Weimin Fang

Next-Generation Sequencing of the Chrysanthemum nankingense (Asteraceae) Transcriptome Permits Large-Scale Unigene Assembly and SSR Marker Discovery

Background Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low. Methodology/Principal Findings Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity. Conclusions/Significance SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera.

Genetic analysis and associated SRAP markers for flowering traits of chrysanthemum (Chrysanthemum morifolium)

The inheritance of two flowering traits of chrysanthemum, initial blooming time and the duration of flowering, was investigated using segregation within an F1 population derived from a cross between the autumn-flowering ‘Yuhualuoying’ and the summer-flowering ‘Aoyunhanxiao’ cultivars. The analysis, based on a single segregating generation and the major gene plus polygene mixed inheritance model, showed that the inheritance of both traits was compatible with the presence of two pairs of major genes displaying additivity–dominance–epistasis, with additivity predominating. As the heritability of both pairs of major genes was high (initial blooming time ~65%, duration of flowering ~72%), it should be possible to select for both traits in early breeding generations. A marker-trait association analysis based on sequence-related amplified polymorphism (SRAP) genotyping uncovered 10 (initial blooming time) and 12 (duration of flowering) markers significantly associated with phenotype, cumulatively explaining, respectively, 46 and 54% of the variation. Some potentially useful markers were identified.

Analysis of Expressed Sequence Tags (ESTs) Collected from the Inflorescence of Chrysanthemum

The expressed sequence tags (ESTs) described in this report were obtained from the inflorescence of chrysanthemum. A complementary DNA (cDNA) library was constructed from the inflorescence of the anemone-type chrysanthemum ‘Zhongshanzigui’. In total, 7,307 cDNA clones were sequenced, representing 4,563 unique sequences and consisting of 3,567 singletons and 996 contigs. Comparison to the GenBank nonredundant (nr) database revealed 57.2% (2,608/4,563) chrysanthemum sequences with homology to genes of known function of other organisms. Approximately 26.67% (1,217/4,563) of the unigenes were clustered into 23 categories by the clusters of orthologous group analysis: Most of the identified transcripts were genes related to metabolism, subcellular localization, protein biosynthesis, and cell wall structure. The ESTs presented here will be a valuable addition to floral development transcriptional database.

A transcriptomic analysis of Chrysanthemum nankingense provides insights into the basis of low temperature tolerance

BackgroundA major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. Yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. Herein, we used RNA-Seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of Chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation.ResultsSix separate RNA-Seq libraries were generated from the RNA samples of leaves and stems from six different temperature treatments, including one cold acclimation (CA), two freezing treatments without prior CA, two freezing treatments with prior CA and the control. At least seven million clean reads were obtained from each library. Over 77% of the reads could be mapped to sets of C. nankingense unigenes established previously. The differentially transcribed genes (DTGs) were identified as low temperature sensing and signalling genes, transcription factors, functional proteins associated with the abiotic response, and low temperature-responsive genes involved in post-transcriptional regulation. The differential transcription of 15 DTGs was validated using quantitative RT-PCR.ConclusionsThe large number of DTGs identified in this study, confirmed the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance.

The constitutive expression of Chrysanthemum dichrum ICE1 in Chrysanthemum grandiflorum improves the level of low temperature, salinity and drought tolerance

AbstractThe quality and productivity of chrysanthemum are severely compromised by various abiotic stresses. Here, we describe the isolation of CdICE1 from Chrysanthemum dichrum using RACE PCR, which shared identical nucleotide of ICE1 ORF from Chrysanthemum grandiflorum variety ‘Jinba’. CdICE1 contains a conserved bHLH domain, a nuclear localization domain, a S-rich motif and a ACT domain. The constitutive expression of CdICE1 in C. grandiflorum improved the tolerance of C. grandiflorum to low temperature/freezing, drought and salinity. When the transgene was inserted in the antisense direction, the expression of the endogenous ICE1 gene was down-regulated, and the level of the plant’s sensitivity to abiotic stress increased. The level of expression of CgDREBa and CgDREBb, activities of superoxide dismutase and peroxidase and the proline content were enhanced in the sense transgenic lines, and lowered in the antisense ones under stresses. In conclusion, CdICE1 represents a promising candidate for a biotechnological approach to improve the level of crop abiotic stress tolerance. Key message Overexpression of CdICE1 in C. grandiflorum confers the stress tolerance via its regulation of CgDREB involved in the oxidative and osmotic homeostasis pathways.

Ambient temperature enhanced freezing tolerance of Chrysanthemum dichrum CdICE1 Arabidopsis via miR398

BackgroundICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance.ResultsUsing EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid assays, we confirmed that CdICE1 binds specifically to the MYC element in the CdDREBa promoter and activates transcription. In addition, overexpression of CdICE1 enhanced Arabidopsis freezing tolerance after transition from 23°C to 4°C or 16°C. We found that after acclimation to 4°C, CdICE1, like Arabidopsis AtICE1, promoted expression of CBFs (CRT/DRE Binding Factor) and their genes downstream involved in freezing tolerance, including COR15a (Cold-Regulated 15a), COR6.6, and RD29a (Responsive to Dessication 29a). Interestingly, we observed that CdICE1-overexpressing plants experienced significant reduction in miR398. In addition, its target genes CSD1 (Copper/zinc Superoxide Dismutase 1) and CSD2 showed inducible expression under acclimation at 16°C, indicating that the miR398-CSD pathway was involved in the induction of freezing tolerance.ConclusionsOur data indicate that CdICE1-mediated freezing tolerance occurs via different pathways, involving either CBF or miR398, under acclimation at two different temperatures.

Rapid Genetic and Epigenetic Alterations under Intergeneric Genomic Shock in Newly Synthesized Chrysanthemum morifolium × Leucanthemum paludosum Hybrids (Asteraceae)

The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5–50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.

The Lateral Suppressor-Like Gene, DgLsL, Alternated the Axillary Branching in Transgenic Chrysanthemum (Chrysanthemum × morifolium) by Modulating IAA and GA Content

The lateral suppressor-like gene DgLsL was transformed by agroinfection into chrysanthemum in both the sense and antisense directions. Sense transformants branched more profusely than the wild-type nontransformant, while branching in the antisense transformants was significantly suppressed. An analysis of DgLsL transcript abundance in the shoot tips revealed that expression was enhanced in the sense transformants and suppressed in the antisense ones. The shoot tip content of indole-3-acetic acid (IAA) was reduced in the sense transformants but enhanced in the antisense ones. The sense transformants had a lower content and the antisense transformants had a higher content of gibberellic acid (GA). Cytokinin content was not affected by the variation in DgLsL expression. We conclude that DgLsL controls shoot branching through its effect on IAA and GA levels.

Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding

Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.

Mapping single-locus and epistatic quantitative trait loci for plant architectural traits in chrysanthemum

Plant architecture is important for chrysanthemum cultivation and breeding. To determine the genetic basis of plant architectural traits in chrysanthemum, a population of 142 F1 plants derived from a cross between the creeping ground-cover chrysanthemum cultivar Yuhualuoying and the erect potted cultivar Aoyunhanxiao was used to detect quantitative trait loci (QTL) associated with plant height, plant width, inter-node length and flower neck length. The broad-sense heritability hB2 for the four plant architectural traits ranged from 0.33 to 0.83, and transgressive segregation was observed. Single-locus QTL analysis revealed a total of five QTL, accounting for 6.0–16.1% of the phenotypic variation. Additionally, 11 pairs of epistatic QTL were identified, explaining 3.5–14.5% of the phenotypic variations. The majority of the interactions detected occurred between background loci. These results indicate that both additive and epistatic effects contribute to phenotypic variation in the plant architecture of chrysanthemum. It is expected that the identified markers associated with the additive QTL and epistatic QTL detected in this study will be of importance in future breeding programs to develop chrysanthemum cultivars exhibiting desirable plant architecture.