CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Crop & Pasture Science   
Crop & Pasture Science
Journal Banner
  Plant Sciences, Sustainable Farming Systems & Food Quality
 
blank image Search
 
blank image blank image
blank image
 
  Advanced Search
   

Journal Home
About the Journal
Editorial Structure
Contacts
Content
Online Early
Current Issue
Just Accepted
All Issues
Special Issues
Research Fronts
Farrer Reviews
Sample Issue
For Authors
General Information
Scope
Submit Article
Author Instructions
Open Access
For Referees
Referee Guidelines
Review an Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates

blue arrow e-Alerts
blank image
Subscribe to our Email Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter LinkedIn

red arrow Farrer Reviews
blank image

Invited Farrer Review Series. More...


red arrow PrometheusWiki
blank image
PrometheusWiki
Protocols in ecological and environmental plant physiology

 

Article << Previous     |     Next >>   Contents Vol 52(12)

Detection and analysis systems for microsatellite markers in wheat

L. R. Rampling, N. Harker, M. R. Shariflou and M. K. Morell

Australian Journal of Agricultural Research 52(12) 1131 - 1141
Published: 15 November 2001

Abstract

This paper describes and discusses strategies for screening microsatellites for use in plant genetic research and illustrates how a subset of useful microsatellites can be optimised for implementation on breeding and research using a range of techniques. Beginning with the initial screening of microsatellites for potential polymorphisms in a core set of potential parental lines, through to scaling up for mapping or breeding purposes, we present a time- and cost-effective approach to microsatellite analysis in wheat lines of interest. Each stage of this process benefits from a fresh examination of the techniques applied in order to increase the efficiency with which key markers can be identified and implemented.

For the primary screening we use primers without modification to prime PCRs in the presence of f-dNTP (fluorescently labelled nucleotide) to provide the basis for high resolution screening for polymorphisms. As markers are defined for use in a breeding program, the focus changes to a smaller set of primer pairs that will be used to screen large numbers of DNA samples either from the analysis of progeny from a cross or the routine checking of cultivar identity in the industry. We then examine appropriate analysis platforms and refinement of PCR primers and conditions in order to identify procedures that can be implemented widely, not just in specialised well-equipped laboratories. In many cases we are able to use lower cost agarose analysis for identified polymorphisms. Where this is not feasable we examine primers for potential redesign to optimise their application either by altering the sequence of the primer itself, based on available sequence information, or by adding tails to the primers. The latter is shown to alter the ‘stutter’ pattern that is commonly observed with wheat microsatellites so that a single band is prominent and thus allows size polymorphisms to be more readily scored. The addition of a generic 5′ tail also provides a method of using a generic fluorescent primer that can be applied to multiple tagged markers in a costeffective fashion. The potential of alternative analytical systems and further refinement of primers to show plus/minus reactions with wheat lines in order to produce simple tests for use in breeding programs are also discussed.

Keywords: microsatellite primers, detection technologies, stutters, modified agarose, 5′ tagged primers, PIGtail primers.



Full text doi:10.1071/AR01027

© CSIRO 2001

blank image
Subscriber Login
Username:
Password:  

 
PDF (1.4 MB) $25
 Export Citation
 Print
  
    
Legal & Privacy | Contact Us | Help

CSIRO

© CSIRO 1996-2015