Polymorphisms in two key anthocyanic genes of clivia (Clivia miniata L.) reveal evidence of selection and possible association with flower pigmentation.

Members of the genus Clivia show considerable variation in flower pigmentation and morphology. Such variation is affected by mutations that emerge in candidate flower development genes over time. Besides population history, mutations can further illuminate the effects of demographic events in populations in addition to population genetic parameters including selection, recombination, and linkage disequilibrium (LD). The current study aimed to find sequence variants in 2 anthocyanin biosynthetic genes (DFR and bHLH) of Clivia miniata and use the data to assess population genetic factors from a random collection of orange/red- and yellow-flowered specimens. Overall, average nucleotide diversity in the 2 anthocyanin genes was moderate (π = 0.00646), whereas haplotypes differed significantly (Hd ≥ 0.9). Gene evolution was seemingly driven by mutations (CmiDFR) or recombinations (CmibHLH001). LD decayed swiftly within the analyzed gene regions and supported the feasibility of assessing trait-variant associations via the association/linkage mapping approach. In the end, most associations were found to be spurious, but 1 haplotype in CmibHLH001 showed a promising correlation to the orange/red flower phenotype in Clivia specimens. In all, the present study is the first to measure gene-level diversity in C. miniata-data that had never been reported so far. Furthermore, the study also identified allelic and haplotypic variants that may be beneficial in future association genetic studies of Clivia. Such studies, however, consider large diverse populations to control for statistical bias intrinsic to the analysis of small datasets.

Reciprocal natural hybridization between Lycoris aurea and Lycoris radiata (Amaryllidaceae) identified by morphological, karyotypic and chloroplast genomic data.

BACKGROUND: Hybridization is considered as an important model of speciation, but the evolutionary process of natural hybridization is still poorly characterized in Lycoris. To reveal the phylogenetic relationship of two new putative natural hybrids in Lycoris, morphological, karyotypic and chloroplast genomic data of four Lycoris species were analyzed in this study. RESULTS: Two putative natural hybrids (2n = 18 = 4 m + 5t + 6st + 3 T) possessed obvious heterozygosity features of L. radiata (2n = 22 = 10t + 12st) and L. aurea (2n = 14 = 8 m + 6 T) in morphology (e.g. leaf shape and flower color), karyotype (e.g. chromosome numbers, CPD/DAPI bands, 45S rDNA-FISH signals etc.) and chloroplast genomes. Among four Lycoris species, the composition and structure features of chloroplast genomes between L. radiata and the putative natural hybrid 1 (L. hunanensis), while L. aurea and the hybrid 2, were completely the same or highly similar, respectively. However, the features of the cp genomes between L. radiata and the hybrid 2, while L. aurea and the hybrid 1, including IR-LSC/SSC boundaries, SSRs, SNPs, and SNVs etc., were significantly different, respectively. Combining the karyotypes and cp genomes analysis, we affirmed that the natural hybrid 1 originated from the natural hybridization of L. radiata (♀) × L. aurea (♂), while the natural hybrid 2 from the hybridization of L. radiata (♂) × L. aurea (♀). CONCLUSION: The strong evidences for natural hybridization between L. radiata (2n = 22) and L. aurea (2n = 14) were found based on morphological, karyotypic and chloroplast genomic data. Their reciprocal hybridization gave rise to two new taxa (2n = 18) of Lycoris. This study revealed the origin of two new species of Lycoris and strongly supported the role of natural hybridization that facilitated lineage diversification in this genus.

Chimeric deletion mutation of rpoC2 underlies the leaf-patterning of Clivia miniata var. variegata.

KEY MESSAGE: The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing chloroplast biogenesis and thylakoid membrane development. Clivia miniata var. variegata (Cmvv) is a common mutant of Clivia miniata but its genetic basis is unclear. Here, we found that a 425 bp deletion mutation of chloroplast rpoC2 underlies the yellow stripes (YSs) of Cmvv. Both RNA polymerase PEP and NEP coexist in seed-plant chloroplasts and the ß″ subunit of PEP is encoded by rpoC2. The rpoC2 mutation changed the discontinuous cleft domain required to form the PEP central cleft for DNA binding from 1103 to 59 aa. RNA-Seq revealed that 28 chloroplast genes (cpDEGs) were all down-regulated in YSs, of which, four involved in chloroplast protein translation and 21 of photosynthesis system (PS)I, PSII, cytochrome b6/f complex and ATP synthase are crucial for chloroplast biogenesis/development. The accuracy and reliability of RNA-Seq was verified by qRT-PCR. Moreover, the chlorophyll (Chl) a/b content, ratio of Chla/Chlb and photosynthetic rate (Pn) of YS decreased significantly. Meanwhile, chloroplasts of the YS mesophyll cells were smaller, irregular in shape, contain almost no thylakoid membrane, and even proplastid was found in YS. These findings indicate that the rpoC2 mutation down-regulated expression of the 28 cpDEGs, which disturb chloroplast biogenesis and its thylakoid membrane development. Thus, there are not enough PSI and II components to bind Chl, so that the corresponding areas of the leaf are yellow and show a low Pn. In this study, the molecular mechanism of three phenotypes of F1 (Cmvv ♀ × C. miniata ♂) was revealed, which lays a foundation for the breeding of variegated plants.

Phylogeny and adaptive evolution of subgenus Rhizirideum (Amaryllidaceae, Allium) based on plastid genomes.

The subgenus Rhizirideum in the genus Allium consists of 38 species worldwide and forms five sections (A. sect. Rhizomatosa, A. sect. Tenuissima, A. sect. Rhizirideum, A. sect. Eduardia, and A. sect. Caespitosoprason), A. sect. Caespitosoprason being merged into A. sect. Rhizomatosa recently. Previous studies on this subgenus mainly focused on separate sections. To investigate the inter-section and inter-subgenera phylogenetic relationships and adaptive evolution of A. subg. Rhizirideum, we selected thirteen representative species, which cover five sections of this subgenus and can represent four typical phenotypes of it. We conducted the comparative plastome analysis with our thirteen plastomes. And phylogenetic inferences with CDSs and complete sequences of plastomes of our thirteen species and another fifty-four related species were also performed. As a result, the A. subg. Rhizirideum plastomes were relatively conservative in structure, IR/SC borders, codon usage, and repeat sequence. In phylogenetic results, the inter-subgenera relationships among A. subg. Rhizirideum and other genus Allium subgenera were generally similar to the previous reports. In contrast, the inter-section relationships within our subgenus A. subg. Rhizirideum were newly resolved in this study. A. sect. Rhizomatosa and A. sect. Tenuissima were sister branches, which were then clustered with A. sect. Rhizirideum and A. sect. Eduardia successively. However, Allium Polyrhizum Turcz. ex Regel, type species of A. sect. Caespitosoprason, was resolved as the basal taxon of A. subg. Rhizirideum. Allium siphonanthum J. M. Xu was also found in clade A. subg. Cyathophora instead of clade A. subg. Rhizirideum. The selective pressure analysis was also conducted, and most protein-coding genes were under purifying selection. At the same time, just one gene, ycf2, was found under positive selection, and another three genes (rbcL, ycf1a, ycf1b) presented relaxed selection, which were all involved in the photosynthesis. The low temperature, dry climate, and high altitude of the extreme habitats where A. subg. Rhizirideum species grow might impose intense natural selection forces on their plastome genes for photosynthesis. In summary, our research provides new insights into the phylogeny and adaptive evolution of A. subg. Rhizirideum. Moreover, we suggest that the positions of the A. subg. Rhizirideum species A. polyrhizum and A. siphonanthum should be reconsidered.

Comparative Analysis of the Complete Chloroplast Genomes in Allium Section Bromatorrhiza Species (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution.

With the development of molecular sequencing approaches, many taxonomic and phylogenetic problems of the genus Allium L. have been solved; however, the phylogenetic relationships of some subgenera or sections, such as section Bromatorrhiza, remain unresolved, which has greatly impeded our full understanding of the species relationships among the major clades of Allium. In this study, the complete chloroplast (cp) genomes of nine species in the Allium sect. Bromatorrhiza were determined using the Illumina paired-end sequencing, the NOVOPlasty de novo assembly strategy, and the PGA annotation method. The results showed that the cp genome exhibited high conservation and revealed a typical circular tetrad structure. Among the sect. Bromatorrhiza species, the gene content, SSRs, codon usage, and RNA editing site were similar. The genome structure and IR regions' fluctuation were investigated while genes, CDSs, and non-coding regions were extracted for phylogeny reconstruction. Evolutionary rates (Ka/Ks values) were calculated, and positive selection analysis was further performed using the branch-site model. Five hypervariable regions were identified as candidate molecular markers for species authentication. A clear relationship among the sect. Bromatorrhiza species were detected based on concatenated genes and CDSs, respectively, which suggested that sect. Bromatorrhiza is monophyly. In addition, there were three genes with higher Ka/Ks values (rps2, ycf1, and ycf2), and four genes (rpoC2, atpF, atpI, and rpl14) were further revealed to own positive selected sites. These results provide new insights into the plastome component, phylogeny, and evolution of Allium species.

Complete genome organization and characterization of Hippeastrum latent virus.

The complete genome sequences of two carlaviruses were determined by high-throughput sequencing of RNA extracted from ringspot and mosaic, disease symptoms on leaves of spider lily plants (Crinum asiaticum, family Amaryllidaceae) growing as landscape plants in Hawaii. One, named Nerine latent virus (NeLV)-Hawaii with a genome of 8281 nucleotide exhibited the highest nucleotide identity and amino acid similarity of 95.5% and 96.0%, respectively, to the genome sequence of an isolate of NeLV from Narcissus sp. in Australia (JQ395044). The second, named Hippeastrum latent virus (HiLV)-Hawaii with a genome of 8497 nucleotides exhibited the highest nucleotide identity and amino acid similarity, 84.3% and 88.7%, respectively, to the sequence of a previously uncharacterized HiLV isolate from a potted flowering plant, Amaryllis (Hippeastrum hybridum Hort) in Taiwan (DQ098905). The amino acid sequence similarities of replicase (Rep) and coat protein (CP) between HiLV-Hawaii and NeLV-Hawaii were 44.8% and 38.4%, respectively. Results of viral protein Rep and CP amino acid sequence comparisons from various carlaviruses provide evidence that HiLV and NeLV, previously classified as synonymous viruses are in fact unique viruses. This is the first report for the complete sequence, organization, and phylogenetic characterization of HiLV and the first detection of HiLV both in C. asiaticum and in the USA.

Comparative Plastome Analysis of Three Amaryllidaceae Subfamilies: Insights into Variation of Genome Characteristics, Phylogeny, and Adaptive Evolution.

In the latest APG IV classification system, Amaryllidaceae is placed under the order of Asparagus and includes three subfamilies: Agapanthoideae, Allioideae, and Amaryllidoideae, which include many economically important crops. With the development of molecular phylogeny, research on the phylogenetic relationship of Amaryllidaceae has become more convenient. However, the current comparative analysis of Amaryllidaceae at the whole chloroplast genome level is still lacking. In this study, we sequenced 18 Allioideae plastomes and combined them with publicly available data (a total of 41 plastomes), including 21 Allioideae species, 1 Agapanthoideae species, 14 Amaryllidoideae species, and 5 Asparagaceae species. Comparative analyses were performed including basic characteristics of genome structure, codon usage, repeat elements, IR boundary, and genome divergence. Phylogenetic relationships were detected using single-copy genes (SCGs) and ribosomal internal transcribed spacer sequences (ITS), and the branch-site model was also employed to conduct the positive selection analysis. The results indicated that all Amaryllidaceae species showed a highly conserved typical tetrad structure. The GC content and five codon usage indexes in Allioideae species were lower than those in the other two subfamilies. Comparison analysis of Bayesian and ML phylogeny based on SCGs strongly supports the monophyly of three subfamilies and the sisterhood among them. Besides, positively selected genes (PSGs) were detected in each of the three subfamilies. Almost all genes with significant posterior probabilities for codon sites were associated with self-replication and photosynthesis. Our study investigated the three subfamilies of Amaryllidaceae at the whole chloroplast genome level and suggested the key role of selective pressure in the adaptation and evolution of Amaryllidaceae.

Use of deep sequencing to profile small RNAs derived from tomato spotted wilt orthotospovirus and hippeastrum chlorotic ringspot orthotospovirus in infected Capsicum annuum.

Virus-derived small RNAs are one of the key factors of RNA silencing in plant defence against viruses. We obtained virus-derived small interfering RNA profiles from Tomato spotted wilt orthotospovirus and Hippeastrum chlorotic ringspot orthotospovirus infected Capsicum annuum XX19 and XY11 by deep sequencing one day after inoculation. The vsiRNAs data were mapped to the TSWV and HCRV genomes, and the results showed that the vsiRNAs measured 19-24 nucleotides in length. Most of the vsiRNAs were mapped to the S segment of the viral genome. For XX19 and XY11 infected with HCRV, the distribution range of vsiRNAs in S RNA was 52.06-55.20%, while for XX19 and XY11 infected with TSWV, the distribution range of vsiRNAs in S RNA was 87.76-89.07%. The first base at the 5' end of the siRNA from TSWV and HCRV was primarily biased towards A, U, or C. Compared with mock-inoculated XX19 and XY11, the expression level of CaRDR1 was upregulated in TSWV- and HCRV-inoculated XX19 and XY11. CaAGO2 and CaAGO5 were upregulated in XY11 against HCRV infection, and CaRDR2 was downregulated in TSWV-infected XY11 and XX19. The profile of HCRV and TSWV vsiRNA verified in this study could be useful for selecting key vsiRNA such as those in disease-resistant varieties by artificially synthesizing amiRNA.

Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox.

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 µM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.

Complete chloroplast genomes shed light on phylogenetic relationships, divergence time, and biogeography of Allioideae (Amaryllidaceae).

Allioideae includes economically important bulb crops such as garlic, onion, leeks, and some ornamental plants in Amaryllidaceae. Here, we reported the complete chloroplast genome (cpDNA) sequences of 17 species of Allioideae, five of Amaryllidoideae, and one of Agapanthoideae. These cpDNA sequences represent 80 protein-coding, 30 tRNA, and four rRNA genes, and range from 151,808 to 159,998 bp in length. Loss and pseudogenization of multiple genes (i.e., rps2, infA, and rpl22) appear to have occurred multiple times during the evolution of Alloideae. Additionally, eight mutation hotspots, including rps15-ycf1, rps16-trnQ-UUG, petG-trnW-CCA, psbA upstream, rpl32-trnL-UAG, ycf1, rpl22, matK, and ndhF, were identified in the studied Allium species. Additionally, we present the first phylogenomic analysis among the four tribes of Allioideae based on 74 cpDNA coding regions of 21 species of Allioideae, five species of Amaryllidoideae, one species of Agapanthoideae, and five species representing selected members of Asparagales. Our molecular phylogenomic results strongly support the monophyly of Allioideae, which is sister to Amaryllioideae. Within Allioideae, Tulbaghieae was sister to Gilliesieae-Leucocoryneae whereas Allieae was sister to the clade of Tulbaghieae- Gilliesieae-Leucocoryneae. Molecular dating analyses revealed the crown age of Allioideae in the Eocene (40.1 mya) followed by differentiation of Allieae in the early Miocene (21.3 mya). The split of Gilliesieae from Leucocoryneae was estimated at 16.5 mya. Biogeographic reconstruction suggests an African origin for Allioideae and subsequent spread to Eurasia during the middle Eocene. Cool and arid conditions during the late Eocene led to isolation between African and Eurasian species. African Allioideae may have diverged to South American taxa in the late Oligocene. Rather than vicariance, long-distance dispersal is the most likely explanation for intercontinental distribution of African and South American Allioideae species.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.

Relative expression of putative genes involved in galanthamine and other Amaryllidaceae alkaloids biosynthesis in Narcissus field and in vitro tissues.

The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20 mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4 mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310 µg Gal/g FW) and bulb (980-1150 µg Gal/g FW) than the culture derived samples; callus (1.0-7.0 µg Gal/g FW) and bulblets (12-215 µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpO4OMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.

Assessment of differences in morphological and physiological leaf lodging characteristics between two cultivars of Hippeastrum rutilum.

BACKGROUND: Environmental lodging stress, which is a result of numerous factors, is characterized by uncertainty. However, several studies related to lodging in cereal crops have reported that lodging in the Hippeastrum rutilum environment is very rare. Hippeastrum rutilum is a garden flower with high ornamental value and abundant germplasm resources. Under past cultivation practices, it was found that the plant types of 'Red Lion', with red flowers, and 'Apple Blossom', with pink flowers, are quite different. The leaves of 'Red Lion' are upright, while the leaves of 'Apple Blossom' show lodging, which seriously affects its ornamental value. The aims of this study were to compare the differences between the two varieties with leaf lodging and upright leaves according to morphological and physiological attributes. In this study, karyotype analysis and phenotypic morphological and physiological characteristics were compared to explore the differences between the two plant types. RESULTS: The karyotype analysis of the two cultivars showed that their chromosome types were both tetraploid plants. The results showed that the lignin content in the leaves of 'Red Lion' was high, the cross-sectional structure of the leaf vascular bundle was more stable, and the chlorophyll content was high. In addition, significantly less energy was transferred to the electron transport chain (ETR) during the photoreaction. Similarly, the results regarding the maximum photosynthetic rate (Fv/Fm), nonphotochemical quenching (NPQ) and effective quantum yield of photosystem II photochemistry (△F/Fm') all indicated that the photosynthetic capacity of "Red Lion" was greater than that of "Apple Blossom", which was affected by leaf lodging. The size of the leaves was significantly smaller, and the leaf sag angle, leaf width, and leaf tip angle presented significantly lower values in 'Red Lion' than in 'Apple Blossom', which exhibits leaf sag. The difference in these factors may be the reason for the different phenotypes of the two cultivars. CONCLUSION: The results of this study proved that lodging affects the photosynthetic capacity of Hippeastrum rutilum and revealed some indexes that might be related to leaf lodging, laying a theoretical foundation for cultivating and improving new varieties.

Comparative Analysis of the Complete Chloroplast Genomes in Allium Subgenus Cyathophora (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution.

Recent advances in molecular phylogenetics provide us with information of Allium L. taxonomy and evolution, such as the subgenus Cyathophora, which is monophyletic and contains five species. However, previous studies detected distinct incongruence between the nrDNA and cpDNA phylogenies, and the interspecies relationships of this subgenus need to be furtherly resolved. In our study, we newly assembled the whole chloroplast genome of four species in subgenus Cyathophora and two allied Allium species. The complete cp genomes were found to possess a quadripartite structure, and the genome size ranged from 152,913 to 154,174 bp. Among these cp genomes, there were subtle differences in the gene order, gene content, and GC content. Seven hotspot regions (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) with nucleotide diversity greater than 0.02 were discovered. The selection analysis showed that some genes have elevated Ka/Ks ratios. Phylogenetic analysis depended on the complete chloroplast genome (CCG), and the intergenic spacer regions (IGS) and coding DNA sequences (CDS) showed same topologies with high support, which revealed that subgenus Cyathophora was a monophyletic group, containing four species, and A. cyathophorum var. farreri was sister to A. spicatum with 100% bootstrap value. Our study revealed selective pressure may exert effect on several genes of the six Allium species, which may be useful for them to adapt to their specific living environment. We have well resolved the phylogenetic relationship of species in the subgenus Cyathophora, which will contribute to future evolutionary studies or phylogeographic analysis of Allium.

CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication.

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant.

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Despite the interest in these compounds, many steps of the biosynthetic pathway have not been elucidated. In this study, we identified the alkaloids produced in different organs of R. bifida under different growth conditions, set up the conditions for in vitro R. bifida regeneration and initiated the molecular characterization of two R. bifida genes involved in alkaloids biosynthesis: the Norbelladine 4'-O-Methyltransferase (RbN4OMT) and the Cytochrome P450 (RbCYP96T). We show that montanine is the main alkaloid produced in the different R. bifida organs and developed a direct organogenesis regeneration protocol, using twin-scale explants cultivated on media enriched with naphthalene acetic acid and benzyladenine. Finally, we analyzed the RbN4OMT and RbCYP96T gene expressions in different organs and culture conditions and compared them to alkaloid production. In different organs of R. bifida young, adult and regenerated plants, as well as under various growing conditions, the transcripts accumulation was correlated with the production of alkaloids. This work provides new tools to improve the production of this important pharmaceutical compound and for future biotechnological studies.

Y2SK2 and SK3 type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants.

Two dehydrins from Agapanthus praecox (ApY2SK2 and ApSK3) show important protective effects under complex stresses. Both ApY2SK2 and ApSK3 contain one intron and consist of a full-length cDNA of 981 bp and 1057 bp encoding 186 and 215 amino acids, respectively. ApY2SK2 and ApSK3 transgenic Arabidopsis thaliana show reduced plasma membrane damage and ROS levels and higher antioxidant activity and photosynthesis capability under salt, osmotic, cold and drought stresses compared with the wild-type. ApY2SK2 and ApSK3 are mainly located in the cytoplasm and cell membrane, and ApY2SK2 can even localize in the nucleus. In vitro tests indicate that ApY2SK2 and ApSK3 can effectively protect enzyme activity during the freeze-thaw process, and ApY2SK2 also exhibits this function during desiccation treatment. Furthermore, ApY2SK2 and ApSK3 can significantly inhibit hydroxyl radical generation. These two dehydrins can bind metal ions with a binding affinity of Co2+> Ni2+> Cu2+> Fe3+; the binding affinity of ApSK3 is higher than that of ApY2SK2. Thus, ApY2SK2 has a better protective effect on enzyme activity, and ApSK3 has stronger metal ion binding function and effect on ROS metabolism. Moreover, plant cryopreservation evaluation tests indicate that ApY2SK2 and ApSK3 transformation can enhance the seedling survival ratio from 23% to 47% and 55%, respectively; the addition of recombinant ApY2SK2 and ApSK3 to plant vitrification solution may increase the survival ratio of wild-type A. thaliana seedlings from 24% to 50% and 46%, respectively. These findings suggest that ApY2SK2 and ApSK3 can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications.

Cloning and characterization of norbelladine synthase catalyzing the first committed reaction in Amaryllidaceae alkaloid biosynthesis.

BACKGROUND: Amaryllidaceae alkaloids (AAs) are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties. Previous investigations on AA biosynthesis have revealed that all AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. Specifically, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis. RESULTS: With the availability of wild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes for a small protein of 19 kDa with an anticipated pI of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PR10/Bet v1 proteins and shared 41% amino acid identity to opium poppy NCS1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry. CONCLUSIONS: Here, we describe a novel enzyme catalyzing the first committed step of AA biosynthesis, which will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.

Comparative transcriptome profiling of genes and pathways involved in leaf-patterning of Clivia miniata var. variegata.

Clivia miniata var. variegata (Cmvv) typically possesses yellow- and green-striped leaves. The striped plant not only has a high ornamental value but also be suitable for photosynthesis and chloroplast development research. Our previous study had revealed that yellow stripes (YSs) of Cmvv leaves contain chlorophyll-less ineffective chloroplasts. However, mechanism of Cmvv variegation is yet to be investigated. In the study, transcriptomes of both the YSs and green stripes (GSs) from single Cmvv leaves were compared using high-throughput sequencing. A total of 688 differential expression genes (DEGs) were identified based on biological replications. The qRT-PCR results indicated that transcriptome profiles accurately reflected global transcriptome differences between YSs and GSs. Subcellular localization analysis suggested that 56 DEG proteins were targeted to chloroplasts, and might be involved in anterograde signaling and leaf patterning. Moreover, the DEGs were mostly enriched in photosynthesis and plant-pathogen interaction KEGG pathways. Meanwhile, there should be coordination interaction between the two pathways. Seven of the eight DEGs involved in photosynthesis KEGG pathway were chloroplast-encoded genes and distributed among different cistrons of chloroplast DNA (cpDNA) large single copy regions (LSC) which are more prone to mutation. It was proposed that the YSs were caused by mutation(s) in cpDNA LSC. Thus, when the primary zygote of Cmvv was chimeric in LSC, leaf might be yellow- and green-striped. The study would give new insights into plant variegation and offers candidate genes to guide future research attempting to breed variegated plants.

Revealing the complex genetic structure of cultivated amaryllis (Hippeastrum hybridum) using transcriptome-derived microsatellite markers.

Although amaryllis (Hippeastrum hybridum) plants are commonly used in physiological and ecological research, the extent of their genomic and genetic resources remains limited. The development of molecular markers is therefore of great importance to accelerate genetic improvements in Hippeastrum species. In this study, a total of 269 unique genes were defined that might regulate the flower spathe development of amaryllis. In addition, 2000 simple sequence repeats (SSRs) were detected based on 171,462 de novo assembled unigenes from transcriptome data, and 66,4091 single nucleotide polymorphisms (SNPs) were also detected as putative molecular markers. Twenty-one SSR markers were screened to evaluate the genetic diversity and population structure of 104 amaryllis accessions. A total of 98 SSR loci were amplified for all accessions. The results reveal that Nei's gene diversity (H) values of these markers ranged between 0.055 and 0.394, whereas the average values of Shannon's Information index (I) ranged between 0.172 and 0.567. Genetic tree analysis further demonstrates that all accessions can be grouped into three main clusters, which can be further divided into two subgroups. STRUCTURE-based analysis revealed that the highest ΔK values were observed when K = 5, K = 6, K = 7 and K = 8. The results of this study enable large-scale transcriptomics and classification of Hippeastrum genetic polymorphisms and will be useful in the future for resource conservation and production.