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Title: Laser Capture and Amplification of a Maize Abnormal Chromosome
10 Translocation Line
Zea mays has ten chromosomes, and in a small percentage of both domesticated maize
and teosinte the normal chromosome 10 (N10) has been replaced with abnormal
chromosome 10 (Ab10). Ab10 is defined cytogenetically by the addition of a terminal
piece of foreign DNA onto the long arm of N10. This piece of DNA includes both
euchromatin and a distinctive, large, heterochromatic knob. The additional DNA is
known as the Ab10 haplotype, and is responsible for neocentromere activity and
preferential segregation (meiotic drive). All chromosomes that are heterozygous for
knobs undergo meiotic drive in the presence of Ab10. This ensures the transmission of
the knobbed chromatid to the progeny at rates of up to 83%. These unique properties of
Ab10 have been studied since the 1940s; however, the genes which control these
properties remain unknown. To address this problem, we are developing a method to
isolate and amplify DNA from individual chromosomes containing the Ab10 haplotype.
Using a chromosome 1/Ab10 translocation line we are able to isolate and collect Ab10
containing chromosomes via laser capture. Whole genome amplification kits will be used
to obtain enough DNA from the isolated chromosomes for high-throughput sequencing
methods. This will allow us to sequence the Ab10 haplotype and uncover the genes
which control neocentromere activity and meiotic drive.
Survey of CentC arrays and CenPC3 in Zea
Maize centromeres are mainly composed of two different repetitive sequences: a
retrotransposon, CRM, and a 156bp tandem repeat, CentC. In domesticated maize CRM
levels are fairly consistent while the amount of CentC varies greatly between inbred lines,
land races and even between chromosomes in the same genus (Kato et al 2004).
However, in Zeamayssspparviglumis and Zeadiploperennisand Zealuxurians there seems
to be abundant, consistent amounts of CentC across chromosomes (Shi unpublished). In
order to function, centromeric DNA has adapted to interact with a large protein complex,
the kinetochore. Kinetochores are composed of an inner layer that interacts with
centromeric DNA, and outer layer that interacts with spindle microtubules, and a middle
layer that bridges the inner and outer layers. The main two inner kinetochore proteins,
CENH3 and CENPC are constitutively expressed and conserved across eukaryotes. In
maize there are two variants of CENPC, and recently Li and Dawe found a third Cenpc
gene, Cenpc3. Interestingly, the Cenpc3 allele found in Zealuxurians and
Zeamayssspparviglumis is different from the Cenpc3 allele in domesticated
maize(unpublished). Centromere maintenance has long been a mystery. However,
previous results and preliminary data indicate that CentC amount correlates with the
Cenpc3 allele present in the genome. Surveying the abundance and distribution of CentC
together with the expression of Cenpc3 alleles in teosintes and maize land races should
shed light on the issue of centromere maintenance.