Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis

Abstract

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-β-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-β-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

IsoSeq transcriptome assembly of C3panicoid grasses provides tools to study evolutionary change in the Panicoideae

“…The average length of consensus sequences ranged from 925 bp to 1,438 kb (Figure S1). The number of consensus transcripts significantly exceeded the expected number of expressed genes, however, this is consistent with other reference genome-free IsoSeq analyses (Li et al, 2017; Kuang et al, 2019; Yan et al, 2019). Inflated numbers of consensus transcripts can result from sequencing of multiple alternatively spliced isoforms of the same gene, sequencing of incompletely processed mRNA molecules (Martin et al, 2014), high sequence error rates preventing multiple sequences from the same transcript being collapsed into a consensus, divergent haplotypes of the same locus present in our clonally propagated, wild collected, or partially inbred starting material, or contamination of the original samples with mRNA from non-target organisms.…”

IsoSeq transcriptome assembly of C3 panicoid grasses provides tools to study evolutionary change in the Panicoideae

“…While H. amplexicaulis exhibited the shortest consensus transcript length, this was not reflected in a reduced number of complete ORFs-those containing both an in-frame ATG and stop codon and occupying at least 60% of the total transcript length. The number of consensus transcripts significantly exceeded the expected number of expressed genes; however, this is consistent with other reference genome-freeIsoSeq analyses(Kuang, Sun, Wei, Li, & Sun, 2019;Li et al, 2017;Yan et al, 2019). Inflated numbers of consensus transcripts can result from sequencing of multiple alternatively spliced isoforms of the same gene, sequencing of incompletely processed mRNA molecules(Martin et al, 2014), high sequence error rates preventing multiple sequences from the same transcript being collapsed into a consensus, divergent haplotypes of the same locus present in our clonally propagated, wild collected, or partially inbred starting material, or contamination of the original samples with mRNA from non-target organisms.…”

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis

“…Moreover, small laboratories require high sequencing costs due to the need for long reads and high-depth short read sequences to be accurate in de novo assembly. Plants with large genomes pose even more difficult as in, for example, the common soybean crop, which has a genome size of ∼1.1Gb [21]. To improve the comprehensive accuracy of gene prediction, there is a need to introduce a new approach, the “Isoform sequencing (Iso-Seq).” Thanks to its long-read technology, Iso-Seq facilitates identifying new isoforms with a high level of accuracy [22].…”