Submitted to: Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/9/2006
Publication Date: 4/13/2006
Citation: Feng, J., Vick, B.A., Lee, M., Zhang, H., Jan, C.C. 2006. Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization. Theoretical and Applied Genetics. 113:23-32. Interpretive Summary: Large-insert BAC and BIBAC libraries have been proven essential tools for modern genomics research in many species. To facilitate genomic research and breeding of sunflower, two complementary BAC and BIBAC libraries were constructed from nuclear DNA of a sunflower inbred line HA89 by using different restriction enzyme and vector systems. 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. The BIBAC library was constructed with HindIII in the pCLD04541 vector and contains 84,864 clones, with an average insert size of about 132 kb. Either of the libraries provides a greater than 99% probability of obtaining a particular DNA sequence from each library. Together, the combined libraries represent about 8.9 equivalents of the sunflower haploid genome (3000 Mb/1C). Using specific probes "overgos" designed from linkage group-specific RFLP clones, we have identified linkage group-specific BAC and BIBAC clones. These selected BAC and BIBAC can be used to identify a set of 17 trisomic genetic stocks having different extra chromosomes of the sunflower genome.
Technical Abstract: Two complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library was constructed with BamHI in the pECBAC1 vector. It contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries together contain a total of 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), providing a greater than 99% probability of obtaining a particular clone from the libraries. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35% and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to chromosomes, one to three single- or low-copy RFLP markers from each linkage group of the sunflower genetic map were used to design pairs of overlapping oligonucleotides (overgos). A total of 36 overgos were designed and pooled as probes to screen a subset (5.1x) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups of the genetic map were identified, including 76 BAC clones and 119 BIBAC clones, which have further verified the genome coverage and utility of the libraries in genome research. Therefore, the BAC and BIBAC libraries and linkage group-specific clones provide resources and tools essential for comprehensive research of the sunflower genome.