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Pollen
Genotyping in Coneflower
Ahmad
Naseer Aziz
Institute of Agricultural and Environmental Research
Seminar Series
Tennessee State University, Nashville, TN
September 9, 2004
Coneflowers
(Echinacea spp.) are important to both the ornamental and
medicinal herb industries, thus efficient methods for their genetic
analysis are needed. Segregation analysis of molecular markers from
individual pollen grains can generate genetic data for linkage mapping
without the need of performing controlled pollinations. These maps can
simplify and enhance breeding efficiencies in coneflowers. In this
presentation the use new approaches for the extraction of DNA using a
pollen germination protocol, the collection of individual pollen grains
using a micromanipulator, the amplification of DNA from individual
pollen grains and the identification of AFLP (amplified fragment length
polymorphism) markers are reported. The limited amount of DNA found in a
pollen grain is not adequate for analysis and needs to be increased by
using primer extension pre-amplification (PEP) protocol. In this
research, Echinacea pollen genome was amplified with an array of
continuous random 15-mer PEP primers. The PEP amplification resulted in
pollen genome distributed in fragments of varied lengths. Restriction
digestion was conducted on PEP products and PCR based AFLP markers were
amplified; but, only short-sized (around 500 base pairs) AFLP markers
were generated. Therefore, MasterAmp™ Extra-Long PCR kit (EPICENTRE®,
Madison, WI) was used to substantially increased the sizes (>600 base
pairs) and numbers of AFLP markers. The parental AFLP markers were
scored for their presence or absences in the pollen samples using Saga™
Generation 2 Version 3.1 (Li-Cor, Lincoln, NE) software. Our findings
indicate these procedures can be used to analyze the segregation of
parental AFLP markers in a pollen population.
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