Characterization, validation, and cross-species transferability of EST-SSR markers developed from Lycoris aurea and their application in genetic evaluation of Lycoris species.

BACKGROUND: The Lycoris genus includes many ornamentally and medicinally important species. Polyploidization and hybridization are considered modes of speciation in this genus, implying great genetic diversity. However, the lack of effective molecular markers has limited the genetic analysis of this genus. RESULTS: In this study, mining of EST-SSR markers was performed using transcriptome sequences of L. aurea, and 839 primer pairs for non-redundant EST-SSRs were successfully designed. A subset of 60 pairs was randomly selected for validation, of which 44 pairs could amplify products of the expected size. Cross-species transferability of the 60 primer pairs among Lycoris species were assessed in L. radiata Hreb, L. sprengeri Comes ex Baker, L. chinensis Traub and L. anhuiensis, of which between 38 to 77% of the primers were able to amplify products in these Lycoris species. Furthermore, 20 and 10 amplification products were selected for sequencing verification in L. aurea and L. radiata respectively. All products were validated as expected SSRs. In addition, 15 SSRs, including 10 sequence-verified and 5 unverified SSRs were selected and used to evaluate the genetic diversity of seven L. radiata lines. Among these, there were three sterile lines, three fertile lines and one line represented by the offspring of one fertile line. Unweighted pair group method with arithmetic mean analysis (UPGMA) demonstrated that the outgroup, L. aurea was separated from L. radiata lines and that the seven L. radiata lines were clustered into two groups, consistent with their fertility. Interestingly, even a dendrogram with 34 individuals representing the seven L. radiata lines was almost consistent with fertility. CONCLUSIONS: This study supplies a pool of potential 839 non-redundant SSR markers for genetic analysis of Lycoris genus, that present high amplification rate, transferability and efficiency, which will facilitate genetic analysis and breeding program in Lycoris.

Complete Chloroplast Genomes and Comparative Analyses of L. chinensis, L. anhuiensis, and L. aurea (Amaryllidaceae).

The genus Lycoris (about 20 species) includes important medicinal and ornamental plants. Due to the similar morphological features and insufficient genomic resources, germplasm identification and molecular phylogeny analysis are very limited. Here, we sequenced the complete chloroplast genomes of L. chinensis, L. anhuiensis, and L. aurea; they have very similar morphological traits that make it difficult to identify. The full length of their cp genomes was nearly 158k bp with the same guanine-cytosine content of 37.8%. A total of 137 genes were annotated, including 87 protein-coding genes, 42 tRNAs, and eight rRNAs. A comparative analysis revealed the conservation in sequence size, GC content, and gene content. Some variations were observed in repeat structures, gene expansion on the IR-SC (Inverted Repeat-Single-Copy) boundary regions. Together with the cpSSR (chloroplast simple sequence repeats), these genetic variations are useful to develop molecular markers for germplasm identification. Phylogenetic analysis showed that seven Lycoris species were clustered into a monophyletic group, and closed to Narcissus in Amaryllidaceae. L. chinensis, L. anhuiensis, and L. longituba were clustered together, suggesting that they were very likely to be derived from one species, and had the same ancestor with L. squamigera. Our results provided information on the study of genetic diversity, origins or relatedness of native species, and the identification of cultivars.

An electrochemical method for plant species determination and classification based on fingerprinting petal tissue.

The identification of plant species not only is a hobby but also has important application value in plant resources science. Traditional plant identification often relies on the experience of botanists. The infrageneric identification of plants is easily mistaken due to similarities in organ features. In this work, we propose an electrochemical method to obtain fingerprints of plant petal tissue. Fourteen species of Lycoris were used as a model for validating this methodology. Pattern and color recognition were established for visualization of electrochemical fingerprints recorded after various solvent extractions. In addition, the infrageneric relationships of these Lycoris species were deduced from the electrochemical fingerprints since the type and content of electroactive compounds in plants are controlled by genes. The results indicate that the electrochemical fingerprints of Lycoris petals are correlated with the infrageneric relationships of native Lycoris species.

Discovery and characterisation of lycorine-type alkaloids in Lycoris spp. (Amaryllidaceae) using UHPLC-QTOF-MS.

INTRODUCTION: Lycorine, one of the most common alkaloids in Lycoris spp., is believed to possess pharmacological activity. OBJECTIVE: To discover and identify lycorine-type alkaloids in the crude extracts of bulbs from six Lycoris spp. by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) detection. METHODOLOGY: A qualitative analytical method with a data mining strategy was utilised. Based on the fragmentation patterns of standards investigated in positive tandem mass spectrometry (MS/MS) mode, the fragmentation rules of lycorine-type alkaloids were summarised. These types of alkaloids were additionally classified as different subtypes based on structural features and MS/MS fragmentation patterns, and the diagnostic ions for characterisation of different subtypes of alkaloids were designated. RESULTS: Thirty-seven lycorine type alkaloids, including 16 previously undescribed compounds, were efficiently screened out and tentatively identified from the crude extracts of six Lycoris spp. Lycoris sprengri may be a preferable species for studying or extracting lycorine-type alkaloids because of elevated relative concentrations and highest diversity of alkaloids. CONCLUSION: The UHPLC-QTOF-MS and MS/MS data-mining strategy proved useful for the detection and tentative identification of lycorine-type alkaloids in bulbs of Lycoris spp. and could be extended to other Amaryllidaceae genera. The consequent profiling of the lycorine-type alkaloids will be useful in the quality control of raw materials of Lycoris species and the exploration of superior species.

Enhanced electrochemical voltammetric fingerprints for plant taxonomic sensing.

Graphene-embedded plant tissues show a high sensitivity to electrochemical signals, which enables a screen-printed electrode to be used for electrochemical fingerprint recording. The electrochemical fingerprints obtained under different conditions can be transformed into multidimensional recognition modes for plant identification. These electrochemical fingerprints reflect the types and quantities of the electrochemically active substances in plant tissues such that the fingerprints can be used for chemotaxonomic investigations. In this paper, five species of Lycoris bulbs, including L. chinensis, L. radiate, L. aurea, L. sprengeri and L. straminea, were successfully recognized by electrochemical fingerprinting. The species's interspecific relationships were also investigated. L. chinensis and L. aurea show highly similar morphology but have a relatively distant relationship. Hybridized L. radiata shows a notably close relationship with L. straminea, suggesting that one of its parents may be L. radiata. In addition, L. chinensis also shows a close relationship with L. straminea, suggesting that the L. straminea may be produced by cross-breeding L. chinensis and L. radiate. The results mentioned above indicate that the proposed electro-chemotaxonomic methodology is an inexpensive and quick taxonomic method that can provide additional evidence for the existing taxonomy system.

Karyotype studies on Lycoris radiata populations from China.

Lycoris radiata is an important medicinal and ornamental plant of China. In the present study, somatic chromosome counts and karyotype analyses, which are important aspects of plant phylogeny and evolution, were performed in 466 individuals from 25 L. radiata populations by root tip squash method. Chromosome counts revealed that 10 populations were diploid (2n = 2x = 22) and 15 were triploid (2n = 3x = 33). Except for one diploid population containing some triploid plants, the remaining 24 populations showed a single cytotype. Karyotype analysis showed that the karyotypes of L. radiata varied in different populations and even within the same population. However, based on the Stebbins' system, the karyotype of all the populations could be classified in 4A classes. The cluster analysis and ordination methods demonstrated that the L. radiata populations grouped in two major clusters. Previous research has shown that the triploid strain of L. radiata is a genetically identical species. However, the cluster analysis revealed that the triploid strains clustered in two groups instead of one, which indicates that these strains may not be identical species, genetically. This study is expected to improve the understanding of the genetic diversity in L. radiata and provide a basis for future studies on species differentiation, speciation, and taxonomy.

Analysis of bioactive Amaryllidaceae alkaloid profiles in Lycoris species by GC-MS.

The genus Lycoris, a group of Amaryllidaceae plants distributed in temperate regions of Eastern Asia, is already known for containing representative alkaloids typical of this botanical family with a wide range of biological activities (for example, lycorine and galanthamine). In the present work, the alkaloid profiles of nine species, L. albiflora, L. aurea, L. chinensis, L. haywardii, L. incarnata, L. longituba, L. radiata, L. sprengeri, and L. squamigera, and one variety (L. radiata var. pumila) have been evaluated by GC-MS. Structures belonging to the lycorine-, homolycorine-, haemanthamine-, narciclasine-, tazettine-, montanine- and galanthamine-series were identified and quantified, with galanthamine- and lycorine-type alkaloids predominating and usually showing a high relative abundance in comparison with other alkaloids of the extracts. Interestingly, L. longituba revealed itself to be a potential commercial source of bioactive alkaloids. In general terms, our results are consistent with the alkaloid profiles reported in the literature for previously studied species.

[Genetic variations in trnL-F sequence and phylogenetic clustering of Lycoris species].

OBJECTIVE: To identify some closely related Lycoris species and evaluate interspecific relationships among them. METHOD: The cpDNA trnL-F sequence of 20 taxa representing 15 species of Lycoris and Narcissus tazaetta var. chinensis as one out-group were determined by using direct sequencing of PCR product, and they were analyzed by means of the software of CLUSTRAL and MEGA. RESULT: The length of trnL-F of all taxa was 905 - 1 036 bp. When the gaps were always treated as missing, there were 14 variable sites and 10 parsim-info sites, which could be used to identify some species of Lycoris. Four nucleotides inserteions/deletions were significant different among Lycoris and two species of Narcissus. Phylogeny tree was constructed with the maximum parsimony methods by bootstrap test. Three infrageneric clades of all Lycoris species were resolved. The classification was basically consistent with that of morphology except for L. longituba, L. aurea, and L. straminea. CONCLUSION: The tree suggested that L. anhuiensis can not be taken as an independent species, while it may be of a variety or a hybrid of L. longituba. Regarding the hybrid origin species, the materal parent of L. rosea and L. haywardii was revealed to be L. sprengeri. There were some variations in the trnL-F sequence, which were good molecular markers for identification species of Lycoris.

Phylogenetic relationships and possible hybrid origin of Lycoris species (Amaryllidaceae) revealed by its sequences.

To examine interspecific relationships and test the hypothesis of hybrid origin within Lycoris species, this study used data from parsimony analyses with nuclear ITS sequences for 19 taxa representing 14 species of Lycoris and two outgroup taxa. The ITS sequences resolved three infrageneric clades. One clade included L. chinensis, L. longituba, L. longituba var. flava, L. anhuiensis, and L. aurea; the second one consisted of L. sprengeri, L. radiata, L. radiata var. radiata, L. radiata var. pumila, L. haywardii, L. rosea, L. sanguinea var. sanguinea, and L. sanguinea var. koreana; and the third included L. caldwellii, L. straminea, L. albiflora, L. flavescens, and two hybrids. The results strongly support the hypothesis that L. straminea originated from hybridization between L. chinensis and L. radiata var. pumila, and the allotriploid L. caldwellii and L. albiflora derived from hybridization between L. chinensis and L. sprengeri. As nucleotide additivity was observed in the artificial hybrids and several presumed hybrids, the likelihood of hybrid origin of Lycoris species is supported.

[Analysis of the inter-species relationships on lycoris Amaryllidaceae) by use of RAPD].

The fingerprints of 13 species in genus Lycoris were generated by use of RAPD method. Forty-one primers were screened from 520 random primers, and a total of 350 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (72.3%) of which were polymorphic. The average number of DNA band produced by each primer was 6.2. Nei's similarity coefficients and genetic distances were calculated by use of the software of TFPGA version 1.3 and dendrogram of Lycoris was constructed using UPGMA. It is indicated that the 13 species of the genus Lycoris were divided into two groups, and five species of the genus including L. rosea, L. haywardii, L. straminea, L. sprengeri and L. radiata with monotype karyotypes (I-shaped) were clustered together respectively. The basic chromosome number was x = 11. The others which have two-types karyotypes (I-shaped and V-shaped) were clustered together respectively. They were L. houdyshelii, L. albiflora, L. chinensis, L. longituba, L. anhuiensis, L. squmigera, L. caldwellii and L. aurea. The closest relationship was between L. rosea and L. haywardii. L. radiata is highly divergent from L. aurea. The results were in consistence with that of the analysis of chromosome karyotype. The present paper discussed the problems whether L. rosea, L. haywardii and L. stramina originated as natural hybrids and taxonomy position of L. albiflora, L. straminea and L. houdyshelii based on the RAPD analysis.