Steps towards map-based cloning Ma(1), a gene controlling photoperiod sensitivity in sorghum.

摘要

Ma1 is important for sorghum production as it has a strong effect on the regulation of flowering photoperiod sensitivity. The goal of this dissertation is to describe the isolation of genetic markers and genomic clones that are linked to Ma1 as steps toward map-based cloning of the gene. AFLP markers were first optimized for amplifying sorghum DNA. A sorghum genetic map using 245 AFLPs was constructed covering 1473 cM. A different population segregating for Ma1 was to identify linked markers. Eighteen markers were identified using bulk segregant analysis, ten of which cosegregate with the gene. The additional markers flank the Ma1 locus by 0.2 cM in one direction and 0.3 cM in the other. Some of these markers were mapped within other sorghum populations. These markers localize Ma1 to linkage group I between RFLP markers umc119 and isu142. Previous comparative mapping between different grass species suggest that this genomic region is strongly involved in controlling photoperiod sensitivity in other cereals besides sorghum. The Ma 1-linked markers may aid in marker-assisted breeding of plants with a desired photoperiod sensitivity.; BAC clones were pooled to allow PCR screening of the library due to difficulties encountered when using the markers as hybridization probes. Our methodology utilizes a six-fold pooling approach. The pools are constructed from a three-dimensional stack of clones which include three additional pools aligned on a diagonal across two orthogonal pools. These pools are more efficient than other pooling strategies when they are sampled using multiplex PCR techniques such as AFLPs. This methodology is also useful in aligning BAC clones for physical map construction. Genomic clones were isolated by hybridization and by PCR screening the pooled BAC clones. Thirteen BAC clones and three lambda clones were isolated using markers that cosegregate with Ma1 and ten BACs flank the borders of the Ma1 locus. One of the BAC clones was a useful probe in BAC FISH experiments. The location of this clone combined with another clone previously localized to linkage group I suggests that the Ma1 locus may be small enough to continue efforts towards the map-based cloning of Ma1.