Sugar beet BAC library construction and assembly of a contig spanning Rf1, a restorer-of-fertility gene for Owen cytoplasmic male sterility

Abstract

Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb.

Unusual and Typical Features of a NovelRestorer-of-FertilityGene of Sugar Beet (Beta vulgarisL.)

“…The sugar beet Rf1 had previously been located to a region delimited by two molecular markers, mP-A16 and mCP-L6 (Hagihara et al 2005b). The region was covered with an array of ordered BAC clones (Hagihara et al 2005b).…”

“…The region was covered with an array of ordered BAC clones (Hagihara et al 2005b). To obtain a nucleotide sequence of this region, we selected three of the clones, 5A3, 9C23, and 33E19 (Hagihara et al 2005b), as sequencing templates.…”

“…The region was covered with an array of ordered BAC clones (Hagihara et al 2005b). To obtain a nucleotide sequence of this region, we selected three of the clones, 5A3, 9C23, and 33E19 (Hagihara et al 2005b), as sequencing templates. To minimize sequence redundancy, we screened a cosmid clone bridging 5A3 and 33E19, from a sublibrary made from 9C23 by using probes made up of 5A3- and 33E19-BAC ends.…”

“…The NK–198 Rf was named Rf1 and mapped to a terminal region of chromosome III, suggesting that the Rf1 was an allele of the X locus (Hagihara et al 2005a). Molecular markers linked to Rf1 were used to isolate BAC clones that covered the Rf1 locus (Hagihara et al 2005b). …”

Construction of a Spinach Bacterial Artificial Chromosome (BAC) Library as a Resource for Gene Identification and Marker Development

“…BAC libraries have been constructed for many major crops, such as rice, maize, wheat, soybean, and cotton (Salimath and Bhattacharyya 1999;O'Sullivan et al 2001;Tomkins et al 2001;Tao et al 2002;Allouis et al 2003). To date, a BAC library is not available for spinach, although libraries have been created for related members of the Chenopodiaceae, such as sugar beet (McGrath et al 2004;Hagihara et al 2005) and quinoa (Chenopodium quinoa) (Stevens et al 2006).…”