Polyploid Plant Species Research Articles

Expression profiling reveals functionally redundant multiple-copy genes related to zinc, iron and cadmium responses in Brassica rapa.

Genes underlying environmental adaptability tend to be over-retained in polyploid plant species. Zinc deficiency (ZnD) and iron deficiency (FeD), excess Zn (ZnE) and cadmium exposure (CdE) are major environmental problems for crop cultivation, but little is known about the differential expression of duplicated genes upon these stress conditions. Applying Tag-Seq technology to leaves of Brassica rapa grown under FeD, ZnD, ZnE or CdE conditions, with normal conditions as a control, we examined global gene expression changes and compared the expression patterns of multiple paralogs. We identified 812, 543, 331 and 447 differentially expressed genes under FeD, ZnD, ZnE and CdE conditions, respectively, in B.rapa leaves. Genes involved in regulatory networks centered on the transcription factors bHLH038 or bHLH100 were differentially expressed under (ZnE-induced) FeD. Further analysis revealed that genes associated with Zn, Fe and Cd responses tended to be over-retained in the B.rapa genome. Most of these multiple-copy genes showed the same direction of expression change under stress conditions. We conclude that the duplicated genes involved in trace element responses in B.rapa are functionally redundant, making the regulatory network more complex in B.rapa than in Arabidopsis thaliana.

Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.

The phylogeographical and population genetic approach to the investigation of the genetic diversity patterns in self-incompatible clonal and polyploidLinnaea borealissubsp.borealis

Surveys of genetic diversity patterns of self-incompatible clonal polyploid plant species are still scarcer than those of diploid plant species. Therefore, I studied the phylogeographical history of Linnaea borealis subsp. borealis to shed light on the colonization history of this clonal self-incompatible polyploid plant in Eurasia using selected regions of plastid DNA and genetic diversity patterns of 22 populations of this species employing AFLP markers. I also addressed the question of whether the genetic diversity patterns in L. borealis subsp. borealis in Eurasia are similar to those of earlier published studies of clonal self-incompatible diploid or polyploid plants. This survey revealed that the shallow phylogeographical history (six plastid haplotypes forming one haplogroup, 100% bootstrap support) and moderate genome-wide diversity estimated using AFLP markers (Fragpoly = 10.8–38.9%, I = 0.060–0.180, FST = 0.289) were general characteristics of L. borealis subsp. borealis in its Eurasian range. The sampling strategy, in most cases at 1–2-m or even 3–5-m intervals, showed that a balance between vegetative and sexual reproduction and limited pollen dispersal among compatible mates can be important for genetic diversity patterns in populations of this taxon. Despite the fact that one-half of the investigated populations were strongly isolated, they still preserved similar levels of genetic diversity across the geographical range. I found no support for the hypothesis that a bottleneck and/or inbreeding had accompanied habitat fragmentation as factors shaping genetic diversity. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 64–76.

The Application of Reverse Genetics to Polyploid Plant Species

Polyploidy events (polyploidization) followed by progressive loss of redundant genome components are a major feature of plant evolution, with new evidence suggesting that all flowering plants possess ancestral genome duplications. Furthermore, many of our most important crop plants have undergone additional, relatively recent, genome duplication events. Recent advances in DNA sequencing have made vast amounts of new genomic data available for many plants, including a range of important crop species with highly duplicated genomes. Along with assisting traditional forward genetics approaches to study gene function, this wealth of new sequence data facilitates extensive reverse genetics-based functional analyses. However, plants featuring high levels of genome duplication as a result of recent polyploidization pose additional challenges for reverse genetic analysis. Here we review reverse genetic analysis in such polyploid plants and highlight key challenges.

Genetic diversity and multiple origins of polyploid Atriplex nummularia Lindl. (Chenopodiaceae)

Few studies have described the genetic diversity within and between populations of polyploid plant species despite the general acceptance of the importance of polyploidy in plant diversification and speciation. The genus Atriplex has a complex evolutionary history in Australia that has included polyploidy and hybridization among perennial forms. The octoploid, dioecious species Atriplex nummularia is proposed to have evolved from an octoploid ancestor in the coastal semi-arid fringe of south-western Australia, and to have spread east and diversified into taxa which occupy edaphically different habitats. Despite interest in the diversification of the genus, and the ecological and economic importance of A. nummularia, there are no descriptions of the genetic structure of the species. Nuclear microsatellite markers and principal coordinate analysis, analysis of molecular variance, Bayesian and phenetic analyses were used to investigate the diversity and taxonomic relationships of two common subspecies of A. nummularia. Genetic diversity was high overall (A = 509, A′ = 42.4, Ho = 0.824, H′ = 2.8), but values were significantly lower in the western subspecies, A. nummularia ssp. spathulata. As in other outbreeding, perennial species, most of the genetic diversity was within populations (FST = 0.125). Clear divergence of subspecies was evident in principal coordinate analysis, neighbor-joining and Bayesian clustering analyses and the differentiation of populations was very low within subspecies (FSC = 0.048). These findings support the taxonomic separation of the two subspecies. Clustering patterns based on Bayesian analyses suggested that the polyploid subspecies of A. nummularia have multiple origins.

Nuclear and Chloroplast DNA Suggest a Complex Single Origin for the Threatened Allopolyploid Solidago houghtonii (Asteraceae) Involving Reticulate Evolution and Introgression

Abstract Solidago houghtonii Torrey & A. Gray ex Gray is a federally threatened polyploid plant species likely of hybrid origin. Several hypothesized combinations of parental species have been suggested but none have been phylogenetically tested. Additionally, it is unclear whether the species is of single or polytopic origin. To study the evolutionary history of S. houghtonii we sequenced four noncoding cpDNA loci (accD-psaI, psbA-trnH, trnL-trnF, rps16-trnQ), and the ITS and 3′ETS regions for four accessions of S. houghtonii, which span its geographic range, and 25 other species of Solidago including all sympatric species. Polymorphisms within the direct nrDNA sequences of all S. houghtonii accessions indicated the presence of multiple homoeologue types. These were separated by molecular cloning of the 3055 bp 3′ETS — ITS region, allowing us to positively link the ETS and ITS homoeologue types. Phylogenetic analyses of the nuclear and chloroplast datasets revealed incongruent topologies. Analysis of clo...

Evolution and polyploid origins in North American ArcticPuccinellia(Poaceae) based on nuclear ribosomal spacer and chloroplast DNA sequences

The proportion of polyploid plant species increases at higher latitudes, and it has been suggested that original postglacial Arctic immigrants of some large groups, including grasses, were polyploid. We analyzed noncoding nuclear and chloroplast DNA of all North American diploid Puccinellia (Poaceae) and a subset of arctic polyploids to hypothesize evolutionary relationships among diploids and to evaluate the parentage of polyploids. Diploids formed three lineages: one uniting arctic species P. arctica and P. banksiensis; a second comprising arctic species P. tenella, P. alaskana, P. vahliana, and P. wrightii; and a third uniting the two temperate species P. lemmonii and P. parishii. The arctic species P. angustata (hexaploid) and P. andersonii (primarily octoploid) apparently derive from the P. arctica-P. banksiensis lineage based on ITS and chloroplast sequences, and share an ancestor with arctic triploid/tetraploid P. phryganodes based on nrDNA sequences. Sequence comparisons also suggest tetraploid P. bruggemannii evolved from two arctic lineages: P. vahliana-P. wrightii and P. arctica-P. banksiensis. These patterns and the predominance of arctic rather than temperate diploid species support the idea that diploid Puccinellia recolonized the Arctic from northern glacial refugia like Beringia, and also formed stabilized polyploid hybrids during these refugial events or subsequently during postglacial colonization.

A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

BackgroundMutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.ResultsTo facilitate the screening for specific homoeologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homoeologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homoeologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homoeologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat lines generated by heavy ion irradiation, we detected multiple mutants with deletions of each TaPFT1 homoeologue, and confirmed these deletions using a CAPS method. We have subsequently designed, optimized, and applied this method for the screening of homoeologous deletions of three additional wheat genes putatively involved in plant disease resistance.ConclusionsWe have developed a method for automated, high-throughput screening to identify deletions of individual homoeologues of a wheat gene. This method is also potentially applicable to other polyploidy plants.

Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

BackgroundThe presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.ResultsThe potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.ConclusionsThe negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 x Q165). The sequencing yielded 96,755 (IJ76-514) and 86,241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP--an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent--with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

Coordinated and Fine-Scale Control of Homoeologous Gene Expression in Allotetraploid Cotton

Within polyploid plant species, it has been demonstrated that homoeologous genes (genes duplicated by polyploidy) often display dynamic expression patterns. To determine if chromosomal location plays a role in establishing these expression patterns, we analyzed the relative levels of homoeolog expression among linked genes from 2 locations in the cotton genome. Genes from the region containing the alcohol dehydrogenase A gene show coordinated expression across several tissues, whereas genes from the region containing cellulose synthase A do not. These results indicate that changes in homoeolog expression may be constrained by linkage in some genomic regions, whereas in other regions, homoeolog expression is largely decoupled from physical proximity. Furthermore, these results suggest that both large- and small-scale regulatory mechanisms may control homoeolog expression patterns.

Oxygen requirement of root meristems in diploid and autotetraploid rye

The oxygen requirement of the root meristems of diploid and tetraploid rye seedlings has been determined. It was found that the polyploid seedlings require 40–50 per cent higher oxygen concentration at the root surface than the diploid seedlings for normal (maximum) respiration in the temperature interval 15–30° C. The critical oxygen concentration, when respiring air atmosphere, occurred at about 22° C in the diploid meristem and at about 15° C in the tetraploid meristem. A hypothesis is presented according to which the radial oxygen diffusion into the root meristems is one of the factors determining the geographical distribution of polyploid plant species.

Homoeologous gene silencing in tissue cultured wheat callus

BackgroundIn contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.ResultsEvidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.ConclusionOur results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

Formation of stable epialleles and their paramutation-like interaction in tetraploid Arabidopsis thaliana.

Polyploidization is found frequently in plants, and species previously considered to be diploid may show remnants of earlier polyploidization events on closer inspection of their genomes. The success of polyploids may lie in increased genetic redundancy supporting subsequent genetic diversification. Although doubling the genome does not generate diversity per se, recent studies show that rapid genomic rearrangements and changes in DNA modification and gene expression patterns are associated with polyploid formation. But recessive modifications will not become phenotypically apparent in early polyploid generations. Here we show that epialleles in tetraploid plants (but not in diploids) interact in trans and lead to heritable gene silencing persisting after segregation from the inactivating allele. This mechanism, resembling paramutation, leads to the establishment of functional epigenetic homozygosity and, thus, to conversion of new recessive alleles into traits expressed in early polyploid generations. Such interactions probably contribute to rapid adaptation and evolution of polyploid plant species.

Genome mapping of polyploid tall fescue (Festuca arundinacea Schreb.) with RFLP markers

Genetic mapping using molecular markers such as restriction fragment length polymorphisms (RFLPs) has become a powerful tool for plant geneticists and breeders. Like many economically important polyploid plant species, detailed genetic studies of hexaploid tall fescue (Festuca arundinacea Schreb.) are complicated, and no genetic map has been established. We report here the first tall fescue genetic map. This map was generated from an F2 population of HD28-56 by 'Kentucky-31' and contains 108 RFLP markers. Although the two parental plants were heterozygous, the perennial and tillering growth habit, high degree of RFLP, and disomic inheritance of tall fescue enabled us to identify the segregating homologous alleles. The map covers 1274 cM on 19 linkage groups with an average of 5 loci per linkage group (LG) and 17.9 cM between loci. Mapping the homoeologous loci detected by the same probe allowed us to identify five homoeologous groups within which the gene orders were found to be generally conserved among homoeologous chromosomes. An exception was homoeologous group 5, in which only 2 of the 3 homoeologous chromosomes were identified. Using 12 genome-specific probes, we were able to assign several linkage groups to one of the three genomes (PG1G2) in tall fescue. All the loci detected by the 11 probes specific to the G1 and/or G2 genomes, with one exception, identified loci located on 4 chromosomes of two homoeologous groups (LG2a, LG2c, LG3a, and LG3c). A P-genome-specific probe was used to map a locus on LG5c. Comparative genome mapping with maize probes indicated that homoeologous group 3 and 2 chromosomes in tall fescue corresponded to maize chromosome 1. Difficulties and advantages of applying RFLP technology in polyploids with high levels of heterozygosity are discussed.

Genomic in situ hybridization reveals the allopolyploid nature ofMilium montianum (Gramineae)

Molecular techniques that “paint” chromosomes offer exciting new opportunities for testing genome relationships.Milium montianum (2n=22) is a grass whose distinctive bimodal karyotype comprises 8 large (L-) and 14 smaller (S-) chromosomes. The proposal thatM. montianum is an allotetraploid, with diploidMilium vernale (2n=8) as the L-chromosome genome donor, has been impossible to confirm by classical means. To test this hypothesis, biotinylated total genomic DNA of diploidM. vernale (2n=8) was hybridized in situ to root tip chromosomes ofM. montianum. TheM. vernale probe hybridized preferentially to all L-chromosomes, but not to the S-chromosomes. These results (i) confirm the allopolyploid nature ofM. montianum, (ii) strongly support the theory that the L-chromosomes ofM. montianum were donated byM. vernale, or a closely related genotype and (iii) show that subsequently the L-chromosomes have largely retained their genomic integrity in the new allopolyploid backgroud. Clearly, genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics. It will often be useful for investigating the origins of wild and cultivated polyploid plant species, especially where conventional methods have failed, for studying introgression, and for understanding the mechanism(s) of origin of bimodal karyotypes.

Assessing the Utility of Isozyme Number for Determining Ploidal Level

In order to evaluate the utility of isozyme number for estimating ploidal level in ancient polyploid (paleopolyploid) plant species, isozyme number was determined for species of the putatively paleopolyploid genus Helianthus with n = 17, and compared with those of a species of Heliomeris with n = 8. Electrophoretic examination of 13 enzymes revealed the presence of nine duplicated isozymes in Helianthus annuus and Helianthus bolanderi and six duplicated isozymes in Heliomeris multiflora. Thus, there is little difference in isozyme number between ploidal levels. It is suggested that the lack of strong concordance between isozyme number and ploidal level may be due to gene silencing in Helianthus and the presence of gene duplications not resulting from polyploidy in both taxa.

GENETIC AND BIOCHEMICAL CONSEQUENCES OF POLYPLOIDY IN TRAGOPOGON.

Most studies of the evolution of polyploid plant species have emphasized phylogenetic issues, for example, identification of diploid progenitors and clarification of polyploid complexes. They have largely utilized evidence from comparative morphology, karyotypes and cytogenetic analysis of interploidal hybrids, as well as biochemical profiles of certain classes of compounds such as flavonoids and seed proteins. Although these studies helped greatly to elucidate the mode of origin and ancestry of many polyploid species, they were not concerned with explaining one of the most intriguing features of polyploidy which is that, in many plant genera, the polyploids are more widely distributed over more habitats than their diploid progenitors. This is a problem of the first rank because at least one-third of the Angiosperms and a higher proportion of the ferns are polyploid. Several recent hypotheses have proposed that the wide capabilities of allopolyploids (we use allotetraploids as an example) are a direct biochemical consequence of their possession of two divergent diploid genomes which provides them with a multiplicity of enzymes relative to both diploid parents as well as a high proportion of novel enzymes (Fincham, 1969; Barber, 1970; Manwell and Baker, 1970). Enzyme multiplicity may extend the range of environments in which normal development can take place and, thereby, might account for the frequently wider distribution of polyploids. This may be true even if the tetraploid as a species is less poly-

Plant density effect on expression of heterosis for yield and its components in wheat and F1 versus F3 yields

The plant density effect on the expression of heterosis for yield and its components in wheat was investigated. It was found that there was no such effect. This was also observed by fellow-researchers. The absence of such a plant density effect might be caused by the buffering capacity of this polyploid plant species. Furthermore it may not hold true for extreme heterotic effects. The yield of the F3 was statistically equal to that of the F1.

The duration of meiosis

A survey of work on meiotic duration in diploid plants shows that the duration is positively correlated with the DNA content per nucleus and with the mitotic cycle time. However, meiotic duration is not solely determined by the DNA content per nucleus but is also affected by chromosomal organization, DNA structure and the developmental pattern of the organism. Thus, in three polyploid plant species meiosis is much shorter and in three animal species it is much longer than would be expected in diploid plant species having corresponding DNA contents. Differences in meiotic duration in plant species are usually the result of proportional differences in all the stages of meiosis. Factors affecting the initiation, control and duration of meiosis are discussed. The consequences of changes in nuclear DNA content on developmental processes and the life cycle in plants are considered. It is suggested that DNA influences development in two ways, first directly through its informational content, and second indirectly by the physical mechanical effects of its mass independent of its informational content.