Nanodrop ultraviolet spectrophotometer

Samples were collected from the Tea Germplasm Resource Park of Wuyi University (N 27°61′01.34″, E 117°96′63.51″) and the Tea Germplasm Resources Reserve of the Fujian Province (N 27°43′42.46″, E 118°0′14.40″); a total of 349 tea trees from 12 provinces in China, including 63 national elite cultivars, 58 provincial elite cultivars and 228 landraces, were used in the study. Six pairs of tea trees from the same variety were used as positive controls for the essentially derived relationship. All samples used are shown in Table S1. Relevant information on tea plant breeding was taken from the book Tea Plant Varieties in China [22 ] and Tea Plant Clonal Varieties in China [23 ].
DNA was extracted using the plant genomic DNA extraction kit (KangWei CW0553S, China), and the concentration and purity of the DNA were determined by NanoDrop ultraviolet spectrophotometer (Thermo Scientific, Carlsbad, CA, USA). DNA integrity was detected by 0.8% agarose electrophoresis. The extracted genomic DNA was stored at −80 ℃.

RNA isolation and purification from JMZ and HD, as well as cDNA library construction and sequencing, were performed as previously described [56 (link)]. Total RNA was extracted from the young shoots of JMZ and HD by using the pBIzol kit (BIOFLUX, Hangzhou Bori Technology, Hangzhou, China). RNA integrity of samples was measured using a Nano-Drop ultraviolet spectrophotometer (Thermo, Waltham, MA, USA) and a Bioanalyzer 2100 System (Agilent, Santa Clara, CA, USA). About 3 μg of RNA per sample was prepared, and sequencing was carried out on an Illumina HiSeq 4000 platform to generate 150 bp paired end reads for each sample. Two test samples were used to construct the transcriptome library and Illumina sequencing in Allwegene Biotechnology Co., Ltd. (Beijing, China).

Trizol reagent (Ambion, USA, Cat No: 15596-026) was used to extract total RNA from control, treated, and transfected cells. The concentration (ng/μL) of microRNA was quantified and evaluated for purity by a NanoDrop ultraviolet spectrophotometer (Thermo Scientific, USA). RNA was reverse-transcribed into cDNA using an mRNA reverse transcription kit and an miRNA reverse transcription kit (TransGen Biotech, Beijing, China, Cat No: M31121) according to the manufacturers' instructions. Quantitative real-time PCR was performed using the primers listed below. Gene specific primers and SYBR Green Master Mix (TransGen Biotech, Beijing, China, Cat No: N10627) were used to detect the expression of miR-103. U6 was used to standardize miRNA. The results are expressed as multiples of U6 and calculated using the 2^-△△CT method.
The primer sequences used were as follows:
U6 (human) F: 5′-CTCGCTTCGGCAGCACA-3′ R: 5′-AACGCTTCACGAATTTGCGT-3′; miR-103 (human) F: 5′-AGAGCAGCATTGTACAGGGCTATGA-3′.

The total microbial DNA of rhizosphere soil samples was extracted with PowerSoil® DNA Isolation Kit (MOBIO, United States). The rhizomes and leaves were chopped and ground with liquid nitrogen, and the total DNA was extracted by PowerSoil® DNA Isolation Kit. The DNA concentration and purity were determined by Nanodrop ultraviolet spectrophotometer (Thermo Scientific, United States). The quality of DNA extract was detected by 1.2% agarose gel electrophoresis, and then the DNA extraction solution was stored at −80°C for later use. The V5-V7 region of fungal 18S rRNA was amplified by using universal primers SSU0817F (5’-TTAGCATGGAATAATRRAATAGGA-3′) and 1196R (5’-TCTGGACCTGGTGAGTTTCC-3′; Jiang et al., 2017 (link)). The V3-V4 region of 16S rRNA was PCR amplified by using universal primers 338\u00B0F (5’-ACTCCTACGGGAGGCAGCA-3′) and 806 R (5’-GGACTACHVGGGTWTCTAAT-3′; Li et al., 2020 (link)). Pfu high-fidelity DNA polymerase from TransGen Biotech was used for PCR amplification with the number of amplification cycles strictly controlled to make cycle number as small as possible and the amplification conditions for the same batch of samples consistent. The PCR products were purified and recovered with the AxyPrep DNA Gel extraction kit. Subsequently, each sample was quantified using BioTek Flx800 microplate reader with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589).

WES and analysis protocols were adapted for genetic analysis. After obtaining the informed consent from the parents, genomic DNA samples were extracted from the whole blood of the patient and her parents with the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany). The concentration and quantity of the DNA samples were measured using a NanoDrop ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [19 (link)]. Genomic DNA fragments were enriched for the target region of the consensus coding sequence exons and sequenced on an Illumina HiSeq 2000 platform (Illumina, San Diego, CA) [19 (link)]. Data analyses were conducted by using ANNOVAR and VEP software in our clinical genetic laboratory by a bioinformatics team. The mutation was confirmed by Sanger sequencing.