Genome characterization of a Eucalyptus natural mutant

Background Eucalyptus genetic improvement is time-consuming mainly because its long time to reach reproductive maturity along with its mixed mating system [6]. As a result, the production of pure lines as in maize is impracticable. The heterozygous status hides deleterious recessive genes that, when in homozygosity can reduce seedling survival due to physiological changes, resulting in considerable loss in seedling production [6]. Therefore, it would be important to identify the gene(s) associated with inbreeding depression. There are very few studies involving the characterization of deleterious recessive genes in Eucalyptus. We detected an anomaly in the offspring obtained from a controlled cross of Eucalyptus grandis. The anomaly appears in a ratio of one abnormal to three normal seedlings, suggestive of a Mendelian segregation. Based on the segregation ratio, we hypothesized that the character is controlled by a single gene, with a homozygous genotype for the recessive allele showing abnormal seedlings. The parents showed normal phenotype and they have no relationship, excluding the possibility of inbreeding depression due to identity by descent. The abnormal seedlings die in a few months and show different characters: high shoot branching, height reduction, leaf area reduction, and changes in leaf shape (Figure 1). In this scenario, we aimed to characterize the genomic and genetic causes of the observed anomaly. Methods The full-sib individuals generated at Suzano Papel and Celulose SA company were divided into normal and abnormal seedlings. The ratio between normal and abnormal seedlings was tested by chi-square analysis. DNA was extracted according to Ferreira and Grattapaglia [1]. The DNA concentration was quantified using a spectrophotometer (Thermo Scientific NanoDropTM 1000), and subjected to electrophoresis in 1% agarose gel and stained with ethidium bromide. DNA from ten normal individuals were randomly selected and mixed, creating the normal bulk, and the same was performed to create the abnormal bulk. The bulks were amplified with random primers (kits AD, AE, AF, AJ, and AK – Operon Technologies, Inc.). Amplicons were submitted to electrophoresis in 1% agarose gel stained with ethidium bromide. Polymorphic markers detected by RAPD were converted to SCAR (Sequence Characterized Amplified Regions) markers. DNA fragments from polymorphic marker were purified from agarose gel using the illustra GFX PCR DNA (GE Healthcare), cloned into vector pGEM-T Easy (Promega) and inserted into UltraMAX DH5a-FT competent Escherichia coli cells (Life Technologies). DNA was sequenced in an ABI3100 Genetic Analyzer (Applied Biosystems). SCAR primers were designed based upon the above mentioned sequencing and further used to amplify DNA fragments from bulks and six individuals: three anomalous, one individual from normal bulk; and two normal individuals of unrelated populations (BAC and Brasuz). Amplicons of the six individuals were purified, cloned into vector pGEM-T Easy (Promega) and inserted into DH5acompetent E. coli cells (Life Technologies). Six colonies from each individual were amplified with the SCAR primerss. Purified product of amplification was sequenced by ABI3100 Genetic Analyzer (Applied Biosystems). SCAR * Correspondence: [email protected] Departamento de Genética, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Distrito de Rubião Jr s/n CEP 18618-000, Botucatu (SP), Brazil Full list of author information is available at the end of the article Fuchs et al. BMC Proceedings 2011, 5(Suppl 7):P65 http://www.biomedcentral.com/1753-6561/5/S7/P65