Comparative transcriptome analysis provides molecular insights into heterosis of waterlogging tolerance in Chrysanthemum indicum.

BACKGROUND: Heterosis breeding is one of the most important breeding methods for chrysanthemum. To date, the genetic mechanisms of heterosis for waterlogging tolerance in chrysanthemum are still unclear. This study aims to analyze the expression profiles and potential heterosis-related genes of two hybrid lines and their parents with extreme differences in waterlogging tolerance under control and waterlogging stress conditions by RNA-seq. RESULTS: A population of 140 F1 progeny derived from Chrysanthemum indicum (Nanchang) (waterlogging-tolerant) and Chrysanthemum indicum (Nanjing) (waterlogging-sensitive) was used to characterize the extent of genetic variation in terms of seven waterlogging tolerance-related traits across two years. Lines 98 and 95, respectively displaying positive and negative overdominance heterosis for the waterlogging tolerance traits together with their parents under control and waterlogging stress conditions, were used for RNA-seq. In consequence, the maximal number of differentially expressed genes (DEGs) occurred in line 98. Gene ontology (GO) enrichment analysis revealed multiple stress-related biological processes for the common up-regulated genes. Line 98 had a significant increase in non-additive genes under waterlogging stress, with transgressive up-regulation and paternal-expression dominant patterns being the major gene expression profiles. Further, GO analysis identified 55 and 95 transgressive up-regulation genes that overlapped with the up-regulated genes shared by two parents in terms of responses to stress and stimulus, respectively. 6,640 genes in total displaying maternal-expression dominance patterns were observed in line 95. In addition, 16 key candidate genes, including SAP12, DOX1, and ERF017 which might be of significant importance for the formation of waterlogging tolerance heterosis in line 98, were highlighted. CONCLUSION: The current study provides a comprehensive overview of the root transcriptomes among F1 hybrids and their parents under waterlogging stress. These findings lay the foundation for further studies on molecular mechanisms underlying chrysanthemum heterosis on waterlogging tolerance.

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.

Genetic architecture and genomic prediction of plant height-related traits in chrysanthemum.

Plant height (PH) is a crucial trait determining plant architecture in chrysanthemum. To better understand the genetic basis of PH, we investigated the variations of PH, internode number (IN), internode length (IL), and stem diameter (SD) in a panel of 200 cut chrysanthemum accessions. Based on 330 710 high-quality SNPs generated by genotyping by sequencing, a total of 42 associations were identified via a genome-wide association study (GWAS), and 16 genomic regions covering 2.57 Mb of the whole genome were detected through selective sweep analysis. In addition, two SNPs, Chr1_339370594 and Chr18_230810045, respectively associated with PH and SD, overlapped with the selective sweep regions from FST and π ratios. Moreover, candidate genes involved in hormones, growth, transcriptional regulation, and metabolic processes were highlighted based on the annotation of homologous genes in Arabidopsis and transcriptomes in chrysanthemum. Finally, genomic selection for four PH-related traits was performed using a ridge regression best linear unbiased predictor model (rrBLUP) and six marker sets. The marker set constituting the top 1000 most significant SNPs identified via GWAS showed higher predictabilities for the four PH-related traits, ranging from 0.94 to 0.97. These findings improve our knowledge of the genetic basis of PH and provide valuable markers that could be applied in chrysanthemum genomic selection breeding programs.

A group VIIIa ethylene-responsive factor, CmERF4, negatively regulates waterlogging tolerance in chrysanthemum.

Ethylene-responsive factors (ERF) play an important role in plant responses to waterlogging stress. However, the function and mechanism of action of ERFVIII in response to waterlogging stress remain poorly understood. In this study, we found that expression of the ERF VIIIa gene CmERF4 in chrysanthemum was induced by waterlogging stress. CmERF4 localized to the nucleus when expressed in tobacco leaves. Yeast two-hybrid and luciferase assays showed that CmERF4 is a transcriptional inhibitor. CmERF4 overexpression in chrysanthemum reduced plant waterlogging tolerance, whereas overexpression of the chimeric activator CmERF4-VP64 reversed its transcriptional activity, promoting higher waterlogging tolerance than that observed in wild-type plants, indicating that CmERF4 negatively regulates waterlogging tolerance. Transcriptome profiling showed that energy metabolism and reactive oxygen species (ROS) pathway-associated genes were differentially expressed between CmERF4-VP64 and wild-type plants. RT-qPCR analysis of selected energy metabolism and reactive oxygen species-related genes showed that the gene expression patterns were consistent with the expression levels obtained from RNA-seq analysis. Overall, we identified new functions of CmERF4 in negatively regulating chrysanthemum waterlogging tolerance by modulating energy metabolism and ROS pathway genes.

Multi-locus genome-wide association study and genomic prediction for flowering time in chrysanthemum.

MAIN CONCLUSION: Multi-locus GWAS detected several known and candidate genes responsible for flowering time in chrysanthemum. The associations could greatly increase the predictive ability of genome selection that accelerates the possible application of GS in chrysanthemum breeding. Timely flowering is critical for successful reproduction and determines the economic value for ornamental plants. To investigate the genetic architecture of flowering time in chrysanthemum, a multi-locus genome-wide association study (GWAS) was performed using a collection of 200 accessions and 330,710 single-nucleotide polymorphisms (SNPs) via 3VmrMLM method. Five flowering time traits including budding (FBD), visible colouring (VC), early opening (EO), full-bloom (OF) and senescing (SF) stages, plus five derived conditional traits were recorded in two environments. Extensive phenotypic variations were observed for these flowering time traits with coefficients of variation ranging from 6.42 to 38.27%, and their broad-sense heritability ranged from 71.47 to 96.78%. GWAS revealed 88 stable quantitative trait nucleotides (QTNs) and 93 QTN-by-environment interactions (QEIs) associated with flowering time traits, accounting for 0.50-8.01% and 0.30-10.42% of the phenotypic variation, respectively. Amongst the genes around these stable QTNs and QEIs, 21 and 10 were homologous to known flowering genes in Arabidopsis; 20 and 11 candidate genes were mined by combining the functional annotation and transcriptomics data, respectively, such as MYB55, FRIGIDA-like, WRKY75 and ANT. Furthermore, genomic selection (GS) was assessed using three models and seven unique marker datasets. We found the prediction accuracy (PA) using significant SNPs identified by GWAS under SVM model exhibited the best performance with PA ranging from 0.90 to 0.95. Our findings provide new insights into the dynamic genetic architecture of flowering time and the identified significant SNPs and candidate genes will accelerate the future molecular improvement of chrysanthemum.

FUL homologous gene CmFL1 is involved in regulating flowering time and floret numbers in Chrysanthemum morifolium.

Flowering time and floret numbers are important ornamental characteristics of chrysanthemums that control their adaptability and inflorescence morphology, respectively. The FRUITFULL (FUL) gene plays a key role in inducing flowering and inflorescence meristem development. In this study, we isolated a homolog of the MADS-box gene FUL, CmFUL-Like 1 (CmFL1), from chrysanthemum inflorescence buds. Quantitative RT-PCR and in situ analyses showed that CmFL1 was strongly expressed in young inflorescence buds. Overexpression of CmFL1 caused early flowering while co-suppression expression of CmFL1 increased the number of florets. Furthermore, the floral promoting factors CmSOC1, CmFDL1, and CmLFY were up-regulated in the shoot tips of transgenic plants. In addition, RNA-seq analysis of the transgenic plants suggested that certain differentially expressed genes (DEGs)-such as MADS-box, homeobox family, and ethylene pathway genes-may be involved in the inflorescence meristem development. GO pathway enrichment analysis showed that the differentially transcribed genes enriched the representation of the carbohydrate metabolic pathway, which is critical for flower development. Overall, our findings revealed the conserved function of CmFL1 in controlling flowering time along with a novel function in regulating the number of florets.

Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a globally important ornamental plant with great economic, cultural, and symbolic value. However, research on chrysanthemum is challenging due to its complex genetic background. Here, we report a near-complete assembly and annotation for C. morifolium comprising 27 pseudochromosomes (8.15 Gb; scaffold N50 of 303.69 Mb). Comparative and evolutionary analyses reveal a whole-genome triplication (WGT) event shared by Chrysanthemum species approximately 6 million years ago (Mya) and the possible lineage-specific polyploidization of C. morifolium approximately 3 Mya. Multilevel evidence suggests that C. morifolium is likely a segmental allopolyploid. Furthermore, a combination of genomics and transcriptomics approaches demonstrate the C. morifolium genome can be used to identify genes underlying key ornamental traits. Phylogenetic analysis of CmCCD4a traces the flower colour breeding history of cultivated chrysanthemum. Genomic resources generated from this study could help to accelerate chrysanthemum genetic improvement.

CmERF5-CmRAP2.3 transcriptional cascade positively regulates waterlogging tolerance in Chrysanthemum morifolium.

Waterlogging stress affects plant growth by limiting root respiration and reducing yield and economic value. Therefore, identifying genes involved in regulating waterlogging stress is vital. This study reports the ethylene-responsive VII transcription factor (CmRAP2.3) in the chrysanthemum. Subcellular localization and transactivation assay analyses revealed that CmRAP2.3 was localized in the nucleus and possessed transactivation activity. Overexpression of CmRAP2.3 in chrysanthemum was found to enhance waterlogging tolerance by decreasing reactive oxygen species (ROS) levels. Furthermore, we found that the transcription factor CmERF5 binds to GCC-like motifs in the CmRAP2.3 promoter region and activates CmRAP2.3 expression. Additionally, CmERF5 overexpression maintained a low ROS level and improved chrysanthemum waterlogging tolerance. Taken together, this study shows a molecular mechanism by which CmERF5 transcriptionally activates CmRAP2.3 to reduce waterlogging stress via the ROS pathway in the chrysanthemum.

The core regulatory networks and hub genes regulating flower development in Chrysanthemum morifolium.

KEY MESSAGE: The study has facilitated important insights into the regulatory networks involved in flower development in chrysanthemum (Asteraceae), and is informative with respect to the mechanism of flower shape determination. Chrysanthemum morifolium, valued as an ornamental species given the diversity of its inflorescence form, is viewed as a model for understanding flower development in the Asteraceae. Yet, the underlying regulatory networks remain largely unexplored. Here, a transcriptomic survey of the Chrysanthemum morifolium variety 'Jinba' was undertaken to uncover the global gene expression profiles and identify the modules of co-transcribed genes associated with flower development. The weighted gene coexpression network analysis revealed important networks and hub genes including ray floret petals-specific coexpression network, disc floret petals-specific network, B and E class genes involved network and CYC2 genes network. Three ray floret petal-specific hub genes were also strongly transcribed in the ray florets of a selection of six diverse varieties and especially so in those which form ligulate ray floret petals. CmCYC2c was strongly transcribed in the distal and lateral regions of the ray floret petals, and also, along with CmCYC2d, in the tubular ray florets. Furthermore, CmOFP, belonging to the family of ovate proteins, was identified in the CYC2 genes network. CmOFP can interact with CmCYC2d that physically interact with CmCYC2c. This work provides important insights into the regulatory networks involved in flower development in chrysanthemum, and is informative with respect to the mechanistic basis of the regulation of flower shape.

Evaluation of a Yeast Two-Hybrid Library by High-Throughput Sequencing.

Yeast two-hybridization (Y2H) is a classical method to study protein-protein interactions in organisms, and the top limitation of Y2H is the high ratio of false negatives and false positives. The most efficient way to reduce this error is to improve the quality of the library. The traditional library quality evaluation method can only inform us of the capacity of the library and a very limited number of library insert lengths. Therefore, we developed a new method to evaluate the library by using only a 10 ng library, amplifying the inserted fragments through 15 cycles of PCR, and then carrying out high-throughput sequencing. This method can eliminate the randomness and one-sidedness of the traditional method and can be used to obtain key indicators, such as the number of inserted genes and gene abundance, to effectively evaluate the quality of the library. In addition, the new library quality assessment method can also reveal the gene sequences of species. This method is expected to greatly accelerate PPI research on nonmodel species.