Boehmeria

A population consisting of 111 F₂ progenies derived from cultivated Zhongsizhu 1 and wild species B. nivea var. tenacissima was used for construction of the genetic map. To detect the SNPs, the clean reads sequenced from the transcriptome of 111 F₂ families and two parents by Illumina sequencing (SRA accession no. SRP182925) were aligned into the ramie genome (accession ID: PHNS00000000) [21 (link)] using Burrows-Wheeler Aligner (BWA) software (version: 0.7.17; settings: mem –t − 4 –k 32 –MR) [30 (link)], and then the alignment files were converted to bam files using SAMtools (settings: –bS –t) [31 (link)]. If there were multiple read pairs mapped to the same position or with external coordinates, only the pair with the highest mapping quality was retained. Thereafter, SNP discovery was performed for the two parents and 111 families using SAMtools [31 (link)]. Because the two parents were heterozygous, the polymorphic markers between them were classified into eight segregation patterns: ef × eg, nn × np, ab × cc, aa × bb, ab × cd, lm × ll, hk × hk, and cc × ab [20 (link), 32 (link), 33 (link)], according to the CP model in JoinMap 4.0 [34 (link)]. The numbers of SNPs, transitions, and transversions were also counted, and a Perl script (uploading in 10.6084/m9.figshare.8010446) was used to filter out the SNPs with more than two genotypes, retaining only polymorphic markers with the ‘aa × bb’ segregation pattern. Finally, retained markers that contained abnormal bases or exhibited significantly distorted segregation (P < 0.001) or non-integrity (missing data in > 30% progenies) were filtered out using JoinMap 4.0 [35 (link)]. The regression algorithm, three circulation sequences, and Kosambi mapping function were used for marker distance calculation [35 (link)], and the linkage map was drawn using mapchart 2.32 [36 (link)], with the default parameters.

Ramie samples at the different growth stages of H5 were collected and the total RNA was isolated from each sample using a Tiangen RNA prep Pure Plant Kit (Tiangen Biomart, Beijing). Twenty micrograms of total RNA from each sample was sent to Novogene Bioinformatics Technology Co. Ltd (Beijing), where the libraries were constructed, and sequenced using the Illumina HiSeq 2000 platform. RNA quality and quantity were determined by a Nano Photometer spectrophotometer (IMPLEN, CA, USA), a Qubit RNA Assay Kit in a Qubit 2.0 Flurometer (Life Technologies, CA, USA) and a Nano 6000 Assay Kit that was part of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 10 µg RNA, 1 µg from each of the ten samples, was used as the input material for the transcriptome library and 3 µg RNA per sample was used for the DGE library. Briefly, the mRNA was purified by poly-T oligo-attached magnetic beads and fragmented by divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5×). Random hexamer primer and M-MuLV Reverse Transcriptase (RNase H) were used for first strand cDNA synthesis. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. These double-stranded cDNA fragments were end-repaired by adding a single ‘A’ base and ligation of adapters. The adaptor modified fragments were selected by gel purification and amplified, through PCR, to create the final cDNA library. Transcriptome sequencing was carried out on an Illumina HiSeq 2000 platform that generated 100 bp paired-end raw reads, while DGE sequencing generated 100 bp single-end raw reads.

Ramie cultivar 1504 (with a fiber finesse of 2800 m/g) was transplanted with pot from our Germplasm Resources Garden and was grown under natural conditions in 2010. Samples from the stem shoot (with about three leaves, sample L), the top part of stem bark (sample T), the middle part of stem bark (sample M) and the bottom part of stem bark (sample B, representing different stages of ramie fiber development, as displayed in Figure 3) were collected when the aboveground stem was turning pitchy in May 2012 (the first season of ramie fiber growth). Each sample was made up from three plants and consisted of samples that were about 5 cm long. They were mixed and frozen in liquid nitrogen for subsequent RNA extraction. Total RNA was separately extracted from the four samples, based on the method described previously [46 (link)]. Afterwards, the RNA quality was confirmed by gel electrophoresis and by NanoDrop 2000 spectrophotometer (Thermo, MA, USA).

Multiple sequence alignments of the R2R3-MYB proteins from ramie and Arabidopsis thaliana were performed using MEGA 7.0 with default parameters (Kumar et al., 2016 (link)) and neighborhood linkage (Guo et al., 2019 (link)) phylogenetic tree constructed with the following parameters: Poisson model; pairwise deletion; and 1,000 bootstrap replications. R2R3-MYB proteins from other species [Arabidopsis (Holub, 2001 (link)), rice (Katiyar et al., 2012 (link)), maize (Du et al., 2012b (link)), tomato (Li et al., 2016 (link)), pineapple (Liu et al., 2017 (link))] were obtained based on the description in corresponding literatures, and adopted same.
To establish WGD events, paralogous genes of ramie were detected using all-vs-all homology searches in BLASTP with an E-value threshold of 1e-5. Syntenic blocks within a genome were identified based on the detected homologous gene pairs using MCscan.

Orthologous relationships of eight selected cadmium responsive MYB regulatory genes were determined between A. thaliana and ramie using OrthoVeen2 (Xu et al., 2019 (link)). Prediction of interactions between BnGMYB proteins and other proteins based on the Arabidopsis homologs was obtained using the online program STRING version 11.5 with high confidence>0.700 (Szklarczyk et al., 2017 (link)), filtered genes were used to construct the correlation network. The interaction network was visualized in Cytoscape v3.8.2.

The detailed chromosome distribution information of each BnGR2R3-MYB genes was obtained from the ramie genome annotation documents. Circos (Krzywinski et al., 2009 (link)) was used to locate all the BnGR2R3-MYB genes on the ramie chromosome. Tandem, segmental duplication and collinearity within species were obtained by using Multiple Collinearity Scantoolkit (MCscanX) (Wang et al., 2012 (link)). Synteny blocks of the orthologous R2R3-MYB genes between ramie and other species were obtained also using MCscanX and both results visualized in TBtools (Chen et al., 2020 (link)). Non-synonymous and synonymous substitution of each duplicated BnGMYB genes were calculated using KaKs_Calculator 2.0 (Wang et al., 2010 (link)).