Management of Genetic Resources of Pacific Northwest Trees

Transcription

1 1 Management of Genetic Resources of Pacific Northwest Trees Draft chapter to be published in Regenerating Pacific Northwest Forests, S. Hobbs and D. Lavender, Editors. Oregon State University Press. In prep. S.N. Aitken. University of British Columbia and J.B. St. Clair, U.S. Forest Service Pacific Northwest Research Station Those forms which possess in some considerable degree the character of species, but which are so closely similar to some other forms, or are so closely linked to them by intermediate gradations, that naturalists do not like to rank them as distinct species, are in several respects the most important to us. - Charles Darwin, 1859 Introduction Foresters and forest managers make many day-to-day decisions that could impact the genetic structure and diversity of forest trees, yet genetics is sometimes erroneously considered a remote and irrelevant science in the management of forests. With any decisions involving seed source, planting stock, planting density, and partial cutting there are opportunities for genetic changes in forest stands. Objectives of increasing forest productivity, improving wood quality, or reducing the impacts of forest pests may be met through genetic solutions. The native forests of the Pacific Northwest contain tree species harbouring some of the highest levels of genetic diversity in the world, and this genetic diversity is a resource that can be utilized but must also be conserved. In this chapter, we will review the basics of inheritance and genetics, describe natural patterns of genetic diversity and how these are maintained in reforestation through seed zones and seed transfer guidelines, discuss selective breeding programs (using examples of Pacific Northwest species), review the opportunities for biotechnology to play a role in silviculture in the Pacific Northwest, and emphasize the need for sound gene conservation programs. Primer on genetic variation What are genes and alleles? Genetic diversity is the fundamental underpinning of all biodiversity. But what is genetic diversity? To understand this, it is necessary to have a basic understanding of genetics and the associated terminology. A gene is the fundamental unit of heredity. It is a DNA sequence that codes for a protein, for molecules associated with the expression of genes, or for molecules that regulates the expression of other genes. Most genes are carried on chromosomes, found within the nucleus of every living cell in a tree. Different species have different numbers of chromosomes. For example, all members of the family Pinaceae (containing the majority of the conifers in the Pacific Northwest including pines (Pinus), spruces (Picea), true firs (Abies) and larches (Larix) have 12 chromosomes (N=12), but Douglas-fir (Pseudotsuga menziesii) has N=13. Red alder (Alnus rubra) has N=14. In the somatic (or vegetative) cells of these species, two copies of every chromosome are found (these cells are called diploid). However, in the gametes produced through meiotic cell divisions (i.e., male and female gametophytes, including pollen and egg cells), there is just one copy of each chromosome (these cells are called haploid). When a pollen cell fertilizes an egg cell, a diploid zygote results. This zygote will then grow into an embryo and ultimately a tree, all the vegetative parts of which will contain cells with identical nuclear DNA (for a review of basic plant genetics, see Raven et al. 2000). Genetic diversity Individual genes are found at specific locations on chromosomes. Since somatic cells

2 2 each have two copies of each chromosome in most cases, each of these cells will have two copies of each gene, one inherited from the maternal (seed cone) parent and one from the paternal (pollen) parent. Alleles are alternate forms, or variants, of genes with slightly different DNA sequences, which may result in different gene products that produce structural or functional changes in the individuals carrying them. An individual might inherit the same allele for a given gene from both parents and be homozygous, or inherit different alleles from each parent for that gene and be heterozygous. The percent of genes for which individual trees are expected to be heterozygous, on average, based on allele frequencies for a subset of genes is a useful overall measure of genetic diversity in a population or species, and is called expected heterozygosity. The more alleles that exist for a gene or the more genes that vary genetically (having two or more alleles), the higher the heterozygosity of that species. For a given number of alleles, heterozygosity will be higher if allele frequencies are intermediate rather than if one allele is common and others rare. Base-line estimates of heterozygosity in wild populations provide a yardstick for natural levels of genetic diversity within species, against which the genetic diversity of managed populations can be compared (Savolainen and Kärkkäinen 1992). For genetic management, it is also useful to know the extent to which populations within a species differ genetically from one another. A statistic called G st (and a very similar statistic called F st ) describes the percentage of total genetic variation that is due to among-population differences (Yeh 2000). A low G st value indicates populations are similar genetically, while a high G st indicates stronger genetic differences among populations. Most temperate forest tree species have fairly low values of G st, due to their often spatially continuous distributions and high levels of gene flow among populations through windborn pollen. The average heterozygosity for woody perennials such as trees and for other categories of plants, are summarized in Table 1, and withinpopulation heterozygosities for some individual Pacific Northwest tree species are included in Table 2. These estimates are based on laboratory analyses of proteins produced from genes coding for alternate forms (alleles) of fundamental metabolic enzymes called isozymes. Long-lived woody plants, including trees and shrubs, have relatively high levels of overall within-species and within-population genetic diversity compared to other types of plants (and to animals), but relatively little variation for isozymes among populations compared to herbaceous annuals or perennials (Hamrick et al. 1992). The reasons for this high level of variation are not entirely known, but may result from large population size, extensive gene flow through wind-born pollen, or even through high mutation rates. One theory suggests that trees may acquire genetic variation through somatic mutations (errors in DNA replication during mitoses) in some of the many cell divisions that occur in the growth of a large tree before sexual reproduction occurs, but supporting empirical evidence is lacking. Isozymes are generally considered to be selectively neutral, with variation reflecting predominately historic population sizes, gene flow among populations, and historic events such as glacial refugia or bottlenecks, rather than reflecting variation in adaptive traits affecting fitness in specific environments. For example, western redcedar has a relatively low level of heterozygosity (Table 2). This is attributed to a population bottleneck during the last glacial period. Douglas-fir has a much higher level of heterozygosity, on average, than western redcedar, and better reflects the typically high average genetic diversity of tree species. It is important to note that the amount of differentiation among populations can be considerably higher for adaptive traits than G st would indicate based on selectively neutral genetic markers, thus it cannot be assumed that populations are adapted to the same environmental conditions if they differ little based on isozymes. Genotype and Phenotype By studying the DNA sequences of genes or the direct protein products of these genes such as isozymes, we can directly assess the genotype, or genetic make-up, of an individual. However, as foresters we are often much more interested in the phenotype than the genotype. The phenotype is the outward appearance or performance of an individual tree or population of

3 3 trees for traits such as growth rate, disease resistance and cold hardiness rather than the DNA sequence of specific genes. Variations in phenotypes caused by genotypic variation are referred to as polymorphisms. The phenotype of an individual tree is the product of its genotype in conjunction with its environment. Some traits of interest are the product of a single gene, but most are polygenic, i.e., the product of many genes working in concert. Typically we do not know what genes, or how many, genetically control a phenotypic trait. For some traits, such as growth rate, the environment has a large effect on the observed phenotype, whereas for other traits, the environment plays a relatively small role in trait expression. Single-gene traits present different opportunities and challenges for selective breeding than polygenic traits. Single-gene traits Genetic polymorphisms resulting from allelic differences for single genes can be observed for traits such as albinism, insect and disease resistance, and cone color. These traits fit the simple one gene, one trait model that many advances in agricultural breeding and medical genetics have relied upon. While many genetically-controlled traits do not fit this simple Mendelian model, some provide good examples. Examples of single-gene traits: Albinism and major-gene resistance Albinism provides a good example of a single-gene trait. When germinating a large seed lot, albino seedlings are often observed at a very low frequency. Albinism is a phenotype typically produced by a recessive allele, thus only homozygotes for the albino allele (genotypes with two copies of the albino allele) lack chlorophyll. These seedlings die soon after germination. Seedlings with just one albino allele have normal, green phenotypes. More important to forestry are genes controlling resistance to forest pests and pathogens. The introduced disease white pine blister rust (Cronartium ribicola) is a major agent of mortality for all white (five-needled) pines in the Pacific Northwest including western white pine, whitebark pine, limber pine and sugar pine. One type of genetic resistance to this disease in both sugar pine and western white pine is a dominant allele for a single gene, called major gene resistance (MGR). Genotypes that are either heterozygous or homozygous for this allele, i.e., have either one or two copies of the resistance allele, are largely resistant to this disease. However, in the case of sugar pine, a more virulent form of white pine blister rust has arisen that can successfully attack the previously resistant MGR genotypes. Thus, breeders typically target insect or disease resistancerelated traits controlled by multiple genes as these are harder for forest pests to overcome quickly through evolution. In the case of western white pine, several types of resistance that appear to be controlled by multiple genes (polygenic) have been identified, and these mechanisms are being combined in breeding populations in Oregon, Idaho and British Columbia. Polygenic traits Most traits of interest to foresters and the wood products industry, as well as traits affecting fitness on which natural selection acts in the wild, are polygenic, controlled by several to many genes, each with a small effect on the phenotype. For most of these traits, the phenotype is the product of both the environment and the genotype (White et al. 2002). We typically have no idea how many genes, or what genes, control these traits, including growth rate, wood density, stem form and adaptation to climate. We do, however, know that they are under genetic control (to varying degrees) because family members, i.e., parents and offspring, or siblings, resemble each other more than unrelated trees when the trees are grown in a uniform environment such as a planted field test. The lack of information on the number of genes controlling these traits is not a major impediment to genetic selection. This is evidenced by the many varieties and breeds of plants and animals developed by humans through selection long before Gregor Mendel discovered how genes are inherited, or before Charles Darwin developed his theory of evolution. Typically, phenotypic traits controlled by many genes exhibit continuous rather than categorical variation (e.g., for growth rate, trees do not fall into clear categories of short or tall), and this variation is normally distributed in a bell-shaped curve.

4 4 Examples of polygenic traits: Height growth and cold hardiness in coastal Douglas-fir Variation in height growth and cold hardiness, both among individual trees and among families within individual trees, in a coastal Douglas-fir population is used to illustrate polygenic variation (Figure 1). Within a relatively small area on the central Oregon Coast, openpollinated seed were collected from 150 parent trees and seedlings planted in a field commongarden experiment. Each family (seedlings with one or two parents in common are called families) was initially represented by 16 offspring at each test site, and there was very little subsequent mortality. The frequency distribution by height class for the offspring of 60 of the parent trees (890 trees in total) is illustrated in Figure 1A, showing an approximately normal, or bell-shaped, distribution. Figure 1B illustrates the large amount of variation both within and among families for these same trees. There is tremendous variation among family means (indicated by horizontal bars), ranging from 576 cm to 788 cm. However, there is also considerable variation among the open-pollinated progeny of individual parent-trees (depicted by the vertical lines). When these same trees were seven years from seed, shoot cuttings were collected from 40 families from this field test site in April (approximately one month prior to bud break) and subjected to artificial freezing at 10 and 14 o C in a programmable freezer (see Anekonda et al for cold hardiness testing methods and Aitken and Adams 1997 for results). There was considerable genetic variation for cold hardiness in the population, with individuals ranging from 1 to 100% cold injury (averaged across the two test temperatures) of stem tissues including the vascular cambium and phloem. Average family cold injury ranged from 2 to 84% (Figure 1C). The polygenic nature of genetic control of cold hardiness was confirmed by a subsequent molecular study. Specific DNA sequences in areas of chromosomes that appear to contain cold hardiness genes has confirmed that a minimum of 11 DNA regions are significantly associated with variation in fall cold hardiness, and 15 are associated with spring cold hardiness, with only one region in common between fall and spring hardiness (Jermstad et al. 2001). The actual number of genes controlling cold hardiness is thought to be at least twice the number detected. Geographic variation in natural populations Adaptation genes and environments Genetic variation for genes or traits that do not affect the fitness of individuals in specific environment (such as isozymes) will be distributed across the landscape in a manner that reflects where new alleles have resulted from mutation, and how new alleles have spread by gene flow via pollen or seed movement (Yeh 2000). Of more interest to foresters are traits that have direct effects on the fitness and economic value of trees, i.e., the ability to survive and grow in specific environments. For traits including the timing of events in the annual cycle of growth and dormancy, such as the initiation of growth in the spring, the cessation of growth in the summer or fall, the cold acclimation of trees in the fall, or the deacclimation in spring, the distribution of genetic variation across the landscape is not random. It is structured spatially at a variety of scales. Within stands there is typically tremendous tree-to-tree variation. However, trees located in close proximity to one another have a greater chance of being genetically similar due to relatedness, i.e., the probability that adjacent trees in a natural stand are siblings with one or possibly both parents in common is somewhat greater than trees at greater distances. This variation is typically random with respect to environmental variation on a local scale (e.g., among stands at the same elevation in a local area). At a regional scale, populations occupying environments that differ substantially in climate are likely to vary genetically for traits relating to response to this climatic variation. Populations that occupy environments with similar climates, even if those environments are located a considerable distance apart, will tend to be genetically similar for traits relating to adaptation to these climates, such as the timing and duration of active growth or level of cold hardiness. The study of relationships between genetic variation and environmental variation is referred to as genecology. When variation among populations is studied across climatic gradients, genetic

5 5 gradients, called clines, are typically observed associated with the climatic gradients. For example, a steep genetic cline was observed for date of bud break against elevation of origin of seed parents in interior spruce seedlings grown in a nursery test (Picea glauca x engelmanii) in southeastern British Columbia (Figure 2). This cline reflects adaptation to temperature regime, including growing season length. Repeated observations of genetic clines for adaptive traits in association with climatic gradients provide strong evidence that natural selection has produced the genetic clines (Endler 1986). Understanding these adaptive patterns of genetic variation across the landscape is the first step of genetic management for reforestation. These patterns are then used to design speciesspecific seed or breeding zones, or limits to seed transfer, to ensure that well-adapted populations of forest trees are regenerated. Just as different tree species have different ecological requirements, species vary in the steepness of genetic clines and patterns of genetic variation. Here we describe how these patterns are assessed, and illustrate clinal patterns of variation for several Pacific Northwest tree species. Provenance testing Because most adaptive traits determining survival, growth and health are polygenic and under strong environmental influence, genotypes originating from different environments must be grown under relatively homogeneous conditions in one or more common garden tests to assess the genetic component of variation. The traditional method of determining patterns of genetic variation for forest tree species is through provenance testing. Provenance means place of origin. For trees, provenance refers to the geographic location where a seedlot, tree or parents of a family originated. Initially, open-pollinated seeds are collected from parent trees in natural stands in different locations throughout a species range or throughout the portion of that range of interest to the organization(s) involved. Seedlings are grown in a nursery, then planted out on several to many test sites across a range of environments throughout the geographic area of interest in a replicated design (see Zobel and Talbert 1984?, White et al. 2002). Sometimes individual provenances are planted in single-provenance blocks rather than in intimate mixtures with other provenances to avoid shading or competitioninduced mortality of less vigorous provenances. These trials are usually designed as long-term tests, with decisions made about zonation after the trees have grown for one quarter to one third of a typical rotation. Survival, growth and health are assessed periodically. Seedling genecology tests Field provenance trials are expensive to install and slow to obtain results from. They are often relatively inaccessible, and while growth is used as a general index of health and vigor, specific biotic agents (e.g., insects and diseases) or abiotic factors (e.g., temperature extremes, drought) causing growth reductions or mortality may go undetected. However, they do provide realistic indications of long-term survival and growth in natural environments. An alternative means of evaluating geographic variation is through the use of seedling common garden experiments. Seeds from different provenances are germinated and seedlings grown for two or three years in a replicated greenhouse or nursery trial. Environmental treatments such as varying temperature or moisture regimes are often imposed. Traits such as the timing of bud burst and bud set can be recorded and studied in detail. Gas exchange traits such as photosynthesis and stomatal behaviour can be monitored (although these traits are extremely labour intensive and difficult to measure on large numbers of seedlings). Tissue samples can be collected and subjected to artificial cold hardiness testing or other biochemical assays. Seedlings can also be exposed to insects (e.g., spruce shoot tip weevil in Sitka or interior spruce) or to diseases (e.g., white pine blister rust, root rot fungi) in a uniform manner. Seedling genecological tests often reveal stronger patterns of genetic variation than provenance trials, and provide information to guide seed transfer in a reasonable time frame. However, results are for seedling traits only, and are more difficult to translate into potential losses in stand-level productivity on field sites than are field-based provenance test results. Ideally, seed transfer guidelines are based on both long-term field provenance trials and short-term, intensive seedling genecological studies.

6 6 Clinal variation along temperature and moisture gradients The Pacific Northwest is characterized by large, steep environmental gradients, particularly for temperature and moisture, and the ranges of many forest tree species span these gradients. Forest trees have evolved different approaches to ensure adaptation to variable climates. Some species, like Douglas-fir, have adopted a specialist approach in which genetic variation is organized into numerous local populations adapted to a relatively narrow subset of environments. Other species, like western white pine and western redcedar, are adaptive generalists. In this case, populations adapt to a broad range of environments through phenotypic plasticity, whereby a given genotype can produce a variety of phenotypes, depending on the environment, either through multiple biochemical pathways associated with high genetic diversity (i.e., heterozygosity) or through natural selection favoring alleles that tolerate broad environmental differences (Rehfeldt 1984). Case study: Douglas-fir Douglas-fir has one of the broadest ranges of any North American conifer, ranging from Mexico to British Columbia, and spanning up to 1,500 m of elevation in local areas such as in the Cascades. When grown in a common environment, Douglas-fir shows a great deal of genetic variation, and much of this variation is found in differences among populations, which in turn is correlated with environmental differences at the seed source. Two varieties of Douglas-fir are recognized, based on morphological, ecological and physiological differences: Coastal Douglas-fir (var. menziesii) and Rocky Mountain or Interior Douglas-fir (var. glauca). Coastal Douglas-fir is a shade-intolerant, fast growing conifer adapted to long growing seasons and relatively high levels of precipitation. Interior Douglas-fir is characterized by greater shade tolerance, greater cold and drought hardiness, and slower growth than the coastal variety. These two forms are thought to have differentiated as many as 10 million years ago (Critchfield 1984(?)). Genecological studies of both the coastal variety and the Rocky Mountain variety indicate that large differences exist among populations in growth, germination, bud phenology, and cold hardiness within varieties. This variation consists of steep clines associated primarily with elevation, but also latitude, longitude, aspect and slope of stand origin (Hermann and Lavender 1968; Campbell and Sorensen 1978; Campbell 1979; Campbell 1986; Rehfeldt 1989; Campbell and Sugano 1993). Temperature appears to be the climatic variable most strongly driving natural selection in Douglas-fir, although moisture may also be important. Figure 3 is a map of genetic variation of a composite trait based on assessments of two-year-old seedlings from parent trees in different geographic locations grown in a common garden experiment. Higher values of the composite trait indicate higher vigor, including larger size, later bud-set, and higher shoot-to-root ratio, but with slower germination (St.Clair et al, in prep). The map resembles a relief map for the region as genetic variation largely follows topographic features. The correlation (r) of average seedling growth vigor with elevation of parent trees was r = 0.75, with December daily minimum temperature r = 0.71, and with the length of frost free growing season r = 0.72 (note that r can range from 1 to 1, with values close to 0 indicating weak or no relationships, and values close to 1 indicating a strong positive relationship between two variables, and values close to 1 indicating a strong negative relationship between variables). The correlation of vigor with annual precipitation was considerably lower, r = In general, Douglasfir populations differing in elevation by approximately 300 m may be considered genetically distinct. Another Pacific Northwestern species that is an adaptive specialist is lodgepole pine. Case study: Western white pine Western white pine also has considerable genetic variation within populations, but in contrast to Douglas-fir, very little variation is found among populations except across very large distances (Rehfeldt et al. 1984). Much of the variation can be attributed to differences between populations from two regions, Pacific

7 7 Northwest populations (Idaho, British Columbia, Washington and Oregon) with high growth potential and low cold hardiness and Sierra Nevada (California) populations with low growth potential and high cold hardiness, with a transition zone in between. Geographic patterns within regions are nonexistent or weak, and no relationship exists between genetic variation and elevation of the seed source. Western white pine appears to have adapted to heterogeneous environments through phenotypic plasticity. Western redcedar is another species exhibiting little genetic variation among populations. Ecotypic variation The term ecotypic variation, in contrast to clinal variation, is used when genetic variation is patchy rather than continuous. This patchy genetic variation usually relates to an underlying patchy distribution of some environmental variable such as soil type. Variation for the vast majority of traits that have been studied for Pacific Northwest tree species is clinal rather than ecotypic. Case study: Weevil resistance in Sitka spruce The distribution of weevil resistance in Sitka spruce provides an example of ecotypic variation, although the underlying cause of this patchy distribution is not entirely clear. The major health problem for Sitka spruce in the Pacific Northwest is the white pine shoot tip weevil (Pissodes strobi). Natural resistance to this endemic pest appears to rely on a number of mechanisms including the size and number of constitutive resin canals in bark, the production of traumatic resin canals in the secondary xylem in response to wounding, and the production of chemical compounds that interfere with the life cycle of the weevil. Two centres of genetic resistance to this insect have been documented, one on Vancouver Island in the vicinity of Big Qualicum, and one in the Fraser Valley near Haney (Figure 4). Resistance was first observed in Sitka spruce provenance trials established in both Europe and British Columbia by the International Union of Forest Research Organizations in the 1970s Initially, these provenance trials were not of great interest to foresters in the Pacific Northwest as planting of Sitka spruce was very low due to the spruce shoot tip weevil. However, when these trials were revisited in the 1990s, the tremendous growth of trees originating from Big Qualicum and Haney compared to that of non-resistant trees from other sources illustrated the potential for managing the shoot tip weevil through genetic selection for resistance. In British Columbia alone, current annual planting of Sitka spruce is around half a million seedlings per year, largely in the Queen Charlotte Islands where Pissodes strobi is absent. It is estimated that ten million Sitka spruce seedlings would be planted per year in British Columbia if weevil-resistant seedlings were widely available (Forest Genetics Council of British Columbia), and reforestation with this species in Washington and Oregon would likely increase as well. Seed zones and seed transfer guidelines Seed zones and seed transfer guidelines are used to ensure that seed collected from wild stands is adapted to the environment of a planting site. Using seed and seedlings for reforestation from within a specific elevation band within a seed zone minimizes the risk of maladaptation. Seed zones and seed transfer guidelines were established in the 1960 s in response to numerous examples around the region of early plantations where growth and survival was poor due to use of seed from unknown origins, but likely from a considerable distance and a different elevation from the planting site (see Isaac 1949). Originally, seed zone maps were developed that were applicable to all species. They were produced by local groups based on knowledge of environmental differences including topography, climate, and tree growth (primarily Douglas-fir), with the assumption that environmental variation and genetic variation in adaptive traits were largely correlated. However, as discussed in the previous section, the degree to which patterns of genetic variation are reflected by environmental differences varies among species. Recently, seed zones in Washington and western Oregon were developed for individual species, taking into account available information on geographic patterns of genetic variation from genecology and provenance studies (Randall 1996; Randall and Berang 2001). These species-specific seed

8 8 zones are presented for Douglas-fir and western white pine (Figure 5). They reflect the extent to which species are adaptive generalists or specialists by nature; thus, Douglas-fir has considerably more zones than western white pine, with each zone divided into elevation bands of 150 to 600 m as compared to fewer zones and a single elevation band for western white pine. In British Columbia, geographic Seed Planning Zones (SPZs) for seed collected from wild stands (Class B seed) are the same for all species (Figure 6), but rules for allowable seed tranfers within SPZs vary among species. Seed transfer restrictions are expressed as maximum transfer distances north, south, east, and west in degrees of latitude and longitude, and up or down in elevation, from the seed collection site to the planting site (Table 3). These guidelines are based on the results of provenance trials, ecological classification and observations of reforestation success. Note: breeding zones are discussed later. Selective Breeding Programs The breeding cycle Selective breeding, also known as tree improvement, is the process of selecting, testing, and interbreeding trees with desirable traits such as faster growth, better wood quality, and greater disease or insect resistance (Zobel and Talbert 1984, White et al. 2002). This process is a continuous cycle with interbreeding creating new genotypes in each generation for further testing and selection for continued improvement of traits of interest (Figure 7). Adaptation to abiotic stresses, including temperature and moisture extremes, is usually ensured in breeding programs by assuming that local, native populations are well-adapted to their environments. Parents from adjacent, environmentally similar areas are thus grouped into breeding zones. Selective breeding programs involve a three-level hiearchy of populations including the gene resource population, the breeding population, and the production population (Figure 8). The gene resource population is the collection of all individuals available in a breeding zone to provide genetic variation for the breeding population, and includes native stands, operational plantations, and individuals in genetic tests or seed orchards. The breeding population is the collection of individuals that is used to create the new genotypes in each generation. The production population consists of the progeny and clones of the best parents that are used for producing planting stock for reforestation, usually in seed orchards. The production population is usually a subset of the breeding population. This three-level hierarchy of populations serves as a means of retaining genetic diversity while capturing genetic gain, with the greatest levels of genetic diversity available in the gene resource population and the greatest level of genetic gain available in the production population. Heritability, selection intensity and genetic gain Genetic gain, also referred to as response to selection, is the amount of change obtained in a target trait after one generation of testing, selection and breeding. Genetic gain (G), depends on two factors: (1) the difference between the selected trees and the average of all trees from which they are being selected, called the selection differential; and (2) the degree to which the trait of interest is inherited, called heritability. The expected value of the progeny of the selected trees is given by the equation G = h 2 S where h 2 is the heritability of the trait of interest, and S is the selection differential. The selection differential depends on the proportion of all trees tested that are selected, called selection intensity, and the degree to which trees differ from one another for the trait of interest, called phenotypic variation. Heritability is the proportion of the phenotypic variation that is genetic, i.e., passed on from parents to offspring. Heritability is given by the equation: h 2 = V G / (V G + V E ) where V G = the genetic component of variation and V E = the environmental component of variation. Heritability is estimated in genetic tests by the degree to which related individuals resemble each other. It is a function of the

9 9 genetic control of a trait, the genetic variation of a trait in a particular population, and the environmental variation of the genetic test. The above equations indicate that genetic gain may be increased either by increasing the selection intensity or by increasing the heritability of a trait. Selection intensity may be increased by selecting fewer individuals or families, or by increasing the number of individuals or families from which to select. Heritability may be increased for a given population by controlling the environmental variation within test environments; for example, by selecting uniform test sites or by appropriate blocking of variation within test sites. Note also that obtaining any genetic gain, whether through breeding or natural selection, depends on having genetic variation, a fact that forms one justification for gene conservation. Genetic testing Selection of parents for the first generation is usually done in native stands, but heritability is low since the environmental component of variation is large (e.g., due to environmental differences in things like temperature, nutrients and moisture as a result of microsite, local stand density, and competition from other species). For this reason, most tree improvement programs involve genetic testing in common environments using a replicated block design. Genetic tests that measure performance of offspring in order to select parents are referred to as progeny tests. The number of sites in the first generation of testing has usually been high, up to 12, but as few as 3 or 4 sites are adequate for estimating the relative performance of families if one does not expect either large family rank changes in different environments due to GxE or the loss of sites due to unforeseen circumstances (Johnson 1997). In the Pacific Northwest, the numbers of trees per family at a site ranges from 10 to 24, and the number of families range from 100 to 900, with more families allowing a greater selection intensity for the next generation while maintaining adequate genetic diversity. Breeding strategies and maintenance of genetic diversity Breeders often face trade-offs between short-term objectives of maximizing genetic gain while maintaining well-adapted trees and longterm objectives of maintaining genetic diversity and avoiding inbreeding. Higher selection intensities in the initial generations may maximize genetic gain, but the resulting smaller breeding populations may result in reduced genetic diversity in later generations, both from selection and from random sampling (genetic drift). Furthermore, inbreeding becomes a problem more quickly after each generation of intermating. Maintaining genetic diversity and avoiding inbreeding may be accomplished by ensuring a sufficient breeding population size in the initial generations, and by structuring the breeding population into subpopulations. Population sizes of 50 may be sufficient to ensure continued gain in a single trait when breeding objectives do not change, but many more may be required to maintain genetic diversity in traits of potential future importance, particularly if those traits are rare (Johnson et al. 2001). Breeding population sizes in the Pacific Northwest are generally between 150 and 450 unrelated individuals, similar to populations sizes used throughout the world (Woods 1993, Johnson et al. 2001). Genetic diversity may be further conserved by subdividing a breeding population into multiple populations. Genetic diversity is conserved in multiple populations both through selection on different traits and due to the fact that different alleles are likely to be lost due to random genetic drift in different populations. Another way to structure populations is called nucleus breeding. In nucleus breeding an elite population of high selection intensity is developed to maximize genetic gain, whereas a main population is used to maintain genetic diversity with less emphasis on short-term gains. A third strategy is sublining, whereby a breeding population is subdivided into unrelated groups of parents for the purpose of controlling inbreeding. Mating within the breeding population is restricted to within sublines, resulting in increased inbreeding within sublines; however, outcrossing is promoted in the production population by restricting matings in the seed orchard to between unrelated sublines. The

10 10 effects of inbreeding are eliminated when unrelated, inbred individuals are crossed. Most programs in the Pacific Northwest have incorporated sublining into their breeding strategy (Woods 1993, Johnson 1998). Propagation methods Once desirable genotypes are identified through testing and selection, these genotypes or their select offspring need to be mass propagated for reforestation. The most common method of propagation in the Pacific Northwest and worldwide remains the production of seed from select parents (or their progeny) in seed orchards. Vegetative propagation, however, may be used to produce propagules for species with seed production or viability problems, or to increase the number of individuals produced from a small amount of seed from valuable parents. There are currently no methods available for the wide-scale vegetative propagation of mature trees; thus, methods for vegetative propagation typically start with seeds or seedlings from desirable parent trees. The most common method of vegatative propagation is to root cuttings. For most tree species, rooting cuttings is fairly straightforward as long as the cutting donors (trees from which cuttings are collected) are juvenile, or are maintained in a juvenile state through frequent hedging. Rooting cuttings is the most common form of propagation for yellow cypress, a species that produces relatively little viable seed. It is also the standard form of propagation for poplars and their hybrids. Recently, rooted cuttings have been used to multiply the number of weevil-resistant Sitka spruce available for reforestation in British Columbia by taking a small amount of seed, growing cutting donors, and obtaining up to 50 rooted cuttings from each donor, thus, replicating each original genotype 50 times. Somatic embryogenesis is a relatively new technology for generating large numbers of embryos from a single seed. Embryos are excised from seeds and placed into tissue culture with the appropriate hormones and nutrients. Initially, the embryos produce callus tissue, an undifferentiated mass of cells. This callus tissue then gives rise to somatic embryos, each of which can be germinated and grown into a somatic seedling (or embling ) for reforestation. Currently, this technology is an expensive method of propagation and is thus not being used extensively in the Pacific Northwest, but it is very useful for producing a large number of plants from a single, high value seed. An additional advantage of somatic embryogenesis is that embryogenic cultures can be stored in liquid nitrogen while clones are tested and desirable phenotypes selected, then retrieved from storage for the production of emblings for reforestation. Deployment strategies Deployment refers to how specific genotypes are distributed across the landscape in reforestation. Issues of deployment include maintaining productive and well-adapted stands given heterogenous environments (i.e., managing genotype x environment interaction) and consideration of stand and landscape level effects of planting mixtures of genotypes versus mosaics of pure family or clonal blocks. Genotype-by-environment interaction Genotype x environment interaction (GxE) is the differential response of genotypes to different environments. For example, families that show the greatest growth in a warm environment may be the ones showing the poorest growth in a cold environment. Large GxE can reduce the overall productivity of stands since the best genotypes are not always deployed on the sites for which they grow best. GxE for adaptive traits (including growth) is managed in seed collected from wild stands by restricting seed movement for reforestation to within seed zones. In breeding programs, GxE can be managed by restricting the area from which parents are chosen (i.e., the breeding zone) and by restricting the area to which seedlings are deployed (also referred to as a breeding zone, but more correctly referred to as a deployment zone), or by selecting genotypes that show stable performance over a wide range of environments. Genecological studies are often used for initial delineation of breeding zones, but the magnitude and nature of GxE is best evaluated with genetic tests established across the full range of expected environments within a deployment zone. The Northwest Tree Improvement Cooperative consolidated first-

11 11 generation breeding programs into larger zones for the second generation based on lower than originally expected GxE observed in both field genetic tests and genecological studies (Figure 9). Another type of GxE of concern to breeders is the interaction of genotypes with different silvicultural treatments, including interactions with spacing, fertilizer, and shade. For example, some managers have expressed concern that families selected in the open, high light environments of typical progeny tests are not appropriate for use in the low-light environments of multiple-storied silvicultural systems. Results with coastal Douglas-fir, however, show little interaction of families with light intensity. Progeny from parents selected when grown in the open (full light) may be expected to result in nearly as much or more genetic gain when grown in the shade as if they had been selected based on performance in the shade (St.Clair and Sniezko 1999). Similarly, family-by-spacing interaction for growth of coastal Douglas-fir is small (Campbell et al. 1986, St.Clair and Adams 1991). Some studies have found a significant interaction of families and different levels of fertilizer in Douglasfir seedlings (Wilson and Anderson 1974, Bell et al. 1979), but not in saplings (DeBell et al. 1986). Pure blocks versus mixed stands Another question of interest to breeders is the consequences of planting genotypes in mixtures versus in mosaics of pure blocks. Clearly, risks of maladaptation and catastrophic loss of stands due to extreme weather or infestations of disease or insects are greater when a single genotype is planted over an extensive area of highly heterogenous environments, but what are the risks of mixtures as compared to mosaics of pure blocks? For example, blocks of resistant genotypes interspersed with blocks of susceptible genotypes may help limit spread of disease or insects, as it becomes difficult to find hosts beyond the block. Pure blocks may also be easier to manage for a uniform product, and may provide incidental information on genetic differences in response to different management regimes. Management decisions on deployment of pure versus mixed blocks will also depend on land ownership, land use objectives and public acceptance, as stand uniformity may be positive for some objectives (e.g., product uniformity) and negative for others (e.g., providing diversity of structure within stands for habitat or aesthetic purposes). Breeding programs in the Pacific Northwest Breeding programs in the Pacific Northwest were generally initiated in the 1960s with the selection and testing of parent trees by several organizations. Most of the effort has focused on coastal Douglas-fir, the dominant species throughout much of the region both ecologically and economically, although considerable effort has also been directed to other species (Table 4). In British Columbia, active programs are underway for a total of nine conifers. The Inland Empire Tree Improvement Cooperative also has breeding underway for several species. Throughout the region, considerable effort has been directed towards breeding western white pine resistant to blister rust. Most coastal Douglas-fir breeding programs, as well as those for western hemlock, western white pine, lodgepole pine and interior spruce, are currently breeding or testing for the second generation. Breeding programs for other species are younger and are still completing the first generation of breeding. Several organizations do not have plans to proceed with later generations at this time for some species and some geographic areas. Genetic considerations for natural regeneration In evaluating the potential genetic implications of silvicultural practices, it is important to consider the effects on average genetic diversity and average genetic quality separately. Any silvicultural practice that decreases the number of potential parent trees for natural regeneration has the potential to reduce genetic diversity. This will only be a substantial effect if very few parents, for example, less than 10, are left to interbreed and regenerate an area. For example, if an area is being regenerated through the use of a seed tree system, and just five seed trees are left per ha, the resulting genetic diversity of the regenerated stand will likely be lower than if

12 12 20 seed trees are left per ha. Any silvicultural practice that selects trees to harvest versus those to leave based on phenotypic criteria, e.g., tree size, form, or branching characteristics, has the potential to change the average genetic quality of natural regeneration. For example, if an evenaged, single species stand is partially cut using diameter limit cutting, whereby the larger trees are cut and the smaller trees are left, this has the potential to decrease the average growth rate of the regenerated stand to the extent that phenotypic differences among trees in size are a function of genetics. There is anecdotal evidence of decreased growth rate or stem form for some species that have repeatedly been highgraded in some parts of the world. Foresters must be conscious of leaving adequate numbers of good quality parents when relying on natural regeneration, to ensure that regeneration is genetically diverse, well-adapted and vigorous. If a tree is being left because it has low economic value due to stem form, quality or size, is it really a tree you want as a parent of the next generation? Juvenile spacing, precommercial and commercial thinning all have the potential to improve or decrease the average genetic quality of stands, depending on whether desirable trees are cut or left. With thinning, however, changes in overall genetic diversity are likely to be inconsequential since typically a large number of trees remain. Biotechnology Our capacity to explore and understand the DNA blueprint of life is growing by leaps and bounds. The biological opportunities at this point to apply this knowledge are greater for hardwoods than for conifers. New technologies may help us understand some of the mysteries of the huge conifer genomes and the genetic diversity they contain. How we apply this increased understanding to forestry problems will depend on the specific ecological and social contexts of managed forests. The current revolution in genetic technologies has taken us from being able to infer genetic differences from visible differences among individuals to being able to explore the DNA sequence of the entire genome of species. How will this biotechnological revolution impact forestry? First, it will greatly enhance our ability to understand how the genome (the entire genetic makeup of the DNA in a cell) is structured and how it functions. Secondly, it will open the door to new applications of genetics for characterizing genetic diversity in natural populations, for selecting on desired traits in conventional breeding programs, and for genetically modifying trees. However, for most species managed for wood production in the Pacific Northwest, it is unlikely to have a major impact. Applications of genetic technology include (from low to high intensity): measuring and monitoring genetic diversity; selective breeding; genetic mapping and marker-assisted selection; genetic engineering (producing geneticallymodified (GM) trees); and genomic sequencing. Appropriate applications of these tools will depend on social, ecological, genetic and economic factors. At one end of the spectrum, opportunities for high-intensity applications will exist for singlegene traits in short-rotation exotic hardwoods on private lands with single primary land-use objectives and large areas of land to be planted. At the other end of the spectrum, opportunities will be limited to relatively low-intensity applications for long-rotation, native conifers on public lands with multiple land-use objectives and relatively small seed zones. Short-rotation hybrid poplar plantations on private land lie towards one end of this spectrum, while management of long-rotation conifers on public lands is at the other. The complete sequencing of the entire genome of a plant species in 2000, a small herbaceous plant in the mustard family called Arabadopsis, will open the door to huge discoveries about gene structure and function in plants. These findings will have particular relevance for hardwoods. Conifers have very large genomes (about 180X Arabadopsis, 40X poplar and 7X that of humans) so it will be a long time before an entire conifer genome is sequenced. However, a large proportion of the genome of conifers does not contain genes. Sequencing only expressed genes (as opposed to total DNA) is well underway for some shortrotation pines and similar efforts are commencing for other species. Initial results indicate that while pine and Arabadopsis share a large number of genes, a substantial number are unique to conifers. Perhaps these genomic efforts will help