Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication

Background

Brassica rapa is one of the most economically important vegetable crops worldwide. Owing to its agronomic importance and phylogenetic position, B. rapa provides a crucial reference to understand polyploidy-related crop genome evolution. The high degree of sequence identity and remarkably conserved genome structure between Arabidopsis and Brassica genomes enables comparative tiling sequencing using Arabidopsis sequences as references to select the counterpart regions in B. rapa, which is a strong challenge of structural and comparative crop genomics.

Results

We assembled 65.8 megabase-pairs of non-redundant euchromatic sequence of B. rapa and compared this sequence to the Arabidopsis genome to investigate chromosomal relationships, macrosynteny blocks, and microsynteny within blocks. The triplicated B. rapa genome contains only approximately twice the number of genes as in Arabidopsis because of genome shrinkage. Genome comparisons suggest that B. rapa has a distinct organization of ancestral genome blocks as a result of recent whole genome triplication followed by a unique diploidization process. A lack of the most recent whole genome duplication (3R) event in the B. rapa genome, atypical of other Brassica genomes, may account for the emergence of B. rapa from the Brassica progenitor around 8 million years ago.

Conclusions

This work demonstrates the potential of using comparative tiling sequencing for genome analysis of crop species. Based on a comparative analysis of the B. rapa sequences and the Arabidopsis genome, it appears that polyploidy and chromosomal diploidization are ongoing processes that collectively stabilize the B. rapa genome and facilitate its evolution.     

Complete reannotation of the Arabidopsis genome: methods, tools, protocols and the final release

Background

Since the initial publication of its complete genome sequence, Arabidopsis thaliana has become more important than ever as a model for plant research. However, the initial genome annotation was submitted by multiple centers using inconsistent methods, making the data difficult to use for many applications.

Results

Over the course of three years, TIGR has completed its effort to standardize the structural and functional annotation of the Arabidopsis genome. Using both manual and automated methods, Arabidopsis gene structures were refined and gene products were renamed and assigned to Gene Ontology categories. We present an overview of the methods employed, tools developed, and protocols followed, summarizing the contents of each data release with special emphasis on our final annotation release (version 5).

Conclusion

Over the entire period, several thousand new genes and pseudogenes were added to the annotation. Approximately one third of the originally annotated gene models were significantly refined yielding improved gene structure annotations, and every protein-coding gene was manually inspected and classified using Gene Ontology terms.     

Evaluation of Genome Sequencing Quality in Selected Plant Species Using Expressed Sequence Tags

Background

With the completion of genome sequencing projects for more than 30 plant species, large volumes of genome sequences have been produced and stored in online databases. Advancements in sequencing technologies have reduced the cost and time of whole genome sequencing enabling more and more plants to be subjected to genome sequencing. Despite this, genome sequence qualities of multiple plants have not been evaluated.

Methodology/Principal Finding

Integrity and accuracy were calculated to evaluate the genome sequence quality of 32 plants. The integrity of a genome sequence is presented by the ratio of chromosome size and genome size (or between scaffold size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of genome sequence was presented by the ratio between matched EST and selected ESTs where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs could be mapped to chromosome and scaffold sequences, respectively. According to the integrity, accuracy and other analysis of each plant species, thirteen plant species were divided into four levels. Arabidopsis thaliana, Oryza sativa and Zea mays had the highest quality, followed by Brachypodium distachyon, Populus trichocarpa, Vitis vinifera and Glycine max, Sorghum bicolor, Solanum lycopersicum and Fragaria vesca, and Lotus japonicus, Medicago truncatula and Malus × domestica in that order. Assembling the scaffold sequences into chromosome sequences should be the primary task for the remaining nineteen species. Low GC content and repeat DNA influences genome sequence assembly.

Conclusion

The quality of plant genome sequences was found to be lower than envisaged and thus the rapid development of genome sequencing projects as well as research on bioinformatics tools and the algorithms of genome sequence assembly should provide increased processing and correction of genome sequences that have already been published.     

Multiple genome alignment for identifying the core structure among moderately related microbial genomes

Background

LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.

Results

We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.

Conclusion

The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.     

A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R

Background

Thellungiella halophila (also known as Thellungiella salsuginea) is a model halophyte with a small plant size, short life cycle, and small genome. It easily undergoes genetic transformation by the floral dipping method used with its close relative, Arabidopsis thaliana. Thellungiella genes exhibit high sequence identity (approximately 90% at the cDNA level) with Arabidopsis genes. Furthermore, Thellungiella not only shows tolerance to extreme salinity stress, but also to chilling, freezing, and ozone stress, supporting the use of Thellungiella as a good genomic resource in studies of abiotic stress tolerance.

Results

We constructed a full-length enriched Thellungiella (Shan Dong ecotype) cDNA library from various tissues and whole plants subjected to environmental stresses, including high salinity, chilling, freezing, and abscisic acid treatment. We randomly selected about 20 000 clones and sequenced them from both ends to obtain a total of 35 171 sequences. CAP3 software was used to assemble the sequences and cluster them into 9569 nonredundant cDNA groups. We named these cDNAs "RTFL" (RIKEN Thellungiella Full-Length) cDNAs. Information on functional domains and Gene Ontology (GO) terms for the RTFL cDNAs were obtained using InterPro. The 8289 genes assigned to InterPro IDs were classified according to the GO terms using Plant GO Slim. Categorical comparison between the whole Arabidopsis genome and Thellungiella genes showing low identity to Arabidopsis genes revealed that the population of Thellungiella transport genes is approximately 1.5 times the size of the corresponding Arabidopsis genes. This suggests that these genes regulate a unique ion transportation system in Thellungiella.

Conclusion

As the number of Thellungiella halophila (Thellungiella salsuginea) expressed sequence tags (ESTs) was 9388 in July 2008, the number of ESTs has increased to approximately four times the original value as a result of this effort. Our sequences will thus contribute to correct future annotation of the Thellungiella genome sequence. The full-length enriched cDNA clones will enable the construction of overexpressing mutant plants by introduction of the cDNAs driven by a constitutive promoter, the complementation of Thellungiella mutants, and the determination of promoter regions in the Thellungiella genome.     

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

Background

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.

Methods

Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.

Results

We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.

General significance

The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.     

Uncovering the novel characteristics of Asian honey bee,Apis cerana,by whole genome sequencing

Background

The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana.

Results

Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes.

Conclusions

This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-16-1) contains supplementary material, which is available to authorized users.     

Evolution of Genome Size and Complexity in Pinus

Background

Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood.

Methodology/Principal Findings

Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA.

Conclusions/Significance

Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.     

AGO1 and AGO2 act redundantly in miR408-mediated Plantacyanin regulation

Background

In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 5′ end, respectively. Because most plant miRNAs have a 5′U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 5′A. AGO2 also participates in antiviral defense in association with viral siRNAs.

Principal Findings

This study reveals that miR408, which has a 5′A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 5′ nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 5′A-MIR408 and a mutant 5′U-MIR408 gene complement a mir408 mutant.

Conclusions/Significance

These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 5′ end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 5′ nucleotide.     

Unravelling the genome of Holy basil: an “incomparable” “elixir of life” of traditional Indian medicine

Background

Ocimum sanctum L. (O. tenuiflorum) family-Lamiaceae is an important component of Indian tradition of medicine as well as culture around the world, and hence is known as “Holy basil” in India. This plant is mentioned in the ancient texts of Ayurveda as an “elixir of life” (life saving) herb and worshipped for over 3000 years due to its healing properties. Although used in various ailments, validation of molecules for differential activities is yet to be fully analyzed, as about 80 % of the patents on this plant are on extracts or the plant parts, and mainly focussed on essential oil components. With a view to understand the full metabolic potential of this plant whole nuclear and chloroplast genomes were sequenced for the first time combining the sequence data from 4 libraries and three NGS platforms.

Results

The saturated draft assembly of the genome was about 386 Mb, along with the plastid genome of 142,245 bp, turning out to be the smallest in Lamiaceae. In addition to SSR markers, 136 proteins were identified as homologous to five important plant genomes. Pathway analysis indicated an abundance of phenylpropanoids in O. sanctum. Phylogenetic analysis for chloroplast proteome placed Salvia miltiorrhiza as the nearest neighbor. Comparison of the chemical compounds and genes availability in O. sanctum and S. miltiorrhiza indicated the potential for the discovery of new active molecules.

Conclusion

The genome sequence and annotation of O. sanctum provides new insights into the function of genes and the medicinal nature of the metabolites synthesized in this plant. This information is highly beneficial for mining biosynthetic pathways for important metabolites in related species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1640-z) contains supplementary material, which is available to authorized users.     

Genome digging: insight into the mitochondrial genome of Homo

Background

A fraction of the Neanderthal mitochondrial genome sequence has a similarity with a 5,839-bp nuclear DNA sequence of mitochondrial origin (numt) on the human chromosome 1. This fact has never been interpreted. Although this phenomenon may be attributed to contamination and mosaic assembly of Neanderthal mtDNA from short sequencing reads, we explain the mysterious similarity by integration of this numt (mtAncestor-1) into the nuclear genome of the common ancestor of Neanderthals and modern humans not long before their reproductive split.

Principal Findings

Exploiting bioinformatics, we uncovered an additional numt (mtAncestor-2) with a high similarity to the Neanderthal mtDNA and indicated that both numts represent almost identical replicas of the mtDNA sequences ancestral to the mitochondrial genomes of Neanderthals and modern humans. In the proteins, encoded by mtDNA, the majority of amino acids distinguishing chimpanzees from humans and Neanderthals were acquired by the ancestral hominins. The overall rate of nonsynonymous evolution in Neanderthal mitochondrial protein-coding genes is not higher than in other lineages. The model incorporating the ancestral hominin mtDNA sequences estimates the average divergence age of the mtDNAs of Neanderthals and modern humans to be 450,000–485,000 years. The mtAncestor-1 and mtAncestor-2 sequences were incorporated into the nuclear genome approximately 620,000 years and 2,885,000 years ago, respectively.

Conclusions

This study provides the first insight into the evolution of the mitochondrial DNA in hominins ancestral to Neanderthals and humans. We hypothesize that mtAncestor-1 and mtAncestor-2 are likely to be molecular fossils of the mtDNAs of Homo heidelbergensis and a stem Homo lineage. The d_N/d_S dynamics suggests that the effective population size of extinct hominins was low. However, the hominin lineage ancestral to humans, Neanderthals and H. heidelbergensis, had a larger effective population size and possessed genetic diversity comparable with those of chimpanzee and gorilla.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.