Youming Wan’s research while affiliated with Chinese Academy of Forestry and other places

Photoperiod-dependent flowering is a critical trait in breeding for flowering time in woody ornamental plants. Circadian clocks are vital for the regulation of photoperiodic flowering in plants, but their molecular regulation pathways in woody perennials remain poorly explored. Here, we identified two circadian clock components LgPSEUDO-RESPONSE REGULATOR 7 (LgPRR7) and LgFLAVIN-BINDING KELCH REPEAT F-BOX 1 (LgFKF1) as key repressors of flowering in Luculia gratissima, a short-day woody ornamental plant with commercial potential. Levels of LgPRR7 and LgFKF1 transcripts exhibited photoperiodic responses and diurnal patterns. Ectopic overexpression of LgPRR7 or LgFKF1 in Arabidopsis thaliana accelerated flowering, whereas silencing LgPRR7 or LgFKF1 in L. gratissima accelerated flowering. Crucially, LgPRR7 directly interacts with LgFKF1, forming a self-reinforcing regulatory module LgPRR7-LgFKF1 to repress flowering in L. gratissima. Furthermore, the observed physical interactions among LgFKF1, LgCONSTANS-LIKE 12 (LgCOL12), and LgREPRESSOR OF ga1-3-LIKE 2 (LgRGL2) implied that they possibly formed a protein complex LgFKF1-LgCOL12-LgRGL2, bridging the circadian clock, photoperiod, and gibberellin signaling pathways to suppress downstream floral integrators. Intriguingly, silencing LgPRR7 and LgFKF1 extended the duration of L. gratissima flowering, a trait of horticultural significance. These results suggest the integration of multi-layered environmental and endogenous signals in the regulation of flowering time. The LgPRR7-LgFKF1 module provides novel targets for molecular improvement to manipulate flowering time and duration in L. gratissima and other economically valuable woody ornamental plants. Our results also support the mediation of flowering convergence in short-day plants through the action of circadian clock genes.

Luculia yunnanensis is a vulnerable species endemic to Yunnan Province, Southwestern China, which has high ornamental value. Its wild population has not been fully protected and utilized for a long time, which is not conducive to the long-term stable development of this species. Genetic diversity assessment is the basis and prerequisite for the conservation of rare species. In this study, 21 phenotypic traits and 17 highly polymorphic EST-SSR markers were used to analyze the genetic diversity and genetic structure of 164 individuals from six L. yunnanensis populations. The coefficient of variation of 21 phenotypic traits ranged from 11.76% to 52.58% (mean=21.72%), and the coefficient of variation of 18 traits was less than 30%. The average values of Ne, I, Ho and He were 1.710, 0.619, 0.384, and 0.352, respectively. The genetic diversity of LLO (Ho = 0.476 and He = 0.426) and LCM (Ho = 0.424 and He = 0.381) populations in Lushui County was highest. The GDX populations (Ho = 0.335 and He = 0.269) isolated by Gaoligong Mountain had the lowest genetic diversity. The AMOVA results showed that 13.04% of the genetic variation was among populations and 86.96% was within populations. The average phenotypic differentiation coefficient of phenotypic traits among populations was 18.69%. The results of phenotypic and genetic variation analysis were consistent, indicating that the most of variation exists within population. Genetic structure, UPGMA clustering and PCA analysis results showed that the populations of L. yunnanensis had obvious geographical divisions, and the populations distributed in the southern region and distributed in the northern region of the Nujiang River clustered into one group respectively. Combining the results of phenotypic and molecular markers, we recommend that give priority to the protection of LLO, LCM and GDX population, in order to ensure the sustainable utilization of L. yunnanensis germplasm resources.

Photoperiod-regulated floral transition is vital to the flowering plant. Luculia gratissima “Xiangfei” is a flowering ornamental plant with high development potential economically and is a short-day woody perennial. However, the genetic regulation of short-day-induced floral transition in L. gratissima is unclear. To systematically research the responses of L. gratissima during this process, dynamic changes in morphology, physiology, and transcript levels were observed and identified in different developmental stages of long-day- and short-day-treated L. gratissima plants. We found that floral transition in L. gratissima occurred 10 d after short-day induction, but flower bud differentiation did not occur at any stage under long-day conditions. A total of 1,226 differentially expressed genes were identified, of which 146 genes were associated with flowering pathways of sugar, phytohormones, photoperiod, ambient temperature, and aging signals, as well as floral integrator and meristem identity genes. The trehalose-6-phosphate signal positively modulated floral transition by interacting with SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 4 (SPL4) in the aging pathway. Endogenous gibberellin, abscisic acid, cytokinin, and jasmonic acid promoted floral transition, whereas strigolactone inhibited it. In the photoperiod pathway, FD, CONSTANS-LIKE 12, and nuclear factors Y positively controlled floral transition, whereas PSEUDO-RESPONSE REGULATOR 7, FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1, and LUX negatively regulated it. SPL4 and pEARLI1 positively affected floral transition. Suppressor of Overexpression of Constans 1 and AGAMOUSLIKE24 integrated multiple flowering signals to modulate the expression of FRUITFULL/AGL8, AP1, LEAFY, SEPALLATAs, SHORT VEGETATIVE PHASE, and TERMINAL FLOWER 1, thereby regulating floral transition. Finally, we propose a regulatory network model for short-day-induced floral transition in L. gratissima. This study improves our understanding of flowering time regulation in L. gratissima and provides knowledge for its production and commercialization.

Rhododendrons are woody plants, famous throughout the world as having high horticultural value. However, many wild species are currently threatened with extinction. Here, we report for the first time a high‐quality, chromosome‐level genome of Rhododendron griersonianum, which has contributed to ~10% of all horticultural rhododendron varieties but which in its wild form has been evaluated as critically endangered. The final genome assembly, which has a contig N50 size of ~34 M and a total length of 677 M, is the highest‐quality genome sequenced within the genus to date, in part due to its low heterozygosity (0.18%). Identified repeats constituted ~57% of the genome, and 38,280 protein‐coding genes were predicted with high support. We further re‐sequenced 31 individuals of R. griersonianum as well as 30 individuals of its widespread relative R. delavayi, and performed additional conservation genomic analysis. The results showed that R. griersonianum had lower genetic diversity (θ = 2.58e‐3; π = 1.94e‐3) when compared not only to R. delavayi (θ = 11.61e‐3, π = 12.97e‐3), but also to most other woody plants. Furthermore, three severe genetic bottlenecks were detected using both Stairway plot and fastsimcoal2 analysis, and are thought to have occurred in the late Middle Pleistocene and the Last Glacial Maximum (LGM) period. After these bottlenecks, R. griersonianum recovered and has maintained a constant effective population size (>25,000) until now. Intriguingly, R. griersonianum has accumulated significantly more deleterious mutations in the homozygous state than R. delavayi, and several deleterious mutations (e.g., in genes involved in the response to heat stress) are likely to have harmed the adaptation of this plant to its surroundings. This high‐quality, chromosome‐level genome and the population genomic analysis of the critically endangered R. griersonianum will provide an invaluable resource as well as conservation insights for future study in this species and in the genus Rhododendron in general.

Phyllanthus emblica L. is a well-known medicinal and edible plant species. Various medicinal compounds in the fruit make it an important medicinal and promising economic material. The plant is widely distributed in Southwestern and Southern China. However, due to massive deforestation and land reclamation as well as deterioration of its natural habitat in recent years, the wild resources of this species have been sharply reduced, and it is rare to see large-scale wild P. emblica forests so far. In order to effectively protect and rationally utilize this species, we investigated the genetic diversity, genetic structure, and population dynamics of 260 individuals from 10 populations of P. emblica sampled from the dry climate area in Yunnan and wet climate area in Guangxi using 20 polymorphic EST-SSR markers. We found high genetic diversity at the species level (He = 0.796) and within populations (He = 0.792), but low genetic differentiation among populations (FST = 0.084). In addition, most genetic variation existed within populations (92.44%) compared with variation among the populations (7.56%). Meanwhile, the NJ tree, STRUCTURE, and hierarchical analysis suggested that the sampled individuals were clustered into two distinct genetic groups. In contrast, the genetic diversity of the dry climate group (He = 0.786, Na = 11.790, I = 1.962) was higher than that of the wet climate group (He = 0.673, Na = 9.060, I = 1.555), which might be attributed to the combined effects of altitude, precipitation, and geographic distance. Interestingly, only altitude and precipitation had significant pure effects on the genetic diversity, and the former was slightly stronger. In addition, DIYABC analysis suggested the effective population size of P. emblica might have contracted in the beginning of the Last Glacial Maximum. These genetic features provided vital information for the conservation and sustainable development of genetic resources of P. emblica, and they also provided new insights and guidelines for ecological restoration and economic development in dry-hot valleys of Yunnan and karst areas in Guangxi.

Background: Photoperiod-regulated floral transition is vital to the flowering plant. Luculia gratissima ‘Xiangfei’ is a flowering ornamental plant with high development potential and is a short-day woody perennial. However, the genetic regulation of short-day-induced floral transition in L. gratissima is unclear. To systematically research the responses of L. gratissima during this process, dynamic changes in morphology, physiology, and transcript levels were observed and identified in different developmental stages of long-day and short-day-treated shoot apexes. Results: The results showed that floral transition in L. gratissima occurred 10 d after short-day induction, but flower bud differentiation did not occur under long-day conditions. A total of 1,226 differentially expressed genes were identified, of which 146 genes were associated with flowering pathways of sugar, phytohormones, photoperiod, ambient temperature, and aging signals, as well as floral integrator and meristem identity genes. The trehalose-6-phosphate signal positively modulated floral transition by interacting with SPL4 in the aging pathway. Endogenous gibberellin, abscisic acid, cytokinin, and jasmonic acid promoted floral transition, whereas strigolactone inhibited it. In the photoperiod pathway, FD, COL12, and NF-Ys positively controlled floral transition, whereas PRR7, FKF1, and LUX negatively regulated it. SPL4 and pEARLI1 positively affected floral transition. SOC1 and AGL24 integrated multiple flowering signals to modulate the expression of FUL/AGL8, AP1, LFY, SEPs, SVP, and TFL1, thereby regulating floral transition. Finally, we propose a regulatory network model for short-day-induced floral transition in L. gratissima. Conclusions: Short-day photoperiod activated systemic responses of morphology, physiology, and transcript levels in L. gratissima and induced the generation of floral transition signals in the photoperiod pathway. Furthermore, multiple flowering signal pathways including phytohormone-, sugar-, temperature-, age-related genes synergistically control this process. This study improves our understanding of flowering time regulation in L. gratissima and provides knowledge for its production and commercialization.

Premise of the Study A novel set of EST‐SSR markers was developed for Phyllanthus emblica (Phyllanthaceae) to investigate the genetic structure and gene flow, identify novel genes of interest, and develop markers for assisted breeding. Methods and Results Based on the transcriptome data of P. emblica, 83 EST‐SSR primer pairs were designed; 52 primer pairs were successfully amplified, with 20 showing polymorphisms in 90 individuals from three populations of P. emblica. The number of alleles per locus varied from 11 to 44. The observed and expected levels of heterozygosity for the 20 loci ranged from 0.240 to 0.868 and 0.754 to 0.933, respectively. Cross‐species amplification was successful for all 20 loci in each of the two related species, P. reticulatus and Leptopus chinensis. Conclusions These markers will be valuable for studying the population genetics and for mining genes of P. emblica, and may be useful for studies of related species.

Premise of the Study To investigate the genetic background and population characteristics of Rhododendron longipedicellatum (Ericaceae), a newly discovered and critically endangered species, expressed sequence tag–simple sequence repeat markers were developed, and transferability was tested in two congeners, R. molle and R. simsii. Methods and Results Based on the transcriptome sequences of R. longipedicellatum, 102 primer sets were designed; 48 primer sets were successfully amplified, with 15 showing polymorphisms in 150 individuals from five extant populations of R. longipedicellatum. The number of alleles per locus ranged from four to 18, and the levels of observed and expected heterozygosity for the 15 loci varied from 0.255 to 0.913 and from 0.306 to 0.851, respectively. All 15 loci were found to amplify in R. molle and R. simsii. Conclusions These polymorphic SSR markers can be used in conservation genetic and phylogeographic studies to elucidate the rarity and origin of R. longipedicellatum.

Rhododendron longipedicellatum is a narrow endemic species and a subject of urgent demand in the domestic market and overseas. Its fascinating shapes, brilliantly gilvous flowers, and unusual flowering time endow this species with extremely high ornamental value. However, only five wild populations of R. longipedicellatum surviving in limestone habitat have been found through elaborate field investigation, and the number of the populations decreases further or is even confronted with risk of extinction due to the damage of human activities. To enhance the protection and utilization of R. longipedicellatum, this study systematically investigated several important aspects of reproductive biology, including floral syndrome, pollen viability and stigma receptivity, petal color reflectance, breeding system, and pollination biology. The results demonstrated that arched styles not only create obvious herkogamy that avoide self-pollination, but also effectively reduce rain damage to the intrinsic characteristics of the stigma surface secretions, promoting the female fitness of R. longipedicellatum in poor weather. Pollen viability maintained a high level over the flowering period. The reflectance spectrum of petals had two peaks at wavelengths of 360 and 580 nm. Tests of OCI, P/O and artificial pollination all indicated that R. longipedicellatum was self-compatible and that the breeding system was mixed mating. Geitonogamy mediated by Bombus braccatus was the primary pollination route in the natural environment, which suggested that the breeding system of R. longipedicellatum might be evolving from selfing to outcrossing. The pollination vector of R. longipedicellatum was very specific, in that only B. braccatus was confirmed to deliver pollen to the stigmas. Visitation frequency was influenced by the activity rhythms and resource requirements of the different castes (i.e., sex). B. braccatus workers were the most effective pollinators because of higher visitation frequency and more effective contribution to fruit production, whereas the presence of B. braccatus males might enhance pollen flow within the population to a certain extent. Finally, these findings not only provided a reliable theoretical basis for hybridization breeding of R. longipedicellatum as parents, but also laid a solid foundation for further molecular biology studies to more broadly reveal the mechanisms of its endangerment in the future.