Giang T. H. Vu

Comparative Genome Analysis Reveals Divergent Genome Size Evolution in a Carnivorous Plant Genus

The C‐value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350‐fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25‐fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion‐biased double‐strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole‐genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.‐Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400–800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral.

Genome Stability and Evolution: Attempting a Holistic View

The reason why the DNA content, chromosome number and shape, and gene content of eukaryotic genomes vary independently remains a matter of speculation. The same is true for the questions of whether there is a general tendency for increase or decrease of genome size and chromosome number and whether genome size and/or chromosome number have an adaptive value and, if so, what this value is. Here we assume that three strategies of genome evolution (shrinkage, expansion, and equilibrium) have developed to find the optimal balance between genomic stability and plasticity. We suggest various modes of DNA double-strand break (DSB) repair in combination with whole-genome duplication (WGD) and dysploid chromosome number alteration to explain the different strategies of genome size and karyotype evolution.

Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

Amplicon sequencing, together with sister chromatid exchange counting at three unique transgenic target loci, enabled quantitative estimation of breakage frequency and of various double-strand break repair pathways in the monocot plant barley. Distinct types of nonhomologous end-joining, even in combination with sequence conversion, occurred frequently and often involved the sister chromatid. DNA double-strand break (DSB) repair mechanisms differ in their requirements for a homologous repair template and in the accuracy of the result. We aimed to quantify the outcome of repair of a single targeted DSB in somatic cells of young barley (Hordeum vulgare) plants. Amplicon sequencing of three reporter constructs revealed 47 to 58% of reads as repaired via nonhomologous end-joining (NHEJ) with deletions and/or small (1 to 3 bp) insertions. Alternative NHEJ revealed 2 to 5 bp microhomology (15.7% of cases) or new replication-mediated short duplications at sealed breaks. Although deletions outweigh insertions in barley, this bias was less pronounced and deleted sequences were shorter than in Arabidopsis thaliana. Between 17 and 33% of reads likely represent restoration of the original sequence. Depending on the construct, 20 to 33% of reads arose via gene conversion (homologous recombination). Remarkably, <1 to >8% of reads apparently display synthesis-dependent strand annealing linked with NHEJ, inserting 4 to 61 bp, mostly originating from the surrounding of breakpoints. Positional coincidence of >81% of sister chromatid exchanges with target loci is unprecedented for higher eukaryotes and indicates that most repair events for staggered DSBs, at least in barley, involve the sister chromatid and occur during S or G2 phase of the cell cycle.

Hidden genetic nature of epigenetic natural variation in plants

Transcriptional gene silencing (TGS) is an epigenetic mechanism that suppresses the activity of repetitive DNA elements via accumulation of repressive chromatin marks. We discuss natural variation in TGS, with a particular focus on cases that affect the function of protein-coding genes and lead to developmental or physiological changes. Comparison of the examples described has revealed that most natural variation is associated with genetic determinants, such as gene rearrangements, inverted repeats, and transposon insertions that triggered TGS. Recent technical advances have enabled the study of epigenetic natural variation at a whole-genome scale and revealed patterns of inter- and intraspecific epigenetic variation. Future studies exploring non-model species may reveal species-specific evolutionary adaptations at the level of chromatin configuration.

Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea.

Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification.

Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species.

In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genliseas leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.

Chromatin features of plant telomeric sequences at terminal vs. internal positions.

Epigenetic mechanisms are involved in regulation of crucial cellular processes in eukaryotic organisms. Data on the epigenetic features of plant telomeres and their epigenetic regulation were published mostly for Arabidopsis thaliana, in which the presence of interstitial telomeric repeats (ITRs) may interfere with genuine telomeres in most analyses. Here, we studied the epigenetic landscape and transcription of telomeres and ITRs in Nicotiana tabacum with long telomeres and no detectable ITRs, and in Ballantinia antipoda with large blocks of pericentromeric ITRs and relatively short telomeres. Chromatin of genuine telomeres displayed heterochromatic as well as euchromatic marks, while ITRs were just heterochromatic. Methylated cytosines were present at telomeres and ITRs, but showed a bias with more methylation toward distal telomere positions and different blocks of B. antipoda ITRs methylated to different levels. Telomeric transcripts TERRA (G-rich) and ARRET (C-rich) were identified in both plants and their levels varied among tissues with a maximum in blossoms. Plants with substantially different proportions of internally and terminally located telomeric repeats are instrumental in clarifying the chromatin status of telomeric repeats at distinct chromosome locations.

The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding.

Genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. Remarkably, the RNA-guided endonuclease technology (RGEN) based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. Here, we review the major technical advances and recent applications of the CRISPR-Cas9 system for manipulation of model and crop plant genomes. We also discuss the future prospects of this technology in molecular plant breeding.

Chromatin organisation in duckweed interphase nuclei in relation to the nuclear DNA content

The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus.

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.