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United States Department of Agriculture

Agricultural Research Service

Research Project: Improved Control of Stripe Rust in Cereal Crops

Location: Wheat Genetics, Quality Physiology and Disease Research

Title: Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

Authors
item Zeng, Q. -
item Yuan, F. -
item Xu, X. -
item Shi, X. -
item Nie, X. -
item Zhuang, H. -
item Chen, Xianming
item Wang, Z. -
item Wang, X. -
item Huang, L. -
item Han, D. -
item Kang, Z. -

Submitted to: BioMed Research International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 18, 2014
Publication Date: May 6, 2014
Repository URL: http://www.hindawi.com/journals/bmri/2014/845806/
Citation: Zeng, Q.D., Yuan, F.P., Xu, X., Shi, X., Nie, X.J., Zhuang, H., Chen, X., Wang, Z.H., Wang, X.J., Huang, L.L., Han, D.J., Kang, Z.S. 2014. Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137. BioMed Research International. 2014:Article 845806. Available: http://www.hindawi.com/journals/bmri/2014/845806/.

Interpretive Summary: For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sub library and 573 clones from the BamHI sub library, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sub library was estimated to have a 3.01-fold coverage and the BamHI sub library a 2.53-fold coverage based on the estimated common wheat genome size of 16,700Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest.

Technical Abstract: For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sub library and 573 clones from the BamHI sub library, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sub library was estimated to have a 3.01-fold coverage and the BamHI sub library a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest.

Last Modified: 10/30/2014
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