|Shaw, Robert - Scott|
Submitted to: Annual Beet Sugar Development Foundation Research Report
Publication Type: Experiment Station
Publication Acceptance Date: 5/1/2004
Publication Date: 6/30/2004
Citation: McGrath, J.M., Shaw, R.S. 2004. Real-time pcr analysis of sugar beet bac library. 2003 Annual Beet Sugar Development Foundation Research Report. p. D8-D9.
Technical Abstract: A 6.1X Bacterial Artificial Chromosome (BAC) library was constructed using US H20 as the source DNA. The average insert size was >100kb. In order to facilitate screening the library, a PCR-based resource was developed with assistance of member companies of the BSDF. All 36,864 individual BAC clone DNAs from 100 384-well microtiter plates were isolated and divided into eight groups. All DNA from each group was pooled, resulting in eight Super Pools (SP). Each Super Pool was further subdivided into one of 36 'Matrix' Pools for subsequent localization of a specific PCR amplified fragment to a specific BAC clone. To test efficacy, Super Pools were screened using a PCR/SYBR Green-based assay with 29 gene-targeted primers and 42 Simple Sequence Repeat (SSR) markers. Seventeen (58.6%) of the gene-target primers and 35 (83.3%) of the SSR markers were able to be located to specific Super Pools. Matrix Pools corresponding with putative positive SP hits were screened with three primers (calmodulin, ABC transporter, BvGer171). Single clones were identified with the calmodulin primers in each of SP7 and SP8, and for ABC transporter (SP5, SP8). Multiple signals were observed in SP1 and SP5 when screened with BvGer171, and these were difficult to assign to a specific clone because of their redundancy since 6 to 12 copies of this or similar sequences are present in the beet genome. Thus, the strategy to assign clones from pools to individual clones appears to be valid for single copy genes or members of small gene families, but more problematic for higher copy number sequences. These pools will be useful, in part, to locate genes to BACs and construct a physical map of sugarbeet.