psbI, petA-psbL, psbE-petL, and rps7-rps12) were highly variable. A phylogenetic analysis showed that C. arborescens and C. opulens were related and clustered together with four other Caragana species. The genera Astragalus and Caragana were relatively closely related.ConclusionsThe present study provides valuable information about the chloroplast genomes of C. arborescens and C. opulens and lays a foundation for future phylogenetic research and molecular marker development.

Multiple configurations of the plastid and mitochondrial genomes of Caragana spinosa.

In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes. Caragana spinosa belongs to the Papilionoidea subfamily and has significant pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled and analyzed the plastome and mitogenome of C. spinosa using the Illumina and Nanopore DNA sequencing data. The plastome of C. spinosa was 129,995bp belonging to the inverted repeat lacking clade (IRLC), which contained 77 protein-coding genes, 29 tRNA genes, and four rRNA genes. The mitogenome was 378,373bp long and encoded 54 unique genes, including 33 protein-coding, three ribosomal RNA (rRNA), and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, alternative conformations mediated by one and four repetitive sequences in the plastome and mitogenome were identified and validated, respectively. The inverted repeat (PDR12, the 12th dispersed repeat sequence in C. spinosa plastome) of plastome mediating recombinant was conserved in the genus Caragana. Furthermore, we identified 14 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. A phylogenetic analysis of protein-coding genes extracted from the plastid and mitochondrial genomes revealed congruent topologies. Analyses of sequence divergence found one intergenic region, trnN-GUU-ycf1, exhibiting a high degree of variation, which can be used to develop novel molecular markers to distinguish the nine Caragana species accurately. This plastome and mitogenome of C. spinosa could provide critical information for the molecular breeding of C. spinosa and be used as a reference genome for other species of Caragana. In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes.

Analysis of Complete Chloroplast Genome: Structure, Phylogenetic Relationships of Galega orientalis and Evolutionary Inference of Galegeae.

Galega orientalis, a leguminous herb in the Fabaceae family, is an ecologically and economically important species widely cultivated for its strong stress resistance and high protein content. However, genomic information of Galega orientalis has not been reported, which limiting its evolutionary analysis. The small genome size makes chloroplast relatively easy to obtain genomic sequence for phylogenetic studies and molecular marker development. Here, the chloroplast genome of Galega orientalis was sequenced and annotated. The results showed that the chloroplast genome of G. orientalis is 125,280 bp in length with GC content of 34.11%. A total of 107 genes were identified, including 74 protein-coding genes, 29 tRNAs and four rRNAs. One inverted repeat (IR) region was lost in the chloroplast genome of G. orientalis. In addition, five genes (rpl22, ycf2, rps16, trnE-UUC and pbf1) were lost compared with the chloroplast genome of its related species G. officinalis. A total of 84 long repeats and 68 simple sequence repeats were detected, which could be used as potential markers in the genetic studies of G. orientalis and related species. We found that the Ka/Ks values of three genes petL, rpl20, and ycf4 were higher than one in the pairwise comparation of G. officinalis and other three Galegeae species (Calophaca sinica, Caragana jubata, Caragana korshinskii), which indicated those three genes were under positive selection. A comparative genomic analysis of 15 Galegeae species showed that most conserved non-coding sequence regions and two genic regions (ycf1 and clpP) were highly divergent, which could be used as DNA barcodes for rapid and accurate species identification. Phylogenetic trees constructed based on the ycf1 and clpP genes confirmed the evolutionary relationships among Galegeae species. In addition, among the 15 Galegeae species analyzed, Galega orientalis had a unique 30-bp intron in the ycf1 gene and Tibetia liangshanensis lacked two introns in the clpP gene, which is contrary to existing conclusion that only Glycyrrhiza species in the IR lacking clade (IRLC) lack two introns. In conclusion, for the first time, the complete chloroplast genome of G. orientalis was determined and annotated, which could provide insights into the unsolved evolutionary relationships within the genus Galegeae.

Characterization of the Chloroplast Genome Structure of Gueldenstaedtia verna (Papilionoideae) and Comparative Analyses among IRLC Species

The genus Gueldenstaedtia belongs to Papilionaceae’s inverted repeat-lacking clade (IRLC) and includes four species distributed throughout Asia. We sequenced the chloroplast genome of G. verna and compared it with those of the IRLC clade. The genome was 122,569 bp long, containing 77 protein-coding genes, 30 tRNAs, and 4 rRNAs. Comparative analyses showed that G. verna lost one inverted repeat region, the rps16 gene, an intron of rpoC1, and two introns of clpP. Additionally, G. verna had four inversions (~50 kb inversion, trnK–psbK; ~28 kb inversion, accD–rpl23; ~10 kb inversion, rps15–trnL; ~6 kb inversion, trnL–trnI) and one reposition (ycf1). Its G + C content was higher than that of other IRLC species. The total length and number of repeats of G. verna were not significantly different from those of the other IRLC species. Phylogenetic analyses showed that G. verna was closely related to Tibetia. A comparison of substitution rates showed that ycf2 and rps7 were higher than one, suggesting that these were positive selection genes, while others were related to purified selection. This study reports the structure of the chloroplast genome of a different type, i.e., with four inversions and one reposition, and would be helpful for future research on the evolution of the genome structure of the IRLC.

Expression and Variation of the Genes Involved in Rhizobium Nodulation in Red Clover.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Transcriptomic Identification of a Unique Set of Nodule-Specific Cysteine-Rich Peptides Expressed in the Nitrogen-Fixing Root Nodule of Astragalus sinicus.

Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The chloroplasts genomic analyses of four specific Caragana species.

Many species of the genus Caragana have been used as wind prevention and sand fixation plants. They are also important traditional Chinese medicine, and ethnic medicine resource plant. Thus, chloroplast genomes (cp-genome) of some of these important species must be studied. In this study, we analyzed the chloroplast genomes of C. jubata, C. erinacea, C. opulens, and C. bicolor, including their structure, repeat sequences, mutation sites, and phylogeny. The size of the chloroplast genomes was between 127,862 and 132,780 bp, and such genomes contained 112 genes (30 tRNA, 4 rRNA, and 78 protein-coding genes), 43 of which were photosynthesis-related genes. The total guanine + cytosine (G+C) content of four Caragana species was between 34.49% and 35.15%. The four Caragana species all lacked inverted repeats and can be classified as inverted repeat-lacking clade (IRLC). Of the anticipated genes of the four chloroplast genomes, introns were discovered in 17 genes, most of which were inserted by one intron. A total of 50 interspersed repeated sequences (IRSs) were found among them, 58, 29, 61, and 74 simple sequences repeats were found in C. jubata, C. bicolor, C. opulens, and C. erinacea, respectively. Analyses of sequence divergence showed that some intergenic regions (between trnK-UUU and rbcl; trnF-GAA and ndhJ; trnL-CAA and trnT-UGU; rpoB and trnC-GCA; petA and psbL; psbE and pebL; and sequences of rpoC, ycf1, and ycf2) exhibited a high degree of variations. A phylogenetic tree of eight Caragana species and another 10 legume species was reconstructed using full sequences of the chloroplast genome. (1) Chloroplast genomes can be used for the identification and classification of Caragana species. (2) The four Caragana species have highly similar cpDNA G+C content. (3) IRS analysis of the chloroplast genomes showed that these four species, similar to the chloroplast genome of most legumes, lost IRLC regions. (4) Comparative cp-genomic analysis suggested that the cp genome structure of the Caragana genus was well conserved in highly variable regions, which can be used to exploit markers for the identification of Caragana species and further phylogenetic study. (5) Results of phylogenetic analyses were in accordance with the current taxonomic status of Caragana. The phylogenetic relationship of Caragana species was partially consistent with elevation and geographical distribution.

The complete chloroplast genome of ornamental and medicinal Callerya dielsiana (Fabaceae)

Callerya dielsiana is a Chinese endemic tropical/subtropical liana. We sequenced the complete chloroplast genome with the Illumina Hiseq X-Ten platform. The genome is obtained with 132,301 bp in length, lacking an inverted repeat (IR) region, contains 4 rRNAs, 30 tRNAs genes, and 76 protein-coding genes. The overall GC content is 33.9%. Based on the whole chloroplast genomes of 14 legume species, a phylogenetic tree is constructed and indicated that C. dielsiana belongs to the well-supported tribe Wisterieae. The tribe is sister to Glycyrrhiza and nested within the IRLC (Inverted Repeat-Lacking Clade) group of the subfamily Papilionoideae (Fabaceae).

Legume Plant Peptides as Sources of Novel Antimicrobial Molecules Against Human Pathogens.

Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30–50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 μM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens.

Characterization of the complete chloroplast genome of Wisteriopsis reticulata (Fabaceae): an IRLC legumes

The inverted repeat‐lacking clade (IRLC) species are characterized by the loss of an IR region in their plastomes, which has long been of great interest. Wisteriopsis reticulata is one of the members of the tribe Wisterieae, which belongs to Fabaceae and is well-known as IRLC. Here, we reported and characterized the complete chloroplast genome of W. reticulata using the genome skimming approach. The chloroplast genome is 132,477 bp in length and lacks one copy of IR region. The genome encoded 112 unique genes including 89 protein-coding genes, 29 transfer RNA genes and four ribosomal RNA genes. Phylogenetic results supported the monophyly of the tribe Wisterieae (IRLC) and confirmed that W. reticulata belongs to the genus Wisteriopsis.

The plastome of Arctic Oxytropis arctobia (Fabaceae) is significantly different from that of O. splendens and other related species.

Anatomical and physiological specializations for plant adaptation to harsh climates result from molecular mechanisms that can be encoded in the nucleus or organelle. In this study, the complete plastomes of an arctic species, Oxytropis arctobia Bunge (Fabaceae), and a closely related temperate species, O. splendens Douglas ex Hook., were assembled, annotated, and analyzed to identify differences that might help explain their adaptation to different environments. This is consistent with the previously sequenced O. bicolor DC. and O. glabra plastomes, O. arctobia and O. splendens plastomes both have the common features of the inverted repeat-lacking clade (IRLC), as well as atpF intron loss, which is unique to the genus. However, significant differences were observed between the plastomes of O. arctobia and O. splendens and other closely related species (Oxytropis spp. and Astragalus spp.), including a 3 kb inversion, two large insertions (>1 kb), significant modifications of the accD gene, and an overall larger size.

Variations in Structure among Androecia and Floral Nectaries in the Inverted Repeat-Lacking Clade (Leguminosae: Papilionoideae).

The vast majority of highly valuable species of the Leguminosae in temperate latitudes belong to the Inverted Repeat-Lacking Clade (IRLC). Despite having a generally conserved monosymmetric floral morphology, members of this group are remarkable with a pronounced diversity of floral sizes, modes of staminal fusion, and pollination strategies. This paper examined androecia and floral nectaries (FNs) in selected genera of the IRLC. External morphology was investigated using stereomicroscopy and scanning electron microscopy. In some cases, the pattern of staminal fusion was additionally examined in transverse sections using light microscopy. Androecia of all selected genera fell into one of four types, viz., monadelphous, pseudomonadelphous, diadelphous or diadelphous reduced (with inner stamens converted into sterile staminodes). However, there was significant variation in the stamens’ mode of contact, as well as the shape and size of the fenestrae providing access to FNs. Some types seemed to arise independently in different genera, thus providing a high level of homoplasy. FNs were more conserved and comprised areas of secretory stomata in the abaxial part of the receptacle and/or hypanthium. Nectariferous stomata could be found in very miniaturized flowers (Medicago lupulina) and could even accompany monadelphy (Galega). This indicates that preferential self-pollination may nevertheless require visitation by insects.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives

BackgroundAlthough plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC.ResultsWe detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences.ConclusionsOur results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Characterization and Comparative Analysis of Complete Chloroplast Genomes of Three Species From the Genus Astragalus (Leguminosae).

Astragalus is the largest genus in Leguminosae. Several molecular studies have investigated the potential adulterants of the species within this genus; nonetheless, the evolutionary relationships among these species remain unclear. Herein, we sequenced and annotated the complete chloroplast genomes of three Astragalus species—Astragalus adsurgens, Astragalus mongholicus var. dahuricus, and Astragalus melilotoides using next-generation sequencing technology and plastid genome annotator (PGA) tool. All species belonged to the inverted repeat lacking clade (IRLC) and had similar sequences concerning gene contents and characteristics. Abundant simple sequence repeat (SSR) loci were detected, with single-nucleotide repeats accounting for the highest proportion of SSRs, most of which were A/T homopolymers. Using Astragalus membranaceus var. membranaceus as reference, the divergence was evident in most non-coding regions of the complete chloroplast genomes of these species. Seven genes (atpB, psbD, rpoB, rpoC1, trnV, rrn16, and rrn23) showed high nucleotide variability (Pi), and could be used as DNA barcodes for Astragalus sp. cemA and rpl33 were found undergoing positive selection by the section patterns in the coded protein. Phylogenetic analysis showed that Astragalus is a monophyletic group closely related to the genus Oxytropis within the tribe Galegeae. The newly sequenced chloroplast genomes provide insight into the unresolved evolutionary relationships within Astragalus spp. and are expected to contribute to species identification.

Phylogenomic framework of the IRLC legumes (Leguminosae subfamily Papilionoideae) and intercontinental biogeography of tribe Wisterieae

The inverted repeat-lacking clade (IRLC) is one of the most derived clades within the subfamily Papilionoideae of the legume family, and includes various economically important plants, e.g., chickpeas, peas, liquorice, and the largest genus of angiosperms, Astragalus. Tribe Wisterieae is one of the earliest diverged groups of the IRLC, and its generic delimitation and spatiotemporal diversification needs further clarifications. Based on genome skimming data, we herein reconstruct the phylogenomic framework of the IRLC, and infer the inter-generic relationships and historical biogeography of Wisterieae. We redefine tribe Caraganeae to contain Caragana only, and tribe Astragaleae is reduced to the Erophaca-Astragalean clade. The chloroplast capture scenario was hypothesized as the most plausible explanation of the topological incongruences between the chloroplast CDSs and nuclear ribosomal DNA trees in both the Glycyrrhizinae-Adinobotrys-Wisterieae clade and the Chesneyeae-Caraganeae-Hedysareae clade. A new name, Caragana lidou L. Duan & Z.Y. Chang, is proposed within Caraganeae. Thirteen genera are herein supported within Wisterieae, including a new genus, Villosocallerya L. Duan, J. Compton & Schrire, segregated from Callerya. Our biogeographic analyses suggest that Wisterieae originated in the late Eocene and its most recent common ancestor (MRCA) was distributed in continental southeastern Asia. Lineages of Wisterieae remained in the ancestral area from the early Oligocene to the early Miocene. By the middle Miocene, Whitfordiodendron and the MRCA of Callerya-Kanburia-Villosocallerya Clade became disjunct between the Sunda area and continental southeastern Asia, respectively; the MRCA of Wisteria migrated to North America via the Bering land bridge. The ancestor of Austrocallerya and Padbruggea migrated to the Wallacea-Oceania area, which split in the early Pliocene. In the Pleistocene, Wisteria brachybotrys, W. floribunda and Wisteriopsis japonica reached Japan, and Callerya cinerea dispersed to South Asia. This study provides a solid phylogenomic for further evolutionary/biogeographic/systematic investigations on the ecologically diverse and economically important IRLC legumes.

Phylogenomics and biogeography of Wisteria: Implications on plastome evolution among inverted repeat‐lacking clade (IRLC) legumes

AbstractThe genus Wisteria (Fabaceae) is disjunctly distributed in eastern Asian and eastern North American temperate deciduous forests, and it is widely cultivated around the world as spectacular garden plants. It is a member of inverted repeat‐lacking clade (IRLC). The IRLC Species are characterized by the loss of an IR region in their plastomes, which has long been of great interest. In this research, we report whole plastome sequences from all four Wisteria species and a Wisteriopsis japonica, combining these with existing data to explore phylogenetic relationships and biogeography of Wisteria, as well as plastome evolution of IRLC species. Phylogenetic analyses recognized a clade containing Glycyrrhiza–Wisteriopsis–Wisteria as sister to the remaining genera of IRLC. North American Wisteria frutescens and the three Asian species formed reciprocal clades, and Wisteria brachybotrys was sister to Wisteria floribunda and Wisteria sinensis. Wisteria may have originated in Japan near the boundary of the Oligocene and Miocene. The disappearance of Bering Land Bridge in the late Miocene might lead to the Eastern Asian–Eastern North American disjunction of Wisteria. Allopatric speciation of Wisteria between the Japanese archipelago and the Asian continent in the Quaternary increased the species richness of eastern Asia in comparison with eastern North America. Synonymous substitution rates (dS) of protein‐coding genes in the IRLC species were around 2‐fold (SC genes) or 11‐fold (IR genes) higher than those of non‐IRLC species. For both SC and IR genes, herbaceous legumes have around 3‐fold higher dS than woody ones. Both loss of one IR region and herbaceous habit elevated substitution rates of the plastomes.