|Shi, Bu-Jun -|
|Button, Jasper -|
|Miyazaki, Junji -|
|Pallotta, Margaret -|
|Gustafson, Nicholas -|
|Zhou, Hui -|
|Langridge, Peter -|
|Collins, Nick -|
Submitted to: Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 21, 2009
Publication Date: June 16, 2009
Citation: Shi, B., Gustafson, J.P., Button, J., Miyazaki, J., Pallotta, M., Gustafson, N., Zhou, H., Langridge, P., Collins, N. 2009. Physical Analysis of the Complex Rye (Secale cereale L.) Alt4 Aluminium (Aluminum) Tolerance Locus Using a Whole-Genome BAC Library of Rye cv. Blanco. Theoretical and Applied Genetics. 119:695-704. Interpretive Summary: Large DNA insert libraries, cloned into Bacterial Artificial Chromosomes (BAC), supply essential tools for use in gene cloning, physical mapping, genome sequencing, and comparative genomics. The first BAC libraries have been shown to be easily maintained and stable. Unfortunately their DNA inserts were rather small in size, and the techniques used for BAC library construction were unable to produce large stable DNA inserts. Currently, BAC libraries exist for all the major cereals except rye, which is known for its tolerance to various abiotic stresses such as drought, cold, and saline and acid soils. The present study designed and established a rye BAC library that contains very large stable DNA inserts. Extensive modifications were made to existing techniques, which were evaluated using rye as a DNA source. The study resulted in the establishment of a technique to create BAC libraries, which could be utilized to produce very large stable DNA inserts from any plant. The study has produced a technique, a large BAC library in rye, and will have a positive impact on the public’s ability to obtain very large DNA libraries for use in characterizing and cloning gene complexes for crop improvement. In addition, the library was used to analyze and characterize a genetic locus controlling aluminum tolerance in rye.
Technical Abstract: Rye is a diploid crop species with many outstanding qualities, and is also important as a source of new traits for wheat and triticale improvement. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies. The library provides a 6 × genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle derived clones. Genetic analysis attributed the aluminium tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R. Several BAC clones containing several allelic forms of genes at or near the Alt4 locus were recovered, revealing heterozygosity within cv. Blanco. BAC contigs providing almost complete coverage of two distinctive Alt4 locus ALMT1 gene cluster haplotypes and two adjacent genes revealed an above-average gene density of one gene per 24 kb in the region.