Plant material and sample preparation
Stolons derived from two-month-old P. vaginatum plants were cultivated hydroponically in plastic containers measuring 90 mm in top diameter, 57 mm in width, and 135 mm in depth. The containers were filled with full-strength Hoagland solution (pH = 6.0). The hydroponic growth occurred within a controlled environment, specifically a growth chamber, maintaining a photoperiod of 16 hrs light and 8 hrs dark, with temperatures set at 30/25°C, a relative humidity of 75%, and a standard light intensity of 1000 µmol m-2 s-1. Subsequent to cleaning the leaves and ensuring dryness using DEPC-treated water, they were promptly subjected to rapid cooling in liquid nitrogen and subsequently preserved in an ultra-low-temperature refrigerator at -80°C.
Mitochondrial genome assembly and structure prediction
Mitochondria from P. vaginatum callus were isolated employing the differential centrifugation method outlined by JL Heazlewood, KA Howell, J Whelan and AH Millar [15]. To achieve a complete mitogenome sequence, a combination of short-read (Illumina) and long-read sequencing (PacBio Sequel II) technologies was employed in this investigation. The short raw reads underwent quality checking with FastQC and subsequent trimming using Trimmomatic (ILLUMINACLIP: TruSeq-PE. fa:2:30:10 LEADING:3 TRAILING:3 MINLEN:75). Meanwhile, the long raw reads were base-called using Albacore v2.1.7 (mean_qscore > 7) with barcode demultiplexing and converted to Fasta format through Samtools Fasta (http://www.htslib.org/doc/samtools.html). Initially, approximately two micrograms of mitochondrial DNA were transformed into SMRTbell libraries using the Express Template Prep Kit 2.0 from PacBio, following the manufacturer's protocol. Subsequently, the libraries were pooled into a single multiplexed library, as per the manufacturer's instructions, and sequenced on an Illumina NovaSeq 6000 with 150 bp paired-end read length.
Mitochondrial genomes in higher plants exhibit marked variability in both structure and sequence content. To enhance assembly reliability, two strategies were employed for the assembly of the P. vaginatum mitogenome. In the first approach, short clean reads were de novo assembled using GetOrganelle v1.6.4. Potential mitochondrial contigs were extracted by aligning against the mitochondrial protein-coding genes from the plant mitogenome database (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/mitochondrion/) with BLAST v 2.8.1+. Subsequently, putative long mitochondrial reads were baited by mapping PacBio long reads to the potential mitochondrial contigs using BLASR v5.1, and finally, these putative long mitochondrial reads were assembled with Canu v2.1.1. In the second strategy, all PacBio long reads were assembled de novo directly using Canu. The draft contigs were improved by mapping short clean reads to them using BWA and refining them with Pilon v1.22. MUMmer 3.23 was then employed to verify whether these contigs were circular. Ultimately, the corrected contigs obtained from the two assembly strategies were aligned using MUMmer. In addition, the raw data and assembled genomes of the P. vaginatum mitogenome are stored in GenBank (access number: PP072315).
Chloroplast DNA sequencing and genome assembly
Young leaves of P. vaginatum were collected, and chloroplasts were isolated from the leaves by using density gradient centrifugation and digested with DNase I (Promega, Madison, USA) to eliminate genomic DNA contamination. To obtain a full-length chloroplast sequence, we used both short-read (Illumina) and long-read sequencing (PacBio Sequel II) technologies in this study. First, approximately two micrograms of chloroplast DNA were made into SMRTbell libraries using the Express Template Prep Kit 2.0 from PacBio according to the manufacturer’s protocol. Samples were pooled into a single multiplexed library, and size was selected using Sage Sciences’ BluePippin, a size selection cutoff of 5000 (BPstart value) was given as instructions. Libraries were sequenced using an Illumina NovaSeq 6000 with 150 bp of paired-end read length.
The short raw reads were checked with FastQC and trimmed by Trimmomatic (ILLUMINACLIP: TruSeq-PE.fa:2:30:10 LEADING:3 TRAILING:3 MINLEN:75). The long raw reads were base-called by using Albacore v2.1.7 (mean_qscore > 7) with barcode demultiplexing, and converted to Fasta format with Samtools Fasta (http://www.htslib.org/doc/samtools.html). To improve assembly reliability, we used two strategies to assemble the XX chloroplast genome. In the first strategy, the short clean reads were de novo assembled with GetOrganelle v1.6.4, and potential chloroplast contigs were extracted by aligning against the chloroplast protein-coding genes from the plant chloroplast database (ftp://ftp.ncbi.nih.gov/refseq/release/plastid/) with BLAST v2.8.1+. Then, the putative long chloroplast reads were baited by mapping the Pacbio long reads to the potential chloroplast contigs using BLASR v5.1. Finally, the putative long chloroplast reads were assembled by Canu v2.1.1. In the second strategy, all Pacbio long reads were assembled de novo by using Canu directly. Subsequently, we used BWA to map the short clean reads to the draft contigs and improved the draft contigs with Pilon v1.22. Then, MUMmer 3.23 was used to check whether these contigs were circular. Finally, the corrected contigs obtained from the above two assembly strategies were aligned with each other using MUMmer, and the result showed that these two contigs were identical. Based on the above assembly steps, we obtained a master circle of the chloroplast genome. In addition, the raw data and assembled genomes of P. vaginatum chloroplast are stored in GenBank (access number: PP072314).
Genome annotation and visualization of genome maps
The genes were annotated through the online GeSeq tool (https://chlorobox.mpimp-golm.mpg.de/geseq.html/), employing default parameters to predict protein-coding genes, transfer RNA (tRNA) genes, and ribosomal RNA (rRNA) genes in mitochondrial and chloroplast genomes. Manual adjustments for start/stop codons and intron/exon boundaries were executed in SnapGene Viewer, referencing the reference mitochondrial and chloroplast genome. The mitochondrial and chloroplast genome maps of P. vaginatum were generated utilizing the OGDRAW tool (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html). A cis-splicing gene map of P. vaginatum was graphed using CPGView (http://www.1kmpg.cn/cpgview/).
Codon preference and repetitive sequence analysis
Utilizing Phylosuite software (http://phylosuite.jushengwu.com/), we extracted protein-coding sequences, and MEGA (v7.0) software was employed to determine the relative synonymous codon usage (RSCU) values for the amino acid composition of protein-coding genes within the mitochondrial and chloroplast genomes. Simple sequence repeats (SSRs) were identified through the MISA software (https://webblast.ipk-gatersleben.de/misa/). Simple sequence repeat and long-repeat in the mitochondrial and genome were scrutinized using TRF (https://tandem.bu.edu/trf/trf.unix.help.html) and REPuter (https://bibiserv.cebitec.unibielefeld.de/reputer/), respectively.
Phylogenetic analysis
The complete mitochondrial and chloroplast genome sequences of maize (Z. mays strain NB) (GenBank: AY506529.1; NCBI Reference Sequence: NC_001666.2), rice (O. sativa) Shen95(FA)A (NCBI Reference Sequence: NC_066488.1; NCBI Reference Sequence: NC_031333.1), and sorghum (Sorghum bicolor L.) (NCBI Reference Sequence: NC_008360.1; NCBI Reference Sequence: NC_008602.1), were used by National Centre for Biotechnology Information (NCBI). Phylogenetic analyses were performed for the three species. Protein-coding genes common to the mitochondrial genomes of all species were extracted to construct phylogenetic trees. Phylogenetic analysis was performed using the maximum likelihood (ML) method using MRBAYES. The final results of the phylogenetic analysis were then visualized using ITOL software.
RNA editing prediction
The RNA editing events were predicted based on the online website PREPACT v3.12.0 (http://www.prepact.de/), and the setting standard is: cutoff value = 0.001.
Comparative analysis of organelles and nuclear genomes
Genomic-scale sequence comparisons were carried out with LASTv7.1.435 to map regions sharing similar sequences within the P. vaginatum genome and to identify the sequences shared between the chloroplast and mitochondrial genomes. The software BLASTN with the e-value of 1e-6 and Tbtools [16] were used to analyse the sequence comparisons between the organelle and nuclear genomes.
Transcriptomic data of the targeted PCGs
The genes encoding for mitochondrial and chloroplast proteins in the leaves of P. vaginatum plants after salinity treatment were retrieved from GEO datasets in the NCBI Gene Expression Omnibus (GEO) under GEO accession number: GSE233155 based on our previous study [17]. We analysed the differentially expressed gene sets (adjusted p-value < 0.05 and |log2FoldChange| > 0.3) using the DESeq2 function in the R package (version 4.1.0).
Statistical analysis and graphics
All of the statistical analyses were performed using a single-factor ANOVA followed by Duncan’s test using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA).