Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome

BMC Genomics Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome Gabino Ros 0 Miguel A Naranjo 0 Domingo J Iglesias 0 Omar Ruiz-Rivero 0 Marion Geraud 0 Antonio Usach 0 Manuel Taln 0 0 Address: Centro de Genomica, Instituto Valenciano de Investigaciones Agrarias , Carretera Moncada-Naquera km 4.5, 46113 Moncada (Valencia) , Spain Background: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background. Results: Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues. Conclusion: In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement. - Background The rapid increase of world population, the field degradation by soil salinization and erosion, and the likely fluctuations in climate caused by global warming will pose new and known challenges to agriculture during this century [1]. Crop improvements required to cope with these challenges could be attained through agronomic advances, leading to a better use of fertilizers, protection agents or soil rescue, and exploitation of recent technologies for plant breeding. Despite the outstanding importance of genetics-based breeding applied to spontaneous mutations and conventional hybrids, molecular and genomic tools are expected to develop their great potential for crop improvement through functional genetics analysis, involving gene and function discovery and genome modification. Citrus, some of the most important fruit crops worldwide, are perennial trees requiring a juvenility period of several years and frequently are parthenocarpic and sexually selfincompatible [2,3], which considerably impairs traditional breeding. Genomic technology, including methods to rapidly identify and manipulate genes of agricultural interest, holds promise of improvements that may be difficult through traditional approaches. In recent years, Citrus has been the target of several genomic developments including large EST collections [4-7], cDNA and oligonucleotide-based microarrays [4,8,9], BAC libraries and BAC end sequencing (BES) (to be published). However, functional studies, i.e. genetic transformation and the capability to perform reverse genetic analyses, are also considerably impaired. In citrus, high throughput transgenic programs such as the generation of RNA interference knockouts, activation tagging through enhancer elements, gene-trap T-DNA insertions, or transposon tagging systems have not been developed yet. Since no efficient tagging or insertional procedures are available in these species, other gene disruption methods including strategies based on genome-wide mutagenesis such as TILLING and fast neutron mutagenesis have been initiated. These approaches are non-transgenic and may have particular interest for the industry where the debate on genetically modified organisms has restricted application of these technologies to crop improvement. Both approaches, however, are of limited usefulness as strategies for reverse genetics because of the lack of knowledge on Citrus genomic sequence and the large amount of space required for the establishment of mutant populations. ECOTILLING on natural citrus variants and microarray-based detection of deletions in fast neutron citrus mutants are apparently very straightforward approaches. In this work we explore the potential of this last idea using two fast neutron Citrus clementina hemizygous mutants from the IVIA collection and a 20K cDNA citrus microarray. Physical mutagenesis through fast neutron irradiation has been reported to cause variable genomic deletions ranging in size from few base pairs to 12 kb in Arabidopsis thaliana [10,11]. Several approaches have been used to characterize plant genomic deletions at the molecular level. These mostly include positional cloning [12], a method applicable to any kind of genetic lesion that, however, needs highly saturated genetic maps; PCR-based reverse genetics techniques [11,13], requiring a previous considerable knowledge of genomic sequence; and genomic subtraction procedures [14-16], which do not need sequence information but are strongly dependent on the gene dosage. Since very little is known about Citrus genome sequence and the Citrus induced deletions are in hemizygous gene dosage, an array-based procedure as the one employed for identifying homozygous gene deletions in Arabidopsis [17] seems more suitable for our purpose than those methods. Although the main application of microarrays is transcriptome profiling analysis, microarrays can also be used to study DNA variation. Oligonucleotide arrays are particularly suited for the detection of single nucleotide mismatches during hybridization, and hence for the discovery of novel DNA variants or the determination of known variants. The origin of this technique relies on a c (...truncated)