Although grain size in cereal crops is under polygenic control, several major genes for this trait have been identified in rice. One such gene, Minute (Mi), was identified in the central region of rice chromosome 3, and found to impart a small-grain phenotype in an incompletely dominant manner (Takeda and Saito, 1977). Mi and other major genes have proved to be useful markers in morphological mapping studies. In this study, we mapped Mi using microsatellite (SSR) markers to place it more precisely on the high-density molecular map of rice.

A line of rice homozygous for Mi, H-343, was crossed with the rice variety IR64 to develop a segregating F₂ population of 113 individuals that was used for mapping. These individuals were evaluated for six different phenotypes: seed length (SL), grain length (GL), seed weight (SWe), grain weight (GWe), seed width (SWi) and grain width (GWi). All individuals were also genotyped using previously published SSR markers on chromosome 3 (Li et al., 2004 and Temnykh et al., 2001).

Using the computer software Mapmaker V2.0 (Lander et al., 1987), an integrated linkage map of chromosome 3 was created and interval analysis was carried out using the Q-gene software package (Nelson, 1997). Empirical significance thresholds for declaring a QTL were similar for all traits evaluated, with an average threshold value of LOD>2.95 at the p<0.01 significance level.

Results of interval mapping for the traits seed length (SL), grain length (GL), seed weight (SWe) and grain weight (GWe) positioned Mi in the interval between markers RM282 and RM16, with peak LOD scores occurring in or within 2 cM of the 3.8 cM interval between markers JL8 and JL14 (Figure 1). Peak LOD scores for SL, GL, SWe and GWe were 39.5, 41.0, 22.2 and 21.4 respectively (Figure 2). Interval mapping for the traits seed width (SWi) and grain width (GWi) indicated that the locus associated with these phenotypes differed from the Mi locus reported above. The peak LOD scores for these traits were 5.6 for SWi and 4.2 for GWi, with both occurring in the interval between markers JL8 and RM186, and falling closer to RM16 than to either of its flanking markers (Figure 2).

These results suggest that there are two separate genes on chromosome 3 that affect grain size: one gene, which is believed to be Mi, has a large effect on grain length and weight, while the other, linked gene has a smaller effect on grain width. These findings are consistent with the conclusion of Kato (1989) that grain length and grain width are generally controlled by different genetic systems. Furthermore, the low LOD scores obtained at the width locus are consistent with results generally seen for polygenes, where any one locus explains only a small portion of the phenotypic variation associated with a quantitative trait. This is in contrast to the much higher LOD scores achieved at the putative Mi locus, which are consistent with those associated with genes exhibiting major effects on a phenotype. Previous work done on Mi

also suggests that this gene is most closely associated with the phenotype grain length (Takeda and Saito, 1977). Thus, results of the interval mapping completed in this study indicate that Mi is most likely located in the 3.8 cM interval between SSR markers JL8 and JL14, both located in the genetically defined centromere region of chromosome 3.

Acknowledgments

We gratefully acknowledge Dr. Itsuro Takamure from Hokkaido University who provided the original Minute seeds and Dr. Neil Rutger at the Dale Bumpers National Research Center in Stuttgart, Arkansas, USA for help with phenotypic evaluation of seeds.

References

Kato, T., 1989. Diallel analysis of grain size of rice (Oryza sativa L.). Japan. J. Breed. 39: 39-45.

Lander, F., P. Green and L. Abrahamson, et al. 1987. Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181.

Li. J., M. Thomson and S. McCouch, 2004. Fine mapping of a grain weight QTL in the pericentromeric region of rice chromosome 3. Genetics: in press.

Nelson, J.C., 1997. Q-gene: software for marker-based genomic analysis and breeding. Mol. Breed. 3: 239-245.

Takeda, K. and K. Saito, 1977. The inheritance and character expression of the minute gene derived from a rice genetic tester Minute. Bull. Fac. Agr. Hirosaki Univ. 27: 1-29. (in Japanese with English summary)

Temnykh, S., G. DeClerck and A. Lukashova, et al. 2001. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, tansposon associations, and genetic marker potential. Genome Res. 11: 1441-1452.