Fine Mapping And Map-based Cloning The Fertility Restorer Gene Rf4 For C-cytoplasmic Male Sterility In Maize

Male sterility, including nuclear sterility and cytoplasmic male sterility(CMS), is a common phenomenon in higher plants. CMS is the major type in practice through“three-line system”. The production of hybrids by“three-line system”avoids artificial emasculation reducing production costs and mechanical mixing improving seed purity and reducing energy waste increasing hybrid yield. CMS can be further classified into CMS-T, CMS-S, and CMS-C types according to the specific restoration of restored gene for sterile lines. Among them, CMS-C is a sporophytic sterility type with stable sterility and widely used in practice. To elucidate the mechanism of fertility restoration of CMS-C and clone the fertility restorer gene is crucial. In this study, we used the near isogenic lines(NILs) of CMS-C, Cms-ES87-1 line with fertility restorer gene Rf4Rf4 and Cms-ES87-l line with male sterility gene rf4rf4 to explore the molecular mechanism of fertility restoration for male sterile line. The Rf4 gene was fine mapped and cloned by polymorphic markers exploitation, BAC library screening, and sequencing. The results were as follows:1. Genetic analysis was performed with the backcross population of 2,800 plants derived from the cross of Cms-ES87-1 NILs: restorer line Cms-ES87-1(Rf4Rf4)and male sterility line Cms-ES87-l(rf4rf4) . Both fertility investigation and pollen microscopy showed that segregation ratio of the fertile to the sterile was l : l, implying one gene controlling the fertility restoration of Cms-ES87-1 male sterile line.2. Rf4 gene was mapped on chromosome 8 between 8.00 bin and 8.01 bin which was narrowed down to primer 28-4 and the telomere of short arm by SSR designed according to the 87-1 BAC sequence. To fine map Rf4 gene, a large segregation population with 42,200 plants was performed. 240 primers including SSR marker, Insertion/deletion marker, etc, were exploited to increase the molecular marker density between 8.00 bin and 8.01 bin and 26 primers were identified as polymorphism. Finally, the Rf4 gene was fine mapped between X-21-1 and X-33 markers.3. These two tightly linked markers were used as probes to screen 87-1 BAC library constructed in our lab. One BAC containing complete Rf4 region was identified and sequenced. The 45,000 Nt between X-21-1 and X-33 markers was p redicted 9 encoding genes by FGENESH software. And expression vector was constructed for each of the three possible candidate genes according to functional prediction.