46. A current RFLP linkage map of rice: Alignment of the molecular map with the classical map

1) Department of Molecular Biology, National Institute of Aerobiological Resources, 2-1-2 Kannondai, Tsukuba City, 305 Japan

2) Department of Low Temperature Technology, Hokkaido National Agric. Exp. Station, Hitsujigaoka, Sapporo, 062 Japan

We previously constructed a detailed genetic map of rice (referred to as "a basic RFLP map"), Oryza sativa L., including 347 loci by linkage analyses of RFLP markers and twelve chromosome-specific genetic markers (isozymes, morphological and physiological markers). By mapping of other three chromosome- specific genetic markers and trisomic analyses, all the linkage groups of the basic RFLP map were completely assigned to the 12 chromosomes in the classical map (Saito et al. 1991). The classical map, however, includes many loci which are

related to agronomically important phenotypes, e.g., pest resistance (Kinoshita 1990). If we can find RFLP markers that are associated with the map loci, they could be useful for the rapid and precise gene tagging.

In this study, we performed RFLP mapping of several chromosome-specific genetic markers to locate the orientation of respective RFLP linkage group within the corresponding linkage group. For this purpose, we used several crosses between indica and japonica types (Table 1) and compared the order of the marker genes common to the two maps (Kishimoto et al., unpublished). A total of six linkage groups in the basic RFLP map were aligned with the corresponding linkage groups in the classical map, as follows (Figs. 1 and 2):

Chr. 1: Two genes for coloration, A and Pn, were mapped between XNpb136 and XNpb121 and between XNpb121 and XNpb165-2, respectively. A morpho-

Fig. 2. Our current RFLP linkage map. of rice. Our RFLP map and the classical map are shown with thick bars and thin bars. Large figures at the top of the bars are the chromosome numbers. Chromosome 1, 9 and 12 are oriented with the short arm on top (Nonomura et al., 1992). Plain figures and letters at the right side of the thick bars indicate RFLP markers (figures only), cloned genes and conventional markers mapped in our RFLP map. Large italic letters indicate the conventional markers placed in relative position to our RFLP map. Open triangles denote RFLP markers linked with one conventional marker at least.

logical gene for panicle, lax, was linked with XNpb113 previously (Saito et al. 1991). This suggests that the basic RFLP map covers the whole chromosome, although the two genes for chlorophyll aberration, v-6 and fs-2. at the terminals in the classical map are not located in the basic RFLP map.

Chr. 2: In our previous study (Saito et al. 1991), two genetic markers, d-30 (a gene for dwarfness) and Lap (an isozyme), were mapped between XNpb388 and XNpb357, and between XNpb227 and XNpb223, respectively. However, this chromosome could not be aligned with the classical linkage groups because lap was not localized in the classical map. The length of this RFLP linkage group was almost the same as that of the classical one. Recently, Ideta et al. (1992) detected linkage relation between tri (a morphological gene for grain) and XNpb 250, and aligned these linkage groups as shown in Fig. 2 (Ideta et al. 1992).

Chr. 3: A chlorophyll aberration gene, chl-1, at a terminal locus in the classical map, was mapped previously at one terminal in the basic RFLP map (Saito et al. 1991). A morphological gene for leaf, dl, at the other terminal region was found to be linked with XNpb394. As the result, the RFLP linkage group was aligned along the classical map (Fig. 2).

Chr. 4: Two marker genes, Ig and Ph, were previously mapped at the terminal region in the basic RFLP map (Saito et al. 1991). This allowed to align the RFLP linkage group along the classical map as shown in Fig. 2. Recent RFLP mapping of Xa-1 (a disease resistance gene) confirmed this orientation (Yoshimura et al. 1992). A-dwarfing gene, d-2, is located on the opposite side to Ph and is separated by about 100 cM from lg in the classical map. However, d-2 is not covered with any RFLP markers. It is not clear why about a half of these classical linkage groups could not be covered by the RFLP map.

Chr. 5: A morphological marker, nl-1, located at the terminal in the classical map, was mapped between XNpb188 and XNpb025 in a terminal region of the basic RFLP map. This allowed us to infer the orientation shown in Fig. 2. Therefore, most of the classical linkage groups may overlap with those of the basic RFLP map.

Chr. 6: Two genetic markers in the classical map, Est-2 and alk, and a cloned gene, cmWx (its locus seems to be identical to the locus of the phenotypic gene, wx) were mapped previously in the basic RFLP map (Saito et al. 1991). The RFLP and the classical linkage groups were aligned by means of the ordering of the three markers. In this study, a chromogen gene, C located as a central locus in the classical map, was mapped between XNpb165-1 and XNpb2OO. This confirmed the location of the C locus between Est-2 and wx on the classical map.

Chr. 7: A gene for coloration of pericarp, Rc, located at the central region in the classical map, was mapped at the terminal in the RFLP map (Saito et al. 1991). In this study, a gene for chlorophyll aberration, rfs, was mapped between XNpb152 and XNpbO2O. These results permitted us to determine the orientation in Fig. 2, and showed that about a half of this classical linkage group (from Rc to d-6; 44 cM) could not be covered by the present RFLP map.

Chr. 8: The classical linkage group is composed of two conventional markers, sug and v-8, which was assigned by the trisomic analysis (Isono et al. 1978; Iwata et al. 1984). These two markers, however, are not linked each other (Khush et al. 1984). The RFLP linkage group of this chromosome was made using sixteen RFLP markers, covering over 126 cM.

Chr. 9: Three genes, Dn-1, drp-2 and dp-2 located in the linkage group in the classical map, were mapped between XNpb339 and XNpb103 of the RFLP map. In addition, a morphological panicle marker, Bp, that was assigned by the trisomic analysis, was mapped between XNpb339 and XNpb112. The other linkage group including gm, I-Bf and lam(t) remains unlocated in the RFLP map.

Chr. 10: A gene for chlorophyll aberration, fgl, located at the center in the classical map, was mapped between XNpb291 and XNpb127 at the center of the RFLP linkage group (Saito et al. 1991). Although the orientation was unclear yet, the comparison of the total map distance of the corresponding linkage group indicates that the RFLP linkage group may cover the whole recion of the classical one.

Chr. 11: In our previous study (Saito et al. 199 1), a morphological gene for panicle (sp) and the cloned maize alcohol dehydrogenase gene, cmADH (its locus seems to be identical to the locus of the isozyme, Adh-1) were mapped between XNpb179 and XNpb044, and between XNpb115 and XNpb180, respectively, although these two genes were linked each other by a so small map distance, (1.0 cM) in the classical map, and we could not align these linkage groups. A morphological gene, la, was found to be linked with XNpb202 by 13.1 cM in this study. These results allowed us to align these linkage groups as in Fig. 2. A total linkage length of the RFLP map is larger than that of the classical map.

Chr. 12: An isozyme marker, Sdh-1, was mapped at the center in the RFLP map in the previous study (Saito et al. 1991). In this study, we mapped two genes, d-33 assigned in one linkage including Sdh-1, and spl-1, in the other linkage group, between XNpb4O2 and XNpb148, and between XNpb154 and XNpb124-1, respectively. The former linkage part was aligned along the RFLP map, while the orientation to the latter part remains unclear. This result is consistent with a map recently reconstructed by Ishikawa et al. (1992).

As a whole, the RFLP map covers most regions of the classical map except parts of chromosomes 4 and 7. This failure may be due to the presence of large heterochromatin regions or homologous regions between indica and japonica types in these chromosomes, rather than limitation of the genomic library made using PstI or HindIII digestions.

The integration of the RFLP map and the classical map containing conventional markers will provide great advantage for mapping unassigned genes on the classical map simply searching for RFLP markers. For example, in the chromosome 4, the rice bacterial blight resistance gene, Xa-1, which was closely placed to Ph in the classical map, was localized on the opposite side of Ph using RFLP markers near Ph (Yoshimura et al. 1991). A restorer gene in chromosome 10, Rf-1, could be rapidly mapped between XNpb291 and XNpb127 (Fukuta et al. 1992). In addition, using the RFLP markers, we succeeded mapping the following new genes that had no map-information: mitochondorial plasmid-like DNAs (Bl-1, Bl-2 and B2; Kanazawa et al. 1991), a gene for the scent of leaf (scl), and a gene controlling glucomannan content in endosperm cell wall of rice (Gmr; Yano et al. 1991a, b).

Although about 500 characters are recognized as genetic phenotypes, only about one third of them have been mapped on the rice chromosomes for over 70 years. Progress in the integration of RFLP and classical maps may facilitate to estimate the coverage of the classical map by the current RFLP map and to identify the actual number of available RFLP markers. More complete integration of the basic RFLP map and the classical map will be necessary to correlate the genetic and physical maps and to apply the basic genetic research to the breeding of rice and the map-based cloning.

We are indebted to many rice researcher, especially to Dr. M. Yano, Dr. M. Kawase, Mr. E. Shimosaka and Mr. S. Matsuura for evaluation and scoring of the genetic markers.

Fukuta, Y., M. Yano and K. Fukui, 1992. Linkage analysis of the restorer gene (Rf-1) in rice using restriction fragment length polymorphism markers. Jpn. J. Breed. 42 (Suppl. 1): 164- 165. (in Japanese)

Ideta, O., A. Yoshimura, H. Tsunematsu, T. Matsumoto and N. Iwata, 1992. Integration of RFLP and conventional linkage maps in rice. In Agricultural Biotechnology (Proc. of Asia-Pacific Conference on Agricultural Biotechnology), C.B. You and Z.L. Chen (eds.), p. 163-166, China Science and Technology Press, Beijing.

Ishikawa, R., T. Harada, M. Niizeki and K. Saito, 1992. Reconstruction of linkage map with isozymes, morphological and physiological markers in rice chromosome 12. Jpn. J. Breed. 42: 235-244.

Isono, Y., H. Satoh and T. Omura, 1978. Characteristics of carbohydrate-synthetic mutants, sugary and shrunken, in rice. Jpn. J. Breed. 28 (Suppl. 1): 130-131. (in Japanese)

Iwata, N. and T. Omura, 1975. Studies on the trisomics in rice plants (Oryza sativa L.), III. Relation between trisomics and genetic linkage groups. Jpn. J. Breed. 25: 363-368.

Iwata, N. and T. Omura, 1984. Studies on the trisomics in rice plants (Oryza sativa L.), VI. An accomplishment of a trisomic series in japonica rice plants. Jpn. J. Genet. 59: 199-204.

Kanazawa, A., W. Sakamoto, N. Kishimoto, M. Yano, N. Tsutsumi, A. Saito and M. Tano, 1991. Linkage analysis of the nuclear homologues of mitochondorial plasmid-like DNAs in rice. Jpn. J. Genet. 66: 597-607.

Khush, G.S., R.J. Singh, S.C. Sur and A.L. Librojo, 1984. Primary trisomics of rice: origin, morphology, cytology, and use in linkage mapping. Genetics 107: 141-163.

Kinoshita, T., 1990. Report of the committee on gene symbolization, nomenclature and linkage groups. RGN 7: 16-50.

McCouch, S.R., G. Kochert, Z.H. Yu, Wang, G.S. Khush, W.R. Coffman and S.D. Tanksley, 1988. Molecular mapping of rice chromosomes. Theor Appl Genet 76: 815-829.

Nonomura, K., A. Yoshimura, N. Kishimoto and N. Iwata, 1992. Rice chromosome mapping by using reciprocal translocations and tertiary trisomics. In Agricultural Biotechnology (Proc. of Asia-Pacific Conference on Agricultural Biotechnology), C.B. You and.Z.L. Chen (eds.), p. 180-183, China Science and Technology Press, Beijing.

Saito, A., M. Yano, N. Kishimoto, M. Nakagahra, A. Yoshimura, K. Saito, S. Kuhara, Y. Ukai, M. Kawase, T. Nagamine, S. Yoshimura, O. Ideta, R. Ohsawa, Y. Hayano, N. Iwata and M. Sugiura, 1991. Linkage map of restriction fracment length polymorphism loci in rice. Jpn. J. Breed. 41: 665-670.

Yano, M., E. Shimosaka, A. Saito and M. Nakagahra, 1991. Linkage analysis on gene for scent in indica rice variety, Surjamkhi, using a restriction fragment length polymorphism markers. Jpn. J. Breed. 41 (Suppl. 1): 338-339. (in Japanese)

Yano, M., R. Zamorski, A. Saito and N. Shibuya, 1991. Linkage analysis of the gene controlling glucomannan content in endosperm cell wall of rice. Jpn. J. Breed. 41 (Suppl. 2): 498-499. (in Japanese)

Yoshimura, S., A. Yoshimura, A. Saito, N. Kishimoto, M. Kawase, M. Yano, M. Nakagahra, T. Oaawa and N. Iwata, 1992. RFLP analysis of introgressed chromosomal segments in three near-isogenic lines of rice for bacterial blight resistance genes, Xa-1, Xa-3, and Xa-4. Jpn. J. Genet. 67: 29-37.

Yu, Z.H., 1991. Molecular mapping of rice (Oryza sativa L.) genes via linkage to restriction fragment length polymorphism (RFLP) markers. Doctor thesis of Cornell University.