Small Genome Size Research Articles

Evolutionary insights from large scale resequencing datasets in Drosophila melanogaster

Drosophila melanogaster has long been used as an evolutionary model system. Its small genome size, well-annotated genome, and ease of sampling, also makes it a choice species for genome resequencing studies. Hundreds of genomic samples from populations worldwide are available and are currently being used to tackle a wide range of evolutionary questions. In this review, we focused on three insights that have increased our understanding of the evolutionary history of this species, and that have implications for the study of evolutionary processes in other species as well. Because of technical limitations, most of the studies so far have focused on SNP variants. However, long-read sequencing techniques should allow us in the near future to include other type of genomic variants that also influence genome evolution.

Genome Size, Ploidy, and Base Composition of Wild and Cultivated Acer

Acer is a large and important genus of woody plants most commonly encountered as small to large trees in urban landscapes. Considerable investigation has been devoted to addressing the taxonomy of maples, but little is known about genome sizes across the genus. Relatively more work has been conducted to determine chromosome numbers and ploidy of more species, but much could be gained by expanding knowledge of genome sizes in combination with traditional cytology. Furthermore, base pair (bp) composition may have implications for a species’ adaptability and also impacts nucleic acid stability at high temperatures. Our objectives were to determine the genome size of 195 accessions of maples, assign ploidy to each using inference as well as cytology, and determine base composition of a subset of 48 accessions. Most species had small genome sizes (1.4–3.5 pg) with the exception of section Rubra, which contains many polyploids. Holoploid genome sizes ranged from 1.39 to 6.10 pg, with the latter being interpreted as 9x. The mean monoploid genome sizes (1Cx) ranged from 0.43 pg in A. carpinifolium (section Indivisa) to 1.66 pg in A. caudatifolium (section Macrantha); mean monoploid genome sizes were significantly different among sections. Forty-four of the 48 accessions measured using both fluorochromes had greater estimates with 4′,6-diamidino-2-phenylindole (DAPI) than propidium iodide (PI). The proportion of the genome composed of guanosine and cytosine (GC%) among the taxa evaluated in this study ranged from just 38.61% to 43.96% and did not appear to be related to ecological adaptability or urban tolerance among these taxa.

Genetic transformation of einkorn (Triticum monococcum L. ssp. monococcum L.), a diploid cultivated wheat species

BackgroundDomesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~ 5.7 GB), low ploidy (2n = 2x = 14, AmAm) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn.ResultsIn this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T1-T2 populations of transgenic einkorn that are resistant to herbicides were selected.ConclusionTo the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

Draft genomes and genomic divergence of two Lepidurus tadpole shrimp species (Crustacea, Branchiopoda, Notostraca).

Crustaceans of the order Notostraca (Branchiopoda) are distributed worldwide and are known for the remarkable morphological stasis between their extant and Permian fossil species. Moreover, these crustaceans show relevant ecological traits and a wide range of reproductive strategies. However, genomic studies on notostracans are fairly limited. Here, we present the genome sequences of two notostracan taxa, Lepidurus arcticus and Lepidurus apus lubbocki. Taking advantage of the small genome sizes (~0.11pg) of these taxa, genomes were sequenced for one individual per species with one run on the Illumina HiSeq X platform. We finally assembled 73.2Mbp (L.arcticus) and 90.3Mbp (L.apus lubbocki) long genomes. Assemblies cover up to 84% of the estimated genome size, with a gene completeness >97% for both genomes. In total, 13%-16% of the assembled genomes consist of repeats, and based on read mapping, L.apus lubbocki shows a significantly lower transposable element content than L.arcticus. The analysis of 2,376 orthologous genes indicates an ~7% divergence between the two Lepidurus taxa, with a nucleotide substitution rate significantly lower than that of Daphnia taxa. Ka /Ks analysis suggests purifying selection in both branchiopod lineages, raising the question of whether the low substitution rate of Lepidurus is correlated with morphological conservation or is linked to specific biological traits. Our analysis demonstrates that, in these organisms, it is possible to obtain high-quality draft genomes from single individuals with a relatively low sequencing effort. This result makes Lepidurus and Notostraca interesting models for genomic studies at taxonomic, ecological and evolutionary levels.

Genome Duplication in Animal Evolution

In this review, the available data on whole genome duplication in different phylogenetic lineages of animals are summarized, and possible mechanisms of the genome duplication and its role in the evolution of animals are considered. Special attention is paid to the problems of studying the first stages of the genome evolution after its duplication and to the search for species the study of which will make it possible to identify the peculiarities of the genome “diploidization” process after its duplication. A group of species of free-living flatworms from the Macrostomum genus is suggested as a promising model object for such studies. According to our data, the genomes of some Macrostomum members (M. lignano, Macrostomum sp. 8) are a result of recent genome duplication and subsequent chromosome rearrangements. In addition, the peculiarities of morphology, life cycle, small genome size, and simply organized karyotype make Macrostomum almost an ideal model object for studying the early stages of duplicated genome reorganization.

Bacterial endosymbiont Cardinium cSfur genome sequence provides insights for understanding the symbiotic relationship in Sogatella furcifera host

BackgroundSogatella furcifera is a migratory pest that damages rice plants and causes severe economic losses. Due to its ability to annually migrate long distances, S. furcifera has emerged as a major pest of rice in several Asian countries. Symbiotic relationships of inherited bacteria with terrestrial arthropods have significant implications. The genus Cardinium is present in many types of arthropods, where it influences some host characteristics. We present a report of a newly identified strain of the bacterial endosymbiont Cardinium cSfur in S. furcifera.ResultFrom the whole genome of S. furcifera previously sequenced by our laboratory, we assembled the whole genome sequence of Cardinium cSfur. The sequence comprised 1,103,593 bp with a GC content of 39.2%. The phylogenetic tree of the Bacteroides phylum to which Cardinium cSfur belongs suggests that Cardinium cSfur is closely related to the other strains (Cardinium cBtQ1 and cEper1) that are members of the Amoebophilaceae family. Genome comparison between the host-dependent endosymbiont including Cardinium cSfur and free-living bacteria revealed that the endosymbiont has a smaller genome size and lower GC content, and has lost some genes related to metabolism because of its special environment, which is similar to the genome pattern observed in other insect symbionts. Cardinium cSfur has limited metabolic capability, which makes it less contributive to metabolic and biosynthetic processes in its host. From our findings, we inferred that, to compensate for its limited metabolic capability, Cardinium cSfur harbors a relatively high proportion of transport proteins, which might act as the hub between it and its host. With its acquisition of the whole operon related to biotin synthesis and glycolysis related genes through HGT event, Cardinium cSfur seems to be undergoing changes while establishing a symbiotic relationship with its host.ConclusionA novel bacterial endosymbiont strain (Cardinium cSfur) has been discovered. A genomic analysis of the endosymbiont in S. furcifera suggests that its genome has undergone certain changes to facilitate its settlement in the host. The envisaged potential reproduction manipulative ability of the new endosymbiont strain in its S. furcifera host has vital implications in designing eco-friendly approaches to combat the insect pest.

Genome Editing in Rice: Recent Advances, Challenges, and Future Implications.

Rice (Oryza sativa L.) is the major food source for more than three billion people of the world. In the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties. However, stagnation in rice yield has been reported in recent decade owing to several factors including the emergence of pests and phyto pathogens, climate change, and other environmental issues posing great threat to global food security. There is an urgent need to produce more rice and associated cereals to satisfy the mammoth task of feeding a still growing population expected to reach 9.7 billion by 2050. Advances in genomics and emergence of multiple genome-editing technologies through use of engineered site-specific nucleases (SSNs) have revolutionized the field of plant science and agriculture. Among them, the CRISPR/Cas9 system is the most advanced and widely accepted because of its simplicity, robustness, and high efficiency. The availability of huge genomic resources together with a small genome size makes rice more suitable and feasible for genetic manipulation. As such, rice has been increasingly used to test the efficiency of different types of genome editing technologies to study the functions of various genes and demonstrate their potential in genetic improvement. Recently developed approaches including CRISPR/Cpf1 system and base editors have evolved as more efficient and accurate genome editing tools which might accelerate the pace of crop improvement. In the present review, we focus on the genome editing strategies for rice improvement, thereby highlighting the applications and advancements of CRISPR/Cas9 system. This review also sheds light on the role of CRISPR/Cpf1 and base editors in the field of genome editing highlighting major challenges and future implications of these tools in rice improvement.

Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations.

Candidate phyla radiation (CPR) bacteria and DPANN (an acronym of the names of the first included phyla) archaea are massive radiations of organisms that are widely distributed across Earth's environments, yet we know little about them. Initial indications are that they are consistently distinct from essentially all other bacteria and archaea owing to their small cell and genome sizes, limited metabolic capacities and often episymbiotic associations with other bacteria and archaea. In this Analysis, we investigate their biology and variations in metabolic capacities by analysis of approximately 1,000 genomes reconstructed from several metagenomics-based studies. We find that they are not monolithic in terms of metabolism but rather harbour a diversity of capacities consistent with a range of lifestyles and degrees of dependence on other organisms. Notably, however, certain CPR and DPANN groups seem to have exceedingly minimal biosynthetic capacities, whereas others could potentially be free living. Understanding of these microorganisms is important from the perspective of evolutionary studies and because their interactions with other organisms are likely to shape natural microbiome function.

Lifestyles of sourdough lactobacilli – Do they matter for microbial ecology and bread quality?

Sourdough is used in production of (steamed) bread as leavening agent (type I sourdoughs) or as baking improver to enhance flavour, texture, and shelf life of bread (type II sourdoughs). The long-term propagation of sourdoughs eliminates dispersal limitation and consistently leads to sourdough microbiota that are composed of host adapted lactobacilli. In contrast, community assembly in spontaneous cereal fermentations is limited by dispersal and nomadic or environmental lactic acid bacteria are the first colonizers of these sourdoughs.Propagation of sourdoughs for use as sole leavening agent (type I sourdoughs) dictates fermentation conditions that select for rapid growth. Type I wheat- and rye sourdoughs are consistently populated by insect-adapted lactobacilli, particularly Lactobacillus sanfranciscensis, which is characterized by a small genome size and a restricted metabolic potential. The diverse fermentation conditions employed in industrial or artisanal Type II sourdough fermentation processes also result in a more diverse microbiota. Nevertheless, type II sourdoughs are typically populated by vertebrate host adapted lactobacilli of the L. delbrueckii and L. reuteri groups. Metabolic traits of host-adapted lactobacilli that enhance competitiveness in intestinal ecosystems also provide technological functionality in bread making. Examples include formation of exopolysaccharides, arginine-, glutamine- and glutamate based mechanisms of acid resistance, and glycosyl hydrolases that reduce FODMAP levels in sourdough and sourdough bread. In conclusion, consideration of the lifestyle of sourdough lactobacilli facilitates the selection of competitive and functional sourdough starter cultures.

Comparative genomic analysis of Enterococcus faecalis: insights into their environmental adaptations

BackgroundEnterococcus faecalis is widely studied as a common gut commensal and a nosocomial pathogen. In fact, Enterococcus faecalis is ubiquitous in nature, and it has been isolated from various niches, including the gastrointestinal tract, faeces, blood, urine, water, and fermented foods (such as dairy products). In order to elucidate the role of habitat in shaping the genome of Enterococcus faecalis, we performed a comparative genomic analysis of 78 strains of various origins.ResultsAlthough no correlation was found between the strain isolation habitat and the phylogeny of Enterococcus faecalis from our whole genome-based phylogenetic analysis, our results revealed some environment-associated features in the analysed Enterococcus faecalis genomes. Significant differences were found in the genome size and the number of predicted open reading frames (ORFs) between strains originated from different environments. In general, strains from water sources had the smallest genome size and the least number of predicted ORFs. We also identified 293 environment-specific genes, some of which might link to the adaptive strategies for survival in particular environments. In addition, the number of antibiotic resistance genes was significantly different between strains isolated from dairy products, water, and blood. Strains isolated from blood had the largest number of antibiotic resistance genes.ConclusionThese findings improve our understanding of the role of habitat in shaping the genomes of Enterococcus faecalis.

Genome Evolution of Bartonellaceae Symbionts of Ants at the Opposite Ends of the Trophic Scale.

Many insects rely on bacterial symbionts to supply essential amino acids and vitamins that are deficient in their diets, but metabolic comparisons of closely related gut bacteria in insects with different dietary preferences have not been performed. Here, we demonstrate that herbivorous ants of the genus Dolichoderus from the Peruvian Amazon host bacteria of the family Bartonellaceae, known for establishing chronic or pathogenic infections in mammals. We detected these bacteria in all studied Dolichoderus species, and found that they reside in the midgut wall, that is, the same location as many previously described nutritional endosymbionts of insects. The genomic analysis of four divergent strains infecting different Dolichoderus species revealed genes encoding pathways for nitrogen recycling and biosynthesis of several vitamins and all essential amino acids. In contrast, several biosynthetic pathways have been lost, whereas genes for the import and conversion of histidine and arginine to glutamine have been retained in the genome of a closely related gut bacterium of the carnivorous ant Harpegnathos saltator. The broad biosynthetic repertoire in Bartonellaceae of herbivorous ants resembled that of gut bacteria of honeybees that likewise feed on carbohydrate-rich diets. Taken together, the broad distribution of Bartonellaceae across Dolichoderus ants, their small genome sizes, the specific location within hosts, and the broad biosynthetic capability suggest that these bacteria are nutritional symbionts in herbivorous ants. The results highlight the important role of the host nutritional biology for the genomic evolution of the gut microbiota—and conversely, the importance of the microbiota for the nutrition of hosts.

Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov., both isolated from freshwater systems.

Strains MWH-EgelM1-30-B4T and MWH-Feld-100T were isolated from the water columns of two freshwater systems. Both strains represent delicate bacteria not easy to work with in laboratory experiments. Phylogenetic analyses of the 16S rRNA genes suggested that both strains were affiliated with the genus Polynucleobacter. Both strains share 16S rRNA gene sequence similarities of >99 % with eight free-living Polynucleobacter type strains, all affiliated with the cryptic species complex PnecC. The full-length 16S rRNA gene sequences of the two strains differ only in two and three positions, respectively, from the sequence of the closest related Polynucleobacter type strain. Genome sequencing of both strains revealed relatively small genome sizes of 2.0 Mbp and G+C contents of 45 mol%. Phylogenetic analyses based on nucleotide sequences of 319 shared protein-encoding genes consistently placed the two strains in taxon PnecC but did not suggest an affiliation with one of the previously described species. Pairwise analyses of whole genome average nucleotide identities (gANI) with representatives of all previously described Polynucleobacter species resulted in both cases throughout in values <80 %. Pairwise comparison of the genomes of the two new strains resulted in gANI values of 83.3 %. All gANI analyses clearly suggested that strains MWH-EgelM1-30-B4T and MWH-Feld-100T represent two novel Polynucleobacter species. We propose for these novel species the names Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov. and strains MWH-EgelM1-30-B4T (=DSM 23911T=LMG 30144T) and MWH-Feld-100T (=DSM 24007T=LMG 29705T) as the type strains, respectively.

Influence of adaptive mutations, from thermal adaptation experiments, on the infection cycle of RNA bacteriophage Qβ

A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qβ using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qβ genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qβ can adapt to higher temperatures, even though Qβ is a simple RNA bacteriophage with a small genome size, encoding only four genes.

Long-read sequencing data analysis for yeasts.

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ∼41 h to generate a complete and well-annotated genome from ∼100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

Genome comparison of different Zymomonas mobilis strains provides insights on conservation of the evolution

Zymomonas mobilis has the special Entner-Doudoroff (ED) pathway and it has excellent industrial characteristics, including low cell mass formation, high-specific productivity,ethanol yield, notable ethanol tolerance and wide pH range, a relatively small genome size. In this study, the genome sequences of NRRL B-14023 and NRRL B-12526 were sequenced and compared with other strains to explore their evolutionary relationships and the genetic basis of Z. mobilis. The comparative genomic analyses revealed that the 8 strains share a conserved core chromosomal backbone. ZM4, NRRL B-12526, NRRL B-14023, NCIMB 11163 and NRRL B-1960 share 98% sequence identity across the whole genome sequences. Highly similar plasmids and CRISPR repeats were detected in these strains. A whole-genome phylogenetic tree of the 8 strains indicated that NRRL B-12526, NRRL B-14023 and ATCC 10988 had a close evolutionary relationship with the strain ZM4. Furthermore, strains ATCC29191 and ATCC29192 had distinctive CRISPR with a far distant relationship. The size of the pan-genome was 1945 genes, including 1428 core genes and 517 accessory genes. The genomes of Z. mobilis were highly conserved; particularly strains ZM4, NRRL B-12526, NRRL B-14023, NCIMB 11163 and NRRL B-1960 had a close genomic relationship. This comparative study of Z. mobilis presents a foundation for future functional analyses and applications.

A staging table for the embryonic development of the brownbanded bamboo shark (Chiloscyllium punctatum).

Background: Studying cartilaginous fishes (chondrichthyans) has helped us understand vertebrate evolution and diversity. However, resources such as genome sequences, embryos, and detailed staging tables are limited for species within this clade. To overcome these limitations, we have focused on a species, the brownbanded bamboo shark (Chiloscyllium punctatum), which is a relatively common aquarium species that lays eggs continuously throughout the year. In addition, because of its relatively small genome size, this species is promising for molecular studies. Results: To enhance biological studies of cartilaginous fishes, we establish a normal staging table for the embryonic development of the brownbanded bamboo shark. Bamboo shark embryos take around 118 days to reach the hatching period at 25°C, which is approximately 1.5 times as fast as the small‐spotted catshark (Scyliorhinus canicula) takes. Our staging table divides the embryonic period into 38 stages. Furthermore, we found culture conditions that allow early embryos to grow in partially opened egg cases. Conclusions: In addition to the embryonic staging table, we show that bamboo shark embryos exhibit relatively fast embryonic growth and are amenable to culture, key characteristics that enhance their experimental utility. Therefore, the present study is a foundation for cartilaginous fish research. Developmental Dynamics 247:712–723, 2018. © 2017 Wiley Periodicals, Inc.

Whole-Genome Sequencing of Suppressor DNA Mixtures Identifies Pathways That Compensate for Chromosome Segregation Defects in Schizosaccharomyces pombe.

Suppressor screening is a powerful method to identify genes that, when mutated, rescue the temperature sensitivity of the original mutation. Previously, however, identification of suppressor mutations has been technically difficult. Due to the small genome size of Schizosaccharomyces pombe, we developed a spontaneous suppressor screening technique, followed by a cost-effective sequencing method. Genomic DNAs of 10 revertants that survived at the restrictive temperature of the original temperature sensitive (ts) mutant were mixed together as one sample before constructing a library for sequencing. Responsible suppressor mutations were identified bioinformatically based on allele frequency. Then, we isolated a large number of spontaneous extragenic suppressors for three ts mutants that exhibited defects in chromosome segregation at their restrictive temperature. Screening provided new insight into mechanisms of chromosome segregation: loss of Ufd2 E4 multi-ubiquitination activity suppresses defects of an AAA ATPase, Cdc48. Loss of Wpl1, a releaser of cohesin, compensates for the Eso1 mutation, which may destabilize sister chromatid cohesion. The segregation defect of a ts histone H2B mutant is rescued if it fails to be deubiquitinated by the SAGA complex, because H2B is stabilized by monoubiquitination.

Small genome size variation across the range of European beech (Fagus sylvatica)

Interpopulation variation of relative and absolute genome size was studied in Fagus sylvatica subsp. sylvatica and subsp. orientalis. The study included 18 populations of beech planted in a common-garden experiment in central Slovakia and three additional populations from the Caucasus. Nuclear DNA content was determined by means of flow cytometry using the AT-specific fluorochrome 4′,6-diamidino-2-phenylindole and non-specific propidium iodide, and its associations with climate, growth, phenology and physiological traits were assessed. The approximate average nuclear DNA content (2C) across all samples was 1.178 ± 0.020 pg. The lowest mean relative genome sizes were observed in the Alpine range, whereas they increased toward the range margins; no clear trend was observed for 2C values. Temperature seasonality and temperature annual range were found to be negatively associated with genome size. Among phenotypic traits, the maximum chlorophyll a fluorescence yield (Fv/Fm) was found to be negatively correlated with relative genome size, whereas phenology and some photosynthetic parameters were correlated with the 2C values.

Extreme haplotype variation in the desiccation-tolerant clubmoss Selaginella lepidophylla

Plant genome size varies by four orders of magnitude, and most of this variation stems from dynamic changes in repetitive DNA content. Here we report the small 109 Mb genome of Selaginella lepidophylla, a clubmoss with extreme desiccation tolerance. Single-molecule sequencing enables accurate haplotype assembly of a single heterozygous S. lepidophylla plant, revealing extensive structural variation. We observe numerous haplotype-specific deletions consisting of largely repetitive and heavily methylated sequences, with enrichment in young Gypsy LTR retrotransposons. Such elements are active but rapidly deleted, suggesting “bloat and purge” to maintain a small genome size. Unlike all other land plant lineages, Selaginella has no evidence of a whole-genome duplication event in its evolutionary history, but instead shows unique tandem gene duplication patterns reflecting adaptation to extreme drying. Gene expression changes during desiccation in S. lepidophylla mirror patterns observed across angiosperm resurrection plants.

Model Legumes: Functional Genomics Tools in Medicago truncatula.

Many researchers have sought along the last two decades a legume species that could serve as a model system for genetic studies to resolve specific developmental or metabolic processes that cannot be studied in other model plants. Nitrogen fixation, nodulation, compound leaf, inflorescence and plant architecture, floral development, pod formation, secondary metabolite biosynthesis, and other developmental and metabolic aspects are legume-specific or show important differences with those described in Arabidopsis thaliana, the most studied model plant. Mainly Medicago truncatula and Lotus japonicus were proposed in the 1990s as model systems due to their key attributes, diploid genome, autogamous nature, short generation times, small genome sizes, and both species can be readily transformed. After more than decade-long, the genome sequences of both species are essentially complete, and a series of functional genomics tools have been successfully developed and applied. Mutagens that cause insertions or deletions are being used in these model systems because these kinds of DNA rearrangements are expected to assist in the isolation of the corresponding genes by Target-Induced Local Lesions IN Genomes (TILLING) approaches. Different M. truncatula mutants have been obtained following γ-irradiation or fast neutron bombardment (FNB), ethyl-nitrosourea (ENU) or ethyl-methanesulfonate (EMS) treatments, T-DNA and activation tagging, use of the tobacco retrotransposon Tnt1 to produce insertional mutants, gene silencing by RNAi, and transient post-transcriptional gene silencing by virus-induced gene silencing (VIGS). Emerging technologies of targeted mutagenesis and gene editing, such as the CRISPR-Cas9 system, could open a new era in this field. Functional genomics tools and phenotypic analyses of several mutants generated in M. truncatula have been essential to better understand differential aspects of legumes development and metabolism.