Construction of a reference transcriptome for the analysis of male sterility in sugi (Cryptomeria japonica D. Don) focusing on MALE STERILITY 1 (MS1)

Abstract

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. As the genome of this species is 11 Gbps, it is too large to assemble within a short timeframe. Transcriptomics is one approach that can address this deficiency. Here we designed a workflow consisting of three stages to de novo assemble transcriptome using Oases and Trinity. The three transcriptomic stage used were independent assembly, automatic and semi-manual integration, and refinement by filtering out potential contamination. We identified a set of 49,795 cDNA and an equal number of translated proteins. According to the benchmark set by BUSCO, 87.01% of cDNAs identified were complete genes, and 78.47% were complete and single-copy genes. Compared to other full-length cDNA resources collected by Sanger and PacBio sequencers, the extent of the coverage in our dataset was the highest, indicating that these data can be safely used for further studies. When two tissue-specific libraries were compared, there were significant expression differences between male strobili and leaf and bark sets. Moreover, subtle expression difference between male-fertile and sterile libraries were detected. Orthologous genes from other model plants and conifer species were identified. We demonstrated that our transcriptome assembly output (CJ3006NRE) can serve as a reference transcriptome for future functional genomics and evolutionary biology studies.

A chromosome-level genome assembly of a model conifer plant, the Japanese cedar,Cryptomeria japonicaD. Don

“…We assembled the RNA-seq data de novo using Trinity 2.13.2 [44]. In addition, we performed reference-based transcriptome assembly using StringTie 2.1.7 [45] with the RNA-seq data, ESTs in GenBank/EMBL/DDBJ [22] and Iso-seq reads previously reported in [23]. Subsequently, the de novo transcriptome assembly, the ESTs, the Iso-seq reads and the gene structures produced by StringTie were input into the alignment assembly pipeline of PASA 2.5.2 [46,47] to generate an integrated gene structure on the contigs.…”

Single-nucleotide substitution determines pollen production in Japanese cedar

“…b , Expression patterns of 67 genes of the MS4 region from male strobili (three samples) or inner bark and leaf tissues (two samples). RNA sequencing (RNA-seq) data were obtained from our previous study 32 and expression levels were categorised into six levels (0, > 0–1, > 1–10, > 10–100, > 100–1,000, or > 1,000 transcripts per million, TPM). Gene no.…”

“…RNA sequencing (RNA-seq) data revealed that CjTKPR1 had the highest expression levels in all three male strobilus samples (183.17-1131.88 transcripts per million (TPM)), but less or no expression in inner bark and leaf tissues (0 and 0.54 TPM, respectively; Fig. 2b and Supplementary Table 3) 32 .…”

“…2a, Supplementary Table 3). Transcript sequences from C. japonica (CJ3006NRE) 32 were mapped against genomic contig sequences using the minimap2 tool and the “-ax splice” option, resulting in 67 transcripts (Supplementary Table 3). An additional marker (CJt085666, Supplementary Table 3) was genotyped for the mapping family using PCR–restriction fragment length polymorphism (RFLP) and the Nde I restriction enzyme, to obtain CjTKPR1 and the genetic markers on either side (Fig.…”

Comparative transcriptome analysis of Cryptomeria japonica between solid and liquid culture

“…The Japanese cedar (Cryptomeria japonica) is an endemic coniferous tree in Japan, occupying the largest forest area in the country. Since the genome size of this species is roughly 11 Gbps, it has been subjected to molecular biological analysis using transcriptomics (Wei et al, 2021). In this study, we compare the transcriptomics of Japanese cedar callus cultured in suspension and solid media.…”