No DNA loss in autotetraploids of Arabidopsis thaliana H. Ozkan 1 , M. Tuna 2 and D. W. Galbraith 3 1 Department of Field Crops, Faculty of Agriculture, University of Cukurova, 01330 Adana, Turkey, E-mail: hozkan@cu.edu.tr; 2 Department of Field Crops, Faculty of Agriculture, University of Tekirdag, Tekirdag-Turkey; 3 Department of Plant Sciences, University of Arizona, 303 Forbes Building, 8572-Tucson, Arizona, USA With 3 tables Received September 5, 2005/Accepted November 17, 2005 Communicated by W. E. Weber Abstract To address the issue of genome evolution in autopolyploids and particularly to investigate whether rapid sequence elimination also occurs in autopolyploids as in allopolyploids, amplified fragment length polymorphism (AFLP) fingerprinting was employed to examine a large number of genomic loci in F 1 hybrids between two different autotetraploids of Arabidopsis thaliana accessions, namely Ler and Col. Using this approach, perfect additivity in the F 1 hybrids was found between the newly-formed autopolyploids when compared with their parental lines. Using flow cytometry, the study was extended in a quantitative manner, in which the nuclear DNA contents in one autotetraploid A. thaliana accession Ler, was determined. The increase in genome size of the autotetraploid line was additive. Taken together, no evidence was found for genome size reduction due to autopolyplo- idization of A. thaliana. The results indicating that there was no DNA loss in autotetraploid A. thaliana suggest that a different type of genome evolution may occur in autopolyploids during the initial stages of their formation when compared with allopolyploids. Key words: Arabidopsis thaliana — autopolyploidy — flow cytometry — genome evolution — AFLP Polyploidy has played an important evolutionary role in flowering plants (Wendel 2000). It occurs in up to 70% of flowering plant species (Masterson 1994), as well as in fish, amphibians and occasionally in mammals (Gallardo et al. 1999). Polyploids are classified either as allopolyploids, derived from inter-specific or inter-generic hybridization between species with distinct genomes followed by chromosome doub- ling, or autopolyploids, originating from the chromosome doubling of genetically similar genomes (Stebbins 1950). In autopolyploids, the two genomes are similar and homologous to that of their diploid ancestors and so multivalents are characteristic of the first meiotic metaphase. The disjunction of multivalents is imperfect, leading to a degree of infertility and inheritance is multisomic. One advantage of autopolyploidy is the capacity to maintain high levels of heterozygosity with multiple alleles per locus or, more rarely, to reach homozyg- osity with multiple dosage of a given allele (Levy and Feldman 2002). It is now clear that autopolyploids are much more common in nature than previously thought, although less prevalent than allopolyploids (Soltis et al. 2003). Arabidopsis thaliana has become the system of choice for many basic studies in plant biology, particularly those invol- ving genetics and molecular biology. It has also been used to study the response of the genome to polyploidy, with most studies examining the behaviour of allopolyploids (Comai et al. 2000) but not of autopolyploids. Little is known about when polyploidy-associated chromosomal changes occur. AFLPs and DNA blot analyses of newly formed wheat allopolyploids have suggested that DNA sequence elimination is an immediate response to allopolyploid formation (Feldman et al. 1997, Ozkan et al. 2001, 2003, Shaked et al. 2001). However, a similar AFLP analysis of cotton allopolyploids failed to detect evidence for sequence elimination (Liu et al. 2001), leaving it unclear whether allopolyploidy-induced changes are a common occurrence. Although the mechanism underlying widespread excision of genomic sequences is unknown, several molecular mechanisms have been outlined by Shaked et al. (2001). The effect of autopolyploidization on genome size (C-value) and on genomic rearrangements has not been investigated. In order to study whether rapid genomic changes might occur in autopolyploids, a large number of genomic loci in F 1 hybrids between two different autote- traploids of A. thaliana accessions [namely Ler (2n ¼ 4x ¼ 20) and Col (2n ¼ 4x ¼ 20)] were examined by amplified fragment length polymorphism (AFLP) fingerprinting. The AFLP survey demonstrated complete genomic additivity in these autopolyploids. In order to address the issue of genome size (C-value) evolution in autopolyploids and particularly to investigate how the genome size might change following genome duplication, diploid A. thaliana accession Ler (2n ¼ 2x ¼ 10) and its autotetraploid deriva- tives (2n ¼ 4x ¼ 20) were examined by flow cytometry. The results indicate that DNA content was additive in synthetic autotetraploid A. thaliana. Materials and Methods Plant materials: The seeds of diploid A. thaliana accessions (Ler and Col) and their autotetraploid derivatives, were kindly provided by the Arabidopsis Biological Resource Center and Prof. Dr Csaba Koncz (Max Planck Institute for Plant Breeding Research), respectively. Reciprocal hybridization was made in a greenhouse at the Max Planck Plant Institute, Cologne, Germany. Genomic DNA extraction and AFLP analyses: Young leaves of A. thaliana were collected, lyophilized and kept at )70°C until used. DNA was extracted using the Qiagen (Hilden, Germany) DNA isolation kit according to the manufacturer’s protocol (DNeasy Plant Mini Handbook for DNA isolation from plant tissue). The AFLP amplification was performed according to Vos et al. (1995) with minor modifications (Ozkan et al. 2005), using nine primer combinations (E AGC /M AGT ,E AGC /M ACT ,E AGC /M AGC ,E ACC /M ACT ,E ACC /M AGG , E ACC /M AAG E ACC /M ACC ,E AGC /M AAC ,E AGC /M AAG ). www.blackwell-synergy.com Plant Breeding 125, 288—291 (2006) Ó 2006 The Authors Journal compilation Ó 2006 Blackwell Verlag, Berlin