[HELICONIUS] W-linked markers

[Frantisek Marec]

Hi Jamie and Alexie,

Brief comments to Alexie's suggestions.

(1) To mlecular approach:
I have never seen chromosomes of H. melpomene but have in my PC almost 1 
GB of digital images of H. erato and H. himera. They both have sex 
chromatin (W chromatin body) in female polyploid nuclei, but rather 
small due to fusion (or translocation) with an autosome. So, laser 
microdissection of the W body followed by cloning is possible but bit 
difficult compared to other Leps species. If you wish, I can send you 
some images. Perhaps, you could test primers for "Kamikaze" transposon. 
We found patiral homology of one sequence accumulated in the W 
chromosome of the codling moth (CpW27 in Fukova et al. 2007, Chromosoma, 
Acc. no. AM292099) with Kamikaze element of Bombyx. Might be also 
conserved in butteflies.

(2) I agree with Alexie, physiological approach will certainly work 
starting 3rd larval instar. It will also work in pupae - look at pupae, 
male and female pupae clearly differ in  morphology of the last 
abdominal segments (see Fukova et al. 2009, J Appl Entomol).

(3) Cytological approach - might be difficult, See (1).

Good luck,
Frantisek

> Hi Jamie
>
> if you sequenced a male, then there will be nothing W-specific in the 
> genome... there will shared elements though which could be 
> over-represented in the W.
>
> your question is much easier though. we've had 3 approaches that worked.
>
> Hard, molecular approach
> Dissecting the w of a butterfly, cloning and sanger sequencing 
> W-specific repeats (Fukova in Applied Entomology 
> <http://www3.interscience.wiley.com/journal/121589905/abstract>)
> Also you can try a AFLP bulk segregation analysis on a family of male 
> and females with the parents. markers on mum and daughters are w linked.
>
> Easy, lethal physiological approach
> Chop it up. There will be two large (yellow or white) bodies in the 
> males. Testis... for us it works well with L3+ even though our moth 
> larvae are much smaller than heliconius larvae
>
> Fiddly, fool-proof cytogenetic approach
> take a spot of hemolymph (no need to kill it). make a squash 
> preparation with lactic orcein. look under 40x. if the nuclei have a 
> big black spot, it's the W body and it's a female... Let me know if 
> you need the protocol (should be in above paper). It's not that fiddly 
> when you learn it and you can process lots of larvae in a day (you can 
> do the microscope work another day)
>
> cheers
> a
>
>
> On Wed, 2010-04-14 at 11:57 +0100, Jamie Walters wrote:
>> Hello all,
>>
>> I'm curious to know if anyone has had any success (or informative  
>> failures...?) with developing W-linked markers for Heliconius (or any  
>> butterfly that would be near enough to be useful).
>>
>> Or, alternatively, if anyone has any particularly clever ideas for  
>> mining the existing genome data for candidate regions I'd be keen to  
>> hear them.
>>
>> I'm simply interested in being able to sex juveniles and pupae.
>>
>>
>> Thanks very much,
>>
>> Jamie
>>
>> _______________________________________________
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>>     
>
>
>   


-- 
************************************
Frantisek Marec
Biology Centre ASCR
Institute of Entomology
Department of Genetics
Branisovska 31
CZ-370 05 Ceske Budejovice
Czech Republic
e-mail: marec at entu.cas.cz
phone: +420-387 775 250
fax: +420-385 310 354
http://www.entu.cas.cz/