Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

Host-Species Variation and Environment Influence Endophyte Symbiosis and Mycotoxin Levels in Chinese Oxytropis Species.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.

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.

Genetic diversity of Oxytropis section Xerobia (Fabaceae) in one of the centres of speciation.

The genetic diversity and phylogenetic relationships of Oxytropis caespitosa, O. grandiflora, O. eriocarpa, O. mixotriche, O. nitens, O. peschkovae and O. triphylla, section Xerobia subgenus Oxytropis, in one of the main speciation centres of the genus Oxytropis (Baikal Siberia and adjacent territories of Northeastern Mongolia) were studied based on sequence analysis of the psbA-trnH, trnL-trnF and trnS-trnG intergenic spacers of cpDNA, as well as the ITS nrDNA. Most populations are characterized by a high level of chloroplast genetic diversity (h varied from 0.327 to 1.000 and π from 0.0001 to 0.0090) due to the ancient origin for some species and to hybridization and polyploidy for others. 67 haplotypes were identified, of which six were shared. Phylogenetic relationships among species could not be satisfactorily resolved. Only the haplotypes of O. triphylla formed a group with rather high support. Probably, O. caespitosa, O. grandiflora, O. mixotriche and O. nitens constitute a single genetic complex. As regards the ITS nrDNA polymorphism, we detected only two ribotypes (RX1, RX2). Both were found in O. caespitosa, O. eriocarpa, O. mixotriche and O. peschkovae, while RX1 was present in O. nitens and O. triphylla, RX2 in O. grandiflora. The absence of diagnostic species-specific variants for the markers studied, together with the sharing of cpDNA haplotypes and nrDNA ribotypes between species, and the resulting polytomies on the phylogenetic trees, confirm the hypothesis on the hybrid origin of some of them. Obviously, the reproductive barriers within the sect. Xerobia are weak. However, morphological differences between the species of the sect. Xerobia are clearly pronounced, even when they grow in sympatry.

Phylogenetic study of the endemic species Oxytropis almaatensis (Fabaceae) based on nuclear ribosomal DNA ITS sequences.

BACKGROUND: Oxytropis almaatensis Bajt. is a rare, narrow endemic species of the Trans-Ili Alatau mountains in Kazakhstan. Up to now, no studies regarding the taxonomy and variation of key morphological traits of O. almaatensis were undertaken. The purpose of this analysis was to evaluate phenotypic variation of O. almaatensis and assess the position of the species within the genus based on nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region. RESULTS: Two populations of O. almaatensis were collected in neighboring gorges of the Trans-Ili Alatau Mountains. The ITS sequences from the samples of two populations of O. almaatensis were identical. The phylogenetic analysis indicated that O. almaatensis is within Oxytropis genetically close to O. glabra as these species formed a separate subclade. The phenotypic variation of populations was assessed using nine morphological traits and compared to descriptions of O. glabra. The range of variation for the traits between two populations was established. A clear morphological difference of O. almaatensis and O. glabra was found in peduncle length to leaf length ratio. This was in O. almaatensis 1.56, while in O. glabra, it was 1.0. CONCLUSIONS: The study provides the first phenotypic description and phylogenetic placement of the rare endemic species O. almaatensis. The morphological traits in two O. almaatensis populations showed a high level of phenotypic variability. Although clearly different from O. glabra, the ITS phylogeny grouped these species in a subclade within the genus.

Molecular characterization of Capra hircus lysosomal α-mannosidase and potential mutant site for the therapy of locoweed poisoning.

Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family 38 and is involved in the biosynthesis and turnover of N-linked glycoproteins process. Locoweeds, which contain swainsonine (SW) that inhibits LAM, are the main poisoning plants in many regions of the world, and thereby resulting in animal poisoning or even death. Based on regions of protein sequence conservation between LAM from Bos taurus and Homo sapiens, we cloned cDNA encoding Capra hircus LAM (chLAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant chLAM was activated 1.6 and 1.2-fold with Zn(2+) and Ca(2+), respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant chLAM is lower than wild type chLAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also of a potential strategy for a novel therapy for locoweed poisoning.

[Genetic variation in six species of the genus Oxytropis DC. (Fabaceae) from Kamchatka Peninsula].

Using the isozyme analysis, genetic variations in six species of the genus Oxytropis DC. (Fabaceae) from Kamchatka was assessed. It was demonstrated that diploid species from the section Arctobia were characterized by a low level of variations typical of endemic plant species. At the same time, polyploid species from the Orobia section demonstrated very high values of the heterozygosity parameters (H(o) varied from 0.200 to 0.274). It has been suggested that the level of polymorphism of the oxytropes from Kamchatka was shaped as a result of the interaction of a number of factors, among which the most important are the ecological confinedness of the species, the specific features of the reproductive system, and gene drift. In the species of Orobia section, it is also the presence of the polyploid genome.

Influence of endophyte genotype on swainsonine concentrations in Oxytropis sericea.

Locoism is a toxic syndrome of livestock caused by the ingestion of a subset of legumes belonging to the Astragalus and Oxytropis genera known as "locoweeds". Locoweeds contain the toxic indolizidine alkaloid swainsonine, which is produced by the endophytic fungi Undifilum species. Previously we reported that swainsonine concentrations differ between populations of Oxytropis sericea. We hypothesized that the genotype of the plant, endophyte, or an interaction of the two may be responsible for the differences in swainsonine concentration between populations of O. sericea. To test this hypothesis, plants derived from seeds collected at each location were grown in a common garden, Undifilum oxytropis isolates from each location were cultured and grown in a common environment, and a plant genotype by endophyte cross inoculation was performed. Here we show that the genotype of the endophyte is responsible for the differences in swainsonine concentrations observed in the two populations of O. sericea.

[Phylogenetic relationships of Oxytropis chankaensis Jurtz. and Oxytropis oxyphylla (Pall.) DC. (Fabaceae) inferred from the data of sequencing of the ITS region of the nuclear ribosomal DNA operon and intergenic spacers of the chloroplast genome].

The autotetraploid Oxytropis chankaensis Jurtz. is the only representative of the section Baicalia (subgenus Oxytropis, Fabaceae) in Primorskii Krai, and its range is restricted to the sandy belt along the western coast of the Khanka Lake. The type species of the section Baicalia is O. oxyphylla (Pall.) DC., and O. chankaensis is regarded by some authors as its synonym. Analysis of the ITS sequences showed that O. chankaensis is phylogenetically closely related to the species O. oxyphylla and O. racemosa Turcz. from the section Baicalia and also to the species O. anertii Nakai., O. ciliata Turcz., and O. inschanica H. C. Fu & S. H. Cheng from the section Xerobia, but this analysis failed to resolve the phylogenetic relationships of the species within the genus Oxytropis. The analysis of the trnH-psbA, trnL-trnF, trnS-trnG, and petG-trnP regions of cpDNA revealed molecular differences between O. chankaensis and O. oxyphylla that are indicative of considerable divergence of their chloroplast genomes. The ancestral lineages of these species diverged nearly 2 million years ago, which confirms the taxonomic validity of O. chankaensis. Taking into account the ecological specificity of O. chankaensis and the present-day distribution range of O. oxyphylla, it can be assumed that they descended from one ancestral maternal lineage that gave rise to the species of the section Baicalia.

The Y-segment of novel cold dehydrin genes is conserved and codons in the PR-10 genes are under positive selection in Oxytropis (Fabaceae) from contrasting climates.

While the arctic flora is particularly threatened by climate changes, the molecular aspects allowing colonization of this harsh environment remain largely enigmatic. Genes with a likely functional or evolutive role for arctic Oxytropis (Fabaceae) were previously discovered given a sharp differential expression between arctic and temperate species, but the evolutionary forces in action were unknown within the respective species. Here, we analyze gene duplication patterns and positive and negative selection between genes from species of contrasting environments, which can reveal potential gene functions. Genes were amplified and sequenced from two arctic (Oxytropis arctobia and O. maydelliana) and two temperate (O. campestris subsp. johannensis and O. splendens) species. Detection of codons under positive or negative selection and phylogenetic analyses were used to further elucidate pathogenesis-related class 10 (PR-10), ripening-related proteins, cold dehydrins gene families and light-harvesting complex (lhcaIII and lhcbI) genes from Oxytropis. Overall, results showed that the three gene families duplicated in tandem prior to the Oxytropis genus diversification; that genes overexpressed in arctic species evolve under higher constraints at the sequence level in these species; that evolving novel protein variants in PR-10 genes were required for initial adaptation to the Arctic, and that Oxytropis cold dehydrins are of a novel (K-like-Y(4)-K-S) structure, where the Y-segment is under stringent evolutive constraints in the arctic species. This suggests a scenario not previously described for arctic plants, where tandem duplications precede gene recruitment that later become both highly expressed and under stringent constraints in the arctic species.

PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic.

In many studied plants, typical responses to cold treatment include up-regulating the hydrophilic COR/LEA genes and down-regulating photosynthesis-related genes, carbohydrate metabolism, GDSL-motif lipase, hormone metabolism and oxidative regulation genes. However, next to nothing is known about gene expression in arctic plants, which are actually adapted to a harsh, cold environment. The molecular mechanisms behind the many specific adaptations of arctic plants, such as slow growth, well-developed root systems and short stature, are not well understood. In this study, we examine whole plantlet transcriptome differences between two arctic and two temperate Oxytropis (Fabaceae) species, grown under their respective controlled environmental conditions. Gene expression differences are analyzed using cDNA library subtraction followed by expressed sequence tags sequencing and annotation. Sequences from a total of nearly 2,000 clones cluster into 121 and 368 unique genes from the arctic and from the temperate plants, respectively. The predominant biological process for genes from the arctic-enriched library is "response to stimulus". A concurrent overexpression of pathogenesis-related class 10 proteins (PR-10), plant defensin and cold dehydrin genes is a novel feature for species adapted to stressful growth environment. The temperate-enriched genes are involved in photosynthesis, translation and nucleosome assembly. Interestingly, both arctic and temperate-enriched libraries also contain genes involved in ribosome biogenesis and assembly, however of different types. Real-time reverse transcription PCR of cold dehydrin and two PR-10 genes, as well as the light harvesting complex b1 genes demonstrates that the gene expression is dependent on species and growth conditions.

High chloroplast haplotype diversity in the endemic legume Oxytropis chankaensis may result from independent polyploidization events.

Oxytropis chankaensis Jurtz. (Fabaceae) is an endangered perennial tetraploid species endemic to the Khanka Lake coast. In Russia, O. chankaensis is distributed across a very restricted zone along the western shore of this lake. To characterise all known populations of this species, we assessed the genetic diversity of four noncoding regions of chloroplast DNA (cpDNA). Variable sites detected within the trnL-trnF, the petG-trnP, and the trnS-trnG regions allowed the identification of seven haplotypes. On the other hand, no variation was found in the trnH-psbA region. O. chankaensis exhibited an overall low level of nucleotide diversity (π = 0.00052) but a marked haplotype diversity (h = 0.718). A combination of three or four haplotypes was found in each population, and most of the cpDNA variation (above 90%) was distributed within populations. The level of genetic structure that we detected in O. chankaensis using maternal plastid DNA markers was much lower (G (ST) = 0.037) than the average that is estimated for angiosperms. We found no evidence for isolation by distance or for phylogeographic structuring in O. chankaensis. Our data suggest that autopolyploidy has arisen more than once in the evolutionary history of this species. Repetitive expansion and contraction during past and ongoing demographic events both seem to be involved in shaping the current genetic structure of O. chankaensis. This study provides valuable information for developing the most appropriate strategy for conserving this endemic species with a narrow habitat range.

[Genetic structure and differentiation of populations of the tetraploid species Oxytropis chankaensis (Fabaceae)].

The population genetic variation of the tetraploid species Oxytropis chankaensis Jurtz. (Fabaceae), a local endemic of the western coast of Khanka Lake (Primorye), was examined. Five populations were analyzed using 28 isozyme loci encoding 16 enzyme systems. Significant allelic heterogeneity among the populations was found for six out of twelve polymorphic loci. The heterozygosity of the samples (total sample size 294 plants) He = 0.301 was considerable higher than the mean values in populations of endemic species (0.076). Based on the results of this study, we identified two groups of O. chankaensis populations (southern and northern), in spite of the absence of marked hiatus between them. Of special interest is the population from Przhewalski Spit, which is a natural reserve of genetic diversity of the species and the putative center of formation of the autotetraploid O. chankaensis.

Population genetics and phylogeography of endangered Oxytropis campestris var. chartacea and relatives: arctic-alpine disjuncts in eastern North America.

Fassett's locoweed (Oxytropis campestris var. chartacea, Fabaceae) is an endangered perennial endemic to Wisconsin. Patterns of genetic variation within and among six remaining populations and their relationship to other members of the O. campestris complex were analysed using AFLPs from 140 accessions across northern North America. Within-population measures of genetic diversity were high (mean expected heterozygosity HE = 0.16; mean nucleotide diversity pi = 0.015) compared with other herbaceous plants. Estimates of among-population differentiation were low (FST = 0.12; PhiST = 0.29), consistent with outcrossing. Genetic and geographical distances between populations were significantly correlated within Fassett's locoweed (r2 = 0.73, P < 0.002 for Mantel test) and O. campestris as a whole (r2 = 0.63, P < 0.0001). Individual and population-based phylogenetic analyses showed that Fassett's locoweed is monophyletic and sister to O. campestris var. johannensis. Morphometric analyses revealed significant differences between Fassett's locoweed and populations of var. johannensis. The first chromosome count for Fassett's locoweed indicates that it is tetraploid (2n = 32), unlike hexaploid var. johannensis. High within-population diversity and relatively low among-population differentiation are consistent with populations of Fassett's locoweed being relicts of a more continuous Pleistocene distribution. Our data support the continued recognition of Fassett's locoweed and protection under federal and state regulations. High levels of genetic diversity within populations suggest that maintain-ing the ecological conditions that favour the life cycle of this plant may be a more pressing concern than the erosion of genetic variation.