Short Single Copy Research Articles

Characterization of the chloroplast genome of Ixonanthes chinensis (Ixonanthaceae)

Ixonanthes chinensis (Ixonanthaceae) is an endangered tree species distributed in South China and Vietnam. The phylogenetic position of Ixonanthes in the order Malpighiales remains controversial. Here we reported and characterized the complete chloroplast genome sequence of I. chinensis assembled from Illumina sequencing reads. The chloroplast genome size is 155,296 bp, containing a large single copy (LSC) region of 83,829 bp, a short single copy (SSC) region of 18,917 bp, and a pair of inverted repeats (IRs) of 26,275 bp. We predicted a total of 139 genes, which included 87 protein-coding genes, 44 tRNA genes, and 8 rRNA genes. Phylogenetic analysis showed that I. chinensis was sister to Linum usitatissimum from the family Linaceae rather than Erythroxylum novogranatense from the family Erythroxylaceae.

Characterization of the chloroplast genome of Osbeckia stellata (Melastomataceae)

Osbeckia stellata (Melastomataceae) is a shrub distributed in the South. Although many species of Osbeckia have important medicinal value, the phylogenetic position of Osbeckia within the family Melastomataceae remains unclear. Osbeckia stellata is one of the most widespread species in this genus. Here, we reported and characterized the complete chloroplast genome sequence of O. stellata assembled from Illumina sequencing reads. The chloroplast genome size is 156,372 bp, containing a large single copy (LSC) region of 85,741 bp, a short single copy (SSC) region of 17,115 bp, and a pair of inverted repeats (IRs) of 26,758 bp. We predicted a total of 136 genes consisting of 86 protein-coding genes, 48 tRNA genes, and 8 rRNA genes. Phylogenetic analysis showed that O. stellata was sister to Melastoma candidum.

Characterization of the complete chloroplast genome sequence of Pueraria montana var. lobata and its phylogenetic implications

Pueraria montana var. lobata is a traditional Chinese herb with various medicinal purposes. In this study, the complete chloroplast genome of the P. montana var. lobata was obtained by using Illumina HiSeq X Ten. The chloroplast genome was 153,411 bp long, which consisted of a large single-copy (LSC) region of 84, 131 bp and a short single-copy (SSC) region of 17,990 bp separated by a pair of inverted repeat (IR) regions of 25,645 bp. The whole chloroplast genome contained 111 gene species including 77 protein-coding genes (PCG), 30 tRNA, and 4 rRNA species, with 19 of them occurring in double copies. Introns were detected in 12 PCG and 6 tRNA species. The overall GC content is 35.4%. Phylogenetic analysis indicated that P. montana var. lobata was relatively close to Pachyrhizus erosus in phylogeny.

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

BackgroundChloroplasts are organelles that conduct photosynthesis in plant and algal cells. The information chloroplast genome contained is widely used in agriculture and studies of evolution and ecology. Correctly assembling chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10–30 kb). Typically, it is simply assumed that the gross structure of the chloroplast genome matches the most commonly observed structure of two single-copy regions separated by a pair of inverted repeats. The advent of long-read sequencing technologies should remove the need to make this assumption by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora, the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long-(Oxford nanopore) and short-(Illumina) reads, different long-read lengths, different assembly pipelines, with a view to determining the most accurate and efficient approach to chloroplast genome assembly.ResultsHybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs (the long single copy, short single copy and inverted repeat regions) of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate. The chloroplast genome of Eucalyptus pauciflora is 159,942 bp, contains 131 genes of known function.ConclusionsOur results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.

Resources for studies of iron walnut (Juglans sigillata) gene expression, genetic diversity, and evolution

Iron walnut (Juglans sigillata Dode) is a temperate deciduous tree indigenous to China. It is distributed mainly in southwestern China, where it is valued for its wood and nuts. Transcriptomic and genomic data for the species are limited. Our goal was to assemble the whole chloroplast genome of J. sigillata, to use transcriptome information from RNA-Seq to understand the gene space in J. sigillata, and to develop polymorphic simple sequence repeats (SSRs, microsatellites) useful for understanding the species’ population genetics. The chloroplast genome consisted of a large single copy (LSC) of 89,872 bp, an inverted region (IR) of 52,072 bp, and a short single copy (SSC) of 18,406 bp. The chloroplast genome consisted of 137 annotated genes, with 71 unique coding regions and eight coding regions that were repeated in the inverted region. De novo assembly of the transcriptome yielded 83,112 unigenes with an average length of 686.9 bp. A search against the Gene Ontology (GO) database identified 19,718 unigenes. We evaluated transcriptome-derived microsatellite markers and chloroplast sequence polymorphisms in 48 J. sigillata individuals from three populations and 66 individuals from five other Chinese walnut (Juglans) species. We found 20 expressed sequence tag-SSRs and four loci in the chloroplast that were polymorphic in J. sigillata. The number of alleles per locus ranged from 3 to 10. The whole chloroplast genome and these 24 informative loci will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species.

IRscope: an online program to visualize the junction sites of chloroplast genomes

Genome plotting is performed using a wide range of visualizations tools each with emphasis on a different informative dimension of the genome. These tools can provide a deeper insight into the genomic structure of the organism. Here, we announce a new visualization tool that is specifically designed for chloroplast genomes. It allows the users to depict the genetic architecture of up to ten chloroplast genomes in the vicinity of the sites connecting the inverted repeats to the short and long single copy regions. The software and its dependent libraries are fully coded in R and the reflected plot is scaled up to realistic size of nucleotide base pairs in the vicinity of the junction sites. We introduce a website for easier use of the program and R source code of the software to be used in case of preferences to be changed and integrated into personal pipelines. The input of the program is an annotation GenBank (.gb) file, the accession or GI number of the sequence or a DOGMA output file. The software was tested using over a 100 embryophyte chloroplast genomes and in all cases a reliable output was obtained. Source codes and the online suit available at https://irscope.shinyapps.io/irapp/ or https://github.com/Limpfrog/irscope.

The complete chloroplast genome sequence annotation for Malania oleifera, a critically endangered and important bioresource tree

In this study, we characterize the chloroplast genome of Malania oleifera Chun et S. Lee, the only species of this genus and member of the Olacaceae family. M. oleifera is an interesting seed oil plant for medicinal purposes. It is also a critically endangered species, which is currently recorded on the International Union for Conservation of Nature (IUCN) Red List and in the “Red Data Book of Chinese Plants: the Rare and Endangered Plants”. Its total chloroplast genome length is 125,050 bp, including a large single copy region of 76,387 bp, a short single copy gene region of 15 bp, and a pair of inverted repeats (IRs) of 24,324 bp. We annotated 80 genes in this chloroplast genome, including 51 protein-coding genes, 4 ribosomal RNAs, 4 pseudogenes and 21 tRNA genes. We found 15 genes duplicated in the IR regions, and the overall GC content was found to be 38.17%. Phylogenetic analysis showed that M. oleifera is closely related to Erythropalum scandens comparing nine chloroplast genomes from Santalales.

Characterization of the complete chloroplast genome of the vulnerable agarwood tree, Aquilaria yunnanensis (Thymelaeaceae)

Aquilaria yunnanensis S. C. Huang is a precious agarwood tree endemic to Yunnan Province of China. It is included in Category II of CITES. In this study, we reported its complete chloroplast genome de novo assembled from Illumina pair-end sequencing data. The plastome was a quadripartite circular with 174,885 bp in length, including one large single copy (LSC) region of 87,508 bp and one very short small single copy (SSC) region of 3351 bp, separated by two extremely expanded inverted repeat (IR) regions of 42,013 bp. The overall GC content of the genome was 36.7%, and the corresponding values of the LSC, SSC and IR regions were 34.9, 29.1 and 38.8%, respectively. The chloroplast genome encoded 142 genes, including 96 protein-coding genes, 38 tRNA genes, and eight rRNA genes. Phylogenetic analysis revealed that sampled species of Thymelaeaceae formed a monophyletic clade, in which A. yunnanensis and A. sinensis formed a sister clade to Daphne kiusiana.

Annotated plastome of the temperate woody vine Muehlenbeckia australis (G.Forst.) Meisn. (Polygonaceae)

We assembled the plastome of the temperate, Southern Hemisphere liana Muehlenbeckia australis from high throughput sequencing data (paired-end Illumina reads) generated from total genomic DNA sequencing libraries. M. australis’ chloroplast genome sequence (GenBank: MG604297) is 163,484 bp in length and composed of long single copy (LSC; 88,166 bp) and short single copy (SSC; 13,486 bp) regions flanked by inverted repeats (IR; 30,916 bp each) typical for angiosperms. The plastome includes 131 genes comprising 83 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, two possible pseudogenes, psbL and rpl23 with internal stop codons, and truncated repeats of ndhF and rps19 at IR boundaries.

The Challenges of Genome-Wide Studies in a Unicellular Eukaryote With Two Nuclear Genomes.

We present here methods to study a eukaryotic microorganism with two nuclear genomes, both originating from the same zygotic genome. Paramecium, like other ciliates, is characterized by nuclear dimorphism, which is the presence of two types of nuclei with distinct organization and functions in the same cytoplasm. The two diploid germline micronuclei (MIC) undergo meiosis and fertilization to transmit the genetic information across sexual generations. The highly polyploid somatic macronucleus (MAC) contains a reduced version of the genome optimized for gene expression. Reproducible programmed DNA elimination of about 30% of the complexity of the 100Mb MIC genome occurs during development of the MAC along with endoreplication to 800 copies. Large regions that contain transposable elements and other repeats are eliminated, and short single copy remnants of transposable elements, which often interrupt coding sequences, are precisely excised to restore functional open reading frames. Genome-wide studies of this process require access to MIC DNA which has long been impossible. The breakthrough with respect to this technical obstacle came with development of a MIC purification protocol involving a critical step of flow cytometry to sort nuclei representing only 0.5% of total genomic DNA. Here, we provide a step-by-step protocol and important tips for purifying nuclei, and present the methods developed for downstream analysis of NGS data.

The plastid genome of Vanillon (Vanilla pompona, Orchidaceae)

This study presents the complete sequence of Vanilla pompona chloroplast genome. This 148,009 bp long genome consist of 107 genes out of which 30 of them are tRNA, 4 rRNA. Fairly long inverted repeat regions (IR) and large single-copy (LSC) of length 29,807 and 86,358 bp, respectively, were detected. This means an exceptionally short single-copy (SSC) region with only 2037 bp. This truncation of the SSC is due to multiple translocation of ndh genes to the mitochondrion as in majority of the Orchidaceae and especially in the genus Vanilla. The phylogeny presented here meaningfully places Vanillon within the orchid family.

The complete plastid genome of the endangered species midget crabapple (Malus micromalus)

The midget crabapple (Malus micromalus) is endemic to Shaanxi and Shanxi Provinces of China. Like many other members of the Rosaceae, it is an important contributor to food production and nutrition. We determined the complete chloroplast genome sequences for M. micromalus using IIumina sequencing. The M. micromalus chloroplast has a total length of 159,834 bp; it consists of a large single copy (LSC) region of 87,950 bp, an inverted region A (IRa) of 26,354 bp, a short single copy (SSC) region of 19,176 bp, and an inverted region B (IRb) of 26,354 bp. The genome encodes 129 annotated genes, including 84 protein-coding genes and 45 tRNA. A phylogenomic analysis revealed that M. micromalus clustered with M. prunifolia within Malus.

The first complete chloroplast genome sequences of Ulmus species by de novo sequencing: Genome comparative and taxonomic position analysis.

Elm (Ulmus) has a long history of use as a high-quality heavy hardwood famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. This is the first report of Ulmaceae chloroplast genomes by de novo sequencing. The Ulmus chloroplast genomes exhibited a typical quadripartite structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The lengths of the chloroplast genomes from five Ulmus ranged from 158,953 to 159,453 bp, with the largest observed in Ulmus davidiana and the smallest in Ulmus laciniata. The genomes contained 137–145 protein-coding genes, of which Ulmus davidiana var. japonica and U. davidiana had the most and U. pumila had the fewest. The five Ulmus species exhibited different evolutionary routes, as some genes had been lost. In total, 18 genes contained introns, 13 of which (trnL-TAA+, trnL-TAA−, rpoC1-, rpl2-, ndhA-, ycf1, rps12-, rps12+, trnA-TGC+, trnA-TGC-, trnV-TAC-, trnI-GAT+, and trnI-GAT) were shared among all five species. The intron of ycf1 was the longest (5,675bp) while that of trnF-AAA was the smallest (53bp). All Ulmus species except U. davidiana exhibited the same degree of amplification in the IR region. To determine the phylogenetic positions of the Ulmus species, we performed phylogenetic analyses using common protein-coding genes in chloroplast sequences of 42 other species published in NCBI. The cluster results showed the closest plants to Ulmaceae were Moraceae and Cannabaceae, followed by Rosaceae. Ulmaceae and Moraceae both belonged to Urticales, and the chloroplast genome clustering results were consistent with their traditional taxonomy. The results strongly supported the position of Ulmaceae as a member of the order Urticales. In addition, we found a potential error in the traditional taxonomies of U. davidiana and U. davidiana var. japonica, which should be confirmed with a further analysis of their nuclear genomes. This study is the first report on Ulmus chloroplast genomes, which has significance for understanding photosynthesis, evolution, and chloroplast transgenic engineering.

Characterization of the complete chloroplast genome of wheel wingnut (Cyclocarya paliurus), an endemic in China

The wheel wingnut (Cyclocarya paliurus) is an endemic species distributed in eastern and central China. Cyclocarya is a woody genus in the Juglandaceae used in medicine and horticulture. The complete chloroplast genome of C. paliurus was sequenced using the Illumina Hiseq 2500 platform. The total genome length was 160,562 bp comprised of a large single copy (LSC) region of 90,007 bp, an inverted region A (IRa) of 26,039 bp, a short single copy (SSC) region of 18,477 bp, and an inverted region B (IRb) region of 26,039 bp. The genome consisted of 137 annotation genes, with 80 unique coding regions and nine coding regions which were repeated in the inverted region. Among these, 40 unique tRNAs with nine duplications, and 4 rRNAs were all duplicated in the IR region. An analysis of chloroplast phylogeny strongly supported the placement of C. paliurus near to Juglans regia.

The complete chloroplast genome of Paeonia anomala subsp. veitchii

The complete chloroplast genome of Paeonia anomala subsp. veitchii., an important traditional Chinese herbal medicine, was sequenced and characterized in this study. The genome size is 152 682 bp in length with 38.42% GC content. Two inverted repeats (25 653 bp) are separated by a large single copy (84 398 bp) and a short single copy (16 978 bp). The genome contains 130 individual genes, including 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Twenty-one genes contain one or two introns. Phylogenetic tree analysis of P. anomala subsp. veitchii and other 12 species belonging to Pentapetalae using MEGA6 with the neighbour-joining algorithm revealed that the chloroplast genome was clustered in subfamily Paeoniaceae.

The complete chloroplast genome sequence of the Taraxacum officinale F.H.Wigg (Asteraceae)

Taraxacum officinale is a distributed weedy plant used as a traditional medicinal herb belonging to the family Asteraceae. The complete chloroplast genome of T. officinale was generated by de novo assembly with whole genome sequence data. The chloroplast genome was 151 324 bp in length, which consisted of a large single copy region of 83 895 bp and a short single copy region of 18 549 bp separated by a pair of inverted repeat regions of 24 440 bp. The chloroplast genome contained 79 protein-coding genes, 29 tRNA genes and four rRNA genes. Phylogenetic analysis revealed that T. officinale was closely related to Lactuca sativa.

The complete chloroplast genome sequence of Indian mustard (Brassica juncea L.)

Brassica juncea L. is the second most important edible oil seed crop of India. In the current study, we report the complete chloroplast genome sequence of Indian mustard, Brassica juncea L. The size of the complete chloroplast genome was found to be 153 483 bp long with an overall GC content of 36.36%. A large single copy (LSC) region of 83 286 bp and a short single copy region (SSC) region of 17 775 bp were separated by a pair of inverted repeat (IR) regions of 26 211 bp. A total of 113 unique genes were annotated, which includes 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Either one or two introns were present in nine protein-coding genes and six tRNA genes. Phylogenetic analysis with the complete chloroplast genomes of other related species revealed that B. juncea is much closer to Brassica rapa.

Localized, non-random differences in chromatin accessibility between homologous metaphase chromosomes.

BackgroundCondensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.ResultsFourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA.ConclusionsThis study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-014-0070-y) contains supplementary material, which is available to authorized users.

A Preliminary Phylogenetic Analysis of the Grass Subfamily Pooideae (Poaceae), with Attention to Structural Features of the Plastid and Nuclear Genomes, Including an Intron Loss in GBSSI

Phylogenetic relationships in the grass family (Poaceae), with specific attention to the internal structure of subfamily Pooideae, are analyzed on the basis of nucleotide sequence variation in plastid-encoded genes (matK, ndhF, ndhH, and rbcL). The resulting phylogenetic hypothesis was examined with attention to the taxonomic distributions of two inversions and an insertion/deletion within ndhF, the absence of intron 10 of the nuclear gene GBSSI (waxy), and positions of the boundaries between the Short Single Copy (SSC) region and the neighboring Inverted Repeat (IR) regions of the plastid genome, relative to the endpoints of ndhF and ndhH, which span these boundaries in some taxa. The PACCAD clade is resolved, and extension of the 3'-end of ndhF from the SSC region into the IR region is interpreted as a synapomorphy of this clade. The BEP clade also is resolved, with Ehrhartoideae placed as the sister of a clade in which Bambusoideae and Pooideae are sister groups. The loss of GBSSI intron 10 is interpreted as a synapomorphy of Poeae s.l., which includes the traditionally defined tribes Poeae, Aveneae, and Hainardieae, and the results support a novel set of relationships among Ihe tribes of Pooideae, including the placement of Brachypodieae, Bromeae, Triticeae, and Poeae s.l. within a clade for which a three-nucleotide inversion in ndhF is interpreted as a synapomorphy, while a six-nucleotide inversion in ndhF marks a clade that includes all sampled members of subtribe Aveninae within Poeae s.l.

Sequence organization of the human genome.

The organization of three sequence classes--single copy, repetitive, and inverted repeated sequences--within the human genome has been studied by renaturation techniques, hydroxylapatite binding methods, and DNA hyperchromism. Repetitive sequence classes are distributed throughout 80% or more of the genome. Slightly more than half of the genome consists of short single copy sequences, with a length of about 2 kb interpersed with repetitive sequences. The average length of the repetitive sequences is also small and approximates the length of these sequences found in other organisms. The sequence organization of the human genome therefore resembles the sequence organization found in Xenopus and sea urchin. The inverted repeats are essentially randomly positioned with respect to both sequence class and sequence arrangement, so that all three sequence classes are found to be mutally interspersed in a portion of the genome.