Huazhong Agricultural University

Mapping cms-S restorer gene Rf3 with RFLPs and RAPDs
--Shi, YG; Zheng, YL; Li, JS and Liu, JL

The use of the cms inbred line as a female parent to produce maize hybrids is a cost-competitive and satisfactory technique since manual detasseling is eliminated. Three types of male-sterile cytoplasms in maize, designated as T, C and S, have been classified by specific nuclear genes that suppress the male-sterility effects of these cytoplasms and restore pollen fertility. S-cytoplasm is conditioned by interaction of the cytoplasm with a single nuclear gene and fertility is restored by a dominant nuclear gene (Rf3) located on the long arm of chromosome 2. It is probably a long term objective to clone the restorer gene (Rf3) to help us understand its function and the mechanism of fertility restoration. However, as the first step of map-based gene cloning, it is a prerequisite to construct a saturated genetic map of rf3 with more closely linked molecular markers.

To map the rf3 gene, a backcross, (Mo17cms-S rf3 rf3 x HZ1N Rf3 Rf3) x Mo17 N rf3 rf3, was used as the mapping population. Two DNA bulks were constructed from each corresponding to the 20 male-sterile and fertile individuals from this segregating population. Bulked segregant analysis (BSA) was employed to identify RFLP and RAPD markers linked to the restorer gene (Rf3). For RFLP analysis, umc36a/HindIII and umc49/PstI were found to cosegregate with the rf3 allele through screening 36 probe/enzyme combinations. Furthermore, 132 random individuals from the segregating population were analyzed to calculate linkage distance. Analysis of the data with JOINMAP reveals that umc36a and umc49 flank rf3 and are separated from rf3 by 4.8 cM and 12.7 cM, respectively (Figs. 1, 2). For RAPD analysis, 340 arbitrary 10-mer oligonucleotide primers were screened on the two paired bulks. Three primers, E08, M02 and O12 were found to produce one polymorphic DNA fragment between bulks in each case associated with the restorer allele (Fig. 3). To determine the map location of the loci represented by the 1.2kb E08 band, 174 individuals from the mapping population were taken as templates to be amplified with primer E08. Figure 1 shows the location of this RAPD allele relative to rf3 and the flanking RFLP markers. The E08 locus lies 2.7 cM from rf3 beyond umc36a. This specific RAPD E08-1.2kb fragment was extracted from the gel and then cloned in the pBluescript SK (M13-) vector. Correct inserts were released by digesting the recombinant plasmid and eventually used as a probe to hybridize with DNA blots. The preliminary result suggests that the amplified fragment should be a medium repetitive copy.

The work of searching for different types of molecular markers (RAPD, RFLP, AFLP, STS and SCARs) to saturate the genetic region near the rf3 locus continues. When the saturated genetic map is established, it will enable us to apply these markers either in marker-assisted breeding programs or in genome walking strategies.

Figure 1. Region of maize chromosome 2 in the vicinity of rf3. Positions are shown for flanking RFLP markers (umc49 and umc36a) and one RAPD markers, with map distances in cM.

Figure 2. Southern analysis of HindIII-digested DNA hybridized with umc36a. (Right to left) DNAs from male-fertile individuals (Rf3 rf3). Lane 4 from right shows a recombinant.

Figure 3. RAPD data for OPE08. (Right to left) Lane 1: Mo17cms-S rf3 rf3 (parent 1); lanes 2 and 4: male-sterile bulk (rf3 rf3); lane 3: male fertile bulk (Rf3 rf3); lane 5: HZ1N Rf3 Rf3 (parent 2); lane 6: 100bp ladder; remaining lanes: male-sterile individuals. Lane 9 from right shows a recombinant. The arrow shows the male-fertile specific RAPD fragment.

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