De-novo whole genome assembly of the orange jewelweed, Impatiens capensis Meerb. (Balsaminaceae) using nanopore long-read sequencing.

The plant family Balsaminaceae comprises only two genera, and they are a study in contrasts. While Impatiens is an impressively prolific genus, with over 1,000 species and more being discovered each year, its sister genus, Hydrocera, has one solitary species, H. triflora. The two genera also differ in geographic distribution and habitat type (Impatiens species are widely distributed in much of the Old World and N. America, while H. triflora is confined to wetlands specific to S. India, Sri Lanka, and SE Asia). Other contrasting features include plant habit, habitat, floral architecture, mode of seed dispersal, and a host of other traits. The family Balsaminaceae is therefore an excellent model for studying speciation and character evolution as well as understanding the proximal and evolutionary forces that have driven the two genera to adopt such contrasting evolutionary paths. Various species of the Impatiens genus are also commercially important in the ornamental flower industry and as sources of phytochemicals that are of medicinal and other commercial value. As a preliminary step towards studying the genomic basis of the contrasting features of the two genera, we have sequenced and assembled, de novo, the genome of an iconic Impatiens species from N. America, namely I. capensis, and report our findings here.

Plastome evolution and phylogenomics of Impatiens (Balsaminaceae).

MAIN CONCLUSION: This study reported seven new plastomes from Impatiens and observed three highly variable regions for phylogeny and DNA barcoding, which resolved the relationships among sections of subgenus Impatiens. Impatiens L. (Balsaminaceae, Ericales) is one of the largest and most diverse genera of angiosperms, widely known for its taxonomic difficulty. In this study, we reevaluated the infrageneric relationships within the genus Impatiens, using complete plastome sequence data. Seven complete plastomes of Impatiens (representing 6 species) were newly sequenced and characterized along with 20 previously published plastomes of other Impatiens species, plus 2 plastomes of outgroups (Hydrocera triflora, Balsaminaceae; Marcgravia coriacea, Marcgraviaceae). The total size of these 29 plastomes ranged from 151,538 bp to 152,917 bp, except 2 samples of Impatiens morsei, which exhibited a shorter length and lost some genes encoding NADH dehydrogenase subunits. Moreover, the number of simple sequence repeats (SSRs) ranged from 51 to 113, and the number of long repeats from 17 to 26. In addition, three highly variable regions were identified (trnG-GCC (The previous one), ndhF-rpl32-trnL-UGA-ccsA, and ycf1). Our phylogenomic analysis based on 80 plastome-derived protein-coding genes strongly supported the monophyly of Impatiens and its two subgenera (Clavicarpa and Impatiens), and fully resolved relationships among the six (out of seven) sampled sections of subgenus Impatiens. Overall, the plastome DNA markers and phylogenetic results reported in this study will facilitate future identification, taxonomic and DNA barcoding studies in Impatiens as well as evolutionary studies in Balsaminaceae.

Comparative chloroplast genome analysis of Impatiens species (Balsaminaceae) in the karst area of China: insights into genome evolution and phylogenomic implications.

BACKGROUND: Impatiens L. is a genus of complex taxonomy that belongs to the family Balsaminaceae (Ericales) and contains approximately 1000 species. The genus is well known for its economic, medicinal, ornamental, and horticultural value. However, knowledge about its germplasm identification, molecular phylogeny, and chloroplast genomics is limited, and taxonomic uncertainties still exist due to overlapping morphological features and insufficient genomic resources. RESULTS: We sequenced the chloroplast genomes of six different species (Impatiens chlorosepala, Impatiens fanjingshanica, Impatiens guizhouensis, Impatiens linearisepala, Impatiens loulanensis, and Impatiens stenosepala) in the karst area of China and compared them with those of six previously published Balsaminaceae species. We contrasted genomic features and repeat sequences, assessed sequence divergence and constructed phylogenetic relationships. Except for those of I. alpicola, I. pritzelii and I. glandulifera, the complete chloroplast genomes ranging in size from 151,366 bp (I. alpicola) to 154,189 bp (Hydrocera triflora) encoded 115 distinct genes [81 protein-coding, 30 transfer RNA (tRNA), and 4 ribosomal RNA (rRNA) genes]. Moreover, the characteristics of the long repeat sequences and simple sequence repeats (SSRs) were determined. psbK-psbI, trnT-GGU-psbD, rpl36-rps8, rpoB-trnC-GCA, trnK-UUU-rps16, trnQ-UUG, trnP-UGG-psaJ, trnT-UGU-trnL-UAA, and ycf4-cemA were identified as divergence hotspot regions and thus might be suitable for species identification and phylogenetic studies. Additionally, the phylogenetic relationships based on Maximum likelihood (ML) and Bayesian inference (BI) of the whole chloroplast genomes showed that the chloroplast genome structure of I. guizhouensis represents the ancestral state of the Balsaminaceae family. CONCLUSION: Our study provided detailed information about nucleotide diversity hotspots and the types of repeats, which can be used to develop molecular markers applicable to Balsaminaceae species. We also reconstructed and analyzed the relationships of some Impatiens species and assessed their taxonomic statuses based on the complete chloroplast genomes. Together, the findings of the current study might provide valuable genomic resources for systematic evolution of the Balsaminaceae species.

Complete chloroplast genomes of Impatiens cyanantha and Impatiens monticola: Insights into genome structures, mutational hotspots, comparative and phylogenetic analysis with its congeneric species.

Impatiens L., the largest genus in the family Balsaminaceae with approximately 1000 species, is a controversial and complex genus that includes many economically important species well known for medicinal and ornamental values. However, there is limited knowledge of molecular phylogeny and chloroplast genomics, and uncertainties still exist at a taxonomic level. In this study, we have assembled four chloroplast genomics specimens of Impatiens cyanantha and Impatiens monticola, which are found at the different altitudes of Guizhou and Yunnan in China, and compared them with previously published three wild Balsaminaceae species (Impatiens piufanensis, Impatiens glandlifera, and Hydrocera triflora). The complete chloroplast genome sequences ranged from 152,236 bp (I. piufanensis) to 154,189 bp (H. triflora) and encoded 115 total distinct genes, of which 81 were protein-coding, 30 were distinct transfer RNA genes(tRNA), and 4 were ribosomal RNA genes (rRNA). A comparative analysis of I. cyanantha (Guizhou) vs. I. cyanantha (Yunnan) and I. monticola (Guizhou) vs. I. monticola (Yunnan) revealed minor changes in lengths; however, similar gene contents, gene orders, and GC contents existed among them. Interestingly, highly coding and non-coding genes, and regions matK, psbK, atpH-atpI, trnC-trnT, petN, psbM, atpE, rbcL, accD, psaL, rps3-rps19, ndhG-ndhA,rpl16, rpoB, ndhB, ndhF, ycf1, and ndhH were found, which could be suitable for identification of species and phylogenetic studies. During the comparison between I. cyanantha (Guizhou) and I. cyanantha (Yunnan), we observed that the rps4, ycf2, ndhF, ycf1, and rpoC2 genes underwent positive selection. Meanwhile, in the comparative study of I. monticola (Guizhou) vs. I. monticola (Yunnan), The accD and ycf1 genes were positively selected. Additionally, phylogenetic relationships based on maximum likelihood (ML) and Bayesian inference (BI) among whole chloroplast genomes showed that a sister relationship with I. monticola (Guizhou) and I. monticola (Yunnan) formed a clade with I.piufanensis proving their close connection. Besides, I.cyanantha (Guizhou) and I. cyanantha (Yunnan) formed a clade with I. glandlifera. Along with the findings and the results, the current study might provide valuable significant genomic resources for systematics and evolution of the genus impatiens in different altitudes of regions.

Comparative Genomics of the Balsaminaceae Sister Genera Hydrocera triflora and Impatiens pinfanensis.

The family Balsaminaceae, which consists of the economically important genus Impatiens and the monotypic genus Hydrocera, lacks a reported or published complete chloroplast genome sequence. Therefore, chloroplast genome sequences of the two sister genera are significant to give insight into the phylogenetic position and understanding the evolution of the Balsaminaceae family among the Ericales. In this study, complete chloroplast (cp) genomes of Impatiens pinfanensis and Hydrocera triflora were characterized and assembled using a high-throughput sequencing method. The complete cp genomes were found to possess the typical quadripartite structure of land plants chloroplast genomes with double-stranded molecules of 154,189 bp (Impatiens pinfanensis) and 152,238 bp (Hydrocera triflora) in length. A total of 115 unique genes were identified in both genomes, of which 80 are protein-coding genes, 31 are distinct transfer RNA (tRNA) and four distinct ribosomal RNA (rRNA). Thirty codons, of which 29 had A/T ending codons, revealed relative synonymous codon usage values of >1, whereas those with G/C ending codons displayed values of <1. The simple sequence repeats comprise mostly the mononucleotide repeats A/T in all examined cp genomes. Phylogenetic analysis based on 51 common protein-coding genes indicated that the Balsaminaceae family formed a lineage with Ebenaceae together with all the other Ericales.

The multiple fuzzy origins of woodiness within Balsaminaceae using an integrated approach. Where do we draw the line?

BACKGROUND AND AIMS: The family Balsaminaceae is essentially herbaceous, except for some woodier species that can be described as 'woody' herbs or small shrubs. The family is nested within the so-called balsaminoid clade of Ericales, including the exclusively woody families Tetrameristaceae and Marcgraviaceae, which is sister to the remaining families of the predominantly woody order. A molecular phylogeny of Balsaminaceae is compared with wood anatomical observations to find out whether the woodier species are derived from herbaceous taxa (i.e. secondary woodiness), or whether woodiness in the family represents the ancestral state for the order (i.e. primary woodiness). METHODS: Wood anatomical observations of 68 Impatiens species and Hydrocera triflora, of which 47 are included in a multigene phylogeny, are carried out using light and scanning electron microscopy and compared with the molecular phylogenetic insights. KEY RESULTS: There is much continuous variation in wood development between the Impatiens species studied, making the distinction between herbaceousness and woodiness difficult. However, the most woody species, unambiguously considered as truly woody shrubs, all display paedomorphic wood features pointing to secondary woodiness. This hypothesis is further supported by the molecular phylogeny, demonstrating that these most woody species are derived from herbaceous (or less woody) species in at least five independent clades. Wood formation in H. triflora is mostly confined to the ribs of the stems and shows paedomorphic wood features as well, suggesting that the common ancestor of Balsaminaceae was probably herbaceous. CONCLUSIONS: The terms 'herbaceousness' and 'woodiness' are notoriously difficult to use in Balsaminaceae. However, anatomical observations and molecular sequence data show that the woodier species are derived from less woody or clearly herbaceous species, demonstrating that secondary woodiness has evolved in parallel.

Diversity and evolution of floral structure among early diverging lineages in the Ericales.

This is a combination of review and original data on floral structure and diversity in the two earliest diverging lineages of the Ericales, i.e. the balsaminoids, comprising Balsaminaceae, Marcgraviaceae and Tetrameristaceae, and the polemonioids, comprising Fouquieriaceae and Polemoniaceae. Each clade is strongly supported in molecular studies, while structural synapomorphies have largely been lacking. For the balsaminoid families, we compare floral morphology, anatomy and histology among selected taxa and find that the entire clade is strongly supported by the shared presence of nectariferous tissue in the floral periphery, thread-like structures on anthers, truncate stigmas, secretion in the ovary, as well as mucilage cells, raphides and tannins in floral tissues. A possible sister group relationship between Balsaminaceae and Tetrameristaceae is supported by the shared presence of post-genital fusion of filaments and ovary and a star-shaped stylar canal. For polemonioids, we document unexpected diversity of floral features in Polemoniaceae, partly providing structural links to Fouquieriaceae. Features include cochlear and quincuncial corolla aestivation, connective protrusions, ventrifixed anthers and nectariferous tissue in the base of the ovary. In addition, we outline future directions for research on floral structure in the Ericales and briefly discuss the general importance of structural studies for our understanding of plant phylogeny and evolution.

[Hairy root induced by wild-type agrobacterium rhizogenes K599 in soybean, cucumber and garden balsam in vivo].

The plantlets of soybean, cucumber and garden balsam were inoculated by wild-type Agrobacterium rhizogenes K599, and hairy root was induced on inoculated sites in vivo. The frequencies of hairy root induction from wound cotyledons of soybean, cucumber and garden balsam were 100%, 65% and 91%, respectively. Moreover, hairy root was induced from healthy cucumber axillary bud with frequency of 10%. PCR analysis of hairy root DNA was conducted using the primers from rolC gene. The PCR results showed that all hairy root lines contained T-DNA. The established system should be ideal for studying soybean and cucumber nematode and garden balsam breeding of flower dwarf architecture.

Conflicting phylogenies of balsaminoid families and the polytomy in Ericales: combining data in a Bayesian framework.

The balsaminoid Ericales, namely Balsaminaceae, Marcgraviaceae, Tetrameristaceae, and Pellicieraceae have been confidently placed at the base of Ericales, but the relations among these families have been resolved differently in recent analyses. Sister to this basal group is a large polytomy comprising all other families of Ericales, which is associated with short internodes. Because there are more than 13 kb of sequences for a large sampling of representatives, a thorough examination of the available data with novel methods seemed in place. Because of its computational speed, Bayesian phylogenetics allows for the use of parameter-rich models that can accommodate differences in the evolutionary process between partitions in a simultaneous analysis. In addition, there are recently proposed Bayesian strategies of assessing incongruence between partitions. We have applied these methods to the current problems in Ericales phylogeny, taking into account reported pitfalls in Bayesian analysis such as model selection uncertainty. Based on our results we infer several, previously unresolved relationships in the order Ericales. In balsaminoid families, we find that the closest relatives of Balsaminaceae are Marcgraviaceae. In the Ericales polytomy, we find strong support for Pentaphylacaceae sensu APG II as the sister group of Maesaceae. In addition, Symplocaceae receive a position as sister to Theaceae and these families form a monophyletic group together with Styracaceae-Diapensiaceae. At the base of this clade are Actinidiaceae and Clethraceae. The positions of Ebenaceae and Lecythidaceae remain uncertain.