Expression Patterns of Key Genes in the Photoperiod and Vernalization Flowering Pathways in Lilium longiflorum with Different Bulb Sizes.

Lilium longiflorum is a wild Lilium, and its flowering transition requires a long period of cold exposure to meet the demand of vernalization. The responses of different sized bulbs to cold exposure and photoperiod are different, and the floral transition pathways of small and large bulbs are different. In this study, small and large bulbs were placed in cold storage for different weeks and then cultured at a constant ambient temperature of 25 °C under long day (LD) and short day (SD) conditions. Then, the flowering characteristics and expression patterns of key genes related to the vernalization and photoperiod pathways in different groups were calculated and analyzed. The results showed that the floral transition of Lilium longiflorum was influenced by both vernalization and photoperiod, that vernalization and LD conditions can significantly improve the flowering rate of Lilium longiflorum, and that the time from planting to visible flowering buds' appearance was decreased. The flowering time and rate of large bulbs were greatly influenced by cold exposure, and the vernalization pathway acted more actively at the floral transition stage. The floral transition of small bulbs was affected more by the photoperiod pathway. Moreover, it was speculated that cold exposure may promote greater sensitivity of the small bulbs to LD conditions. In addition, the expression of LlVRN1, LlFKF1, LlGI, LlCO5, LlCO7, LlCO16, LlFT1, LlFT3 and LlSOC1 was high during the process of floral transition, and LlCO13, LlCO14 and LlCO15 were highly expressed in the vegetative stage. The expression of LlCO13 and LlCO14 was different under different lighting conditions, and the flowering induction function of LlCO9 and LlFT3 was related to vernalization. Moreover, LlFKF1, LlGI, LlCO5, LlCO16, LlSOC1 and LlFT2 were involved in the entire growth process of plants, while LlCO6, LlCO16 and LlFT1 are involved in the differentiation and formation of small bulblets of plants after the inflorescence stage, and this process is also closely related to LD conditions. This study has great significance for understanding the molecular mechanisms of the vernalization and photoperiod flowering pathways of Lilium longiflorum.

Analysis of miRNA-mediated regulation of flowering induction in Lilium × formolongi.

BACKGROUND: MicroRNAs play pivotal roles in plant vegetative phase change and flowering induction via integrating into multiple flowering pathways. Lilium × formolongi is an important ornamental lily cultivar that can flower within one year after sowing. However, it remains unresolved how miRNA-mediated regulation networks contribute to the L. × formolongi characteristics of a short vegetative growth period and rapid flowering. RESULTS: In this study, the small RNA libraries and one degradome library were constructed for L. × formolongi during vegetative growth and flowering initiation, and 366 conserved miRNAs and 32 novel miRNAs were identified. Additionally, 84 miRNAs were significantly differentially expressed during development. A total of 396 targets of 185 miRNAs were identified and validated through degradome sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that functions of the targets were top enriched in the cold and cadmium ion responses, pentose phosphate pathway and carbon fixation in photosynthetic organisms. Furthermore, among 23 differentially expressed miRNA-target pairs, the miR156s-LfSPL2, miR172a-LfAP2 and miR164a-LfNAC pairs as well as miR159a-LfSPL2 were found to be relevant to flowering based on the correlation analysis of expression profiles in the miRNA libraries, degradome and transcriptome. A coexpression regulatory network focused on differentially expressed pairs was also constructed by WGCNA, and 14 miRNAs were considered putative key miRNAs during vegetative development and flowering induction. miR156a/ d/ e showed particularly strong relationships with other miRNAs in the coexpression network. CONCLUSIONS: This study provides cues for the further exploration of the regulatory mechanisms of short vegetative development and flowering in L. × formolongi.

Cryptochrome 2 from Lilium × formolongi Regulates Photoperiodic Flowering in Transgenic Arabidopsis thaliana.

The photoperiodic flowering pathway is essential for plant reproduction. As blue and ultraviolet-A light receptors, cryptochromes play an important role in the photoperiodic regulation of flowering. Lilium × formolongi is an important cut flower that flowers within a year after seed propagation. Floral induction is highly sensitive to photoperiod. In this study, we isolated the CRYPTOCHROME2 gene (LfCRY2) from L. × formolongi. The predicted LfCRY2 protein was highly homologous to other CRY2 proteins. The transcription of LfCRY2 was induced by blue light. LfCRY2 exhibits its highest diurnal expression during the floral induction stage under both long-day and short-day photoperiods. Overexpression of LfCRY2 in Arabidopsis thaliana promoted flowering under long days but not short days, and inhibited hypocotyl elongation under blue light. Furthermore, LfCRY2 was located in the nucleus and could interact with L. × formolongi CONSTANS-like 9 (LfCOL9) and A. thaliana CRY-interacting basic-helix-loop-helix 1 (AtCIB1) in both yeast and onion cells, which supports the hypothesis that LfCRY2 hastens the floral transition via the CIB1-CO pathway in a manner similar to AtCRY2. These results provide evidence that LfCRY2 plays a vital role in promoting flowering under long days in L. × formolongi.

Functional and Evolutionary Characterization of the CONSTANS-like Family in Lilium�formolongi.

Lilium�formolongi is a facultative long-day (LD) plant. Aiming to dissect the molecular regulation of the photoperiodic pathway, largely unknown in Lilium, we explored the CONSTANS/FLOWERING LOCUS T (CO/FT) module, a major regulatory factor in the external coincidence model of the photoperiodic flowering pathway in lily. We identified eight CONSTANS-LIKE (COL) family members in L.�formolongi, which could be divided into three types, according to their zinc-finger (B-box) protein domains. Type I included only LfCOL5, containing two B-box motifs. Type II contained six LfCOLs members that had only one B-box motif. Type III contained only LfCOL9 that showed a normal B-box and a second divergent B-box motif. Phylogenic analyses revealed that LfCOL5 was the closest to Arabidopsis CO. LfCOL5, LfCOL6 and LfCOL9 were up-regulated at the flowering induction stage under LDs, coinciding with an increase in LfFT1 expression. LfCOL5, LfCOL6 and LfCOL9 also showed obvious diurnal expression pattern for 3 d under LDs. However, under short-day (SD) conditions, the expression patterns of LfCOL5, LfCOL6 and LfCOL9 were variable and complex, with regard to the developmental stages and circadian rhythm. LfCOL5, LfCOL6 and LfCOL9 complemented the late flowering phenotype of the co mutant in Arabidopsis. Taken together, the results suggest that LfCOL5, LfCOL6 and LfCOL9 are involved in triggering flowering induction under LDs. LfCOL6 and LfCOL9 belong to types different from functional COL homologs in other plant species, illustrating the variation in phylogeny, evolution and gene function among LfCOL family members.

Analysis of global gene expression profiles during the flowering initiation process of Lilium × formolongi.

The onset of flowering is critical for the reproductive development of plants. Lilium × formolongi is a lily hybrid that flowers within a year after sowing. We successfully identified four important stages during vegetative growth and flowering initiation of L. × formolongi under long day conditions. The plant tissues from the four stages were used in a genome-wide transcriptional analysis to investigate stage-specific changes of gene expression in L. × formolongi. In total, the sequence reads of the four RNA-sequencing libraries were assembled into 52,824 unigenes, of which 37,031 (70.10%) were differentially expressed. The global expression dynamics of the differentially expressed genes were predominant in flowering induction phase I and the floral differentiation stage, but down-regulated in flowering induction phase II. Various transcription factor families relevant to flowering were elucidated, and the members of the MADS-box, SBP and CO-like transcription factor families were the most represented. There were 85 differentially expressed genes relevant to flowering. CONSTANS-LIKE, FLOWERING LOCUS T, TREHALOSE-6-PHOSPHATE SYNTHASE and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE homologs were discovered and may play significant roles in the flowering induction and transition process of L. × formolongi. A putative gene regulatory network, including photoperiod, age-dependent and trehalose-6-phosphate flowering pathways, was constructed. This is the first expression dataset obtained from a transcriptome analysis of photoperiod-mediated flowering pathway in lily, and it is valuable for the exploration of the molecular mechanisms of flowering initiation and the short vegetative stage of L. × formolongi.

De novo transcriptome characterization of Lilium 'Sorbonne' and key enzymes related to the flavonoid biosynthesis.

Lily is an important cut-flower and bulb crop in the commercial market. Here, transcriptome profiling of Lilium 'Sorbonne' was conducted through de novo sequencing based on Illumina platform. This research aims at revealing basic information and data that can be used for applied purposes especially the molecular regulatory information on flower color formation in lily. In total, 36,920,680 short reads which corresponded to 3.32 GB of total nucleotides, were produced through transcriptome sequencing. These reads were assembled into 39,636 Unigenes, of which 30,986 were annotated in Nr, Nt, Swiss-Prot, KEGG, COG, GO databases. Based on the three public protein databases, a total of 32,601 coding sequences were obtained. Meanwhile, 19,242 Unigenes were assigned to 128 KEGG pathways. Those with the greatest representation by unique sequences were for ''metabolic pathways'' (5,406 counts, 28.09 %). Our transcriptome revealed 156 Unigenes that encode key enzymes in the flavonoid biosynthesis pathway including CHS, CHI, F3H, FLS, DFR, etc. MISA software identified 2,762 simple sequence repeats, from which 1,975 primers pairs were designed. Over 2,762 motifs were identified, of which the most frequent was AG/CT (659, 23.86 %), followed by A/T (615, 22.27 %) and CCG/CGG (416, 15.06 %). Based on the results, we believe that the color formation of the Lilium 'Sorbonne' flower was mainly controlled by the flavonoid biosynthesis pathway. Additionally, this research provides initial genetic resources that will be valuable to the lily community for other molecular biology research, and the SSRs will facilitate marker-assisted selection in lily breeding.

Molecular phylogeny and genetic variation in the genus Lilium native to China based on the internal transcribed spacer sequences of nuclear ribosomal DNA.

We present a comprehensive phylogeny derived from nuclear ribosomal DNA (nrDNA) for 214 samples representing 98 species and five varieties, including 44 species and five varieties native to China. Our collection of 25 species and five varieties (44 samples) covering all five sections of the genus (Comber) distributed in China also were included in the internal transcribed spacer (ITS) database. This study incorporates previous research with an emphasis on Chinese species, including the controversial subsection, Sinomartagon 5c Comber. In the phylogenetic tree obtained by maximum parsimony (PAUP) and maximum likelihood (RAxML) analyses, the samples were divided into four major groups. Our results suggest that the subsection (subsect.) 5c Comber should be classified into the true subsect. 5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum I, subsect. Lophophorum II, and subsect. Lophophorum III). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, we report that L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion 6a. Our results support Comber's subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.

Effective Induction of Fertile Gametes in Oriental × Trumpet (OT) Lily by High Temperatures.

Lily is a very important bulb crop, and interspecific distant hybridization is a crucial method of lily breeding. However, F1 interspecific hybrids tend to be highly sterile due to low levels of genetic homozygosity. This can be addressed by meiotic polyploidization, which has the advantage of reducing breeding time and being able to promote genetic recombination resulting in many variant progenies. High temperatures have been proven to induce 2n gametes via hindering a spindle formation in several plants, but little has been reported in lilies. In the present study, after observing the correlation between the development of the pollen mother cells (PMCs) and the length of the buds, 28-31 mm long buds were selected as the experimental material, which were at the stage of prophase I-metaphase I. Individual buds were induced at different temperatures (40 °C, 42 °C, and 44 °C) and durations (4 h and 6 h) using self-made multiwire heating equipment, and successfully induced fertile male gametes.. The best results were achieved with treatment of 42 °C for 4 h, reaching a maximum fertile pollen induction rate of 36.64%, while bud mortality was 40%. Two chemicals, colchicine and oryzalin, were also used by injection, and only the treatment with oryzalin obtained fertile gametes, with the highest fertile gamete rate of 15.39% at a concentration of 0.005%, while the bud mortality was 36.67%. This suggests that high temperatures have a superior effect on lily 2n gamete induction. In addition, the pollen obtained from the 6 h induction of high temperature was significantly larger than that from the 4 h induction, with an average diameter of 138.64 µm and 107.88 µm, respectively, 2.35 and 1.84 times wider than haploid pollen. The fertile pollen was crossed with four cultivars and two species, and a total of 267 embryonic seeds were obtained, with the highest embryonic rate of 4.52% in OT lily 'Mister Cas' as the parent, which had a germination rate of 26.27%. This suggests that the method of high-temperature induction for fertile gametes probably has important significance for ploidy and distant hybrid breeding in lilies.

Identification of trehalose-6-phosphate synthase (TPS)-coding genes involved in flowering induction of Lilium× formolongi.

Lilium × formolongi, a facultative long-day (LD) plant, can complete the floral transition within one year after sowing under LD conditions. In addition to the photoperiod, the molecular mechanisms by which other flowering regulators, such as sugar, participate in juvenile development and flowering induction in L. × formolongi remain elusive. Therefore, based on the investigation of seedling development under different day length conditions, we explored the growth and nonstructural carbohydrate (NSC) contents of leaves and underground bulbs. Furthermore, the expression profiles of trehalose-6-phosphate synthase (TPS)-coding genes, LfTPSs, and miR156 were also determined. Three putative LfTPS genes, LfTPS1, LfTPS3 and LfTPS5, displayed high expression levels at the juvenile vegetative stage under different day length conditions. Among them, LfTPS1 maintained gradually elevated expression until the visible bud stage under short-day (SD) conditions. Additionally, the expression levels of LfTPS3 and LfTPS5 increased with the exogenous sucrose concentration. In contrast, the expression of miR156 rapidly decreased under the same sucrose treatments. Overexpression of LfTPS1/3/5 hastened flowering and the decline in miR156 expression levels to varying degrees in transgenic Arabidopsis. Taken together, the results demonstrate that LfTPS1, LfTPS3 and LfTPS5 modulate both juvenile vegetative development and flowering induction controlled by sugars in L. × formolongi.

Molecular cloning of cryptochrome 1 from Lilium×formolongi and the characterization of its photoperiodic flowering function in Arabidopsis.

Lilium × formolongi is an important cut flower species that is able to flower within a year following seed propagation, with flower induction that is very sensitive to the photoperiod. Cryptochromes are blue/UV-A light receptors that regulate many important plant growth and development processes, including photoperiodic flowering. In this study, we isolated the cryptochrome 1 (CRY1) gene from L. × formolongi and analyzed its function in transgenic Arabidopsis. The predicted LfCRY1 protein was strongly homologous to other CRY1 proteins. The transcription of LfCRY1 was induced by blue light, with LfCRY1 exhibiting its highest expression and diurnal expression patterns during the flowering-induction stage under both long-day (LD) and short-day (SD) photoperiods. Overexpression of LfCRY1 in Arabidopsis promoted flowering under LDs but not SDs and inhibited hypocotyl elongation under blue light. The LfCRY1 protein was located in both the nucleus and cytoplasm. LfCRY1 interacted with the important flowering activator LfCOL9 in both yeast and onion cells. These results provide functional evidence for the role of LfCRY1 in controlling photoperiodic flowering under LDs and indicate that LfCRY1 may be a counterpart of AtCRY1. Understanding the role of LfCRY1 in photoperiodic flowering is beneficial for the molecular breeding of lilies with shorter vegetative stages.

Stomata and ROS changes during Botrytis elliptica infection in diploid and tetraploid Lilium rosthornii Diels.

Polyploid plants often show improved resistance against many diseases, but whether they show increased resistance to grey mould, a devastating disease caused by Botrytis spp. fungi, is seldom reported. Stomata and reactive oxygen species (ROS) play dual roles in defence against grey mould, and it is unclear how their roles change after polyploidization. We addressed these questions in diploid and colchicine-induced Lilium rosthornii after B. elliptica infection. Tetraploids were less susceptible to grey mould, with lower morality rates in naturally infected plants. Before the stomata closed in artificially infected leaves, tetraploids, with larger stomatal apertures, were more easily invaded by the pathogen than diploids. However, the lesion area increased more slowly in tetraploids than in diploids, which may be explained by three causes based on histological and physiological characteristics. First, the pathogen required more time to penetrate the epidermis and closed stomata in tetraploids than in diploids. Second, the pathogen penetrated the reopened stomata more easily than the epidermis, and stomatal density was lower in tetraploids than in diploids. Third, tetraploids showed faster ROS accumulation, a more effective ROS-scavenging system and less malondialdehyde (MDA) accumulation than diploids. Stomatal starch and abnormal guard cell nuclei were present in the infected leaves. This phenomenon may be caused by oxalic acid, a pathogenic factor for many pathogens that promotes stomatal starch degradation and stomatal reopening in Sclerotinia spp., a pathogen closely related to Botrytis spp. This suggestion was primarily confirmed by immersing healthy leaves in oxalic acid solution.

Functional analysis and expression patterns of members of the FLOWERING LOCUS T (FT) gene family in Lilium.

Lilium is an important commercial flowering species, and there are many varieties and more than 100 species of wild Lilium. Lilium × formolongi is usually propagated from seedlings, and the flowering of these plants is driven mainly by the photoperiodic pathway. Most of the other lily plants are propagated via bulblets and need to be vernalized; these plants can be simply divided into pretransplantation types and posttransplantation types according to the time at which the floral transition occurs. We identified three Lilium FLOWERING LOCUS T (LFT) family members in 7 Lilium varieties, and for each gene, the coding sequence of the different varieties was identical. Among these genes, the LFT1 gene of Lilium was most homologous to the AtFT gene, which promotes flowering in Arabidopsis. We analyzed the expression patterns of LFT genes in Lilium × formolongi seedlings and in different Lilium varieties, and the results showed that LFT1 and LFT3 may promote floral induction. Compared with LFT3, LFT1 may have a greater effect on floral induction in Lilium, which is photoperiod sensitive, while LFT3 may play a more important role in the floral transition of lily plants, which have a high requirement for vernalization. LFT2 may be involved in the differentiation of bulblets, which was verified by tissue culture experiments, and LFT1 may have other functions involved in promoting bulblet growth. The functions of LFT genes were verified by the use of transgenic Arabidopsis thaliana plants, which showed that both the LFT1 and LFT3 genes can promote early flowering in Arabidopsis. Compared with LFT3, LFT1 promoted flowering more obviously, and thus, this gene could be an important promoter of floral induction in Lilium.

The lre-miR159a-LrGAMYB pathway mediates resistance to grey mould infection in Lilium regale.

Grey mould is one of the most determinative factors of lily growth and plays a major role in limiting lily productivity. MicroRNA159 (miR159) is a highly conserved microRNA in plants, and participates in the regulation of plant development and stress responses. Our previous studies revealed that lre-miR159a participates in the response of Lilium regale to Botrytis elliptica according to deep sequencing analyses; however, the response mechanism remains unknown. Here, lre-miR159a and its target LrGAMYB gene were isolated from L. regale. Transgenic Arabidopsis overexpressing lre-MIR159a exhibited larger leaves and smaller necrotic spots on inoculation with Botrytis than those of wild-type and overexpressing LrGAMYB plants. The lre-MIR159a overexpression also led to repressed expression of two targets of miR159, AtMYB33 and AtMYB65, and enhanced accumulation of hormone-related genes, including AtPR1, AtPR2, AtNPR1, AtPDF1.2, and AtLOX for both the jasmonic acid and salicylic acid pathways. Moreover, lower levels of H2 O2 and O2- were observed in lre-MIR159a transgenic Arabidopsis, which reduced the damage from reactive oxygen species accumulation. Taken together, these results indicate that lre-miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response.

Analysis of WRKY transcription factors and characterization of two Botrytis cinerea-responsive LrWRKY genes from Lilium regale.

A major constraint in producing lilies is gray mold caused by Botrytis elliptica and B. cinerea. WRKY transcription factors play important roles in plant immune responses. However, limited information is available about the WRKY gene family in lily plants. In this study, 23 LrWRKY genes with complete WRKY domains were identified from the Botrytis-resistant species Lilium regale. The putative WRKY genes were divided into seven subgroups (Group I, IIa-e, and III) according to their structural features. Sequence alignment revealed that LrWRKY proteins have a highly conserved WRKYGQK domain and a variant, the WRKYGKK domain, and these proteins generally contained similar motif compositions throughout the same subgroup. Functional annotation predicted they might be involved in biological processes related to abiotic and biotic stresses. A qRT-PCR analysis confirmed that expression of six LrWRKY genes in L. regale or the susceptible Asian hybrid 'Yale' was induced by B. cinerea infection. Among these genes, LrWRKY4, LrWRKY8 and LrWRKY10 were expressed at a higher level in L. regale than 'Yale', while the expression of LrWRKY6 and LrWRKY12 was lower in L. regale. Furthermore, LrWRKY4 and LrWRKY12 genes, which also respond to salicylic acid (SA) and methyl jasmonate (MeJA) treatments, were isolated from L. regale. Subcellular localization analysis determined that they were targeted to the nucleus. Constitutive expression of LrWRKY4 and LrWRKY12 in Arabidopsis resulted in plants that were more resistant to B. cinerea than wild-type plants. This resistance was coupled with the transcriptional changes of SA and JA-responsive genes. Overall, our study provides valuable information about the structural and functional characterization of LrWRKY genes that will not only deepen our understanding of the molecular mechanisms underlying the defense of lily against B. cinerea but also offer potential targets for cultivar improvement via biotechnology.

Evaluation of resistance to Botrytis elliptica in Lilium hybrid cultivars.

Gray mold disease, caused by the fungus Botrytis elliptica, is one of the major diseases affecting Lilium species, and it has become a limiting factor in the production of ornamental lilies. To support selecting and breeding Botrytis-resistant cultivars, a total of 50 Lilium cultivars belonging to seven hybrid types were evaluated using a detached leaf technique for resistance to B. elliptica. Through resistance evaluations, Oriental × Trumpet and Oriental hybrid cultivars were classified as resistant lines, while Asiatic and Trumpet hybrids were classified as susceptible lines. A highly resistant (HR) Oriental hybrid, 'Sorbonne', and a highly susceptible (HS) Asiatic hybrid, 'Tresor', were selected for further study of early host-parasite interactions. After infection, B. elliptica grew faster and more easily on the leaves of 'Tresor' than on those of 'Sorbonne' during initial infection; when 'Tresor' leaves were completely infected, only a few lesions were observed on 'Sorbonne' leaves. Biochemical differences between these two cultivars were found following inoculation with B. elliptica, as shown by studies of reactive oxygen species (ROS) and the enzymatic antioxidant system. Rapid accumulation of H2O2 and ·O2- to trigger a defense response was detected in HR 'Sorbonne'. Meanwhile, higher levels of antioxidant activity, including SOD, POD and CAT, were found in HR 'Sorbonne' than in HS 'Tresor' before 48 h post-inoculation, but antioxidant activity was reduced with subsequent infection progress. These large and timely increases in ROS and antioxidant activities could be the main contributors to the high resistance of the 'Sorbonne' cultivar.

Genome Size Diversity in Lilium (Liliaceae) Is Correlated with Karyotype and Environmental Traits.

Genome size (GS) diversity is of fundamental biological importance. The occurrence of giant genomes in angiosperms is restricted to just a few lineages in the analyzed genome size of plant species so far. It is still an open question whether GS diversity is shaped by neutral or natural selection. The genus Lilium, with giant genomes, is phylogenetically and horticulturally important and is distributed throughout the northern hemisphere. GS diversity in Lilium and the underlying evolutionary mechanisms are poorly understood. We performed a comprehensive study involving phylogenetically independent analysis on 71 species to explore the diversity and evolution of GS and its correlation with karyological and environmental traits within Lilium (including Nomocharis). The strong phylogenetic signal detected for GS in the genus provides evidence consistent with that the repetitive DNA may be the primary contributors to the GS diversity, while the significant positive relationships detected between GS and the haploid chromosome length (HCL) provide insights into patterns of genome evolution. The relationships between GS and karyotypes indicate that ancestral karyotypes of Lilium are likely to have exhibited small genomes, low diversity in centromeric index (CVCI) values and relatively high relative variation in chromosome length (CVCL) values. Significant relationships identified between GS and annual temperature and between GS and annual precipitation suggest that adaptation to habitat strongly influences GS diversity. We conclude that GS in Lilium is shaped by both neutral (genetic drift) and adaptive evolution. These findings will have important consequences for understanding the evolution of giant plant genomes, and exploring the role of repetitive DNA fraction and chromosome changes in a plant group with large genomes and conservation of chromosome number.

Transcriptome-Wide Analysis of Botrytis elliptica Responsive microRNAs and Their Targets in Lilium Regale Wilson by High-Throughput Sequencing and Degradome Analysis.

MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B. elliptica treatment or B. elliptica infection for 6 and 24 h. By high-throughput sequencing, 71 known miRNAs belonging to 47 conserved families and 24 novel miRNA were identified, of which 18 miRNAs were downreguleted and 13 were upregulated in response to B. elliptica. Moreover, based on the lily mRNA transcriptome, 22 targets for 9 known and 1 novel miRNAs were identified by the degradome sequencing approach. Most target genes for elliptica-responsive miRNAs were involved in metabolic processes, few encoding different transcription factors, including ELONGATION FACTOR 1 ALPHA (EF1a) and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 2 (TCP2). Furthermore, the expression patterns of a set of elliptica-responsive miRNAs and their targets were validated by quantitative real-time PCR. This study represents the first transcriptome-based analysis of miRNAs responsive to B. elliptica and their targets in lily. The results reveal the possible regulatory roles of miRNAs and their targets in B. elliptica interaction, which will extend our understanding of the mechanisms of this disease in lily.

Evaluation of putative reference genes for quantitative real-time PCR normalization in Lilium regale during development and under stress.

Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression, Lilium, and particularly L. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely 18S rRNA, ACT, BHLH, CLA, CYP, EF1, GAPDH, SAND and TIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms, BHLH was superior to the other candidates when all the experimental treatments were analyzed together; CLA and EF1 were also recommended by two of the three algorithms. As for specific conditions, EF1 under various developmental stages, SAND under biotic stress, CYP/GAPDH under drought stress, and TIP41 under salinity stress were generally considered suitable. All the algorithms agreed on the stability of SAND and GAPDH under cold stress, while only CYP was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of LrLOX in leaves inoculated with B. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding of Lilium.

Lilium spp. pollen in China (Liliaceae): taxonomic and phylogenetic implications and pollen evolution related to environmental conditions.

Recent molecular and karyologic studies have significantly modified delimitation of Lilium. However, despite the importance of pollen evolution in the genus comprehensive studies with electron microscopy and evaluation of pollen evolution are lacking. Therefore, we studied pollen morphology in a sample of 65 individuals from 37 taxa covering all the sections distributed in the world, using scanning electron microscopy. Our collection of 49 individuals from 21 taxa covering all five sections in China was also included in the database. We found pollen tetrads in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, we propose the following hypothesis: evolution of an exine sculpture shows pollen type trends from Martagon → Callose → Concolor → Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions. Evolution trends in exine sculpture do not appear to be definitively correlated with pollen size and shape.