Chrysanthemum

The Agrobacterium strain GV3101 containing TRV-VIGS vectors was grown at 28 °C in Luria–Bertani medium supplemented with 10mM MES, 20mM acetosyringone, 50mg l^–1 gentamicin, and 50mg l^–1 kanamycin for ~24h. Agrobacterium cells were harvested and suspended in the infiltration buffer (10mM MgCl₂, 200mM acetosyringone, and 10mM MES, pH 5.6). A mixture of Agrobacterium cultures containing pTRV1 and pTRV2 and its derivatives, in a ratio of 1:1 (v/v), were placed in the dark at room temperature for 4h before inoculation. For vacuum infiltration, Silwet L-77 was added to a final concentration of 0.01% (v/v).
Nicotiana benthamiana infiltration was performed as described by Liu et al. (2002a). Agrobacterium cultures containing pTRV1 and pTRV2 or its derivatives (1:1, OD₆₀₀=1.0) were injected into the lower leaf of four-leaf stage plants by using a 1ml needleless syringe. For axil injection, 20 μl of bacterial suspension (1:1, OD₆₀₀=4.0) were injected per plant into the leaf axil using a 1ml syringe with a needle at the four-leaf stage period.
Since rose leaves and axils are hard to inject, the vacuum infiltration method was used for rose cuttings, and seedlings were infiltrated as described previously (Ma et al., 2008 (link); Yan et al., 2012 (link)). Four-week-old rose cuttings were removed from the soil, the roots were rinsed with distilled water, and whole plants were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD₆₀₀=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. This was repeated once. Treated plants were briefly washed with distilled water and then planted in pots. For seedlings, the seeds generated were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD₆₀₀=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. The infiltrated seeds were briefly washed with distilled water and placed on wet filter paper for several days, and then the seedlings were planted in pots.
Strawberry was vacuum infiltrated in the same way as rose cuttings and seedlings. After the release of the vacuum, the plants were briefly washed with distilled and planted in pots.
Arabidopsis and chrysanthemum infiltration was performed as described by Burch-Smith et al. (2006) (link). A mixture of Agrobacterium culture containing pTRV1 and pTRV2-GFP (OD₆₀₀=1.5) was infiltrated into the two leaves of 2-week-old Arabidopsis plants and the lower leaves of six- to eight-leaf-stage chrysanthemum plants using a needleless syringe. The infected chrysanthemum plants were transferred into pots.

The translated protein sequences of CSE_r1.1_cds were clustered by OrthoMCL with those of A. thaliana (Araport11, https://www.araport.org/data/araport11/), S. lycopersicum (tomato, ITAG3.2),¹⁷ (link)Lactuca sativa (lettuce, Lsativa_467_v5, http://lgr.genomecenter.ucdavis.edu/)²³ (link) and Helianthus annuus (sunflower, Ha412v1r1_port_v1.0)²⁴ (link) with the parameters c = 0.6 and aL = 0.9. The phylogenetic analysis was performed by MEGA 7.0.9 beta and TIMETREE based on the single copy genes conserved in C. seticuspe and the four species. The divergence time of 104 MYA between A. thaliana and S. lycopersicum was used for calibration. The published sequence read archive (SRA) transcripts for six cultivated chrysanthemum (C. × morifolium) varieties were mapped onto CSE_r1.0 by TopHat v2.1.1 (Supplementary Table S1b) in order to investigate the mapped ratios and identify variants. Variants were called based on the mapping result using SAMtools 0.1.19 and subsequently filtered using VarScan 2.3. SNP effects on gene function were predicted by using SnpEff ver.4.0.

The C. nankingense and other germplasm used are maintained by the Chrysanthemum Germplasm Resource Preserving Centre, Nanjing Agricultural University, China. For the RNA required for the transcriptome sequencing, stems and leaves were harvested from three 30 day old C. nankingense cuttings rooted on MS media and grown at a constant temperature of 25°C and a 16 h photoperiod (provided by cool white fluorescent lamps producing 36 µmol m⁻² s⁻¹).
A Total RNA Isolation System (Takara, Japan) was employed to extract RNA from the plant tissue, following the manufacturer’s instructions. The quality of the RNA (RNA Integrity Number (RIN)>8.5 and 28S:18S>1.5) was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA) and its concentration ascertained using an ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE). The standards applied were 1.8≤ OD_260/280≤2.2 and OD_260/230≥1.8. At least 20 µg of RNA was pooled in an equimolar fashion from each of the three sample plants.

Our previously established transgenic chrysanthemum (Chrysanthemum × morifolium) harboring TcCHS-promoter-driven GFP and tobacco (Nicotiana tabacum) harboring TcGLIP-promoter-driven GUS were treated with MeJA (Sultana et al., 2015 (link)). The plants were sprayed with 5 ml of 300 μM MeJA dissolved in 0.8% ethanol as a single foliar application. Leaves were collected in triplicate at 0 (control) and 12 h after treatment, and were immediately frozen in liquid nitrogen. The GFP expression level was determined by quantitative real-time PCR (qRT-PCR) analysis. The GUS activity was measured as previously described (Luo et al., 2013 (link)).

The reservoir of 1785 putative low-copy nuclear genes used in this study was identified from the previous study (Cheng et al., 2022b (link)). Generally, OGs were identified with OrthoFinder v2.0.0 (Emms and Kelly, 2019 (link)) through 11 genomes of 8 families. The 11 genomes include Lactuca sativa (Reyes-Chin-Wo et al., 2017 (link)), Chrysanthemum seticuspe (Hirakawa et al., 2019 (link)), Daucus carota (Iorizzo et al., 2016 (link)), Solanum lycopersicum (Hosmani et al., 2019 (link)), Capsicum annuum (Kim et al., 2014 (link)), CSS ‘Shuchazao’ (Wei et al., 2018 (link)), CSA ‘Yunkang 10’ (Xia et al., 2017 (link)), Actinidia chinensis (Wu et al., 2019 (link)), Primula veris (Nowak et al., 2015 (link)), Vitis vinifera (Jaillon et al., 2007 (link)), and Aquilegia coerulea (Filiault et al., 2018 (link)). The resulting 1785 OGs were used as source genes to obtain the corresponding putative orthologs (E-value < 1e-20) from 94 new assemblies of transcriptomes in HaMStR v13.2.6 (Ebersberger et al., 2009 (link)). The numbers of low-copy nuclear genes identified by HaMStR from those 1785 OGs were described in Supplementary Table S1.