SUnSeT: spectral unmixing of hyperspectral images for phenotyping soybean seed traits.

KEY MESSAGE: Hyperspectral features enable accurate classification of soybean seeds using linear discriminant analysis and GWAS for novel seed trait genes. Evaluating crop seed traits such as size, shape, and color is crucial for assessing seed quality and improving agricultural productivity. The introduction of the SUnSet toolbox, which employs hyperspectral sensor-derived image analysis, addresses this necessity. In a validation test involving 420 seed accessions from the Korean Soybean Core Collections, the pixel purity index algorithm identified seed- specific hyperspectral endmembers to facilitate segmentation. Various metrics extracted from ventral and lateral side images facilitated the categorization of seeds into three size groups and four shape groups. Additionally, quantitative RGB triplets representing seven seed coat colors, averaged reflectance spectra, and pigment indices were acquired. Machine learning models, trained on a dataset comprising 420 accession seeds and 199 predictors encompassing seed size, shape, and reflectance spectra, achieved accuracy rates of 95.8% for linear discriminant analysis model. Furthermore, a genome-wide association study utilizing hyperspectral features uncovered associations between seed traits and genes governing seed pigmentation and shapes. This comprehensive approach underscores the effectiveness of SUnSet in advancing precision agriculture through meticulous seed trait analysis.

Map-Based Cloning and Characterization of a Major QTL Gene, FfR1, Which Confers Resistance to Rice Bakanae Disease.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, is a serious threat to rice production worldwide. Breeding elite rice varieties resistant to BD requires the identification of resistance genes. Previously, we discovered a resistant quantitative trait locus (QTL), qFfR1, in a Korean japonica rice variety, Nampyeong. In this study, we fine-mapped qFfR1 with a Junam*4/Nampyeong BC3F3 population and delimited its location to a 37.1 kb region on chromosome 1. Complementation experiments with seven candidate genes in this region revealed that OsI_02728 is the gene for qFfR1. This gene encodes a protein with a typical leucine-rich repeat (LRR) receptor-like protein structure. RNA-sequencing-based transcriptomic analysis revealed that FfR1 induces the transcription of defense genes, including lignin and terpenoid biosynthesis genes, pathogenesis-related genes, and thionin genes. These results may facilitate investigations into the molecular mechanisms underlying BD resistance, including molecular patterns of Fusarium fujikuroi interacting with FfR1 and players working in signal transduction pathways downstream of FfR1, and the breeding of new BD-resistant varieties by providing a BD resistance gene with its precise selection marker. This will contribute to efficient control of BD, which is becoming more prevalent according to temperature rises due to climate change.

Genome-wide analysis of KIX gene family for organ size regulation in soybean (Glycine max L.).

The KIX domain, conserved among various nuclear and co-activator factors, acts as a binding site that interacts with other transcriptional activators and co-activators, playing a crucial role in gene expression regulation. In plants, the KIX domain is involved in plant hormone signaling, stress response regulation, cell cycle control, and differentiation, indicating its potential relevance to crop productivity. This study aims to identify and characterize KIX domains within the soybean (Glycine max L.) genome to predict their potential role in improving crop productivity. The conservation and evolutionary history of the KIX domains were explored in 59 plant species, confirming the presence of the KIX domains in diverse plants. Specifically, 13 KIX domains were identified within the soybean genome and classified into four main groups, namely GmKIX8/9, GmMED15, GmHAC, and GmRECQL, through sequence alignment, structural analysis, and phylogenetic tree construction. Association analysis was performed between KIX domain haplotypes and soybean seed-related agronomic traits using re-sequencing data from a core collection of 422 accessions. The results revealed correlations between SNP variations observed in GmKIX8-3 and GmMED15-4 and soybean seed phenotypic traits. Additionally, transcriptome analysis confirmed significant expression of the KIX domains during the early stages of soybean seed development. This study provides the first characterization of the structural, expression, genomic haplotype, and molecular features of the KIX domain in soybean, offering a foundation for functional analysis of the KIX domain in soybean and other plants.

Image-Based High-Throughput Phenotyping in Horticultural Crops.

Plant phenotyping is the primary task of any plant breeding program, and accurate measurement of plant traits is essential to select genotypes with better quality, high yield, and climate resilience. The majority of currently used phenotyping techniques are destructive and time-consuming. Recently, the development of various sensors and imaging platforms for rapid and efficient quantitative measurement of plant traits has become the mainstream approach in plant phenotyping studies. Here, we reviewed the trends of image-based high-throughput phenotyping methods applied to horticultural crops. High-throughput phenotyping is carried out using various types of imaging platforms developed for indoor or field conditions. We highlighted the applications of different imaging platforms in the horticulture sector with their advantages and limitations. Furthermore, the principles and applications of commonly used imaging techniques, visible light (RGB) imaging, thermal imaging, chlorophyll fluorescence, hyperspectral imaging, and tomographic imaging for high-throughput plant phenotyping, are discussed. High-throughput phenotyping has been widely used for phenotyping various horticultural traits, which can be morphological, physiological, biochemical, yield, biotic, and abiotic stress responses. Moreover, the ability of high-throughput phenotyping with the help of various optical sensors will lead to the discovery of new phenotypic traits which need to be explored in the future. We summarized the applications of image analysis for the quantitative evaluation of various traits with several examples of horticultural crops in the literature. Finally, we summarized the current trend of high-throughput phenotyping in horticultural crops and highlighted future perspectives.

QTL Mapping of Tiller Number in Korean Japonica Rice Varieties.

Tiller number is an important trait associated with yield in rice. Tiller number in Korean japonica rice was analyzed under greenhouse conditions in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae and Unbong40 to identify quantitative trait loci (QTLs). A genetic map comprising 239 kompetitive allele-specific PCR (KASP) and 57 cleaved amplified polymorphic sequence markers was constructed. qTN3, a major QTL for tiller number, was identified at 132.4 cm on chromosome 3. This QTL was also detected under field conditions in a backcross population; thus, qTN3 was stable across generations and environments. qTN3 co-located with QTLs associated with panicle number per plant and culm diameter, indicating it had pleiotropic effects. The qTN3 regions of Odae and Unbong40 differed in a known functional variant (4 bp TGTG insertion/deletion) in the 5' UTR of OsTB1, a gene underlying variation in tiller number and culm strength. Investigation of variation in genotype and tiller number revealed that varieties with the insertion genotype had lower tiller numbers than those with the reference genotype. A high-resolution melting marker was developed to enable efficient marker-assisted selection. The QTL qTN3 will therefore be useful in breeding programs developing japonica varieties with optimal tiller numbers for increased yield.

Comparison of Various Drought Resistance Traits in Soybean (Glycine max L.) Based on Image Analysis for Precision Agriculture.

Drought is being annually exacerbated by recent global warming, leading to crucial damage of crop growth and final yields. Soybean, one of the most consumed crops worldwide, has also been affected in the process. The development of a resistant cultivar is required to solve this problem, which is considered the most efficient method for crop producers. To accelerate breeding cycles, genetic engineering and high-throughput phenotyping technologies have replaced conventional breeding methods. However, the current novel phenotyping method still needs to be optimized by species and varieties. Therefore, we aimed to assess the most appropriate and effective phenotypes for evaluating drought stress by applying a high-throughput image-based method on the nested association mapping (NAM) population of soybeans. The acquired image-based traits from the phenotyping platform were divided into three large categories-area, boundary, and color-and demonstrated an aspect for each characteristic. Analysis on categorized traits interpreted stress responses in morphological and physiological changes. The evaluation of drought stress regardless of varieties was possible by combining various image-based traits. We might suggest that a combination of image-based traits obtained using computer vision can be more efficient than using only one trait for the precision agriculture.

Development and Application of a Target Capture Sequencing SNP-Genotyping Platform in Rice.

The development of efficient, robust, and high-throughput SNP genotyping platforms is pivotal for crop genetics and breeding. Recently, SNP genotyping platforms based on target capture sequencing, which is very flexible in terms of the number of SNP markers, have been developed for maize, cassava, and fava bean. We aimed to develop a target capture sequencing SNP genotyping platform for rice. A target capture sequencing panel containing 2565 SNPs, including 1225 SNPs informative for japonica and 1339 SNPs informative for indica, was developed. This platform was used in diversity analysis of 50 rice varieties. Of the 2565 SNP markers, 2341 (91.3%) produced useful polymorphic genotype data, enabling the production of a phylogenetic tree of the 50 varieties. The mean number of markers polymorphic between any two varieties was 854. The platform was used for QTL mapping of preharvest sprouting (PHS) resistance in an F8 recombinant inbred line population derived from the cross Odae × Joun. A genetic map comprising 475 markers was constructed, and two QTLs for PHS resistance were identified on chromosomes 4 and 11. This system is a powerful tool for rice genetics and breeding and will facilitate QTL studies and gene mapping, germplasm diversity analysis, and marker-assisted selection.

Genomic Variation in Korean japonica Rice Varieties.

Next-generation sequencing technologies have enabled the discovery of numerous sequence variations among closely related crop varieties. We analyzed genome resequencing data from 24 Korean temperate japonica rice varieties and discovered 954,233 sequence variations, including 791,121 single nucleotide polymorphisms (SNPs) and 163,112 insertions/deletions (InDels). On average, there was one variant per 391 base-pairs (bp), a variant density of 2.6 per 1 kbp. Of the InDels, 10,860 were longer than 20 bp, which enabled conversion to markers resolvable on an agarose gel. The effect of each variant on gene function was predicted using the SnpEff program. The variants were categorized into four groups according to their impact: high, moderate, low, and modifier. These groups contained 3524 (0.4%), 27,656 (2.9%), 24,875 (2.6%), and 898,178 (94.1%) variants, respectively. To test the accuracy of these data, eight InDels from a pre-harvest sprouting resistance QTL (qPHS11) target region, four highly polymorphic InDels, and four functional sequence variations in known agronomically important genes were selected and successfully developed into markers. These results will be useful to develop markers for marker-assisted selection, to select candidate genes in map-based cloning, and to produce efficient high-throughput genome-wide genotyping systems for Korean temperate japonica rice varieties.

High-Throughput Phenotyping Methods for Breeding Drought-Tolerant Crops.

Drought is a main factor limiting crop yields. Modern agricultural technologies such as irrigation systems, ground mulching, and rainwater storage can prevent drought, but these are only temporary solutions. Understanding the physiological, biochemical, and molecular reactions of plants to drought stress is therefore urgent. The recent rapid development of genomics tools has led to an increasing interest in phenomics, i.e., the study of phenotypic plant traits. Among phenomic strategies, high-throughput phenotyping (HTP) is attracting increasing attention as a way to address the bottlenecks of genomic and phenomic studies. HTP provides researchers a non-destructive and non-invasive method yet accurate in analyzing large-scale phenotypic data. This review describes plant responses to drought stress and introduces HTP methods that can detect changes in plant phenotypes in response to drought.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

Development of 454 New Kompetitive Allele-Specific PCR (KASP) Markers for Temperate japonica Rice Varieties.

Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.

High-throughput phenotyping platform for analyzing drought tolerance in rice.

MAIN CONCLUSION: A new imaging platform was constructed to analyze drought-tolerant traits of rice. Rice was used to quantify drought phenotypes through image-based parameters and analyzing tools. Climate change has increased the frequency and severity of drought, which limits crop production worldwide. Developing new cultivars with increased drought tolerance and short breeding cycles is critical. However, achieving this goal requires phenotyping a large number of breeding populations in a short time and in an accurate manner. Novel cutting-edge technologies such as those based on remote sensors are being applied to solve this problem. In this study, new technologies were applied to obtain and analyze imaging data and establish efficient screening platforms for drought tolerance in rice using the drought-tolerant mutant osphyb. Red-Green-Blue images were used to predict plant area, color, and compactness. Near-infrared imaging was used to determine the water content of rice, infrared was used to assess plant temperature, and fluorescence was used to examine photosynthesis efficiency. DroughtSpotter technology was used to determine water use efficiency, plant water loss rate, and transpiration rate. The results indicate that these methods can detect the difference between tolerant and susceptible plants, suggesting their value as high-throughput phenotyping methods for short breeding cycles as well as for functional genetic studies of tolerance to drought stress.

QTL mapping for pre-harvest sprouting resistance in japonica rice varieties utilizing genome re-sequencing.

Pre-harvest sprouting (PHS) leads to serious economic losses because of reductions in yield and quality. To analyze the quantitative trait loci (QTLs) for PHS resistance in japonica rice, PHS rates on panicles were measured in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae (PHS resistant) and Unbong40 (PHS susceptible) under two different environmental conditions-field (summer) and greenhouse (winter) environments. Genome re-sequencing of the parental varieties detected 266,773 DNA polymorphisms including 248,255 single nucleotide polymorphisms and 18,518 insertions/deletions. We constructed a genetic map comprising 239 kompetitive allele-specific PCR and 49 cleaved amplified polymorphic sequence markers. In the field environment, two major QTLs, qPHS-3FD and qPHS-11FD, were identified on chromosomes 3 and 11, respectively, whereas three major QTLs, qPHS-3GH, qPHS-4GH, and qPHS-11GH, were identified on chromosomes 3, 4, and 11, respectively, in the greenhouse environment. qPHS-11GH and qPHS-11FD had similar locations on chromosome 11, suggesting the existence of a gene conferring stable PHS resistance effects under different environmental conditions. The QTLs identified in this study can be used to improve the PHS resistance of japonica varieties, and they may improve our understanding of the genetic basis of PHS resistance.

High Throughput Phenotyping for Various Traits on Soybean Seeds Using Image Analysis.

Data phenotyping traits on soybean seeds such as shape and color has been obscure because it is difficult to define them clearly. Further, it takes too much time and effort to have sufficient number of samplings especially length and width. These difficulties prevented seed morphology to be incorporated into efficient breeding program. Here, we propose methods for an image acquisition, a data processing, and analysis for the morphology and color of soybean seeds by high-throughput method using images analysis. As results, quantitative values for colors and various types of morphological traits could be screened to create a standard for subsequent evaluation of the genotype. Phenotyping method in the current study could define the morphology and color of soybean seeds in highly accurate and reliable manner. Further, this method enables the measurement and analysis of large amounts of plant seed phenotype data in a short time, which was not possible before. Fast and precise phenotype data obtained here may facilitate Genome Wide Association Study for the gene function analysis as well as for development of the elite varieties having desirable seed traits.

Mutations in the microRNA172 binding site of SUPERNUMERARY BRACT (SNB) suppress internode elongation in rice.

BACKGROUND: Internode elongation is an important agronomic trait in rice that determines culm length, which is related to lodging, panicle exsertion, and biomass. sui4 (shortened uppermost internode 4) mutants show reduced internode length and a dwarf phenotype due to shortened internodes; the uppermost internode is particularly severely affected. The present study was performed to identify the molecular nature and function of the SUI4 gene during internode elongation. RESULTS: Our previous study showed that the SUI4 gene was mapped to a 1.1-Mb interval on chromosome 7 (Ji et al. 2014). In order to isolate the gene responsible for the sui4 phenotype, genomic DNA resequencing of sui4 mutants and wild-type plants and reciprocal transformation of wild-type and mutant alleles of the putative SUI4 gene was performed. The data revealed that the causative mutation of sui4 was a T to A nucleotide substitution at the microRNA172 binding site of Os07g0235800, and that SUI4 is a new allele of the previously reported gene SUPERNUMERARY BRACT (SNB), which affects flower structure. In order to understand the effect of this mutation on expression of the SUI4/SNB gene, SUI4/SNB native promoter-fuzed GUS transgenics were examined, along with qRT-PCR analysis at various developmental stages. In sui4 mutants, the SUI4/SNB gene was upregulated in the leaves, culms, and panicles, especially when internodes were elongated. In culms, SUI4/SNB was expressed in the nodes and the lower parts of elongating internodes. In order to further explore the molecular nature of SUI4/SNB during internode elongation, RNA-seq and qRT-PCR analysis were performed with RNAs from the culms of sui4 mutants and wild-type plants in the booting stage. The data showed that in sui4 mutants, genes deactivating bioactive gibberellins and cytokinin were upregulated while genes related to cell expansion and cell wall synthesis were downregulated. CONCLUSION: In summary, this paper shows that interaction between SUI4/SNB and microRNA172 could determine internode elongation during the reproductive stage in rice plants. Due to a mutation at the microRNA172 binding site in sui4 mutants, the expression of SUI4/SNB was enhanced, which lowered the activities of cell expansion and cell wall synthesis and consequently resulted in shortened internodes.

The opening of phenome-assisted selection era in the early seedling stage.

Faster and more efficient breeding cycle is not an option to deal with unpredictable and fast global climate changes. Phenomics for collecting huge number of individuals in accurate manner could be an answer to solve this problem. We collected image data to measure plant height and manual data for shoot length to be compared. QTLs clustered of plant height and shoot length were detected in 2-week old seedlings, which was consistent with many other reports using various genetic resources in matured stage. Further, these traits are highly correlated with yield by pleiotropism or tight linkage of those traits. It implies the "phenome-assisted selection" can be applied for yield trait in rice in the very early stage to shorten the breeding cycle significantly in fast but low-cost manner.

Rice Genome Resequencing Reveals a Major Quantitative Trait Locus for Resistance to Bakanae Disease Caused by Fusarium fujikuroi.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, 'Samgwang' and 'Junam', respectively. Resequencing of 'Junam' and 'Samgwang' genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24-7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the 'Junam' and 'Samgwang' genome sequence data, and are possibly candidate genes for qFfR9; therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties.

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

The complete mitochondrial genome sequence of Schisandra chinensis (Austrobaileyales: Schisandraceae).

Chinese magnolia vine (Schisandra chinensis) is an economically important oriental medicinal plant that belongs to the Schisandraceae family. The complete mitochondrial genome sequence of S. chinensis was 946,141 bp in length. A total of 45 genes was annotated, including 30 protein-coding genes, 12 tRNA genes, and 3 rRNA genes. A phylogenetic tree based on the mitochondrial genome demonstrated that S. chinensis was most closely related to Schisandra sphenanthera of the Schisandraceae family.

Assembly of the Mitochondrial Genome in the Campanulaceae Family Using Illumina Low-Coverage Sequencing.

Platycodongrandiflorus (balloon flower) and Codonopsislanceolata (bonnet bellflower) are important herbs used in Asian traditional medicine, and both belong to the botanical family Campanulaceae. In this study, we designed and implemented a de novo DNA sequencing and assembly strategy to map the complete mitochondrial genomes of the first two members of the Campanulaceae using low-coverage Illumina DNA sequencing data. We produced a total of 28.9 Gb of paired-end sequencing data from the genomic DNA of P.grandiflorus (20.9 Gb) and C.lanceolata (8.0 Gb). The assembled mitochondrial genome of P.grandiflorus was found to consist of two circular chromosomes; the master circle contains 56 genes, and the minor circle contains 42 genes. The C.lanceolata mitochondrial genome consists of a single circle harboring 54 genes. Using a comparative genome structure and a pattern of repeated sequences, we show that the P.grandiflorus minor circle resulted from a recombination event involving the direct repeats of the master circle. Our dataset will be useful for comparative genomics and for evolutionary studies, and will facilitate further biological and phylogenetic characterization of species in the Campanulaceae.