Plant mini kit

From the stem tip, five 5- to 8-cm long shoots were arbitrarily selected from each of the total 96 replicate samples. For each shoot, ‘Vardar Valley’ had an average of 20 leaves, and ‘Justin Brouwers’ had an average of 40 leaves. We used liquid nitrogen to homogenize the shoot samples. The extraction of genomic DNA followed Qiagen Plant Mini Kit (Hilden, Germany) with minor modifications. Specifically, approximately 200 mg ground tissue was further homogenized using a MP FastPrep™ 24 (MP Bio, Irvine, CA, USA) at the speed of 4/s for 1 min in a sterilized zirconium bead tube (500 um garnet and 6 mm zirconium, PFMM 500-100-25U, OPS Diagnostics, Lebanon, NJ, USA) prefilled with 400 µl of AP1 buffer. Four µl of RNase A was added and vortexed for 3 s. Nuclease free water was used to dissolve DNA from the MB Spin Column membrane. The DNA was stored at -20°C.

Total RNA was extracted from the seedling of Chinese cabbage and from the leaves of tobacco using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using the FavorPrep Plant Total RNA Mini Kit (Favorgen, Changzhi, Taiwan), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 μg total RNA using the amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA).
Genomic DNA was extracted from the leaves of Chinese cabbage using the Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

To confirm endosymbiotic algal clades, restriction fragment length polymorphism (RFLP) was performed. We used symbiotic polyps of 1- and 4-months old after inoculation with Symbiodinium. Three polyps were homogenized and DNA was extracted using a Plant Mini Kit (Qiagen, Valencia, CA). The SSU rDNA was amplified using the primers ss5z and ss3z according to Rowan and Powers (1991) [20] . For RFLP analysis of the SSU rDNA, 10 µL of product from each PCR amplification was digested with 1 µL TaqI enzyme (Takara, Ohtstu, Japan) for 3 h at 65°C in 10 µL of distilled H₂O and 2 µL enzyme buffer solution. Reaction fragments were separated by electrophoresis and stained with ethidium bromide.

Total RNA was isolated from P. tomentosa Carr. using the Plant Mini Kit (Qiagen, Germany). First-strand cDNAs were synthesized from 2 μg of total RNA in 20 μl of reaction mixture using the RT-AMV transcriptase Kit (TaKaRa, Dalian, China). The coding sequence (CDS) of PtoMYB156 was amplified by gene-specific primers (Supplementary Table S1). Thermal cycler programmes were as follows: 96 °C for 3 min followed by 32 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 50 s, and a final extension step at 72 °C for 10 min. The amplification products were cloned into the plant binary vector pCXSN as previously described35 (link).
The amino acid sequences of R2R3 MYB transcription factors in other species were obtained by BLAST searchers (http://www.phytozome.com). The deduced amino acid sequences were aligned with the program DNAMAN7.0 (Lynnon Corporation, USA). Phylogenetic analysis based on amino acid sequences was preformed using the Neighbor-Joining (NJ) method through MAGE 5.036 (link).

Citrus leaf samples were collected from the six Citrus plants in USHRL's (USDA Horticultural Research Laboratory, Fort Pierce, Florida): C. sinensis (sweet, navel orange), C. aurantium (karum jamir, sour orange), C. reticulata (mandarin orange), C. clementina (clementina), C. aurantiifolia (sweet lime), C. japonica (Yuzu, kumquat), and C. maxima (pomelo). Total DNA was extracted from leaf midribs following the Plant Mini Kit standard protocol from Qiagen Inc. (Valencia, CA), followed by DNA quantity and quality evaluation with Nanodrop. We chose the NBS gene, Cs1g09350, which was conserved in C. clementina and C. sinensis for validating the conservation of NBS gene among different Citrus genomes. Primers used in this study were designed using Oligo 7.23 (Molecular Biology Insights, Inc., Cascade, CO, USA). DNA Polymerase (Invitrogen, Carlsbad, CA, USA) was used to amplify the NBS-LRR genes from Citrus DNA. For PCR, 20 μL reactions using standard conditions provided by the manufacturer for DNA Polymerase. PCR was performed using an initial denaturation at 95°C for 3 minutes, 35 cycles of 94°C for 20 seconds, 50–52°C for 20 seconds (specified by different primer sets) and 68°C for 3 minutes, follow by final extension at 68°C for 10 minutes. The cloning and sequencing analysis of amplified PCR products were conducted as previously described [43 (link)]

Two milliliters of cells were collected from the samples on days 0, 1, 5, and 9 in the TAP-N medium and RNA was extracted using a QIAGEN Plant Mini Kit. The concentration and purity of the extracted RNA were measured using a UV spectrophotometer. Single stranded cDNA was synthesized from 2 μg of DNA-digested total RNA following the reverse transcription protocol provided by the manufacturer (Promega™). Transcriptional levels of genes related to lipid accumulation were detected using real-time quantitative PCR. Primers for quantitative real-time RT-PCR were designed using the Primer 5 software and are shown in Table 1. Real-time quantitative PCR was performed as stipulated by the manufacturer of the SYBR Green real-time PCR Master Mix Kit (TOYOBO™, Japan). The actin gene from C. reinhardtii was used as an internal control to normalize the differences between the loading amounts of the template (Makarova et al., 2007 (link)). Each PCR reaction contained 1 μL (8 ng) of cDNA, 10 μL of SYBR Green 2 × Master Mix, and 1 μL of each gene-specific primer pair (10 mM) to a final volume of 20 μL. PCR was performed as follows: 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 60°C for 1 min, and 72°C for 30 s. PCR products were analyzed using the Dissociation Curves Software of ABI. The 2^−ΔΔct method was used to calculate the fold changes of differentially expressed genes.

Fresh and health leaf materials were obtained from living plants and transplanted at Fujian Agriculture and Forestry University (Fujian, China) and Shanghai Chenshan Botanical Garden (Shanghai, China). Voucher specimens were deposited in Fujian Agriculture and Forestry University (FJFC). After detailed morphological comparison, Dr. Liang Ma and Prof. Zhong-Jian Liu performed plant material identification. All plant materials were identified in their flowering period. Based on the previous study [22 (link)], a total of 37 taxa from 22 genera were sampled, which included six Luisia species. The voucher details and GenBank accessions for the five newly sequenced Luisia species are listed in Supplementary Table S1.
DNA extraction from leaf materials was carried out using the Plant Mini Kit (Qiagen, Valencia, CA, USA), following the protocol of the manufacturer. DNA degradation was assessed using 1% agarose gels. Illumina libraries were constructed according to the manufacturer’s protocol and paired-end sequencing was executed on the Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA). Each species obtained approximately 10 Gb of clean data.