A reproductive barrier plays an indispensable role for evolution and speciation of organisms. Few genes responsible for a reproductive barrier have been identified in animals (Wu and Ting. 2004), but none in plants due to the difficulties in mapping of reproductive barrier loci and detecting interactive loci.

We developed a method of mapping of reproductive barrier loci on the whole genome in rice (Harushima et al. 2001), and are developing a method to detect interacting reproductive barrier loci in F₂ population by an independence test for segregation (author(s) in preparation). During these studies, we detected a pair of interactive reproductive barrier loci in a cross between a japonica cultivar Nipponbare and an indica cultivar Kasalath. Here, we report an action of a set of the interacting reproductive barrier loci at around 45.4 cM of chromosome 1, and at around 15.8 cM of chromosome 6 and their fine mapping.

We first examined whether the barrier was zygotic or gametophytic, and also male-dependent or female-dependent, using reciprocal backcrossings between Nipponbare and the F₁. Crossing of the F₁ (♀) and Nipponbare (♂) showed independent segregation of chromosomes 1 and 6 using 237 progenies, whereas crossing of Nipponbare (♀) and the F₁ (♂) showed clear dependent segregation(χ² = 40.9). The significant reduction of progenies was observed when the reproductive barrier locus on chromosome 1 was heterozygous and that on chromosome 6 was Nipponbare homozygous (Table. 1). This indicated specific elimination of pollens carrying the Kasalath allele on chromosome 1 and the Nipponbare allele on chromosome 6. Thus, this is a male gametophytic barrier caused by the interaction between chromosome 1 (Kasalath allele, 45.4 cM) and chromosome 6 (Nipponbare allele 15.8 cM).

For fine mapping, we have developed PCR markers, i27556 (36.9 cM) and i18100 (50.8cM) on chromosome 1. Twenty plants that have recombination between these markers with Nipponbare homozygous allele at the interactive barrier locus on chromosome 6 were selected from 474 lines of BC₁F₁ and 294 lines of F₂. The genotypes of the 20 recombinant lines at the barrier locus were determined by their progeny analysis of chromosome 1. The genotypes of the 20 recombinants determined by newly developed internal 12 PCR markers showed that the barrier locus was mapped at 45.4 cM between markers i3068_2 and i7872 spanning 220 kb on chromosome 1. Markers S11214, i12296, i6342_2 and i267_2 were co-segregated with the male gametophytic barrier locus (Fig. 1).

For fine mapping of the interactive locus on chromosome 6, 16 recombinants homozygous for the Kasalath allele at the barrier locus on chromosome 1 were selected from 294 F₂ plants using PCR markers, i2121 (10.4cM) and i4790 (32.7cM). Genotypes of the 16 lines with internal 9 PCR markers and progeny (F₃) analysis showed that the barrier locus was mapped at 15.8 cM located between markers i2121 (10.4 cM) and i4099 (18.0 cM) spanning 2.5Mb on chromosome 6. Markers S1520, i3635, i316, i1404_2 and i11601_2 were co-segregated with the male gametophytic barrier locus (Fig. 1). Further fine mapping, sequencing and transgenic experiments would identify the barrier genes.

References

Harushima Y., M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki, T. Yamamoto, S. Y. Lin, B. A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G. S. Khush and T. Sasaki, 1998. A high-density rice genetic linkage map with 2275 markers using a single F2 population. Genet. 148: 479-94.

Harushima Y., M. Nakagahara, M. Yano, T. Sasaki and N. Kurata, 2001. A genome-wide survey of reproductive barriers in an interspecific hybrid. Genet. 159: 883-892.

Wu C.-I. and C.-T. Ting, 2004. Genes and speciation. Genet. 5: 114-122.