Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley

Abstract

A new strategy has been devised and used for the physical localization of genetically mapped restriction fragment length polymorphism (RFLP) clones to barley chromosomes. Morphologically distinct translocation chromosomes from synchronized root-tip meristems were microisolated and their DNA was used as a template for polymerase chain reaction with sequence-specific primers. Four RFLP clones were assigned to cytologically defined segments of chromosome 5. This related approximately one-third of the map length of linkage group 5 to approximately one-fifth of the mitotic metaphase length of chromosome 5. The technique may substantially contribute to the connection of the RFLP-based genetic linkage maps with cytological markers of the barley chromosomes.

Dissection of barley chromosome 5H in common wheat

“…In barley, physical RFLP maps have been constructed by two different methods. One utilizes microdissected translocation chromosomes as templates for PCR (polymerase chain reaction) with primers designed from sequenced RFLP probes (Sorokin et al, 1994;Künzel et al, 2000). The other is deletion mapping using deletions and translocations involving a dissected barley chromosome in common wheat (Serizawa et al, 2001).…”

Chromosomal assignment and deletion mapping of barley EST markers

“…Such distortions of gene loci can be remedied by physical, or cytological, mapping. In barley, two cytogenetical methods have been used for the physical mapping of RFLP markers: One uses microisolated translocation chromosomes as the templates for PCR with primers designed from sequenced RFLP probes (Sorokin et al, 1994;Künzel et al, 2000); the other is deletion mapping using structural changes of a barley chromosome added to wheat (Serizawa et al, 2001a). The latter type of mapping employs common wheat stocks carrying structural aberrations of barley chromosomes that have been induced in wheat-barley addition lines using the gametocidal system (Shi and Endo, 1997, 1999, 2000.…”

Genetic induction of structural changes in barley chromosomes added to common wheat by a gametocidal chromosome derived from Aegilop Scylindrica.

“…However, cytogenetic stocks such as aneuploids and deletion lines are either very difficult or impossible to maintain in barley as it is a diploid. To date, various techniques have been used to conduct physical mapping in barley, such as PCR mediated localization of DNA sequences on microisolated translocation chromosomes (Sorokin et al 1994), pulsed field gel electrophoresis (Siedler and Graner, 1991), and in situ hybridization (Butnaru et al, 1998). However, these methods are technically cumbersome and have their limitations.…”