Artificial chromosome formation in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) |
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Authors: | Evgueni V Ananiev Chengcang Wu Mark A Chamberlin Sergei Svitashev Chris Schwartz William Gordon-Kamm Scott Tingey |
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Institution: | (1) Pioneer Hi-Bred Int’l, 7300 NW62nd Avenue, Johnston, IA 50131, USA;(2) DuPont Crop Genetics Research, Wilmington, DE 19880, USA;(3) Present address: Lucigen Corporation, 2120 West Greenview Drive, Middleton, WI 53562, USA |
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Abstract: | We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins
of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize
immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes
in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited
centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated
to be 15–30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes
contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes
were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous
cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal
replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide
a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics
and breeding.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Evgueni V. Ananiev, deceased
Evgueni V. Ananiev and Chengcang Wu contributed equally to this work.
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