Developing an efficient DNA barcoding system to differentiate between Lilium species

Background Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. Results To develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). Conclusions A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Metabolome-Based Discrimination Analysis of Five Lilium Bulbs Associated with Differences in Secondary Metabolites

The bulbs of several Lilium species are considered to be both functional foods and traditional medicine in northern and eastern Asia. Considering the limited information regarding the specific bioactive compounds contributing to the functional properties of these bulbs, we compared the secondary metabolites of ten Lilium bulb samples belonging to five different species, using an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)-based secondary metabolomics approach. In total, 245 secondary metabolites were detected; further, more metabolites were detected from purple Lilium bulbs (217 compounds) than from white bulbs (123–171 compounds). Similar metabolite profiles were detected in samples within the same species irrespective of where they were collected. By combining herbal analysis and screening differential metabolites, steroid saponins were considered the key bioactive compounds in medicinal lilies. Of the 14 saponins detected, none were accumulated in the bulbs of L. davidii var. willmottiae, also called sweet lily. The purple bulbs of L. regale accumulated more secondary metabolites, and, notably, more phenolic acid compounds and flavonoids. Overall, this study elucidates the differential metabolites in lily bulbs with varying functions and colors and provides a reference for further research on functional foods and the medicinal efficacy of Lilium species.

Transcriptome wide SSR discovery cross-taxa transferability and development of marker database for studying genetic diversity population structure of Lilium species

Lily belongs to family liliaceae, which mainly propagates vegetatively. Therefore, sufficient number of polymorphic, informative, and functional molecular markers are essential for studying a wide range of genetic parameters in Lilium species. We attempted to develop, characterize and design SSR (simple sequence repeat) markers using online genetic resources for analyzing genetic diversity and population structure of Lilium species. We found di-nucleotide repeat motif were more frequent (4684) within 0.14 gb (giga bases) transcriptome than other repeats, of which was two times higher than tetra-repeat motifs. Frequency of di-(AG/CT), tri-(AGG/CTT), tetra-(AAAT), penta-(AGAGG), and hexa-(AGAGGG) repeats was 34.9%, 7.0%, 0.4%, 0.3%, and 0.2%, respectively. A total of 3607 non-redundant SSR primer pairs was designed based on the sequences of CDS, 5′-UTR and 3′-UTR region covering 34%, 14%, 23%, respectively. Among them, a sub set of primers (245 SSR) was validated using polymerase chain reaction (PCR) amplification, of which 167 primers gave expected PCR amplicon and 101 primers showed polymorphism. Each locus contained 2 to 12 alleles on average 0.82 PIC (polymorphic information content) value. A total of 87 lily accessions was subjected to genetic diversity analysis using polymorphic SSRs and found to separate into seven groups with 0.73 to 0.79 heterozygosity. Our data on large scale SSR based genetic diversity and population structure analysis may help to accelerate the breeding programs of lily through utilizing different genomes, understanding genetics and characterizing germplasm with efficient manner.

Comparative Chloroplast Genomics of Fritillaria (Liliaceae), Inferences for Phylogenetic Relationships between Fritillaria and Lilium and Plastome Evolution

Fritillaria is a genus that has important medicinal and horticultural values. The study involved the most comprehensive chloroplast genome samples referring to Old and New World clades of Fritillaria for marker selection and phylogenetic studies. We reported and compared eleven newly sequenced whole-plastome sequences of Fritillaria which proved highly similar in overall size (151,652–152,434 bp), genome structure, gene content, and order. Comparing them with other species of Liliales (6 out of 10 families) indicated the same similarity but showed some structural variations due to the contraction or expansion of the inverted repeat (IR) regions. A/T mononucleotides, palindromic, and forward repeats were the most common types. Six hypervariable regions (rps16-trnQ, rbcL-accD, accD-psaI, psaJ-rpl33, petD-rpoA, and rpl32-trnL) were discovered based on 26 Fritillaria whole-plastomes to be potential molecular markers. Based on the plastome data that were collected from 26 Fritillaria and 21 Lilium species, a phylogenomic study was carried out with three Cardiocrinum species as outgroups. Fritillaria was sister to Lilium with a high support value, and the interspecies relationships within subgenus Fritillaria were resolved very well. The six hypervariable regions can be used as candidate DNA barcodes of Fritillaria and the phylogenomic framework can guide extensive genomic sampling for further phylogenetic analyses.

New Insights on Lilium Phylogeny Based on a Comparative Phylogenomic Study Using Complete Plastome Sequences

The genus Lilium L. is widely distributed in the cold and temperate regions of the Northern Hemisphere and is one of the most valuable plant groups in the world. Regarding the classification of the genus Lilium, Comber’s sectional classification, based on the natural characteristics, has been primarily used to recognize species and circumscribe the sections within the genus. Although molecular phylogenetic approaches have been attempted using different markers to elucidate their phylogenetic relationships, there still are unresolved clades within the genus. In this study, we constructed the species tree for the genus using 28 Lilium species plastomes, including three currently determined species (L. candidum, L. formosanum, and L. leichtlinii var. maximowiczii). We also sought to verify Comber’s classification and to evaluate all loci for phylogenetic molecular markers. Based on the results, the genus was divided into two major lineages, group A and B, consisting of eastern Asia + Europe species and Hengduan Mountains + North America species, respectively. Sectional relationships revealed that the ancestor Martagon diverged from Sinomartagon species and that Pseudolirium and Leucolirion are polyphyletic. Out of all loci in that Lilium plastome, ycf1, trnF-ndhJ, and trnT-psbD regions are suggested as evaluated markers with high coincidence with the species tree. We also discussed the biogeographical diversification and long-distance dispersal event of the genus.

Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.