Cross species/genera transferability of simple sequence repeat markers, genetic diversity and population structure analysis in gladiolus (Gladiolus × grandiflorus L.) genotypes.

Background: Genetic analysis of gladiolus germplasm using simple sequence repeat (SSR) markers is largely missing due to scarce genomic information. Hence, microsatellites identified for related genera or species may be utilized to understand the genetic diversity and assess genetic relationships among cultivated gladiolus varieties. Methods: In the present investigation, we screened 26 genomic SSRs (Gladiolus palustris, Crocus sativus, Herbertia zebrina, Sysirinchium micranthum), 14 chloroplast SSRs (Gladiolus spp., chloroplast DNA regions) and 25 Iris Expressed Sequence Tags (ESTs) derived SSRs across the 84 gladiolus (Gladiolus × grandiflorus L.) genotypes. Polymorphic markers detected from amplified SSRs were used to calculate genetic diversity estimates, analyze population structure, cluster analysis and principal coordinate analysis (PCoA). Results: A total of 41 SSRs showed reproducible amplification pattern among the selected gladiolus cultivars. Among these, 17 highly polymorphic SSRs revealed a total of 58 polymorphic alleles ranging from two to six with an average of 3.41 alleles per marker. Polymorphic information content (PIC) values ranged from 0.11 to 0.71 with an average value of 0.48. A total of 4 SSRs were selectively neutral based on the Ewens-Watterson test. Hence, 66.66% of Gladiolus palustris, 48% of Iris spp. EST, 71.42% of Crocus sativus SSRs showed cross-transferability among the gladiolus genotypes. Analysis of genetic structure of 84 gladiolus genotypes revealed two subpopulations; 35 genotypes were assigned to subpopulation 1, 37 to subpopulation 2 and the remaining 12 genotypes could not be attributed to either subpopulation. Analysis of molecular variance indicated maximum variance (53.59%) among individuals within subpopulations, whereas 36.55% of variation among individuals within the total population. The least variation (9.86%) was noticed between two subpopulations. Moderate (FST = 0.10) genetic differentiation between two subpopulations was observed. The grouping pattern of population structure was consistent with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on simple matching dissimilarity coefficient and PCoA. Conclusion: SSR markers from the present study can be utilized for cultivar identification, conservation and sustainable utilization of gladiolus genotypes for crop improvement. Genetic relationships assessed among the genotypes of respective clusters may assist the breeders in selecting desirable parents for crossing.

Complete Chloroplast Genome of Gladiolus gandavensis (Gladiolus) and Genetic Evolutionary Analysis.

Gladiolus is an important ornamental plant that is one of the world's four most-grown cut flowers. Gladiolus gandavensis has only been found in the Cangnan County (Zhejiang Province) of China, which is recorded in the "Botanical". To explore the origin of G. gandavensis, chloroplast genome sequencing was conducted. The results indicated that a total of 151,654 bp of circular DNA was obtained. The chloroplast genome of G. gandavensis has a quadripartite structure (contains a large single-copy (LSC) region (81,547 bp), a small single-copy region (SSC) (17,895 bp), and two inverted repeats (IRs) (IRa and IRb, 52,212 bp)), similar to that of other species. In addition, a total of 84 protein-coding genes, 8 rRNA-encoding genes, and 38 tRNA-encoding genes were present in the chloroplast genome. To further study the structural characteristics of the chloroplast genome in G. gandavensis, a comparative analysis of eight species of the Iridaceae family was conducted, and the results revealed higher similarity in the IR regions than in the LSC and SSC regions. In addition, 265 simple sequence repeats (SSRs) were detected in this study. The results of the phylogenetic analysis indicated that the chloroplast genome of G. gandavensis has high homology with the Crocus cartwrightianus and Crocus sativus chloroplast genomes. Genetic analysis based on the rbcl sequence among 49 Gladiolus species showed that samples 42, 49, 50, and 54 had high homology with the three samples from China (64, 65, and 66), which might be caused by chance similarity in genotypes. These results suggest that G. gandavensis may have originated from South Africa.

4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.

BACKGROUND: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis. RESULTS: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae). CONCLUSIONS: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.

Identification and Evaluation of Aromatic Volatile Compounds in 26 Cultivars and 8 Hybrids of Freesia hybrida.

Freesia hybrida is a group of cultivars in the genus Freesia with a strong floral scent composed of diverse volatile organic compounds (VOCs). In this study, the VOCs of 34 F. hybrida were extracted and analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 164 VOCs whose relative contents were higher than 0.05% were detected. The numbers of VOCs in all germplasms differed between 11 to 38, and the relative contents ranged from 32.39% to 94.28%, in which most germplasms were higher than 80%. Terpenoids, especially monoterpenes, were the crucial type of VOCs in most germplasms, of which linalool and D-limonene were the most frequently occurring. Principal component analysis (PCA) clearly separated samples based on whether linalool was the main component, and hierarchical clustering analysis (HCA) clustered samples into 4 groups according to the preponderant compounds linalool and (E)-ß-ocimene. Comparison of parental species and hybrids showed heterosis in three hybrids, and the inherited and novel substances suggested that monoterpene played an important role in F. hybrida floral scent. This study established a foundation for the evaluation of Freesia genetic resources, breeding for the floral aroma and promoting commercial application.

Genetic melting pot and importance of long-distance dispersal indicated in the Gladiolus imbricatus L. populations in the Polish Carpathians.

The genetic diversity in 11 populations of Gladiolus imbricatus in five mountain ranges, including the Tatra, Pieniny, Gorce, Beskid Niski (Western Carpathians) and Bieszczady Mts (Eastern Carpathians), was studied with inter-simple sequence repeat (ISSR) markers. The species is a perennial plant occurring in open and semi-open sites of anthropogenic origin (meadows and forest margins). We checked a hypothesis on the microrefugial character of the plant populations in the Pieniny Mts, a small calcareous Carpathian range of complicated relief that has never been glaciated. Plant populations in the Tatra and Pieniny Mts had the highest genetic diversity indices, pointing to their long-term persistence. The refugial vs. the non-refugial mountain ranges accounted for a relatively high value of total genetic variation [analysis of molecular variance (AMOVA), 14.12%, p = 0.003]. One of the Pieniny populations was of hybridogenous origin and shared genetic stock with the Tatra population, indicating there is a local genetic melting pot. A weak genetic structuring of populations among particular regions was found (AMOVA, 4.5%, p > 0.05). This could be an effect of the frequent short-distance and sporadic long-distance gene flow. The dispersal of diaspores between the remote populations in the Western Carpathians and Eastern Carpathians could be affected by the historical transportation of flocks of sheep from the Tatra to Bieszczady Mts.

Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida.

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7', 8' double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.

MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida.

Floral anthocyanin has multiple ecological and economic values, its biosynthesis largely depends on the conserved MYB-bHLH-WD40 (MBW) activation complex and MYB repressors hierarchically with the MBW complex. In contrast to eudicots, the MBW regulatory network model has not been addressed in monocots because of the lack of a suitable system, as grass plants exhibit monotonous floral pigmentation patterns. Presently, the MBW regulatory network was investigated in a non-grass monocot plant, Freesia hybrida. FhMYB27 and FhMYBx with different functional manners were confirmed to be anthocyanin related R2R3 and R3 MYB repressors, respectively. Particularly, FhMYBx could obstruct the formation of positive MBW complex by titrating bHLH proteins, whereas FhMYB27 mainly defected the activator complex into suppressor via its repression domains in C-terminus. Furthermore, the hierarchical and feedback regulatory loop was verified, indicating the synergistic and sophisticated regulatory network underlying Freesia anthocyanin biosynthesis was quite similar to that reported in eudicot plants.

The Conserved and Particular Roles of the R2R3-MYB Regulator FhPAP1 from Freesia hybrida in Flower Anthocyanin Biosynthesis.

Anthocyanin biosynthesis is mainly controlled by MYB-bHLH-WD40 (MBW) complexes that modulate the expression of anthocyanin biosynthetic genes (ABGs). The MYB regulators involved in anthocyanin biosynthesis arose early during plant evolution and thus might function divergently in different evolutionary lineages. Although the anthocyanin-promoting R2R3-MYB regulators in eudicots have been comprehensively explored, little consensus has been reached about functional discrepancies versus conservation among MYB regulators from different plant lineages. Here, we integrated transcriptome analysis, gene expression profiles, gain-of-function experiments and transient protoplast transfection assays to functionally characterize the monocot Freesia hybrida anthocyanin MYB regulator gene FhPAP1, which showed correlations with late ABGs. FhPAP1 could activate ABGs as well as TT8-clade genes FhTT8L, AtTT8 and NtAN1 when overexpressed in Freesia, Arabidopsis and tobacco, respectively. Consistently, FhPAP1 could interact with FhTT8L and FhTTG1 to form the conserved MBW complex and shared similar target genes with its orthologs from Arabidopsis. Most prominently, FhPAP1 displayed higher transactivation capacity than its homologs in Arabidopsis and tobacco, which was instantiated in its powerful regulation on ABGs. Moreover, we found that FhPAP1 might be the selected gene during the domestication and rapid evolution of the wild Freesia species to generate intensive flower pigmentation. These results showed that while the MBW complex was highly evolutionarily conserved between tested monocot and core eudicot plants, participating MYB regulators showed functional differences in transactivation capacity according to their activation domain and played important roles in the flower coloration domestication and evolution of angiosperms.

A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynthesis in both Freesia hybrida and Arabidopsis thaliana.

The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH) and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC) assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L) to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.

GhTCP19 Transcription Factor Regulates Corm Dormancy Release by Repressing GhNCED Expression in Gladiolus.

Dormancy is one of the least understood phenomena in plant biology; however, bud/corm dormancy is an important economic trait in agricultural/horticultural breeding. In this study, we isolated an ABA biosynthesis gene, GhNCED, from the transcriptome database of corm dormancy release (CDR), and characterized its negative role in regulating CDR. To understand transcriptional regulation of GhNCED, yeast one-hybrid screening was conducted and GhTCP19 was identified and shown to regulate GhNCED expression directly. An in planta assay showed that GhTCP19 negatively regulates GhNCED expression. GhTCP19 is dramatically induced by exogenous cytokinins (CKs) and is induced during CDR. Silencing of GhTCP19 in dormant cormels delayed CDR, resulting in higher expression of GhNCED and ABA levels. Meanwhile, endogenous CK biosynthesis and signaling were inhibited in GhTCP19-silenced cormels. Taken together, our results reveal that GhTCP19 is a positive regulator of the CDR process by repressing expression of an ABA biosynthesis gene (GhNCED), promoting CK biosynthesis (GhIPT) and signal transduction (GhARR) as well as inducing cyclin genes. This study expands our knowledge on CDR which is mediated by TCP family members.

GhNAC83 inhibits corm dormancy release by regulating ABA signaling and cytokinin biosynthesis in Gladiolus hybridus.

Corm dormancy is an important trait for breeding in many bulb flowers, including the most cultivated Gladiolus hybridus. Gladiolus corms are modified underground stems that function as storage organs and remain dormant to survive adverse environmental conditions. Unlike seed dormancy, not much is known about corm dormancy. Here, we characterize the mechanism of corm dormancy release (CDR) in Gladiolus. We identified an important ABA (abscisic acid) signaling regulator, GhPP2C1 (protein phosphatase 2C1), by transcriptome analysis of CDR. GhPP2C1 expression increased during CDR, and silencing of GhPP2C1 expression in dormant cormels delayed CDR. Furthermore, we show that GhPP2C1 expression is directly regulated by GhNAC83, which was identified by yeast one-hybrid library screening. In planta assays show that GhNAC83 is a negative regulator of GhPP2C1, and silencing of GhNAC83 promoted CDR. As expected, silencing of GhNAC83 decreased the ABA level, but also dramatically increased cytokinin (CK; zeatin) content in cormels. Binding assays demonstrate that GhNAC83 associates with the GhIPT (ISOPENTENYLTRANSFERASE) promoter and negatively regulates zeatin biosynthesis. Taken together, our results reveal that GhNAC83 promotes ABA signaling and synthesis, and inhibits CK biosynthesis pathways, thereby inhibiting CDR. These findings demonstrate that GhNAC83 regulates the ABA and CK pathways, and therefore controls corm dormancy.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida.

The development of flower scents was a crucial event in biological evolution, providing olfactory signals by which plants can attract pollinators. In this study, bioinformatics, metabolomics, and biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of volatile components in two cultivars of Freesia x hybrida, Red River® and Ambiance, which release different categories of compounds. We found that terpene synthase (TPS) genes were the pivotal genes determining spatiotemporal release of volatile compounds in both cultivars. Eight FhTPS genes were isolated and six were found to be functional: FhTPS1 was a single-product enzyme catalyzing the formation of linalool, whereas the other four FhTPS proteins were multi-product enzymes, among which FhTPS4, FhTPS6, and FhTPS7 could recognize geranyl diphosphate and farnesyl diphosphate simultaneously. The FhTPS enzymatic products closely matched the volatile terpenes emitted from flowers, and significant correlations were found between release of volatile terpenes and FhTPS gene expression. Graphical models based on these results are proposed that summarize the biosynthesis of Freesia floral volatile terpenes. The characterization of FhTPS genes paves the way to decipher their roles in the speciation and fitness of Freesia, and this knowledge could also be used to introduce or enhance scent in other plants.

Characterization of Gladiolus Germplasm Using Morphological, Physiological, and Molecular Markers.

Estimation of variability and genetic relationships among breeding materials is one of the important strategies in crop improvement programs. Morphological (plant height, spike length, a number of florets/spike), physiological (chlorophyll content, chlorophyll fluorescence, and rapid light curve parameters) and Directed amplification of minisatellite DNA (DAMD) markers were used to investigate the relationships among 50 Gladiolus cultivars. Cluster analysis based on morphological data, physiological characteristics, molecular markers, and cumulative data discriminated all cultivars into seven, five, seven, and six clusters in the unweighted pair-group method using arithmetic mean (UPGMA) dendrogram, respectively. The results of the principal coordinate analysis (PCoA) also supported UPGMA clustering. Variations among the Gladiolus cultivars at phenotypic level could be due to the changes in physiology, environmental conditions, and genetic variability. DAMD analysis using 10 primers produced 120 polymorphic bands with 80% polymorphism showing polymorphic information content (PIC = 0.28), Marker index (MI = 3.37), Nei's gene diversity (h = 0.267), and Shannon's information index (I = 0.407). Plant height showed a positive significant correlation with Spike length and Number of florets/spike (r = 0.729, p < 0.001 and r = 0.448, p = 0.001 respectively). Whereas, Spike length showed positive significant correlation with Number of florets/spike (r = 0.688, p < 0.001) and Chlorophyll content showed positive significant correlation with Electron transport rate (r = 0.863, p < 0.001). Based on significant morphological variations, high physiological performance, high genetic variability, and genetic distances between cultivars, we have been able to identify diverse cultivars of Gladiolus that could be the potential source as breeding material for further genetic improvement in this ornamental crop.

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae.

The plastid genome regions of two intergenic spacers, psbA-trnH and trnL-trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA-trnH region was higher than trnL-trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA-trnH and trnL-trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative's lineage. Present study revealed that psbA-trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL-trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.

The complete chloroplast genome sequences of Iris sanguinea donn ex Hornem.

The complete chloroplast genome of Iris sanguinea was sequenced newly in this study. The total chloroplast genome size of I. sanguinea was 152 408 bp, its structure and gene contents were well conserved as typical chloroplast characteristics. Large single copy (LSC) and small single copy (SSC) of 82 340 bp and 18 016 bp, respectively, were separated from a pair of inverted repeats (IRA and IRB) of 26 026 bp. A total of 112 genes, i.e. 78 protein-coding genes, 30 tRNA genes, and four rRNA genes, were encoded in the chloroplast genome of I. sanguinea. Overall GC content of I. sanguinea was 36.83%. Phylogenetic analysis with the reported chloroplast genomes revealed that I. sanguinea is most closely related to I. gatesii.

Isolation and characterization of microsatellite loci in Sisyrinchium (Iridaceae) and cross amplification in other genera.

Recent phylogenetic studies on Sisyrinchium strongly suggest that species classified in section Hydastylus and section Viperella belong to a single group of plants in recent adaptive radiation (Clade IV). These species neither present clear morphological differentiation among them nor show clear identification using DNA barcode markers. Thus, the main goal of this study was to develop a set of polymorphic microsatellite markers compatible for representative species of both sections to ensure variability that could be revealed by SSR markers. Therefore, microsatellite primers were isolated and characterized for Sisyrinchium palmifolium and S. marchioides. In addition, transferability of the developed primers was tested in Iridoideae, primarily in closely related species of Sisyrinchium. Sixteen microsatellite loci were developed from enriched genomic libraries, of which ten were polymorphic. GST values indicated higher differentiation among subpopulations of S. palmifolium than those from S. marchioides. Major transferability was obtained using primers isolated from S. marchioides. All primers exhibited higher rates of cross-amplification for species belonging to Clade IV of Sisyrinchium, as well as to the genera Calydorea and Herbertia. These developed microsatellite markers can be used as an efficient tool for characterization of genetic variability in species belonging to Iridoideae, as well as for studies on population dynamics, genetic structure, and mating system in other Sisyrinchium species.

Two IIIf Clade-bHLHs from Freesia hybrida Play Divergent Roles in Flavonoid Biosynthesis and Trichome Formation when Ectopically Expressed in Arabidopsis.

The MBW complex, comprised by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40, is a single regulatory protein complex that drives the evolution of multiple traits such as flavonoid biosynthesis and epidermal cell differentiation in plants. In this study, two IIIf Clade-bHLH regulator genes, FhGL3L and FhTT8L, were isolated and functionally characterized from Freesia hybrida. Different spatio-temporal transcription patterns were observed showing diverse correlation with anthocyanin and proanthocyanidin accumulation. When overexpressed in Arabidopsis, FhGL3L could enhance the anthocyanin accumulation through up-regulating endogenous regulators and late structural genes. Unexpectedly, trichome formation was inhibited associating with the down-regulation of AtGL2. Comparably, only the accumulation of anthocyanins and proanthocyanidins was strengthened in FhTT8L transgenic lines. Furthermore, transient expression assays demonstrated that FhGL3L interacted with AtPAP1, AtTT2 and AtGL1, while FhTT8L only showed interaction with AtPAP1 and AtTT2. In addition, similar activation of the AtDFR promoter was found between AtPAP1-FhGL3L/FhTT8L and AtPAP1- AtGL3/AtTT8 combinations. When FhGL3L was fused with a strong activation domain VP16, it could activate the AtGL2 promoter when co-transfected with AtGL1. Therefore, it can be concluded that the functionality of bHLH factors may have diverged, and a sophisticated interaction and hierarchical network might exist in the regulation of flavonoid biosynthesis and trichome formation.

[INTRASPECIFIC CHROMOSOME POLYMORPHISM OF IRIS PUMILA L. FROM THE TERRITORY OF UKRAINE].

Cytogenetic analysis of Iris pumila L. plants from different habitats in Ukraine revealed the intrachromosomal polymorphism. Chromosome number 2n = 32 was established for plants of this species. Root meristem of I. pumila seedlings of all populations showed high level of mixoploidy. The differences in the percentage of aneuploid cells and anaphase aberration were found between the sampled populations. Anaphase analysis revealed the level of chromosomal rearrangements that reached 9.2% in some populations reached.

Transcriptome Profiling of Louisiana iris Root and Identification of Genes Involved in Lead-Stress Response.

Louisiana iris is tolerant to and accumulates the heavy metal lead (Pb). However, there is limited knowledge of the molecular mechanisms behind this feature. We describe the transcriptome of Louisiana iris using Illumina sequencing technology. The root transcriptome of Louisiana iris under control and Pb-stress conditions was sequenced. Overall, 525,498 transcripts representing 313,958 unigenes were assembled using the clean raw reads. Among them, 43,015 unigenes were annotated and their functions classified using the euKaryotic Orthologous Groups (KOG) database. They were divided into 25 molecular families. In the Gene Ontology (GO) database, 50,174 unigenes were categorized into three GO trees (molecular function, cellular component and biological process). After analysis of differentially expressed genes, some Pb-stress-related genes were selected, including biosynthesis genes of chelating compounds, metal transporters, transcription factors and antioxidant-related genes. This study not only lays a foundation for further studies on differential genes under Pb stress, but also facilitates the molecular breeding of Louisiana iris.

Flower color polymorphism maintained by overdominant selection in Sisyrinchium sp.

Negative frequency-dependent selection derived from positive frequency-dependent foraging is the best-known selection force maintaining genetic polymorphism within a population. However, in flowering plants, positive frequency-dependent foraging by pollinators is expected to accelerate the loss of low-frequency morphs by conferring a fitness advantage to the common morph, leading to monomorphism. In Japan, a non-native species, Sisyrinchium sp., exhibits conspicuous flower color polymorphism within a population comprising both purple morphs (homozygous recessive) and white morphs (heterozygous or homozygous dominant). Here we quantified genotype-specific reproductive success in order to reveal the contribution of overdominant selection on the maintenance of flower color polymorphism in this species. In artificial pollination experiments using individuals with identified genotypes, female reproductive success was higher in the heterozygote than in either homozygote. The frequency of purple morphs in natural populations (ca. 31%) is similar to the frequency predicted by overdominant selection (25%). Our results suggest that overdominant selection contributes to the maintenance of color morphs in the natural population of this species.