Poster: Late and Moved Abstracts

Abs # 1009: Segregation of Parental AFLP Markers in Isolated Echinacea Pollen Grains

Presenter:	Aziz, Ahmad Naseer, anaqureshi@yahoo.com
Authors	Aziz, Ahmad Naseer (A)   Sauve, Roger J. (A)
Affiliations:	(A): Institute of Agriculture and Environmental Research

Because coneflowers (Echinacea spp.) are important to both the ornamental and medicinal herb industries, efficient methods for genetic analysis are needed. Segregation analysis of molecular markers from individual pollen grains can generate genetic data for mapping without the need of performing controlled pollinations. These maps can simplify and enhance breeding efficiencies in coneflowers. In this paper, we report on the use new approaches for the extraction of DNA using a pollen germination protocol, the collection of individual pollen grains using micromanipulator, the amplification of DNA from individual pollen grains and the identification of AFLP (amplified fragment length polymorphism) markers. 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.