Repeated DNA sequences isolated by microdissection. I. Karyotyping of barley (Hordeum vulgare L.)

Abstract

We report on microdissection, cloning and sequence, and Southern and fluorescence in situ hybridization (FISH) analysis of one moderately and one highly amplified repetitive DNA element, pHvMWG2314 and pHvMWG2315, respectively, isolated from barley (Hordeum vulgare L.) chromosome arm 3HL. The pHvMWG2315 sequence hybridizes to all 14 telomeric or subtelomeric regions of the barley chromosomes as determined by FISH. The 50 different hybridization sites that include intercalary signals allow the discrimination of all 14 chromosome arms and the construction of a karyotype of barley. The tandemly repeated subtelomeric element of 331 bp exists in all Triticeae species tested (H. vulgare, Agropyron elongatum, Secale cereale, Triticum tauschii, T. turgidum, and T. aestivum). It is AT rich (66%), exhibits 84% sequence homology to subfragments of the D genome "specific" 1-kb element pAsl of T. tauschii and 75% homology to the interspersed genome-specific DNA sequence pHcKB6 from H. chilense. The repetitive sequence pHvMWG2314 is moderately amplified in barley and highly amplified in hexaploid wheat. The in situ experiments revealed no distinct signals on barley chromosomes, indicating a dispersed character for the sequence. The significance of the results for the identification of chromosomes and chromosome aberrations in FISH experiments are discussed.Key words: karyotype, fluorescence in situ hybridization, FISH, DNA sequencing.