Construction of a barley bacterial artificial chromosome library suitable for cloning genes for boron tolerance, sodium exclusion and high grain zinc content

摘要

P>Tolerance to soil boron toxicity, salinity and zinc deficiency is required for maintaining adequate yields on many soils, while a high content of zinc in the grain is desirable for human nutrition. To facilitate the identification of genes for these traits in barley, we constructed a bacterial artificial chromosome (BAC) library of the barley-doubled haploid line CS134 derived from a cross between the Australian malting variety 'Clipper' and the Algerian landrace Sahara 3771. CS134 contains tolerance alleles from Sahara 3771 at four boron tolerance loci, genes at two loci conferring high zinc content in seeds, and a gene from 'Clipper' that limits shoot sodium accumulation. The library contains 221184 clones and has an average insert size of around 120 kb, giving a 4.9-fold coverage of the barley genome. Clones were recovered from the library using probes closely linked to the boron tolerance locus on chromosome 4H. One of the positive BAC clones facilitated isolation and sequencing of the boron tolerance gene (Bot1) at this locus. Therefore, the BAC library represents a useful tool that could be used to isolate several genes of agronomic interest. In the process of constructing this library, we identified several technical modifications that improve the efficiency of BAC library construction.