Copy number polymorphism in plant genomes

Copy number variants (CNVs) are genomic rearrangements resulting from gains or losses of DNA segments. Typically, the term refers to rearrangements of sequences larger than 1 kb. This type of polymorphism has recently been shown to be a key contributor to intra-species genetic variation, along with single-nucleotide polymorphisms and short insertion-deletion polymorphisms. Over the last decade, a growing number of studies have highlighted the importance of copy number variation (CNV) as a factor affecting human phenotype and individual CNVs have been linked to risks for severe diseases. In plants, the exploration of the extent and role of CNV is still just beginning. Initial genomic analyses indicate that CNVs are prevalent in plants and have greatly affected plant genome evolution. Many CNV events have been observed in outcrossing and autogamous species. CNVs are usually found on all chromosomes, with CNV hotspots interspersed with regions of very low genetic variation. Although CNV is mainly associated with intergenic regions, many CNVs encompass protein-coding genes. The collected data suggest that CNV mainly affects the members of large families of functionally redundant genes. Thus, the effects of individual CNV events on phenotype are usually modest. Nevertheless, there are many cases in which CNVs for specific genes have been linked to important traits such as flowering time, plant height and resistance to biotic and abiotic stress. Recent reports suggest that CNVs may form rapidly in response to stress.     

Exploring plant genomes by RNA-induced gene silencing

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms--for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.     

Dynamic gene copy number variation in collinear regions of grass genomes

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.     

High rate of chimeric gene origination by retroposition in plant genomes

Retroposition is widely found to play essential roles in origination of new mammalian and other animal genes. However, the scarcity of retrogenes in plants has led to the assumption that plant genomes rarely evolve new gene duplicates by retroposition, despite abundant retrotransposons in plants and a reported long terminal repeat (LTR) retrotransposon-mediated mechanism of retroposing cellular genes in maize (Zea mays). We show extensive retropositions in the rice (Oryza sativa) genome, with 1235 identified primary retrogenes. We identified 27 of these primary retrogenes within LTR retrotransposons, confirming a previously observed role of retroelements in generating plant retrogenes. Substitution analyses revealed that the vast majority are subject to negative selection, suggesting, along with expression data and evidence of age, that they are likely functional retrogenes. In addition, 42% of these retrosequences have recruited new exons from flanking regions, generating a large number of chimerical genes. We also identified young chimerical genes, suggesting that gene origination through retroposition is ongoing, with a rate an order of magnitude higher than the rate in primates. Finally, we observed that retropositions have followed an unexpected spatial pattern in which functional retrogenes avoid centromeric regions, while retropseudogenes are randomly distributed. These observations suggest that retroposition is an important mechanism that governs gene evolution in rice and other grass species.     

Delineation of metabolic gene clusters in plant genomes by chromatin signatures

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.     

Parallel selection on gene copy number variations through evolution of three-spined stickleback genomes

Background

Understanding the genetic basis of adaptive evolution is one of the major goals in evolutionary biology. Recently, it has been revealed that gene copy number variations (GCNVs) constitute significant proportions of genomic diversities within natural populations. However, it has been unclear whether GCNVs are under positive selection and contribute to adaptive evolution. Parallel evolution refers to adaptive evolution of the same trait in related but independent lineages, and three-spined stickleback (Gasterosteus aculeatus) is a well-known model organism. Through identification of genetic variations under parallel selection, i.e., variations shared among related but independent lineages, evidence of positive selection is obtained. In this study, we investigated whole-genome resequencing data from the marine and freshwater groups of three-spined sticklebacks from diverse areas along the Pacific and Atlantic Ocean coastlines, and searched for GCNVs under parallel selection.

Results

We identified 24 GCNVs that showed significant differences in the numbers of mapped reads between the two groups, and this number was significantly larger than that expected by chance. The derived group, i.e., freshwater group, was typically characterized by larger gene-copy numbers, which implied that gene duplications or multiplications helped with adaptation to the freshwater environment. Some of the identified GCNVs were those of multigenic family genes, which is consistent with the theory that fatal effects due to copy-number changes of multigenic family genes tend to be less than those of single-copy genes.

Conclusion

The identification of GCNVs that were likely under parallel selection suggests that contribution of GCNVs should be considered in studies on adaptive evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-735) contains supplementary material, which is available to authorized users.     

Large-scale sequencing of plant genomes

The large number of ESTs generated for Arabidopsis and rice in recent years now act as an important complement to whole genome sequencing projects. The Arabidopsis Genome Initiative has begun a coordinated effort to sequence the entire genome and, as a result, increasing numbers of large sequence entries can be found in the public databases. In addition, the mitochondrial genome of Arabidopsis has been completely sequenced. Genome sequencing studies and the public sequence databases have begun to influence the direction of diverse areas of research from physiology to evolution.     

Feast and famine in plant genomes

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a one-way ticket to genomic obesity, as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.     

Retrotransposon evolution in diverse plant genomes

Retrotransposon or retrotransposon-like sequences have been reported to be conserved components of cereal centromeres. Here we show that the published sequences are derived from a single conventional Ty3-gypsy family or a nonautonomous derivative. Both autonomous and nonautonomous elements are likely to have colonized Poaceae centromeres at the time of a common ancestor but have been maintained since by active retrotransposition. The retrotransposon family is also present at a lower copy number in the Arabidopsis genome, where it shows less pronounced localization. The history of the family in the two types of genome provides an interesting contrast between "boom and bust" and persistent evolutionary patterns.     

High-throughput genotyping of unknown genomic terrain in complex plant genomes: lessons from a case study

Novel high-throughput genotyping technologies have facilitated rapid genotyping of single nucleotide polymorphisms in non-model organisms. Most plant species have complex genomes with a large proportion of their genes having one or more paralogous copies due to single gene duplications and ancient or recent polyploidization events. These paralogous gene copies are potential sources of genotyping errors, and hence genotyping of plant genomes is inherently difficult. Here we present a case study that exemplifies paralog-related problems in high-throughput genotyping of plant genomes. We used the MassARRAY genotyping platform to genotype the LpIRI locus in L. perenne populations; this gene is thought to be involved in low-temperature stress tolerance. The dissection of the molecular genetics underlying the genotyping results provides a good example of how unknown paralogs can mask the true genotype of the locus, instructive to the non-specialist plant researcher and breeder.     

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.     

Finding the functional gems in plant genomes

A report on the joint second Plant Genomics European Meeting (Plant GEMs) and fourth Genomic Arabidopsis Resource Network (GARNet) meeting, York, UK, 3-6 September 2003.     

Discovering the seeds of diversity in plant genomes

A report on the Keystone Symposium 'Comparative Genomics of Plants', Taos, USA, 4-9 March 2004.