Defining autopolyploidy: Cytology, genetics, and taxonomy.

Autopolyploidy is taxonomically defined as the presence of more than two copies of each genome within an organism or species, where the genomes present must all originate within the same species. Alternatively, "genetic" or "cytological" autopolyploidy is defined by polysomic inheritance: random pairing and segregation of the four (or more) homologous chromosomes present, with no preferential pairing partners. In this review, we provide an overview of methods used to categorize species as taxonomic and cytological autopolyploids, including both modern and obsolete cytological methods, marker-segregation-based and genomics methods. Subsequently, we also investigated how frequently polysomic inheritance has been reliably documented in autopolyploids. Pure or predominantly polysomic inheritance was documented in 39 of 43 putative autopolyploid species where inheritance data was available (91%) and in seven of eight synthetic autopolyploids, with several cases of more mixed inheritance within species. We found no clear cases of autopolyploids with disomic inheritance, which was likely a function of our search methodology. Interestingly, we found seven species with purely polysomic inheritance and another five species with partial or predominant polysomic inheritance that appear to be taxonomic allopolyploids. Our results suggest that observations of polysomic inheritance can lead to relabeling of taxonomically allopolyploid species as autopolyploid and highlight the need for further cytogenetic and genomic investigation into polyploid origins and inheritance types.

Conservation and trans-regulation of histone modification in the A and B subgenomes of polyploid wheat during domestication and ploidy transition.

BACKGROUND: Polyploidy has played a prominent role in the evolution of plants and many other eukaryotic lineages. However, how polyploid genomes adapt to the abrupt presence of two or more sets of chromosomes via genome regulation remains poorly understood. Here, we analyzed genome-wide histone modification and gene expression profiles in relation to domestication and ploidy transition in the A and B subgenomes of polyploid wheat. RESULTS: We found that epigenetic modification patterns by two typical euchromatin histone markers, H3K4me3 and H3K27me3, for the great majority of homoeologous triad genes in A and B subgenomes were highly conserved between wild and domesticated tetraploid wheats and remained stable in the process of ploidy transitions from hexaploid to extracted tetraploid and then back to resynthesized hexaploid. However, a subset of genes was differentially modified during tetraploid and hexaploid wheat domestication and in response to ploidy transitions, and these genes were enriched for particular gene ontology (GO) terms. The extracted tetraploid wheat manifested higher overall histone modification levels than its hexaploid donor, and which were reversible and restored to normal levels in the resynthesized hexaploid. Further, while H3K4me3 marks were distally distributed along each chromosome and significantly correlated with subgenome expression as expected, H3K27me3 marks showed only a weak distal bias and did not show a significant correlation with gene expression. CONCLUSIONS: Our results reveal overall high stability of histone modification patterns in the A and B subgenomes of polyploid wheat during domestication and in the process of ploidy transitions. However, modification levels of a subset of functionally relevant genes in the A and B genomes were trans-regulated by the D genome in hexaploid wheat.

Extensive allele-level remodeling of histone methylation modification in reciprocal F1 hybrids of rice subspecies.

Epigenetic mechanisms play a major role in heterosis, partly as a result of the remodeling of epigenetic modifications in F1 hybrids. Based on chromatin immunoprecipitation-sequencing (ChIP-Seq) analyses, we show that at the allele level extensive histone methylation remodeling occurred for a subset of genomic loci in reciprocal F1 hybrids of Oryza sativa (rice) cultivars Nipponbare and 93-11, representing the two subspecies japonica and indica. Globally, the allele modification-altered loci in leaf or root of the reciprocal F1 hybrids involved ˜12-43% or more of the genomic regions carrying either of two typical histone methylation markers, H3K4me3 (>21 000 genomic regions) and H3K27me3 (>11 000 genomic regions). Nevertheless, at the total modification level, the majority (from ˜43 to >90%) of the modification-altered alleles lay within the range of parental additivity in the hybrids because of concerted alteration in opposite directions, consistent with an overall attenuation of allelic differences in the modifications. Importantly, of the genomic regions that did show non-additivity in total modification level by either marker in the two tissues of hybrids, >80% manifested transgressivity, which involved genes enriched in specific functional categories. Extensive allele-level alteration of H3K4me3 alone was positively correlated with genome-wide changes in allele-level gene expression, whereas at the total level, both H3K4me3 and H3K27me3 remodeling, although affecting just a small number of genes, contributes to the overall non-additive gene expression to variable extents, depending on tissue/marker combinations. Our results emphasize the importance of allele-level analysis in hybrids to assess the remodeling of epigenetic modifications and their relation to changes in gene expression.

Dynamic chromatin changes associated with de novo centromere formation in maize euchromatin.

The inheritance and function of centromeres are not strictly dependent on any specific DNA sequence, but involve an epigenetic component in most species. CENH3, a centromere histone H3 variant, is one of the best-described epigenetic factors in centromere identity, but the chromatin features required during centromere formation have not yet been revealed. We previously identified two de novo centromeres on Zea mays (maize) minichromosomes derived from euchromatic sites with high-density gene distributions but low-density transposon distributions. The distribution of gene location and gene expression in these sites indicates that transcriptionally active regions can initiate de novo centromere formation, and CENH3 seeding shows a preference for gene-free regions or regions with no gene expression. The locations of the expressed genes detected were at relatively hypomethylated loci, and the altered gene expression resulted from de novo centromere formation, but not from the additional copy of the minichromosome. The initial overall DNA methylation level of the two de novo regions was at a low level, but increased substantially to that of native centromeres after centromere formation. These results illustrate the dynamic chromatin changes during euchromatin-originated de novo centromere formation, which provides insight into the mechanism of de novo centromere formation and regulation of subsequent consequences.

Heritable alteration of DNA methylation induced by whole-chromosome aneuploidy in wheat.

Aneuploidy causes changes in gene expression and phenotypes in all organisms studied. A previous study in the model plant Arabidopsis thaliana showed that aneuploidy-generated phenotypic changes can be inherited to euploid progenies and implicated an epigenetic underpinning of the heritable variations. Based on an analysis by amplified fragment length polymorphism and methylation-sensitive amplified fragment length polymorphism markers, we found that although genetic changes at the nucleotide sequence level were negligible, extensive changes in cytosine DNA methylation patterns occurred in all studied homeologous group 1 whole-chromosome aneuploid lines of common wheat (Triticum aestivum), with monosomic 1A showing the greatest amount of methylation changes. The changed methylation patterns were inherited by euploid progenies derived from the aneuploid parents. The aneuploidy-induced DNA methylation alterations and their heritability were verified at selected loci by bisulfite sequencing. Our data have provided empirical evidence supporting earlier suggestions that heritability of aneuploidy-generated, but aneuploidy-independent, phenotypic variations may have an epigenetic basis. That at least one type of aneuploidy - monosomic 1A - was able to cause significant epigenetic divergence of the aneuploid plants and their euploid progenies also lends support to recent suggestions that aneuploidy may have played an important and protracted role in polyploid genome evolution.

Formation of a functional maize centromere after loss of centromeric sequences and gain of ectopic sequences.

The maize (Zea mays) B centromere is composed of B centromere-specific repeats (ZmBs), centromere-specific satellite repeats (CentC), and centromeric retrotransposons of maize (CRM). Here we describe a newly formed B centromere in maize, which has lost CentC sequences and has dramatically reduced CRM and ZmBs sequences, but still retains the molecular features of functional centromeres, such as CENH3, H2A phosphorylation at Thr-133, H3 phosphorylation at Ser-10, and Thr-3 immunostaining signals. This new centromere is stable and can be transmitted to offspring through meiosis. Anti-CENH3 chromatin immunoprecipitation sequencing revealed that a 723-kb region from the short arm of chromosome 9 (9S) was involved in the formation of the new centromere. The 723-kb region, which is gene poor and enriched for transposons, contains two abundant DNA motifs. Genes in the new centromere region are still transcribed. The original 723-kb region showed a higher DNA methylation level compared with native centromeres but was not significantly changed when it was involved in new centromere formation. Our results indicate that functional centromeres may be formed without the known centromere-specific sequences, yet the maintenance of a high DNA methylation level seems to be crucial for the proper function of a new centromere.

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.

Characterization of two CENH3 genes and their roles in wheat evolution.

Wheat evolution is complex as a result of successive rounds of allopolyploidization and continuous selection during domestication. Diploid and tetraploid wheat species (Triticum spp.) were used as model systems in which to study the role of centromere-specific histone H3 variant (CENH3) in wheat evolution. We characterized two types of CENH3 genes, named αCENH3 and ßCENH3, each of which has three slightly different copies derived from the AA, BB and DD genomes. Specific antibodies were raised against the two CENH3 proteins and were co-localized to centromeres with subtle differences. In most tetraploid wheat species, CENH3 genes are more highly expressed from the AA genome. In wild tetraploids, ßCENH3 has a much lower expression level than αCENH3, while in cultivated tetraploids ßCENH3 transcripts are enhanced to near αCENH3 levels. Comparison of the CENH3 proteins in wild and cultivated tetraploids revealed that the histone folding domain (HFD) of only ßCENH3 is under positive selection, especially in the region responsible for targeting of CENH3 to the centromere. Taken together, positive selection of ßCENH3 and its increased expression in tetraploid cultivars are indicative of adaptive evolution. Furthermore, the differences in localization between αCENH3 and ßCENH3 observed using fiber fluorescence in situ hybridization (FISH) and immunodetection and in developmental phenotypes resulting from virus-reduced gene silencing imply their functional diversification during wheat evolution.

Molecular cytogenetic characterization of a new wheat-rye 4R chromosome translocation line resistant to powdery mildew.

Rye is an important and valuable gene resource for wheat improvement. However, due to extensive growing of cultivars with disease resistance genes from short arm of rye chromosome 1R and coevolution of pathogen virulence and host resistance, these cultivars successively lost resistance to pathogens. Identification and deployment of new resistance gene sources in rye are, therefore, of especial importance and urgency. A new wheat-rye line, designated as WR41-1, was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. It was proved to be a new wheat-rye T4BL·4RL and T7AS·4RS translocation line using sequential genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and expressed sequence tag-simple sequence repeat (EST-SSR) marker analysis. WR41-1 showed high levels of resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 13 of 23 Bgt isolates tested at the seedling stage. According to its resistant pattern to 23 different Bgt isolates, WR41-1 may possess new gene(s) for resistance to powdery mildew, which differed from previously identified and known powdery mildew genes from rye (Pm7, Pm8, Pm17, and Pm20). In addition, WR41-1 was cytologically stable, had a desirable fertility, and is expected to be useful in wheat improvement.

Genetic factors inherited from both diploid parents interact to affect genome stability and fertility in resynthesized allotetraploid Brassica napus.

Established allopolyploids are known to be genomically stable and fertile. However, in contrast, most newly resynthesized allopolyploids are infertile and meiotically unstable. Identifying the genetic factors responsible for genome stability in newly formed allopolyploid is key to understanding how 2 genomes come together to form a species. One hypothesis is that established allopolyploids may have inherited specific alleles from their diploid progenitors which conferred meiotic stability. Resynthesized Brassica napus lines are often unstable and infertile, unlike B. napus cultivars. We tested this hypothesis by characterizing 41 resynthesized B. napus lines produced by crosses between 8 Brassica rapa and 8 Brassica oleracea lines for copy number variation resulting from nonhomologous recombination events and fertility. We resequenced 8 B. rapa and 5 B. oleracea parent accessions and analyzed 19 resynthesized lines for allelic variation in a list of meiosis gene homologs. SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 3 individuals per line. Self-pollinated seed set and genome stability (number of copy number variants) were significantly affected by the interaction between both B. rapa and B. oleracea parental genotypes. We identified 13 putative meiosis gene candidates which were significantly associated with frequency of copy number variants and which contained putatively harmful mutations in meiosis gene haplotypes for further investigation. Our results support the hypothesis that allelic variants inherited from parental genotypes affect genome stability and fertility in resynthesized rapeseed.

Assessing Diversity in the Camelina Genus Provides Insights into the Genome Structure of Camelina sativa.

Camelina sativa (L.) Crantz an oilseed crop of the Brassicaceae family is gaining attention due to its potential as a source of high value oil for food, feed or fuel. The hexaploid domesticated C. sativa has limited genetic diversity, encouraging the exploration of related species for novel allelic variation for traits of interest. The current study utilized genotyping by sequencing to characterize 193 Camelina accessions belonging to seven different species collected primarily from the Ukrainian-Russian region and Eastern Europe. Population analyses among Camelina accessions with a 2n = 40 karyotype identified three subpopulations, two composed of domesticated C. sativa and one of C. microcarpa species. Winter type Camelina lines were identified as admixtures of C. sativa and C. microcarpa Eighteen genotypes of related C. microcarpa unexpectedly shared only two subgenomes with C. sativa, suggesting a novel or cryptic sub-species of C. microcarpa with 19 haploid chromosomes. One C. microcarpa accession (2n = 26) was found to comprise the first two subgenomes of C. sativa suggesting a tetraploid structure. The defined chromosome series among C. microcarpa germplasm, including the newly designated C. neglecta diploid née C. microcarpa, suggested an evolutionary trajectory for the formation of the C. sativa hexaploid genome and re-defined the underlying subgenome structure of the reference genome.

The Capacity to Buffer and Sustain Imbalanced D-Subgenome Chromosomes by the BBAA Component of Hexaploid Wheat Is an Evolved Dominant Trait.

Successful generation of pentaploid wheat (genome, BBAAD) via interspecific hybridization between tetraploid wheat (BBAA) and hexaploid wheat (BBAADD) holds great promise to mutually exchange desirable traits between the two cultivated wheat species, as well as providing a novel facet for evolutionary studies of polyploid wheat. Taking advantage of the viable and fertile nature of an extracted tetraploid wheat (ETW) with a BBAA genome that is virtually identical with the BBAA component of a hexaploid common wheat, and a synthetic hexaploid wheat, we constructed four pentaploid wheats with several distinct yet complementary features, of which harboring homozygous BBAA subgenomes is a common feature. By using a combined FISH/GISH method that enables diagnosing all individual wheat chromosomes, we precisely karyotyped a larger number of cohorts from the immediate progenies of each of the four pentaploid wheats. We found that the BBAA component of hexaploid common wheat possesses a significantly stronger capacity to buffer and sustain imbalanced D genome chromosomes and appears to harbor more structural chromosome variations than the BBAA genome of tetraploid wheat. We also document that this stronger capacity of the hexaploid BBAA subgenomes behaves as a genetically controlled dominant trait. Our findings bear implications to the known greater than expected level of genetic diversity in, and the remarkable adaptability of, hexaploid common wheat as a staple crop of global significance, as well as in using pentaploidy as intermediates for reciprocal introgression of useful traits between tetraploid and hexaploid wheat cultivars.

Site-specific transfer of chromosomal segments and genes in wheat engineered chromosomes.

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited. Here, telomere-mediated truncation was successfully performed in common wheat (Triticum aestivum) to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.

Molecular cytogenetic characterization and stem rust resistance of five wheat-Thinopyrum ponticum partial amphiploids.

Partial amphiploids created by crossing common wheat (Triticum aestivum L.) and Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey are important intermediates in wheat breeding because of their resistance to major wheat diseases. In this study, we examined the chromosome compositions of five Xiaoyan-series wheat-Th. ponticum partial amphiploids (Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, and Xiaoyan 7631) using GISH, multicolor-GISH, and multicolor-FISH. We found several chromosome changes in these lines. For example, wheat chromosomes 1B and 2B were added in Xiaoyan 68 and Xiaoyan 7430, respectively, while wheat chromosome 6B was eliminated from Xiaoyan 693 and Xiaoyan 7631. Chromosome rearrangements were also detected in these amphiploids, including an interspecific translocation involving chromosome 4D and some intergenomic translocations, such as A-B and A-D translocations, among wheat genomes. Analysis of the Th. ponticum chromosomes in the amphiploids showed that some lines shared the same alien chromosomes. We also evaluated these partial amphiploids for resistance to nine races of stem rust, including TTKSK (commonly known as Ug99). Three lines, Xiaoyan 68, Xiaoyan 784, and Xiaoyan 7430, exhibited excellent resistance to all nine races, and could therefore be valuable sources of stem rust resistance in wheat breeding.

Alteration of terminal heterochromatin and chromosome rearrangements in derivatives of wheat-rye hybrids.

Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres. Furthermore, a mitotically unstable dicentric chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese Spring-Imperial rye 3R addition line. An unstable multicentric chromosome was found in the progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including movement and disappearance of terminal heterochromatin occurred in the progeny of wheat-rye addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were observed in the progeny of a monosomic 4R addition line, a ditelosomic 5RS addition line and a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric DNA (TTTAGGG)n, derived from a monosomic 4R addition line are stable and transmissible to the next generation. The results indicated that centromeres and terminal heterochromatin can be profoundly altered in wheat-rye hybrid derivatives.

Molecular cytogenetic characterization of wheat--Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight.

Thinopyrum elongatum (2n=2x=14, EE), a wild relative of wheat, has been suggested as a potentially novel source of resistance to several major wheat diseases including Fusarium Head Blight (FHB). In this study, a series of wheat (cv. Chinese Spring, CS) substitution and ditelosomic lines, including Th. elongatum additions, were assessed for Type II resistance to FHB. Results indicated that the lines containing chromosome 7E of Th. elongatum gave a high level of resistance to FHB, wherein the infection did not spread beyond the inoculated floret. Furthermore, it was determined that the novel resistance gene(s) of 7E was located on the short-arm (7ES) based on sharp difference in FHB resistance between the two 7E ditelosomic lines for each arm. On the other hand, Th. elongatum chromosomes 5E and 6E likely contain gene(s) for susceptibility to FHB because the disease spreads rapidly within the inoculated spikes of these lines. Genomic in situ hybridization (GISH) analysis revealed that the alien chromosomes in the addition and substitution lines were intact, and the lines did not contain discernible genomic aberrations. GISH and multicolor-GISH analyses were further performed on three translocation lines that also showed high levels of resistance to FHB. Lines TA3499 and TA3695 were shown to contain one pair of wheat-Th. elongatum translocated chromosomes involving fragments of 7D plus a segment of the 7E, while line TA3493 was found to contain one pair of wheat-Th. elongatum translocated chromosomes involving the D- and A-genome chromosomes of wheat. Thus, this study has established that the short-arm of chromosome 7E of Th. elongatum harbors gene(s) highly resistant to the spreading of FHB, and chromatin of 7E introgressed into wheat chromosomes largely retained the resistance, implicating the feasibility of using these lines as novel material for breeding FHB-resistant wheat cultivars.