RFLP analysis of maize mtDNA using important function genes as probes
--Jiang, YH; Zhang, FD; Xia, T; Zheng, YL
Cytoplasmic male sterility (CMS) in maize has been classified into three groups (T, C and S) based on the differential restoration by nuclear fertility restorer genes. Unique chimeric genes in mitochondria are always thought to be responsible for the male sterility phenotype. T-urf13 and atp6-C are postulated to be the causes of CMS-T and CMS-C correspondingly and they are both chimeric genes: T-urf13 is rearranged in rrn26 and an unknown nucleotide sequence; atp6-C is a triple gene fusion product comprised of DNAs derived from atp9, atp6 and an open reading frame of unknown origin. CMS-S is the most sufficient cytoplasm in CMS of maize, but there is less knowledge whether it is due to chimeric genes.
To identify mitochondrial genes that are potentially rearranged with respect to the N cytoplasm, RFLP analysis was conducted using available clones of mitochondrial genes which have important functions. The mtDNAs from 18 kinds of maize including N (normal) and CMS-T, C, S were digested with HindIII, BamHI, EcoRI, PstI and hybridized with mitochondrial gene probes: coxI, coxII, cob, atp6, atp9, atp-alpha, rrn26, rrn5-18. All of these genes are frequently involved in chimeric genes according to the results from rice, rapeseed, sorghum, sunflower, etc. RFLP maps show abundant polymorphisms among different cytoplasms. Analysis of data suggests that there may be two copies of atp-alpha in N and CMS-S, one copy from CMS-S may be the same as that from N, but the others display significant differences. CMS-C and CMS-T mitochondria only possess one copy of atp-alpha. When mtDNAs were digested with HindIII and hybridized with cob, special bands about 4.4kb appeared in CMS-S (Figure 1). In coxI/HindIII, bands about 4.4kb also were found specially in CMS-S (Figure 2). These three genes: coxI, cob, atp-alpha, were assumed to be related with CMS-S. atp6 gene was reported to be involved in CMS-C. It is interesting that variance was observed among the CMS-C group in atp6/EcoRI combination (Figure 3). This provides a probable way to elucidate the nature of subgrouping.
Figure 1. RFLP maps of cob/HindIII. From left to right the order is: Mo17-N, T group (T), C group (RB, EL, C), S group (TANGXU, SHUANG, J) and 77-N, T group (T), C group (EL, C), S group (VG, TANGXU, SHUANG, JANG, S, RJIN). DNA markers lambda/HindIII are located at the left of the photo. The arrows show the 4.4kb bands in S group and the 6.5kb bands in the other cytoplasms.
Figure 2. RFLP map of coxI/HindIII. The arrow shows the special bands about 4.4kb in S group.
Figure 3. The arrow shows bands in Mo17-EL and 77-EL among C group in atp6/EcoRI combination.
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