53. Characterization of monosomic alien chromosomes of O. punctata transferred to O. sativa using RFLP markers

H. YASUI, A. YOSHIMURA and N. IWATA

Faculty of Agriculture, Kyushu University, Fukuoka, 812 Japan

Monosomic alien addition lines (MAAL) are useful in identifying the location of new genes transferred from wild species. Thirteen MAALs of O. punctata, previously classified on the basis of morphological features (Yasui and Iwata 1991), were examined with 20 DNA clones (Saito et al. 1991) assembled into the 10 of 12 RFLP linkage groups.

Japonica rice cultivar Nipponbare (2n=2x=AA), W1514, a strain of 0. punctata (2n=2x=BB), interspecific hybrids (2n=2x=BB), interspecific hybrids (2n=3x=AAB) and 13 MAALs derived from the cross between the two species were used for DNA extraction. The plant materials, their chromosome number and genome constitutions are shown in Table 1. Methods for DNA digestion, electrophoresis and Southern blotting were based on Saito et al. (1991).

Polymorphism for Southern blot pattern between Nipponbare and W1514 was detected in 19 of 20 DNA clones hybridized. As some of the unique sequences in O. sativa were also observed in O. punctata, the 19 DNA clones are useful to characterize alien chromosomes of addition line.

The Southern blot pattern of O. punctata for each clone was detected only in related MAAL except for MAALs 5 and 7 (Type 1) (Table 2), indicating the possible homoeologous relationship between the chromosomes of A and B genomes. MAAL 1 was characterized by 3 DNA clones, Npb121, 93 and 54 (Fig. 1a), located on the chromosome 1 in O. sativa. MAALs 2 and 8 were characterized each by a single DNA clone, Npb 42 (Fig. Ib) or 126 located on the O. sativa chromosomes 2 and 8, respectively. Similarly, MAALs 4, 6, 7 (Type 11), 10 and 12 were characterized by two species of DNA clones assigned into each related the O. sativa chromosomes 4, 6 (Fig. 1c), 7, 10 and 12. 

Table 1. Plant materials used in the study
===============================================================================
Line              Type  2n=  Genome
                             constitution
===============================================================================
MAAL 1              0    25  AA+b\1\
MAAL 2              N    25  AA+b\2\
MAAL 4              E    25  AA+b\4\
MAAL 5              L    25  AA+b\5\
MAAL 6              B    25  AA+b\6\
MAAL 7 (Type I)    F-I   25  AA+b\7\
MAAL 7 (Type II)   F-II  25  AA+b\7\
MAAL 8              D    25  AA+b\8\
MAAL 10             C    25  AA+b\10\
MAAL 11 (Type I)   G-I   25  AA+b\11\
MAAL 11 (Type II)  G-II  25  AA+b\11\
MAAL 12             A    25  AA+b\12\
MAAL  ?             X    25  AA+b?
Nipponbare               24  AA
W 1514                   24  BB
Interspecific hybrids    36  AAB
==============================================================================

Table 2. Types of monosomic addition line and DNA clones showing the same
Southern blot pattern of interspecific hybrids between O.  sativa and O.
punctata
==============================================================================
MAAL           Type  Restriction  Gene No.    Assigned
                     enzyme         (XNpb)  chromosome
==============================================================================
1              0     DraI           54,121           1
1              0     BamHI              93           1
2              N     DraI               42           2
4              E     DraI              114           4
4              E     BamHI             120           4
5              L     BamHI              25           5
5              L     HindIII            81           5
5              L     BamHI              32          10
6              B     HindIII            12           6
6              B     BamHI              27           6
7  (Type  I)   F-I   DraI           54,121           1
7  (Type  I)   F-I   BamHI              93           1
7  (Type  I)   F-I   DraI               31           7
7  (Type  I)   F-I   HindIII            50           7
7 (Type II)    F-11  DraI               31           7
7 (Type II)    F-11  HindIII            50           7
8              D     BamHI             126           8
10             C     BamHI              32          10
10             C     HindIII            37          10
1 1 (Type  I)  G-I   HindIII            52          11
11 (Type II)   G-II  HindIII            52          11
12             A     DraI               79          12
12             A     BamHI             148          12
?              X     BamHI             148          12
==============================================================================
On the other hand, MAAL 5 was characterized by Npb 32 located on the O. sativa chromosome 10 as well as Npb 25 and 81 located on the O. sativa chromosome 5. Therefore, the alien chromosome of MAAL 5 has orientation between XNpb 25 and 81 on the O. sativa chromosome 5 and the region near the XNpb 32 on the O. sativa chromosome 10. Similarly, MAAL 7 (Type I) was characterized by Npb 54 (Fig. 1a), 93 and 121 located on the O. sativa chromosome 1 as well as Npb 31 and 50 located on the O. sativa chromosome 7. The alien chromosome of MAAL 7 (Type I), therefore, has orientation between XNpb3l and 50 on the O. sativa chromosome 7 and the region XNpb121-XNpb93-XNpb54 on the O. sativa chromosome 1. Each extra chromosome in MAALs 5 and 7 (Type 1) could have originated from break and fusion mechanism in the process of producing MAALS.


Fig. 1. Southern hybridization patterns of Oryza sativa cultivar Nipponbare (P\1\), Oryza punctata W1514 strain (P2), their interspecific hybrid (F\1\) and 13 monosomic alien addition lines. (a): 12 MAALs digested with DraI and probed with Npb 54. MAALs 1(O) and 7 (Type 1) (F-1) show detection of the band from O. punctata as well as interspecific hybrid. (b): 12 MAALs digested with DraI and probed with Npb 42. MAAL 2(N) shows detection of the band from O. punctata as well as interspecific hybrid. (c): 13 MAALs digested with HindIII and probed with Npb 12. The band derived from O. punctata for XNpb 12 shifted to lower molecular weight. (d): 14 MAALs digested with DraI and probed with Npb 78. The bands derived from O. punctata for XNpb 78 were not detected in any MAALS.

MAALs 11 (Type I and II) were characterized by one of two DNA clones assigned into each related the O. sativa chromosome 11. XNpb52 was detected on MAALs 11 (Type I and II), but not XNpb78 (Fig. 1d), which is located on the distal end of the O. sativa chromosome 11. Deletion in the distal end of chromosome 11 might have occurred in both of the MAALS.

Finally, autoradiogram pattern derived from interspecific hybrids and MAAL 6 hybridized by DNA clone, Npb 12, were modified from that of W1514 (Fig. 1c). The band from O. punctata in interspecific hybrid and MAAL 6 shifted to a lower molecular weight. This suggested that micro insertion should have occurred in the chromosome 6 on interspecific hybrid and MAAL 6.

This study showed that similar orientation of RFLP markers exist in each homoeologous chromosomes on O. sativa and O. punctata, although some exceptions were found on MAALs 5, 7 (Type 1) and 11 (Type I and II). In such cases, chromosome rearrangement and modification might have occurred in the process of transferring alien chromosomes to cultivated rice.

References

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 fragment length polymorphism loci in rice. Jpn. J. Breed. 41: 665-670.

Yasui, H. and N. Iwata, 1991. Production of monosomic alien addition lines of Oryza sativa having a single O. punctata chromosome. IRRI, Rice genetics II: 147-155.