Submitted to: Proceedings Sunflower Research Workshop
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2005
Publication Date: 3/15/2005
Citation: Feng, J., Jan, C.C., Vick, B.A., Zhang, H. 2005. Construction of two sunflower (Helianthus annuus L.) BAC and BIBAC libraries and their application to fluorescence in situ hybridization (FISH). Proceedings Sunflower Research Workshop. 27th Sunflower Research Workshop, January 12-13, 2005, Fargo, ND. Available: http://www.sunflowernsa.com/research/research-workshop/documents/Feng_BIBAC_05.PDF Interpretive Summary: Sunflower (Helianthus annuus L.) is an economically important oilseed crop, but the molecular biology and cytogenetics in sunflower have still lagged behind many other major crops including wheat, maize, tomato, rice, and barley. BAC library have been proven essential tools for modern genomics research in many species. To facilitate genomic research and breeding of sunflower, we constructed a BAC library and a plant-transformation-competent BIBAC library for sunflower. The two libraries together contain a total of 192,000 clones, representing about 8.9 equivalents of the sunflower haploid genome (3000 Mb/1C), and provides a greater than 99% probability of obtaining a particular clone from each library. Using a random clone from the BIBAC library as a probe, a fluorescence in situ hybridization (FISH) was conducted in cultivated sunflower. The preliminary analysis of FISH strongly suggests that BAC and BIBAC clones can be successfully used as FISH probes in sunflower and will make it possible to directly order the BAC and BIBAC clones on chromosomes.
Technical Abstract: The BAC and BIBAC libraries were constructed from megabase-size DNA isolated from nuclei preparations of HA89, a widely used sunflower inbred line. The megabase-size DNA was partially digested with HindIII or BamHI, and then ligated into either HindIII-digested BIBAC vector pCLD04541 or BamHI-digested vector pECBAC1, respectively. Ligated DNA was transformed into E. coli strain ElectroMAX DH10B competent cells by electroporation. White colonies were manually arrayed into 384-well microtiter plates. The BAC library was constructed with BamH1 in the pECBAC1 vector and contains 107,136 clones, with an average insert size of about 140 kb and equivalent to 5.0 x of the sunflower haploid genome (3000 Mb/1C). The BIBAC library was constructed with HindIII in the pCLD04541 vector and contains 84,864 clones, with an average insert size of about 137 kb and equivalent to 3.9x of the sunflower haploid genome. Together, the combined libraries represent about 8.9 equivalents of the sunflower haploid genome. A random clone, JF497A2, from the BIBAC library was selected as a probe, and then hybridized with the HA89 chromosome slides by using FISH. The result showed that this BIBAC clone produced six strong signals on three pairs of satellited chromosomes, and is likely related with a Nucleolus organizing region(NOR). Our findings suggest that FISH of BAC clones is an efficient technique for individual chromosome identification and for development of a physical map of the sunflower genome.