Published in Probe Volume 4(1-2): July 1993-July 1994
Claire Kinlaw and David Harry
Institute of Forest Genetics, USDA, Forest Service
Berkeley, CA 94701
Forest trees and their molecular genetics were the focus of a recent conference (Prouts Neck, Maine, USA, May 20-23, 1994) organized by Michael Greenwood and Keith Hutchison (University of Maine, USA). A group of 70 researchers gathered to discuss progress toward understanding and manipulating molecular processes within this diverse group of economically and ecologically important species.
The biology of forest trees provides both distinct challenges and unique opportunities. Compared to previous meetings of this research community, significant progress has been made in several areas. Research teams continue to isolate and characterize new genes, while transgenic plants, especially in Populus, are increasingly being used to study gene function in vivo. Genome mapping has also matured. In earlier meetings, presentations described the construction of genetic linkage maps, while at this meeting maps were presented as tools to identify and dissect quantitative trait loci (QTL).
Advantages of the haploid genetics offered by conifer gametophytes continue to be exploited for mapping work and for population surveys. Recent advances in model organisms continue to influence studies in forest trees. Homeotic genes, for example, are being sought for flower and cone development. Other research focuses on processes that either are unique to trees or are simply more important in trees than in other organisms. For example, lignin and its related biosynthetic pathways are affected by wounding and stress in trees as in other plants, while in trees alone, lignin also is a major component of wood.
Gene Expression Patterns
As highlighted by Olof Olsson (Swedish University of Agricultural Sciences, Sweden), woody angiosperms show fluctuations in gene expression during annual cycles of quiescence and "reesence" in addition to changes observed during development and in response to environmental stresses. With the goal of increasing pulp production efficiency, Wout Boerjan's laboratory (Universiteit Gent, Belgium) has produced transgenic Populus plants containing antisense constructs for the lignin biosynthetic enzymes O-methyltransferase (OMT) and cinnamyl alcohol dehydrogenase (CAD). Work on conifer lignin and its role in development continues despite being hindered by the lack of reliable transformation and regeneration methods. Working on Pinus taeda, John Mackay (North Carolina State Biotechnology Group, USA, directed by Ron Sederoff) described the isolation and characterization of several genes encoding lignin biosynthetic enzymes including phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and 4-coumarate:CoA ligase (4-CL).
A slightly different tack toward understanding xylogenesis is being taken by Mackay's colleague, Malcolm Campbell. Because myb-like genes play an important role in signal transduction pathways in other organisms, such genes may also control conifer xylem development. Campbell has initiated cloning of Pinus taeda homologues. A similar rationale underlies the strategy of Sharon Regan and Bob Rutledge (Petawawa National Forestry Institute, Canada) in their efforts to characterize MADS box homeotic genes controlling cone development in Picea mariana.
With the goal of understanding the role of flavonoids in root formation, Lise Jouanin's laboratory (INRA, Versailles Cedex, France) has produced transgenic Populus and Juglans containing altered levels of chalcone synthase (CHS). Carmen Diaz-Sala, Keith Hutchison, and Mike Greenwood (University of Maine, USA) are investigating the cellular and molecular changes associated with the organization of root primordia in Pinus taeda. In particular, they are addressing how the cytoskeleton orients the plane of cellular divisions and nuclear reorganization.
Engineering for pest resistance using proteinase inhibitors is being done by several groups including those of Ned Klopfenstein (USDA-FS Center for Semiarid Agroforestry, USA), Lise Jouanin, and Seguin Armand (Universite Laval, Canada). Jouanin has found a cysteine protease inhibitor to be particularly effective against pests which contain high levels of cysteine proteases.
Genes responding to major environmental stresses have been identified in stress-specific cDNA libraries. Genes induced by drought stress in Pinus taeda (Shujun Chang et al., Texas A&M University, USA) include caffeoyl CoA, SAM synthetase, chitinase, and a protein similar to an animal skin matrix component. Genes induced by ozone (Dieter Ernst, Institut fur Biochemische Pflanzenpathologie, Germany) in Pinus sylvestris include CAD, stilbene synthase, hydroxymethylglutaryl-CoA-synthase, and polyubiquitin. As found in angiosperms, certain conifer genes appear to be induced by a number of environmental stresses. For example chitinase is induced by wounding, fungal infection, and drought (Haiguo Wu, Craig Echt, and John Davis, University of Florida, USA).
To expand upon the identification of new conifer genes, Claire Kinlaw (Institute of Forest Genetics, USA) has initiated "single-pass" sequencing efforts of Pinus taeda seedling cDNAs used as markers by David Neale and co-workers (Institute of Forest Genetics, USA) for genetic maps. These identified sequences will provide molecular tools for studying conifer genome organization and evolution. Early results have been encouraging in that a variety of genes have been identified including those encoding photosynthetic proteins, translation factors, glycolytic enzymes, and stress-response proteins.
With a systemic point of view, Gary Coleman (Oregon State University, USA) proposed a model to explain how trees regulate autumn nitrogen storage and spring remobilization in response to nitrogen availability and photoperiod. In this model, bark and leaves communicate with each other using a bark storage protein (BSP) and a leaf protein encoded by Win4. During short days or high levels of nitrogen, BSP accumulates in bark parenchyma while the Win4-encoded protein is repressed. During long day shoot growth, BSP is degraded while the Win4-encoded protein accumulates.
Genetic Maps and Quantitative Trait Loci
Genetic maps using two alternative approaches are being used to identify QTLs. Lively discussions of the merits and disadvantages of these two alternate approaches accompanied formal presentations. Mitch Sewell (Institute of Forest Genetics, USA) described the integration of restriction fragment length polymorphism (RFLP)-based linkage maps from two Pinus taeda pedigrees. This work will further efforts by Neale and his co-workers to dissect wood quality traits and to understand conifer genome organization and evolution. Several members of the Forest Biotechnology Group at North Carolina State University (USA) presented RAPD-based maps including those from Eucalyptus (Dario Grattapaglia) for the identification of QTL controlling sprouting and rooting.
New Markers
Several laboratories are exploring the use of length polymorphism among simple sequence repeats (SSRs). Craig Echt, (USDA Forest Service, Rhinelander, USA) has observed that approximately 0.7% of the Pinus strobus genome is comprised of SSRs. Of the primer pairs tested from the flanking sequences of Pinus strobus SSR loci, approximately 65% reliably amplify DNA. A high proportion show size polymorphisms, and a significant number amplify DNA from other conifer taxa. In apparent contrast to these observations, Keith Hutchison (University of Maine, USA) has observed a low level of size polymorphisms among SSR alleles in Larix laricina.
With a similar goal of developing co-dominant PCR-based markers, but using a different approach, David Harry (Institute of Forest Genetics, USA) is designing and testing primers based upon sequences of specific Pinus taeda cDNAs. Approximately 75% of the primer pairs reliably amplify genomic DNA, with a high proportion revealing Mendelian polymorphisms following restriction enzyme digestion. Some primers amplify only hard pines, others amplify all pines, and still others amplify DNA from other conifer taxa. Hisato Okuizumi presented an application of restriction landmark genomic scanning (RLGS) to large genomes by including a restriction trapper. High-speed scanning of entire genomes and the construction of genetic maps of individual trees from a single run with several hundred loci are made possible. As an example, a profile of Pinus koraiensis was shown.
Describing Genome Flux and Evolution
Because seed plants represent an ancient lineage, and because woody plants have long generation times, mechanisms of genetic mutation and genome evolution, as well as rates of species evolution, continue to be important areas of study. In seeming contrast to low levels of observed SSR polymorphism in Larix laricina, Hutchison and coworkers have found relatively high levels of sequence variation within genomic regions encoding proteins.
In addition, they have observed segregation distortion of alleles under different environments. The apparent contrast between the high levels of polymorphism among coding regions and low levels of polymorphism among SSR regions may indicate that conifers have a relatively efficient mismatch repair mechanism and may thus partially account for the stability of conifer karyotypes.
Jean Bousquet (Universite Laval, Canada) and his co-workers are investigating ancient events during the evolution of seed plants. After carefully calibrating a molecular clock, they established that modern gymnosperms derived from a single lineage, and they estimated divergence times to have occurred as follows:
liverworts from vascular plants
440 mya
ferns from seed bearing plants
400 mya
flowering from cone bearing plants
290 mya
monocots from dicots
200 mya
Pinus from Pseudotsuga
140 mya
Ross Whetten (North Carolina State University, USA) is exploiting the idea that a tree's crown represents a common lineage of shoots with known separation times. Using visible phenotypes in peach, Whetten estimated the somatic excision rate of a transposable element. The rate is relatively higher than rates reported for annual species and varies among meristematic layers. This notion that different shoots within the crown of a tree can be genetically distinct might help explain how long-lived trees endure pathogens and insect pests with shorter generation times.
Genetic Diversity and Population Structure
In addition to their use in mapping, RAPDs have been used by a number of laboratories for estimating genetic diversity and describing population structure. Natalie Isabel (Universite Laval, Canada) compared estimates of genetic variation within and among populations of Picea mariana using RAPDs and allozymes. Results from these two types of markers were similar. Linda DeVerno (Petawawa National Forestry Institute, Canada) surveyed Pinus resinosa using 400 RAPD primers and found no polymorphism. Again this data supports earlier conclusions based upon allozymes.
1995 Meeting
The next tree molecular geneticists meeting will take place at
Universiteit Gent, Belgium, in combination with the IUFRO Somatic Cell
Genetics Working Party. Those wishing more information are encouraged
to contact Wout Boerjan
(Boerjan%research%RUG.genetica@genwet1.rug.ac.be).