43. Identification of a rice BB 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

Analysis of the genus Oryza has characterized six diploid genome types, AA, BB, CC, DD, EE, and FF (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). In this communication, we report the isolation and characterization of a BB genome type-spcific repetitive DNA sequence from O. punctata (BB genome type). This repetitive sequence, BB-513, is present as long tandem arrays with a repeat unit length of 513 bp. The genome type-specific clones can be used as molecular markers in simple hybridization experiments to distinguish rice species of unknown genome types. This repetitive sequence probe can also be used by rice breeders as a simple method to determine the extent of introgression of DNA from one genome type to another in genetic crosses, especially in wide crosses or in protoplast fusion.

Rice varieties were obtained from T. T. Chang of the International Rice Research Institute (IRRI, Manila, Philippines). The cloning of repetitive se- quences from O. punctata (accession # 103897) was carried out by digesting the total DNA with Hincll. A distinct DNA band, corresponding to a DNA fragment of approximately 500 bp, was found, and the DNA was eluted from the gel and cloned into the HincII site of pUC19. After transforming E. coli JM101 cells, the recombinant clones were screened with ³²P-labeled total rice DNA from O. punctata. Several strongly hybridizing clones were analyzed, and one clone, pBB-513, was further characterized.

To determine the distribution of this repetitive DNA in different rice genome types, the cloned pBB-513 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 only with O. punctata DNA from species of the BB genome type. Southern blot analysis was performed to examine the presence and arrangement of this repetitive sequence (Fig. 1). The results show that pBB-513 is BB genome type-specific. The imperfect ladder pattern suggests that a substantial

Fig. 1. Genomic blot analysis of different rice genome types using pBB-513 DNA as a probe. Total rice DNA (5 pg/lane) from different genome types was digested to completion with restriction eyzyme HincII and fractionated electrophoretically on a 1% (w/v) agarose gel. The DNA samples were transferred to a NYTRAN filter and hybridized with ³²P-labeled pBB-513 DNA. Genomic DNA from different rice species was loaded in different lanes as follows: Lane 1, O. meridionalis (accession # 101 147); Lane 2, O. longistaminata (acce ssion # 100930); Lane 3, O. punctata (accession 103897); Lane 4, O. officinalis (accession # 103286); Lane 5, O. alta (accession # 100161); Lane 6, O. latifolia (accession # 100165); Lane 7, O. australiensis (accession # 101467); Lane 8, O. brachyantha (accession # 101236).

amount of this repetitive sequence is present as long tandem repeats. The copy number of the pBB-513-related sequence in O. punctata 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 pBB-513 showed that it contained a 513-bp sequence as the basic repeat unit (Fig. 2). Within each 513-bp repeat unit, there are copies of a 33-bp perfect repeat between positions 56 to 90 and 118 to 150, and two copies of a 27-bp repeat, one from position 46 to 72 which overlaps the first 33-bp repeat, the other from position 92 to 118 which is within the second 33-bp repeat. There are also copies of a 17-bp repeat between positions 56 to 72, 102 to 118, and to 134. No significant sequence similarity to the pBB-513 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.

Fig. 2. The DNA sequence of pBB-513 from O. punctata. The accession number of the O. punctata used was 103897. The 33-bp perfect repeat is indicated by heavy arrows above the sequences; two copies of a 27-bp perfect repeat are marked with underlines and the letter (a); three copies of a 17-bp perfect repeat are marked with underlines and the letter (b); two copies of a 7-bp perfect repeat are marked with underlines and the letter (c). The sequence for pBB-513 has been deposited in the Genome Sequence Data Base, National Center for Genome Resources, under the accession number L36560.

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.