Genetic Parameters and Improvement Strategies for the Pinus Elliottii Var

Genetic parameters and improvement strategies for the Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis hybrid in Queensland, Australia

Dominic Paul Kain

March 2003

A thesis submitted for the degree of Doctor of Philosophy at the Australian National University Chapter 1 - 1

Chapter 1 - Introduction

This thesis addresses two complementary issues in tree improvement: the genetic improvement of artificial interspecific forest tree hybrid populations, and the genetic improvement of wood properties.

The context of hybrid tree improvement A hybrid is an offspring of genetically dissimilar parents. Hybrids can arise from crosses between species (interspecific hybrids), or between types (eg varieties, cultivars, provenances, land races) within a species. Hybrids often display "hybrid vigour", or heterosis: an increase in vigour and yield above the mid-parent value, in one or more traits (Falconer and Mackay 1996). The favourable properties of some hybrids have been known for millenia: native North American Indian tribes, for example, exploited heterosis by ceremonially exchanging maize seeds during meetings between tribes - a practice known to maintain plant vigour and yield (Rife 1965). The utility of other traditional hybrids such as the mule and palomino horse is well known.

The use of hybrids in industrialised agriculture, however, did not commence until the beginning of the 20th Century, with the work of Shull (1908) in maize. From 1930 to 1963, the average yield per acre of maize in the United States increased more than threefold, largely due to the substitution of hybrid seed for purebred seed selected using conventional methods (Hallauer 1999). Although numerous scattered tree hybridisation experiments occurred prior to this (Duffield 1981), it was largely the spectacular success of maize hybrids that inspired organised research in tree hybridisation directed at industrial deployment (Wright 1976).

While many of the resulting tree hybrids demonstrated favourable characteristics, and these and various putative hybrids were sometimes deployed as unselected composite varieties (eg Venkatesh 1982; Ferreira and dos Santos 1997), it was primarily the difficulty of propagating the vigorous first generation (F1, filial 1) hybrid genotypes (eg Shelboume and Danks 1963; Potts and Dungey 2001) that thwarted organised hybrid breeding and deployment on an industrial scale. More recently, these Chapter 1 - 2 constraints have been overcome many taxa, through improved vegetative propagation (Brandao 1984; Walker et 1996) and pollination (Harbard et 1974; Nikles 1996) technologies.

Largely as a result, artificial interspecific forest tree hybrids are currently enjoying a resurgence of interest in industrial plantation forestry programs globally (Griffin et al. 2000; V erryn 2000; Zobel et al. 1987), often yielding results reminiscent of those in maize: "The greatest advance in industrial plantation forestry of the past 20 years has undoubtedly been in the clonal deployment of hybrid genotypes" (Griffin et al. 2000). Increases in stem volume of 20-100% over preferred parental species are not uncommon in interspecific tree hybrids (eg Baltunis et al. 1998 in Larix, Rockwood and Nikles 2000 in Pinus), although enormous gains are often reported in some hardwoods (eg 600% for stem volume in Populus, Li and Wu 1997). Often more importantly than improvements in vigour, however, the gains from preferential inheritance of favourable parental characteristics in a broad range of traits have become an important motivation for adopting 'complementary' hybrid taxa (Nikles and Griffin 1992; de Assis 2000). Additionally, the often strong adaptive characteristics of tree hybrids (eg Potts and Dungey 2001, Verryn 2000) have allowed many forestry agencies to expand into marginal environments that would otherwise be unprofitable (Denison and Kietzka 1993).

The number of organised tree hybrid breeding programs has burgeoned in recent years, with efforts in Eucalyptus in the Congo, South Africa and Brazil (Vigneron 1991; Verryn 2000; de Assis 2000), Pinus in Australia, North America and South Africa, and Larix and Populus in North America and Canada (Dungey and Nikles 2000). Large, recently established estates of hybrid Eucalyptus in Brazil, the Congo

and South Africa form the bulk of an estimated c 0.5 million ha of hybrids currently in plantation internationally (Dungey and Nikles 2000).

However, the focus of hybrid forestry, as in early maize hybridisation, has been on exploiting the gains from hybrid performance rather than seeking to understand its genetic basis or investigate the possibility of further improvement. Research efforts have mainly focussed on pre-first generation hybrid issues, such as useful hybrid combinations and propagation methods. Due to the high risk of uneconomic outcomes, hybridisation has usually been performed in small research experiments Chapter 1 - 3 ancillary to pure species breeding programs: as a result, hybrid breeding populations in most taxa have been small and poorly structured (Payne and Miller 2000). The lack of structured hybrid experiments incorporating pure species controls has contributed to the lack of empirical and theoretical genetic information available to support decisions in many breeding programs now seeking to improve interspecific tree hybrids. The recent inception of hybrid forestry on a large scale, in many countries, has created a strong need for genetic research in aid of evaluating and developing recurrent improvement strategies suitable for tree hybrids.

Issues in improvement of interspecific hybrid trees The recurring central issue in interspecific hybrid tree improvement is the high cost of genetic gain per unit capital per unit time invested, relative to pure species breeding. This is largely due to the involvement of multiple populations in hybrid improvement, yet only a single population in pure species improvement. Hence, while a large genetic gain may be made very quickly upon creating and deploying hybrids, the genetic gain per year achieved from recurrent selection thereafter is likely to be less than that achievable using pure species breeding under the same resource constraints.

In addressing this central issue, a primary consideration is to ensure that the basis of hybrid superiority is well understood in terms of the hybrid's performance relative to its parental species, in the traits of greatest economic importance. A second, increasingly critical consideration is to revise, improve, and develop cost-effective interspecific hybrid tree improvement strategies based on appropriate genetic parameter estimates, other empirical evidence and practical considerations.

To these ends, four key research priorities may be identified as: 1. Assess and understand the basis of hybrid superiority relative to pure species, in terms of trait values; 2. Assess the importance of hybrid testing relative to pure species testing, and thereby choose between existing breeding strategy options, for Fr hybrid improvement; 3. Investigate methods for reducing hybrid breeding cycle interval and expense through early and indirect selection for traits of economic importance, and; Chapter 1 - 4 4. Investigate the modes of gene action contributing to hybrid performance, and use this information to assess the potential for advanced generation hybrid breeding strategies.

1. The basis of hybrid superiority The assessment of hybrid superiority is a primary consideration in hybrid development. The expense of hybrid improvement necessitates careful assessment of benefits from the choice of a hybrid taxon over pure species alternatives. This choice must ultimately be justified based on the economic value of, or profit from, products obtained from the taxa. Although profit can rarely be measured directly, comparison of taxa based on measurable characters known to affect profit can provide a useful indication of superiority. Growth, stem form,. disease and frost resistance are examples of traits commonly assessed for this purpose. Wood density (Harding et al. 2000; Greaves et al. 2000; Borralho et al. 1993) and wood variability (Malan 1997; Wright et al. 1996; Zobel and Sprague 1998) have been shown to strongly affect the value of many wood products, yet have rarely contributed to decisions between taxa in forestry (Zobel and Jett 1995). The inclusion of such traits in taxon comparisons may be critical in some instances. Where taxa differ only slightly in economic value, the choice of pure species improvement may yield large cost efficiencies in breeding, and be preferable to hybrid forestry (Shelbourne 2000; Potts and Dungey 2001). Where the hybrid is clearly economically superior, an understanding of wood variation patterns in the hybrid and parental taxa, and their possible environmental and genetic causes, is likely to be critical for sustained genetic improvement of product value.

2. The choice among breeding strategies for F 1 hybrid improvement In hybrids, the task of further genetic improvement beyond the Ft becomes more complex. While in pure species, improvement can be achieved simply by forward selection in the traits of interest (Cotterill and Dean 1990), this practice has traditionally been avoided in hybrids due to "hybrid breakdown": the deterioration of hybrid performance in forward selected Ft progeny, evidenced from early breeding experiments in model organisms (eg Shull 1908). Consequently, the conventional approach in crops and trees has been recurrent improvement of vigorous F1 hybrid populations. Breeding strategies for this purpose require the concurrent improvement of one or both parental populations, and an F1 deployment population. The value of Chapter 1 - 5 these complicated strategies involving both hybrid crossing testing and pure species recombination and testing within a single breeding cycle has been well demonstrated by realised gain maize in short rotation Eucalyptus (eg Moll and Hanson 1984; Vigneron et 2000). However, these strategies (eg Shelboume 1993, Fig.1, Fig.3) may commonly take 20 years to complete a breeding cycle in trees, whereas a cycle of pure species breeding under comparable conditions can be completed in 9-10 years. Ascertaining the need for hybrid testing is therefore critical. Where hybrid performance and pure species performance are both heritable and well correlated genetically, it may be possible to improve the hybrid through pure species selection, thereby halving the breeding cycle interval. The efficiency of pure species selection for hybrid performance depends principally upon the genetic relationship between pure species and hybrids, which is poorly understood: while moderate to strong relationships have been indicated by some studies (eg Powell and Nikles 1996b; Rockwood et al. 1991), in other instances the two appear uncorrelated (eg Dieters and Nikles 1998). Improvement of hybrids through pure species selection holds promise, but prediction of its efficacy awaits further estimates of critical genetic parameters.

3. Efficient assessment of traits Reduction of the breeding cycle interval and expense of hybrid improvement may also be achieved through more efficient assessment of traits. Determining the optimum age for early selection, and identifying cheaper methods for indirectly assessmg expensive-to-measure traits such as wood properties, are two useful ways of improving efficiency. Although a common problem in pure species tree breeding, the issue of measurement expense is exacerbated in hybrid improvement - firstly, as the need to shorten the long breeding cycle interval is of foremost priority, and secondly, as traits must often be measured in two or more breeding populations. The theoretical efficiency of early selection has been demonstrated in a variety of taxa and traits, often suggesting high genetic gains from selection at juvenile ages. However, few estimates have been published for wood properties, and early selection efficiency predictions are calculated from genetic parameters that are often highly sensitive to experimental conditions such as the taxon, age, site and silviculture, and so cannot be directly compared between studies. As existing research results are insufficient to allow generalisations, direct assessment of early selection efficiency becomes necessary in the populations of interest. Similarly, although some studies of field Chapter 1 - 6 screening methods such as the Pilodyn (Hoffmeyer 1978) and Protractor (Harris 1984) have shown potential for highly efficient genetic prediction of stem density and spiral (eg Cown and Andrew 1979; Greaves et 1995; Sorensson et al. 1997a), results in other studies have been inconsistent (eg Muneri and Raymond 2000, Sorensson, pers. comm.). The usefulness of these tools may be confined to certain populations, environments or operating methods, and extensive testing in the populations of interest may be necessary to ascertain their reliability (Cown and Andrew 1979). Further investigation into early selection and field testing methods has the potential to significantly reduce the duration of hybrid breeding cycles, and the expense of improving hybrid wood properties, respectively.

4. Advanced generation hybrid breeding strategies and their dependence on gene action A fourth key issue in hybrid forestry is the possibility of advanced generation hybrid breeding strategies. Early tree hybrid breeders were strongly influenced by theory in maize hybrids, and most of the strategies yet proposed in trees have been borrowed from the maize literature (eg Comstock et al. 1949), with only slight modifications (eg Shelbourne 1993). While their proven usefulness in model species is re-assuring, their long cycle intervals, and the need to concurrently improve multiple breeding populations, create impracticalities when applied to trees. This has fuelled recent interest in the possibility of alternative, simpler hybrid improvement strategy options in trees (eg Li and Wyckoff 1994; Nikles and Griffin 1992; Verryn 2000).

Early experiments in crops indicated declining performance in second generation (F2) and higher generation (Fn) hybrids derived from crosses amongst individuals of the F1 generation. Typically however, these F1 individuals were genetically closely related, and the advanced generation hybrids, consequently inbred (eg Neal 1935; Morris et al. 1999; Hadley and Openshaw 1980). Conversely, selection experiments in outcrossed, genetically variable composite (also called synthetic) crop and animal hybrid populations have demonstrated steady genetic gain from successive generations of recurrent selection (Bourdon 1999; Martin and Russell 1984). Strategies employing forward selection and recombination in hybrid populations - often referred to as Advanced Generation Hybridisation (AGH), or Composite Breeding strategies, have recently been widely and successfully used in animal breeding (Kinghorn 2000). However, they have rarely been considered by forest tree Chapter 1 - 7 breeders (Dieters et al. 1995c). Possible explanations for this are firstly, the lack of broad base populations of F1 hybrids, secondly, expense of tree breeding and consequent lack of margin for risk in breeding strategy. The first issue is being resolved as organised hybrid breeding programs amass unrelated F 1 hybrid crosses. The second issue relates to the risk of the advanced generation hybrids being inferior to theFt hybrids. However, accumulating empirical evidence suggests that selected, outcrossed advanced generation hybrids may be capable of improving on F 1 performance in a broad range of forest tree hybrid taxa (eg Braun 2000; Hyun 1974; Paques 2000; Powell and Nikles 1996a; Potts et al. 2000). AGH presents two main advantages over conventional hybrid breeding: firstly, a large reduction in costs due to shortened breeding cycle interval and the need for only a single breeding population and, secondly, the ability to select directly for traits of interest in the population of interest (Li and Wyckoff 1994).

In theory, the risk of producing degenerate progeny from AGH depends largely upon the importance of dominance, overdominance (and related epistatic) gene action relative to additive (and additive-related epistatic) gene action in the genetic control of economically important traits (Namkoong 1979, p.97). While the conventional statistical genetic model (eg Fisher 1918; Comstock and Robinson 1948) has proved useful for estimating pooled statistical effects of genes, the relationship between these statistical effects and physiological (Mendelian) gene action is clear only under very specific circumstances, which are not met in hybrid populations (eg Gardner 1963; Gordon 1999). Physiological gene action at the level of individual loci or linkage groups is difficult to assess in polygenic traits due to the concurrent effects of multiple linkage groups. Recent increases in computing capacity have allowed the implementation of theoretical statistical genetic models based at the level of the individual locus or linkage group (eg Fernando et al. 1994; Li and Wu 1996; Pong­ Wong et al. 1998; Wu et al. 2001). These models hold particular promise for describing the primary modes of gene action responsible for hybrid performance, and hence, for suggesting the most appropriate hybrid breeding strategies.

The Queensland case Amongst the pioneering efforts towards successful large-scale operational deployment of hybrids backed by organised hybrid breeding was the work of Nikles and co­ workers in South-East Queensland (SE QLD), Australia (eg Nikles 1996; Bowyer Chapter 1 - 8 1985). Today, the plantation area of Pinus elliottii var. elliottii x P. caribaea var. hondurensis (PEExPCH) hybrid exceeds 26 000 ha, and is anticipated to exceed 100 000 ha, in SE QLD alone (Nikles 2000). The PEExPCH hybrid is also planted operationally in South Africa (van der Sijde and Roelofsen 1986; Malan 1995), and has demonstrated increased stem volume, and sometimes improved stem straightness, over preferred plantation species in Zimbabwe (Gwaze 1999) and Florida (Rockwood and Nikles 2000), on certain sites. It has also performed well in Argentina, China and Uruguay (Nikles 2000).

The focus in development of the PEExPCH hybrid in Queensland has been exclusively on selection of parents for superior growth and stem form, and although an active program of investigation and screening of wood properties has been in progress for several decades, no operational selection for wood properties has yet been carried out. Genetic gains in growth and form traits have been made through 1-2 generations of family and within-family selection in the parental populations (Toon et al. 1996; Dieters 1999), and recently, through a round of clonal hybrid testing and selection, also for stem volume and form traits (Dieters, pers. comm.). The resulting fast growth and superior form of clonal hybrid plantations has made possible the production of merchantable sized logs within a 20-year rotation from most sites in the SE QLD estate, for the current principal market of sawn structural timber (Haines 2000). However, this planned reduction in rotation age entails a substantial increase in the proportion of juvenile wood in the final harvest, and a series of utilisation studies conducted in QLD (reviewed by Harding et al. 2000) have found undesirable juvenile wood properties for structural grade sawn timber in a large proportion of representative samples from the PEExPCH hybrid and both parental populations.

Due to the impracticalities of improving juvenile wood properties through silviculture (eg Clark and Saucier 1991; Kucera 1994), genetic improvement, demonstrated to yield economically useful gains in other taxa (eg Borralho et al. 1992, Reck 1974), is likely to be the most feasible approach for wood quality improvement. The breeding objective is maximum revenue from a dressed sawn structural timber end product, with selection criteria of MAl, wood density, butt sweep and spiral grain (Greaves et al. 2000). Chapter 1 - 9 Serious impracticalities and suspected inefficiency of existing hybrid breeding strategies have created a need to critically examine phenotypic and genetic parameters in the PEExPCH hybrid and parental species, and re-assess strategy options for maximising genetic gain in the breeding objective per unit capital per unit time. A unique opportunity to examine PEExPCH and parental taxa has been made available in Queensland Forestry Research Institute (QFRI) experiment EXP674, a large trial incorporating pure species and hybrid progeny of common pedigree, generated from controlled crosses, replicated across four sites in SE QLD. The hierarchical taxon, parent-within-taxon and family-within-parent structure of the dataset allows estimation of means and variances at different levels. The age of the trial at the time of assessment, 11 years, allows sampling of important wood and growth traits at mid­ rotation.

The subject matter of this thesis This thesis uses data from QFRI EXP674 to address four key issues in hybrid breeding strategy, using four sets of analyses conducted on the dataset, with the following objectives:

1. Evaluate and compare variation in wood, growth and form within and among

F1 and F2 PEExPCH hybrids and parental species populations, and discuss results with reference to current and future markets for the taxa in SE QLD; 2. Estimate genetic parameters for wood, growth and form traits in PEExPCH Ft hybrid and parental populations, and examine their implications for breeding

strategies for F1 hybrid improvement; 3. Estimate the optimum selection age for wood and growth traits, and investigate effective indirect selection methods for wood traits in the PEExPCH hybrid and parental populations; 4. Elucidate the biological genetic mechanisms contributing to PEExPCH hybrid performance in wood, growth and form traits, and discuss the implications for hybrid genetic improvement and alternative breeding strategies.

To achieve these objectives, phenotypic measurements of whole-tree growth traits and form, tree basal area and wood density measured on annual ring increments, and

spiral grain angle measured on selected ring increments, were carried out on the F1

hybrid, and on selected traits in the parental species and F2 hybrid, in QFRI EXP674. Chapter 1 - 10 The following analyses were conducted to achieve each of the four objectives, respectively:

1. Trait means and variances were calculated and compared among PEE, PCH, F1 and F2 populations. The degree of hybrid vigour was calculated and compared among traits. Hybrid vigour and within-family variation were compared between the F1 and F2 populations, to provide indications of changes in mean and variance, respectively, in an advanced generation hybrid population. Taxon means in economically important wood traits were discussed in relation to QFRI wood improvement objectives for current and future markets.

2. REML (Residual Maximum Likelihood, Patterson and Thompson 1971) analysis of variance was used to partition genetic and environmental variance components in each trait, in the pure species and hybrid populations separately; these variances were used to estimate genetic parameters and predicted genetic gain from family selection in these populations. The genetic correlations among traits within each population, and the genetic correlation between the same trait in pure and hybrid populations (rph), were estimated. The selection efficiency of indirect selection for hybrid performance based on pure species performance was estimated using calculated parameters. The results were discussed in reference to the decision between hybrid breeding strategies under the assumption of deploying Fr hybrids.

3. Heritability and juvenile-mature genetic correlations for basal area growth, wood density and spiral grain angle were estimated for each juvenile year through to maturity, in each of the three taxa. These parameters were used to calculate the efficiency of early selection at each age, and to identify the optimum age for selection in each trait in each taxon. The indirect selection efficiency of pilodyn pin penetration for wood density, and bubble protractor field measurements for spiral grain traits, were also assessed. The results were discussed in relation to cost-effective incorporation of economically important wood properties into the QFRI hybrid improvement programme.

4. A novel quantitative genetic model developed at NC State University was used to model hybrid population genotypic values at the level of the individual locus or genetic factor, for growth and wood traits. The model enabled estimation of the Chapter 1 - 11 relative contribution of different modes of gene action to hybrid performance, as well as the number of genetic factors contributing to hybrid performance. Implications of the results were discussed in relation to future genetic improvement strategies in the PEExPCH hybrid, and in relation to the likely usefulness of alternatives to conventional hybrid improvement strategies in both PEExPCH and in tree hybrids more generally.

The four research objectives and analyses described above address the four key issues for tree hybridisation research discussed earlier in this chapter, and are fully documented in chapters 3, 4, 5 and 6 respectively. These investigations pertain to the PEExPCH hybrid and pure species experiments examined, but the results have broader implications, which are discussed with reference to existing literature and evidence relevant to the key issues identified. Chapter 2 reviews the context of these four issues in greater detail, and addresses analytical issues associated with the study of wood properties and interspecific hybrids. Chapter 2 - 12

Chapter 2 - Issues in hybrid tree improvement

This chapter reviews general literature providing the background and context of the four key issues identified in Chapter 1, and analytical issues associated with the investigation of these issues in PEExPCH with respect to analysis of QFRI EXP674. Four main sections: 1. Hybrids and pure species: assessing variation at the taxon level; 2. Quantitative genetic analysis of interspecific forest tree hybrid data: application in hybrid breeding strategies; 3. Quantitative genetic analysis of longitudinal wood property data; 4. The genetic architecture of tree hybrid populations: implications for conventional and novel hybrid breeding strategies; discuss background literature for chapters 3, 4, 5 and 6, respectively. Other issues in hybrid tree improvement not directly relevant to the aims of this thesis are addressed in the previous reviews of Martin (1989), Dungey (1999), Dungey (2001) and Potts and Dungey (2001).

2.1 Hybrids and pure species: assessing variation at the taxon level

Hybridisation commonly results in improved performance in a variety of traits, relative to the parental populations. Many hybrids of tree species have been found to exhibit favourable characteristics for industrial forestry. Deciding between hybrids and pure species for forestry, and optimising the value of further genetic improvements in hybrids, requires an understanding of the basis of hybrid superiority in the various traits that contribute to profit, or product value. Suitable parameters are ones that provide practically useful and meaningful comparisons among pure species and hybrid taxa. Many forest tree hybrids in industrial use, and many more of potential use, may surpass both parents in their economic value to industry, without surpassing either parent in any individual trait (selection criterion). Consequently, simple measures of hybrid superiority used in crops (eg Crow 1952) may not be appropriate in trees. Additionally, while hybrid forestry and improvement has generally concentrated on the F1 (1st filial) generation of hybrids, some hybrid tree breeding programs have entered advanced generations, and second generation hybrids and backcrosses have in some cases surpassed both pure and F1 hybrid taxa in their Chapter 2 - 13 performance. However, many advanced generation hybrid experiments trees have suffered inbreeding, fair comparisons with parental taxa have not been made and reviewed literature proper consideration of their genetic history. Further issues are that comparisons between hybrid and pure species performance are often strongly affected by genotype-environment interaction, ontogeny, and crossing incompatibilities between populations, and must be interpreted carefully with respect to these factors. Discussion of parameters for comparing pure species and hybrids will be followed by a review of hybrid performance in a variety of taxa, and additional considerations for assessing hybrid populations.

2.1.1 Parameters for comparing pure species and hybrid populations

Hybrids are of little use industrially if they do not surpass their parental genotypes. Numerous measures of hybrid performance relative to parental performance have been proposed and often assigned conflicting definitions in the crop and tree genetics literature, so clarification is necessary.

Better parent heterosis, or better parent hybrid vigour (herein referred to as HVB), is defined as the deviation, either positive or negative, of hybrid performance in a metric trait from the performance of its better parent in that trait (eg Kearsey and Pooni 1996). To justify the additional expense of hybrid breeding, hybrids must display, at very least, significant positive HVB for overall profit or revenue. In such cases, a general term 'hybrid superiority' (Nikles 1994) is used to denote greater economic value of the hybrid. In practice, however, the value of wood products from different taxa can rarely be evaluated directly, and a comparison based on characteristics known or expected to contribute to product value may be the best available information on which to base the choice of taxon.

In many operationally deployed forest tree hybrids, particularly in complementary and adaptive hybrids, hybrid performance exceeds the mid-parent value in most economically important traits, but does not exceed the better parent in any trait, although when all traits are considered together, the hybrid is preferable (eg Martin 1989; Nikles and Griffin 1992). Hence at the individual-trait level, the concept of mid-parent heterosis, or hybrid vigour (herein referred to as HV), is a more useful reference point for comparing the hybrid and pure species populations, where HV is Chapter 2 - 14 defined as the deviation, either positive or negative, of the hybrid from the mid­ parental value, in a metric trait (Falconer and Mackay 1996) expressed as a percentage,

[2.1] where: HV% =percent hybrid vigour (heterosis) relative to the mid-parent; F 1 =mean value of the F1 hybrid generation MP = mid-parent value;

Note: Although HV here is defined for F 1 hybrids, in practice it may also be used to compare the mean of F2 and more advanced generation hybrids to pure species parental population means.

The mid-parent also provides a more useful reference point for quantitative genetic theory, being the expectation of hybrid performance under completely additive gene action (discussed further in Section 2.4.6). For these reasons, the definition of hybrid vigour, or heterosis, used in this thesis will be midparent hybrid vigour (HV) or heterosis. However, statistically significant HVB is an important observation, and should be noted where it occurs. Additionally, measurement of HV is not always possible, for example where one parental species will not grow in the environment of choice, as is the case with Pinus rigida x P. taeda in Korea (Hyun 1976) and Larix decidua x L. leptolepis in France (Paques 2000). In such cases, better parent heterosis is a more appropriate statistic. The assignment of positive and negative heterosis is arbitrary, and may be ambiguous in some traits such as wood density, where lower density is desirable for some end products (eg Smook 1992); in these cases, the orientation of positive and negative values should be specified.

2.1.2 The uses of hybrids in forestry

Understanding the various reasons for using hybrids is useful to aid the decision between pure and hybrid taxa, and to guide hybrid improvement strategies. The principal reasons identified in the literature for preferring interspecific hybrid to pure species taxa are complementarity, adaptability and better parent heterosis (Stettler et al. 1996), although the superiority of many hybrid combinations is determined by two or more of these factors: Chapter 2 - 1. Complementarity is an attribute of hybrids combine favourable characteristics traits importance from both parental populations, creating on average, a more desirable phenotype. The PEExPCH developed in South-East Queensland, Australia (eg Nikles 1996), is a good example of such a hybrid, exhibiting complementary performance in a variety of traits. Although the hybrid does not, on most sites, exceed the better parent in performance in any trait, it consistently improves on the mid-parent in wind-firmness, growth, stem form, branch size and angle, bark thickness, stem taper and dry wood weight yield, among other traits, and derives its overall superiority from these complementary characteristics variously contributed by the two parental species (Harding and Hagan 1990; Nikles 2000). Complementary hybrids attain superiority from generating novel combinations of genes that could not be achieved through pure species improvement.

2. Adaptability is often confused with complementarity, although it may in most part result from complementarity between species with different adaptive characteristics, as in the E. nitens x gunnii hybrid used in Tasmania (Manson and Potts 1995). Another example is the Larix decidua x L. kaempferi hybrid planted in France and Scotland, whose intermediate growth, cold tolerance, stem form and wood density result in hybrid superiority to both parents on a variety of low elevation sites in northern Europe (Paques 1989). However, in addition to these complementary effects for adaptive traits, hybrid adaptability often appears to be reinforced by a 'buffering effect' of the high genetic variability in the hybrid population (Lerner 1954), which in theory acts to stabilise hybrid performance across sites due to the greater diversity of genes adapted to a wider variety of environments (Rieseberg and Carney 1998).

3. Better parent heterosis is where hybrid performance in a single measurable trait exceeds the performance of either parent in that trait. An example is the Populus tremula x P. tremuloides hybrid, which shows increases in stem volume of up to 600% over the better parent (Stettler et al. 1996). In forest trees, better parent heterosis in economically important traits is less commonly a cause of hybrid superiority than it is in crops; more commonly, the decision to use tree hybrids is motivated by their complementary and adaptive characteristics (Nikles and Griffin 1992; Verryn 2000). Chapter 2 - 16 2.1.3 Why isn't everyone using hybrids?

The useful characteristics of tree hybrids have been known for over a century (eg Klotsch 1871 cited in Duffield 1981) and have stimulated a large range of interspecific hybridisation experiments in a variety of tree taxa (reviewed by Critchfield 1973; Potts and Dungey 2001; Paques 1989; Zsuffa 1973). In many cases these hybrids have outperformed their parents (eg Powell and Nikles 1996a; de Assis 2000), while in other cases hybrids are inferior, or only superior on some sites (eg Verryn 2000). The deployment of superior hybrids in industrial forestry has been precluded by the difficulty of propagating them - a result of low seed yields and lack of vegetative propagation technology (Zobel and Talbert 1984). However, emerging technologies for vegetative propagation such as micropropagation and tissue culture in conifers (eg Steltzer et al. 1998; Park et al. 1993) and minicuttings (de Assis 2001) in eucalypts are now making the exploitation of heterosis increasingly accessible to tree breeders. The search for ways to cost-effectively exploit hybrid superiority is likely to strongly influence future research in tree improvement, as opportunities become available to mass-propagate interspecific crosses of known and potential merit.

2.1.4 Observations of heterosis in forest tree hybrids

Although the degree of hybrid superiority (in terms of value or profit) is of greatest interest as an index of hybrid performance, in practice it can rarely be quantified. The most commonly used measure of hybrid performance is the degree of heterosis in several economically important traits, which typically provides a useful indication of hybrid superiority.

Investigations of heterosis have focussed on five genera: Pinus, Eucalyptus, Populus, Larix and Picea, although research has also been undertaken in Salix, Alnus, Juglans and Betula (see Martin 1989, Table 1), and more recently, Acacia (Dungey and Nikles 2000). Interspecific forest tree crosses typically do not display the large better parent heterosis found in crop variety hybrids of inbred lines (eg Moll and Stuber 1971), but often achieve hybrid superiority through moderate heterosis in several important traits. Chapter 2 - 17 A serious problem in assessing heterosis in tree hybrids is the general lack of hybrid experiments with parental controls (Dungey 1999). Even where parental controls are present, parental and hybrid material is usually of different pedigree, making comparisons difficult, and possibly misleading in the case of small population samples involving few parents from each population (Paques 1989).

In comparing the performance of interspecific hybrid and pure species tree taxa, two major trends stand out, although there are notable exceptions to each. The first trend is that of greater heterosis in some types of traits than others: typically, it is greatest in traits related to adaptive fitness, such as growth and reproductive characters (Rieseberg and Carney 1998), and lowest in evolutionarily neutral traits such as most wood properties and morphological characteristics. This trend is not confined to trees but appears common to all organisms (eg Kinghorn and Atkins 1986, Swan and Kinghorn 1992 in livestock; Levin 1978 in plants). A second trend is that of greater heterosis in some genera than in others: in general, heterosis tends to be highest in Populus and Salix, and generally somewhat lower in most hybrids of Eucalyptus, Larix, and Pinus. Variation in heterosis among traits appears to be a more general trend, with broader implications than variation among taxa, and so this review will emphasise trait-related differences in heterosis.

2.1.4.1 Growth, reproductive and adaptive traits A strong trend in the performance of hybrid trees, and also in hybrids of other organisms, is noticeably larger heterosis in the fitness-related traits of growth, vigour and reproductive characteristics, in contrast to much lower heterosis in most wood properties and morphological traits, which typically appear to be evolutionarily neutral (Zobel and Rhodes 1956; Rieseberg and Carney 1998). This phenomenon is thought to be due to stronger natural selection for alleles displaying directional dominance in adaptive traits than in non-adaptive traits (Hahn and Haber 1978), and corresponds with the generally higher non-additive genetic variance observed in growth, fecundity and adaptive characteristics than in wood and morphological traits, in pure species populations (eg Zobel and Jett 1995).

The expectation of larger heterosis in fitness-related traits is well supported by empirical evidence in trees. Traits such as growth, flowering, disease resistance and Chapter 2 - 18 insect resistance often display strong and somewhat unpredictable heterosis. For example, in Populus and Salix, very strong heterosis is commonly reported: Li and Wu (1997) found better parent heterosis of 190% and 70% for stem volume in Populus tremuloides x P. tremula on a high and low quality site, respectively; Stettler et al. (1988) similarly found better parent heterosis of 201% and 295% for stem volume in Populus trichocarpa x P. deltoides on two sites. These results concur with those of Heilman and Stettler (1985) and Weber et al. (1985) in Populus, and are likely to be reliable because the experiments were replicated progeny tests incorporating multiple parents, with pure species and F1 progeny of common pedigree.

Moderate to strong heterosis for growth has also been reported in some hybrids within Eucalyptus. Endo and Lambeth (1992) report better parent heterosis of approximately 50% for wood volume in a trial comparing E. grandis with the E. grandis x E. urophylla 'urograndis' hybrids in Colombia. Although this estimate is questionable as the genetic relationship between the pure species and hybrid material was unknown, the magnitude of the estimate appears consistent with other studies of this genetically distant (inter-sectional) cross within Eucalyptus (eg Vigneron 1991, approximately 25% better parent heterosis), exceeding that typically found in other interspecific crosses within the genus (reviewed by Potts and Dungey 2001). In a study by Darrow (1995) in South Africa, E. grandis x E. camaldulensis and E. grandis x E. tereticornis each displayed approximately 20% better parent heterosis,

although this was on a marginal site for the pure species. Studies of E .. nitens x E. globulus, and E. gunnii x E. globulus in Tasmania have generally shown hybrid intermediacy, and in some cases slight heterosis for growth (Potts and Dungey 2001). Studies in Larix and Pinus typically show at least statistically significant mid-parent heterosis, and often better parent heterosis (eg Powell and Nikles 1996a; Rockwood and Nikles 1996; Baltunis et al. 1998; Li and Wyckoff 1994; Paques 1989)

In many interspecific crosses, however, little hybrid vigour is evident for any trait. For example, Schmitt (1968) summarized the performance of southern pine hybrids tested in Southern Mississippi; these were generally intermediate in growth rate between the parents. Chapter 2 - 19 In addition to growth and vigour, adaptive and reproductive traits often show very strong heterosis. The common success of F1 hybrids between species with good growth and frost tolerance (eg Eucalyptus gunnii x E. globulus in Tasmania, Manson and Potts 1995; Pinus rigida X P. taeda in Korea, Hyun 1976), and species with good growth and good disease or insect resistance (eg Pinus coulteri x P. jeffreyi, Zobel and Talbert 1984, Pinus strobus x P. griffithii, Blada 1992) suggest dominant properties of genes conferring frost and disease tolerance, consistent with expectations for traits subjected to strong natural selection pressure. The high cold tolerance of F1 P. rigida x P. taeda hybrids, yet poor cold tolerance of the less heterozygous second generation hybrids, strongly suggests the role of dominance or dominance-related epistasis in the control of this trait in the hybrid. In other taxa however, strong negative heterosis has been documented in these traits; for example, Dungey et al. (2000a) report that Eucalyptus gunnii x E. globulus hybrids in Tasmania are favoured by defoliating insects, while the data of Rockwood and Nikles (1996) show greatly increased susceptibility of the PEExPCH hybrid to fusiform rust, relative to PEE. In summary, it appears heterosis for such traits is typically high, but of unpredictable sign.

Hybrid reproductive traits also commonly deviate markedly from the mid-parent expectation. Venkatesh and Sharma (1979) report strong heterosis, sometimes better­ parent heterosis, for the reproductive traits lignotuber development, flowering precocity and fecundity, and also for growth, but intermediacy for stem form, leaf length, and most flower morphological characteristics, in an Eucalyptus tereticornis x E. grandis cross, and a reciprocal cross of different pedigree. Similarly, Venkatesh (1982) reports strong heterosis for diameter, height, flowering precocity, flower and seed productivity, seed weight, germinability and germination vigour in E. tereticornis x E. camaldulensis. These results require cautious interpretation as they are based on a single full-sib hybrid cross (FRI-4), albeit widely deployed in India. However, in the context of evolution of Australian species, strongly dominant alleles for traits such as lignotuber development in species such as E. tereticornis seem congruent with the likely strong selection pressure exerted on this trait by Aboriginal burning regimes over the past c 40 000 years (Attiwill 1994). The ability to produce coppice from stumps in some pines (eg Pinus rigida) is similarly likely to be of adaptive importance, and is similarly preferentially inherited in the P. rigida x P. Chapter 2 - 20 taeda hybrid in Korea, in which most or all individuals exhibit coppice sprouting, while pure taeda does not (Wright 1976). Flowering precocity and fecundity clearly bear relation to fitness, and strong heterosis has also been reported PEExPCH in Queensland, which typically flowers at age while its parental species flower at 5-8 years (Dieters et al. 1995c).

2.1.4.2 Wood properties and morphological characters

A strong trend in hybrid wood properties and most morphological characters relative to many other traits is that of hybrid intermediacy between the parental populations. Hybrid performance in these characters, in which variation typically has little effect on the functioning of the organism or its reproductive success, tends to be much more predictable (ie, inherited in a more additive fashion) than growth and reproductive characters. Even hybrids that exhibit strong heterosis for wood volume typically display intermediacy, often with a slight tendency towards the lower performing parent, in wood properties. Examples from extensive tests in Brazil reported by de Assis (2000) demonstrated significant, often strong, better parent heterosis stem

volume in urophylla x E. maidenii, E. saligna X E. maidenii and E. saligna x E. tereticomis, while wood density, percentage lignin, percentage ash and pulp yield almost uniformly equated to the mid-parent value in all three taxa. Similarly, the PEExPCH hybrid in South-East Queensland, Australia, displays wood density characteristics similar to or slightly lower than the mid-parent on a variety of sites, while often displaying slight better-parent heterosis for growth characteristics (Harding and Copley 2000); similar properties of the same taxa have been reported in South Africa (Malan 1995; van der Syde and Slabbert 1980). Very similar trends in both growth and wood density have been observed in Larix decidua x L. leptolepis and L. x eurolepis (Li and Wyckoff 1994; Nanson and Sacre 1978; Paques 1992; Wyckoff et al. 1992). Strongly intermediate hybrid wood properties have also been reported in Eucalyptus, for E. grandis hybrids in South Africa (Denison and Kietzka 1993; Malan 1993). Wood density in hybrids of nearly all taxa tends to be around the mid-parent value, or slightly lower. Very few studies report hybrid population means for wood density that fall outside the range of the parental species; very few also report densities higher than the mid-parent, and no known studies report higher hybrid wood density than the higher parent. Hybrid populations also tend to exhibit Chapter 2 - 21 intermediate values for morphological traits such as stem straightness and bark thickness (Powell and Nikles 1996a; Nanson and Sacre 1978).

2.1.5 Issues in assessing heterosis

Of the many evaluations of hybrid taxa, few have provided reliable estimates of heterosis, particularly in growth traits, at rotation age. Besides the need to include genetically representative pure species controls in hybrid experiments, F1 hybrid inviability, heterosis x environment interactions, and heterosis x age interactions are three common and key considerations in comparing pure species and hybrid taxa, and hence in generating meaningful estimates of heterosis.

2.1.5.1 F 1 hybrid inviability One definition of a species is a group of organisms that are capable of interbreeding to produce fertile offspring (Schwindlein 2001). Crossing incompatibilities between species would seem a natural consequence of this definition, and are commonly observed in trees (eg Griffin et al. 2000; Critchfield 1973). Although prezygotic incompatibilities are of little consequence for the measurement of heterosis, postzygotic inviability is a genetic characteristic of the progeny population, which affects comparison with other populations. Ft hybrid inviability, and resulting increased population variability due to the presence of runts and malformed trees, presents two serious issues in the phenotypic comparison of hybrid and pure species performance.

Firstly, there is the problem of which trees to count as part of the hybrid population. For example, in Eucalyptus nitens x E. globulus hybrid populations described by Volker (1995), one E. globulus provenance consistently produced a high proportion of deformed and runt-like hybrid progeny, resulting in a strongly left-skewed distribution of the F1 progeny population relative to the parental species populations,

although when these were excluded, the F1 distribution was intermediate to the parents and heterosis became positive. There are strong arguments for excluding measurements of runts and otherwise suppressed or stunted trees suffering genetically related malformations from taxon comparisons involving hybrids (eg Powell2001) on the basis that they are unlikely to contribute to the harvested crop. They will also Chapter 2 - 22 artificially inflate F1 population variance and are likely to result in a skewed distribution, which may violate the assumptions of subsequent analyses (Searle et al. 1992).

A second problem in comparing pure species and hybrids affected by runts is the increased environmental variability and reduced competition in hybrid blocks with high proportions of runts, relative to pure species blocks with few or no runts. Where there are few runts, thinning regimes may be adjusted to produce a comparable stand density in blocks of all taxa, although this may cause imbalances where a family structure exists. Where there are many runts, valid comparison of pure and hybrid taxa becomes difficult beyond the onset of competition in the pure species - in such cases, Potts (pers. comm.) has suggested and implemented single-tree plots of pure species and hybrids randomised within blocks, in an attempt to randomly distribute the variation due to competition effects among taxa. This approach is likely to result in high random error but provide the best available estimate of taxon means under some circumstances. Another option is nearest neighbour analysis, although this tends to remove genetic effects when strong imbalance is present (Dutkowski pers. comm.) The use of separate blocks for comparison of taxa is statistically far preferable wherever possible. Where the proportion of runts is noticeably higher than in pure species taxa, their occurrence should be recorded and considered in the interpretation of taxa comparisons (eg Nikles et al. 1999).

2.1.5.2 Heterosis x environment interaction

Heterosis, and hybrid superiority, are usually strongly dependent on environment. As noted by Martin (1989): "Heterosis cannot be considered without taking into account its interaction with the environment where the hybrid is tested". Although tree hybrids commonly show reduced levels of heterosis on sites ideal for either pure species parent (Martin 1989), hybrids are still used in such cases to extend the range of pure species onto sites that are marginal, often due to frost or drought (Potts and Dungey 2001). For example, the use of Eucalyptus hybrids in South Africa appears to be largely confined to sites intermediate to those suitable for favoured pure species such as E. grandis, E. nitens, E. tereticomis and E. camaldulensis (Darrow 1995; Verryn et al. 1996); hybrids between these species typically only display better parent heterosis for growth on marginal sites for the pure species. These are clearly Chapter 2 - 23 'adaptive' hybrids, in terminology of Stettler et (1996), for which hybrid superiority is more likely to be more strongly dependent on environment than in types hybrids. Such hybrids are likely to enjoy increasing attention with the push of forestry onto land considered marginal for traditionally important species.

Other hybrid taxa in other environments are often preferable to pure species parents over a broader range of site types including those suited to the parental species (eg Populus tremuloides x P. tremula hybrids in the Northern USA, Li and Wu 1997; Pinus elliottii x P. caribaea hybrids in South-East Queensland, Nikles 1996). In these examples, the hybrid retains superiority over a broad range of site types, including most of those suitable for each pure species parent. In such instances, although the choice of taxon may be less sensitive to environmental parameters, the evaluation of hybrids on different sites is still necessary to obtain reliable estimates of the degree of heterosis and hybrid superiority.

According to the concept of genetic homeostasis, proposed by Lerner (1954), hybrid performance is likely to be more stable across environments than are more homozygous populations such as pure species, due to a suggested 'buffering' effect of heterozygosity against environmental variation. Existing evidence for this theory is inconclusive in trees (eg Li and Wu 1997; Wu and Stettler 1997) as in other organisms (Barlow 1981; Pani and Lasley 1972), mainly as replicated trials incorporating hybrids and pure species controls on multiple sites are rare. The lack of theory to predict the performance of interspecific hybrids necessitates testing with pure species controls on a representative sample 'Of the site types of interest for production forestry, if the most appropriate taxa are to be matched to the right sites for the most valuable end products (Turner 2001).

2.1.5.3 Heterosis and ontogeny

Ontogeny is the process of an organism's development towards biological maturity. As the ontogeny of hybrids often differs from expectations based on pure species parents, the age at which hybrids are assessed may be of key importance for determining heterosis and hybrid superiority. Numerous hybrids exhibiting strong heterosis for growth at early ages (eg Populus tremuloides x P. tremula, Stettler et al. 1988; Eucalyptus grandis x., Denison and Kietzka 1993) may sometimes slow down Chapter 2 - 24 dramatically in later age growth, even to the point of being overtaken by their pure species parents before maturity. Denison and Kietzka (1993) have categorised these hybrids as 'sprinters', and recommend waiting until at least half rotation age to evaluate Eucalyptus hybrids. It has been suggested that 'sprinting' may be more commonly a characteristic of highly heterotic hybrids than complementary hybrids (Zobel and Talbert 1984). However, as few tree improvement experiments are ever retained to maturity, ontogeny-related changes in heterosis or even in taxon rankings are often not detected. In a long term hybrid trial, Namkoong (1963) found that while Pinus taeda x P. palustris displayed better parent heterosis for volume at an early age, it was soon outgrown by P. taeda; yet by age 40, P. palustris was superior to both. In the rare cases where mature experiments are available, the evidence is sometimes inconclusive due to experimental design issues. For example, the common large number of runts and other deformations in interspecific F1 hybrid tree populations sometimes biases assessments of heterosis at later ages because of higher mortality in hybrid plots (Nikles et al. 1999; Potts and Dungey 2001). Additionally, results are inconclusive where experiments sample unrepresentative or different sites for pure species and hybrids, or use genetically unrelated and/or small samples of pure and hybrid germplasm. These factors may be responsible for inconsistent results in Larix europaea x L. leptolepis in Europe. Gothe (1987) in Germany found early heterosis, but declining hybrid performance relative to parental species after 20 years; these results were supported by those of Keiding (1980) in Denmark and Reck (1980) in Germany, but contradicted by those of Ferrand and Bastien (1985) in France, where a constant level of better parent heterosis for stem volume was retained to 26 years. These studies illustrate the importance of long-term experiments to examine hybrid performance at or near rotation age using relevant sites and genotypes. Such data is currently scarce for most tree hybrids (Paques 1989).

2.1.6 F1 vs advanced generation hybrid performance

Traditionally, most comparisons of hybrids with pure species have been made using

the F1 hybrid generation- the first hybrid generation resulting from pure species

parental crossing. Early experiments in F2 hybrids, partly motivated by the difficulty

of seed production in F1 hybrids, demonstrated (eg Cook 1969 cited in Holst 1974; Hyun 1974) that Fz and other advanced generation hybrid seed could be produced in abundance, prompting interest in deployment of these taxa (Wright 1976). Advanced Chapter 2 - 25 generation hybrids are defined in this thesis as any population descended in any way from an interspecific F1 hybrid. These may be F2, F3 ..• Fn generations, backcrosses (hybrids crossed back to either of the parental pure species), three-way or four-way crosses (hybrids involving multiple species), and a variety of other genotypic configurations. Because of the length of time required to create them, crosses of more than two species have rarely been used in forestry, although this option has potential as an element of long-term, organised hybrid strategies (Griffin et al. 2000). The term 'Composite' is used to denote a base population resulting from a hybrid cross (eg Hallauer and Miranda 1988, p. 358), and in this thesis refers to any hybrid population descended from the F1, in which continued recombination is to take place as a part of organised breeding.

Tree breeders appear to have been deterred from serious consideration and evaluation of composite breeding by early experiments in crops (eg Wright 1922; Neal1935) and putative tree hybrids (eg Venkatesh 1982; Kulkarni et al. 2001), which commonly showed declining performance and increased variance in composite hybrid populations. A review of the literature indicated that the observed declining mean performance, sometimes termed 'hybrid breakdown', and increased variability, in advanced generation hybrids appears to have been due in many cases to inbreeding depression resulting from matings amongst related individuals. Inbreeding depression is the decline in vigour observed with inbreeding, thought to result from increased homozygosity and exposure of deleterious recessive alleles (Falconer and Mackay 1996; Wright 1976).

For example, the hybrid breakdown reported by Venkatesh (1982) in Eucalyptus tereticomis x E. camaldulensis is not surprising given that the seven F2 open­ pollinated 'half-sib families' examined in this paper were in fact seedlots harvested from seven mass selected trees in a stand comprised of a single F1 hybrid full-sib cross. The same paper proposes an advanced generation hybrid breeding strategy in this population based on recurrent selection and recombination of open-pollinated progeny in F2, F3 .• Fn generations. The failure of a similar breeding strategy in another advanced generation hybrid, 'Mysore Gum', putatively involving E. tereticomis, also in India, was due to a strong and steady decline in performance over several generations (Varghese et al. 2000), and can similarly be attributed to inbreeding, both from the narrow genetic base of the population and from the partial Chapter 2 - 26 inbreeding mating system common in open-pollinated Eucalyptus (Eldridge et al. 1993). In warning against the "exceedingly dangerous practice" of creating composite hybrid populations, Zobel and Talbert (1984) cite the example of early hybrids involving Eucalyptus grandis in Brazil, where putative hybrid seeds from several individuals in an arboretum were collected and found to produce high yielding plantations. A steady decline in productivity, and increase in variability, was observed in plantations from several generations of selection of open-pollinated seed from these plantations (Brune and Zobel 1981). As in the Indian examples, this can be explained by inbreeding, resulting from several generations of recombination, with very little selection, in a population derived from as few as two parents. Similar dysgenic changes would almost certainly occur in a pure species population under such circumstances (eg Wu et al. 1998), and would correctly be attributed to inbreeding; it is therefore clear that the Brazilian and Indian examples provide evidence against inbreeding, not against composite breeding.

Some experiments have compared outcrossed and inbred F2 hybrids. In a small but unique experimental trial of Pinus elliottii x P. taeda in South-East Queensland,

Australia, outcrossed F2 progeny outperformed inbred Fz progeny of common pedigree, and had lower population variance, in stem diameter and height Nikles et al.

(1999). In a similar experiment, outcrossed F2 progeny of a Eucalyptus gunnii x E. globulus F1 hybrid parent outperformed selfed progeny of the same parent (Potts et al. 2000); subsequent molecular genetic mapping and analysis indicated that high occurrence of a semi-lethal abnormal phenotype in the selfed Fz was due to a deleterious recessive allele inherited from the E. gunnii parent (Vaillancourt et al. 1995).

Where advanced generation hybrids in forest trees and in other organisms (addressed in Section 2.4) have been generated by outcrossing, their performance has often been

very similar to that of F1 hybrids, even where selection was not applied. In Korea, the

Pinus rigida x P. taeda outcrossed F2 hybrid, generated by crossing Fts from different

seed sources, performed at least as well as its F1 parents on average, and had lower population variance, in a replicated experiment on a low latitude site (Hyun 1974).

On a colder, higher latitude site, the F2 had slightly slower growth than the Ft. and on

the coldest (highest latitude) of three sites, the F1 outperformed the F2, yet seedlings

from wind-pollinated F1 hybrids (a mixture of F2 hybrids and backcrosses) Chapter 2 - 27 outperformed both the F1 and F2. The similarity of F1 and F2 hybrids at low and intermediate latitudes and greatly increased seed yields in F2 than F1 crosses underpinned the decision to deploy F2 hybrids at these latitudes, while wind­ pollinated F1 hybrids were to be deployed in the northernmost latitudes (Hyun 1974). P. rigida x P. taeda F2 hybrids have since also been used successfully by Westvaco Corporation in North America (McCutchan1 pers. comm.). In the Pinus elliottii x P. caribaea hybrid in South-East Queensland, Australia, outcrossed F2 progeny are typically alinost indistinguishable from the Ft. both visually and statistically (eg Powell and Nikles 1996a, Harding et al. 1996), and have been used to successfully afforest an area of over 12 000 Ha (Nikles 2000). Similarly in Larix, outcrossed advanced generation hybrid populations have demonstrated comparable vigour to the Ft. and have maintained better parent heterosis over the parental populations, in at least one case through to the F3 generation, both in the Northern USA (Cook 1969 cited in Holst 1974; Li and Wyckoff 1994; Holst 1974) and in France (Lacaze and Birot 1974; Paques 1989; Paques 2000; Vincent and Fer 1965). Although the hybridisation of Eucalyptus for production forestry is a relatively recent phenomenon, a successful trial of outcrossed F2 hybrids of Eucalyptus urophylla x E. grandis has been reported in recent conference proceedings by Hardiyanto and Tridasa (2000) in Indonesia (clonal deployment), and in China using genetic material of the same origin (seedling deployment), by Zheng Bai cited in Potts and Dungey (2001). However, in E. nitens x E. globulus, a poorly-performing outcrossed F2 hybrid was reported by Potts et al. (2000): although F2 hybrid mortality was much lower than in the Ft hybrid, the F2 hybrid had much lower mean stem basal area than both parental species, both backcrosses and the F1 hybrid. It should be noted however that in this study even the F1 hybrid had lower stem basal area than either of the parental species. In Populus, F2 hybrids have consistently performed poorly (eg Stettler et al. 1988). The utility of F2 hybrids is clearly taxon-specific and is likely to depend mainly on the predominant modes of gene action contributing to hybrid performance, which will be addressed in Section 2.4.3.

Evidence for the potential of outcrossed composite hybrids in trees can be found in the success of outcrossed composite maize hybrid populations derived from multiple breeding lines, referred to as 'synthetic' populations (eg Lonnquist and McGil11956,

1 McCutchan, B.G., Quantitative Geneticist, Westvaco Research Centre, Covington USA. Chapter 2 - 28 Kinman and Sprague 1945). While severe inbreeding depression results when small numbers of lines are used to generate the synthetic population (Neal 1935), when larger numbers of lines are used, F1 heterosis can be almost completely retained by random mating in a stabilised synthetic population (Hallauer and Miranda 1988; Kinman and Sprague 1945). Since the concept was first proposed by Jenkins (1940), synthetic populations such as "Iowa Stiff Stalk Synthetic" and "Dawes Synthetic" have formed the genetic base for many generations of genetic gain through recurrent selection (Martin and Russell1984).

Theoretical considerations suggest that inbreeding can be more easily avoided in genetically variable forest tree hybrid composites than in composites of highly selected crop populations (eg Kinman and Sprague 1945). Additionally, the relatively low cost of F2 hybrid seed production prompted some early theorists (eg Allard 1960;

Wright 1962) to suggest further investigation of F2 hybrids, on the basis that uniformity of stem size is not as critical in trees as crops, due to silvicultural techniques such as thinning. In the event that the population mean did decrease

slightly relative to the F1, and variability increased, the better stems could be retained. Zsuffa (1973) and Wright (1976) argued that increased variability in composite populations may be an advantage where vegetative propagation technology allows the multiplication of favourable genotypes through clonal forestry.

Besides composite hybrid populations of the F2, F3 and beyond, backcrossing may be a useful tool for incorporating specific desired genes (eg Eucalyptus gunnii for frost tolerance) into well-established genetic backgrounds (eg Eucalyptus globulus for good growth and pulping properties; Manson and Potts 1995). For example, the hybrid between Pinus jeffreyi and P. coulteri may be improved by backcrossing to fast growing P. jeffreyi parents (Zobel and Talbert 1984). The backcross has better

form than the F1 hybrid, and grows slightly faster, while still incorporating the pine reproduction weevil resistance of Coulter pine. On cooler sites in Northern Florida, backcrosses of PEExPCH to PEE show promise in conferring the good frost resistance of PEE while retaining the good growth of PEExPCH (Rockwood and Nikles 2000).

Existing evidence supporting the possibility of outcrossed composite hybrid breeding strategies has not been followed by much further investigation and investment Chapter 2 - 29 because of theoretical evidence in crops (eg Wright 1922; Morris et al. 1999), empirical evidence from maize inbred advanced generation hybrids (Neal 1935), and inbred composite hybrid trees. This evidence has apparently been interpreted as contradictory to the empirical evidence in many outcrossed advanced generation hybrids of both trees and maize, which suggest strong potential for composite hybrid breeding in trees in some taxa. It appears that composite hybrid breeding in trees has not eventuated due to the lack of understanding of the genetic architecture and specifically, modes of gene action, in forest tree hybrid populations: this issue will be addressed in Section 2.4.

2.1.7 Hybrids for wood improvement

Wood properties have often been an afterthought when selecting taxa, yet may have a profound effect on product value. For example, Eucalyptus nitens pulpwood may fetch only 65-75% of the price of E. globulus pulpwood (eg Raga 2001), although the species grow on similar sites. Hybridisation, possibly involving backcrossing, may allow development of families or individuals with both the frost tolerance of E. nitens and the good pulping characteristics of E. globulus. Given the typically additive inheritance of wood traits, both in pure species and in hybrids (eg Zobel and Jett 1995; de Assis 2000), and rarity of better parent heterosis, the wood characteristics of novel hybrids appear to be more predictable than growth, survival and reproductive characteristics, which often show large heterosis, and great variance, among different hybrid crosses. Wood improvement using hybrids is most likely to be successful by combining species with complementary wood characteristics - for example, the combination of high wood density from one species with uniform wood density from another, where uniform high density is desirable. Although wood characteristics have rarely made a serious contribution to the choice between pure and hybrid taxa, the emergence of market premiums for higher quality wood, and increasing international competition have stimulated increasing wood improvement efforts; the introgression of frost-, drought- or pathogen- hardy genes to high wood quality species using hybridisation may be an increasingly useful, if complicated, option. Chapter 2 - 30 2.1.8 Summary

The choice of appropriate taxa for afforestation is a critical one for the profitability of forestry enterprises. However, the economic value of taxa is difficult to measure directly. In forest trees, where numerous traits typically contribute to product value and hybrid heterosis is usually moderate, estimates of mid-parent heterosis in important traits are likely to be more informative than estimates of better parent heterosis, although assessment of the latter remains important. Recent investment in developing a variety of propagation technologies is likely to increasingly provide opportunities for the deployment of hybrid germplasm, although hybrid superiority must first be ascertained, with attention to GxE interaction, ontogeny and experimental design issues specific to hybrid populations. The generally strong performance of outcrossed advanced generation hybrids warrants developing and evaluating these taxa in other hybrid breeding programs on at least an experimental basis, and further theoretical investigation of composite hybrid breeding.

2.2 Quantitative genetic analysis of interspecific forest tree hybrid data

Of the two phenomena that affect quantitative traits, breeding strategy in most organisms of industrial interest has relied on recurrent selection rather than inbreeding and hybridisation for genetic improvement (Rife 1965). A large body of selection theory has been developed for panmictic (random-mating) pure species populations, based on their property of linkage equilibrium, the condition of random association of alleles at different loci within a gamete (Falconer and Mackay 1996). This useful population property confers the critical characteristic of population stability from generation to generation, enabling the use of quantitative genetic models of population genetic variation for predicting the response to different types of intra­ population forward selection (selection of progenies) following recombination (eg Falconer and Mackay 1996; White and Hodge 1992). The conventional quantitative genetic model for panmictic populations is based on that proposed by Fisher 1918, and extensions of it by Wright 1935 (see Cockerham 1963 for a full description), and will simply be referred to as the conventional model. Predictions of selection response using this model in pure species populations have generally been good (eg Hill and Caballero 1992; Turelli and Barton 1994; Carson et al. 2000 in trees). Conversely, in inter-population hybrids, particularly in inter-specific hybrids of Chapter 2 - 31 outcrossing species, a state of severe linkage disequilibrium violates the assumptions of the pure species biometrical model, biasing genetic variances and prediction of the response to forward selection where it is applied (eg Moll and Stuber 1971). In the absence of useful selection theory for the general case of hybrid populations (Schnell 1963; Stuber and Cockerham 1966), hybrid breeding strategy can be guided by the types and relative amounts of physiological gene action responsible for heterosis (Li and Wyckoff 1994; Namkoong et al. 1988); however, the conventional model is unable to provide this information. Some statistical parameters from the analysis of variance on which the conventional model is based can be used to direct some basic decisions in hybrid breeding strategy. Limitations of the conventional model will be addressed first, followed by a discussion of those conventional statistical parameters with applications in hybrid breeding. The estimation of physiological gene action and its use in hybrid breeding will be addressed in Section 2.4.

2.2.1 Hardy-Weinberg and linkage equilibrium

Due to their fundamental importance in understanding hybrid genetics, it is useful to first discuss the concepts of Hardy-Weinberg equlibrium (HWE) and linkage equilibrium (LE). Pure species populations that have been random-mating for some generations are expected to be in a state of genetic equilibrium (Falconer and Mackay 1996). This refers to the condition of Hardy-Weinberg equilibrium, where genotype

frequencies are at equilibrium with gene frequencies (in the ratio p2, 2pq and l, for a hi-allelic locus), and linkage equilibrium (LE), where alleles at different loci in a gamete are distributed independently of each other. Disequilibrium, or deviation from equilibrium, results when deviations from random mating, such as inbreeding or hybridisation, occur. Disequilibrium can be severe, as caused by hybridisation, or mild, as caused by assortative mating (Falconer and Mackay 1996). In an F1 hybrid, one homologue of each chromosome contains only species a alleles, and the other homologue contains only species b alleles: an F1 hybrid population is therefore in Hardy-Weinberg disequilibrium, as there is an excess of heterozygotes, and also in linkage disequilibrium, as alleles at different loci are not distributed independently of each other in gametes forming the F1 population.

A major consequence of disequilibrium is instability of the population's genetic structure from generation to generation, when random mating is re-introduced. Chapter 2 - 32

Although HWE is restored in the first generation of random mating after the F1 hybrid, LE can take several to several hundred generations to reach, following a hybridisation event. For practical purposes, evidence in maize suggests that a population approximates LE after 5 to 8 generations of random mating in the hybrid population (Gardner 1963). Instability in early generation hybrid populations (eg F1 - Fs) presents problems for selection theory, which relies on genetic equilibrium from generation to generation in order to reliably predict the outcome of forward selection and recombination.

2.2.2 Assumptions of analyses based on the conventional model

The purpose of the conventional biometrical genetic model of gene effects and variances is twofold: to provide an indication of the modes of gene action underlying a quantitative trait, and to provide a means of predicting the response to forward selection (Cockerham 1963). Comstock and Robinson (1948) presented mating designs ("NC designs I and II") that allow estimation of biometrical genetic variances based on empirical measurements of among-family genetic variances (within-family genetic covariances). These derivations were based on the infinitesimal model and covariances among relatives introduced by Fisher (1918). In populations in genetic equilibrium, the NCI and NCII designs allow estimation of the variance of additive gene effects (causal components of genetic variance) based on family variances (observational components of variance), using the following formulae (for design II):

2 4 2 0' A = 0'GCA [2.2] 2 4 2 0' D = 0' SCA [2.3]

Where: 0'~ = variance due to additive gene effects; a; = variance due to dominance gene effects; O'~cA =variance due to general combining ability (or parental effect); a;cA =variance due to specific combining ability (or cross effect);

Tree breeders use these formulae (with appropriate modifications for inbreeding), and the model on which they are based, almost exclusively in all breeding methodology. The assumptions underlying this model (referred to as the conventional model) in order for biometrical additive and dominance genetic variance to provide a reliable Chapter 2 - 33 estimate of relative importance of additive and dominance gene action are given by Gardner (1963), as follows:

1. Random choice of individuals mated for production of experimental progenies; 2. Random distribution of genotypes relative to variations in environment; 3. No non-genetic maternal effect; 4. Regular diploid behaviour at meiosis; 5. No multiple alleles; 6. No correlation of genotypes at separate loci. This implies no linkage among genes affecting the character studied or that, if linkages exist, the distribution of genotypes is at equilibrium with respect to coupling and repulsion phases; 7. No epistasis, ie, the effect on variation in genotype at any single locus is not modified by genes at other loci; 8. For estimating degree of dominance, gene frequencies of one half (p=q=0.5) at all loci where there is segregation (not necessary for design III).

These assumptions may be approximately satisfied in advanced generation synthetic hybrids originating from inbred lines, commonly used in maize breeding (see Hallauer and Miranda 1988); in early generations of interspecific hybrids of outcrossing species however, assumptions 1, 5, 6 and 7 are likely to be seriously violated (Cockerham 1963; Gordon 1999; Keirn et al. 1989; Li and Wu 1996; Stokoe et al. 2000). Most importantly, and implicit in most of these assumptions, interspecific hybrids of outcrossing species violate the assumption that the parents being evaluated are random samples from a single random-mating population in linkage equilibrium. The conventional biometrical model is therefore likely to be inappropriate both for estimating gene action, and for predicting response to selection, in hybrid populations (Gordon 1999; Wei et al. 1991). To demonstrate this, it is necessary to describe the basis of the relationship between Mendelian gene action and biometrical genetic variances, and to demonstrate how hybrids violate the assumptions of this correspondence; secondly, to demonstrate the problems with applying the conventional biometrical genetic model to hybrid selection and breeding. Chapter 2 - 34 2.2.3 Mendelian vs biometrical concepts

A review of the quantitative genetic literature in trees, both in pure species and in hybrids, revealed some confusion between the concepts of Mendelian additive and dominance gene action, and biometrical additive and dominance genetic variances. This confusion is understandable because of the overlapping terminology, but must be resolved before further discussing hybrid genetics and breeding, in which modes of gene action are of critical importance, yet biometrical genetic variances are largely inappropriate (Gordon 1999). The issue of the relationship between the two concepts rarely surfaces in pure species breeding due to the heavy use of selection methodology (eg White and Hodge 1992), which is based on statistical properties of genes in populations, not simply on gene action.

At the single-locus level, the main difference between Mendelian and biometrical genetics is that while the Mendelian concepts of additivity and dominance are defined relative to individual-locus genotypic values, biometrical 'additive' and 'dominance' genetic variances at a locus are defined relative to both genotypic values and allele frequencies in the population (Falconer and Mackay 1996). To estimate biometrical gene effects, a regression of genotypes on genotypic values weighted by genotype frequency is first constructed at a single locus (see Fig. 2.1, taken from Falconer and Mackay, Fig. 7.2, p. 117), under assumptions 1-8 (Section 2.2.2)- most importantly, that the study material is a pure species population in Hardy-Weinberg equilibrium. The slope of this regression is used to calculate a, the 'average effect of a gene substitution', based on which within-locus additive genetic variance is calculated (see Figure 2.1 ). The residuals from the regression are called the 'dominance deviation', and are used to calculate the within-locus genetic variance due to dominance. Chapter 2 - 35

·tl· ·~·

Figure 2.1 The relationship between Mendelian gene action and biometrical gene effects (taken from Falconer and Mackay 1996). Note: closed circles represent genotypic values; open circles represent breeding values, of the genotypes at a locus with two alleles, A 1 and A2. with frequencies p and q. The resulting genotypes A 1A h A 1A2 and A2A2 are at Hardy-Weinberg Equilibrium frequencies. On the vertical axis, on the left hand side are arbitrary individual-locus genotypic values representing measures of gene action; on the right are the deviations of breeding values from the population mean, which are biometrical genetic effects. a is the average effect of an allele substitution, an additive genetic effect. The deviations of the genotypic values from the breeding values are functions of d, the dominance deviation. a and d are biometrical measures of additive and dominance gene action.

The extension of this model across multiple loci is a simple matter of summing the variances across all n loci controlling a quantitative trait, assuming negligible linkage disequilibrium and no epistasis:

[2.4] i=l n (J~ =I 4d2 p2q2 [2.5] i=l Chapter 2 - 36 Inherent regression-based model in Figure 1, yet rarely acknowledged,

IS the additive and dominance variances cannot be estimated independently of other; as a result, biometrical estimates of additive dominance genetic variance are both influenced by a combination of additive and dominance Mendelian gene action, unless gene action is completely additive (Cockerham 1963). Furthermore, these variances cannot be estimated independently of gene frequency, which exerts a strong influence on their absolute and relative values (eg Falconer and · · Mackay:Fii~~~l," p.128). Although under the assumptions of the Comstock and Robinson (1948) designs NCI and NCII, when p=q=0.5, the biometric and Mendelian concepts of gene action correspond exactly at the within-locus level (eg Cockerham 1963), at other gene frequencies the conespondence is in fact very poor (eg Falconer and Mackay Fig 8.1, p.128). Additionally, both additive and dominance genetic variance are likely to be contributed to heavily by epistatic gene action (eg Lush 1945; Cheverud and Routman 1995).

It can be seen that genetic variances estimated from the conventional model provide useful estimates of gene action only under very specific and unusual circumstances: even carefully controlled laboratory experiments (eg Barker 1974, 1979), assumptions 1-8 listed above are unlikely to be met, much less in operational breeding programs. Hence even in pure species populations, biometrical genetic variances cannot be directly related to, or be considered to provide a reliable guide of, gene action; rather, they are related to aggregate statistical effects of genes in a population (Holland 1999).

In interspecific hybrid populations, the biometrical (conventional) model departs even further from reliable representation of gene action, because of disequilibrium at both the within-locus and among-loci levels.

1. Within-locus level. In the F 1 generation, the population is in severe Hardy­ Weinberg disequilibrium. Note the genotype frequencies along the X-axis in Figure 2.1. They represent the Hardy-Weinberg Equilibrium genotype

frequencies of the gene frequencies on the Y-axis. F1 hybrid populations contain exclusively heterozygotes at many or most loci, and sd there is no basis for construction of the regression. Additionally, the assumption of only two alleles at frequencies of 0.5 is likely to be violated in interspecific hybrids of outcrossing species, where each species may contribute a different set of Chapter 2 - 37 alleles at different frequencies, at each locus (eg Keirn et al. 1989; Stokoe et al. 2000; Vaillancourt et al. 1995). 2. Among-loci level. The direct summation across loci of the within-locus additive and dominance variance (see Equations 2.4 and 2.5), for generalising to the whole-genome level, assumes that the occurrence of alleles at different loci is randomly distributed with respect to other alleles (that the population is linkage equilibrium). Non-random distribution caused by hybridisation strongly biases biometrical genetic variances, causing them to misrepresent the underlying gene action, in early generations of the hybrid population (reviewed by Gardner 1963).

The above factors seriously frustrate the correspondence between gene action and biometrical genetic variances in interspecific hybrids, particularly in hybrids of outcrossing species. The conventional model is still reflective of gene action in the limited sense that if dominance gene action is absent, there will be no dominance genetic variance (but not necessarily vice versa), and similarly for epistatic gene action and epistatic genetic variance (Cockerham 1963). However, beyond this generalisation, no useful information about gene action is provided. Analytical methods in development that relax some of the limiting assumptions of the conventional model to estimate modes of gene action will be addressed in Section 2.4 and in Chapter 6.

2.2.4 Response to selection in hybrid populations

The second and more common use of the conventional genetic model is to estimate the response to selection. To discuss selection, it is necessary to define additivity in the biometrical sense: an additive genetic effect, in the biometrical sense, is the average effect of an allele in a population, at an individual locus level, and the mean contribution of a parent to its offspring performance (the GCA) at the whole-genome level. As discussed above, additive genetic biometrical effects are likely to be contributed to by all types of gene action, not just additive (Cockerham 1963; Holland 1999). Progeny tested half-sib families have proven the worth of their genes with respect to a random sample of genes from the inference population; their contributions, or GCA effects, can be said to be additive, in the biometrical sense, because we expect that after backward selection and crossing among the best parents, Chapter 2 - 38 an additive improvement will be obtained equivalent to the mean of their GCA values, subject to measurement error. Prediction of the degree of improvement from backward selection of parents is hence purely a statistical matter, and can be done with no assumptions on the genetic architecture of the progeny population, in either pure species or hybrid populations, using the equation:

[2.6] where:

11G F = genetic gain from backward selection of half-sib families; i =selection intensity (expressed in standard deviations from the population mean); h; =half-sib family heritability; a F =phenotypic standard deviation of half-sib family means.

In pure species populations, it can additionally be shown (Falconer and Mackay 1996, p. 151), subject to assumptions 1, 2, 3, 4 and 6 listed in Section 2.2.2 and in Cockerham (1963), that the variance due to half-sib families is related to the total additive genetic variance in the population according to Equation 2.7:

[2.7] where: O'~CA = variance due to half-sib families, or GCA variance; p and q (=1-p) are the gene frequencies giving rise to the genotype frequencies at the reference locus in Figure 2.1; a = the average effect of an allele substitution, as defined in Figure 2.1; 1 - pqa 2 = the additive genetic variance at the reference locus, derived as the variance of the genotypic 2 values in Figure 2.1 around the mean, weighted by the genotypic frequencies; n = the number of loci affecting the trait measured; 0'~ = the total additive genetic variance in the population.

However, the average effect of an allele substitution ~ and hence the additive genetic variance at a locus, and hence the additive genetic variance, are not defined in first generation (F1) hybrid populations, because they are in Hardy-Weinberg disequilibrium. As a consequence, the result of forward selection (eg mass selection, within-family selection) in such populations cannot be predicted, because it depends upon the population additive genetic variance. This has been well demonstrated in practice by the poor correspondence between predicted and realised gain in such populations (eg Gardner 1963; Moll and Stuber 1971), whereas this correspondence is typically strong in pure species populations (Hill and Caballero 1992). Similarly, dominance genetic variance is not defined in F1 hybrid populations (Gordon 1999). Chapter 2 - 39

In Fz hybrids derived from random mating of the F1, Hardy-Weinberg Equilibrium is reached or approximately reached (Falconer and Mackay 1996), and so the regression depicted in Figure 2.1 may be defined. However, the persistence of linkage disequilibrium in the Fz still prevents generalising from this single-locus model to the full genome model necessary for phenotypic selection. The biasing effect of linkage disequilibrium in hybrid populations on biometrical genetic variances was best demonstrated in several classic experiments in maize reported by Gardner (1963). The findings are reproduced in Table 2.1. The effects of linkage disequilibrium biased estimates of genetic variance until after the 6th generation of recombination in the hybrid population. Linkage disequilibrium is the most important impediment to unbiased prediction of the genetic gain from forward selection in hybrid populations.

Table 2.1 Comparison of estimates of average degree of dominance (ratio of dominance genetic variance to additive genetic variance) obtained in an F2 variety hybrid of maize with those in more advanced generations (individual experiments pooled). Reproduced from Gardner (1963). Generation Population CI21 xNCi M14 X 187-22 Fz 1.68 1.98 F4 1.04 Fs 1.24 0.72 F13 1.09 F16 0.62 1 from Lindsey, M.F. (1960) PhD. thesis, North Carolina State University 2 from Gardner and Lonnquist (1959).

In summary, the prediction of genetic gain from backward selection in hybrid populations is a simple matter, because it is based on Galton and Pearson's statistical concept of regression, which preceded Fisher's 1918 biometrical model of gene effects, and does not invoke its assumptions. However, theory to predict the genetic gain from forward selection in hybrids has not yet been developed, and prediction using existing methods is likely to provide misleading results. With existing theory, additive and dominance genetic variances are inappropriate concepts for hybrids of highly divergent and genetically variable populations because of violation of the assumptions of the model on which they are based. Chapter 2 - 40 2.2.5 Applications of conventional quantitative genetic parameters in hybrid improvement

While genetic theory is available to predict genetic gain from many types of selection options in pure species, only the component of this selection theory not relying on restrictive assumptions associated with random-mating populations will provide unbiased gain estimates in hybrids.

.·. Genetic parameter estimates are not of value without a clear understanding of both their underlying assumptions and practical uses. The applications of various genetic parameters in pure species tree improvement have been well defined (see Cotterill and Dean 1990; Mullin and Park 1992; Shelbourne 1992; White and Hodge 1992). Conversely, the practical applications of conventional genetic parameters in tree hybrid improvement have not been defined in the tree improvement literature, although breeding theory developed in maize hybrids (reviewed by Hallauer and Miranda 1988) has been used as a guide in some instances. Most papers examining family variation in forest tree hybrids, present parameters such as the individual tree heritability, apparently inappropriate for hybrids, with no discussion of their limitations or applications in hybrid improvement. Individual-tree heritability, within-family heritability and broad sense heritability estimated in hybrids are biased because they are based on conventional concepts of population additive and dominance genetic variance and are used to predict gain from forward selection. Family heritabilities such as the half-sib (see Equation 2.4) and full-sib family heritability, however, are defined in respect to backward selection, and so can be used to predict gain from selection in hybrid populations. The GCA variance and SCA variance may also be useful, providing indications of the relative importance of half­ sib and full-sib family variance, and possibly providing approximate indications of the relative importance of additive and dominance variance (Li and Wu 1997). The numerous estimates of narrow-sense heritability (h2) and dominance ratio (d2) in tree hybrids in the literature (see Dungey 2001) are subject to the issues discussed above; they do provide useful estimates of half-sib and full-sib family variation, but this information is comparatively inconsequential for hybrid breeding strategy relative to other parameters.

An important statistical parameter specific to hybrid breeding is the correlation between parental performance in pure species and hybrid combination, denoted rph Chapter 2 - 41

{Dieters Dungey 2000). This parameter IS critical determining whether intraspecific or interspecific testing be most efficient for hybrid improvement

{Vigneron et 2000). Where rph is low, the testing of interspecific progeny is likely to be necessary for effective hybrid population improvement; where rph is high, it is likely that cost-effective improvement of the hybrid population can be achieved simply through correlated gains from improvement of the pure species populations separately.

The genetic correlation can also be used to indicate the genetic association between traits. In hybrid populations, although additive genetic variance cannot be reliably estimated, the genetic correlation can be estimated as the half-sib family correlation, and can be applied to predict the correlated response in one trait from backward family selection on another trait. This parameter cannot reliably be applied between generations (eg to predict indirect selection response in a trait from forward selection on another trait), as linkage disequilibrium can create false, transient correlations between traits that change after successive recombination events.

The practical usefulness of parameters is best discussed in reference to their application in improvement strategies. Ignoring, for the moment, organised advanced generation hybridisation strategies, which have not yet been formally applied in trees, conventional hybrid breeding strategies in crops and trees have focussed on recurrent improvement and deployment of the generally vigorous F 1 hybrid generation. These will be referred to as F 1R strategies, to denote the practice of recurrently improving the F1 hybrid. While numerous variants have been proposed (see Shelboume 1993

and Dungey et al. 1999 for brief reviews in trees), F1R strategies can be broadly dichotomised into strategies based on hybrid testing, and strategies based on pure species testing. An example of each will be provided, with examples of relevant genetic parameters. Key to determining the relevance of parameters is avoidance of assumptions underlying the pure species biometrical model of Fisher (1918).

2.2.5.1 F 1R strategies: Reciprocal Recurrent Selection (RRS)

Reciprocal Recurrent Selection {RRS, Comstock et al. 1949) is the classic inter­ population recurrent improvement strategy, developed in maize. Numerous variations of RRS have been developed. Half-sib RRS (HS-RRS) selects for half-sib family performance (interspecific GCA) in the hybrid population; Full-sib RRS (FS-RRS) Chapter 2 - 42 selects based on full-sib performance in the hybrid population. Although SCA effects cannot be recurrently selected for, they can be exploited through backwards selection and deployment. Statistical parameters help decide between these two options are the ratio of GCA to SCA variance, and the genetic gain from direct selection of full-sib families vs. that from selection of half-sibs (eg Nikles and Toon 1993). The correlation between half-sib and full-sib family breeding values has also been used to provide a broad indication (eg Dieters and Nikles 1998). The decision between half-sib RRS and full-sib RRS is also influenced by practical considerations such as the expense and difficulty of conducting full-sib crosses relative to polycrosses, and whether or not juvenile propagules can be obtained from mature selections, for immediate clonal testing (eg stump sprouts from some Eucalyptus species).

An example of a full-sib RRS strategy applied to Pinus interspecific hybrids by Nikles (1993) is presented in Figure 2.2. The strategy involves making and testing hybrid crosses of pure species parents, then backward selecting the best parents, before making intraspecific crosses among the selected parents in each species separately, to form the next generation of parents for hybrid testing and selection.

For estimating the genetic gain from backward selection of parents of the best full-sib families in the hybrid population, the full-sib family heritability in the hybrid

population is an appropriate statistical parameter ( h:s ). For half-sib RRS, which

involves backward selection of parents of the best half-sib families in the hybrid population, the male and female half-sib family heritabilities are appropriate ( h!sm and

h!sJ, Hallauer and Miranda 1988). An approximation of the genetic gain from HS­

RRS is calculated in the following way (see Hallauer and Miranda 1988, p. 183-197 for approximate genetic gain equations for these strategies):

[2.8] Where:

!::.G HI = genetic gain in the F1 hybrid population from selection on species 1 interspecific half-sib family means;

h! 1 =half-sib family mean heritability of interspecific crosses in species 1;

iH1 =selection intensity in half-sib families of interspecific crosses in species 1;

a P(HI) =phenotypic standard deviation of half-sib families of interspecific crosses in species 1. Chapter 2 - 43 The genetic gam from selection among half-sib families of interspecific crosses involving species 2 ( !1G HZ) is calculated in the same fashion, and an approximate estimate of the total gain from a single cycle of HS-RRS can be obtained as

!1.GT = !1G Hl + !1G HZ •

Other RRS variants (eg Shelboume 1993) incorporate clonal testing and selection. Although genetic gain from clonal selection can be predicted in a clonal hybrid trial without using causal components of genetic variance, it should be noted that the genetic gain from selection of hybrid clones cannot reliably be predicted based on

broad sense heritability (H2) calculated from seedling hybrid trials, as this requires the use of causal genetic components of variance (eg Mullin and Park 1992). Chapter 2 - 44

Figure 2.2 Reciprocal Recurrent Selection, as suggested by Nikles (1993) for PEExPCH hybrids: a hybrid improvement strategy applying selection on pure species parents based on the performance of interspecific crosses (taken from Shelboume 1993).

2.2.5.2 F 1R strategies: Recurrent Selection for GCA (RSGCA)

An alternative strategy for hybrid trees, first used in maize, and proposed in eucalypts by Shelboume, is recurrent selection for general combining ability (RSGCA; Shelboume 1993). The advantage of RSGCA over RRS is its relative simplicity: while RRS requires using the same parents to make both interspecific crosses, for selection, and intraspecific crosses, for recombination, RSGCA simply selects parents based on their pure species cross performance, eliminating the need for hybrid crossing except for deployment. While omitting the step of hybrid crossing and Chapter 2 - 45 testing reduces the breeding generation interval, it also introduces several elements of risk into hybrid breeding. Firstly, it relies on a high value of rph (the correlation between parental performance in intraspecific and interspecific combination) in order to make genetic gains. Secondly, it forfeits any extra genetic gain from backward selecting for SCA in the hybrid population. An example of an RSGCA strategy, applied by Tony Shelbourne in Eucalyptus in South Africa, is presented in Figure 2.3.

The genetic gain from RSGCA can be calculated as the genetic gain from indirect selection for hybrid performance based on pure species performance. The genetic gain in the hybrid is the average of the indirect gain from selection in species 1 and the indirect gain from selection in species 2. To calculate the indirect gain from selection in species 1, the conventional genetic gain formula for indirect selection can be applied, using the half-sib family heritability in both the species 1 and hybrid populations, the correlation at the half-sib family level between the pure and hybrid populations, the selection intensity and the phenotypic variation of half-sib family means in the hybrid population:

[2.9]

Where:

L1G1 HI =correlated genetic gain in the F 1 hybrid population from selection on species 1 intraspecific half-sib family means; ~ = square root of the half-sib family mean heritability of intraspecific crosses in species 1;

hH 1 = square root of the half-sib family mean heritability of interspecific crosses in species 1;

I{J,HI) =half-sib family correlation (calculated equivalently to the genetic correlation) between intraspecific and interspecific crosses;

i1 = selection intensity of intraspecific half-sib family means;

a P(H!) = phenotypic standard deviation of interspecific half-sib family means.

Being based entirely on observational components of variance from genetic test analysis, this gain prediction relies only on statistical properties of the linear regression and the normal distribution, and does not invoke genetic assumptions other than that of continuous polygenic variation. Where selection is also carried out in

species 2 intraspecific crosses, the genetic gain from indirect selection ( L1G2,H2 ) is calculated in the same way, and the indirect gain from each species contributes half of

the total genetic gain in theFt hybrid ( L1GT = L1G1,H1 + L1G2.H2 ). Chapter 2 - 46 Where appropriate genetic parameters are available, the genetic gain calculations above provide a quantitative basis for the choice between half-sib RRS, full-sib RRS and RSGCA. The genetic gain per year from each strategy can be estimated by dividing its genetic gain prediction by the number of years in its breeding cycle. It should be noted that where existing experiments have been structured appropriately for estimating these parameters, the numbers of parents involved have usually been small, and so it may be unreasonable to assume the results apply to the broader breeding population. Some recommended cost-effective trial designs for estimating these parameters efficiently based on a larger sample of parents are: • Replicated progeny trials incorporating a broad set of half-sib families of species 1 tested with both an unrelated species 1 polymix and a species 2 polymix, and a broad set of half-sib families of species 2 tested with the same species 1 and (unrelated) species 2 polymixes (estimates breeding values or GCA, family heritability in species 1, species 2 and hybrid populations, and the genetic correlation between pure species and hybrid performance);

• Factorial or half-diallel mating design in the F1 hybrid, (estimates the relative importance of GCA and SCA, and the correlation between half-sib and full-sib family performance - to assist the decision between half-sib RRS and full-sib RRS). Chapter 2 - 4 7

0~%>""'1:--~

~-.,.,~..,.t t!'!'SI

lt\tl' t<.y~!l!f

Figure 2.3 Recurrent selection for GCA (taken from Shelboume 1993): a strategy for genetically improving the hybrid population based on recurrent improvement in the pure species populations. The strategy is based on pure line selection in maize, with minor adaptations to the biology of forest trees.

2.2.6 Summary

Both theoretical and empirical evidence indicates that the conventional biometrical genetic model of Fisher (1918) is unsuitable for both intraspecific and interspecific hybrids. Parameters estimated using this model in hybrid populations are expected to be of very limited use for either the theory or practice of tree hybrid improvement. Although the choice between hybrid improvement via strategies selecting for interspecific performance (eg RRS) and strategies selecting for intraspecific performance (eg RSGCA) ultimately depends on the predominant mode of gene Chapter 2 - 48 action governing the selection hybrid population, this choice can also be made using some basic statistical parameters the pure and hybrid populations - most importantly, the half-sib family correlation between them. However, no biometrical genetic parameters are currently available to reliably predict the results of forward selection in hybrids. Prediction of advanced generation hybrid performance requires a detailed understanding of genetic architecture at the level of the individual linkage group or genetic factor - particularly, the predominant mode of gene action influencing hybrid performance. Recent advances in quantitative and molecular genetic methods have begun to provide this information, which will be reviewed with particular attention to forest tree populations in Section 2.4.

2.3 Quantitative genetic analysis of longitudinal wood property data

Although not unique to hybrids, an issue of increasing impm1ance in hybrid breeding is wood property improvement. Wood property data are unique in that at the individual-tree level, the trait of interest is usually comprised of multiple measurements (eg a separate measurement in each growth ring), whereas other individual-tree traits of economic importance such as growth and form characters can be wholly represented by a scalar value. Two examples of common analyses where acknowledgement of the multivariate, or more accurately, longitudinal, nature of wood property data in analysis may be appropriate are in estimating genetic parameters for within-tree wood variability, and in the estimation of variances and covariances between wood properties at different ages such as for prediction of early selection age. Both applications involve analysis of repeated measurements taken on the same trees; however, both have traditionally been addressed using univariate or

bivariate analyses (eg Vargas-Hernandez and Adams 1992; Belanger 1998). Two issues of concern associated with these analyses are that repeated measures data may violate some of their assumptions, and that they do not make use of all available information on the experimental units. A re-appraisal of these approaches, and consideration of new methods for quantitative genetic analysis of wood traits that recognise the longitudinal structure of the data, are necessary in the light of recent developments in theoretical methodology (reviewed by Davidian and Giltinan 1995) and computer software (eg Gilmour et al. 2001). Chapter 2 - 49

Within-tree wood variability is often of high economic importance, particularly young conifers (eg Zobel and van Buijtenen 1989; Wright and Burley 1990), but needs to be statistically characterised somehow in order to assess the potential for its genetic improvement. Wood scientists and tree breeders have traditionally avoided recognising the longitudinal structure of wood property data due to the lack of theory and methods for appropriate analyses, and the statistical complications involved. Typically, analysts have followed a 'two-stage' approach (Davidian and Giltinan 1995) to characterising wood property variation in genetic tests. In the first 'stage', individual-tree indices of pith-to-bark variation in wood properties (for example weighted mean wood density (Brazier 1965) or variance of spiral grain angles (Harding et al. 2000) are constructed. This serves to condense data vectors of multiple within-tree measurements into scalar values that can then (in the second stage) be subjected to straightforward univariate analyses such as univariate analysis of variance, as commonly used in quantitative genetic analysis of genetic tests. The two-stage method has the advantage of flexibility, simplicity of calculation and interpretation, and ease of practical application (eg Brazier 1967; Harding et 2000).

Various first-stage indices have been proposed, though many have been either too simple (eg Harris 1969), or possibly too complex (eg Olson and Arganbright 1977; Rozenberg et al. 2000) to be of practical use in the genetic improvement of wood properties. The most commonly studied wood trait is wood density. Breast height increment core density is generally a satisfactory predictor of whole-tree density (eg Downes et al. 1997; Mandaltsi 1977; McKinnell 1970; Ong 1978; Sardinha 1974); this relationship may be assumed to hold for radial density variability. Early attempts at developing density variability indices have been summarised by Kanowski (1985), and focussed on developing scalar indices of density variability from pith to bark. These early efforts were dominated by statistical and theoretical considerations (eg Olson and Arganbright 1977); few studies considered the relevance of their index to wood utilisation, or its applicability in breeding. Wood utilisation and genetics studies have rarely adopted these indices, more commonly applying simpler indices such as the density differential between arbitrary groups of growth rings nearest the pith and nearest the bark (eg Belanger 1998; Hodge and Purnell 1993; Harding et al. 2000). Some papers (eg Vargas-Hernandez et al. 1993; Hodge and Purnell 1993, Chapter 2 - 50 Rozenberg et al. 2000) have developed more detailed indices for either within-ring variation or for pith-to-bark variation, although no papers appear to have constructed a general index to explicitly partition these two distinct types of variability in recognition of their separate and potentially important impacts on wood processing.

2.3.2 Structured multivariate (longitudinal) models for characterising wood property variation

While simple indices have served well as a component of 'two-stage' models for assessing the genetics of wood variability (eg Bannister and Vine 1981; Hodge and Purnell 1993), recent theoretical and computing advances allow index development to be incorporated into the analysis of variance methodology, through the use of longitudinal models. These models, in essence, integrate the 'two-stage' approach into a 'one-stage' approach, by simultaneously estimating indices for individual tree parameters and population variances. An example of this is the Random Regression (RR) model (see Meyer 1998), which can be used to model both the individual trajectory of each experimental unit over time (eg the density trajectory across rings) and the variances of statistical parameters describing these trajectories, within a single analysis (eg Jarnrozik and Schaeffer 1997; Davidian 1998).

These approaches can be thought of as 'structured multivariate' analyses, as they specifically recognise that tree ring data are multivariate, but that the data for the variables (rings) are not distributed entirely independently of each other, as recognised by the use of structured covariance matrices or covariance functions which describe the additive genetic and residual correlations between rings. The main analytical advantage of this over purely multivariate analyses is one of efficiency, in that where a structure is assumed, the number of off-diagonal elements to be estimated in covariance matrices is reduced. For characterising data, the main advantage of longitudinal analyses over both univariate and multivariate methods is that they are more efficient at using all available information, particularly where measurements of some variables on some experimental units are missing (Apiolaza and Garrick 2001a). Additionally, the residual, or error, covariance between ring measurements can be modelled, which is assumed absent in univariate methods of estimating the genetic covariance between measurements (eg Kempthome 1957). The longitudinal structure of data can thereby be accounted for in models that would Chapter 2 - 51 otherwise be overparameterised usmg multivariate techniques unstructured covariance matrices.

Some statistical deficiencies in 'two-stage' models relative to longitudinal data analysis models have been pointed out and can be summarised as follows for the specific case of estimating variance components for pith-to-bark radial variability in wood properties: 1. Taking the example of a pith-to-bark wood density trajectory, or slope, being estimated for each individual, as in Hodge and Purnell (1993): a slope parameter /J; or other parameter estimated from a regression of this type is in subsequent analyses of variance treated as the 'true' fk The uncertainty of the slope estimate is therefore not taken into account, though it would be using an RR model, for example. This is particularly of issue where only small numbers of observations (eg rings) are available on each experimental unit (individual); 2. Similarly, where there are missing data for some rings in some trees, but other trees have all rings present, the /J; estimated from a 'first-stage' index will more closely approximate fh in trees with complete records than in trees with incomplete records; subsequent 'second-stage' analyses this is not accounted for, whereas in an RR model, for example, it is (Davidian 1998); 3. The two-stage method ignores the covariance structure of the individual measurements; longitudinal methods try and model, or rather, include more

realistic guesses, of these covariance structures in the analysis (eg using an autoregressive covariance structure on the presumption that rings closer together are more likely to be autocorrelated - see Apiolaza and Garrick (2001a) for an example); 4. For the above reasons, longitudinal methods may be better able to extrapolate observed patterns to predict variability in rings outside the ones measured (eg to predict pith-to-bark density gradient for mature trees based on measurements of juvenile trees).

These statistical arguments are valid in theory, but may or may not make much difference in practice. The few existing studies in trees have found little difference between variance components (eg Lu et al. 2001; Dieters et al. 2000) and coefficients Chapter 2 - 52 of genetic prediction (for extrapolating observed trends; Apiolaza et al. 2000; Apiolaza and Garrick 2001a) estimated from univariate and multivariate methods on the same data. The results of these studies, particularly Lu et al. (2001) and Apiolaza et al. (2000) suggest that of the four points mentioned above, only (2) - the presence of missing data points - appear to provide any serious advantage to multivariate or structured multivariate analyses in reducing the bias of parameter estimates relative to univariate methods. Further studies are needed to extend Apiolaza' s work to . investigate the relative advantages of the various longitudinal models available, in datasets that vary in the characteristics identified in issues 1-4 above.

Additional considerations in the adoption of longitudinal over two-stage methods for characterising wood variation are practical issues in breeding such as the incorporation of wood variability into economic breeding objectives, which presents challenges to longitudinal data analysis. For example, measuring the relationship between economic value and traits assessed in a multivariate or longitudinal sense is likely to be fraught with complications, since even with univariate traits such analyses often become complicated. Simple two-stage methods provide a practical advantage for this purpose.

2.3.3 Age trends in genetic parameters: univariate vs longitudinal methods

Due to the longevity of trees, it is desirable to select at an age prior to maturity, in order to speed the process of recurrent genetic improvement. Traits for early selection have been a research priority in tree improvement, however wood characteristics measured at different ages in the same trees have generally been considered as separate, univariate traits, with few exceptions (eg Kremer and Magnussen 1993; Apiolaza 2001). The efficiency of early selection is a commonly estimated statistical parameter calculated based on the juvenile-mature correlation between a juvenile and mature age considered as two separate univariate traits, and the heritability of these two traits (the juvenile-mature covariance is often obtained from univariate analysis of the sum of the traits, as cov(j,m) = [var(j+m) - Var(j) - Var(m)]/2 ). There are several potential problems with the use of covariances estimated in this manner. Firstly, univariate analyses assume an uncorrelated residual variance-covariance structure between the pair of repeated measurements, which may be unrealistic (and therefore may bias estimates of error variance), particularly where measurements are Chapter 2 - 53 closely spaced time. Secondly, selection based on a wood property measurement at a single juvenile age forfeits information about the trajectory of measurements from measurements other rings which may be useful predicting mature age wood properties (Apiolaza et al. 2000; Davidian 1998).

In spite of the theoretical arguments in favour of longitudinal methods, there are numerous arguments supporting the use of univariate or bivariate analyses to support early selection in many instances. Firstly, with large numbers of sequential measurements (eg successive growth rings), purely multivariate analyses suffer from poor statistical power and often, inestimable solutions. This is because the size of the among-ring variance-covariance matrices fitted for both the genetic effect and the residual effect increases sharply and non-linearly with the number of repeated measurements included, and so the models quickly become overparameterised (Davidian and Giltinan 1995). This is particularly problematic in wood traits, where data are often available in each ring, and datasets tend to be small. Longitudinal data analysis offers some improvement on this overparameterisation by imposing assumed structures in the additive genetic and residual variance-covariance matrices (eg Wolfinger 1996). However, only a limited range of structures are available, and the choice among them involves substantial guesswork (Davidian 1998). In the particular case of estimating the optimum age for early selection, imposing a predefined structure on the among-age genetic variance-covariance matrix does not seem a desirable approach given that the main purpose of the analysis is to estimate the genetic covariances among ages. Bivariate analyses of pairwise age combinations (eg Balocchi 1993) avoid this issue.

Secondly, existing empirical evidence suggests these models may in most instances yield little or no difference in variance component magnitude or prec1s10n over univariate/two-stage techniques, where data are fairly well balanced. Several recent studies of forest trees have obtained roughly equivalent variance component estimates (eg Lu et al. 2001; Dieters et al. 2000) and coefficients of genetic prediction for early selection (Apiolaza et al. 2000; Apiolaza and Garrick 2001), and similar standard errors, from univariate and multivariate/longitudinal methods in a variety of traits. Importantly however, Dieters et al. (2000) and Lu et al. (2001) both found that the multivariate and longitudinal methods were more precise than univariate methods in unbalanced datasets, yielding lower standard errors of variance components. Chapter 2 - 54

In a well-balanced dataset, the marginal genetic gain from using longitudinal analysis to extrapolate multiple juvenile measurements, over the use of a single juvenile measurement for early selection, was minimal in the study of Apiolaza et al. 2000). The discrepancy between the methods is likely to be small for wood property data, as the advantage of repeated measures/multivariate approaches for this purpose diminishes with increasing genetic within-individual (co)variances, decreasing environmental within-individual covariances (Apiolaza et al. 2000), and decreasing incidence of missing values within-individuals (Davidian and Giltinan 1995).

Although the issue is in need of further investigation and ought form the subject matter for a PhD or similar study in quantitative genetics, it seems unlikely that variance and covariance components estimated using two-stage methods would be seriously biased, particularly in the case of wood property data.

While the conventional univariate analyses described for estimating juvenile-mature correlations may not be theoretically ideal for estimating juvenile-mature correlations, it appears likely that where data are well balanced, juvenile and mature selection ages are sufficiently far apart to minimise environmental autocorrelation, and genetic juvenile-mature correlations are reasonably high, longitudinal data analyses are unlikely to substantially improve the accuracy of genetic prediction over computationally simpler univariate methods, particularly in small datasets. More research is needed on a variety of datasets to further compare univariate and longitudinal/multivariate methods for early selection.

2.3.4 Summary and issues with longitudinal data analysis techniques

In summary, the arguments for using longitudinal models to analyse wood property data collected on sequential rings are strongest where: 1. the number of repeated measurements (n) is small; 2. some observations on some individuals are missing; 3. among-ring genetic covariances are low; environmental covariances are high; 4. data have been collected at distinct intervals, eg in each growth ring. Chapter 2 - 55 In most assessments of trends in wood properties across a stem (eg Harding 1996; Hodge and Purnell 1993) these arguments are not particularly strong in that:

1. usually a large number of rings are assessed, with the exception of very young samples; 2. sometimes the last ring or part thereof will be missing, but in general either the full sample is there or it's not; 3. most wood data is likely to have high genetic covariances among rings; 4. in many tropical taxa, rings cannot be identified: using first-stage indices, parameters can be calculated easily from densitometry pixel data, while longitudinal data analysis would necessitate breaking the data up into arbitrary 'chunks';

Additionally, there are some problems with the implementation of longitudinal methods: • Where n is large, analyses may not converge due to the large number of off­ diagonal elements to estimate in covariance structures; • They are computationally very complex, and so require additional statistical training for analysts and considerable guesswork in imposing appropriate covariance structures (Davidian 1998); • They are less flexible than two-stage models in the types of within-tree variation that can be described: for example, it would be difficult to analyse both across­ ring and within-ring variation within a multivariate or longitudinal framework; • They can only handle a limited amount of data; for example, raw densitometric traces may include several thousand point-estimates of density, and some prior calculations are required to reduce the dimensionality of the data before longitudinal models can be applied.

Longitudinal data analysis techniques need further investigation in tree breeding, particularly as new statistical methodologies, such as improved methods for characterising among-measurement covariances (eg Meyer 1998), become available. With currently available methods, there are still many instances where methods using univariate analysis of variance are preferable, providing advantages in both simplicity and flexibility. Chapter 2 - 56 genetic of hybrid populations: implications

pure species breeding, conventional biometrical genetics and selection theory provide a means of estimating the genetic gain from different breeding strategies, allowing choices between strategies to be made on a quantitative basis. Section 2.2 addressed the inapplicability of the conventional quantitative genetic model and popular statistical tools that incorporate it, such as the "individual-tree model" (eg Mrode 1996), for estimating the genetic gain from forward selection in hybrid populations. Examples of breeding strategies using backward selection for F 1 hybrid improvement, and applicable selection theory were presented and discussed. These strategies (eg RRS) are designed conservatively, to accumulate genetic gains that can be predicted using simple statistical parameters that make no serious genetic assumptions other than polygenic inheritance. However, they are typically complicated and expensive strategies with generation intervals up to twice those of pure species breeding: a severe handicap in long-lived trees. Additionally, strategies recurrently backward selecting for F1 interspecific cross performance, such as RRS, may excessively restrict the genetic base of the population through over-reliance on family selection (Shelboume 1993).

Mounting evidence suggests that in some taxa, strategies using recurrent forward selection in hybrid populations may be able to achieve similar genetic gains to more conservative hybrid improvement strategies, yet with greatly reduced generation intervals and the flexibility and simplicity of pure species breeding strategy. Several examples of successful outcrossed F2 generation tree hybrid populations were presented in Section 2.1. Despite these examples, and the widespread use of composite, or synthetic breeding in various applications in crop and livestock improvement (eg Martin and Russell 1984; Bourdon 1999), hybrid tree breeders have been reluctant to invest in advanced generation hybridisation experiments. The general literature, and a recent five-day conference on tree hybrid genetics and breeding (Dungey et al. 2000b ), suggested two main reasons why tree breeders have been deterred from advanced generation hybridization: Chapter 2 - 57 1. A common perception that hybrids are "genetic dead (Shelboume 2000), which appears to be based partly on experience (Hallauer and Miranda 1988; Wright 1976 p.43) also partly on examples mean performance and increased variation in some tree hybrid composites have typically been of highly restricted genetic base (reviewed in Section 2.1); 2. The lack of genetic theory to predict the genetic change resulting from forward selection hybrids.

This review will discuss the theoretical basis of these two concerns, where possible providing relevant empirical evidence. An overview of the genetic architecture of forest trees and their interspecific hybrids will precede two main sections presenting the case for two main arguments, respectively: 1. That interspecific forest tree hybrid breeding populations in general are likely to be less susceptible to hybrid breakdown than most crop hybrids and other less genetically variable populations; 2. That the choice of hybrid breeding strategy trees largely depends upon the mode of gene action governing hybrid performance.

An important objective of this review is to outline the theoretical considerations for successful composite breeding in trees.

2.4.1 Genetic architecture of interspecific forest tree hybrid populations

It is first necessary to define the concept of the genetic architecture of a population. The parameters of the genetic architecture have been described comprehensively by Mackay (2001), although the abbreviated description by Barker (1995) pertaining to a single trait is more amenable to discussion. Assuming diploidy, the genetic architecture of a population for any given trait is defined by the following parameters:

1. Number of alleles segregating at each locus; 2. Allele frequencies; 3. Nature and magnitudes of allelic effects at each locus; 4. Effects of dominance; 5. Number of loci contributing to heritable variation; 6. Linkage relations among loci; 7. Nature and magnitudes of inter-locus interactions; Chapter 2 - 58 8. Mutation rates.

Knowledge of these parameters can assist greatly predicting the response of a population to different types of selection and hybridisation, and hence guide choices among breeding strategies. Several common and key differences in genetic architecture between crop and tree hybrid populations are likely to result in. important differences in their response to different types of genetic improvement strategies, and in the type of assumptions appropriate in theoretical models. These differences can be summarised as follows:

1. Interspecific hybrids are most commonly used in trees, while intraspecific hybrids are the norm in crops. Tree species used to generate interspecific hybrids may commonly possess completely different allelic systems (eg Groover et al. 1994; Keirn et al. 1989; Stokoe et al. 2000), while crop varieties are expected to have the same, or very similar, sets of alleles that differ between varieties only in their frequency. 2. Allelic diversity is typically greater tree breeding populations m crop breeding populations (Hamrick et al. 1979; Ledig 1986), largely due to

differences in their histories of domestication (70 years; 0-4 generations vs 10 000 years; several thousand generations). 3. Trees tend to have greater additive genetic variation than crop populations, as a combined result of their short history of artificial selection, outcrossing mating system and high frequency of mutations (Ledig 1986). Additionally, where traits are subjected to consistent and strong selection over many generations as has occurred in many crops, non-additive genetic variation (dominance and inter-locus interactions) is expected to be of greater importance than in relatively unselected taxa such as trees (Holland 1999).

The importance of these factors will be discussed in the two main sections of this review, in relation to expectations of hybrid breakdown, and the effect of gene action on breeding strategy, respectively. Chapter 2 - 59 ·causes crosses

Hybrid respect to fitness is the phenomenon of reduced viability or fertility F2, backcross, or later generation hybrids, relative to the F 1 (Avise 1994). In breeding, hybrid breakdown manifests as a deterioration in mean population performance, usually accompanied by an increase in population variability, in a trait. The term 'hybrid breakdown' is variously and often ambiguously defined in the literature, and will be used here to denote both a decrease in mean and increase in variance in an advanced generation hybrid population relative to its ancestral F1 hybrid population, in accordance with the concept of advanced generation hybrid breakdown used by Levin (1978). Hybrid breakdown results from the process of segregation (separation of chromosomes during meiosis) and recombination (crossing-over between chromosomes) in a highly heterozygous hybrid population, and while in some populations a change in population mean and/or variance is strongly apparent, in others it is not observed at all. This discrepancy results from differences among populations in the basic parameters of genetic architecture listed above. Considering the effects of loci individually, the extent to which random­ mating advanced generation hybrids maintain F1 performance will be affected by the degree of dominance exhibited, the number of alleles and allele frequencies in the hybrid population. Considering the joint effects of multiple loci, it will additionally depend on the number of loci and distribution of their effects, linkage relationships between loci, and epistatic interactions among loci. The effect of each of these parameters on hybrid breakdown will be discussed with reference to known general trends in these parameters in tree and crop breeding populations, where possible comparing the potential for hybrid breakdown in these taxa. The utility of predicting hybrid breakdown is obvious: selection will not be worthwhile where hybrid breakdown is likely to cancel out any gains achieved. In such cases, advanced generation hybrids can be avoided in breeding programs.

2.4.2.1 Segregation: within-locus effects

At the individual-locus level, the most obvious consequence of segregation within a

random-mating F 1 hybrid population is the breaking apart of heterozygous genotypes,

causing an increase in the proportion of homozygotes in the F2 and subsequent generations. The resulting exposure of homozygous recessive deleterious alleles is a Chapter 2 - 60 commonly suggested cause of hybrid breakdown (eg Davenport 1908; Crow 1999). The extent of hybrid breakdown due to this cause depends on the degree of dominance exhibited by alleles, the number of alleles, and allele frequencies. The degree of dominance exhibited in the F1 is positively related to hybrid breakdown, and where dominance interactions are absent, hybrid breakdown does not occur (unless caused by deleterious epistatic effects). As in hybrids of other organisms, the importance of dominance gene action in tree hybrids varies strongly among taxa, with strong dominance detected in some crosses such as Populus tremuloides x P. tremula (eg Li and Wu 1996), yet probable lower importance of dominance in most Pinus hybrids (reviewed by Dungey 2001). In accordance with these findings, Stettler et al.

(1988) and others have reported strong hybrid breakdown in outcrossed F2 poplar hybrids, while outcrossed F2 hybrids in Pinus have typically shown little or no hybrid breakdown (eg Hyun 1974, 1976; Dungey 1999). The profound effect of dominance and other types of gene action on advanced generation hybrid performance, but variability among taxa in its incidence, motivates a separate and more comprehensive discussion of gene action in Section 2.4.3 of this review.

Multi-allelism is rarely considered or accounted for in genetic models (Li and Wu 1996; Mackay 2001) yet it is a common feature of tree populations (Groover et al. 1994; Ledig 1986) and the degree of allelic variability in hybrid populations may strongly influence hybrid breakdown. According to single-locus theory developed by Wright (1922) and extended by Eberhardt et al. (1967), regardless of the level of polymorphism (allelic diversity), allele frequencies or modes of gene action, Fz

heterosis is expected to be half of F1 heterosis, where only two parental populations contribute to the hybrid. This result is insensitive to the parameters of genetic architecture at the single-locus level, and in itself suggests limited potential for advanced generation hybridisation in trees, where there are typically two parental populations. However, if heterosis is small, as it often is in 'complementary' tree hybrids, a reduction of half the heterosis may not be of critical importance. A more useful way of measuring hybrid breakdown in practice may be as the percentage

change in F2 hybrid performance relative to F1 hybrid performance. This parameter is sensitive to the parameters of genetic architecture. Assuming that the presence of homozygotes of deleterious recessive alleles causes a reduction in Fz population

performance relative to the F1, and that there is some dominance, increased frequency

of heterozygotes in the F2 population is likely to improve F2 performance. Under Chapter 2 - 61 random mating, the degree of heterozygosity in the F2 degree genetic in the populations were crossed to the

. The effect of degree of allelic polymorphism the populations on F2 heterozygosity is demonstrated Table 2.2.

Table 2.2 Frequency of intraspecific and interspecific heterozygotes in parental species, two-way F 1 and F2 hybrid populations, for parental populations with varying degrees of allelic diversity, for the example of a single locus. Number of polymorphisms in 1 2 3 4 5 each parental population Parental 0 0.5 0.667 0.75 0.8 f.1...... _ (i~~~E~P~~ifi~~~t~~?~:Y~?.~~?) .. 1 1 1 1 1 F2 (interspecific heterozygotes) 0.5 0.5 0.5 0.5 0.5 (intraspecific heterozygotes) 0 0.25 0.333 0.375 0.4 (total heterozygotes) 0.5 0.75 0.833 0.875 0.9 Note: This example assumes different allelic systems in the two parental species, equal allelic frequencies, random mating, Hardy-Weinberg Equilibrium in the parental and F2 populations, and no segregation distortion.

Comparing the columns of Table 2.2 illustrates how greater allelic variation in the original parental populations results in a higher proportion of intraspecific heterozygotes in the F2 hybrid. Therefore, where dominance (eg over deleterious recessive alleles) is present in intraspecific heterozygotes, composite populations generated from highly genetically variable parental populations will maintain a higher proportion of F 1 mean performance, and exhibit lower variation due to segregation of homozygotes, than composites derived from genetically restricted parental populations. While this example assumed random mating, the proportion of intraspecific heterozygotes can be increased still further by deliberately making outcrossed F2 hybrids. Some empirical support for the above conclusions can be found in trees, where sustained performance has often been found in advanced generation hybrids generated from genetically variable parental populations (eg Hyun 1974; Powell and Nikles 1996a), while dramatic reductions in performance have often been found in advanced generation hybrids derived from genetically restricted parental populations (eg Brune and Zobel 1981; Kulkarni et al. 2001; Nikles et al. 1999).

Further evidence for the expected negative relationship between genetic variability in the parental populations and advanced generation hybrid breakdown comes from a recent review of transgressive segregation (the appearance of extreme phenotypes in segregating advanced generation hybrid populations, causing increased population Chapter 2 - 62 variance). Of a total of 579 adaptive and non-adaptive traits in 113 studies reviewed by Rieseberg et al. (1999), transgressive segregation occurred most frequently (in 92% of traits observed) in intraspecific crosses of inbred, domesticated populations, mainly crops and plants. It occurred least frequently (in 38% of traits observed) in interspecific crosses of outcrossing, wild populations (mainly trees and some wild plants). The authors commented on the agreement of this result with theoretical expectations based on the relative expected amounts of genetic variability in the two types of populations. In summary, hybrid breakdown is less likely where the parental populations are genetically diverse, as expected in most wild populations of trees (eg Hamrick 1983).

Hybrid breakdown is also affected by allele frequency. The results reported in Table 2.2 have assumed intermediate allele frequencies; this may not be realistic in some populations, and homozygosity will increase nonlinearly with increasing allele frequency. Populations in which many alternative forms of a gene are carried at low frequency are unlikely to make a significant contribution towards maintaining heterozygosity unless the frequencies of the rare alleles are increased by selection.

As noted above, mating systems affect genotype frequencies. Outcrossing will increase the frequencies of heterozygotes; inbreeding will decrease them, having the same effect as reducing the genetic variability in the parental populations. An experiment in Pinus elliottii X P. taeda hybrids (Nikles et al. 1999) found that hybrid breakdown was increased by inbreeding: slightly greater variability and decreased mean growth were found in mildly inbred relative to outcrossed Fz progeny of common parents.

In summary, by inference from the genotype frequencies in Table 2.2, at the single­ locus level it can be seen that where the following conditions are fulfilled: • The degree of dominance in genotypes that are unique to the hybrid (or that are at higher frequencies in the hybrid than in the parental populations) is similar to or not a lot greater than the degree of dominance in heterozygous genotypes that occur within the parental populations; • Parental species populations are genetically variable, and; • Fz hybrids are deliberately outcrossed; Chapter 2 - 63

performance may closely approximate F 1 performance. absence of deleterious epistatic effects, conclusion can be generalised from single-locus to the multi-locus level. The same conclusions apply to types of hybrids such as inter-provenance hybrids.

2.4.2.2 Segregation and recombination: effects across multiple loci

Considering the joint effects of multiple loci, the maintenance of heterozygosity, and hence the maintenance of heterosis, in composite populations also depends upon the number of loci and the distribution of their effects, linkage relationships between loci (or recombination fraction r), and epistatic interactions among loci. To demonstrate the effect of the number of loci on composite performance, Wright (1976) provided a simplistic but illustrative table of expectations for the "recovery ratio" of individuals homozygous for the most favourable allele at every locus affecting a trait, where different numbers of unlinked loci control the trait, in a segregating population. The table is reproduced in Table 2.3; the computations assume two alleles at every locus, each with frequency of one-half, equal effects of loci and no linkage:

Table 2.3 Recovery ratios of individuals homozygous for the most favourable allele at every locus, for traits affected by different numbers of biallelic loci (taken from Wright 1976). Number of loci 1 2 3 4 5 6 7 ratio 1116 1/64 11256 111024 1/4096 1/16384

The greatly decreasing probability of purely homozygous segregants with increasing number of loci, or linkage groups, acts to reduce the probable importance of hybrid breakdown due to recessiveness where more loci affect a trait, or where linkage is looser. The model in Table 2.3 is an over-simplification, because in reality loci are usually unequal in effect (Bost et al. 1999); where the effects of loci are distributed unevenly, major genes are likely to increase the importance of segregation variance relative to the completely polygenic case. High segregation variance in a trait, even in

the absence of inbreeding (eg ramicoms in Pinus elliottii xP. caribaea F2 hybrid, Dieters unpublished data) may indicate the presence of a major gene. However, the polygenic component of genetic variation is generally thought to be of relatively strong importance in most forest tree traits aside from stem defects and disease resistance (reviewed by van Buijtenen 2001). Polygenic inheritance will result in Chapter 2 - 64 reduced segregation variance at the relative to taxa oligogenic traits are

In addition to segregation, hybrid breakdown can result deleterious epistatic effects such as the dismantling of "co-adapted gene complexes" present in populations of common evolutionary background (Dobzhansky 1937; Wright 1968). This refers to the dismantling of favourable epistatic combinations of alleles present in pure species and F1 hybrid populations, due to recombination in F2, backcross, or other advanced generation hybrids (Levin 1978). However, the contribution of this factor to hybrid breakdown is very difficult to ascertain, due to the difficulty of measuring epistasis, further compounded by the presence of linkage disequilibrium. Both theoretical and empirical evidence point to the likely greater importance of co­ adapted gene complexes in species with inbreeding mating systems. A classic experiment in backcrosses of Gossypium interspecific hybrids demonstrated selective elimination of alleles from the donor species at the gametic stage (Stephenson 1949). Observed hybrid breakdown in the backcross population could then be attributed to deleterious interactions between combinations of genes from the two parental populations. As co-adapted gene complexes are thought to be less frequently of importance in outcrossing species than in inbreeding taxa such as Gossypium (reviewed by Holland 1999), deleterious epistatic effects in advanced tree hybrid generations may be less likely to contribute to hybrid breakdown. This argument is supported by studies demonstrating slightly increased yield in advanced generation hybrids of maize varieties (Lonnquist and McGill 1956), and in Fz and F3 generations of hybrid larch (Holst 1974; Li and Wyckoff 1994), although in both these examples, mild selection was applied. The very similar performance of F1 and unselected Fz hybrids of Pinus rigida x P. taeda in Korea (Hyun 1974) and PEExPCH in Queensland (Powell and Nikles 1996a) suggest that breakdown of co-adapted gene complexes is unlikely to be a serious problem in these hybrids. Co-adapted gene complexes may be more important in crosses of distantly related species, such as the intersectional Eucalyptus grandis x E. globulus, but published comparisons of F1 and advanced generation hybrid performance in this and other intersectional crosses could not be found. Direct assessment of the importance of epistasis in co-adapted gene complexes in hybrid trees awaits the application of molecular genetic techniques to structured hybrid populations. Although several such populations exist (eg Vaillancourt et al. 1995; Bradshaw and Stettler 1995), population sizes are typically Chapter 2 - insufficient to detect even the effects individual QTL, detection epistatic effects requires statistical

This brief overv1ew of factors affecting advanced generation hybrid breakdown suggests that low importance of dominance gene action in heterozygote genotypes unique to the hybrid population, high allelic polymorphism with intermediate allele frequency, polygenic inheritance and low importance of co-adapted gene complexes may reduce the severity of hybrid breakdown. Theoretical and empirical evidence suggests that tree hybrids in some taxa may be able to sustain F 1 hybrid performance through forward selection into advanced generations. Allelic variability and gene action appear likely to be the most important factors affecting hybrid breakdown, and while large, genetically variable base populations can ensure allelic variability, the mode of gene action affecting hybrid performance varies considerably among taxa, and must be estimated directly in the hybrid population of interest. Gene action is therefore the most useful theoretical parameter for deciding between breeding strategies for improving interspecific hybrids of forest trees; for this reason it will be examined in a separate and more comprehensive discussion, in the next section. The current review has not considered the effects of selection, focussing instead on the expected performance of populations under random mating. The prospects for selective improvement of composite hybrid populations will be addressed in discussion of gene action and breeding strategy, in the next section.

2.4.3 Gene action and hybrid performance

Among the numerous factors that affect the expected performance of hybrids of vanous generations, the predominant mode of gene action affecting hybrid performance is likely to be the most important. While numerous hypotheses of gene action have been proposed and investigated, mainly in taxa other than forest trees, the predominant type of gene action in many hybrid populations is yet unclear. A full consideration of the types of gene action that could underly hybrid population performance, and their potential effects on different breeding strategies, is necessary in order to design appropriate methods of detecting them in specific populations. A discussion of the types of gene action hypothesised to contribute to heterosis will be Chapter 2 - 66 followed by a description of their effects on breeding strategy, and a brief appraisal of some conventional and novel methods for assessing gene action in interspecific hybrids.

2.4.3.1 Genetic causes of heterosis

The predominant mode of gene action governing heterosis has been one of the most actively contested topics in genetics over the past century (Crow 1998). While determining gene action in simple Mendelian traits is a straightforward matter, ascertaining the relative importance of different modes of gene action in heterosis has been complicated by the likelihood that functionally related loci are both numerous and unequal in their effects, in quantitative traits (reviewed by van Buijtenen 2001 in trees), interact to some degree in their contribution towards hybrid phenotypes (Wright 1968; Goodnight 1999) and are in severe linkage disequilibrium in hybrid populations (Weir et al. 1980).

Heterosis itself, being the deviation from mid-parent performance, cannot be due purely to additive gene action, as under completely additive gene action, hybrid performance would equate to the midparent. Numerous hypotheses attributing heterosis to various types of non-additive gene action have been proposed, under the assumption of diploid inheritance (appropriate for most hybrids). These hypotheses were separated by Hayes (1952) into Component I and Component II, pertaining to the effects of within-locus interactions and among-locus interactions, respectively.

Component 1: Within-locus interactions

The major genetic consequence of hybridisation IS to increase the proportion of heterozygotes in the hybrid relative to the parental populations. Based on Mendelian principles, early theorists hypothesised that within-locus interactions, known to occur at heterozygous loci in Mendelian traits, were responsible for heterosis. The dominance of linked factors (Jones 1917) and overdominance (Shull 1908, East 1936) hypotheses were proposed as possible causal mechanisms. The dominance hypothesis explains hybrid vigour in terms of deleterious recessive alleles from one parent being suppressed by dominant alleles from the other parent, yielding an increase in mean performance. This hypothesis depends upon recessiveness being related to detrimental effect, and dominance being related to beneficial effect - a consistent trend observed a few studies (Davenport 1908; Mackay overdominance hypothesis proposes a non-Mendelian explanation: heterosis results the superiority of heterozygous genotypes to homozygous genotypes, at individual locus level.

Almost a century of subsequent research has debated the relative importance of these two mechanisms in contributing to heterosis (Crow 1998). Overdominance was intuitively suggested in early observations of enormous heterosis found in some crosses, as reported by East and Jones (1919) of a radish x cabbage hybrid: "A single plant filled an entire greenhouse and grew out the roof'. However, the occurrence of overdominance has never been proven conclusively - partly because it is statistically impossible to distinguish true overdominance from pseudo-overdominance: a type of epistasis caused by linkage of dominant alleles in repulsion phase, resulting in heterozygote superiority at the level of the individual linkage group (Richey 1942; Falconer and Mackay 1996). While some studies have reported overdominance in some hybrids (eg Li and Wu 1996 in poplars; Stuber et al. 1992 maize), a detailed re-analysis of Stuber et al.'s data by Cockerham and Zeng (1996) suggested pseudo­ overdominance. A molecular genetic analysis of data examined by Li and Wu, by Bradshaw and Stettler (1995) similarly also suggested pseudo-overdominance, demonstrating that even many sophisticated molecular genetic techniques and biometrical analyses cannot conclusively distinguish between these two types of gene action (Falconer and Mackay 1996).

The relative popularity of the dominance and overdominance hypotheses has varied greatly over the course of the past century. Early theorists (eg Jones 1917) favoured the dominance hypothesis as it incorporates a Mendelian explanation for heterosis. Overdominance gained favour after the publication of East (1936) and during and after the hybrids conference of 1950 (Gowen 1952). More recently, the lack of solid evidence for true overdominance in molecular genetic studies, and strong empirical evidence for the importance of dominance in various taxa (notably, a conclusive study in tobacco by Pooni et al. 1994; a classic line hybridisation experiment by Sprague 1983, and a molecular genetic experiment in rice by Xiao et al. 1995) have resulted in the greater body of opinion today supporting the dominance hypothesis as the most likely and common cause of heterosis (Allendorf and Leary 1986; Brewbaker and Sun 1999; Crow 1998; Ledig 1986; Rieseberg and Carney 1998). Chapter 2 - 68

Component Although epistatic interactions at are undoubtedly ubiquitous, formed much of subject matter for a recent conference on heterosis in crops (Coors and Pandey 1999), the practical importance of assessing them is often less clear. In summarizing the conference, James Crow stated: "Is there epistasis? Of course. Can we safely ignore it? Often, yes. But it is there, and perhaps ways can be found to exploit it" (Crow 1999). The most serious problem in studies of epistasis is the lack of experimental material/statistical analyses powerful enough to detect its presence with any confidence, due to the almost infinite number of possible interactions among loci. Most studies have ignored possible contributions from among-locus interactions, due to the difficulty of modelling them. The possible importance of epistatic interactions in both pure species and hybrid populations thereby seems to have been underestimated in the literature, by assuming them absent.

A review of studies of epistasis in maize using biometrical methods highlights the importance of choice of method, and the need for statistically powerful methods, when studying epistasis. While low and insignificant levels of epistatic genetic variance are usually reported in studies of variance components estimated from mating designs (eg in four studies reviewed in Hallauer and Miranda 1988), biometrical methods using more statistically powerful techniques such as generation mean comparisons and triple testcross analysis have commonly indicated importance of epistasis in contributing to heterosis (eg Stuber and Moll1971; Lamkey et al. 1995; Wolf and Hallauer 1997). The poor correspondence between biometrical genetic variances and underlying gene action, as discussed in Section 2.2.1, has been suggested responsible for the failure of methods using mating designs to detect epistasis (Goodnight 1999). Lush (1945) and Cheverud and Routman (1995) demonstrated through simulation that biometrical models tend to partition variance due to epistatic effects into additive and dominance components, even where variation is purely epistatic (Lush 1945).

Recent investigations using molecular genetic techniques (eg Yu et al. 1997) and some advanced biometrical techniques (eg Wu and Li 1999) have circumvented problems with the traditional analytical approach, allowing new insights into epistasis at the level of the individual locus, or linkage group. The results of these studies Chapter 2 - 69 increasingly support prediction of Mac (1976) epistasis tends to be greater autogamous (self-fertilising) plant species (eg wheat, Busch et 1974; Cregan Busch 1978), oat Munkvold 1999; and Frey 1991), rice (Gravois et 1997; Yu et 1997) others, than has generally been detected in outcrossing species. Epistasis has also more commonly been detected in elite material (eg Yu et al. 1997; Wolf and Hallauer 1998; Pooni et al. 1994) than in routine germplasm. These findings are consistent with the fixing of additive x additive epistatic effects in stable, homozygous populations and in highly selected populations, whereas epistatic effects might less easily be fixed in random mating populations, in which dominance interactions are expected to be of greater importance (Mac Key 1976).

However, multiplicative epistatic effects, or epistasis resulting from multiplicative interactions among loci, have been hypothesised to play an important role in outcrossing species and their hybrids, and have recently been modelled biometrically, by Schnell and Cockerham (1992) and Wu and (1999). Table 2.4 uses a hypothetical numerical example to demonstrate multiplicative epistasis for the trait tree height, which is, in both hypothetical species A and B, determined by the product of the traits average internode length (IL) and average number of internodes (IN) (example after Schnell and Cockerham 1992). The second-last column shows that the mid-parent heterosis for height is much greater than for either IL or IN, demonstrating a large multiplicative epistatic contribution to heterosis in tree height. Similarly, multiplicative epistasis involving complex component physiological traits may in turn contribute to the observed heterosis for the traits IL and IN. The last column shows the mid-parent heterosis for each taxon, in the hypothetical instance that genetic control of the two traits IL and IN is solely additive. This serves to demonstrate that heterosis -albeit probably of a small magnitude (Richey 1942; Schnell and Cockerham 1992) can occur in the absence of dominance variance (ie due to additivexadditive epistatic interaction), as demonstrated theoretically for two loci by Minvielle (1987). This heterosis (MP HV%A=2.9%, Table 2.4) is in fact simply due to the mathematical disparity between the mean of products and the product of means. Chapter 2 - 70 Table 2.4 A hypothetical example of multiplicative epistasis contributing to heterosis in the F1 hybrid. Trait Species A species B MPHV% MPHV%A (observed) internode length 0.8 1.35 1.65 53.5 0 (IL) no. internodes 12.1 15.2 18.4 34.8 0 (IN) tree height 9.68 20.52 30.36 101.1 2.9 (IL X lN) Note: vol= stem volume, strt= stem straightness, volxstrt= the product of the two traits, MP HV% = observed mid-parent hybrid vigour, MP HV% A= mid-parent hybrid vigour if the genetic control of vol and strt were solely additive.

Multiplicative epistasis can also involve additivexdominance and dominancexdominance epistatic interactions. It may make a particularly strong contribution to heterosis in traits governed by strong dominance or overdominance at a number of loci, where the effects of loci act multiplicatively (Wu and Li 1999). Multiplicative epistasis is thought to play an important role in the complex physiological systems of trees (Brewbaker and Sun 1999).

Although the modelling and measurement of epistasis provides a valuable contribution to our understanding of the genetic basis of heterosis, the practical applications of information about epistasis, for example in making breeding strategy decisions, are yet unclear (Crow 1999). Definitive unravelling of epistatic effects at the the level of individual loci may reveal some useful information for breeding, but awaits the results of fine-scale mapping in large, targeted experiments (Ahn and Tanksley 1996; Mitchell-Olds 1995; Yu et al. 1997).

Summary - gene action In summary, the majority of evidence suggests dominance as the most likely and common cause of heterosis. However, most studies of gene action in hybrids have been in intraspecific hybrids, and in highly domesticated crops and model species; there is a dearth of studies examining the genetic basis of heterosis in interspecific hybrids of trees and other relatively 'wild' populations. While studies in some hybrids (eg poplars, maize) have suggested overdominance, the statistical impossibility of distinguishing overdominance from pseudo-overdominance creates difficulties in proof. The importance of epistasis has been suggested in some studies, particularly in highly selected, elite germplasm and in autogamous species, but also in Chapter 2 - 71 multiplicative epistasis been investigated trees. However, general it is likely the effect of epistasis, aside from specific co-adapted gene complexes elite material, will be to exaggerate existing additive or dominance genetic effects. Therefore, the distinctions between overdominance and pseudo­ overdominance due to epistasis, or additive and additive-related epistatic effects, for example, may not be critical ones for most practical decisions in breeding and deployment strategy. Until fine scale genetic mapping can reveal the true influence of epistatic effects, the dominance hypothesis appears to have gained acceptance as the most common and important cause of heterosis.

2.4.3.2 Hybrid breeding strategy and its dependence on gene action

The choice among the complete range of available hybrid breeding strategy options in trees is likely to depend to a large extent upon the types of gene action operating in the hybrid taxon of interest.

The most basic decision hybrid breeding strategy is that of which genotypic configuration to deploy. This question has traditionally not been of particular interest to hybrid breeders, as emphasis has been on exploiting heterosis in the vigorous F1 hybrid, and so recurrent improvement efforts naturally aimed to improve the F 1. In forest tree improvement, the long generation interval and large investment required to recurrently improve the F 1 hybrid have stimulated interest in the deployment and improvement of advanced generation hybrid taxa through forward selection in the F 1•

Where dominance, overdominance, pseudo-overdominance or other dominance­ related epistatic effects (collectively referred to as dominance-related gene action) strongly influence hybrid population performance, deployment of the F1 hybrid in preference to advanced generation hybrids is likely to be necessary to avoid hybrid breakdown (Li and Wyckoff 1994; Namkoong et al. 1988). In such cases, hybrid

testing and direct selection for F1 hybrid performance is likely to be necessary. Where dominance-related gene action is of small importance relative to the additive effects of alleles, a range of lower cost breeding strategy alternatives to direct

selection for F1 hybrid improvement are available, selecting on additive and additive­ related epistatic gene action. These strategies include recurrent selection for general

combining ability (RSGCA, or F 1 hybrid improvement by indirect selection on Chapter 2 - 72 species breeding Nikles and recurrent in a population. Backcrossing is a special case, appropriate for introgressing specific desirable genes from one parental population population, although backcrossed genotypes are not likely to surpass those of the maximally heterozygous F1, where dominance-related gene action makes a large contribution to hybrid performance.

Reciprocal Recurrent Selection (RRS) The most conservative breeding strategy available is Reciprocal Recurrent Selection (RRS) (Comstock et al. 1949). This strategy makes genetic gains regardless of the modes of gene action influencing hybrid performance, through testing and direct selection for F 1 hybrid family or parental means. Even in the absence of non-additive gene action, RRS will make equivalent gains per generation to RSGCA and composite strategies using family selection, yet at much higher cost. Given the lack of understanding of gene action in most hybrids until recent definitive studies in some taxa (eg dominance in tobacco, Pooni et al. 1994; pseudo-overdominance in aspen, Bradshaw and Stettler 1995; and Wu 1996), RRS has been recommended trees to ensure genetic gains from investments in breeding (Vigneron 1991). However, the high expense of RRS places a research priority on estimating predominant modes of gene action in the taxa and traits of interest and thereby assessing the likely viability of alternative strategies. A comparison of the features of RRS, RSGCA and Composite strategies is presented in Table 2.5. Chapter 2 - 73 Features of some recurrent strategies interspecific hybrids trees.

strategy (eg Composite Shelboume 1993) (eg Nikles 1991) type of gene action all types mainly additive mainly additive & exploited additive-related epistatic; adversely affected by strong dominance-related gene action number of populations to 3 3 1 +any populations maintain and improve for infusion breeding cycle length long: twice as short: similar or short: similar to long as pure sp. slightly longer pure sp. or shorter, than pure sp. depending on flowering 12recocity recurrent selection of F1 yes no no hybrid crosses? (difficult with crossing incom2atibilities) type of selection family only indirect family direct family and possible and within- within-family family start-u12 time reguired intermediate short long size of F1 population intermediate small large

Table 2.5 suggests that for the practical purpose of breeding strategy design, the most critical and useful distinction is likely to be that between hybrids with predominantly additive gene action, and hybrids where dominance-related gene action strongly contributes to hybrid performance. Fine distinctions such as that between overdominance and pseudo-overdominance (Richey 1942), are not likely to be critical for hybrid breeding strategy. Their effects on performance differ only in that

overdominance will largely disappear in the F2 generation, while pseudo­ overdominance will decay more gradually over successive advanced hybrid generations as linkage equilibrium is approached (Namkoong 1979). Both

overdominance and pseudo-overdominance are likely to require deployment of F1 hybrids, and neither type of gene action is conducive to recurrent genetic gain in advanced generation hybrids. Similarly, strong dominance and dominance-related epistatic effects will have synonymous implications for breeding strategy. Chapter 2 - 74

Where gene action is predominantly additive, within between pure species hybrid populations, the strategy of RSGCA (eg Nikles has been proposed, where selection is performed in pure species without the need for testing hybrid crosses. This strategy offers a greatly reduced generation interval relative to RRS, and may be of interest where improvement of a pure species parent is also required, for deployment on certain sites. However, Namkoong et al. (1988) have pointed out that under additive inheritance, indirect selection for hybrid performance based on pure species family performance is less efficient than direct combined family and within-family selection in the composite hybrid population. If the predominance of additive-related gene action can be confirmed, a more efficient strategy would be to separately improve the composite hybrid population and the pure species parental population of interest (Li 2, pers. comm.).

Composite breeding (COMP) Where additive gene action predominates, optimal genotypes are most likely to be found in recurrently improved segregating composite hybrid populations (Li and Wyckoff 1994). Strategies selecting forwards in composite populations can utilise a large range of variation in the combined interspecies population by applying both family and within-family selection, although emphasis on family selection may be necessary in early generations due to linkage disequilibrium. Composite strategies may be particular! y useful where clonal deployment allows the capture of unique allelic combinations such as transgressive segregants (Nikles and Griffin 1992), although these are expected to be rare in genetically variable and outcrossed hybrid populations (Rieseberg et al. 1999). However, in taxa such as aspen and poplar hybrids, (eg Populus tremuloides x P. tremula), where overdominance or pseudo­ overdominance make a strong contribution to hybrid performance (Li and Wu 1996), advanced generation hybridisation is likely to result in seriously deteriorating performance, as demonstrated in Populus trichocarpa x Populus deltoides hybrids by Stettler et al. (1988). Understanding the mode of gene action affecting hybrid performance is therefore an important prerequisite for applying advanced generation hybridisation.

2 Associate Professor Bailian Li, Department of Forestry, North Carolina State University, Raleigh USA. Chapter 2 - 75 Gene action and breeding strategy: empirical studies in maize Some empirical evidence concerning gene action and breeding strategy can be found in the literature on maize. A series of classic empirical selection experiments in maize populations since the early 1970s (eg Moll and Stuber 1971; Tragesser 1991) clearly demonstrate that similar genetic gains can be achieved by capitalising on different genetic mechanisms within the same populations. Certainly the gains from different methods depend on critical genetic parameters such as the importance of dominance-related gene effects in the hybrid population. However, even in maize hybrids, where dominance and even overdominance are considered to contribute substantially to hybrid performance (Gowen 1952; Stuber et al. 1992), selection experiments have realised similar genetic gains by selecting for other types and combinations of genetic effects including additive, dominant and epistatic gene action. For example, Moll and Stuber (1971) in a maize (Zea mays) selection experiment spanning 6 generations, compared the realised genetic gain from RRS with that from pure line selection (PLS, analogous to RSGCA in trees). These strategies yielded very similar gains (statistically NSD) in the variety hybrid

population, even though the rph (pure-hybrid correlation) was only 0.63 for the 'Jarvis' variety and 0.72 for the 'Indian Chief' variety. An important outcome of this experiment was that similar genetic gains were achieved using different genetic mechanisms. Table 2 from Moll and Stuber (1971) is reproduced here as Table 2.6, and lists the mid-parent heterosis of hybrid crosses at each generation, for each selection method. Note that in the hybrids made from the pure-line-selected material, heterosis stays constant or slightly decreases after 6 generations of selection, while in hybrids made from varieties subjected to RRS, the heterosis increases markedly over the 6 generations of selection. This suggests that RRS had achieved genetic gain by increasing the frequency of alleles displaying dominance and/or epistatic effects, while PLS had achieved similar genetic gain by capitalising primarily on additive or additive epistatic effects. Chapter 2 - 76 Table 2.6 Percentage heterosis in grain yield, in the Zea mays variety hybrid (Jarvis x Indian Chief) before and after PLS (pure-line selection) and RRS (reciprocal recurrent selection) (reproduced in full from Moll and Stuber 1971). Kind of selection selection cycle Full-sib (PLS) RRS 0 19.2 19.2 3 14.8 4 18.2 24.2 6 15.4 30.2 Note: Although RRS and PLS produced similar overall genetic gains after 6 cycles of selection, a much larger proportion of the gains from RRS was due to heterosis.

These results were confirmed by subsequent selection experiments (Moll et al. 1978 and Moll and Hanson 1984) in the same maize populations. Genetic gains achieved in the hybrid from ten generations of PLS and RRS were approximately equivalent and N.S.D., even though heterosis in the hybrids developed using RRS was 96% while those from PLS was only 42% (Moll et al. 1978). More recent selection experiments by Holthaus and Lamkey (1995) and Tragesser (1991) in other maize varieties have verified these results, similarly achieving comparable genetic gains from additive and non-additive genetic mechanisms.

The strong genetic gain in the hybrid from additive genetic effects accumulated by pure line selection in these experiments suggests that similar gain could have been made from combining the species in the first generation, and conducting simple recurrent selection (SRS) in the composite hybrid population thereafter (Namkoong et al. 1988). The preponderance of additive genetic variation in most recently domesticated forest tree species populations, and the strong performance of

outcrossed F2 hybrids, suggests that hybrid breeding in forest tree species might best focus on accumulating additive genetic effects, provided that overdominance or pseudo-overdominance gene action does not strongly influence economically important traits. Novel methods for estimating gene action in interspecific hybrids of outcrossing species may provide a quantitative basis for tree hybrid breeding strategy decisions. Chapter 2 - 77 gene

NJJmerous genetic analyses been used to infer gene population measurements. As discussed Section 2.2.1, genetic variances estimated using conventional quantitative genetic methods are unlikely to provide useful representations of gene action in interspecific hybrid populations. However, simple manipulations using some conventional statistical methods may provide rough indications of gene action some instances.

The comparison of parental variance between pure species and hybrid populations may be informative in some instances. Where the GCA (General Combining Ability) variance in the F 1 hybrid far exceeds the GCA variance in either pure species parental population (eg Volker 1995), this may indicate epistatic gene action in the hybrid (Comstock 1955), though in interspecific hybrids, it could also be caused by dominance interactions between different alleles in the two species. Likewise for SCA variance, although high SCA variance in the hybrid is more likely to indicate dominance or dominance-related epistasis. The genetic correlation between parental performance pure species combination and hybrid combination has also been used to infer modes of gene action; high correlations with both parental species may suggest importance of additive gene action in determining hybrid performance; high correlations with only one species may indicate that alleles contributed by that species are dominant. The interpretation of low correlations is not as clear.

A more commonly made genetic inference from observational studies involves the comparison of hybrid and pure species means. The widespread use of this inference warrants further discussion of its basis and possible problems. The condition of hybrid intermediacy between its pure species parents, or hybrid equivalence to the mid-parent, is often interpreted as evidence of additive gene action, and deviations

from intermediacy as evidence of dominance gene action (eg Potts and Dungey 2001). While this is true of a Mendelian trait, in polygenic traits many other factors contribute to taxon means. Ambidirectional dominance, segregation distortion, extranuclear genetic effects, ploidy level and epistasis are five key issues for consideration when comparing the hybrid to the mid-parental value: Although dominance 1s usually (consistently occurs one direction or the other at loci), it may also be ambidirectional (having no consistent (eg Stoddard Herath 2001 in beans), at different loci affecting a trait. Equivalence of a hybrid population mean to its mid-parent mean may thereby result from cancellation of dominance effects rather than completely additive gene action, although directional dominance is usually assumed to be the norm (Falconer and Mackay 1996).

2. Segregation distortion. Segregation distortion refers to any deviation from expected segregation ratios based on Mendelian rules of inheritance. The result of segregation distortion is that the progeny do not inherit a random sample of alleles from their parents, at each locus. Causes, in interspecific hybrid populations, may include crossing or chromosomal incompatibilities during species cross-fertilisation (Khurana and Khosla 1998; Saylor and Smith 1966), and deleterious epistatic interactions such as synthetic lethals (Mackay 1996). While inter-sectional crosses are largely impossible in both Eucalyptus and Pinus, with some notable exceptions in the eucalypts (eg Griffin et 2000), strong reproductive barriers and hybrid inviability are common even in within-series or within-subsection crosses (Potts and Dungey 2001; Critchfield 1973). In some cases, these may cause severe segregation distortion, as in the (inter-sectional) cross of E. grandis x E. globulus, where Griffin et al. (2000) reported that only 0.15% of seed produced 'normal' trees after 2 years of growth the field. This is perhaps the most extreme example; in a review of 17 studies of various inter-specific, within-series crosses in Eucalyptus, Potts and Dungey (200 1) found that the normal seedlings as a percentage of seeds sown ranged from 20% to 84%, with a median of 63%, and commented that a figure in excess of 80% would be expected in intra-specific crosses.

Consistent with these results, a molecular genetic study observed distorted segregation ratios of RAPD markers in F2 progeny of E. globulus x E. gunnii (Vaillancourt et al. 1995). However, no other molecular genetic studies of segregation distortion in hybrids appear to have been published in trees. Evidence from studies of crop plants suggests that segregation distortion is much more prevalent in inter-specific than in intra-specific crosses. Zamir and Tadmor (1986) report segregation distortion at 54% of loci in inter-specific crosses in Lenz, Capsicum and Lycopersicon, but only 13% of loci in intra-specific crosses; similar results were Chapter 2 - 79 obtained in Helianthus (Rieseberg and Carney 1998). In an interspecific hybrid of pearl millet (Pennisetum), segregation ratios in an advanced generation hybrid were biased toward one of the species by a 12:1 ratio (Liu et al. 1996). Where segregation ratios are skewed, as in Liu et al. (1996), hybrid progeny may receive more alleles from, and therefore more closely resemble, one pure species parent over the other. In such cases, segregation distortion may be an important factor influencing heterosis.

3. Extranuclear genetic effects Extranuclear genetic effects may play a role in influencing the performance of interspecific hybrids. Extranuclear genetic control is most commonly observed in the form of maternal genetic effects (DeVerno et al. 1993), where reciprocal crosses perform differently under the same experimental conditions. Possible causes may be greater importance of extranuclear DNA effects in one species than the other, or disruption of co-adapted complexes involving nuclear and extranuclear genes, due to hybridisation. The lack of significant maternal effects in most hybrids and for most traits appears to be the norm (eg in Larix decidua x L. leptolepis, Paques 1989; Li and Wu 1997 in Populus tremuloides x P. tremula); in the few observed cases of maternal effects, the evidence is inconclusive due to the small numbers of families involved, and experimental design issues (eg de Assis 2000; Blada 2000b; Siarot 1991; Gothe 1987). Griffin et al. (2000) consider that reciprocal effects are generally likely to be of little consequence in hybrids, consistent with the generally low incidence of maternal effects in intra-specific crosses in trees (eg van Wyk 1990; Wu and Matheson 2001 ).

4. Ploidy level Most forest tree hybrids, eg in Pinus (Williams et al. 2002) and Eucalyptus (Grattapaglia and Bradshaw 1994), appear to be diploid. However, some tree hybrids, such as in Populus, exhibit triploidy or other types of polyploidy (eg Wu 1995, Bradshaw and Stettler 1993). While polyploidy is relatively rare in forest trees in general (reviewed by Khoshoo 1958), it is more common in hybrids than in pure species, and so requires consideration in genetic analyses (eg Wu 1995), particularly as ploidy levels often have a dramatic effect on vigour. Chapter 2 - 80 5. Despite a diverse suite of methods proposed for detecting epistatic effects (eg et 1999; Wolf Hallauer 1998; Powers 1950), contribution to hybrid performance remains poorly understood, because among-locus interactions are potentially complex and unpredictable. Although expected to be of greater importance highly selected hybrid germplasm than in unselected populations (Wright 1968; Yu et al. 1997), conclusive empirical evidence for the presence of epistasis hybrids has been presented by Gardner (1963) (epistasis due to linkage, detected as decreasing genetic variances in successive advanced hybrid generations). However, despite increasing research into the importance of epistasis, partly summarized in the recent symposium on heterosis (Coors and Pandey 1999) and also reviewed by Holland (1999), its exact influence on hybrid performance is very uncertain. It has been suggested that multiplicative epistatic interactions in composite traits (eg total tree height) may reinforce the effects of dominance in the component traits (eg mean internode length and internode number, which multiply to make tree height) and exaggerate the expected importance of dominance, where epistatic interactions have not been considered. However, for the design breeding strategies in practice, the distinction between overdominance, strong dominance and dominance-related epistatic gene effects may not be critical: in all such cases, strategies targeting non-additive gene effects are likely to be necessary.

The inference that hybid equivalence to the mid-parent indicates additive gene action may be tenable, if it can be assumed that the above factors are inconsequential. The validity of this assumption is likely to be highly dependent upon the taxon of interest. Attributing deviations of hybrid performance from the mid-parent simply to dominance gene action is likely to be an over-simplification, but may be a necessary assumption of genetic models in situations where epistasis cannot be accounted for. The absence of segregation distortion is not likely to be a reasonable assumption in hybrids with very low crossing success; in these taxa, statistical measures of populations are unlikely to yield any useful information about their genetic architecture. In summary, taxon means may provide some clues to gene action under some circumstances, but must be interpreted with respect to known or likely genetic characteristics of the specific taxa of interest.

Over the past decade, significant new molecular genetic and quantitative genetic methods have provided new tools for examining gene architecture in artificial hybrid Chapter 2 - 81 populations. While molecular genetic tools have provided very useful characterisations of QTL and their effects, their ability to estimate gene action at individual loci is still in development (eg Knott et al. 1997, Sewell et al. 2000). Additionally, these methods are typically very expensive, and require unconventional experimental materials at a mature age (eg Grattapaglia et al. 1995).

A new variety of biometrical methods modelling gene action at the level of the individual locus (eg Li and Wu 1996; Pong-Wong et al. 1998; Wu et al. in press), provide methods of mining existing quantitative genetic datasets to obtain estimates of gene action and the distribution of QTL effects. These methods vary in their assumptions and in the analytical techniques used, and the field is in the early stages of its development. Pong-Wong et al. (1998) use a Bayesian approach to estimate additive and dominance genetic variance using a finite locus model, with genetic assumptions appropriate to intraspecific populations of livestock (only two allelic variants possible at each locus, adjustments allowed for inbreeding/complex pedigrees). The models developed by Li and Wu (1996) and Wu and Li (1999) are the first finite locus models specifically developed for interspecific forest tree hybrids, incorporating the assumption of possible different allelic systems between the parental populations suggested by molecular genetic studies of tree populations (eg Hamrick et al. 1979; Ledig 1986; Stokoe et al. 2000). Results from preliminary application of the model to hybrid poplar (Li and Wu 1996) corresponded well with an investigation of material from the same dataset using molecular genetic techniques (Bradshaw and Stettler 1995). This model has the potential to provide estimates of gene action in tree hybrid populations, but awaits data from progeny tests involving full-sib crosses of hybrids and pure species of common ancestry, and trials additionally incorporating clonal replication of individuals-within-families (Li and Wu 1996; Wu and Li 1999, respectively).

2.4.4 Summary

In general, the genetic characteristics of large populations of outcrossing tree species and hybrids between them: high degree of polymorphism, large additive genetic variation and polygenic control of economically important traits, are likely to result in less heterosis, less segregation and less hybrid breakdown than is commonly found in hybrids of less genetically diverse organisms such as intensively bred crops and Chapter 2 - 82 inbred trees. These characteristics suggest potential breeding strategies advanced generation hybridisation to introgress tree populations complementary characteristics, accumulate genetic gains through selection for additive and additive-related epistatic gene effects. However, the type of gene action governing heterosis has been known to vary between taxa (eg Cockerham and Zeng 1996, pseudo-overdominance; Yu et al. 1999, epistasis; Li and Wu 1996, overdominance; Pooni et al. 1994, dominance) and needs to be investigated to provide a sound theoretical premise on which to base breeding strategy recommendations. The capacity of advanced generation hybridisation strategies to make positive genetic gains is likely to be strongly influenced by the types of gene action contributing to hybrid performance. Where dominance-related gene action is important, AGH strategies are likely to be inappropriate, and more expensive strategies such as RRS selecting directly for hybrid performance may be necessary. Recently developed approaches to determining gene action in interspecific hybrids may provide a useful practical framework for breeding strategy decisions in hybrid trees, but must incorporate realistic genetic assumptions appropriate to the particular genetic characteristics of tree populations. Epistatic effects such as epistasis-related hybrid inviability and hybrid breakdown due to co-adapted gene complexes violate assumptions of negligible segregation distortion and single-locus theory, respectively, and provide serious challenges to both quantitative and molecular genetics in hybrid breeding.

2.5 Conclusion

Hybridisation has the potential to make a strong and continuing contribution to tree improvement in many taxa. This review has discussed the general background of hybrid improvement, reviewing some general knowledge of hybrids, highlighting current impediments to progress, and suggesting analyses to advance understanding of hybrid trees. A re-iteration of the key points sets the context for the remainder of this work: 1. The basis of hybrid superiority needs to be well defined, to justify the use of the hybrid in favour of competing taxa. Wood properties appear to be more predictable than growth and most other economically important traits in many cases, yet with the growing importance of

wood properties, assessment is critical. F2 and other outcrossed Chapter 2 - 83 advanced generation shown promise of use forest tree breeding general warrants more systematic

2. Selection theory available to hybrid breeders is limited, due to violation of the conventional genetic model developed by Fisher (1918), on which most pure species breeding is based. The meaning of conventional concepts of additive and non-additive genetic variance is unclear in hybrid populations, and they have been shown to be misleading when applied to forward selection. Useful statistical

parameters for recurrent F 1 hybrid breeding include the pure-hybrid genetic correlation, family heritabilities in the pure species and hybrid, the relative importance of GCA and SCA variance in the hybrid, and genetic correlations among traits and across sites. 3. The utility of multivariate and longitudinal data analysis has not been well explored in tree improvement. These analyses may be more appropriate than conventional univariate methods in the case of wood traits, where changes the through the stem are likely to be of interest. However, these methods are less flexible for indexing wood variation than many univariate methods, and are only likely to be preferable under specific circumstances, not often met in wood densitometry studies. 4. The feasibility of alternative breeding strategies for hybrids needs to be investigated, with the aim of reducing the cost of recurrent improvement. While the genetic architecture of hybrid populations appears to be generally conducive to the low-cost option of advanced generation hybridisation, study of the modes of gene action at the level of the individual locus, or genetic factor, is necessary to determine the

influence of dominance gene action on F1 hybrid performance.

Chapters 3, 4&5, and 6 will address the issues raised in Sections 1, 2&3, and 4 above, respectively, in Pinus elliottii var. elliottii X Pinus caribaea var. hondurensis hybrids in south-east Queensland, Australia.