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Manipulations with Filamentous Fungi

Brett Tyler bmtyler at UCDAVIS.EDU
Mon Oct 17 10:02:28 EST 1994

At  8:41 AM 10/15/94 -0700, John J Weiland wrote:
>I'm kind of new to these beasts, and am looking for some input into the 
>following questions.  The fungus I'm working with is a pathogen of barley 
>called Pyrenophora teres.
>1)      We currently store long-term cultures of the fungus on silica 
>gel.  Is this a standard long term storage media?  Can spores be stored 
>in glycerol at -80 degrees C.  Can they be stored as stabs in agar (like 
>many bacterial isolates)?
>2)      Conidia of P. teres can be collected from mycelia grown in 
>culture.  Ascospores, however, result from the mating of two opposite 
>mating types of the fungus on barley plants (but not in culture).  Has 
>anyone seen mention of mutations in the fungus, or culture conditions, 
>that may induce productive mating of such a fungus in culture, rather 
>than on its normal host plant?
>3)      Any papers out there on the "bulk segregation analysis" of traits 
>in haploid fungi using PCR?   Did Michelmore's group do it with the 
>fungus, or just with the plant traits?
>Thanks in advance!
>John Weiland            jweiland at badlands.nodak.edu 

Regarding 1)  In our experience with Neurospora, silica gel is in fact the
best long term storage method.  Slants or glycerol can be used, depending
on the fungus, but can be unreliable.  Liquid nitrogen storage is another
excellent long term storage method.

Regarding 3) We are using bulked segregant analysis for Phytophthora sojae.
 There is no reason at all why this method can't be used on any genetic
progeny showing random segregation of genes.  In haploids, BSA should work
even better than diploids because dominance and recessiveness (especially
of the RAPDs themselves) is not a problem.  The most important
considerations for BSA are (i) that the trait of interest is determined by
a single locus, (ii) that you have at least  8-10 progeny for each bulk
(you should have much more than this if the genetics have been done well),
(iii) that you are absolutely sure that none of the chosen progeny have
been mis-scored and (iv) that there is a sufficient level of DNA sequence
polymorphism between the parents (at least 5% of all RAPD bands should
differ between the two parents, otherwise you will have a real struggle).

Can't help with 2).

Brett Tyler
bmtyler at ucdavis.edu

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