Waterlogging tolerance (WT) is a major objective in chrysanthemum breeding programs, and although certain genotypes with different tolerance levels have been identified, their value as parents for WT breeding is unknown. Here, twelve F1 crosses derived from an incomplete diallel mating scheme were conducted to investigate combining ability and heterosis for WT and their relationships with parental genetic distance. The results showed that the membership function value of waterlogging (MFVW) was controlled by additive and non-additive gene effects, whereas other growth and biomass traits were mainly controlled by non-additive gene effects. The estimated broad and narrow sense heritabilities of the MFVW were 97.5 and 51.5%, respectively. Combining ability analyses indicated that ‘Nannong Xuefeng’ showing the largest general combining ability (GCA) effect for the MFVW was the best combiner, and identified several best cross combinations with high positive specific combining ability (SCA) effects for most WT-related traits. Mid- and high-parent heterosis occurred widely. The three distance measures, based on phenotypic traits (PD), molecular markers (GD) and markers linked with quantitative trait loci (QTL-GD), presented a non-significant correlation with combining ability except for the GD with GCA for the relative root fresh weight. The correlations between the QTL-GD and heterosis were significant for certain traits and generally higher than that of the PD or GD and heterosis. The SCA effects were positively correlated with heterosis for most of the WT traits (0.51 ≤ r ≤ 0.80). The findings benefit understanding the inheritance mode and then achieving desirable improvement for WT in chrysanthemum.

Whole genome duplication has a major effect on the phenotype and physiology of higher plants. A comparison between the diploid and tetraploid forms of Chrysanthemum nankingense showed that the latter’s leaf contained a higher content of chlorophyll a/b and harbored a larger number of chloroplasts per cell, leading to an enhancement in its photosynthetic capacity and an improved level of productivity with respect to biomass. A transcriptomic analysis of the two ploidy level forms revealed 21,559 differentially transcribed genes. Compared with diploid progenitor, a number of genes associated with chlorophyll synthesis and those encoding components of photosystems I and II were up-regulated in the tetraploid form, while those associated with chlorophyll degradation were down-regulated. These results indicated that whole genome duplication can directly affect chlorophyll synthesis/degradation and photosynthesis pathways associated with plant growth ratio and biomass accumulation.

Auxin regulates chrysanthemum petal elongation by promoting cell elongation. Transcriptomic analysis shows that auxin signal transduction may connect with other transcription factors by TCPs to regulate chrysanthemum petal elongation.As an ornamental species, Chrysanthemum morifolium has high ornamental and economic value. Petal size is the primary factor that influences the ornamental value of chrysanthemum, but the mechanism underlying the development of C. morifolium petals remains unclear. In our study, we tracked the growth of petals and found that the basal region of ‘Jinba’ petals showed a higher elongation rate, exhibiting rapid cell elongation during petal growth. During petal elongation growth, auxin was demonstrated to promote cell elongation and an increase in cell numbers in the petal basal region. To further study the molecular mechanisms underlying petal growth, the RNA-seq (high-throughput cDNA sequencing) technique was employed. Four cDNA libraries were assembled from petals in the budding, bud breaking, early blooming and full blooming stages of ‘Jinba’ flower development. Analysis of differentially expressed genes (DEGs) showed that auxin was the most important regulator in controlling petal growth. The TEOSINTEBRANCHED 1, CYCLOIDEA and PCF transcription factor genes (TCPs), basic helix-loop-helix-encoding gene (bHLH), glutaredoxin-C (GRXC) and other zinc finger protein genes exhibited obvious up-regulation and might have significant effects on the growth of ‘Jinba’ petals. Given the interaction between these genes in Arabidopsis thaliana, we speculated that auxin signal transduction might exhibit a close relationship with transcription factors through TCPs. In summary, we present the first comprehensive transcriptomic and hormone analyses of C. morifolium petals. The results offer direction in identifying the mechanism underlying the development of chrysanthemum petals in the elongated phase and have great significance in improving the ornamental characteristics of C. morifolium via molecular breeding.

•Phenotypic variants of C. nankingense were first identified by morphological traits.•C. nankingense was high in genetic diversity.•The total DNA methylation level ranged from 54.6% to 62.6%.•Most of the MSAP-methylated fragments were polymorphic in the lines.The diploid species Chrysanthemum nankingense (Anthemideae, Asteraceae) is closely related to the commercially important hexaploid ornamental species Chrysanthemum morifolium and is well adapted to poor environments. In this study, phenotypic variants of C. nankingense were first identified by morphological traits. Using EST-SSR (simple sequence repeat) analysis, we detected some absent EST-SSRs. The percentage of AFLP (amplified fragment length polymorphism) polymorphic fragments was 78.2%, indicating high genetic diversity. To evaluate the genome methylation level and methylation polymorphism, we used the MSAP (methylation-sensitive amplification polymorphism) technique to analyze the 30 C. nankingense lines. The total DNA methylation level ranged from 54.6% to 62.6%. Most of the MSAP-methylated fragments (97%) were polymorphic in the lines. The U-values associated with hemi-methylation were larger than those associated with full methylation in four of the 30 lines, and six individual values were statistically significant (U > 1.96). The high genomic diversity as well as the high methylation polymorphism may be responsible for the morphological polymorphism. There was no significant correlation between the phenotypic and genetic diversity among the lines.

Seedlings of Chrysanthemum, cultivar ‘Puma Sunny’, were grown under a range of shading regimes (natural full sunlight, 55, 25, and 15% of full sunlight) for 18 days. Here, we characterized effects of varying light regimes on plant morphology, photosynthesis, chlorophyll fluorescence, anatomical traits, and chloroplast ultrastructure. We showed that leaf color was yellowish-green under full sunlight. Leaf area, internode length, and petiole length of plants were the largest under 15% irradiance. Net photosynthetic rate, water-use efficiency, PSII quantum efficiency, and starch grain were reduced with decreasing irradiance from 100 to 15%. Heavy shading resulted in the partial closure of PSII reaction centers and the CO2 assimilation was restricted. The results showed the leaves of plants were thinner under 25 and 15% irradiance with loose palisade tissue and irregularly arranged spongy mesophyll cells, while the plants grown under full sunlight showed the most compact leaf palisade parenchyma. Irradiance lesser than 25% of full sunlight reduced carbon assimilation and led to limited plant growth. Approximately 55% irradiance was suggested to be the optimal for Chrysanthemum morifolium.

•This study describes the isolation of a TCP-P transcription factor CmTCP14 from Chrysanthemum.•Heterologous expression studies showed that CmTCP14 suppresses organ size, delays leaf senescence and prolong flowering time.•Overexpression of CmTCP14 down regulated some cell-cycle related and up regulated chlorophyll synthesis-related genes.•Y2H assay indicated CmTCP14 can interact with CmFTL2 and some CmDELLAs.TCP transcription factors are important for plant growth and development, but their activity in chrysanthemum (Chrysanthemum morifolium) has not been thoroughly explored. Here, a chrysanthemum TCP-P sequence, which encodes a protein harboring the conserved basic helix-loop-helix (bHLH) motif, was shown to be related phylogenetically to the Arabidopsis thaliana gene AtTCP14. A yeast-one hybrid assay showed that the encoding protein had no transcriptional activation ability, and a localization experiment indicated that it was localized in the nucleus. Transcription profiling established that the gene was most active in the stem and leaf. Its heterologous expression in A. thaliana down-regulated certain cell cycle-related genes, reduced the size of various organs and increased the chlorophyll and carotenoid contents of the leaf which led to delayed senescence and a prolonged flowering period. Moreover, by screening the cDNA library of chrysanthemum, we found that the CmTCP14 can interact with CmFTL2 and some CmDELLAs.

Forty-five molecular markers were detected significantly associated with chrysanthemum’ waterlogging tolerance, and four favorable parental lines were identified as potential donors for improving waterlogging tolerance in chrysanthemum.The productivity of chrysanthemum is downgraded by waterlogging soils, which has driven a search for germplasm showing an enhanced level of waterlogging tolerance (WT). As yet little is known regarding the mode of inheritance of WT in chrysanthemum. The study set out to characterize the extent of genetic variation for WT represented in a collection of one hundred chrysanthemum accessions by testing them under both greenhouse and field conditions. A membership function value of waterlogging (MFVW), which integrated a wilting index, a chlorosis score and the proportion of dead leaf in waterlogged plants, was used as a measure of WT. The variation for MFVW among plants grown in the greenhouse (two experiments) was generally higher than that generated in field-grown (one experiment) plants. The MFVW broad sense heritability was 0.82, and the phenotypic coefficient of variation (31.8 %) was larger than the genetic one (28.8 %). Association mapping (AM) identified 45 markers related to WT: 25 by applying the general linear model (GLM) + principal component (PC) model, 16 by applying the mixed linear model (MLM), 31 by applying the MLM + Q matrix model and 12 by applying the MLM + PC model. Of the associated markers, eight and two were predictive in two and three experiments within all models, respectively; the proportion of the phenotypic variance explained by the eight associations ranged from 6.3 to 16.4 %. On the basis of their harboring all four of the leading markers E2M16-2, SSR150-6, E19M16-1 and E10M10-12, the varieties ‘Nannong Xuefeng’, ‘Qx097’, ‘Nannong Xunzhang’ and ‘Finch’ were identified as potential donors for future improvement of WT in chrysanthemum.

Characterizing the genetic diversity present in a working set of plant germplasm can contribute to its effective management and genetic improvement. The cut flower chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important ornamental species. With the repeated germplasm exchange and intensive breeding activities, it remains a major task in genetic research. The purpose of the present study was to characterize the genetic diversity and the population structure of a worldwide collection of 159 varieties, and to apply an association mapping approach to identify DNA-based markers linked to five plant architecture traits and six inflorescence traits. The genotyping demonstrated that there was no lack of genetic diversity in the collection and that pair-wise kinship values were relatively low. The clustering based on a Bayesian model of population structure did not reflect known variation in either provenance or inflorescence type. A principal coordinate analysis was, however, able to discriminate most of the varieties according to both of these criteria. About 1 in 100 marker pairs exhibited a degree of linkage disequilibrium. The association analysis identified a number of markers putatively linked to one or more of the traits. Some of these associations were robust over two seasons. The findings provide an in-depth understanding of genetic diversity and population structure present in cut flower chrysanthemum varieties, and an insight into the genetic control of plant architecture and inflorescence-related traits.

•Localized 45S and 5S rDNA loci in 12 species of Chrysanthemum and related genera via FISH.•Characterized karyotypes of the twelve species.•Inferred phylogenetic relationships among the 12 species based on FISH and karyotypes.•The results faciliated future improvement for cultivated chrysanthemum via wide hybridization.The phylogenetic relationships among Chrysanthemum and its related genera (Anthemideae, Asteraceae) is poorly understood. In the present study, these relationships were investigated using 45S and 5S ribosomal DNA (rDNA)-targeted fluorescent in situ hybridization. The results showed that there were two 45S rDNA signals present in Crossostephium chinense, four 45S rDNA signals in Cercidiphyllum japonicum, Artemisia sieversiana, Artemisia annua and Artemisia absinthium, six 45S rDNA signals in Chrysanthemum boreale and Pyrethrum parthenium, eight 45S rDNA signals in Chrysanthemum nankingense, Chrysanthemum dichrum, Chrysanthemum lavandulifolium and Tanacetum vulgare, and ten 45S rDNA signals in Ajania przewalskii. For the 5S rDNA locus, two 5S rDNA signals were present in C. nankingense, C. dichrum, C. lavandulifolium, C. boreale, C. japonicum, C. chinense and P. parthenium, four in A. sieversiana, A. annua, A. absinthium and A. przewalskii, and six 5S in T. vulgare. In addition, karyotypes of the 12 species were investigated. From this study, we inferred that Chrysanthemum was closely related to Ajania, and that Chrysanthemum species originating from China and Japan may have evolved differently. These findings add a new level to the understanding of the phylogenetic relationships of Chrysanthemum and related genera.

Branching is an important horticultural trait that influences plant architecture and production of cut chrysanthemum; however, its genetic determinism is poorly understood. In this study, an F1 mapping population derived from a cross between two spray cut chrysanthemum cultivars was used to determine QTL underlying branching. The branching traits in the F1 mapping population exhibited over-parent segregation and relatively high heritability (0.68–0.92). Based on the branching traits across two consecutive years, 16 additive QTL, including five QTL for primary branch number, each four QTL for branch height, and branch angle, and three QTL for primary branch length, were identified, with each accounting for 6.9–24.4 % of the phenotypic variation. Of these, four QTL expressed stable, located in the same genomic regions across two consecutive years. Results from this study add new understanding of the genetic basis of branching in spray cut chrysanthemum, and the putative major QTL detected here are valuable for future breeding cut chrysanthemum that exhibits desirable branching type.

CmWRKY17was induced by salinity in chrysanthemum, and it might negatively regulate salt stress in transgenic plants as a transcriptional repressor.WRKY transcription factors play roles as positive or negative regulators in response to various stresses in plants. In this study, CmWRKY17 was isolated from chrysanthemum (Chrysanthemum morifolium). The gene encodes a 227-amino acid protein and belongs to the group II WRKY family, but has an atypical WRKY domain with the sequence WKKYGEK. Our data indicated that CmWRKY17 was localized to the nucleus in onion epidermal cells. CmWRKY17 showed no transcriptional activation in yeast; furthermore, luminescence assay clearly suggested that CmWRKY17 functions as a transcriptional repressor. DNA-binding assay showed that CmWRKY17 can bind to W-box. The expression of CmWRKY17 was induced by salinity in chrysanthemum, and a higher expression level was observed in the stem and leaf compared with that in the root, disk florets, and ray florets. Overexpression of CmWRKY17 in chrysanthemum and Arabidopsis increased the sensitivity to salinity stress. The activities of superoxide dismutase and peroxidase and proline content in the leaf were significantly lower in transgenic chrysanthemum than those in the wild type under salinity stress, whereas electrical conductivity was increased in transgenic plants. Expression of the stress-related genes AtRD29, AtDREB2B, AtSOS1, AtSOS2, AtSOS3, and AtNHX1 was reduced in the CmWRKY17 transgenic Arabidopsis compared with that in the wild-type Col-0. Collectively, these data suggest that CmWRKY17 may increase the salinity sensitivity in plants as a transcriptional repressor.

Intergeneric hybridization is an effective way of improving crop resistance against biotic and tolerance to abiotic stresses. Here, we describe an intergeneric hybrid between Chrysanthemummorifolium ‘Nannongxiaoli’ and Artemisia vulgaris ‘Variegata’ using ovule rescue. Chromosome counting confirmed that out of the 124 regenerated plantlets, 19 were genuine hybrids, carrying the expected hybrid number of somatic chromosomes (45, representing 27 from ‘Nannongxiaoli’ and 18 from ‘Variegata’). The 19 genuine hybrids were vigorous, flowered normally and possessed the cut flower characteristics of ‘Nannongxiaoli’. The hybrids were more resistant against both aphid infestation and Alternaria leaf spot inoculation than the ‘Nannongxiaoli’ parent, but less than the ‘Variegata’ parent.

Using embryo rescue, we generated an intergeneric hybrid between Chrysanthemum × morifolium ‘Maoyan’ and Artemisia japonica Thunb. Cytological tests confirmed that regenerated plantlets were all genuine hybrids possessing 45 chromosomes, with 27 chromosomes inherited from C. × morifolium (2n = 6x = 54) and the other 18 derived from A. japonica (2n = 4x = 36). Hybrid plant flowered normally. The shape and color of the hybrid flowers and leaves resembled those of chrysanthemum, while leaf width, leaf length, plant height, and inflorescence diameter were intermediate between those of the parents. Hybrid plant had higher levels of chlorophyll and free proline, and lower concentrations of malondialdehyde and Na+, than the maternal parent (C. × morifolium), and these levels were correlated with the hybrid’s enhanced salt tolerance. These results clearly demonstrate that intergeneric hybridization is an effective method of cultivar improvement in chrysanthemum.

Ovule rescue was used to obtain the intergeneric hybrid Chrysanthemum indicum L. Des Moul. x Opisthopappus taihangensis (Ling) Shih. The hybridity of the seven independent progeny was confirmed by morphology, chromosome counting and GISH analysis. Plant height, crown width, leaf length, leaf width, epidermal hair height and epidermal hair length of the hybrid were all intermediate between those of the parents. However, their petiole length and epidermal hair density exceeded those of either parent. GISH analysis of the hybrid was able to distinguish between the parental genomes.Highlights► Intergeneric hybrid was obtained between C. indicum and O. taihangensis. ► The hybrids’ hybridity was identified by morphology, chromosome counting and GISH. ► Genomes of D. indicum and O. taihangensis are distantly related to one another.

Knowledge on genes related to plant responses to adverse growth conditions and development is essential for germplasm improvement. In this study, a chrysanthemum R2R3-MYB transcription factor gene, designated CmMYB2 (GenBank accession No. JF795918), was cloned and functionally characterized. Expression of CmMYB2 in chrysanthemum leaves was up-regulated in response to drought, salinity and cold stress, as well as by treatment with exogenous abscisic acid (ABA). When the gene was constitutively expressed in Arabidopsisthaliana, it increased plant sensitivity to ABA and reduced stomatal aperture. Plant survival under drought was improved than in the wild type, as was the plants’ salinity tolerance. The level of expression of a number of genes associated with the stress response, including RD22, RD29A, RAB18, COR47, ABA1 and ABA2, was raised in the CmMYB2 transgenic Arabidopsis plants. CmMYB2 transgenic Arabidopsis plants were also delayed in flowering. The expression of CONSTANS (CO), FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY) and APETALA1 (AP1) genes involved in flowering was down-regulated in the CmMYB2 transgenics. Together, these results suggest that CmMYB2 may be a promising gene for the drought and salt tolerance improvement and flowering-time modulation.

The cis-acting elements present in gene promoters are important for promoter function. Thermal Asymmetric Interlaced PCR (TAIL-PCR) provides a simple means for isolating promoter sequences, but the necessary primers can be troublesome to design. Here, we describe an approach, which targets cis-acting elements for TAIL-PCR. The method combines the advantages of TAIL-PCR and SiteFinding-PCR. The new method proved successful for isolating a number of plant promoter sequences.

The character of branching for two chrysanthemum (Chrysanthemum × morifolium) cvs. Jinghai and Jingyun was observed, and the changes of endogenous hormones in apical and lateral buds were investigated to determine the relationship between the pattern of hormone distribution, apical dominance, and lateral bud outgrowth. The growth rate of Jinghai lateral buds was higher than that of Jingyun. In vegetative growth stage, IAA level in apical buds of Jingyun was significantly higher than in Jinghai. After flower induction, IAA level in apical buds of two cultivars decreased remarkably, but the IAA level decreased in Jingyun faster than in Jinghai. These results showed that the higher was the IAA level in apical buds the stronger was inhibition of lateral bud outgrowth. An increase in IAA and iP/iPA and a decrease in ABA concentrations were closely associated with lateral bud growth alterations in chrysanthemum.

The chrysanthemum (Dendranthema morifolium) variety ‘Yuhuajinhua’ has a creeping growth habit. An ELISA-based assessment of the content and distribution of IAA in the stem of ‘Yuhuajinhua’ showed that there was an IAA concentration gradient across the stem segment between the 2nd and 4th nodes, counting from the apex, before the creeping growth, while this difference disappeared after the initiation of creeping growth. An immunohistochemical assay for IAA showed that auxin was concentrated in the epidermis and cortex of the proximal side of the stem, particularly in the first few hours after gravitational stimulation was applied. The bending of the stem was generated by the asymmetric elongation of the epidermal cells in proximal side of the stem, especially upon to 6 h after gravistimulation, and this was probably mediated by an alteration in the IAA gradient across the stem. Exogenously applied 1-naphthaleneacetic acid (NAA) or 2,3,5-triiodobenzoic acid (TIBA) played converse effects on the gravitropic stem curvature. The data support the idea that IAA plays a crucial regulatory role in the formation of the creeping habit in the chrysanthemum variety ‘Yuhuajinhua’.

The 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.

In order to provide additional information on the coloration of chrysanthemum flowers, the flavonoid composition and the expression of six structural genes involved in anthocyanin pathway in the ray florets of a pink flowering (cv. H5) and two white flowering (cvs. Keikai and Jinba) Chrysanthemum grandiflorum cultivars were examined. HPLCDAD/ESI-MSn analysis showed that cyanidin 3-O-(6″-O-malonylglucoside) and cyanidin 3-O-(3″,6″-O-dimalonylglucoside) were the two major flavonoids presented in H5, while white flowering cultivars contained flavones instead of anthocyanins. Nine flavone derivatives were detected in the three cultivars, the amount of each flavone varied upon cultivars, and seven of these were identified as luteolin 7-O-arabinosylglucuronide, apigenin 7-O-glucoside, luteolin 7-O-malonylglucoside, apigenin 7-O-malonylglucoside, chrysoeriol 7-O-malonylglucoside, acacetin 7-O-rutinoside and acacetin 7-O-malonylglucoside. The two white flowering cultivars showed similar total flavonoid content, which was about two fold higher than that in H5. A high expression of the genes encoding dihydroflavonol 4-reductase and 3-O-glucosyltransferase was detected only in H5 but not in Keikai or Jinba. Chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and flavonoid 3′-hydroxylase were expressed in all flowers, suggesting that the lack of anthocyanin in white flowering cultivars cannot be due to any blockage of their expression.

The garden chrysanthemum (Chrysanthemum × morifolium) variety “Aoyunhuoju” (2n = 6x = 54) was crossed as female with Ajania pacifica (2n = 10x = 90) to produce intergeneric F1 hybrids, which were used both as the source of F2 generation and as the parent for a first back-cross with “Aoyunhuoju”. The morphology of all of the F1 hybrids and hybrid derivatives was intermediate with respect to the two parents, although the BC1 progenies resembled “Aoyunhuoju” more closely than any of the F1 and F2 progenies did. In the F1 generation, the density of silvery hairs on the lower leaf surface and along the margin of the leaf was lower than in A. pacifica, while that in the BC1 generation, this trait was less prominent than in the F1. The somatic chromosome number of the F1, F2 (with an exception of F2-6 of a mainly 63) and BC1 generations was 2n = 8x = 72, 2n = 8x = 72 and 2n = 7x = 63 respectively, as expected. The hybrids and their derivatives retained a variable degree of fertility. There was a low frequency of meiotic chromosome pairing failure in all three hybrid generations, with most of the chromosomes involved as bivalents. Some BC1 individuals show potential for commercialization thanks both to their flower shape and the inheritance of the silvery leaf trait from A. pacifica.

A 1,474-bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB site, GT-1, and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream 430 and 351 nt. This 80-bp fragment enhanced GUS expression under salinity stress when fused to −90/+8 CaMV 35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature-responsive element was located between positions −430 and −390, and a salinity inducible one between −385 and −351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.

The effect of exogenously applied nitric oxide on the heat tolerance of Chrysanthemum morifolium was investigated by applying the NO donor sodium nitroprusside (SNP). We found that the SNP partially alleviated the heat stress by slowing down the reduction of photosynthetic pigment content and net photosynthetic rate. SNP treatment also lowered the increase in the non-photochemical quenching of fluorescence and malondialdehyde content and maintained higher activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase.

Disease assessment to measure severity of alternaria leaf spot (Alternaria tenuissima (Fr.) Wiltsh.) was performed in 32 wild species of Compositae family by seedling inoculation. It was found that two species were resistant, four were moderately resistant, and others were susceptible to various degrees. Some leaf morphological traits of two resistant and two highly susceptible wild species were studied. Trichome density and height and wax content were found to be associated with plant passive resistance, while stomata density was not associated. After A. tenuissima inoculation, enzymes phenylalanine ammonia-lyase, β-1,3-glucanase, and chitinase were also assayed. Their higher activities in the selected resistant wild species and lower activities in the highly susceptible ones were found. This suggests the possibility that these enzymes are not only constituents of resistance but also could be used as biochemical markers for screening Compositae wild species for alternaria leaf spot resistance.

The floral organs of typical eudicots such as Arabidopsis thaliana are arranged in four characteristic whorls, namely the sepal, petal, stamen and carpel, and the “ABC” floral organ identity model has been based on this arrangement. However, the floral organs in most basal angiosperms are spirally arranged with a gradual transition from the inside to outside, and an alternative model referred to as “fading borders” was developed to take account of this. The flower morphology of the water lily was tested against the “fading borders” model by determining the expression profile of the six primary floral organ identity genes AP2, AGL6, AP3, PI, AG and SEP1 in two cultivars showing contrasting floral morphology. In addition, to get accurate floatation of the genes expression level from outer to inner, we divided the floral organs into eight whorls according to morphological features. All these genes were expressed throughout all whorls of the flower, but their expression level changed gradually from the outside of the flower to its inside. This pattern was consistent with the “fading borders” model.

Chrysanthemum (Dendranthema morifolium) is an economically important ornamental species and comprises a large proportion of the flower industry in south-east Asian and European countries. In this study, a segregating population of 142 F1 progeny of the cross between the two chrysanthemum cultivars ‘Yuhualuoying’ and ‘Aoyunhanxiao’ was used to construct two separate genetic linkage maps via a double pseudo-testcross mapping strategy. Genotyping was performed using 500 SRAP primer combinations, of which about 50% were informative. This allowed the definition of 896 SRAP loci, of which about 23% showed some segregation distortion. The ‘Yuhualuoying’ map consisted of 333 testcross markers arranged into 57 linkage groups (LGs). It covered >1,900 cM with a mean inter-marker distance of 6.9 cM. The map constructed from ‘Aoyunhanxiao’ comprised 342 test cross markers arranged into 55 LGs. It spanned nearly 1,900 cM, with a mean inter-marker distance of 6.6 cM. The markers were distributed rather uniformly along both maps. A quantitative trait loci analysis was conducted to investigate the pattern of inheritance of three inflorescence traits. This led to the detection of 12 putative loci at a LOD score >2.5, of which four each specified flower diameter, ray floret layer number, and ray floret length. This study provides molecular mapping information on marker-assisted selection programs for the improvement of multiple traits of interest.

Five intergeneric hybrids between the chrysanthemum cultivar ‘Zhongshanjingui’ (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While ‘Zhongshanjingui’ bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids’ flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from ‘Zhongshanjingui’ and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.

Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1α (elongation factor 1α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.

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.

The interspecific cross between Chrysanthemum × grandiflorum (Ramat.) Tzvel. ‘rm20-12’ (R, 2n = 54) and C. makinoi Matsum., and Nakai (M, 2n = 18) was achieved using embryo rescue, and a single backcross progeny using C. × grandiflorum ‘rm20-12’ as paternal parent was obtained. The morphology of the two independent F1 hybrids (MR1 and MR2) differed from that of both parents. MR1 had a larger inflorescence diameter along with narrow leaves and a reduced number of ray and tubular florets. MR2 was shorter and its inflorescence developed fewer tubular florets than either M or R. The BC1F1 hybrid was similar to its maternal plant MR2 in terms of leaf length and width, inflorescence diameter and the number of ray florets, while it produced fewer tubular florets than either MR2 or R. The flower color in both F1 hybrids was lavender, while the BC1F1 plant bore purple flowers. The aphid resistance and heat tolerance of MR1, MR2 and the BC1F1 hybrid were both significantly superior to that of C. × grandiflorum ‘rm20-12’. Interspecific hybridization followed by backcrossing shows clear potential for cultivar improvement in chrysanthemum.

The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1α) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.

Ovary rescue was employed to create six interspecific hybrids from the cross between Dendranthema morifolium (Ramat.) Kitamura ‘rm20-12’ (2n = 54) and its wild diploid relative D. nankingense (Nakai) Tzvel. (2n = 18). The morphology of the hybrids differed from that of either parent. The leaf length and width of all three D. morifolium × D. nankingense hybrids exceeded that of the parents, as did the plant height of two and the inflorescence diameter of another of the hybrids. One of the reciprocal hybrids was heterotic for leaf length and width. All the hybrids bore yellow flowers. The cold tolerance of five hybrids was significantly superior to that of their D. morifolium parent. Interspecific hybridization clearly provides an effective means of cultivar improvement in chrysanthemum.

Aphids represent the most destructive of chrysanthemum pests to cultivation. Reliable variety sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. An intergeneric hybrid between Dendranthema morifolium (chrysanthemum) variety ‘Zhongshanjingui’ and Artemisia vulgaris (mugwort) ‘Variegata’ was attempted. Most of the hybrid embryos aborted at an early developmental stage. Embryo rescue allowed the generation of hybrid plants, whose hybridity was confirmed by a combination of morphological, cytological and GISH analysis. The hybrids were vigorous, flowered normally, and their flower and leaf shape resembled those of the chrysanthemum more than those of the mugwort parent. The hybrids showed much higher resistance to chrysanthemum aphid (Macrosiphoniellasanbourni) than maternal chrysanthemum by inoculation test. The leaves of the hybrid developed a higher density of trichomes and secretory glands compared to the maternal chrysanthemum. GC–MS analysis revealed that ~51% of the essential oil in the hybrid leaves were monoterpenoids and sesquiterpenoids, while the proportion in the chrysanthemum was ~37%, and in the mugwort was ~90%. It is inferred that higher aphid resistance in the hybrid mainly owed to the leaf micromorphology and bioactive essential oil content.

In order to reveal the causes of low seed set in the intergeneric cross between Chrysanthemum grandiflorum (Ramat.) Kitam. ‘Zhongshanjingui’ (female parent) and Ajania przewalskii Poljak. (male parent), we systematically investigated megasporogenesis and megagametogenesis of female parents, pollen germinability of male parents, pollen–pistil interaction and hybrid embryo development using paraffin section and light, fluorescence and scanning electron microscopy. The results indicate that development of megagametophyte of female parent belongs to Polygonum type and 84.6% ovules developed to normal 8-nucleate embryo sac at the stage of pollination; the pollen germinability in vitro of male parent was 39.3%; a large number of pollen grains germinated normally and no callose deposited on the female stigmas within 24 h after pollination. Furthermore, about 75% ovules were fertilized, whereas only 17.2% ovules developed to normal maturing embryos at 15 days after pollination due to abortion at various developmental stages. Taken together, these results suggest that sterile ovules of female parent, pollen viability and pollen–pistil interaction have no significant influence on seed set in the cross. A high percentage of embryo abortion or degeneration, i.e. post-fertilization barrier, may be the main factor for low seed yield.

In order to improve resistance to pathogens in chrysanthemum, we generated transgenic chrysanthemum plants heterogeneously expressing hpaGXoo gene from Xanthomonas oryzae pv. oryzae via leaf-disc mediated Agrobacterium tumefaciens (EHA105) transformation. Polymerase chain reaction (PCR), Southern blot, and Reverse transcription PCR (RT-PCR) analysis showed that the hpaGXoo gene was successfully integrated into the chrysanthemum genome and expressed in transgenic lines. An inoculation assay indicated that the presence of the transgene was associated with an improved level of resistance to alternaria leaf spot. The transgenic introduction of pathogen elicitor-encoding genes has a considerable potential for improving the disease resistance of ornamental plants. Furthermore, transgenic chrysanthemums flowered significantly earlier than the wild-type plants did, suggesting that the hpaGXoo gene probably accelerated chrysanthemum development.

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.

Although most of the hybrid embryos aborted at an early developmental stage, a 2n = 27 true intergeneric hybrid between Dendranthema indica (L.) Des Moul (2n = 36) as ♀ and Crossostephium chinense (L.) Makino (2n = 18) as ♂ was produced following pollination and ovule rescue. The morphology of the resulting adult putative hybrid and the two parents differed significantly from one another in seven of the nine traits measured, the exceptions being leaf width and leaf length, for which the putative hybrid was indistinguishable from the maternal plant. Genomic in situ hybridization experiments were able to successfully distinguish the genomic origin of both mitotic and meiotic metaphase chromosomes in the hybrid. In addition, the 18 D. indica chromosomes were paired as bivalents at meiotic metaphase in the hybrid.

To better understand the evolutionary history, intergeneric relationships and circumscription of Chrysanthemum and Ajania and the taxonomic position of some small Asian genera (Anthemideae, Asteraceae), the sequences of the nuclear ribosomal internal transcribed spacer (nrDNA ITS) and the chloroplast trnL-F intergenic spacer (cpDNA IGS) were newly obtained for 48 taxa and combined with those already deposited in GenBank. Phylogenies with an emphasis on Chrysanthemum, Ajania and its allies, by both maximum parsimony and Bayesian analysis, were constructed using either the ITS sequence alone, the IGS sequence alone or combined sequences. The IGS sequence was low phylogenetically informative, but some deletions and insertions were informative for interspecific and intergeneric delimitations. The ITS and the ITS/IGS phylogenies both suggested the presence of two major clades. The monophyly of subtribe Artemisiinae (clade A) could be retrieved when the phylogenetic positions of some ambiguous taxa were renewedly considered. Subtribe Artemisiinae was chiefly divided into two groups, (1) one corresponding to Chrysanthemum, Arctanthemum, Ajania, Opisthopappus and Elachanthemum (the Chrysanthemum group), (2) another to Artemisia, Crossostephium, Neopallasia and Sphaeromeria (the Artemisia group). Within the Chrysanthemum group, ChrysanthemumArctanthemum and Ajania were closely related to each other, and the generic circumscription was ambiguous; Phaeostigma was excluded from this group that was also confirmed by the 6-bp insertion in the IGS sequence; radiate or rare discoid Brachanthemum was excluded, and discoid Elachanthemum without ray florets was added to this group; at the same time, Opisthopappus in subtribe Tanacetinae should be transferred to subtribe Artemisiinae and became one of the components of the Chrysanthemum group. Based on the molecular phylogenetic framework, the evolution of pollen and capitulum characters was inferred.

An intergeneric cross has been made between Dendranthema crassum (kitam.) kitam. (2n = 90; ♀) and Crossostephiumchinense (L.) Makino (2n = 18; ♂). Most of the hybrid embryos aborted at an early developmental stage. Using ovule rescue, it was possible to establish a single intergeneric hybrid plant showing 2n = 54 chromosomes. The leaf length, leaf width and epidermal hair density of the hybrid were all intermediate between those of the parents. However the flower diameter, number of tubular florets, epidermal hair height and epidermal hair length exceeded those of both parents. A genomic in situ hybridization approach was able to distinguish between the parental genomes in the hybrid plant.

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.

Meiotic behavior in pollen mother cells and in vitro pollen germination were examined in a sample of small-flowered anemone type chrysanthemum cultivars. Bivalent formation predominated at metaphase I in all the cultivars although some univalents and multivalents (mainly quadrivalents) were observed. Lagging chromosomes, chromosome bridges, micronuclei and polyads were observed at anaphase I/telophase I and anaphase II/telophase II. Pollen germinability ranged from 0.3 to 25.6% and was below 10% in 15 of the 22 cultivars. A significant positive correlation was established between disk floret length (tube + lobe)/style length and the frequency of chromosome bridges at anaphase II/telophase II, and negative ones both between disk floret length/style length and pollen germinability, and between the frequency of univalents at metaphase I and pollen germinability.

The chrysanthemum cultivar ‘Yuhuajinhua’ has a creeping habit. The anatomy and distribution of amyloplasts within ‘Yuhuajinhua’ stems were compared to those typical of non-creeping cultivars. ‘Yuhuajinhua’ stems are similar to those of conventional cultivars; but except for the pith, the proportion of the various tissues present in the upper side of stems was higher than that in the lower side. Most of the amyloplasts lie in the centre of the endodermal cells of the ‘Yuhuajinhua’ stems, rather than at the bottom, as is typical for non-creeping cultivars. When ‘Yuhuajinhua’ plants were oriented horizontally and kept in the dark, the stems retained their original growth direction, and the endodermis amyloplasts sedimented according to the gravitational direction. The endodermis amyloplasts responded rapidly to gravistimulation. The content of IAA in the upper side of the ‘Yuhuajinhua’ stems was higher than that in the lower side, associated with the assymetric growth of the stems.

•12 SPL genes were isolated and characterized in chrysanthemum.•Conserved motifs in the SBP-box proteins were shared by Arabidopsis and chrysanthemum.•6 CmSPLs contained a miR156 target site, while 5 CmSPLs were targeted by miR157.•The hormone and stress treatments arouse the expression of 12 CmSPLs variously.SQUAMOSA promoter-binding protein (SBP) transcription factors are known to function in a number of processes in plants. Here, we have characterized twelve SBP-like (SPL) genes in the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of twelve distinct sequences were isolated and amplified based on transcriptomic sequences. Phylogenetic analysis identified two pairs of orthologous proteins for Arabidopsis and chrysanthemum and two pairs of paralogous proteins in chrysanthemum. Conserved motifs in the SPL proteins shared by Arabidopsis and chrysanthemum were scanned using MEME. A bioinformatics analysis revealed that six of these genes contained a miR156 target site, while five CmSPLs were targeted by miR157. Moreover, we used 5′ RLM-RACE to map the cleavage sites in CmSPL2 and CmSPL3. The expression of these twelve genes in response to a variety of phytohormone treatments and abiotic stresses was characterized. This work improves our understanding of the various functions of SPL gene family members in the stress response.

The drought and salinity tolerances of a creeping ground-cover chrysanthemum variety ‘Yuhuaxunzhang’ were compared to the performance of its two derived transgenic lines carrying a drought-responsive element binding (DREB) factor from chrysanthemum designated as CgDREBa. The over-expression of CgDREBa conferred a measure of tolerance to both stresses. The transgenic lines showed a higher survival rate and were better able to retain fresh weight in the presence of stress. Activities of superoxide dismutase and peroxidase and the proline content were all higher in the leaves of the transgenic plants after a prolonged period of stress, but they accumulated less malondialdehyde. CgDREBa appears to function as a transcription activator of genes within the oxidative and osmotic homeostasis transduction pathways, and represents a promising candidate for a biotechnological approach to improve the level of abiotic stress tolerance in plants.Highlights• The over-expression of CgDREBa conferred drought and salinity tolerance of chrysanthemum. • Activities of superoxide dismutase and peroxidase were higher in the transgenic plants. • The transgenic plants accumulated higher proline while less malondialdehyde than control. • CgDREBa regulates the oxidative and osmotic homeostasis pathways in chrysanthemum.

We report the production of an intergeneric hybrid between Dendranthema nankingense and Tanacetum vulgare via ovule rescue. Out of 420 cultured ovules, four gave rise to a total of 27 plantlets. A genomic in situ hybridization analysis confirmed that regenerated plantlets were all genuine hybrids. The leaf length, petiole length, number of tubular florets and epidermal hair density of the hybrids were all intermediate between those of the two parents. However, both the leaf width and epidermal hair length of the hybrids were less than those of the parents. The number of ligulate florets is significantly greater than those of the parents. Flower diameter and epidermal hair height is similar to that of paternal parent. GISH was able to distinguish the parental genomes in the hybrids, suggested the two genera is taxonomically distant from each other.Highlights► Intergeneric hybrid between Dendranthema nankingense and Tanacetum vulgare was produced. ► Genomic in situ hybridization analysis confirmed that all hybrids are genuine. ► GISH analysis suggested the two genera is taxonomically distant from each other.

The drought tolerance of the chrysanthemum (Chrysanthemum morifolium Ramat.) ‘Zhongshanjingui’, Ajania przewalskii Poljakov and two independent hybrid lines of ‘Zhongshanjingui’ × A. przewalskii were compared. The intergeneric hybrids and paternal parent (A. przewalskii) showed higher tolerance to drought than the maternal parent (Zhongshanjingui) during continuous drought stress conditions. After 10 days of drought treatment, when the water content of the growth medium decreased to 0.8%, the female parent exhibited the most severe wilting (the maximum index value of 3.0), whereas the male parent and two hybrid lines showed moderate wilting (wilting indices of 1.9, 2.2 and 2.4, respectively). Twelve hours after rewatering of the drought-stressed plants, the chrysanthemum leaves failed to recover, whereas leaves of the hybrids and A. przewalskii recovered turgor. Under drought conditions, the hybrids and A. przewalskii showed superior root growth to that of chrysanthemum with respect to both the rate of relative root growth and root dry weight per plant. Enhanced drought tolerance was also correlated with an increase in free proline and a decrease in malondialdehyde contents. The results indicated that the hybrids showed morphological and physiological responses to drought conditions inherited from A. przewalskii, and that intergeneric hybridization is a favorable means to introduce multiple resistance characters of relative germplasm into chrysanthemum.Highlights► We studied drought tolerance of hybrids between chrysanthemum and Ajania przewalskii. ► Hybrids showed higher drought tolerance than the chrysanthemum parent. ► Wilting was temporary in hybrids and A. przewalskii but permanent in chrysanthemum. ► A larger root system in the hybrids was a morphological response to drought. ► Increased proline and decreased MDA were synthesized in the hybrids under drought.

Opisthopappus taihangensis is an endangered species endemic to China and represents an important genetic resource for chrysanthemum improvement. We describe here its basic reproductive characteristics. The anthers are tetrasporangiate and the anther wall is composed of an epidermis, endothecium, middle layer and tapetum. The middle layer is lost by the microspore tetrad stage, and the tapetum disintegrates at the trinucleate pollen stage. Meiosis in the microspore mother cells is of the simultaneous type, and the tetrad is tetrahedral in shape. Mature pollen grains have three germinal apertures, two sperm nuclei and one vegetative nucleus. The in vitro pollen germination rate is only ∼10%. The ovule is anatropous, dual-integument, tenuinucellatae and the development of the embryo sac follows the Oenothera pattern. The archesporial cell below the nucellus epidermis functions as the megaspore mother cell and forms a linear tetrad. The embryo passes through a globular, heart and torpedo stage before maturing into a cotyledon embryo. The endangerment of O. taihangensis may be associated with low reproductive capacity, as a consequence of poor pollen viability.

A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F1 progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.

•The inheritance and molecular markers were first investigated for aphid resistance in chrysanthemum.•Aphid resistance was controlled with two pairs of major genes with additivity and high heritability.•Useful markers were detected for aphid resistance with marker-trait analysis, BSA, and QTL mapping.•The findings would facilitate MAS for aphid resistance in future.Aphids have caused great damage to chrysanthemum production, and it's crucial to breed new chrysanthemums with strong aphid resistance. However, little information is available regarding the inheritance of aphid resistance in chrysanthemum. The inheritance pattern of chrysanthemum aphid resistance within an F1 segregating population was dissected with the major gene plus polygene mixed inheritance model and molecular markers. The result showed that aphid resistance of chrysanthemum is a quantitative trait with a moderate coefficient of variation >50%. The mixed inheritance model based on a single segregating generation suggested the inheritance of aphid resistance was controlled by two pairs of major genes with additive effect 0.68 and 0.39, respectively. The heritability of major gene was calculated at 89.21%. Marker-trait analysis based on one-way variance analysis uncovered 11 markers significantly associated with phenotype, cumulatively explaining ∼74% variation. Bulked segregant analysis (BSA) based on the gene bulks of high resistant and susceptive genotypes in F1 population identified two SSR markers, SSR145-93 and SSR197-205, linked to high aphid resistance (r > 0.4). The QTL analysis detected 5 putative QTL for aphid resistance in two successive years, with individual QTL explaining the phenotypic variation of 14.30–28.00%. The inheritance model and molecular markers identified for aphid resistance facilitate the ongoing breeding activities in chrysanthemum.

Anther wall development, microsporogenesis and microgametogenesis were compared between a normal male fertile chrysanthemum cultivar ‘NJAU04-29-2’ and the two male sterile selections ‘rm20-11’ (anther indehiscent) and ‘NJAU05-52-2’ (anther aborted). In both of the two male sterile types, the tapetum enlarged abnormally and showed signs of disorganization of walls at the onset of meiosis, the pollen was aborted, the anthers appeared shrunken, and the anther vascular bundle and connective tissue were degenerated by anthesis. In ‘rm20-11’, the two smaller pollen sacs began to degenerate at the microsporogenesis stage, so that only one or two microsporangia developed, while in ‘NJAU05-52-2’, only one or two microsporangia were formed following the archesporial cell stage, and most of the microspore mother cells degenerated during the course of meiosis.Research highlights▶ Two types of male sterility in the chrysanthemum are documented. ▶ Malfunctioning of the tapetum appears in both of two types of male sterility. ▶ Absence of endothecial cell wall thickening plays a key role in anther indehiscent. ▶ Anther abortion is mainly resulted from the lack of microsporangia.

•The transcriptome of chrysanthemum under nitrogen limitation was sequenced.•1510 and 686 different expressed genes were identified in the root and leaf.•Molecular basis of response to nitrogen stress in chrysanthemum was characterized.•Our results will help to enhance tolerance of chrysanthemum in nitrogen deficiency.The diploid Chrysanthemum L. species Chrysanthemum nankingense is a native plant in China, which is related to the commercially important polyploidy ornamental species Chrysanthemum morifolium (the garden chrysanthemum). As nitrogen is a limited element for plant growth, finding out genes responding to low nitrogen stress is important for improving the nitrogen-use efficiency. The transcriptome of C. nankingense was sequenced using roots and leaves of plants grown in the presence of either 4 mM or 0.04 mM nitrate. About 7,000,000 clean reads were generated from each of the four libraries. A total of 1510 and 686 differentially transcribed genes were identified in the roots and leaves, respectively. Many of the differentially transcribed genes were involved in photosynthesis and nitrogen metabolism, along with a number of transcription factors and genes encoding protein kinases. qPCR results of 15 randomly selected genes validated the RNA-Seq output. The molecular basis of the response to nitrogen stress in C. nankingense was comprehensively characterized. Overall, our results may be useful for breeding new chrysanthemum varieties with enhanced tolerance to nitrogen deficiency.

The tetraploid of Dendranthema nankingense (Nakai) Tzvel. was induced by the colchicine treatment using nodal segments. Ploidy level was determined by an analysis of flow cytometry and chromosome counting. The morphological characteristics such as the stomata, leaves, flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. The tolerance responses of the diploid and tetraploid were compared under the imposition of heat, cold, drought and salinity stress. Semi-lethal temperatures suggest that cold tolerance is improved by polyploidization, but the heat tolerance is reduced. Under drought and salt stress, the activity of peroxidase (POD) and relative water content (RWC) in the tetraploid were higher than those in the diploid. Accordingly its malondialdehyde (MDA) content maintained a lower level. The content of chlorophyll (a + b) in the tetraploid was higher, and decrease of its content was postponed in tetraploid compared with the diploid under salt stress. It suggested that polyploidization could alleviate oxidative stress, maintain good water balance and higher chlorophyll (a + b) content, thereby enhanced the drought and salt tolerance in colchicine induced tetraploid.Research highlights▶ The tetraploid of Dendranthema nankingense (Nakai) Tzvel. was induced by colchicines. ▶ Stomata, leaves, flowers and pollen grains of the tetraploid were larger. ▶ Tetraploid showed better cold, drought and salt tolerance, but worse heat tolerance. ▶ Polyploidization could alleviate oxidative stress. ▶ Tetraploid maintained good water balance and higher chlorophyll (a + b) content.

Chrysanthemum multicaule is an annual herbaceous ornamental species. The inflorescence is gynomonoecious and consists of bisexual tubular florets and female ray florets. The pistils consist of two stigmas which are of the open type with a hollow stylar canal. At the base of the tubular floret style, the pistil is surrounded by oil gland cells. The anthers are tetrasporangiate and the young anther wall is composed of epidermis, endothecium, middle layer and tapetum. The mature anther wall comprises only thickened endothecium after the release of the pollens. In the tubular florets, simultaneous microsporocyte meiotic cytokinesis results in mostly tetrahedral with a small proportion of decussate tetrads. The mature pollen grain is tricellular. The ovules are unitegmic and tenuinucellate, and the nucellus degenerates during the development of the megasporocyte. The development of the embryo sac follows the Polygonum type. At 4–6 days after blooming, the embryos reached the globular stage, thereafter passing through the heart- and torpedo-shape stages before maturing into the cotyledon embryos. From blooming to seed maturity, it takes about 3–4 weeks under our conditions.

APETALA2 (AP2) genes are ancient and widely distributed among the seed plants, and play an important role during the plant life cycle, acting as key regulators of many developmental processes. In this study, an AP2 homologue, NsAP2, was characterized from water lily (Nymphaea sp. cv. ‘Yellow Prince’) and is believed to be rather primitive in the evolution of the angiosperms. In situ RNA hybridization showed that NsAP2 transcript was present in all regions of the floral primordium, but had the highest level in the emerging floral organ primordium. After the differentiation of floral organs, NsAP2 was strongly expressed in sepals and petals, while low levels were found in stamens and carpels. The NsAP2 protein was suggested to be localized in the cell nucleus by onion transient expression experiment. Overexpression of NsAP2 in Arabidopsis led to more petal numbers, and Arabidopsis plants expressing NsAP2 exhibited higher plant height, which may be a result of down-regulated expression of GA2ox2 and GA2ox7. Our results indicated that the NsAP2 protein may function in flower organogenesis in water lily, and it is a promising gene for plant height improvement.

The rooting ability and resistance to alternaria root spot of the chrysanthemum (Chrysanthemum grandiflorum (Ramat.) Kitamura) variety ‘Zhongshanjingui’, the mugwort (Artemisia vulgaris L.) variety ‘Variegata’ and ten independent ‘Zhongshanjingui’ × ‘Variegata’ intergeneric hybrids were compared. Although the rooting ability of the hybrid cuttings was not as high as those taken from mugwort itself, it was higher than that of chrysanthemum with respect to the rate of initiation of both adventitious and lateral roots. By 15 d after cutting, the hybrids produced a mean of ∼22 adventitious roots per cutting (mean length 23.4 mm), whereas ‘Zhongshanjingui’ produced only ∼14 (14.5 mm). The ability of the cuttings to recover from dehydration also differed: hybrid cuttings were able to form ∼9 lateral roots per adventitious root, while those from ‘Zhongshanjingui’ could form only 4.1. ‘Zhongshanjingui’ was susceptible to alternaria leaf spot infection, the hybrid was moderately resistant and ‘Variegata’ was highly resistant. An assay based on essential oils extracted from the leaf showed that both the hybrid and ‘Variegata’ leaves possessed more antifungal activity than extracts from ‘Zhongshanjingui’ leaf.Highlights► The intergeneric hybrid between chrysanthemum and mugwort was used as plant material. ► The rooting ability of the hybrid was higher than that of chrysanthemum. ► The hybrid was moderately resistant to alternaria leaf spot. ► The hybrid's essential oils are of higher antifungal activity to Alternaria sp.