Ann A. Ferguson

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Pack-Mutator–like transposable elements (Pack-MULEs) induce directional modification of genes through biased insertion and DNA acquisition

In monocots, many genes demonstrate a significant negative GC gradient, meaning that the GC content declines along the orientation of transcription. Such a gradient is not observed in the genes of the dicot plant Arabidopsis. In addition, a lack of homology is often observed when comparing the 5′ end of the coding region of orthologous genes in rice and Arabidopsis. The reasons for these differences have been enigmatic. The presence of GC-rich sequences at the 5′ end of genes may influence the conformation of chromatin, the expression level of genes, as well as the recombination rate. Here we show that Pack-Mutator–like transposable elements (Pack-MULEs) that carry gene fragments specifically acquire GC-rich fragments and preferentially insert into the 5′ end of genes. The resulting Pack-MULEs form independent, GC-rich transcripts with a negative GC gradient. Alternatively, the Pack-MULEs evolve into additional exons at the 5′ end of existing genes, thus altering the GC content in those regions. We demonstrate that Pack-MULEs modify the 5′ end of genes and are at least partially responsible for the negative GC gradient of genes in grasses. Such a unique and global impact on gene composition and gene structure has not been observed for any other transposable elements.

What makes up plant genomes: The vanishing line between transposable elements and genes

The ultimate source of evolution is mutation. As the largest component in plant genomes, transposable elements (TEs) create numerous types of mutations that cannot be mimicked by other genetic mechanisms. When TEs insert into genomic sequences, they influence the expression of nearby genes as well as genes unlinked to the insertion. TEs can duplicate, mobilize, and recombine normal genes or gene fragments, with the potential to generate new genes or modify the structure of existing genes. TEs also donate their transposase coding regions for cellular functions in a process called TE domestication. Despite the host defense against TE activity, a subset of TEs survived and thrived through discreet selection of transposition activity, target site, element size, and the internal sequence. Finally, TEs have established strategies to reduce the efficacy of host defense system by increasing the cost of silencing TEs. This review discusses the recent progress in the area of plant TEs with a focus on the interaction between TEs and genes.

Selective Acquisition and Retention of Genomic Sequences by Pack- Mutator -Like Elements Based on Guanine-Cytosine Content and the Breadth of Expression

Pack-MULE transposable elements selectively duplicate bona fide genes. The process of gene duplication followed by sequence and functional divergence is important for the generation of new genes. Pack-MULEs, nonautonomous Mutator-like elements (MULEs) that carry genic sequence(s), are potentially involved in generating new open reading frames and regulating parental gene expression. These elements are identified in many plant genomes and are most abundant in rice (Oryza sativa). Despite the abundance of Pack-MULEs, the mechanism by which parental genes are captured by Pack-MULEs remains largely unknown. In this study, we identified all MULEs in rice and examined factors likely important for sequence acquisition. Terminal inverted repeat MULEs are the predominant MULE type and account for the majority of the Pack-MULEs. In addition to genic sequences, rice MULEs capture guanine-cytosine (GC)-rich intergenic sequences, albeit at a much lower frequency. MULEs carrying nontransposon sequences have longer terminal inverted repeats and higher GC content in terminal and subterminal regions. An overrepresentation of genes with known functions and genes with orthologs among parental genes of Pack-MULEs is observed in rice, maize (Zea mays), and Arabidopsis (Arabidopsis thaliana), suggesting preferential acquisition for bona fide genes by these elements. Pack-MULEs selectively acquire/retain parental sequences through a combined effect of GC content and breadth of expression, with GC content playing a stronger role. Increased GC content and number of tissues with detectable expression result in higher chances of a gene being acquired by Pack-MULEs. Such selective acquisition/retention provides these elements greater chances of carrying functional sequences that may provide new genetic resources for the evolution of new genes or the modification of existing genes.

Transposition of a Rice Mutator-Like Element in the Yeast Saccharomyces cerevisiae

In this system, modification of the transposase alters the transposition frequency of Mutator-like elements, and the element size and target site duplication affect the frequency of precise excision. Mutator-like transposable elements (MULEs) are widespread in plants and are well known for their high transposition activity as well as their ability to duplicate and amplify host gene fragments. Despite their abundance and importance, few active MULEs have been identified. In this study, we demonstrated that a rice (Oryza sativa) MULE, Os3378, is capable of excising and reinserting in yeast (Saccharomyces cerevisiae), suggesting that yeast harbors all the host factors for the transposition of MULEs. The transposition activity induced by the wild-type transposase is low but can be altered by modification of the transposase sequence, including deletion, fusion, and substitution. Particularly, fusion of a fluorescent protein to the transposase enhanced the transposition activity, representing another approach to manipulate transposases. Moreover, we identified a critical region in the transposase where the net charge of the amino acids seems to be important for activity. Finally, transposition efficiency is also influenced by the element and its flanking sequences (i.e., small elements are more competent than their large counterparts). Perfect target site duplication is favorable, but not required, for precise excision. In addition to the potential application in functional genomics, this study provides the foundation for further studies of the transposition mechanism of MULEs.

Mutator-like elements with multiple long terminal inverted repeats in plants.

Mutator-like transposable elements (MULEs) are widespread in plants and the majority have long terminal inverted repeats (TIRs), which distinguish them from other DNA transposons. It is known that the long TIRs of Mutator elements harbor transposase binding sites and promoters for transcription, indicating that the TIR sequence is critical for transposition and for expression of sequences between the TIRs. Here, we report the presence of MULEs with multiple TIRs mostly located in tandem. These elements are detected in the genomes of maize, tomato, rice, and Arabidopsis. Some of these elements are present in multiple copies, suggesting their mobility. For those elements that have amplified, sequence conservation was observed for both of the tandem TIRs. For one MULE family carrying a gene fragment, the elements with tandem TIRs are more prevalent than their counterparts with a single TIR. The successful amplification of this particular MULE demonstrates that MULEs with tandem TIRs are functional in both transposition and duplication of gene sequences.

Pack-MULEs: Recycling and reshaping genes through GC-biased acquisition.

The availability of genomic sequences provided new opportunities to decipher how plant genomes evolve. One recent discovery about plant genomes is the abundance of Pack-MULEs, a special group of transposable elements that duplicate, amplify, and recombine gene fragments in many species at a very large scale. Despite the widespread occurrence of Pack-MULEs, their function remains an enigma. Our analysis using maize, rice, and Arabidopsis genomic sequences indicates that the acquisition of genic sequences by Pack-MULEs is not random. Pack-MULEs in grasses specifically acquire and amplify GC-rich gene fragments. The resulting GC-rich elements have the ability to form independent transcripts with negative GC gradient, which refers to the decline of GC content along the orientation of transcription of genes. In other cases, Pack-MULEs insert near the 5’ region of “normal” genes, and consequently form additional 5’ exons or replace the original 5’ exon of genes. In this manner, Pack-MULEs raise the GC content of the 5’ termini of genes, modify the gene structure, and contribute to the increased number of genes with negative GC gradient in grasses. The possible consequence of such activity is discussed.

Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) - eScholarship

BackgroundSacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResultsThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.ConclusionsThe slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.

Nelumbo nucifera [data set]

BackgroundSacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResultsThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.ConclusionsThe slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.

Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.)

BackgroundSacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResultsThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.ConclusionsThe slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.