27. Annual and perennial differentiation in wild rice: Analysis by direct sequencing of PCR-amplified DNA

Pascale BARBIERI and Akira ISHIHAMA

National Institute of Genetics, Department of Molecular Genetics, Mishima, 411 Japan.

1) Present address: Service de Biochimie, Centre d'Etudes Nucleaires de Saclay, 91191 Gif sur Yvette, France.

The Asian wild rice species, Oryza rufipogon, is thought to be the progenitor species of the cultivated rice, 0. sativa. It is differentiated into annual and perennial ecotypes. Although they differ clearly in phenotypic traits, little difference has been found in isozyme markers (Oka 1988, p.66; Barbier 1989a, 1989b). In order to investigate the phylogenetical relationships among these strains, we initiated an analysis of the variation in DNA sequence of nuclear genes for eight different strains, three annuals and five perennials, of 0. rufipogon collected ftom various geographic regions in Asia (Table 1), and one outgroup reference strain from the related African species 0. longistaminata.

For DNA sequencing, we employed a direct method, in which target genes were amplified by the polymerase chain reaction (PCR) and PCR products were directly subjected to dideoxy chain termination reactions. This method allows rapid sequencing without gene cloning and detection of heterozygosity, hetero-

Table 1. Rice strains used

================================================================
Species/Acc. code    Life  cycle       Origin
================================================================
0.rufipogon,
W106                     Annual     Orissa, India
W2008                    Annual     Gudjarat, India
NE4                      Annual     Central plain, Thailand
W108                     Perennial  Orissa, India
W180                     Perennial  North region, Thailand
CP20                     Perennial  Central plain, Thailand
NE88                     Perennial  Central plain, Thailand
W2025                    Perennial  Kalimantan, Indonesia
0.longistaminata,
W1444                    Perennial  Ivory Coast, Africa
O. sativa
Nipponbare(NB)           Annual     Japan
================================================================

Table 2.  Variations in DNA sequence of the genes for prolamin and
phytochrome

====================================================================
Strain       Prolamin                 Phytochrome
(Acc. code)          ===============================================
                     Intron 2 Exon 3 Intron 3 Exon 4 Intron 4 Exon 5
           =========================================================
                              Sequence  analyzed  (bp)
               326    201      186      276     117    100     192
====================================================================
W106            0     2(+1)      0        0       0      0      0
W2008           1     O(+1)      0        0       0      1      0
NE4             0     1(+1)      0        0       0      0      0
W108            0     2(+1)      0        0       1      0      0
W180            0     1(+1)      0        0       0      0      0
CP20            0     O(+1)      0        0       0      1      0
NE88            2     O(+1)      0        1       0      0      0
W2025           0     O(+1)      0        2       0      1      0
====================================================================
W1444         4(+6)   7(+4)      1     13(-62)    2    4(-1)    2
Note: Total numbers of base substitutions are shown, compared with the published sequences of 10 kDa prolamin (strain NB) and phytochrome (strain IR36), respectively. The total numbers of deletions (-) or insertions (+) are shown in parentheses. Details of the prolamin and phytochrome sequences are described in Barbier and Ishihama (1990) and Barbier et al. (1990), respectively.

plasmy and heterogeneity within gene families. Up to now, we determined the DNA sequence for the genes encoding 10 kDa prolamin, a rice storage protein, and phytochrome apoprotein. Primers for PCR were designed based on the respective published DNA sequences determined for 0. sativa (Masumura et al. 1989; Kay et al. 1989).

Variation in the 1O kDa prolamin gene family was probed by sequencing PCR-amplified DNA fragments of about 350 bp in length. No intron was found in this gene for all the strains examined. Within eight 0. rufipogon strains, variants were detected only in two strains: W2008, a strain from West India (one base substitution) and NE88, a strain from Thailand (two base substitutions) (Table 2). In contrast, large differences were found between Asian and African strains, including four base substitutions (two amino acid substitutions) and three base insertions at two points which led to the duplication of Leu at amino acid residue 17 (within leader peptide) and Met at 108 (for details see Barbier and Ishihama 1990). Southern hybridization analysis revealed a complexity similar to other prolamin gene families. However, no detectable heterogeneity among amplified copies of 10 kDa prolamin gene family was found for all the strains analyzed.

As to the phytochrome gene, we sequenced about 600 bp introns (intron 2, 3 and 4) and about 500 bp flanking exon borders (exon 3, 4 and 5) (Barbier et al., 1990). The variation in these regions is localized mostly in the introns. Within O. rufipogen, 14 base substitutions (and a single insertion) exist in the introns while the exons contain only one base substitution, as summarized in Table 2.An analysis of the pattern of base substitutions within the phytochrome introns showed that 0. rufipogon and 0. longistaminata differ by about 0.042 substitutions per site, whereas strains within 0. rufipogon differ only by 0.0017-0.0050 substitutions per site (roughly one tenth the level of interspecies difference). The extent of polymorphism within species (number of polymorphic sites/site sampled) is slightly lower than the extent of polymorphism found in Drosophila melanogaster, presumably due to a smaller population size. In so far as our sampling is representative, this result suggests that extant strains of Asian wild rice have all diverged as recently as a few hundred thousand years ago, compared to an estimated 5-6 million years ago for the divergence between Asian 0. rufipogon and African 0. longistaminata.

This result could also imply that extensive gene flow took place among 0. rufipogon strains a few hundred years ago, possibly during the last glaciation, which probably reduced the distribution of this species. Another result is that, within Asian wild rice, the annual-perennial differentiation seems to be even more recent than the geographic differentiation.

References

Barbier, P., 1989a. Genetic variation and ecotypic differentiation in the wild rice Oryza rufipogon. I. Population differentiation in life-history traits and isozymic loci. ipn. J. Genet. 64: 259- 271.

Barbier, P., 1989b. Genetic variation and ecotypic differentiation in the wild rice Oryza rufipogon, II. Influence of the mating system and life-history on the genetic structure of populations. Jpn. J. Genet. 64: 273-285.

Barbier, P. and A. Ishihama, 1990. Variation in the nucleotide sequence of a prolamin gene family in wild rice. Plant Mol. Biol. 15: 191-195.

Barbier, P., H. Morishima and A. Ishihama, 1990. Phylogenetic relationships of annual and perennial wild rice: Probing by direct DNA sequencing. Theor. Appl. Genet., in press.

Kay, S. A., B. Keith, K. Shinozaki and N. H. Chua, 1989. The sequence of the rice phytochrome. Nucleic Acids Res. 17: 2865-2866.

Masumura, T., D. Shibata, T. Hibino, K. KaNk,abe, G. Takeba, K. Tanaka and S. Fujii, 1989. cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds. Plant Mol. Biol. 12: 123-130.

Oka, H. I., 1988. Origin of cultivated rice. Elsevier/Jpn. Sci. Soc. Press, Amsterdam/Tokyo.