We are producing the double haploids in Maize and we need to identify the double haploids in early stage of the plants. I want to know what are the best and accurate method for identification of double haploids, haploids at molecular level.
Because haploids and dihaploids differ by ploidy, sequence-based methods will not work. Any technique for measuring ploidy differences will work - chromosome counts (as Yuan-Yeu suggested), flow cytometry (as you suggested), and quantifying the signal of DAPI stained nuclei might also work. Making an epidermal impression with nail polish/varnish for measuring stomate size (larger with increased ploidy) is another quick method - paint the leaf surface with nail polish/varnish, peel off the dried polish, and measure stomate size under brightfield microscopy.
Dear Anand,
Please have a look at these useful PDF attachments.
Good luck
you can to use anther as explant for producing of hapliody plants and cholchisine for polyploidy chromosomes
@ Aravind Singh,
Thank you for your response, I have these articles with me and I am exploring them. My question is will these markers differentiate between the haploids and double haploids?
@ Yuan-Yeu Yau,
Ya you are right, but I want to differentiate haploids and double haploids using molecular markers either SSR/SNP's.
@ Anand,
I doubt that molecular markers can be used to distinguish hapolid from a DH line derived from it. Becuase a real 'double haploid' is just two sets of the same thing the haploid plant has (but just one set).
@ Yuan-Yeu Yau,
Ya, you got the point. what about flowcytometry?. Let me know if any other methods.
Because haploids and dihaploids differ by ploidy, sequence-based methods will not work. Any technique for measuring ploidy differences will work - chromosome counts (as Yuan-Yeu suggested), flow cytometry (as you suggested), and quantifying the signal of DAPI stained nuclei might also work. Making an epidermal impression with nail polish/varnish for measuring stomate size (larger with increased ploidy) is another quick method - paint the leaf surface with nail polish/varnish, peel off the dried polish, and measure stomate size under brightfield microscopy.
@ Randall P Niedz ,
Dear Sir,
Thank you for picking the right point and suggesting me. As per your suggestion I want to "quantify the signal of DAPI stained nuclei " Kindly let me know if you have protocol regarding this.
I think root tip chormosome count is the cheapest ,easiest and quick method to differentiate haploids and diploids.
Attached are some papers where ploidy level was determined by microfluorimetry of DAPI-stained cells. The Aust J Bot paper uses acid and enzyme digestion to more evenly spread the DAPI-stained cells for standard chromosome counting.
Flow cytometry is the fastest and the easiest to perform method to classify different plant ploidy levels, as long as you have access to the instrument. All you need is a petri dish, razor blade, nuclei extraction buffer with a dye (e.g. DAPI) and a standard, usually diploid plant tissue of the same species (e.g. the parental form of your haploids/DHs).
@ Randall P Niedz ,
Dear Sir,
Thank you for for the response and doing the needful.
Dear Anand,
If you developed DH from a cross involving differing parents, you can identify DH progenies by the expression of homozygous recessive allele based trait. If you have a few such recessive traits distributed in both the parents, you can identify more and more DHs. If the plant of interrest is heterozygous, this would end up in two alleles in the fingerprint, whereas homozygous plants (such as DH) should have only half of the alleles derived from their parent(s).
Also, parental polymorphisms in many SSRs can identify your DHs. But try to look for codominant SSRs which will make your job easier. Any parent like anther wall product can be detectable and dispensed using above said methods. There should be a lot of protocols and SSR sequences in the literature. I remember something about kits already suitable for DH identification.
Flow cytometry and chromosome counting do not really help you to differentiate DHs from other diploid contaminations like parents.
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