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Construction and Characterization of a BAC Library of a Cold‐Tolerant Hexaploid Wheat Cultivar
Author(s) -
Ratnayaka Indira,
Båga Monica,
Fowler D. Brian,
Chibbar Ravindra N.
Publication year - 2005
Publication title -
crop science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.76
H-Index - 147
eISSN - 1435-0653
pISSN - 0011-183X
DOI - 10.2135/cropsci2004.0543
Subject(s) - biology , insert (composites) , genome , locus (genetics) , plasmid , genomic library , bacterial artificial chromosome , ploidy , genetics , dna , cultivar , genome size , botany , gene , base sequence , mechanical engineering , engineering
A bacterial artificial chromosome (BAC) library was constructed from DNA of a commercially grown winter wheat cultivar ( Triticum aestivum L. cv. Norstar) that is highly tolerant to low temperatures (LT 50 = −24°C). The library was produced from nuclear leaf DNA that was partially digested with Hin dIII and inserted into pCC1BAC vector. In excess of 1.2 × 10 6 clones propagated in E. coli were obtained, archived in 384‐well microtitre plates and stored at −80°C. More than 97.1% of the recombinant plasmids contained inserts and were free from organelle DNA contamination. Analysis of 119 randomly isolated clones revealed insert sizes ranging from 17 to 262 kb with 26% of the inserts exceeding 100 kb. The average insert size was 75 kb and the genome coverage of the library was theoretically 5.5 times the haploid genome equivalent. This corresponds to a 99.6% probability of recovering any specific Norstar DNA sequence from the library. Screening of the library with nine simple sequence repeat (SSR) locus‐specific markers supported a >4.4‐fold genome coverage. The BAC library was gridded onto high‐density filters and will be used for isolation of genetic loci associated with cold‐tolerance and grain quality traits.