Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato.
We obtained DNA clones specific to tomato chromosome 2 from a small number of chromosomes collected by flow sorting. Suspensions of metaphase chromosomes were prepared from 3-month-old tomato cell cultures of Lycopersicon pennellii. Isolated chromosomes stained with chromomycin A3 and Hoechst 33258 were analyzed on an EPICS 753 flow cytometer using a UV laser to excite Hoechst fluorescence and a 458 nm laser to excite chromomycin A3 fluorescence. Chromosomes from well-resolved peaks on a bivariate flow karyotype were sorted directly onto membrane filters for spot-blot analysis. The filters were processed and hybridized with chromosome-specific repetitive DNA probes. In this way tomato chromosome 1 and chromosome 2 were assigned to peaks in the bivariate flow karyotypes. One thousand copies of the putative chromosome 2 were flow-sorted directly into microfuge tubes. DNA specific to chromosome 2 was amplified by a polymerase chain reaction (PCR) technique using universal 22mer degenerate oligonucleotide primers (DOP) sequences. DOP-PCR yields a smear of fragments of various sizes from 250 to 1600 bp. Amplified products were cloned into the Bluescript plasmid vector. Approximately 11% of the clones contained sequences with highly repetitive elements, and 85% contained only low-copy-number sequences. Eleven clones containing low-copy-number sequences that detect restriction fragment length polymorphisms were placed on the molecular linkage map of tomato. All showed linkage to chromosome 2.
Arumuganathan
K
K
Department of Plant Breeding, Cornell University, Ithaca, NY 14853-1902.
Martin
G B
GB
Telenius
H
H
Tanksley
S D
SD
Earle
E D
ED
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002875:Chromosomes; D003001:Cloning, Molecular; D015342:DNA Probes; D005434:Flow Cytometry; D017343:Genes, Plant; D015698:Genomic Library; D007621:Karyotyping; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014675:Vegetables / Q000235:genetics*
D015342:DNA Probes