Construction of a binary BAC library for an apomictic monosomic addition line of Beta corolliflora in sugar beet and identification of the clones derived from the alien chromosome

Abstract

A plant-transformation-competent binary BAC library was constructed from the genomic DNA of the chromosome 9 monosomic addition line of Beta corolliflora Zoss. in sugar beet ( B. vulgaris. L). This monosomic addition line (designated M14) is characterized by diplosporic reproduction caused by the alien chromosome carrying the gene(s) responsible for diplospory. The library consists of 49,920 clones with an average insert size of 127 kb, representing approximately 7.5 haploid genome equivalents and providing a greater than 99% probability of isolating a single-copy DNA sequence from the library. To develop the scaffold of a physical map for the alien chromosome, B. corolliflora genome-specific dispersed repetitive DNA sequences were used as probes to isolate BAC clones derived from the alien chromosome in the library. A total of 2,365 positive clones were obtained and arrayed into a sublibrary specific for B. corolliflora chromosome 9 (designated bcBAC-IX). The bcBAC-IX sublibrary was further screened with a subtractive cDNA pool generated from the ovules of M14 and the floral buds of B. vulgaris by the suppression subtractive hybridization method. One hundred and three positive binary BACs were obtained, which potentially contain the genes of the alien chromosome specifically expressed during the ovule and embryo development of M14, and may be associated with apomictic reproduction. Thus, these binary BAC clones will be useful for identification of the genes for apomixis by genetic transformation.

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

“…These markers provide one tool required for map-based cloning. This report describes the construction of a sugar beet BAC library, which is another resource necessary for the cloning strategy (Gindullis et al 2001;Hohmann et al 2003;Fang et al 2004;McGrath et al 2004). We also describe the use of the BAC library in the assembly of a 250 kb contig spanning Rf1.…”

A BAC library of Beta vulgaris L. for the targeted isolation of centromeric DNA and molecular cytogenetics of Beta species

“…Therefore, it remains open whether the organelle DNA sequences found in the B. vulgaris BAC library originate from chloroplasts and mitochondria or from transferred copies. So far, several B. vulgaris BAC libraries have been published comprising in total 193,000 clones and a larger than 30-fold genome coverage (Gindullis et al 2001a;Hohmann et al 2003;Fang et al 2004;McGrath et al 2004). The PAT2 BAC library presented consists of 36,096 clones with a 5.7-fold genome coverage and will extend the genomic resources available for B. vulgaris to 36 genome equivalents.…”

“…Another strategy for the isolation of a desired gene or sequence is the screening of a second library constructed using a different restriction enzyme. However, for sugar beet, only Fang et al (2004) reported a BAC library using BamHI as the cloning enzyme.…”

“…However, large-insert clones originating from wild relatives of crops (Moullet et al 1999;Lin et al1999;Chen et al 2004) or crops containing alien genomic fragments are rare, and no large-insert library of any wild beet has been reported although they can be used as important donors of agronomically desired traits (Van Geyt et al 1990). Large-insert clones from a monosomic Beta corolliflora chromosome in a sugar beet hybrid have been reported by Fang et al (2004) aiming to investigate the genetic basis for apomixis. Recently, we have established a BAC library of PRO1, a monosomic addition line of sugar beet carrying a chromosome fragment of Beta procumbens and selected clones originating from the wild beet fragment (Gindullis et al 2001a).…”

Identification of a sugar beet BvM14-MADS box gene through differential gene expression analysis of monosomic addition line M14

“…Fang and colleagues reported the construction of a large-insert plant transformation-competent binary BAC library for M14 and the identification of BAC clones covering the alien chromosome (Fang et al, 2004). Li et al (2009) used the comparative proteomic method to analyze the flowers of M14 and B. vulgaris using 2-DE and LC-MS/MS.…”