Fangpu Han

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.

Construction and behavior of engineered minichromosomes in maize

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.

Reactivation of an Inactive Centromere Reveals Epigenetic and Structural Components for Centromere Specification in Maize

Stable maize (Zea mays) chromosomes were recovered from an unstable dicentric containing large and small versions of the B chromosome centromere. In the stable chromosome, the smaller centromere had become inactivated. This inactive centromere can be inherited from one generation to the next attached to the active version and loses all known cytological and molecular properties of active centromeres. When separated from the active centromere by intrachromosomal recombination, the inactive centromere can be reactivated. The reactivated centromere regains the molecular attributes of activity in anaphase I of meiosis. When two copies of the dicentric chromosome with one active and one inactive centromere are present, homologous chromosome pairing reduces the frequency of intrachromosomal recombination and thus decreases, but does not eliminate, the reactivation of inactive centromeres. These findings indicate an epigenetic component to centromere specification in that centromere inactivation can be directed by joining two centromeres in opposition. These findings also indicate a structural aspect to centromere specification revealed by the gain of activity at the site of the previously inactive sequences.

Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb

We have reported previously that introgression by Zizania latifolia resulted in extensive DNA methylation changes in the recipient rice genome, as detected by a set of pre-selected DNA segments. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines relative to their rice parent at ∼2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as inter-conversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology. Possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding are discussed.

Telomere-mediated chromosomal truncation in maize

Direct repeats of Arabidopsis telomeric sequence were constructed to test telomere-mediated chromosomal truncation in maize. Two constructs with 2.6 kb of telomeric sequence were used to transform maize immature embryos by Agrobacterium-mediated transformation. One hundred seventy-six transgenic lines were recovered in which 231 transgene loci were revealed by a FISH analysis. To analyze chromosomal truncations that result in transgenes located near chromosomal termini, Southern hybridization analyses were performed. A pattern of smear in truncated lines was seen as compared with discrete bands for internal integrations, because telomeres in different cells are elongated differently by telomerase. When multiple restriction enzymes were used to map the transgene positions, the size of the smears shifted in accordance with the locations of restriction sites on the construct. This result demonstrated that the transgene was present at the end of the chromosome immediately before the integrated telomere sequence. Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends. These results demonstrate that telomere-mediated chromosomal truncation operates in plant species. This technology will be useful for chromosomal engineering in maize as well as other plant species.

Persistent whole-chromosome aneuploidy is generally associated with nascent allohexaploid wheat

Allopolyploidization has been a driving force in plant evolution. Formation of common wheat (Triticum aestivum L.) represents a classic example of successful speciation via allopolyploidy. Nevertheless, the immediate chromosomal consequences of allopolyploidization in wheat remain largely unexplored. We report here an in-depth investigation on transgenerational chromosomal variation in resynthesized allohexaploid wheats that are identical in genome constitution to common wheat. We deployed sequential FISH, genomic in situ hybridization (GISH), and homeolog-specific pyrosequencing, which enabled unequivocal identification of each of the 21 homologous chromosome pairs in each of >1,000 individual plants from 16 independent lines. We report that whole-chromosome aneuploidy occurred ubiquitously in early generations (from selfed generation S1 to >S20) of wheat allohexaploidy although at highly variable frequencies (20–100%). In contrast, other types of gross structural variations were scant. Aneuploidy included an unexpected hidden type, which had a euploid chromosome number of 2n = 42 but with simultaneous loss and gain of nonhomeologous chromosomes. Of the three constituent subgenomes, B showed the most lability for aneuploidy, followed by A, but the recently added D subgenome was largely stable in most of the studied lines. Chromosome loss and gain were also unequal across the 21 homologous chromosome pairs. Pedigree analysis showed no evidence for progressive karyotype stabilization even with multigenerational selection for euploidy. Profiling of two traits directly related to reproductive fitness showed that although pollen viability was generally reduced by aneuploidy, the adverse effect of aneuploidy on seed-set is dependent on both aneuploidy type and synthetic line.

Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines

BackgroundAlteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.ResultsMulti-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.ConclusionsOur results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.

Minichromosome Analysis of Chromosome Pairing, Disjunction, and Sister Chromatid Cohesion in Maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.

Extensive and Heritable Epigenetic Remodeling and Genetic Stability Accompany Allohexaploidization of Wheat

Allopolyploidy has played a prominent role in organismal evolution, particularly in angiosperms. Allohexaploidization is a critical step leading to the formation of common wheat as a new species, Triticum aestivum, as well as for bestowing its remarkable adaptability. A recent study documented that the initial stages of wheat allohexaploidization was associated with rampant genetic and epigenetic instabilities at genomic regions flanking a retrotransposon family named Veju. Although this finding is in line with the prevailing opinion of rapid genomic instability associated with nascent plant allopolyploidy, its relevance to speciation of T. aestivum remains unclear. Here, we show that genetic instability at genomic regions flanking the Veju, flanking a more abundant retroelement BARE-1, as well as at a large number of randomly sampled genomic loci, is all extremely rare or nonexistent in preselected individuals representing three sets of independently formed nascent allohexaploid wheat lines, which had a transgenerationally stable genomic constitution analogous to that of T. aestivum. In contrast, extensive and transgenerationally heritable repatterning of DNA methylation at all three kinds of genomic loci were reproducibly detected. Thus, our results suggest that rampant genetic instability associated with nascent allohexaploidization in wheat likely represents incidental and anomalous phenomena that are confined to by-product individuals inconsequential to the establishment of the newly formed plants toward speciation of T. aestivum; instead, extensive and heritable epigenetic remodeling coupled with preponderant genetic stability is generally associated with nascent wheat allohexaploidy, and therefore, more likely a contributory factor to the speciation event(s).

De novo centromere formation on a chromosome fragment in maize

The centromere is the part of the chromosome that organizes the kinetochore, which mediates chromosome movement during mitosis and meiosis. A small fragment from chromosome 3, named Duplication 3a (Dp3a), was described from UV-irradiated materials by Stadler and Roman in the 1940s [Stadler LJ, Roman H (1948) Genetics 33(3):273–303]. The genetic behavior of Dp3a is reminiscent of a ring chromosome, but fluoresecent in situ hybridization detected telomeres at both ends, suggesting a linear structure. This small chromosome has no detectable canonical centromeric sequences, but contains a site with protein features of functional centromeres such as CENH3, the centromere specific H3 histone variant, and CENP-C, a foundational kinetochore protein, suggesting the de novo formation of a centromere on the chromatin fragment. To examine the sequences associated with CENH3, chromatin immunoprecipitation was carried out with anti-CENH3 antibodies using material from young seedlings with and without the Dp3a chromosome. A novel peak was detected from the ChIP-Sequencing reads of the Dp3a sample. The peak spanned 350 kb within the long arm of chromosome 3 covering 22 genes. Collectively, these results define the behavior and molecular features of de novo centromere formation in the Dp3a chromosome, which may shed light on the initiation of new centromere sites during evolution.