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Polyploidy occurs in some animals and all flowering plants, including important crops such as wheat. The consequences of polyploidy in crops remain elusive, partly because their progenitors are unknown. Using two resynthesized wheat allotetraploids Sl Sl AA and AADD with known diploid progenitors, we analyzed mRNA and small RNA transcriptomes in the endosperm, compared transcriptomes between endosperm and root in AADD, and examined chromatin changes in the allotetraploids. In the endosperm, there were more non-additively expressed genes in Sl Sl AA than in AADD. In AADD, non-additively expressed genes were developmentally regulated, and the majority (62-70%) were repressed. The repressed genes in AADD included a group of histone methyltransferase gene homologs, which correlated with reduced histone H3K9me2 levels and activation of various transposable elements in AADD. In Sl Sl AA, there was a tendency for expression dominance of Sl over A homoeologs, but the histone methyltransferase gene homologs were additively expressed, correlating with insignificant changes in histone H3K9me2 levels. Moreover, more 24-nucleotide small inferring RNAs (siRNAs) in the A subgenome were disrupted in AADD than in Sl Sl AA, which were associated with expression changes of siRNA-associated genes. Our results indicate that asymmetrical changes in siRNAs, chromatin modifications, transposons and gene expression coincide with unstable AADD genomes and stable Sl Sl AA genomes, which could help explain the evolutionary trajectories of wheat allotetraploids formed by different progenitors.

In plants, many mRNAs and non-coding RNAs are cleaved by RNA-induced silencing complexes. After cleavage, only a limited number of RNAs are processed into trans-acting siRNAs (tasiRNAs). One reason is that 22 nt small RNAs, but not the more common 21 nt small RNAs, can efficiently trigger tasiRNA formation. The characteristics of the target transcripts may also affect tasiRNA production. Here we report the effects of target site location and sequence complementarity on tasiRNA formation. A synthetic sequence that included a miR173 target site and two siRNAs targeting an endogenous mRNA encoding PHYTOENE DESATURASE3 was introduced into a protein-coding (GFP) gene in the coding region or 3' UTR. tasiRNAs were generated in the transgenic seedlings, and the PDS3 mRNA level was reduced, leading to a photobleaching phenotype. It was found that tasiRNAs were most efficiently produced when the miR173 target site was placed immediately after the stop codon. Introducing premature stop codons caused a dramatic reduction of tasiRNAs and over-accumulation of 3' cleavage products, suggesting positive effects of translation on processing the 3' cleavage products into tasiRNAs. By systematically mutating the miR173 target site, we found that perfect complementarity between the 3' end of miR173 and the 5' end of the target sequence was crucial. Mismatches at that position abolished tasiRNA formation, but mismatches at the 5' end of miR173 had less effect. These data suggest important roles for translation and specific sequence complementarity in tasiRNA formation, providing new insights into tasiRNA biogenesis as well as a strategy for improving the efficiency of RNA interference (RNAi) using tasiRNAs.

Two fundamental types of polyploids are known: allopolyploids, in which different parental chromosome sets were combined by ancestral hybridization and duplication; and autopolyploids, which derive from multiplication of the same chromosome set. In autopolyploids, changes to the nuclear environment are not as profound as in allopolyploids, and therefore the effects of genome doubling on gene regulation remain unclear. To investigate the consequences of autopolyploidization per se, we performed a microarray analysis in three equivalent lineages of matched diploids and autotetraploids of Arabidopsis thaliana. Additionally, we compared the expression levels of GFP transgenes driven by endogenous enhancer elements (enhancer traps) in diploids and autotetraploid of 16 transgenic lines. We expected that true ploidy-dependent changes should occur in independently derived autopolyploid lineages. By this criterion, our microarray analysis detected few changes associated with polyploidization, while the enhancer-trap analysis revealed altered GFP expression at multiple plant life stages for 25% of the lines tested. Genes on individual traps were coordinately regulated while endogenous gene expression was not affected except for one line. The unique sensitivity of enhancer traps to ploidy, in contrast to the observed stability of genes, could derive from lower complexity of regulatory pathways acting on traps versus endogenous genes.

Polyploidy is a major evolutionary feature of many plants and some animals (Grant, 1981; Otto & Whitton, 2000). Allopolyploids (e.g. wheat, cotton, and canola) were formed by combination of two or more distinct genomes, whereas autopolyploids (e.g. potato, sugarcane, and banana) resulted from duplication of a single genome. Both allopolyploids and autopolyploids are prevalent in nature (Tate et al., 2004). Recent research has shown that polyploid genomes may undergo rapid changes in genome structure and function via genetic and epigenetic changes (Fig. 1) (Levy & Feldman, 2002; Osborn et al., 2003; Chen, 2007). The former include chromosomal rearrangements (e.g. translocation, deletion, and transposition) and DNA sequence elimination and mutations, whereas epigenetic modifications (chromatin and RNA-mediated pathways) give rise to gene expression changes that are not associated with changes in DNA sequence. Over time, polyploids may become ‘diploidized’ so that they behave like diploids cytogenetically and genetically. Comparative and genome sequence analyses indicate that many plant species, including maize, rice, poplar, and Arabidopsis, are recent or ancient diploidized (paleo-) polyploids. Fig. 1 Diagram of allopolyploid formation and evolution The consequences of polyploidy have been of long-standing interest in genetics, evolution, and systematics (Wendel, 2000; Soltis et al., 2003). Research interest in polyploids has been renewed in the past decade following the discovery of multiple origins and patterns of polyploid formation (Soltis et al., 2003) and rapid genetic changes in resynthesized allotetraploids in Brassica (Song et al., 1995) and wheat (Feldman et al., 1997). Rapid technological advances have also facilitated genomic-scale investigation of polyploids and hybrids (Wang et al., 2006). Many ongoing studies are focused on investigation of: (i) the evolutionary consequence of gene and genome duplications in polyploids; (ii) genomic and gene expression changes in resynthesized allotetraploids; (iii) genetic and gene expression variation in natural populations of polyploids; and (iv) comparison of genetic and gene expression changes in resynthesized and natural polyploids (Wendel, 2000; Osborn et al., 2003; Soltis et al., 2003; Comai, 2005; Chen, 2007). The presentations given at the Polyploidy workshop, Plant and Animal Genome XV Conference (http://www.intl-pag.org/), reflected these current research themes, reporting on ancient polyploidy events in Glycine, expression evolution of duplicate genes in Arabidopsis, gene expression changes in resynthesized Brassica and wheat allopolyploids, hybridization barriers in Arabidopsis, and tissue-specific and stress-induced expression patterns of duplicate genes in cotton and hybrid Populus. ‘… expression of duplicate genes in response to developmental programs is more strongly correlated than that of duplicate genes in response to environmental stresses, suggesting rapid evolution of duplicate genes in response to external factors’

Upland cotton (Gossypium hirsutum L.) has a narrow germplasm base, which constrains marker development and hampers intraspecific breeding. A pressing need exists for high-throughput single nucleotide polymorphism (SNP) markers that can be readily applied to germplasm in breeding and breeding-related research programs. Despite progress made in developing new sequencing technologies during the past decade, the cost of sequencing remains substantial when one is dealing with numerous samples and large genomes. Several strategies have been proposed to lower the cost of sequencing for multiple genotypes of large-genome species like cotton, such as transcriptome sequencing and reduced-representation DNA sequencing. This paper reports the development of a transcriptome assembly of the inbred line Texas Marker-1 (TM-1), a genetic standard for cotton, its usefulness as a reference for RNA sequencing (RNA-seq)-based SNP identification, and the availability of transcriptome sequences of four other cotton cultivars. An assembly of TM-1 was made using Roche 454 transcriptome reads combined with an assembly of all available public expressed sequence tag (EST) sequences of TM-1. The TM-1 assembly consists of 72,450 contigs with a total of 70 million bp. Functional predictions of the transcripts were estimated by alignment to selected protein databases. Transcriptome sequences of the five lines, including TM-1, were obtained using an Illumina Genome Analyzer-II, and the short reads were mapped to the TM-1 assembly to discover SNPs among the five lines. We identified >14,000 unfiltered allelic SNPs, of which ∼3,700 SNPs were retained for assay development after applying several rigorous filters. This paper reports availability of the reference transcriptome assembly and shows its utility in developing intraspecific SNP markers in upland cotton.

Gene expression during the early stages of fiber cell development and in allopolyploid crops is poorly understood. Here we report computational and expression analyses of 32 789 high-quality ESTs derived from Gossypium hirsutum L. Texas Marker-1 (TM-1) immature ovules (GH_TMO). The ESTs were assembled into 8540 unique sequences including 4036 tentative consensus sequences (TCs) and 4504 singletons, representing approximately 15% of the unique sequences in the cotton EST collection. Compared with approximately 178 000 existing ESTs derived from elongating fibers and non-fiber tissues, GH_TMO ESTs showed a significant increase in the percentage of genes encoding putative transcription factors such as MYB and WRKY and genes encoding predicted proteins involved in auxin, brassinosteroid (BR), gibberellic acid (GA), abscisic acid (ABA) and ethylene signaling pathways. Cotton homologs related to MIXTA, MYB5, GL2 and eight genes in the auxin, BR, GA and ethylene pathways were induced during fiber cell initiation but repressed in the naked seed mutant (N1N1) that is impaired in fiber formation. The data agree with the known roles of MYB and WRKY transcription factors in Arabidopsis leaf trichome development and the well-documented phytohormonal effects on fiber cell development in immature cotton ovules cultured in vitro. Moreover, the phytohormonal pathway-related genes were induced prior to the activation of MYB-like genes, suggesting an important role of phytohormones in cell fate determination. Significantly, AA sub-genome ESTs of all functional classifications including cell-cycle control and transcription factor activity were selectively enriched in G. hirsutum L., an allotetraploid derived from polyploidization between AA and DD genome species, a result consistent with the production of long lint fibers in AA genome species. These results suggest general roles for genome-specific, phytohormonal and transcriptional gene regulation during the early stages of fiber cell development in cotton allopolyploids.