Avraham A. Levy

Sequence Elimination and Cytosine Methylation Are Rapid and Reproducible Responses of the Genome to Wide Hybridization and Allopolyploidy in Wheat

Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between Ae. sharonensis × Ae. umbellulata, 14% of the loci from Ae. sharonensis were eliminated compared with only 0.5% from Ae. umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between Ae. longissima × T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in ∼13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.

Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops–Triticum) Group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.

Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat

Retrotransposons are a principal component of most eukaryotic genomes, representing roughly 40% of the human genome and 50–80% of some grass genomes. They are usually transcriptionally silent but can be activated under certain stresses. Despite their considerable contribution to genome structure, their impact on the expression of adjacent genes is not well understood. The steady-state transcript levels originating from Wis 2-1A retrotransposons are much higher in newly synthesized wheat amphiploids (two or more diverged genomes in the same nucleus). On activation, both Wis 2-1A long terminal repeats drive the readout synthesis of new transcripts from adjacent sequences including the antisense or sense strands of known genes. Here we report that activation of these antisense or sense transcripts is associated with silencing or activation of the corresponding genes, respectively. These data, together with the abundance of retrotransposons in genomes and their ability to be activated by various signals, support the view of transposons as potential controlling elements.

A Yeast Hybrid Provides Insight into the Evolution of Gene Expression Regulation

Gene Expression in Hybrids A large proportion of genes differ in their expression patterns between closely related species, and this divergence is believed to be an important driver of phenotypic evolution. However, little is known about the genetic basis of this divergence. Tirosh et al. (p. 659) use allele-specific expression profiling of two yeast species and their hybrid to identify which genes diverged in expression owing to changes in their genomic loci (cis) and which genes diverged owing to changes in their regulators (trans). The data can be used to explain the distinct gene expression pattern observed in the hybrid. Analysis of two yeast species and their hybrid reveals the role of cis and trans effects in the evolution of gene expression. During evolution, novel phenotypes emerge through changes in gene expression, but the genetic basis is poorly understood. We compared the allele-specific expression of two yeast species and their hybrid, which allowed us to distinguish changes in regulatory sequences of the gene itself (cis) from changes in upstream regulatory factors (trans). Expression divergence between species was generally due to changes in cis. Divergence in trans reflected a differential response to the environment and explained the tendency of certain genes to diverge rapidly. Hybrid-specific expression, deviating from the parental range, occurred through novel cis-trans interactions or, more often, through modified trans regulation associated with environmental sensing. These results provide insights on the regulatory changes in cis and trans during the divergence of species and upon hybridization.

The Impact of Polyploidy on Grass Genome Evolution

Polyploidy is an evolutionary process whereby two or more genomes are brought together into the same nucleus, usually by hybridization followed by chromosome doubling. As a result, the new polyploid is genetically isolated from its diploid progenitor(s) and a new species is formed. The importance of

Allopolyploidy – a shaping force in the evolution of wheat genomes

Recent studies have shown that allopolyploidy accelerates genome evolution in wheat in two ways: (1) allopolyploidization triggers rapid genome changes (revolutionary changes) through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations, and (2) the allopolyploid condition facilitates sporadic genomic changes during the life of the species (evolutionary changes) that are not attainable at the diploid level. The revolutionary changes comprise (1) non-random elimination of coding and non-coding DNA sequences, (2) epigenetic changes such as DNA methylation of coding and non-coding DNA leading, among others, to gene silencing, (3) activation of genes and retroelements which in turn alters the expression of adjacent genes. These highly reproducible changes occur in the F1 hybrids or in the first generation(s) of the nascent allopolyploids and were similar to those that occurred twice in nature: first in the formation of allotetraploid wheat (∼0.5 million years ago) and second in the formation of hexaploid wheat (∼10,000 years ago). Elimination of non-coding sequences from one of the two homoeologous pairs in tetraploids and from two homoeologous pairs in hexaploids, augments the differentiation of homoeologous chromosomes at the polyploid level, thus providing the physical basis for the diploid-like meiotic behavior of allopolyploid wheat. Regulation of gene expression may lead to improved inter-genomic interactions. Gene inactivation brings about rapid diploidization while activation of genes through demethylation or through transcriptional activation of retroelements altering the expression of adjacent genes, leads to novel expression patterns. The evolutionary changes comprise (1) horizontal inter-genomic transfer of chromosome segments between the constituent genomes, (2) production of recombinant genomes through hybridization and introgression between different allopolyploid species or, more seldom, between allopolyploids and diploids, and (3) mutations. These phenomena, emphasizing the plasticity of the genome with regards to both structure and function, might improve the adaptability of the newly formed allopolyploids and facilitate their rapid and successful establishment in nature.

How plants make ends meet: DNA double-strand break repair

DNA double-strand breaks (DSBs) lead to serious genomic deficiencies if left unrepaired. Recent studies have provided new insight into the mechanisms, the mutants and the genes involved in DSB repair in plants. These studies indicate that high fidelity DSB repair via homologous recombination is less frequent than non-homologous end-joining. Interestingly, non-homologous end-joining in plants is more error-prone than in other species, being associated with various rearrangements that often include deletions and insertions (filler DNA). We discuss the mechanism of error-prone DSB repair, which is probably an important driving force in plant genome evolution.

Elimination of selection markers from transgenic plants

Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed.

Tomato mutants as tools for functional genomics.

Tomato mutants have been used in genetic studies and breeding for decades, yet only a few tomato mutants have been characterized at the molecular level. Similarly, a wealth of sequence information for tomato is now available but the functions of only a few genes are known. New developments - such as the use of saturated mutant populations, new methods for the detection of mutants and new sequence data - are bridging the gap between tomato genes and their functions.

Functional Analyses of Two Tomato APETALA3 Genes Demonstrate Diversification in Their Roles in Regulating Floral Development

The floral homeotic APETALA3 (AP3) gene in Arabidopsis thaliana encodes a MADS box transcription factor required for specifying petal and stamen identities. AP3 is a member of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. Although Arabidopsis lacks genes in the paralogous Tomato MADS box gene 6 (TM6) lineage, tomato (Solanum lycopersicum) possesses both euAP3 and TM6 genes, which have functionally diversified. A loss-of-function mutation in Tomato AP3 (TAP3) resulted in homeotic transformations of both petals and stamens, whereas RNA interference–induced reduction in TM6 function resulted in flowers with homeotic defects primarily in stamens. The functional differences between these genes can be ascribed partly to different expression domains. When overexpressed in an equivalent domain, both genes can partially rescue the tap3 mutant, indicating that relative levels as well as spatial patterns of expression contribute to functional differences. Our results also indicate that the two proteins have differing biochemical capabilities. Together, these results suggest that TM6 and TAP3 play qualitatively different roles in floral development; they also support the ideas that the ancestral role of AP3 lineage genes was in specifying stamen development and that duplication and divergence in the AP3 lineage allowed for the acquisition of a role in petal specification in the core eudicots.