Multi-locus genome-wide association studies reveal the dynamic genetic architecture of flowering time in chrysanthemum.

KEY MESSAGE: The dynamic genetic architecture of flowering time in chrysanthemum was elucidated by GWAS. Thirty-six known genes and 14 candidate genes were identified around the stable QTNs and QEIs, among which ERF-1 was highlighted. Flowering time (FT) adaptation is one of the major breeding goals in chrysanthemum, a multipurpose ornamental plant. In order to reveal the dynamic genetic architecture of FT in chrysanthemum, phenotype investigation of ten FT-related traits was conducted on 169 entries in 2 environments. The broad-sense heritability of five non-conditional FT traits, i.e., budding (FBD), visible coloring (VC), early opening (EO), full-bloom (OF) and decay period (DP), ranged from 56.93 to 84.26%, which were higher than that of the five derived conditional FT traits (38.51-75.13%). The phenotypic variation coefficients of OF_EO and DP_OF were relatively large ranging from 30.59 to 36.17%. Based on 375,865 SNPs, the compressed variance component mixed linear model 3VmrMLM was applied for a multi-locus genome-wide association study (GWAS). As a result, 313 quantitative trait nucleotides (QTNs) were identified for the non-conditional FT traits in single-environment analysis, while 119 QTNs and 67 QTN-by-environment interactions (QEIs) were identified in multi-environment analysis. As for the conditional traits, 343 QTNs were detected in single-environment analysis, and 119 QTNs and 83 QEIs were identified in multi- environment analysis. Among the genes around stable QTNs and QEIs, 36 were orthologs of known FT genes in Arabidopsis and other plants; 14 candidates were mined by combining the transcriptomics data and functional annotation, including ERF-1, ACA10, and FOP1. Furthermore, the haplotype analysis of ERF-1 revealed six elite accessions with extreme FBD. Our findings contribute to the understanding of dynamic genetic architecture of FT and provide valuable resources for future chrysanthemum molecular breeding programs.

Genome-Wide Characterization of Chrysanthemum indicum Nuclear Factor Y, Subunit C Gene Family Reveals the Roles of CiNF-YCs in Flowering Regulation.

Nuclear Factor Y, Subunit C (NF-YC) transcription factors are conserved in most plants, and play essential roles in plant growth and development, especially in flowering regulation. Chrysanthemums are important commercial plants, and their market value is strongly impacted by flowering time. Until now, no details regarding the NF-YC family in the Chrysanthemum genus have been available. In this study, five NF-YC genes were cloned from Chrysanthemum indicum. Multiple alignments showed that CiNF-YCs had the highly conserved characteristic regions. Phylogenetic analyses identified a pair of paralogue NF-YC proteins in chrysanthemums. Gene structure and conserved motifs were also analyzed for functional understanding. According to the results of the expression experiments, CiNF-YC1 and CiNF-YC5 were mainly expressed in leaves or flowers, and their expression levels varied greatly from the seedling to flower bud differentiation stage. Arabidopsis overexpressing CiNF-YC1 and CiNF-YC3 showed significantly delayed flowering, accompanied by other morphological alterations. RT-qPCR analysis revealed that genes associated with photoperiod, vernalization, aging, and gibberellin pathways were downregulated in CiNF-YC1-OX lines, relative to the wild type, whereas in CiNF-YC3-OX lines, only SHORT VEGETATIVE PHASE (AtSVP), the key factor in the ambient temperature pathway, was upregulated. Taken together, these findings suggest that CiNF-YC1 and CiNF-YC3 negatively regulate flowering in Arabidopsis via different flowering pathways.

Genomic identification and expression analysis of the BBX transcription factor gene family in Petunia hybrida.

The B-box proteins (BBXs) are a class of zinc finger transcription factors containing one or two B-BOX domains that play important roles in plant growth, development and stress response. The petunia (Petunia hybrida) is a model ornamental plant, and its draft genome has been published. However, no systematic study of the BBX gene family in Petunia has been reported. In this study, a total of 28 BBX members from the Petunia genome were identified. We performed analyses of their phylogenetic relationships, structures, conserved motifs, promoter regions, and expression patterns. Based on the phylogenetic relationship, the PhBBXs were divided into six groups. Analysis of the gene structures and conserved motifs further confirmed the closer relationships in each group. Based on the RNA-seq data, the transcript abundance of PhBBXs in different tissues were divided into two major groups. The analysis of cis-elements showed that many stress responsive elements appeared in the promoter region of most PhBBX genes. The stress response patterns of PhBBXs were detected under drought, salinity, cold and heat treatments. Based on the RNA-seq data, we found that 3 genes responded to drought, 8 genes responded to salt, 18 genes responded to cold, and 15 genes responded to heat. In conclusion, this study may facilitate further functional studies of BBXs in Petunia.

Comprehensive Analyses of Four PhNF-YC Genes from Petunia hybrida and Impacts on Flowering Time.

Nuclear Factor Y (NF-Y) is a class of heterotrimeric transcription factors composed of three subunits: NF-A, NF-YB, and NF-YC. NF-YC family members play crucial roles in various developmental processes, particularly in the regulation of flowering time. However, their functions in petunia remain poorly understood. In this study, we isolated four PhNF-YC genes from petunia and confirmed their subcellular localization in both the nucleus and cytoplasm. We analyzed the transcript abundance of all four PhNF-YC genes and found that PhNF-YC2 and PhNF-YC4 were highly expressed in apical buds and leaves, with their transcript levels decreasing before flower bud differentiation. Silencing PhNF-YC2 using VIGS resulted in a delayed flowering time and reduced chlorophyll content, while PhNF-YC4-silenced plants only exhibited a delayed flowering time. Furthermore, we detected the transcript abundance of flowering-related genes involved in different signaling pathways and found that PhCO, PhGI, PhFBP21, PhGA20ox4, and PhSPL9b were regulated by both PhNF-YC2 and PhNF-YC4. Additionally, the transcript abundance of PhSPL2, PhSPL3, and PhSPL4 increased only in PhNF-YC2-silenced plants. Overall, these results provide evidence that PhNF-YC2 and PhNF-YC4 negatively regulate flowering time in petunia by modulating a series of flowering-related genes.

Genome-Wide Identification and Expression Profile Analysis of the NF-Y Transcription Factor Gene Family in Petunia hybrida.

Nuclear Factor Ys (NF-Ys) are a class of heterotrimeric transcription factors that play key roles in many biological processes, such as abiotic stress responses, flowering time, and root development. The petunia (Petunia hybrida) is a model ornamental plant, and its draft genome has been published. However, no details regarding the NF-Y gene family in petunias are available. Here, 27 NF-Y members from the petunia genome were identified, including 10 PhNF-YAs, 13 PhNF-YBs, and 4 PhNF-YCs. Multiple alignments showed that all PhNF-Y proteins had clear conserved core regions flanked by non-conserved sequences. Phylogenetic analyses identified five pairs of orthologues NF-YB proteins from Petunia and Arabidopsis, and six pairs of paralogues NF-Y proteins in Petunia. Analysis of the gene structure and conserved motifs further confirmed the closer relationship in each subfamily. Bioinformatics analysis revealed that 16 PhNF-Ys could be targeted by 18 miRNA families. RNA-seq results showed that expression patterns of PhNF-Ys among four major organs (leaf, stem, flower, and root) were clustered into six major groups. The stress response pattern of PhNF-Ys was identified under cold, heat, drought, and salinity treatments. Based on the RNA-seq data, we found that 3 genes responded to drought, 4 genes responded to salt, 10 genes responded to cold, and 9 genes responded to hot. In conclusion, this study provides useful information for further studying the functions of NF-Ys in stress response.

Acclimation of tropical tree species to hurricane disturbance: ontogenetic differences.

We investigated acclimation responses of seedlings and saplings of the pioneer species Cecropia schreberiana Miq. and three non-pioneer species, Dacryodes excelsa Vahl, Prestoea acuminata (Willdenow) H.E. Moore var. montana (Graham) Henderson and Galeano, and Sloanea berteriana Choisy ex DC, following a hurricane disturbance in a lower montane wet forest in Puerto Rico. Measurements were made, shortly after passage of the hurricane, on leaves expanded before the hurricane (pre-hurricane leaves) and, at a later time, on recently matured leaves that developed after the hurricane (post-hurricane leaves) from both seedlings and saplings at sites that were severely damaged by the hurricane (disturbed sites) and at sites with little disturbance (undisturbed sites). Pre-hurricane leaves of the non-pioneer species had relatively low light-saturated photosynthetic rates (A(max)) and stomatal conductance (g(s)); neither A(max) nor g(s) responded greatly to the increase in irradiance that resulted from the disturbance, and there were few significant differences between seedlings and saplings. Pre-hurricane leaves of plants at undisturbed sites had low dark respiration rates per unit area (R(d)) and light compensation points (LCP), whereas pre-hurricane leaves of plants at disturbed sites had significantly higher R(d) and LCP. Post-hurricane leaves of plants at disturbed sites had significantly higher A(max) and R(d) than plants at undisturbed sites. Compared with seedlings, saplings had higher A(max) and R(d) and showed greater acclimation to the increase in irradiance that followed the disturbance. Post-hurricane leaves of the non-pioneer species had significantly lower A(max) and were less responsive to changes in irradiance than the pioneer species C. schreberiana. Variation in A(max) across light environments and stages was strongly related to differences in leaf mass per unit area (LMA), especially in the non-pioneer species. As indicated by V(cmax) or J(max) per unit nitrogen, light acclimation of A(max) was determined by leaf morphology (LMA) for the non-pioneer species and by both leaf morphology and leaf biochemistry for C. schreberiana. Ontogenetic changes in A(max) were attributable to changes in leaf morphology. The ontogenetic component of variation in A(max) across light environments and stages differed among species, ranging from 36 to 59% for the non-pioneer species (D. excelsa, 59.3%; P. acuminata var. montana, 44.7%; and S. berteriana, 36.3%) compared with only 17% in the pioneer species C. schreberiana.