Huazhong Agricultural University
Structure and sequence analysis of the R region in S-type CMS maize --Zhang, FD, Xiao, HL, Zheng, YL
The S-type cytoplasmic male sterility (CMS) was reported to be associated with the R region in the mitochondrial genome. A fragment designated as R' was obtained by PCR from the R region. The R' used as a probe was hybridized to BamHI and/or PstI digests of the mitochondrial DNA (mtDNA) from seventeen maize lines, which included N, T, C, and S types of cytoplasms within two nuclear backgrounds of Mo17 and 77. The hybridization pattern of R'/BamHI (Figure 1) demonstrated that there was no fragment of the R region in the C group, while different hybridization bands appeared in the N, T, and S groups. As for the T group, only a fragment of about 0.5kb was found in the hybridization pattern of R'/BamHI+PstI (Figure 2). Maybe there is only a partial R segment in the T group.

Figure 1. R'/BamHI RFLP pattern. At far left is DNA/HindIII. From top to bottom are 23.1kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb. From left to right the others are the 17 types of maize lines: 1.Mo17N; 2.Mo17CMS-T; 3.Mo17CMS-C; 4.Mo17CMS-RB; 5.Mo17CMS-EL; 6.Mo17CMS-TangXu; 7.Mo17CMS-Shung; 8.MO17CMS-J; 9.77N; 10.77CMS-T; 11.77CMS-C; 12.77CMS-EL; 13.77CMS-TangXu; 14.77CMS-Vg; 15.77CMS-Jiang; 16. 77CMS-RJing; 17.77CMS-S. These maize lines of No.6, 7, 8, 13, 14, 15, 16, 17 all belong to the S group.

Figure 2. R'/BamHI +PstI RFLP patterns.

Three bands were found in the Rí/BamHI hybridizaton pattern of the S group, of which the size was 6.7kb, 4.5kb and 1.8kb respectively. We presumed the DNA fragments of 6.7kb and 4.5kb were in the circular mitochondrial genome. They all contained the R region and recombined with S1 or S2 plasmids through the IR. This leads to linearization of the mitochondrial chromosome, which produced the linear end fragments of 1.8kb. There is a PstI site in the IR region, so only one form of DNA bands appeared in the R'/BamHI+PstI RFLP pattern in the S group (Fig.2). The structure and recombination model of the R region was shown in Figure 3.

Figure 3. Structure and recombination model of the R region. S1 and S2, which only exist in S cytoplasms, can recombine with the mitochondrial main genome through the IR. This leads to linearization of mitochondrial chromosome. Three types of R can be seen in R'/BamHI, including the linear end 1.8kb fragments. The coxI gene was located at 5' upstream of the R region of pV48 and coxII was found at the 5' flank region of R of pB39.

Meanwhile, the BAC mitochondrial genome library from Mo17CMS-J was constructed. After screening it with the R' probe, several clones containing the R region were detected. One of these clones, B39, was found to contain the intact 6.7kb fragment. After digestion with BamHI, it was subcloned and named pB39. Another clone, V48, containing the complete 4.8kb fragment, was also subcloned. pB39 and pV48 were sequenced, and the results were submitted to the Gene Bank. The accession numbers are AF545834 and AF542203, respectively. The nucleotide sequence showed that both of them contained orf355 and orf77, which were considered to be the main sequence in the R region. In the clone of pV48, the coxI gene was located at the 5' flank region of R, which was coincident with the former report. In the clone of pB39, the first exon of coxII was found in the upstream area of the R region. Up to now, this kind of R region has not been reported. Both cox1 and coxII were in the opposite direction of the orf355 and orf77. So the structure of the R region in this study is different from the other types. We did not know the exact relationship between the cytoplasmic male sterility, the R region and its upstream genes. Maybe this unsteady structure is responsible for the unstableness of CMS-S, because obviously the promoter of the R region is easily changed in different nuclear backgrounds (data not shown). In depth research on the transcription and protein products of the R region is underway.

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