ARS | Publication request: BAC Libraries from Wheat Chromosome 7D – Efficient Tool for Positional Cloning of Aphid Resistance Genes

Submitted to: Journal of Biomedicine and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 26, 2010
Publication Date: December 18, 2010
Citation: Simkova, H., Safar, J., Kubalakova, M., Suchankova, P., Cihalikova, J., Robert-Quatre, H., Azhaguvel, P., Weng, Y., Peng, J.H., Lapitan, N.L., Ma, Y., You, F.M., Luo, M., Bartos, J., Dolezel, J. 2010. BAC Libraries from Wheat Chromosome 7D – Efficient Tool for Positional Cloning of Aphid Resistance Genes. Journal of Biomedicine and Biotechnology. Available: http://www.hindawi.com/journals/jbb/2011/302543.html.

Interpretive Summary: Cloning of genes through the map-based cloning strategy (positional cloning) in bread wheat is a tedious task due to its huge genome size and polyploid nature. BAC libraries with larger insert DNA fragments represent an essential tool for positional cloning. However, wheat BAC libraries often comprise more than one million clones, which make their screening very laborious. Here we present a targeted approach based on using chromosome arm-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library derived from the short arm (7DS) consists of 49,152 clones with 113 kb mean insert size and represents 12.1 arm equivalents whereas a library from the long arm (7DL) comprises 50,304 clones of 116 kb providing 14.9 × arm coverage. The 7DS library was PCR screened with markers for Russian wheat aphid resistance gene DnCI2401, whereas the 7DL library was screened by hybridization targeting greenbug resistance gene Gb3. Small number of clones combined with high coverage made the screening highly efficient and cost-effective.

Technical Abstract: Positional cloning in bread wheat is a tedious task due to its huge genome size (~17 Gbp) and polyploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which make their screening very laborious. Here we present a targeted approach based on using chromosome-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library derived from the short arm (7DS) consists of 49,152 clones with 113 kb mean insert size and represents 12.1 arm equivalents whereas a library from the long arm (7DL) comprises 50,304 clones of 116 kb providing 14.9× arm coverage. The 7DS library was PCR screened with markers for Russian wheat aphid resistance gene DnCI2401, the 7DL library was screened by hybridization targeting greenbug resistance gene Gb3. Small number of clones combined with high coverage made the screening highly efficient and cost-effective.