Three near-complete genome assemblies reveal substantial centromere dynamics from diploid to tetraploid in Brachypodium genus.

BACKGROUND: Centromeres are critical for maintaining genomic stability in eukaryotes, and their turnover shapes genome architectures and drives karyotype evolution. However, the co-evolution of centromeres from different species in allopolyploids over millions of years remains largely unknown. RESULTS: Here, we generate three near-complete genome assemblies, a tetraploid Brachypodium hybridum and its two diploid ancestors, Brachypodium distachyon and Brachypodium stacei. We detect high degrees of sequence, structural, and epigenetic variations of centromeres at base-pair resolution between closely related Brachypodium genomes, indicating the appearance and accumulation of species-specific centromere repeats from a common origin during evolution. We also find that centromere homogenization is accompanied by local satellite repeats bursting and retrotransposon purging, and the frequency of retrotransposon invasions drives the degree of interspecies centromere diversification. We further investigate the dynamics of centromeres during alloploidization process, and find that dramatic genetics and epigenetics architecture variations are associated with the turnover of centromeres between homologous chromosomal pairs from diploid to tetraploid. Additionally, our pangenomes analysis reveals the ongoing variations of satellite repeats and stable evolutionary homeostasis within centromeres among individuals of each Brachypodium genome with different polyploidy levels. CONCLUSIONS: Our results provide unprecedented information on the genomic, epigenomic, and functional diversity of highly repetitive DNA between closely related species and their allopolyploid genomes at both coarse and fine scale.

Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.

Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.

A happy accident: a novel turfgrass reference genome.

Poa pratensis, commonly known as Kentucky bluegrass, is a popular cool-season grass species used as turf in lawns and recreation areas globally. Despite its substantial economic value, a reference genome had not previously been assembled due to the genome's relatively large size and biological complexity that includes apomixis, polyploidy, and interspecific hybridization. We report here a fortuitous de novo assembly and annotation of a P. pratensis genome. Instead of sequencing the genome of a C4 grass, we accidentally sampled and sequenced tissue from a weedy P. pratensis whose stolon was intertwined with that of the C4 grass. The draft assembly consists of 6.09 Gbp with an N50 scaffold length of 65.1 Mbp, and a total of 118 scaffolds, generated using PacBio long reads and Bionano optical map technology. We annotated 256K gene models and found 58% of the genome to be composed of transposable elements. To demonstrate the applicability of the reference genome, we evaluated population structure and estimated genetic diversity in P. pratensis collected from three North American prairies, two in Manitoba, Canada and one in Colorado, USA. Our results support previous studies that found high genetic diversity and population structure within the species. The reference genome and annotation will be an important resource for turfgrass breeding and study of bluegrasses.

Non-B-form DNA tends to form in centromeric regions and has undergone changes in polyploid oat subgenomes.

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non-B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non-B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non-B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non-B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non-B-form DNA in the oat genome and reveals its potential role in centromeres.

Genomic imbalance modulates transposable element expression in maize.

Genomic imbalance refers to the more severe phenotypic consequences of changing part of a chromosome compared with the whole genome set. Previous genome imbalance studies in maize have identified prevalent inverse modulation of genes on the unvaried chromosomes (trans) with both the addition or subtraction of chromosome arms. Transposable elements (TEs) comprise a substantial fraction of the genome, and their reaction to genomic imbalance is therefore of interest. Here, we analyzed TE expression using RNA-seq data of aneuploidy and ploidy series and found that most aneuploidies showed an inverse modulation of TEs, but reductions in monosomy and increases in disomy and trisomy were also common. By contrast, the ploidy series showed little TE modulation. The modulation of TEs and genes in the same experimental group were compared, and TEs showed greater modulation than genes, especially in disomy. Class I and II TEs were differentially modulated in most aneuploidies, and some superfamilies in each TE class also showed differential modulation. Finally, the significantly upregulated TEs in three disomies (TB-7Lb, TB9Lc, and TB-10L19) did not increase the proportion of adjacent gene expression when compared with non-differentially expressed TEs, indicating that modulations of TEs do not compound the effect on genes. These results suggest that the prevalent inverse TE modulation in aneuploidy results from stoichiometric upset of the regulatory machinery used by TEs, similar to the response of core genes to genomic imbalance.

MicroRNAs play regulatory roles in genomic balance.

Classic genetics studies found that genomic imbalance caused by changing the dosage of part of the genome (aneuploidy) has more detrimental effects than altering the dosage of the whole genome (ploidy). Previous analysis revealed global modulation of gene expression triggered by aneuploidy across various species, including maize (Zea mays), Arabidopsis, yeast, mammals, etc. Plant microRNAs (miRNAs) are a class of 20- to 24-nt endogenous small noncoding RNAs that carry out post-transcriptional gene expression regulation. That miRNAs and their putative targets are preferentially retained as duplicates after whole-genome duplication, as are many transcription factors and signaling components, indicates miRNAs are likely to be dosage-sensitive and potentially involved in genomic balance networks. This review addresses the following questions regarding the role of miRNAs in genomic imbalance. (1) How do aneuploidy and polyploidy impact the expression of miRNAs? (2) Do miRNAs play a regulatory role in modulating the expression of their targets under genomic imbalance?

Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants.

Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.

Dosage-sensitive miRNAs trigger modulation of gene expression during genomic imbalance in maize.

The genomic imbalance caused by varying the dosage of individual chromosomes or chromosomal segments (aneuploidy) has more detrimental effects than altering the dosage of complete chromosome sets (ploidy). Previous analysis of maize (Zea mays) aneuploids revealed global modulation of gene expression both on the varied chromosome (cis) and the remainder of the genome (trans). However, little is known regarding the role of microRNAs (miRNAs) under genomic imbalance. Here, we report the impact of aneuploidy and polyploidy on the expression of miRNAs. In general, cis miRNAs in aneuploids present a predominant gene-dosage effect, whereas trans miRNAs trend toward the inverse level, although other types of responses including dosage compensation, increased effect, and decreased effect also occur. By contrast, polyploids show less differential miRNA expression than aneuploids. Significant correlations between expression levels of miRNAs and their targets are identified in aneuploids, indicating the regulatory role of miRNAs on gene expression triggered by genomic imbalance.

Prospects of Feral Crop De Novo Redomestication.

Modern agriculture depends on a narrow variety of crop species, leaving global food and nutritional security highly vulnerable to the adverse effects of climate change and population expansion. Crop improvement using conventional and molecular breeding approaches leveraging plant genetic diversity using crop wild relatives (CWRs) has been one approach to address these issues. However, the rapid pace of the global change requires additional innovative solutions to adapt agriculture to meet global needs. Neodomestication-the rapid and targeted introduction of domestication traits using introgression or genome editing of CWRs-is being explored as a supplementary approach. These methods show promise; however, they have so far been limited in efficiency and applicability. We propose expanding the scope of neodomestication beyond truly wild CWRs to include feral crops as a source of genetic diversity for novel crop development, in this case 'redomestication'. Feral crops are plants that have escaped cultivation and evolved independently, typically adapting to their local environments. Thus, feral crops potentially contain valuable adaptive features while retaining some domestication traits. Due to their genetic proximity to crop species, feral crops may be easier targets for de novo domestication (i.e. neodomestication via genome editing techniques). In this review, we explore the potential of de novo redomestication as an application for novel crop development by genome editing of feral crops. This approach to efficiently exploit plant genetic diversity would access an underutilized reservoir of genetic diversity that could prove important in support of global food insecurity in the face of the climate change.

The non-Mendelian behavior of plant B chromosomes.

B chromosomes, also known as supernumerary chromosomes, are dispensable elements in the genome of many plants, animals, and fungi. Many B chromosomes have evolved one or more drive mechanisms to transmit themselves at a higher frequency than predicted by Mendelian genetics, and these mechanisms counteract the tendency of non-essential genetic elements to be lost over time. The frequency of Bs in a population results from a balance between their effect on host fitness and their transmission rate. Here, we will summarize the findings of the drive process of plant B chromosomes, focusing on maize and rye.

The multiple fates of gene duplications: Deletion, hypofunctionalization, subfunctionalization, neofunctionalization, dosage balance constraints, and neutral variation.

Gene duplications have long been recognized as a contributor to the evolution of genes with new functions. Multiple copies of genes can result from tandem duplication, from transposition to new chromosomes, or from whole-genome duplication (polyploidy). The most common fate is that one member of the pair is deleted to return the gene to the singleton state. Other paths involve the reduced expression of both copies (hypofunctionalization) that are held in duplicate to maintain sufficient quantity of function. The two copies can split functions (subfunctionalization) or can diverge to generate a new function (neofunctionalization). Retention of duplicates resulting from doubling of the whole genome occurs for genes involved with multicomponent interactions such as transcription factors and signal transduction components. In contrast, these classes of genes are underrepresented in small segmental duplications. This complementary pattern suggests that the balance of interactors affects the fate of the duplicate pair. We discuss the different mechanisms that maintain duplicated genes, which may change over time and intersect.

Effect of aneuploidy of a non-essential chromosome on gene expression in maize.

The non-essential supernumerary maize (Zea mays) B chromosome (B) has recently been shown to contain active genes and to be capable of impacting gene expression of the A chromosomes. However, the effect of the B chromosome on gene expression is still unclear. In addition, it is unknown whether the accumulation of the B chromosome has a cumulative effect on gene expression. To examine these questions, the global expression of genes, microRNAs (miRNAs), and transposable elements (TEs) of leaf tissue of maize W22 plants with 0-7 copies of the B chromosome was studied. All experimental genotypes with B chromosomes displayed a trend of upregulated gene expression for a subset of A-located genes compared to the control. Over 3000 A-located genes are significantly differentially expressed in all experimental genotypes with the B chromosome relative to the control. Modulations of these genes are largely determined by the presence rather than the copy number of the B chromosome. By contrast, the expression of most B-located genes is positively correlated with B copy number, showing a proportional gene dosage effect. The B chromosome also causes increased expression of A-located miRNAs. Differentially expressed miRNAs potentially regulate their targets in a cascade of effects. Furthermore, the varied copy number of the B chromosome leads to the differential expression of A-located and B-located TEs. The findings provide novel insights into the function and properties of the B chromosome.

Gene-dosage issues: a recurrent theme in whole genome duplication events.

Two recent studies have addressed the long-term consequences of whole genome duplications (WGD). Specifically, they analyzed transcriptomes of the plant Arabidopsis thaliana and of four salmonids to assess the impact of WGD on gene expression. These studies point to commonalities in gene expression adjustments after polyploidization that we outline and discuss below.

Prospects and challenges of epigenomics in crop improvement.

BACKGROUND: The advent of high-throughput epigenome mapping techniques has ushered in a new era of multiomics with powerful tools now available to map and record genomic output at different levels. Integrating the different components of the epigenome from these multiomics measures allows investigations of cis-regulatory elements on a genome-scale. Mapping of chromatin state, chromatin accessibility dynamics, and higher-order chromatin structure enables a new level of understanding of cell fate determination, identity and function in normal growth and development, disease resistance, and yield. OBJECTIVE: In this paper, the recent advances in epigenomics research of rice, maize, and wheat are reviewed, and the development trends of epigenomics of major crops in the coming years are projected. METHODS: We highlight the role of epigenomics in regulating growth and development and identifying potential distal cis-regulatory elements in three major crops, and discuss the prospects and challenges for new epigenetics-mediated breeding technologies in crop improvement. CONCLUSION: In this review, we summarize and analyze recent epigenomic advances in three major crops epigenomics and discuss possibilities and challenges for future research in the field.

The supernumerary B chromosome of maize: drive and genomic conflict.

The supernumerary B chromosome of maize is dispensable, containing no vital genes, and thus is variable in number and presence in lines of maize. In order to be maintained in populations, it has a drive mechanism consisting of nondisjunction at the pollen mitosis that produces the two sperm cells, and then the sperm with the two B chromosomes has a preference for fertilizing the egg as opposed to the central cell in the process of double fertilization. The sequence of the B chromosome coupled with B chromosomal aberrations has localized features involved with nondisjunction and preferential fertilization, which are present at the centromeric region. The predicted genes from the sequence have paralogues dispersed across all A chromosomes and have widely different divergence times suggesting that they have transposed to the B chromosome over evolutionary time followed by degradation or have been co-opted for the selfish functions of the supernumerary chromosome.

One Hundred Years of Gene Balance: How Stoichiometric Issues Affect Gene Expression, Genome Evolution, and Quantitative Traits.

A century ago experiments with the flowering plant Datura stramonium and the fruit fly Drosophila melanogaster revealed that adding an extra chromosome to a karyotype was much more detrimental than adding a whole set of chromosomes. This phenomenon was referred to as gene balance and has been recapitulated across eukaryotic species. Here, we retrace some developments in this field. Molecular studies suggest that the basis of balance involves stoichiometric relationships of multi-component interactions. This concept has implication for the mechanisms controlling gene expression, genome evolution, sex chromosome evolution/dosage compensation, speciation mechanisms, and the underlying genetics of quantitative traits.

De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays).

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.