Isolation and characterization of TrsC family of tandem repeats from species in the 0. officinalis complex

We have isolated and characterized repetitive sequences from the different diploid species of the 0. officinalis complex. For this purpose we constructed the libraries of genomic DNA from all diploid species of the complex using HaeIII restriction endonuclease. Libraries were searched with the labeled total DNA of corresponding species as a probe. As a result 15 clones were found to have a strong hybridization signal with DNA of the diploid CC group of species (0. officinalis, 0. rhizomatis, 0. eichingeri). These clones gave cross-hybridization signals with each species of the group and no hybridization with BB and EE diploid species. Primary structure analysis revealed that these CC-specific clones represent the TrsC family of tandem repeats which was previously found to be highly amplified within 0. officinalis (Nakajima et a!. 1996).

The repetitive unit of TrsC repeat is 366 bp long. It can be divided into two HaeIII subfragments of 171 and 195 bp, respectively. Both subfragments were found among the cloned sequences. The level of homology of the TrsC sequences within and between species is similar and ranges from 90 to 95% (data not presented). The high conservation of the both Haeffl subfragments could be explained by the recent amplification of TrsC in the common ancestor of the CC group of species.

The genomic organization of TrsC family within species of the 0. officinalis complex was analyzed by the blot-hybridization of probe p01.88 containing full TrsC unit with genomic DNA digested with various restriction enzymes. 30 accessions representing all taxa of this complex were chosen for the study, most materials were kindly supplied by IRRI. All diploid CC species have the same pattern typical of tandemly repeated sequences after digestion with Hinfi enzyme (Fig. 1). A similar type of pattern appeared with AvaIl, HindIII and RsaI enzymes (data not shown). In order to detect sequence divergence the probe was hybridized at two different stringencies (65°C and 55°C). No additional fragments were detected at low stringency (55°C).

Diploid 0. punctata (BB) and 0. australiensis (EE) showed no hybridization with the probe p01.88 (Fig. 1). This indicates that TrsC was most likely amplified within the ancestor of the CC genome after its divergence from BB and EE genomes.

Among tetraploid species only 0. punctata (BBCC) and 0. officinalis (syn. 0. snalampuzhaensis) (BBCC) hybridized to the probe. 0. minuta (BBCC) as well as CCDD species 0. grandiglumis, 0. latVolia and 0. alta did not hybridize to p01 .88 even at low stringency. These data imply a closer relationship between the first two tetraploids and CC genome. The absence of hybridization signal in the other tetraploids can be explained by deletion, high divergence of the TrsC family during speciation, or speciation prior to amplification of the TrsC family in the CC genome donor to these allopolypoids. Whichever explanation is correct the implication is that the polyploid event leading to 0. minuta and CCDD species occurred earlier than for 0. punctata (BBCC) and 0. officinalis

(BBCC).

The copy number of TrsC was determined by the slot-blot hybridization method at high stringency in those species that showed different intensities of signal on Southern blots (Table 1). The genome sizes were taken from Uozu et a!. (1997). The copy number of TrsC in 0. officinalis with CC genome was 7.6 x 104 per diploid genome, which is similar to the number reported by Nakajima et al. (1996). Among the tetraploids 0. punctata and 0. officinalis having the BBCC genome the copy number was about 10 and 3 times less, respectively.

Our results demonstrate that the TrsC family is characterized by a conserved primary structure and genome organization among 0. officinalis complex species having the CC genome. On the other hand, we found large quantitative differences between species that implies intensive processes of amplification or deletion of this repeat especially among allotetraploid species.