45. RFLP mapping of nuclear DNA sequences homologous to organellar DNAs.

National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602, Japan.

Nuclear DNA sequences homologous to organellar DNAs have been reported in plant species including rice, as well as animal and microbial species (Blanchard and Lynch, 2000). However little is known about the distribution of sequences homologous to the organellar DNAs on the rice nuclear genome. Here we report the rice loci for some of the organellar DNA homologues determined by RFLP mapping and the aggregated distribution of the loci on a rice linkage map.

First we obtained 11 organellar DNA clones from the DNA libraries which were made of rice total DNA (c.v. 'Nipponbare') to construct an RFLP map of rice (Saito et al., 1991), based on DNA sequence analysis of ca.300-400bp of each insert in these DNA clones. Next we found that some of the hybridizing faint bands produced by these DNA clones showed RFLPs between an indica rice 'Kasalath' and a japonica line 'FL134' and segregated in the F2 population derived from the crossing of these two lines. The F2 population is the same population used to construct an RFLP map of rice (Saito et al., 1991). In scoring for segregating genotype in the autoradiograms from probed F2 blotting filters, we have given the definition of a single locus as described below; when all the F2 individuals have either one of two bands (A or B) or both (A and B), these two bands indicate a pair of alleles on a single locus; when at least one individual has neither of these two bands, the bands (A and B) indicate two different loci. RFLP segregation data of these faint bands were analyzed using MAPL (Ukai et al., 1991) to determine the loci of these bands on the rice RFLP map (Saito et al., 1991).

Table 1 shows the segregation ratios of 19 loci identified with the 11 organellar clones. Five

of the 11 clones (XNpb060, XNpb478, XNpb483, XNpb538 and XNpb543) detected multiple loci. Two of the 19 loci exhibited significant deviation from Mendelian inheritance (Table 1). The 19 loci were mapped on 9 chromosomes. The multiple loci detected with a single clone did not cluster each other, and scattered either on separate chromosomes or on the same chromosome without significant linkage (Figure 1). The 19 loci showed random distribution on the 12 chromosomes and aggregated distribution at 20-cM intervals ('Goodness of fit test' of observed frequency distribution of the 19 loci at the 12 chromosomes to binomial distribution: chi2 = 0.790, p > 0.5, the number of the loci = 19, df = 2; 'Goodness of fit test' of observed frequency distribution of the 19 loci at 20-cM intervals to Poisson distribution: chi2 = 5.120, p < 0.025, the number of the intervals = 100, df = 1). Ten of the 19 loci formed four clusters on three chromosomes (chr. 1, 10 and 12). A cluster on chr. 1 consisted of 2 loci, XNpb460 and XNpb466, which were linked with a distance of 3.4 cM. Another cluster, located on chr. 10, consisted of 3 loci, XNpb483-4, XNpb467 and XNpb060-1, which were linked with distances of 3.6 cM and 2.2 cM in this linear order, respectively. The third and fourth clusters were located on chr. 12. The third one consisted of 2 loci, XNpb543-2 and XNpb060-2, which were tightly linked with 0 cM distance. The fourth one consisted of 3 loci, XNpb538-2 - XNpb478-1 - XNpb457, which were linked with distances of 2.8 cM and 0 cM in this linear order, respectively. Also other two loci, XNpb538-1 and XNpb537, showed close linkage to the loci

of sequences homologous to mitochondrial plasmid-like DNAs (B1 to B4; Kanazawa et al., 1993), XNpbB1-1 and XNpbB4 on chr. 1 and XNpbB2 and XNpbB3 on chr. 8, respectively.

We have performed preliminary blast search using rice genome databases of a japonica line 'Nipponbare' made by RGP and TIGR to see if there are nuclear DNA regions similar to the whole mitochondrial DNA (Accession: AB076665 and AB076666 by Notsu et al., 2002) at the two pairs of linked loci with 0 cM distance on chr. 12 which detected with mitochondrial DNA clones in this study. So far we have found no BAC/PAC clones containing long sequences similar to the whole mitochondrial DNA from chr. 12 though there are BAC clones carrying sequences similar to many mitochondrial DNA fragments some of which are more than 10 kpb (Kishimoto et al., unpubulished).

Getting together, clustering of the loci for sequences homologous to organellar DNAs suggests that there may be chromosome regions tending to be inserted by organellar-originated DNAs. In addition, The 19 loci identified in this study had a tendency to be located near deduced centromere regions (Figure 1), suggesting organellar-originated DNAs might be liable to be transferred to heterochromatic regions, especially those near centromeres.


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