44. Identification of a rice CCDD genome type-specific repetitive DNA sequence

1) Field of Botany

2) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA

Cytogenetic analysis of the genus Oryza has characterized six diploid genome types, AA, BB, CC, DD, EE, and FF and two tetraploid genome types, BBCC and CCDD (Chang 1984). We have already reported the isolation and characterization of four genome type-specific repetitive DNA sequences, which are of the AA, CC, EE and FF genome types in the genus Oryza (Zhao et al. 1989), and a BB genome type-specific repetitive DNA (see accompanying paper). In this communication, we report the isolation and characterization of a CCDD genome type-specific repetitive DNA sequence from O. alta (CCDD genome type; note that the CCDD genome type is a tetraploid that contains both CC and DD genome types). This repetitive sequence, CCDD-827, is present as an interspersed sequence with a repeat unit length of 827 bp. The genome type-specific clones can be used as molecular markers in simple hybridization experiments to distinguish rice species of unknown genome types.

Rice varieties were obtained from T. T. Chang of the International Rice Research Institute (IRRI, Manila, Philippines). The cloning of repetitive sequences from O. alta (accession # 100161) was carried out by digesting the total DNA with HincII. A distinct DNA band, corresponding to a DNA fragment of approximately 800 bp, was found, and the DNA was eluted from the gel cloned into the HincII site of pUC19. After transforming E. coli JM101 cells, the recombinant clones were screened with 32 P-labeled total rice DNA from O. alta. A strong hybridizing clone, pCCDD-827, was further characterized.

To verify the distribution of this repetitive DNA in different rice entries, the cloned pCCDD-827 was used as the probe for slot-blot hybridization using 39 rice varieties, both domestic and wild plants, including several entries from the genome types BB, BBCC, CC, CCDD, and EE. Strong hybridization was observed, mainly with O. alta and O. latifolia DNAs rom species of the CCDD genome type. The pCCDD-827 sequence or a related sequence is also present, but at much lower copy number, in species of the AA and CC genome types, and in some species of the BBCC genome types (data not shown). Southern blot analysis was performed and the results show that pCCDD-827 hybridized only with DNA from the CCDD genome type, but not with DNA from BBCC and CC genome types (Fig. 1). Thus, the pCCDD-827 related sequence can be considered CCDD genome type-specific. The copy number of the pCCDD-827-related sequence in O. alta was estimated to be 1000 per haploid genome by using a quantitative slot-blot hybridization method described by Zhao et al. (1989).

Sequence analysis of the pCCDD-827 showed that it contained an 827-bp

Fig. 1. Genomic blot analysis of DNA from different genome types of rice using pCCDD-827 as a probe. Total rice DNA (5 pg/lane) from different genome types (as indicated at bottom) was digested to completion with the restriction enzyme HincII and fractionated electrophoretically on a 1% (w/v) agarose gel. The DNA samples were transferred to a NYTRAN filter and hybridized with 32 P-labeled pCCDD-827. Genomic DNA from different rice species was loaded in different lanes as follows: Lane 1, O. punctata (accession # 103897); Lane 2, O. malampuzhaensis (accession # 100957); Lane 3, O. minuta (accession # 101125); Lane 4, O. officinalis (accession # 103286); Lane 5, O. collina (accession # 103410); Lane 6, O. minuta (accession # 101089); Lane 7, O. alta (accession # 100161); Lane 8, O. latifolia (accession # 100165); Lane 9, O. alta (accession 101395).

Fig. 2. The DNA sequence of pCCDD-827 from O. alta. The accession number of the O. alta used was 100161. Perfect repeats of 7 bp or more in length (a to g) are indicated with lines either above or below the sequences. The sequence for pCCDD-827 has been deposited in the Genome Sequence Data Base, National Center for Genome Resources, under the accession number L36561.

sequence (Figure 2). Within this sequence, there are six perfect repeats, each of which is more than 7 bp in length. There are also several short, imperfect repeats within the 827-bp unit. The pCCDD-827 is comprised of 60% A+T residues. No significant sequence similarity to the pCCDD-827 sequence was found among sequences listed in the EMBL/GenBank database of 1992.

This research was supported by a research grant, 91-37300-6567, from the USDA Plant Genome Award.

References

Chang, T. T., 1984. Conservation of rice genetic resources: Luxury or necessity? Science 224: 251- 256.

Zhao, X., T. Wu, Y. Xie and R. Wu, 1989. Genome specific repetitive sequences in the genus Oryza. Theor. Appl. Genet. 76: 835-840.