David Neale and Ronald Sederoff
Institute of Forest Genetics (IFG)
USDA Forest Service
Berkeley, CA
Genome mapping in pine and other forest trees began in the 1970's with the construction of maps based on isozymes. Isozymes are enzyme polymorphisms resulting from amino acid substitutions, which can be detected by gel electrophoresis.
These were the first genetic maps for conifers; large numbers of phenotypic markers do not exist in conifers to construct classical maps. Some of the earliest maps were constructed by Tom Adams at the University of New Hampshire and Oregon State University, Tom Conkle at the USDA Forest Service in Berkeley, and Ray Guries at Yale University and the University of Wisconsin.
Isozyme linkage maps have now been created for as many as 25 conifer species. These maps were generated rather quickly due to a unique aspect of the genetics of conifer seeds. The nutritive tissue of conifer seeds, the megagametophyte, is haploid and genetically identical to the egg. Thus, linkages between isozyme loci could be established by scoring haploid segregations from individual mother trees, eliminating the need for crosses.
Although the isozyme markers have proven to be extremely useful for population genetic studies, the 30 or so isozyme loci only map a very small segment of the genome. This limitation could easily be overcome with the application of new molecular markers, developed since the 1980's, restriction fragment length polymorphisms (RFLP's), and random amplified polymorphic DNA (RAPD's).
RFLP Map for Loblolly Pine
In 1988, David Neale's lab at the Institute of Forest Genetics (IFG) in Placerville, CA, received funding from the Forest Biology Program, USDA Competitive Research Grants Office (CRGO), to begin constructing an RFLP map for loblolly pine.
Their approach is to map RFLP's from a single segregating family derived from three generations of outbred matings. The genotypic information on the grandparent trees of the pedigree assists in determining phases in the parent trees. Their map now has 100 RFLP markers located on it, some of which are shown in Figure 1.
In collaboration with Claire Williams and Bob McGraw at the Weyerhaeuser Company, the IFG lab is now mapping on a second 3- generation loblolly pine pedigree with the goal of mapping quantitative trait loci (QTL) coding for wood specific gravity. Specific gravity is a trait of commercial importance and is thought to be under the control of just a small number of genes. The IFG lab is also working with Weyerhaeuser to construct a map for Douglas-fir, another species of commercial importance.
Constructing RAPD Maps
In April 1990, the RAPD mapping technique was introduced by the DuPont Company. RAPD markers are assayed using the polymerase chain reaction (PCR) technique, which is inherently simpler and faster than the Southern blot procedure required for RFLP's.
The drawback of RAPD's is that they are dominant markers as opposed to RFLP's which are codominant. Forest tree mapping researchers quickly realized, however, that the problem of dominance could be overcome by taking advantage of the haploid megagametophytes; RAPD maps could be constructed in the same way as isozyme maps.
Several labs have begun to construct RAPD maps following this approach but Ron Sederoff, David O'Malley, and co-workers at North Carolina State University (NCSU) have made the most progress. In fact, the NCSU lab was able to generate a 191-marker RAPD map for an elite loblolly pine tree in just 2 months (Fig. 2).
Their project was recently funded by the Plant Genome Program of the USDA National Research Initiative Competitive Grants Program (NRICGP). They are planning to develop a theory for mapping QTL's in open-pollinated families from individual trees. This would eliminate the need for making crosses, a very lengthy process in trees.
Third Mapping Effort Funded
The IFG lab has also received funding from the Plant Genome Program to map a major gene for resistance to white pine blister rust. Mike Devey, David Neale, and Bro Kinloch are using the RAPD approach to generate the map from a single mother tree that is heterozygous for the dominant resistance gene. This tree was mated to a homozygous recessive male, therefore, the progeny will segregate 1:1 for resistance and susceptibility. This genetic system will permit mapping the dominant resistance gene based on haploid segregations from the mother tree.
In summary, the three mapping efforts funded by the USDA's CRGO and the NRICGP, in pine are beginning to make significant progress toward mapping the genomes of this very important group of plants. Other pine mapping projects are emerging and will contribute to this effort during the next few years.