RAPD mapping

Ross Whetten rosswhet at forbt2.nrrc.ncsu.edu
Mon Sep 28 09:47:22 EST 1992

 samodena at csemail.cropsci.ncsu.edu (S. A. Modena) writes in message <1992Sep24.072411.114 at ncsu.edu>:

>Why are RAPDs of special importance in trees, rather than RFLPs....are
>RFLPs worth the effort in trees?

One of the arguments against using molecular markers to assist tree breeding has been
that highly outbred wild populations of trees are likely to be in linkage equilibrium,
ie a particular marker will be linked to a "bad" allele at some quantitative trait locus
just as frequently as it is linked to a "good" allele, and a map for one tree or cross will
not be useful for other trees or crosses. The advantage of RAPDs is that a map can be made for
an individual tree relatively quickly. Last summer, a group of 5 full-time and 5 part-time
workers mapped 200 markers in 12 linkage groups in 60 days, and with standard laboratory 
automation, we think we could do it in three or four weeks now. Association of QTLs with these
markers is then a matter of measuring the progeny set for the trait of interest and analyzing
the data, and then you have a useful map for that particular tree. I should point out that conifers
are particularly well suited to RAPD because the megagametophyte is retained in the seed, giving us
access to haploid tissue representing the maternal contribution to the zygote. Megagametophytes
obtained from open-pollinated seed of a single tree can be used to generate a map of that tree,
without harming the embryos in the seeds. Those open-pollinated progeny are then available for
planting, and after a suitable period of growth, can be scored for various traits of interest
and the data analyzed in conjunction with the molecular marker data from the same seeds.
The reason this is valuable for RAPDs is the dominant nature of RAPD markers -  in a haploid tissue,
there is no confusion about homozygous dominant versus heterozygote genotypes.

The short answer to the above question is likely to be "No, RFLPS are not worth the effort for QTL
mapping for practical tree breeding purposes", because of the linkage equilibrium problem.

>We have a German visiting scientist here who gave a seminar on RAPDs
>and barley genetic/QTL analysis.  A main point (in response to one
>of my questions) was that search for suitable RAPD primers is arduous.
>And, further, once a set has been developed for analysis of a particular
>set of genetic materials, one most likely will have to toss that set
>and start again for a different set of genetic materials (of the
>same species, of course).

>Would that be true in trees?  I.E., having found a RAPD primer that
>illuminates a particular trait via unique banding pattern, it might
>fail during a broadbased survey?


As mentioned above, we consider breeding populations of trees likely to be at linkage equilibrium,
and therefore _expect_ to have to make a new map for every individual tree. The real question is,
how hard is it to screen primers and make maps? For the mapping project mentioned above, it took about two weeks to identify 100 primers that would be useful in mapping, ie they gave reaction products that
segregated 1:1 in accordance with Mendelian expectations. Given the current state of automation in our
lab, we could repeat that same screening procedure in about four days, which does not strike me as
arduous compared to the months and years people spend identifying informative RFLP markers.

I should add that I am not arguing against the use of RFLPs in crop plants in which the markers have
already been identified, and for which linkage disequilibrium is present. RFLPS have advantages as
codominant markers which RAPDs lack. I would argue, however, that for plant species where no molecular
or classical genetic work has been done, RAPDs are the easiest cheapest fastest way to get to a complete
linkage map. A student in our group has been working part-time over the past five or six months,
between classes, teaching requirements, field work etc, on a map of an interspecific Eucalyptus hybrid.
He can't get haploid tissue, but he finds so many polymorphisms that he can choose to work only with
those for which one parent is heterozygous and the other is homozygous null. This "pseudo-backcross"
scheme resulted in two maps, one for each of the parents, each with over 200 markers. He is now 
propagating the progeny set used for the mapping (clonal propagation is well established for eucalypts)
for establishment of replicated field trials in three different environments, to see if QTLs
for economically important traits can be identified and to test the hypothesis that some QTLs will be
found only in some environments but not in others. It is too early to say that RAPD maps have had 
an effect on practical breeding in trees, but it seems safe to guess that they will, in the next five to
ten years. 


Ross Whetten       (nwhett at ncsumvs.ncsu.edu or rosswhet at unity.ncsu.edu) 
Research Assistant Professor of Forestry
Forest Biotechnology Group
North Carolina State University
Raleigh, North Carolina 27695-8008  USA
telephone or fax (919)515-7801


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