Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

Muoz-Amatrian et al. Genome Biology Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome Mara Muoz-Amatrian 0 Steven R Eichten Thomas Wicker Todd A Richmond Martin Mascher 1 Burkhard Steuernagel 1 Uwe Scholz 1 Ruvini Ariyadasa 1 Manuel Spannagl Thomas Nussbaumer Klaus FX Mayer Stefan Taudien Matthias Platzer Jeffrey A Jeddeloh Nathan M Springer Gary J Muehlbauer 0 Nils Stein 1 0 Department of Agronomy and Plant Genetics, University of Minnesota , St. Paul, MN 55108 , USA 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) , Gatersleben, D-06466 , Germany Background: There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. Results: A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as diseaseresistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. Conclusions: We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes. Barley; Copy number variation; Comparative genomic hybridization; Disease-resistance genes; Doublestrand break repair mechanisms - Background The identification and prevalence of copy number variation (CNV) among the genomes of individuals within a species has provided the rationale to redefine genomes as dynamic entities. Copy number variants (CNVs) are currently defined as unbalanced changes in the genome structure and include deletions, insertions, and duplications of >50 bp in size [1]. The first studies documenting the existence of numerous CNVs throughout the human genome and their relation with genetic disorders [2,3] were followed shortly by the completion of the first CNV map of the human genome [4]. Since then, an increasing number of human studies have produced evidence for the association of CNV with complex diseases, environmental response, and population diversity (reviewed in [1]). Other large-scale studies showed that CNV is common in other animal genomes including chimpanzee and other great apes [5,6], cattle [7,8], rat [9], dog [10,11], and Drosophila [12] among others. CNV is also a common feature of plant genomes and several recent studies provided insights into the extent of this type of intraspecific structural variation in plants. High levels of CNV have been found distributed throughout the maize genome, with a tendency for variants to be located near the ends of the chromosomes and the existence of high- and low-diversity regions [13-15]. The undomesticated progenitor of maize (teosinte) exhibits high levels of CNV and shares most of the variants with modern maize [15]. There is evidence that prevalent CNV in maize plays an important role in contributing to phenotypic variation as it overlaps loci associated with important traits related to stress and stimulus responses [16]. Studies in other plant species including Arabidopsis [17,18], wheat [19], sorghum [20], rice [21,22], and soybean [23,24], also demonstrated that CNV contributes to the genetic diversity of their genomes. Genes affected by CNV in soybean are enriched for annotations related to stress and plant defense responses [24]. There are several examples demonstrating a causal relationship between CNV and plant phenotypes. CNV at the Rhg1 locus in soybean increases the resistance to the cyst nematode Heterodera glycines [25]. In barley, increased copy number at the boron transporter gene (Bot1) confers borontoxicity tolerance to the African barley landrace Sahara [26]. CNV at the MATE1 transporter gene in maize is associated with increased aluminum tolerance [27]. CNV can arise from a variety of molecular mechanisms including: non-allelic homologous recombination (NAHR) at regions of extensive sequence similarity (synonymous with unequal crossing-over); non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ), which are associated with DNA repair at regions with very limited or no homology; replication-error mechanisms such as fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR); and transposable element (TE)mediated mechanisms [28-31]. CNV could also arise from the segregation of non-allelic homologs (SNH) among F2 siblings or recombinant inbred lines (RILs) [32,33]. NAHR is one of the best studied recombination-based mechanisms in humans, known to cause recurrent rearrangements in hotspots of homologous recombination, while replication mechanisms are a major contributor to non-recurrent CNVs [31]. In contrast, our understanding of the most prevalent contributors to CNV in plants is more limited. Barley (Hordeum vulgare L.) is one of the first crops domesticated by humans approximately 10,000 years ago [34] and currently ranks fourth among cereals in terms of harvested area [35]. It is also considered a model for the Triticeae tribe, which includes other agronomically-important species such as wheat and rye. CNV is known to affect some genes with important adaptive functions in barley. As mentioned above, increased copy number of a boron transporter gene (Bot1) confers boron-toxicity tolerance [26]. CBF (C-Repeat Binding Factor) gene copy number variation at the Frost Resistant-2 locus (FR-2) is associated with low-temperature tolerance [36]. These examples, together with the recent discovery of CNV affecting two major genes controlling flowering time in wheat, Ppd-B1 and Vrn-A1 [37], suggest CNV as a potential source of agronomically important phenotypic variation in barley and other Triticeae crops. In the present study, we developed and used a barley comparative genomic hybridization (CGH) array containing 2.1 M probes covering approximately 50 Mbp of repeat-masked barley sequence (cv. Morex). Four (...truncated)