- Comparative genomics showed that the sequence of the Fagales species cp genome was relatively conserved, but there were still some high variation regions that could be used as molecular markers. - The simple sequence repeat (SSR) analysis showed that there were 105 SSRs in the cp genome of B. - RNA editing sites recognition indicated that at least 80 RNA editing events occurred in the cp genome. - Most of the substitutions were C to U, while a small proportion of them were not. - For synonymous conversion, most of them increased the relative synonymous codon usage (RSCU) value of the codons. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Furthermore, we recognized RNA editing sites in the whole cp genome of B. - The qualities of the clean reads were checked using FastQC (v0.11.5). - To identify the cp sequences, all of the clean reads, which included se- quences from both the nucleus and organelles, were mapped to the complete cp genome sequences of 2670 plant species, which were downloaded from the NCBI Organelle Genome Resources database (www.ncbi.nlm.. - After that, GapCloser (v1.12) was used to close most of the gaps, and Sanger sequencing was used to fill residual gaps. - To examine the deviation in synonymous codon usage while avoiding the influence of the amino acid composition, the relative synonymous codon usage (RSCU) was calculated with MEGA 7 software (version 7.0.18).. - Three cp genes (rps19, psbC and ndhD) were annotated with the Non-ATG start codon in the B. - Then, the sequence logos of the first 10 bp of the three genes across the species were created using the WebLogo 3 application (http://weblogo.threeplusone.. - EMBOSS Stretcher, a modifica- tion of the Needleman-Wunsch algorithm that allows larger sequences to be aligned globally, was used to align these cp genome sequences to obtain accurate identity and similarity.. - More information is summarized in the Additional file 1: Table S3.. - Further scaffolding with all of the paired-end and mate-pair reads resulted in a single scaf- fold under the guidance of the reference sequences.. - After using GapCloser to close most of the gaps, only two gaps remained. - Finally, with the aid of Sanger se- quencing, we filled the gaps, identified both ends of the sequence and obtained a circular cp genome.. - The overall GC content of the B. - platyphylla cp genome was 36.06%, and the GC contents of the LSC and the SSC re- gions were 33.66 and 29.76%, respectively. - Because each IR region contained relatively abundant GC-rich rRNA and tRNA genes, the GC content of the IR region was 42.48%, which was much higher than that of the LSC and SSC regions.. - A total of 129 genes were predicted to be encoded in the B. - Among them, 95 genes were unique, and 17 genes were duplicated in the IR regions. - By calculating the GC content of the genes, we found that it was higher in the rRNAs (54.89%) and tRNAs (53.20%) than in the protein-coding genes (36.93. - All of the gen- ome and annotation information is shown in Fig. - C-terminal exons 2 and 3 of rps12 were located in each IR region, but exon 1 was located in the LSC, ap- proximately 28 kbp downstream of the nearest copy of exon 2, which was located in one of the IR regions and 61 kbp away from the other copy of exon 3, which was located in the other IR region. - Prediction of the B. - Regarding photosynthesis, a subset of the genes synthesizes the large Rubisco subunit and thylakoid proteins. - Table 2 shows the gene functions and groups in the B. - We further analyzed the codon usage frequency and RSCU value in the B. - There were 84 protein-coding genes in the B. - In the B. - GTG was the dominant initiation codon in rps19, but not in psbC, and about half of the species took ACG as the start codon in ndhD. - To investigate the similarities and differences of the cp gen- ome sequences between B. - IR expansions and contractions are common in cp ge- nomes, which results in the variation in cp genome size.. - Here, we selected four representative species of the four families in Fagales and compared their sizes and the junctions of their LSC, SSC and IR regions. - Although the lengths of the IR regions, ranging from 25,701 bp to 26,056 bp, varied little among the four species, some differences in the IR expansions and contractions were observed. - Although the rpl2 genes of the four species were all located completely within the IRb regions, the gene in B.. - platyphylla was at the farthest from the left boundary of the IR region. - the others were all located in the IRb regions. - Of particular interest, the ψrps19 pseudogene only existed in the B. - platyphylla genome, which is located along the right boundary of the IRa region. - In summary, we found that the IR regions of the B. - platyphylla cp genome were slightly expanded compared with that of the other three species.. - A total of 105 SSRs were detected in the B. - platyphylla cp genome (Additional file 1: Table S5). - A majority of the mononucleotides (98.1%) were composed of A/T, and most of the dinucleotides (84.2%) were AT Table 2 Group of genes within the B. - 2 Sequence logo and RNA-Seq mapping of the three genes. - a: Sequence logo of the first 10 bp of the three genes across the species. - RNA-Seq mapping of the first 10 bp of the three genes in B. - The read alignment rates of the three bio- logical repetitions to the reference B. - However, a 95.3% region of the cp genome was covered with reads, and the average sequencing depth was over 650x. - Finally, we identi- fied 80 RNA editing events in the whole B. - Among all of the edited genes, most of them were protein-coding genes, while 3 of them were rRNA genes. - We did not discover any editing events that occurred in the tRNA transcripts. - In protein-coding transcripts, editing events mainly occurred in the codon region, with only 1 in the 5’ UTR and 4 in introns. - The largest number of editing sites was recognized in the ndhB gene (10 editing sites).. - After com- parison with the codon usage frequency and RSCU value at the editing sites, except for stop codon conversion, we found that all 6 other amino acid synonymous conversions increased the RSCU value of the codons, whereas the other nonsynonymous conversions did not show the same trend.. - For several editing sites located in the Intergenic region, nucleotide changes, including G to A, C to U and A to C (genome sequence direction as a positive chain), were ob- served. - However, the editing sites located in the rRNA were the most specific. - 5, the C to T transitions at the 1406th base of the accD gene and at the 155th base of the matK gene were noticeable. - After careful observation of the cDNA region in Fig. - 5, it is not difficult to see that there is a weak cytosine (C) base signal under the peak of the corresponding editing site.. - In the latest APG IV system, Fagales includes seven fam- ilies (Nothofagaceae, Fagaceae, Myricaceae, Juglanda- ceae, Ticodendraceae, Betulaceae, Casuarinaceae). - nana of the genus Betula and family Betulaceae, B. - 4 Comparison of the borders of LSC, SSC and IR regions among the four Fagales genomes. - However, some of the phenomena attracted our attention.. - Chloroplast microsatellites are some of the most com- mon cp molecular markers. - In our study, we found that there were more than 100 cpSSR loci in the B.. - RNA editing in the B. - platyphylla cp genome is also one of the concerns of our study. - How- ever, it clearly improved the accuracy of the recognition.. - Finally, we identified 80 editing sites in the whole cp gen- ome, including coding regions, noncoding regions and intergenic regions. - Most of the substitutions were C to U, but a small number of other types that have never been reported in flowering plants were also detected. - It should be noted that most of the rRNAs were removed in the process of library con- struction. - Obviously, these studies are insufficient to elucidate the essence of the problem, espe- cially for those other than for C-to-U editing. - Because rRNA is the main component of the ribosome and catalyzes peptide bond formation, these editing sites may change the ribosome structure or influ- ence protein synthesis. - Although the sequence logo clearly shows the trend of initiation codon selection in different spe- cies, most of the current cp genome annotations were based on software prediction, which may be different from the real situation. - platyphylla cp, the initi- ation codons of the ndhD transcripts were obviously. - The cp genome of B. - In the future, we will focus on molecular mecha- nisms that are involved in transcriptional regulation and translational modification of the cp by using new technolo- gies and methods. - Taxa and ID of the selected species. - Taxa and ID of the selected Fagales. - Codon usage frequency and RSCU value of the B. - Amino acid composition of protein-coding gene in the B. - The funding bodies had no role in the design of the study, collection, analysis, or interpretation of data or in the writing of the manuscript.. - As researches of the laboratory, we are allowed to use these materials for research. - The Illustrated Encyclopedia of Trees and Shrubs: An Essential Guide to Trees and Shrubs of the World. - The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. - SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome. - An evaluation of the PacBio. - organization and evolution of the largest and most highly rearranged chloroplast genome of land plants. - ChloroSeq, an optimized chloroplast RNA-Seq Bioinformatic pipeline, reveals remodeling of the Organellar transcriptome under heat stress. - A review of the prevalence, utility, and caveats of using chloroplast simple sequence repeats for studies of plant biology. - Phylogenetic investigation of the complex evolutionary history of dispersal mode and diversification rates across living and fossil Fagales. - Reverse U-to-C editing exceeds C-to-U RNA editing in some ferns – a monilophyte-wide comparison of chloroplast and mitochondrial RNA editing suggests independent evolution of the two processes in both organelles. - Identification and analysis of RNA editing sites in the chloroplast transcripts of Aegilops tauschii L. - Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing. - Full transcription of the chloroplast genome in photosynthetic eukaryotes. - Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium
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