Plant Genomics
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Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex

The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes.
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Abstract (provisional)Background

The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called "African", "Asian" and "American" Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex.

Results

Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi ("Asian" Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets.

Conclusion

The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.

 
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The regulatory role of Pcf11-similar-4 (PCFS4) in Arabidopsis development by genome-wide physical interactions with target loci

The yeast and human Pcf11 functions in both constitutive and regulated transcription and pre-mRNA processing.
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Background

The yeast and human Pcf11 functions in both constitutive and regulated transcription and pre-mRNA processing. The constitutive roles of PCF11 are largely mediated by its direct interaction with RNA Polymerase II C-terminal domain and a polyadenylation factor, Clp1. However, little is known about the mechanism of the regulatory roles of Pcf11. Though similar to Pcf11 in multiple aspects, Arabidopsis Pcf11-similar-4 protein (PCFS4) plays only a regulatory role in Arabidopsis gene expression. Towards understanding how PCFS4 regulates the expression of its direct target genes in a genome level, ChIP-Seq approach was employed in this study to identify PCFS4 enrichment sites (ES) and the ES-linked genes within the Arabidopsis genome.

Results

A total of 892 PCFS4 ES sites linked to 839 genes were identified. Distribution analysis of the ES sites along the gene bodies suggested that PCFS4 is preferentially located on the coding sequences of the genes, consistent with its regulatory role in transcription and pre-mRNA processing. Gene ontology (GO) analysis revealed that the ES-linked genes were specifically enriched in a few GO terms, including those categories of known PCFS4 functions in Arabidopsis development. More interestingly, GO analysis suggested novel roles of PCFS4. An example is its role in circadian rhythm, which was experimentally verified herein. ES site sequences analysis identified some over-represented sequence motifs shared by subsets of ES sites. The motifs may explain the specificity of PCFS4 on its target genes and the PCFS4[prime]s functions in multiple aspects of Arabidopsis development and behavior.

Conclusions

Arabidopsis PCFS4 has been shown to specifically target on, and physically interact with, the subsets of genes. Its targeting specificity is likely mediated by cis-elements shared by the genes of each subset. The potential regulation on both transcription and mRNA processing levels of each subset of the genes may explain the functions of PCFS4 in multiple aspects of Arabidopsis development and behavior.

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Identification, expression, and comparative genomic analysis of the IPT and CKX gene families in Chinese cabbage (Brassica rapa ssp. pekinensis)

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Abstract (provisional)Background

Cytokinins (CKs) have significant roles in various aspects of plant growth and development, and they are also involved in plant stress adaptations. The fine-tuning of the controlled CK levels in individual tissues, cells, and organelles is properly maintained by isopentenyl transferases (IPTs) and cytokinin oxidase/dehydrogenases (CKXs). Chinese cabbage is one of the most economically important vegetable crops worldwide. The whole genome sequencing of Brassica rapa enables us to perform the genome-wide identification and functional analysis of the IPT and CKX gene families.

Results

In this study, a total of 13 BrIPT genes and 12 BrCKX genes were identified. The gene structures, conserved domains and phylogenetic relationships were analyzed. The isoelectric point, subcellular localization and glycosylation sites of the proteins were predicted. Segmental duplicates were found in both BrIPT and BrCKX gene families. We also analyzed evolutionary patterns and divergence of the IPT and CKX genes in the Cruciferae family. The transcription levels of BrIPT and BrCKX genes were analyzed to obtain an initial picture of the functions of these genes. Abiotic stress elements related to adverse environmental stimuli were found in the promoter regions of BrIPT and BrCKX genes and they were confirmed to respond to drought and high salinity conditions. The effects of 6-BA and ABA on the expressions of BrIPT and BrCKX genes were also investigated.

Conclusions

The expansion of BrIPT and BrCKX genes after speciation from Arabidopsis thaliana is mainly attributed to segmental duplication events during the whole genome triplication (WGT) and substantial duplicated genes are lost during the long evolutionary history. Genes produced by segmental duplication events have changed their expression patterns or may adopted new functions and thus are obtained. BrIPT and BrCKX genes respond well to drought and high salinity stresses, and their transcripts are affected by exogenous hormones, such as 6-BA and ABA, suggesting their potential roles in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. The appropriate modulation of endogenous CKs levels by IPT and CKX genes is a promising approach for developing economically important high-yielding and high-quality stress-tolerant crops in agriculture.

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Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

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Abstract (provisional)Background

Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes.

Results

We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs).

Conclusions

Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3.

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RNA-Seq effectively monitors gene expression in Eutrema salsugineum plants growing in an extreme natural habitat and in controlled growth cabinet conditions

Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

The investigation of extremophile plant species growing in their natural environment offers certain advantages, chiefly that plants adapted to severe habitats have a repertoire of stress tolerance genes that are regulated to maximize plant performance under physiologically challenging conditions. Accordingly, transcriptome sequencing offers a powerful approach to address questions concerning the influence of natural habitat on the physiology of an organism. We used RNA sequencing of Eutrema salsugineum, an extremophile relative of Arabidopsis thaliana, to investigate the extent to which genetic variation and controlled versus natural environments contribute to differences between transcript profiles.

Results

Using 10 million cDNA reads, we compared transcriptomes from two natural Eutrema accessions (originating from Yukon Territory, Canada and Shandong Province, China) grown under controlled conditions in cabinets and those from Yukon plants collected at a Yukon field site. We assessed the genetic heterogeneity between individuals using single-nucleotide polymorphisms (SNPs) and the expression patterns of 27,016 genes. Over 39,000 SNPs distinguish the Yukon from the Shandong accessions but only 4,475 SNPs differentiated transcriptomes of Yukon field plants from an inbred Yukon line. We found 2,989 genes that were differentially expressed between the three sample groups and multivariate statistical analyses showed that transcriptomes of individual plants from a Yukon field site were as reproducible as those from inbred plants grown under controlled conditions. Predicted functions based upon gene ontology classifications show that the transcriptomes of field plants were enriched by the differential expression of light- and stress-related genes, an observation consistent with the habitat where the plants were found.

Conclusion

Our expectation that comparative RNA-Seq analysis of transcriptomes from plants originating in natural habitats would be confounded by uncontrolled genetic and environmental factors was not borne out. Moreover, the transcriptome data shows little genetic variation between laboratory Yukon Eutrema plants and those found at a field site. Transcriptomes were reproducible and biological associations meaningful whether plants were grown in cabinets or found in the field. Thus RNA-Seq is a valuable approach to study native plants in natural environments and this technology can be exploited to discover new gene targets for improved crop performance under adverse conditions.

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Draft genome of the wheat A-genome progenitor Triticum urartu

Draft genome of the wheat A-genome progenitor Triticum urartu | Plant Genomics | Scoop.it
Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging.
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Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGAmAm), is central to wheat evolution, domestication and genetic improvement1. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu2, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome3) and Ae. tauschii (the donor of the D genome4), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

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Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapa ssp. pekinensis)

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Abstract (provisional)Background

Chinese cabbage (Brassica rapa ssp. pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence from Arabidopsis thaliana (13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species. Arabidopsis is therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed in Brassica species. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms in Brassica rapa ssp. pekinensis.

Results

In the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage and Arabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively.

Conclusions

The evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.

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Analysis of the global transcriptome of longan (Dimocarpus longan Lour.) embryogenic callus using Illumina paired-end sequencing

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Abstract (provisional)Background

Longan is a tropical/subtropical fruit tree of great economic importance in Southeast Asia. Progress in understanding molecular mechanisms of longan embryogenesis, which is the primary influence on fruit quality and yield, is slowed by lack of transcriptomic and genomic information. Illumina second generation sequencing, which is suitable for generating enormous numbers of transcript sequences that can be used for functional genomic analysis of longan.

Results

In this study, a longan embryogenic callus (EC) cDNA library was sequenced using an Illumina HiSeq 2000 system. A total of 64,876,258 clean reads comprising 5.84 Gb of nucleotides were assembled into 68,925 unigenes of 448-bp mean length, with unigenes >=1000 bp accounting for 8.26% of the total. Using BLASTx, 40,634 unigenes were found to have significant similarity with accessions in Nr and Swiss- Prot databases. Of these, 38,845 unigenes were assigned to 43 GO sub-categories and 17,118 unigenes were classified into 25 COG sub-groups. In addition, 17,306 unigenes mapped to 199 KEGG pathways, with the categories of Metabolic pathways, Plant-pathogen interaction, Biosynthesis of secondary metabolites, and Genetic information processing being well represented. Analyses of unigenes >=1000 bp revealed 328 embryogenesis-related unigenes as well as numerous unigenes expressed in EC associated with functions of reproductive growth, such as flowering, gametophytogenesis, and fertility, and vegetative growth, such as root and shoot growth. Furthermore, 23 unigenes related to embryogenesis and reproductive and vegetative growth were validated by quantitative real time PCR (qPCR) in samples from different stages of longan somatic embryogenesis (SE); their differentially expressions in the various embryogenic cultures indicated their possible roles in longan SE.

Conclusions

The quantity and variety of expressed EC genes identified in this study is sufficient to serve as a global transcriptome dataset for longan EC and to provide more molecular resources for longan functional genomics.

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Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape

Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

Aspartic proteases (APs) are a large family of proteolytic enzymes found in almost all organisms. In plants, they are involved in many biological processes, such as senescence, stress responses, programmed cell death, and reproduction. Prior to the present study, no grape AP gene(s) had been reported, and their research on woody species was very limited.

Results

In this study, a total of 50 AP genes (VvAP) were identified in the grape genome, among which 30 contained the complete ASP domain. Synteny analysis within grape indicated that segmental and tandem duplication events contributed to the expansion of the grape AP family. Additional analysis between grape and Arabidopsis demonstrated that several grape AP genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grape and Arabidopsis. Phylogenetic relationships of the 30 VvAPs with the complete ASP domain and their Arabidopsis orthologs, as well as their gene and protein features were analyzed and their cellular localization was predicted. Moreover, expression profiles of VvAP genes in six different tissues were determined, and their transcript abundance under various stresses and hormone treatments were measured. Twenty-seven VvAP genes were expressed in at least one of the six tissues examined; nineteen VvAPs responded to at least one abiotic stress, 12 VvAPs responded to powdery mildew infection, and most of the VvAPs responded to SA and ABA treatments. Furthermore, integrated synteny and phylogenetic analysis identified orthologous AP genes between grape and Arabidopsis, providing a unique starting point for investigating the function of grape AP genes.

Conclusions

The genome-wide identification, evolutionary and expression analyses of grape AP genes provide a framework for future analysis of AP genes in defining their roles during stress response. Integrated synteny and phylogenetic analyses provide novel insight into the functions of less well-studied genes using information from their better understood orthologs.

 
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Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range

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Abstract (provisional)Background

Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime.

Results

To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306.

Conclusions

Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

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De novo characterization of Larix gmelinii (Rupr.) Rupr. transcriptome and analysis of its gene expression induced by jasmonates

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Abstract (provisional)

 

Background

 

Larix gmelinii is a dominant tree species in China's boreal forests and plays an important role in the coniferous ecosystem. It is also one of the most economically important tree species in the Chinese timber industry due to excellent water resistance and anti-corrosion of its wood products. Unfortunately, in Northeast China, L. gmelinii often suffers from serious attacks by diseases and insects. The application of exogenous volatile semiochemicals may induce and enhance its resistance against insect or disease attacks; however, little is known regarding the genes and molecular mechanisms related to induced resistance.

 

Results

 

We performed de novo sequencing and assembly of the L. gmelinii transcriptome using a short read sequencing technology (Illumina). Chemical defenses of L. gmelinii seedlings were induced with jasmonic acid (JA) or methyl jasmonate (MeJA) for 6 hours. Transcriptomes were compared between seedlings induced by JA, MeJA and untreated controls using a tag-based digital gene expression profiling system. In a single run, 25,977,782 short reads were produced and 51,157 unigenes were obtained with a mean length of 517 nt. We sequenced 3 digital gene expression libraries and generated between 3.5 and 5.9 million raw tags, and obtained 52,040 reliable reference genes after removing redundancy. The expression of disease/insect-resistance genes (e. g., phenylalanine ammonialyase, coumarate 3-hydroxylase, lipoxygenase, allene oxide synthase and allene oxide cyclase) was up-regulated. The expression profiles of some abundant genes under different elicitor treatment were studied by using real-time qRT-PCR.

 

The results showed that the expression levels of disease/insect-resistance genes in the seedling samples induced by JA and MeJA were higher than those in the control group. The seedlings induced with MeJA elicited the strongest increases in disease/insect-resistance genes.

 

Conclusions

 

Both JA and MeJA induced seedlings of L. gmelinii showed significantly increased expression of disease/insect-resistance genes. MeJA seemed to have a stronger induction effect than JA on expression of disease/insect-resistance related genes. This study provides sequence resources for L. gmelinii research and will help us to better understand the functions of disease/insect-resistance genes and the molecular mechanisms of secondary metabolisms in L. gmelinii.

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Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger

Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

A major part of second generation biofuel production is the enzymatic saccharification of lignocellulosic biomass into fermentable sugars. Many fungi produce enzymes that can saccarify lignocellulose and cocktails from several fungi, including well-studied species such as Trichoderma reesei and Aspergillus niger, are available commercially for this process. Such commercially-available enzyme cocktails are not necessarily representative of the array of enzymes used by the fungi themselves when faced with a complex lignocellulosic material. The global induction of genes in response to exposure of T. reesei to wheat straw was explored using RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to wheat straw.

Results

In T. reesei, levels of transcript that encode known and predicted cell-wall degrading enzymes were very high after 24 h exposure to straw (approximately 13% of the total mRNA) but were less than recorded in A. niger (approximately 19% of the total mRNA). Closer analysis revealed that enzymes from the same glycoside hydrolase families but different carbohydrate esterase and polysaccharide lyase families were up-regulated in both organisms. Accessory proteins which have been hypothesised to possibly have a role in enhancing carbohydrate deconstruction in A. niger were also uncovered in T. reesei and categories of enzymes induced were in general similar to those in A. niger. Similarly to A. niger, antisense transcripts are present in T. reesei and their expression is regulated by the growth condition.

Conclusions

T. reesei uses a similar array of enzymes, for the deconstruction of a solid lignocellulosic substrate, to A. niger. This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi. This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate. The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

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Genome-wide analysis of the Populus Hsp90 gene family reveals differential expression patterns, localization, and heat stress responses

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Background

Members of the heat shock protein 90 (Hsp90) class of proteins are evolutionarily conserved molecular chaperones. They are involved in protein folding, assembly, stabilization, activation, and degradation in many normal cellular processes and under stress conditions. Unlike many other well-characterized molecular chaperones, Hsp90s play key roles in signal transduction, cell-cycle control, genomic silencing, and protein trafficking. However, no systematic analysis of genome organization, gene structure, and expression compendium has been performed in the Populus model tree genus to date.

Results

We performed a comprehensive analysis of the Populus Hsp90 gene family and identified 10 Populus Hsp90 genes, which were phylogenetically clustered into two major groups. Gene structure and motif composition are relatively conserved in each group. In Populus trichocarpa, we identified three paralogous pairs, among which the PtHsp90-5a/PtHsp90-5b paralogous pair might be created by duplication of a genome segment. Subcellular localization analysis shows that PtHsp90 members are localized in different subcellular compartments. PtHsp90-3 is localized both in the nucleus and in the cytoplasm, PtHsp90-5a and PtHsp90-5b are in chloroplasts, and PtHsp90-7 is in the endoplasmic reticulum (ER). Furthermore, microarray and semi-quantitative real-time RT-PCR analyses show that a number of Populus Hsp90 genes are differentially expressed upon exposure to various stresses.

Conclusions

The gene structure and motif composition of PtHsp90s are highly conserved among group members, suggesting that members of the same group may also have conserved functions. Microarray and RT-PCR analyses show that most PtHsp90s were induced by various stresses, including heat stress. Collectively, these observations lay the foundation for future efforts to unravel the biological roles of PtHsp90 genes.

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Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers are feasible provided that a multidimensional pooling strategy is implemented.
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Abstract (provisional)Background

Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers are feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate(R) Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome.

Results

A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina's GoldenGate(R) Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated.

Conclusions

Illumina's GoldenGate(R) Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome.

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Microbes, metagenomes and marine mammals: enabling the next generation of scientist to enter the genomic era

The revolution in DNA sequencing technology continues unabated, and is affecting all aspects of the biological and medical sciences.
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Background

The revolution in DNA sequencing technology continues unabated, and is affecting all aspects of the biological and medical sciences. The training and recruitment of the next generation of researchers who are able to use and exploit the new technology is severely lacking and potentially negatively influencing research and development efforts to advance genome biology. Here we present a cross-disciplinary course that provides undergraduate students with practical experience in running a next generation sequencing instrument through to the analysis and annotation of the generated DNA sequences.

Results

Many labs across world are installing next generation sequencing technology and we show that the undergraduate students produce quality sequence data and were excited to participate in cutting edge research. The students conducted the work flow from DNA extraction, library preparation, running the sequencing instrument, to the extraction and analysis of the data. They sequenced microbes, metagenomes, and a marine mammal, the Californian sea lion, Zalophus californianus. The students met sequencing quality controls, had no detectable contamination in the targeted DNA sequences, provided publication quality data, and became part of an international collaboration to investigate carcinomas in carnivores.

Conclusions

Students learned important skills for their future education and career opportunities, and a perceived increase in students' ability to conduct independent scientific research was measured. DNA sequencing is rapidly expanding in the life sciences. Teaching undergraduates to use the latest technology to sequence genomic DNA ensures they are ready to meet the challenges of the genomic era and allows them to participate in annotating the tree of life.

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Identification, expression, and comparative genomic analysis of the IPT and CKX gene families in Chinese cabbage (Brassica rapa ssp. pekinensis)

Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

Cytokinins (CKs) have significant roles in various aspects of plant growth and development, and they are also involved in plant stress adaptations. The fine-tuning of the controlled CK levels in individual tissues, cells, and organelles is properly maintained by isopentenyl transferases (IPTs) and cytokinin oxidase/dehydrogenases (CKXs). Chinese cabbage is one of the most economically important vegetable crops worldwide. The whole genome sequencing of Brassica rapa enables us to perform the genome-wide identification and functional analysis of the IPT and CKX gene families.

Results

In this study, a total of 13 BrIPT genes and 12 BrCKX genes were identified. The gene structures, conserved domains and phylogenetic relationships were analyzed. The isoelectric point, subcellular localization and glycosylation sites of the proteins were predicted. Segmental duplicates were found in both BrIPT and BrCKX gene families. We also analyzed evolutionary patterns and divergence of the IPT and CKX genes in the Cruciferae family. The transcription levels of BrIPT and BrCKX genes were analyzed to obtain an initial picture of the functions of these genes. Abiotic stress elements related to adverse environmental stimuli were found in the promoter regions of BrIPT and BrCKX genes and they were confirmed to respond to drought and high salinity conditions. The effects of 6-BA and ABA on the expressions of BrIPT and BrCKX genes were also investigated.

Conclusions

The expansion of BrIPT and BrCKX genes after speciation from Arabidopsis thaliana is mainly attributed to segmental duplication events during the whole genome triplication (WGT) and substantial duplicated genes are lost during the long evolutionary history. Genes produced by segmental duplication events have changed their expression patterns or may adopted new functions and thus are obtained. BrIPT and BrCKX genes respond well to drought and high salinity stresses, and their transcripts are affected by exogenous hormones, such as 6-BA and ABA, suggesting their potential roles in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. The appropriate modulation of endogenous CKs levels by IPT and CKX genes is a promising approach for developing economically important high-yielding and high-quality stress-tolerant crops in agriculture.

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Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing

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Abstract (provisional)Background

Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed.

Results

A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The selection regions were not distributed randomly or uniformly throughout the genome. Instead, clusters of selection hotspots in certain genomic regions were observed. Moreover, a set of candidate genes (4.38% of the total annotated genes) significantly affected by selection underlying soybean domestication and genetic improvement were identified.

Conclusions

Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes/loci underlying agronomically important traits.

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Genome-wide analysis of the Hsp20 gene family in soybean: comprehensive sequence, genomic organization and expression profile analysis under abiotic and biotic stresses

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Abstract (provisional)Background

The Hsp20 genes are associated with stress caused by HS and other abiotic factors, but have recently been found to be associated with the response to biotic stresses. These genes represent the most abundant class among the HSPs in plants, but little is known about this gene family in soybean. Because of their apparent multifunctionality, these proteins are promising targets for developing crop varieties that are better adapted to biotic and abiotic stresses. Thus, in the present study an in silico identification of GmHsp20 gene family members was performed, and the genes were characterized and subjected to in vivo expression analysis under biotic and abiotic stresses.

Results

A search of the available soybean genome databases revealed 51 gene models as potential GmHsp20 candidates. The 51 GmHsp20 genes were distributed across a total of 15 subfamilies where a specific predicted secondary structure was identified. Based on in vivo analysis, only 47 soybean Hsp20 genes were responsive to heat shock stress. Among the GmHsp20 genes that were potentials HSR, five were also cold-induced, and another five, in addition to one GmAcd gene, were responsive to Meloidogyne javanica infection. Furthermore, one predicted GmHsp20 was shown to be responsive only to nematode infection; no expression change was detected under other stress conditions. Some of the biotic stress-responsive GmHsp20 genes exhibited a divergent expression pattern between resistant and susceptible soybean genotypes under M. javanica infection. The putative regulatory elements presenting some conservation level in the GmHsp20 promoters included HSE, W-box, CAAT box, and TA-rich elements. Some of these putative elements showed a unique occurrence pattern among genes responsive to nematode infection.

Conclusions

The evolution of Hsp20 family in soybean genome has most likely involved a total of 23 gene duplications. The obtained expression profiles revealed that the majority of the 51 GmHsp20 candidates are induced under HT, but other members of this family could also be involved in normal cellular functions, unrelated to HT. Some of the GmHsp20 genes might be specialized to respond to nematode stress, and the predicted promoter structure of these genes seems to have a particular conserved pattern related to their biological function.

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Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation | Plant Genomics | Scoop.it
About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T.
Biswapriya Biswavas Misra's insight:

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD)1, 2. Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker’s flour2. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

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De novo transcriptome assembly of drought tolerant CAM plants, Agave deserti and Agave tequilana

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Abstract (provisional)Background

Agaves are succulent monocotyledonous plants native to xeric environments of North America. Because of their adaptations to their environment, including crassulacean acid metabolism (CAM, a water-efficient form of photosynthesis), and existing technologies for ethanol production, agaves have gained attention both as potential lignocellulosic bioenergy feedstocks and models for exploring plant responses to abiotic stress. However, the lack of comprehensive Agave sequence datasets limits the scope of investigations into the molecular-genetic basis of Agave traits.

Results

Here, we present comprehensive, high quality de novo transcriptome assemblies of two Agave species, A. tequilana and A. deserti, built from short-read RNA-seq data. Our analyses support completeness and accuracy of the de novo transcriptome assemblies, with each species having a minimum of approximately 35,000 protein-coding genes. Comparison of agave proteomes to those of additional plant species identifies biological functions of gene families displaying sequence divergence in agave species. Additionally, a focus on the transcriptomics of the A. deserti juvenile leaf confirms evolutionary conservation of monocotyledonous leaf physiology and development along the proximal-distal axis.

Conclusions

Our work presents a comprehensive transcriptome resource for two Agave species and provides insight into their biology and physiology. These resources are a foundation for further investigation of agave biology and their improvement for bioenergy development.

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De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach

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Abstract (provisional)Background

Application of Single Nucleotide Polymorphism (SNP) marker technology as a tool in sunflower breeding programs offers enormous potential to improve sunflower genetics, and facilitate faster release of sunflower hybrids to the market place. Through a National Sunflower Association (NSA) funded initiative, we report on the process of SNP discovery through reductive genome sequencing and local assembly of six diverse sunflower inbred lines that represent oil as well as confection types.

Results

A combination of Restriction site Associated DNA Sequencing (RAD-Seq) protocols and Illumina paired-end sequencing chemistry generated high quality 89.4 M paired end reads from the six lines which represent 5.3 GB of the sequencing data. Raw reads from the sunflower line, RHA 464 were assembled de novo to serve as a framework reference genome. About 15.2 Mb of sunflower genome distributed over 42,267 contigs were obtained upon assembly of RHA 464 sequencing data, the contig lengths ranged from 200 to 950 bp with an N50 length of 393 bp. SNP calling was performed by aligning sequencing data from the six sunflower lines to the assembled reference RHA 464. On average, 1 SNP was located every 143 bp of the sunflower genome sequence. Based on several filtering criteria, a final set of 16,467 putative sequence variants with characteristics favorable for Illumina Infinium Genotyping Technology (IGT) were mined from the sequence data generated across six diverse sunflower lines.

Conclusion

Here we report the molecular and computational methodology involved in SNP development for a complex genome like sunflower lacking reference assembly, offering an attractive tool for molecular breeding purposes in sunflower.

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De novo assembly and characterization of fruit transcriptome in Litchi chinensis Sonn and analysis of differentially regulated genes in fruit in response to shading

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Abstract (provisional)Background

Litchi (Litchi chinensis Sonn.) is one of the most important fruit trees cultivated in tropical and subtropical areas. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying fruit set and fruit development in litchi. Shading during early fruit development decreases fruit growth and induces fruit abscission. Here, high-throughput RNA sequencing (RNA-Seq) was employed for the de novo assembly and characterization of the fruit transcriptome in litchi, and differentially regulated genes, which are responsive to shading, were also investigated using digital transcript abundance(DTA)profiling.

Results

More than 53 million paired-end reads were generated and assembled into 57,050 unigenes with an average length of 601 bp. These unigenes were annotated by querying against various public databases, with 34,029 unigenes found to be homologous to genes in the NCBI GenBank database and 22,945 unigenes annotated based on known proteins in the Swiss-Prot database. In further orthologous analyses, 5,885 unigenes were assigned with one or more Gene Ontology terms, 10,234 hits were aligned to the 24 Clusters of Orthologous Groups classifications and 15,330 unigenes were classified into 266 Kyoto Encyclopedia of Genes and Genomes pathways. Based on the newly assembled transcriptome, the DTA profiling approach was applied to investigate the differentially expressed genes related to shading stress. A total of 3.6 million and 3.5 million high-quality tags were generated from shaded and non-shaded libraries, respectively. As many as 1,039 unigenes were shown to be significantly differentially regulated. Eleven of the 14 differentially regulated unigenes, which were randomly selected for more detailed expression comparison during the course of shading treatment, were identified as being likely to be involved in the process of fruitlet abscission in litchi.

Conclusions

The assembled transcriptome of litchi fruit provides a global description of expressed genes in litchi fruit development, and could serve as an ideal repository for future functional characterization of specific genes. The DTA analysis revealed that more than 1000 differentially regulated unigenes respond to the shading signal, some of which might be involved in the fruitlet abscission process in litchi, shedding new light on the molecular mechanisms underlying organ abscission.

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Transcriptome deep-sequencing and clustering of expressed isoforms from Favia corals

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Abstract (provisional)Background

Genomic and transcriptomic sequence data are essential tools for tackling ecological problems. Using an approach that combines next-generation sequencing, de novo transcriptome assembly, gene annotation and synthetic gene construction, we identify and cluster the protein families from Favia corals from the northern Red Sea.

Results

We obtained 80 million 75 bp paired-end cDNA reads from two Favia adult samples collected at 65 m (Fav1, Fav2) on the Illumina GA platform, and generated two de novo assemblies using ABySS and CAP3. After removing redundancy and filtering out low quality reads, our transcriptome datasets contained 58,268 (Fav1) and 62,469 (Fav2) contigs longer than 100 bp, with N50 values of 1,665 bp and 1,439 bp, respectively. Using the proteome of the sea anemone Nematostella vectensis as a reference, we were able to annotate almost 20% of each dataset using reciprocal homology searches. Homologous clustering of these annotated transcripts allowed us to divide them into 7,186 (Fav1) and 6,862 (Fav2) homologous transcript clusters (E-value <= 2e-30). Functional annotation categories were assigned to homologous clusters using the functional annotation of Nematostella vectensis. General annotation of the assembled transcripts was improved 1-3% using the Acropora digitifera proteome. In addition, we screened these transcript isoform clusters for fluorescent proteins (FPs) homologs and identified seven potential FP homologs in Fav1, and four in Fav2. These transcripts were validated as bona fide FP transcripts via robust fluorescence heterologous expression. Annotation of the assembled contigs revealed that 1.34% and 1.61% (in Fav1 and Fav2, respectively) of the total assembled contigs likely originated from the corals' algal symbiont, Symbiodinium spp.

Conclusions

Here we present a study to identify the homologous transcript isoform clusters from the transcriptome of Favia corals using a far-related reference proteome. Furthermore, the symbiont-derived transcripts were isolated from the datasets and their contribution quantified. This is the first annotated transcriptome of the genus Favia, a major increase in genomics resources available in this important family of corals.

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Solanum torvum responses to the root-knot nematode Meloidogyne incognita

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Abstract (provisional)Background

Solanum torvum Sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. The little information on Solanum torvum (hereafter Torvum) resistance mechanisms, is mostly attributable to the lack of genomic tools (e.g. dedicated microarray) as well as to the paucity of database information limiting high-throughput expression studies in Torvum.

Results

As a first step towards transcriptome profiling of Torvum inoculated with the nematode M. incognita, we built a Torvum 3' transcript catalogue. One-quarter of a 454 full run resulted in 205,591 quality-filtered reads. De novo assembly yielded 24,922 contigs and 11,875 singletons. Similarity searches of the S. torvum transcript tags catalogue produced 12,344 annotations. A 30,0000 features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and 14 dpi (day post inoculation) with Meloidogyne incognita-infected roots samples resulting in 390 differentially expressed genes (DEG). We also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species Solanum melongena. An in-silico validation strategy was developed based on assessment of sequence similarity among Torvum probes and eggplant expressed sequences available in public repositories. GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.

The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.

Conclusions

By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. In addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. The availability of the long sequence tags in S. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species.

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Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae

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Background

Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes.

Results

Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species.

Conclusion

This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of microalgae was proposed which may be generally applicable to other microalgal genera and should serve as a valuable tool in the expanding algal biotechnology industry.

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