Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome

Abstract

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

Chloroplast Genome Sequence of Pigeonpea (Cajanus cajan (L.) Millspaugh) and Cajanus scarabaeoides (L.) Thouars: Genome Organization and Comparison with Other Legumes

“…Like in pigeonpea cp genome, the frequency of editing sites is similar to that observed in other legumes like Pea (Inada et al, 2004) and V. radiata (Lin et al, 2015). Generally the editing sites vary between 20 and 37 in angiosperms (Hirose et al, 1999; Corneille et al, 2000; Lutz and Maliga, 2001).…”

“…We analyzed chloroplast SSRs (cpSSRs) with the MISA perl script and a total of 280 and 292 cpSSRs were identified in C. scarabaeoides and C. cajan respectively. The number was higher than that of cpSSRs identified in V. radiata, Sesamum indicum and Camellia species (Yi and Kim, 2012; Huang et al, 2014; Lin et al, 2015). Of the 280 repeats identified in C. scarabaeoides : 71.07% (199 SSRs) were located in the LSC region, 17.85% (50 SSRs) in the SSC region and 31% (11.07 SSRs) in the IR regions.…”

Variation in plastid genomes and transcriptomes in the gynodioecious species Silene vulgaris

“…We compared S. vulgaris editing sites with eight angiosperm species, for which plastid editome had comprehensively been studied: Amborella trichopoda, Cucumis sativus [25], Spirodela polyrhiza [26], Aegilops tauschii [27], Arabidopsis thaliana [28], Hevea brasiliensis [29], Nicotiana tabacum [30], Vigna radiata [31] ( Table 3) where C is present in the second position of the coding region. However, all KRA and KOV plants were edited < 15% in this position, which means that only a small portion of the ndhD transcripts encoded a functional protein.…”

The complete mitochondrial genome of wild mung bean (Vigna radiata var. sublobata TC1966)

“…Researchers commonly use the variety sublobata (wild mung bean) as reference to study the breeding and domestication issues of mung bean. To date, several entire mitochondrial genomes of mung bean cultivars, such as Berken (Alverson et al 2011) and NM92 (Lin et al 2015), were reported, but that of wild mung bean is lacking. Here, we complete an entire mitochondrial genome sequence of wild mung bean to provide genomic resource for further researches.…”

“…sublobata TC1966) was obtained from AVRDC -The World Vegetable Center, Taiwan, and then was germinated and planted in the greenhouse of the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan. The extraction and sequencing of wild mung bean genomes were reported in our previous study (Lin et al 2015). The 29.17 million clean Illumina paired-reads were employed for assembling the mitogenome of wild mung bean using MITObim v1.7 (Hahn et al 2013), with that of the congener cultivar NM92 (accession number: AP014716) (Lin et al 2016) as the initial reference.…”