Genome assembly of the pioneer species Plantago major L. (Plantaginaceae) provides insight into its global distribution and adaptation to metal-contaminated soil.

Plantago is a major genus belonging to the Plantaginaceae family and is used in herbal medicine, functional food, and pastures. Several Plantago species are also characterized by their global distribution, but the mechanism underpinning this is not known. Here, we present a high-quality, chromosome-level genome assembly of Plantago major L., a species of Plantago, by incorporating Oxford Nanopore sequencing and Hi-C technologies. The genome assembly size was approximately 671.27 Mb with a contig N50 length of 31.30 Mb. 31,654 protein-coding genes were identified from the genome. Evolutionary analysis showed that P. major diverged from other Lamiales species at ~62.18 Mya and experienced two rounds of WGD events. Notably, many gene families related to plant acclimation and adaptation expanded. We also found that many polyphenol biosynthesis genes showed high expression patterns in roots. Some amino acid biosynthesis genes, such as those involved in histidine synthesis, were highly induced under metal (Ni) stress that led to the accumulation of corresponding metabolites. These results suggest persuasive arguments for the global distribution of P. major through multiscale analysis. Decoding the P. major genome provides a valuable genomic resource for research on dissecting biological function, molecular evolution, taxonomy, and breeding.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the ß-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Comprehensive analysis of complete chloroplast genome sequence of Plantago asiatica L. (Plantaginaceae).

Plantago asiatica L. is a representative individual species of Plantaginaceae, whose high reputation is owed to its edible and medicinal values. However, the phylogeny and genes of the P. asiatica chloroplast have not yet been well described. Here we report the findings of a comprehensive analysis of the P. asiatica chloroplast genome. The P. asiatica chloroplast genome is 164,992 bp, circular, and has a GC content of 37.98%. The circular genome contains 141 genes, including 8 rRNAs, 38 tRNAs, and 95 protein-coding genes. Seventy-two simple sequence repeats are detected. Comparative chloroplast genome analysis of six related species suggests that a higher similarity exists in the coding region than the non-coding region, and differences in the degree of preservation is smaller between P. asiatica and Plantago depressa than among others. Our phylogenetic analysis illustrates P. asiatica has a relatively close relationship with P. depressa, which was also divided into different clades with Plantago ovata and Plantago lagopus in the genus Plantago. This analysis of the P. asiatica chloroplast genome contributes to an improved deeply understanding of the evolutionary relationships among Plantaginaceae.

A chromosome-level genome assembly of Plantago ovata.

Plantago ovata is cultivated for production of its seed husk (psyllium). When wet, the husk transforms into a mucilage with properties suitable for pharmaceutical industries, utilised in supplements for controlling blood cholesterol levels, and food industries for making gluten-free products. There has been limited success in improving husk quantity and quality through breeding approaches, partly due to the lack of a reference genome. Here we constructed the first chromosome-scale reference assembly of P. ovata using a combination of 5.98 million PacBio and 636.5 million Hi-C reads. We also used corrected PacBio reads to estimate genome size and transcripts to generate gene models. The final assembly covers ~ 500 Mb with 99.3% gene set completeness. A total of 97% of the sequences are anchored to four chromosomes with an N50 of ~ 128.87 Mb. The P. ovata genome contains 61.90% repeats, where 40.04% are long terminal repeats. We identified 41,820 protein-coding genes, 411 non-coding RNAs, 108 ribosomal RNAs, and 1295 transfer RNAs. This genome will provide a resource for plant breeding programs to, for example, reduce agronomic constraints such as seed shattering, increase psyllium yield and quality, and overcome crop disease susceptibility.

Arbuscular mycorrhizal fungi influence host infection during epidemics in a wild plant pathosystem.

While pathogenic and mutualistic microbes are ubiquitous across ecosystems and often co-occur within hosts, how they interact to determine patterns of disease in genetically diverse wild populations is unknown. To test whether microbial mutualists provide protection against pathogens, and whether this varies among host genotypes, we conducted a field experiment in three naturally occurring epidemics of a fungal pathogen, Podosphaera plantaginis, infecting a host plant, Plantago lanceolata, in the Åland Islands, Finland. In each population, we collected epidemiological data on experimental plants from six allopatric populations that had been inoculated with a mixture of mutualistic arbuscular mycorrhizal fungi or a nonmycorrhizal control. Inoculation with arbuscular mycorrhizal fungi increased growth in plants from every population, but also increased host infection rate. Mycorrhizal effects on disease severity varied among host genotypes and strengthened over time during the epidemic. Host genotypes that were more susceptible to the pathogen received stronger protective effects from inoculation. Our results show that arbuscular mycorrhizal fungi introduce both benefits and risks to host plants, and shift patterns of infection in host populations under pathogen attack. Understanding how mutualists alter host susceptibility to disease will be important for predicting infection outcomes in ecological communities and in agriculture.

Comparative transcriptome mining for terpenoid biosynthetic pathway genes in wild and cultivated species of Plantago.

Plantagos are important economical and medicinal plants that possess several bioactive secondary metabolites, such as phenolics, iridoids, triterpenes, and alkaloids. Triterpenoids are the ubiquitous and dynamic secondary metabolites that are deployed by plants for chemical interactions and protection under biotic/abiotic stress. Plantago ovata, a cultivated species, is the source of psyllium, while Plantago major, a wild species, has significant therapeutic potential. Wild species are considered more tolerant to stressful conditions in comparison to their cultivated allies. In view of this, the present study aimed to decipher the terpenoid biosynthetic pathway operative in P. ovata and P. major using a comparative transcriptomics approach. Majority of terpenoid biosynthetic genes were observed as upregulated in P. major including rate limiting genes of MVA (HMGR) and MEP (DXR) pathways and genes (α-AS, BAS, SM, and CYP716) involved in ursolic acid biosynthesis, an important triterpenoid prevalent in Plantago species. The HPLC output further confirmed the higher concentration of ursolic acid in P. major as compared to P. ovata leaf samples, respectively. In addition to terpenoid biosynthesis, KEGG annotation revealed the involvement of differentially expressed unigenes in several metabolic pathways, aminoacyl-tRNA biosynthesis, biosynthesis of antibiotics, and biosynthesis of secondary metabolites. MYB was found as the most abundant transcription factor family in Plantago transcriptome. We have been able to generate valuable information which can help in improving terpenoid production in Plantago. Additionally, the present study has laid a strong foundation for deciphering other important metabolic pathways in Plantago.

Quantifying the effect of genetic, environmental and individual demographic stochastic variability for population dynamics in Plantago lanceolata.

Simple demographic events, the survival and reproduction of individuals, drive population dynamics. These demographic events are influenced by genetic and environmental parameters, and are the focus of many evolutionary and ecological investigations that aim to predict and understand population change. However, such a focus often neglects the stochastic events that individuals experience throughout their lives. These stochastic events also influence survival and reproduction and thereby evolutionary and ecological dynamics. Here, we illustrate the influence of such non-selective demographic variability on population dynamics using population projection models of an experimental population of Plantago lanceolata. Our analysis shows that the variability in survival and reproduction among individuals is largely due to demographic stochastic variation with only modest effects of differences in environment, genes, and their interaction. Common expectations of population growth, based on expected lifetime reproduction and generation time, can be misleading when demographic stochastic variation is large. Large demographic stochastic variation exhibited within genotypes can lower population growth and slow evolutionary adaptive dynamics. Our results accompany recent investigations that call for more focus on stochastic variation in fitness components, such as survival, reproduction, and functional traits, rather than dismissal of this variation as uninformative noise.

Plastomes from tribe Plantagineae (Plantaginaceae) reveal infrageneric structural synapormorphies and localized hypermutation for Plantago and functional loss of ndh genes from Littorella.

Tribe Plantagineae (Plantaginaceae) comprises ~ 270 species in three currently recognized genera (Aragoa, Littorella, Plantago), of which Plantago is most speciose. Plantago plastomes exhibit several atypical features including large inversions, expansions of the inverted repeat, increased repetitiveness, intron losses, and gene-specific increases in substitution rate, but the prevalence of these plastid features among species and subgenera is unknown. To assess phylogenetic relationships and plastomic evolutionary dynamics among Plantagineae genera and Plantago subgenera, we generated 25 complete plastome sequences and compared them with existing plastome sequences from Plantaginaceae. Using whole plastome and partitioned alignments, our phylogenomic analyses provided strong support for relationships among major Plantagineae lineages. General plastid features-including size, GC content, intron content, and indels-provided additional support that reinforced major Plantagineae subdivisions. Plastomes from Plantago subgenera Plantago and Coronopus have synapomorphic expansions and inversions affecting the size and gene order of the inverted repeats, and particular genes near the inversion breakpoints exhibit accelerated nucleotide substitution rates, suggesting localized hypermutation associated with rearrangements. The Littorella plastome lacks functional copies of ndh genes, which may be related to an amphibious lifestyle and partial reliance on CAM photosynthesis.

The composition of Australian Plantago seeds highlights their potential as nutritionally-rich functional food ingredients.

When wetted, Plantago seeds become covered with a polysaccharide-rich gel called mucilage that has value as a food additive and bulking dietary fibre. Industrially, the dry husk layer that becomes mucilage, called psyllium, is milled off Plantago ovata seeds, the only commercial-relevant Plantago species, while the residual inner seed tissues are either used for low value animal feed or discarded. We suggest that this practice is potentially wasting a highly nutritious resource and here describe the use of histological, physicochemical, and chromatographic analyses to compare whole seed composition/characteristics of P. ovata with 11 relatives already adapted to harsh Australian conditions that may represent novel commercial crop options. We show that substantial interspecific differences in mucilage yield and macromolecular properties are mainly a consequence of differences in heteroxylan and pectin composition and probably represent wide differences in hydrocolloid functionality that can be exploited in industry. We also show that non-mucilage producing inner seed tissues contain a substantial mannan-rich endosperm, high in fermentable sugars, protein, and fats. Whole seed Plantago flour, particularly from some species obtained from harsh Australian environments, may provide improved economic and health benefits compared to purified P. ovata psyllium husk, by retaining the functionality of the seed mucilage and providing additional essential nutrients.

Establishment and elicitation of transgenic root culture of Plantago lanceolata and evaluation of its anti-bacterial and cytotoxicity activity.

Hairy root induction in Plantago lanceolata was optimized to take advantage of transformed root cultures. The highest frequency of transformation was achieved using leaf explant, A4 strain, pre-cultivation of explant, 150 µM Acetosyringone, 5 min inoculation, half-strength Murashige and Skoog basal medium as co-cultivation, and half-strength Gamborg's basal medium as a selective medium with 3% sucrose. Among the studied compound encompassing gallic acid, catalpol and apigenin, only the production of gallic acid in hairy roots was affected by 20 mg L-1 AgNO3 and 100 mg L-1 chitosan at 24 hr which yielded 7.63, 4.76-fold increase in its content, respectively. The methanolic extracts of hairy roots elicited by 20 mg L-1 AgNO3 exhibited anti-bacterial activity (MIC and MBC = 25 mg mL-1) against Klebsiella pneumoniae, Proteus vulgaris and Salmonella typhi and anti-bacterial potential of non-elicited hairy roots of P. lanceolata (MIC = 25 mg mL-1 and MBC = 35 mg mL-1) were more active against Klebsiella pneumoniae and P. vulgaris than other bacteria. The methanolic extracts of the P. lanceolata hairy roots demonstrated significant cytotoxic activity on colorectal carcinoma cell line (SW-480) with IC50 = 250.65 ± 6.8 µg mL-1 in comparison to human embryonic kidney (HEK-293) with IC50 = 5263.65 ± 4.6 µg mL-1. Plantago lanceolata hairy roots showed important biological activity explaining its role in traditional medicine.

Intraspecific host variation plays a key role in virus community assembly.

Infection by multiple pathogens of the same host is ubiquitous in both natural and managed habitats. While intraspecific variation in disease resistance is known to affect pathogen occurrence, how differences among host genotypes affect the assembly of pathogen communities remains untested. In our experiment using cloned replicates of naive Plantago lanceolata plants as sentinels during a seasonal virus epidemic, we find non-random co-occurrence patterns of five focal viruses. Using joint species distribution modelling, we attribute the non-random virus occurrence patterns primarily to differences among host genotypes and local population context. Our results show that intraspecific variation among host genotypes may play a large, previously unquantified role in pathogen community structure.

The methylome is altered for plants in a high CO2 world: Insights into the response of a wild plant population to multigenerational exposure to elevated atmospheric [CO2 ].

Unravelling plant responses to rising atmospheric CO2 concentration ([CO2 ]) has largely focussed on plastic functional attributes to single generation [CO2 ] exposure. Quantifying the consequences of long-term, decadal multigenerational exposure to elevated [CO2 ] and the genetic changes that may underpin evolutionary mechanisms with [CO2 ] as a driver remain largely unexplored. Here, we investigated both plastic and evolutionary plant responses to elevated [CO2 ] by applying multi-omic technologies using populations of Plantago lanceolata L., grown in naturally high [CO2 ] for many generations in a CO2 spring. Seed from populations at the CO2 spring and an adjacent control site (ambient [CO2 ]) were grown in a common environment for one generation, and then offspring were grown in ambient or elevated [CO2 ] growth chambers. Low overall genetic differentiation between the CO2 spring and control site populations was found, with evidence of weak selection in exons. We identified evolutionary divergence in the DNA methylation profiles of populations derived from the spring relative to the control population, providing the first evidence that plant methylomes may respond to elevated [CO2 ] over multiple generations. In contrast, growth at elevated [CO2 ] for a single generation induced limited methylome remodelling (an order of magnitude fewer differential methylation events than observed between populations), although some of this appeared to be stably transgenerationally inherited. In all, 59 regions of the genome were identified where transcripts exhibiting differential expression (associated with single generation or long-term natural exposure to elevated [CO2 ]) co-located with sites of differential methylation or with single nucleotide polymorphisms exhibiting significant inter-population divergence. This included genes in pathways known to respond to elevated [CO2 ], such as nitrogen use efficiency and stomatal patterning. This study provides the first indication that DNA methylation may contribute to plant adaptation to future atmospheric [CO2 ] and identifies several areas of the genome that are targets for future study.

Expanded inverted repeat region with large scale inversion in the first complete plastid genome sequence of Plantago ovata.

Plantago ovata (Plantaginaceae) is an economically and medicinally important species, however, least is known about its genomics and evolution. Here, we report the first complete plastome genome of P. ovata and comparison with previously published genomes of related species from Plantaginaceae. The results revealed that P. ovata plastome size was 162,116 bp and that it had typical quadripartite structure containing a large single copy region of 82,084 bp and small single copy region of 5,272 bp. The genome has a markedly higher inverted repeat (IR) size of 37.4 kb, suggesting large-scale inversion of 13.8 kb within the expanded IR regions. In addition, the P. ovata plastome contains 149 different genes, including 43 tRNA, 8 rRNA, and 98 protein-coding genes. The analysis revealed 139 microsatellites, of which 71 were in the non-coding regions. Approximately 32 forward, 34 tandem, and 17 palindromic repeats were detected. The complete genome sequences, 72 shared genes, matK gene, and rbcL gene from related species generated the same phylogenetic signals, and phylogenetic analysis revealed that P. ovata formed a single clade with P. maritima and P. media. The divergence time estimation as employed in BEAST revealed that P. ovata diverged from P. maritima and P. media about 11.0 million years ago (Mya; 95% highest posterior density, 10.06-12.25 Mya). In conclusion, P. ovata had significant variation in the IR region, suggesting a more stable P. ovata plastome genome than that of other Plantaginaceae species.

Global gene flow releases invasive plants from environmental constraints on genetic diversity.

When plants establish outside their native range, their ability to adapt to the new environment is influenced by both demography and dispersal. However, the relative importance of these two factors is poorly understood. To quantify the influence of demography and dispersal on patterns of genetic diversity underlying adaptation, we used data from a globally distributed demographic research network comprising 35 native and 18 nonnative populations of Plantago lanceolata Species-specific simulation experiments showed that dispersal would dilute demographic influences on genetic diversity at local scales. Populations in the native European range had strong spatial genetic structure associated with geographic distance and precipitation seasonality. In contrast, nonnative populations had weaker spatial genetic structure that was not associated with environmental gradients but with higher within-population genetic diversity. Our findings show that dispersal caused by repeated, long-distance, human-mediated introductions has allowed invasive plant populations to overcome environmental constraints on genetic diversity, even without strong demographic changes. The impact of invasive plants may, therefore, increase with repeated introductions, highlighting the need to constrain future introductions of species even if they already exist in an area.

Two reproductive traits show contrasting genetic architectures in Plantago lanceolata.

In many species, temperature-sensitive phenotypic plasticity (i.e., an individual's phenotypic response to temperature) displays a positive correlation with latitude, a pattern presumed to reflect local adaptation. This geographical pattern raises two general questions: (a) Do a few large-effect genes contribute to latitudinal variation in a trait? (b) Is the thermal plasticity of different traits regulated pleiotropically? To address the questions, we crossed individuals of Plantago lanceolata derived from northern and southern European populations. Individuals naturally exhibited high and low thermal plasticity in floral reflectance and flowering time. We grew parents and offspring in controlled cool- and warm-temperature environments, mimicking what plants would encounter in nature. We obtained genetic markers via genotype-by-sequencing, produced the first recombination map for this ecologically important nonmodel species, and performed quantitative trait locus (QTL) mapping of thermal plasticity and single-environment values for both traits. We identified a large-effect QTL that largely explained the reflectance plasticity differences between northern and southern populations. We identified multiple smaller-effect QTLs affecting aspects of flowering time, one of which affected flowering time plasticity. The results indicate that the genetic architecture of thermal plasticity in flowering is more complex than for reflectance. One flowering time QTL showed strong cytonuclear interactions under cool temperatures. Reflectance and flowering plasticity QTLs did not colocalize, suggesting little pleiotropic genetic control and freedom for independent trait evolution. Such genetic information about the architecture of plasticity is environmentally important because it informs us about the potential for plasticity to offset negative effects of climate change.

Genetic polymorphism of Plantago major populations from the radioactive and chemical polluted areas.

The variability of nine microsatellite loci was studied for Plantago major L. populations from radioactive (East-Ural Radioactive Trace, EURT) and chemical (Karabash Copper Smelter, KCS) contaminated areas (Urals, Russia). The absorbed dose rates in the EURT area were 178-1455 times higher than background, and the indices of the total toxic load in the KCS area were 13-42 times higher than background values. In total, 65 alleles were identified in P. major populations, while the number of alleles per locus in the EURT and KCS samples was lower than in the background samples. The expected heterozygosity in all loci significantly exceeded the observed, indicating a high level of inbreeding. The largest number of rare alleles (11-21) was found in background samples, of which 3-7 alleles were private. In the technogenically disturbed zones, 8-11 rare alleles (1-2 private) were noted. The Bayesian analysis (K = 3) showed that no unique groups were found in any of the areas; descendants of all founders (pioneers) were represented in each population, but in different proportions. However, only 4.1% of the variability was distributed between local P. major populations (FST = 0.041) and 95.9% was concentrated within the samples. A pairwise comparison revealed genetic differentiation between all EURT samples. In the KCS area, there was no significant differentiation in pairs of samples that were at a distance of 3-4 km from each other. For samples from the KCS and background sites, the Mantel test showed a statistically significant relationship between geographical and genetic distances, therefore, the intensity of migration flows between these areas is high. For samples from the EURT and background areas, no such dependence was found. In both impact zones, P. major populations showed reduced genetic diversity. This article discusses the causes of this phenomenon.

Bioactive amines changes during the ripening and thermal processes of bananas and plantains.

Bioactive amines are found in food and can be relevant for the assessment of fruits shelf life and nutritional quality. The pulp and peel of 20 banana and plantain were analyzed and the bioactive amine content varied according to the genotype, ripening stage, fruit tissue and thermal processing. In most of the analyzed genotypes, tyramine, histamine, dopamine, serotonin, spermidine, and spermine were decreased during the ripening process in the pulps. By contrast, there was an increase in putrescine level. In many genotypes of plantains, the serotonin and dopamine contents in pulp decreased until stage 5 and increased at stage 7. Peels contain higher levels of serotonin, dopamine, histamine and tyramine than pulps. Additionally, thermal processing affects the content of amines present in fruit. Boiling with the peel should be preferred in domestic preparations, regardless of the genotype used.

The application of high-throughput sequencing for taxonomy: The case of Plantago subg. Plantago (Plantaginaceae).

Plantago is a cosmopolitan genus including over 250 species, concentrated in temperate and high-elevation tropical regions. The taxonomy of Plantago is very difficult, mainly because of its reduced morphology, which features relatively few characters for species classification. Consequently, the infrageneric classification of the genus remains controversial and inadequate. In this study we applied high-throughput plastid genome skimming to provide powerful phylogenetic resolution to clarify the relationships within subg. Plantago, which is the largest, most broadly distributed and poorest understood subgenus of Plantago. Ninety-four samples covering ~56% of all species and representing all sections of subg. Plantago as well as an outgroup were successfully sequenced. The resulting phylogenetic topology was used, complemented by field and herbarium studies, to revise the sectional classification of subg. Plantago and present a complete listing of the accepted species in the subgenus. Our phylogenetic results were also tested for their usefulness in clarifying the taxonomic placement of some taxonomically complicated species in the subgenus. We conclude that a combination of morphological studies and state-of-the art high-throughput DNA data provide a useful toolbox for resolving outstanding taxonomic puzzles exemplified by the genus Plantago.

Arms races with mitochondrial genome soft sweeps in a gynodioecious plant, Plantago lanceolata.

Biological situations involving conflict can create arms race situations with repeated fixations of different functional variants, producing selective sweeps and lowering neutral diversity in genome regions linked to the functional locus. However, they can sometimes lead to balancing selection, potentially creating long coalescent times for sites with functionally different variants, and, if recombination occurs rarely, for extended haplotypes carrying such variants. We tested between these possibilities in a gynodioecious plant, Plantago lanceolata, in which cytoplasmic male-sterility factors conflict with nuclear restorers of male fertility. We find low mitochondrial diversity, which does not support very long-term coexistence of highly diverged mitochondrial haplotypes. Interestingly, however, we found a derived haplotype that is associated with male fertility in a restricted geographic region, and that has fixed differences from the ancestral sequence in several genes, suggesting that it did not arise very recently. Taken together, the results suggest arms race events that involved "soft" selective sweeps involving a moderately old-established haplotype, consistent with the frequency fluctuations predicted by theoretical models of gynodioecy.

Origin, Behaviour, and Transmission of B Chromosome with Special Reference to Plantago lagopus.

B chromosomes have been reported in many eukaryotic organisms. These chromosomes occur in addition to the standard complement of a species. Bs do not pair with any of the A chromosomes and they have generally been considered to be non-essential and genetically inert. However, due to tremendous advancements in the technologies, the molecular composition of B chromosomes has been determined. The sequencing data has revealed that B chromosomes have originated from A chromosomes and they are rich in repetitive elements. In our laboratory, a novel B chromosome was discovered in Plantago lagopus. Using molecular cytogenetic techniques, the B chromosome was found to be composed of ribosomal DNA sequences. However, further characterization of the chromosome using next generation sequencing (NGS) etc. revealed that the B chromosome is a mosaic of sequences derived from A chromosomes, 5S ribosomal DNA (rDNA), 45S rDNA, and various types of repetitive elements. The transmission of B chromosome through the female sex track did not follow the Mendelian principles. The chromosome was found to have drive due to which it was perpetuating in populations. The present paper attempts to summarize the information on nature, transmission, and origin of B chromosomes, particularly the current status of our knowledge in P. lagopus.