Impact of selection and breeding on the genetic ... - Springer Link

Biodiversity and Conservation 5, 795--813 (1996)

Impact of selection and breeding on the genetic diversity in Douglas-fir YOUSRY A. EL-KASSABY Pacific Forest Products Limited, Saanich Forestry Centre, Saanichton, BC, V8M 1K1 and Faculty of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada KERMIT RITLAND Department of Botany, University of Toronto, Ontario, M5S 3B2, Canada Received 7 August 1994; revised and accepted 4 May 1995 Genetic changes following domestication of Douglas-fir were studied using isozyme data derived from two generations of seed orchards and their 49 wild progenitor populations. In addition, the breeding, production, and infusion populations used in the seed orchards were compared to their wild counterparts. Several parameters of gene diversity were measured (number of alleles per locus Na, per cent of polymorphic loci PLP, and expected heterozygosityH, and population divergence D). These measures were similar or higher in the domesticated populations compared to their natural progenitors, indicating that early selection and breeding of a highly polymorphic species does not significantly reduce genetic variation. The two generations of seed orchards also did not differ, indicating that genetic variation may remain stable over future generations of forest plantations. Interestingly, compared to the natural populations, heterozygosity was higher in the seed orchards, probably due to pooling of widely distributed natural populations; however, rare localized or private alleles seemed to be less frequent in the domesticated populations. Differentiation values were not significant between the first generation orchards and the natural populations, but significant differences were observed between the second generation orchards and the wild progenitor populations, probably due to the interbreeding that forms the advanced generation seed orchards. Keywords: genetic diversity; Douglas-fir; isozymes;natural breeding; production populations

Introduction

The genetic structure of natural forests has evolved over many generations via the processes of selection, migration, mutation, and random genetic drift. In many commercial species, this genetic organization will be altered by regeneration and tree breeding programmes, which involve the production of seedlings using cultivated populations termed seed orchards. By the year 2000, seed orchards in British Columbia are expected to produce about two-thirds of the 180 million seedlings used each year in the British Columbia provincial artificial regeneration programme (Cuthbert, 1992). However, the breeding process may reduce the genetic base of natural populations (Francis, 1981); examples of agricultural crop plant domestication have illustrated this process well (Brown and Clegg, 1983; Ellstrand and Marshall, 1985). Monitoring of the genetic diversity in tree breeding and production populations is needed, not only for gauging the impacts of breeding and production activities on reforestation stock, but also for effective genetic conservation of existing natural populations (Millar and Marshall, 1991; EI-Kassaby and Namkoong, 1994; EI-Kassaby et al., 1994). 0960-3115 © 1996Chapman& Hall

796

EI-Kassaby and Ritland

Forest tree improvement programmes, by their very nature, create bottlenecks of population size (Burdon, 1988). In coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), 800 parent trees were selected for the British Columbia low-elevation breeding programme from natural populations with aggregate size perhaps of order 1 0 9 . From these, 373 parent trees were selected for the breeding and testing programme and 465 were incorporated in the 12 first-generation seed orchards. The early results from breeding and testing efforts have been used to rogue (thin) the first-generation seed orchards, further reducing the number of parent trees within orchards. Eventually, most of the firstgeneration seed orchards will be phased out, and at present, four second-generation seed orchards with 223 parents have been established and are planned to provide all seed for planting in this zone. Tree breeders are aware of the population bottlenecks involved in this process, and often introduce new trees (infuse) into breeding populations as a means of enriching the genetic base. This process is illustrated in Fig. 1. In this paper, levels of isozyme genetic diversity and their changes are inferred in domesticated populations of low-elevation, coastal Douglas-fir in British Columbia. We expect progressively reduced diversity from wild populations to breeding populations to production (seed orchards) populations (genetic diversity in wild populations was described in E1-Kassaby and Ritland, 1996). We study this process on two levels: (i) the strategic (involving breeding, production, and infusion populations) and (ii) the tactical (involving operational seed production populations, also known as seed orchards). The maintenance of genetic diversity in breeding and production populations of commercially valuable species is a priority of all breeding programmes, but has seldom been verified by direct genetic assays of levels of variation. However, the problems with identifying rare alleles and the general limitations of isozymes for characterizing genetic variation will limit our inferences. Materials and methods

Origin of populations and their sampling Six groups of populations were included in this study. The relationship among these populations and the effect of breeding and testing on the genetic structure of the seed orchards are illustrated in Fig. 1. These groups were: (i) Natural populations (NPs), consisting of 49 Douglas-fir natural populations (see E1-Kassaby and Ritland, 1996 for more information). The observed levels of genetic diversity in these populations provided a 'bench mark' for comparing diversity between wild and domesticated populations. (ii) Breeding population (BP), consisting of 373 parent trees from the Province of British Columbia Douglas-fir breeding and testing programme (Heaman, 1985). Subsets of these trees were included in several first-generation seed orchards. (iii) Production population (PP), of size 465, consisting of the 373 trees in the breeding population plus 92 parent trees not included in the breeding and testing programme. These 465 trees represent all the parent trees used in the establishment of the 12 first-generation seed orchards in British Columbia (see below). (iv) Infusion population (IP), consisting of 66 parent trees tested in provenance trials. (v) First-generation seed orchards (1GSOs), consisting of 12 seed orchards (see Table 1, for their sizes). Since all 12 seed orchards were derived from the production population (of size 465), overlap in the genetic material is expected to occur. The genetic

Selection, breeding and genetic diversity in Douglas-fir

797

structure of these seed orchards have been altered either by genetic roguing (removal of selected parents) or expansion (Table 1 and Fig. 1). (vi) Second-generation seed orchards (2GSOs), consisting of four seed orchards. Their parental contribution is either through forward or backward selections from the breeding population (orchards 2a, b, d, Table 1) or from additional selections made from material tested outside the breeding population (orchard 2c, Table 1). Dormant vegetative buds were collected from 373 parent trees representing the BP and 66 trees representing the IP (Fig. 1). The geographic range of the parent trees is highlighted in (Fig. 1; E1-Kassaby and Ritland, 1996). Clonal material for the parent trees are grown in the British Columbia Ministry of Forests' Douglas-fir clonal archives at Mesachie Lake Research Station (Cowichan Lake, BC). Twelve, first-generation seed orchards (1GSOs) were included in this study (identified as 1-12). The original establishment reports of these 12 orchards were obtained from various governmental and/or coastal forest industries. Dormant vegetative buds were collected from the various orchard sites or from the British Columbia Ministry of Forests' Douglas-fir clonal archives. The number of parent trees per seed orchard ranged from 53 to 129 (Table 1). Four 2GSOs (identified as 2a-d) were sampled similarly, the number of parent trees ranging from 40 to 85 (Table 1). The early information generated from the breeding and testing programme was used by seed orchardists to upgrade the genetic quality of their seed orchards (i.e. genetic roguing). Thus the number of parent trees in most of these first-generation seed orchards have changed (Table 1). Following this, a list of original (i.e. before roguing) and updated (i.e. after roguing) parent trees was made for each seed orchard.

lsozyme analysis of populations The genotype of every tree was determined for a total of 20 allozyme loci using the methods described in E1-Kassaby and Ritland (1996). These allozyme loci were: phosphoglucose isomerase (PGI1, PGI2), phosphoglucomutase (PGM1, PGM2), fructose diphosphatase (FDP2), 6-phosphogluconic dehydrogenase (6PG1, 6PG2), shikimate dehydrogenase (SKD1), aspartate amino-transferase (AAT1, AAT2), malate dehydrogenase (MDH1, MDH2, MDH3), leucine aminopeptidase (LAP1, LAP2), aconitase (ACO1, ACO2), isocitrate dehydrogenase (IDH), glutamate dehydrogenase (GDH), and glucose-6-phosphate dehydrogenase (G6P). Gene and genotypic frequencies were compiled for each of the BP, PP, and IP, and for each of the 12 1GSOs before and after genetic roguing, as well as the four 2GSOs. Data for the 49 natural populations was obtained from E1-Kassaby and Ritland (1996). For each population and/or seed orchard, the following was then calculated: (i) the proportion of polymorphic loci (PLP) for the 95% criterion, (ii) the average number of alleles per locus Na, (iii) the expected heterozygosity H and (iv) population divergence D, equivalent to Wright's (1965) Fsr (the 'population divergence' D is defined as the excess homozygosity in a population caused by genetic divergence via drift and selection from a larger, 'reference' population; see E1-Kassaby and Ritland, 1996). In addition, Nei's gene diversity statistics (Nei, 1973) were computed for the 12 1GSOs before and after genetic roguing, and the four 2GSOs. These were compared to those obtained for the 49 NPs studied by EI-Kassaby and Ritland (1996). The SE of heterozygosity was found using the methods of Nei and Roychoudhury (1974). To portray the structure of genetic relatedness among the 12 1GSOs both before and after genetic roguing, the four 2GSOs, and the 49 NPs, principal

798


components analysis (PCA) was performed on the matrix of inferred relationship among all 77 populations (for more information see E1-Kassaby and Ritland, 1996). Results and discussion

Natural breeding, production, and infusion populations Average allelic frequencies of the 49 NPs, BP, PP, and IP are presented in Table 2. The number of polymorphic loci varied slightly among these groups of populations, with 17 for the NPs and BP, 18 for the PP, and 15 for the IP (Table 2). The loss and gain of alleles in these populations is important in assessing the impact of breeding and selection. Loss of alleles is expected due to the reductionist approach of breeding, and a gain of alleles could result from the inclusion of new parent trees in the PP and IP. The difference in the sampling breadth between the 49 NPs, the 373 and the 465 trees in PP could also result in gain of alleles. This is because, although the sampling of natural populations was extensive, it is usually limited to a relatively small area, such that trees sampled for breeding may include other alleles not originally sampled. This is also concordant with the observed clustering of similar genotypes within natural forest tree populations (Brunel and Rodolphe, 1985; E1-Kassaby et al., 1987). Following Marshall and Brown (1975), we classified alleles as either common (gene frequency greater than 0.05) or rare (less than 0.05). In addition, we classified alleles as either widespread (found in more than one natural population), or local (found in few of the 49 NPs), or private (found in only one of the 49 NPs). The loss of alleles from the NPs to BP and PP and the gain of alleles from the IP or the inclusion of untested parent trees in the PP occurred as follows with regards to their being common/rare and widespread/local: (i) Loss from the BP, PP, and IP: five alleles (IDH-7, PGM1-5, MDH1-3, MDHI-4, and AAT1-2); all were rare and either local or private. (ii) Loss from the BP and PP but recovered in IP: two rare and local alleles (FEST2-3 and MDH3-3), (iii) Gain: two alleles (MDH2-2 and LAP2-3) were detected in the BP and PP bul were not present in both the NPs or IP. In summary, five alleles were lost but two restored through the infusion; all these alleles belonged to the rare and local class (Table 2). Table 3 shows that the average number of alleles per locus, N,, showed a steady increase from the 49 NPs (2.14) to the BP (2.50) to the PP (2.65). N ofor the IP (2.30) was lower than that observed for the BP and PP, but higher than that observed for the NPs (Table 3). The observed increase in the N~, between the BP and PP is caused by the inclusion of the 92 additional parent trees excluded from the breeding programme. The observed range of N,, for these four populations (2.14-2.65) was higher than that reported for gymnosperms (1.83 _+ 0.58), temperate-zone species (1.81 +_ 0.06), and species with wind-pollinated outcrossing mating system (1.84 + 0.05) (Hamrick et al., 1992). The proportion of polymorphic loci by the 95% criterion, PLPg~, was lower in the NPs than in the BP, PP, and IP (53 vs. 65) (Table 3). In general, the PLP~ in all populations is similar to that reported for gymnosperms (53.4 _+2.4), temperate-zone species (49.2 _+2.2 ). and species with wind-pollinated outcrossing mating system (53.0 +_ 2.2) (Hamrick et al., 1992). Table 4 indicates no significant differences among the NPs, BP. PP. and IP for expected

Selec~on, breedingandgeneticdive~i~in Doug~s~r RECURRENT

SELECTION

BREEDING

799

PROGRAM

PRODUCTION

]

NATURAL STANDS

a,~] I st

GENERATION SEED ORCHARDS

[ [

P'-I PLANTATIONS 1

/ / / / SELECTION

TESTING

/

Ist GENERATION ROGUED SEED ORCHARDS

.._1 PLANTATIONS ,=,,..--i

]

2nd GENERATION SEED ORCHARDS

~'~=,,.~[PLANTATIONS

]

BREEDING

J"-] SELECTION

TESTING

BREEDING

Y

Figure 1. Diagram depicting the Douglas-fir tree breeding programme showing the relationship between natural populations and first- and second-generation seed orchards as well as the infusion population. heterozygosity, H. This parameter ranged from 0.151 - 0.041 (IP) to 0.176 _+ 0.038 (BP). Thus the heterozygosity present in NPs was retained during the early stages of the breeding process of the species. Also, these H values, which range from 0.151 to 0.176, are similar to those found in gymnosperms (0.151 _ 0.008), temperate-zone species (0.166 ___0.008), and species with wind-pollinated outcrossing mating system (0.173 _ 0.007) (Hamrick et al., 1992). In addition, all three diversity statistics are comparable to those reported from

El-Kassaby and Ritland

800

Table 1. The number of parent trees included in the 12 first-generation seed orchards before and after roguing as well as the four second-generation seed orchards Before roguing After roguing First-generation seed orchards Orchard I 71 Orchard 2 74 Orchard 3 S7 Orchard 41 64 Orchard 5 54 Orchard 6 08 Orchard 7 71 Orchard 8 08 Orchard 9 129 Orchard tO 53 Orchard 11 114 Orchard 12 ~3

45 72 85 94 35 58 58 63 68 37 54 62

Second-generation seed orchards Orchard 2a 43 Orchard 2b 55 Orchard 2c: 85 Orchard 2d 40 ~Theno. of parent trees has increased due to orchard expansion. "This orchard includes tested parent trees from outside the breeding population.

studies of other Douglas-fir natural populations, which found PLP95 in the range 45-82%, N, in the range 1.8-2.5, and H in the range 0.155-0.191 (Yeh and O'Malley, 1980: E1-Kassaby and Sziklai, 1982: Markel and Adams, 1987: Li and Adams, 1989: Moran and Adams, 1989). The estimates of population divergence (D) for the four groups of populations (NPs, BP~ PP, and IP) ranged between 0.032 _+ 0.015 (PP), 0.061 _+ 0.025 (BP), and were nol significantly different from each other (Table 4). The changes in the D from one population to another were small and non-significant, indicating that early selection and breeding did not cause any measurable genetic divergence among the four populations. In addition, the infusion of new parents to the BP did not cause any significant increase of genetic divergence (Table 4).

Seed orchards Evaluation of changes of diversity parameters of the 1GSOs and 2GSOs with that observed from the 49 NPs is important, since the seed orchards contribute substantially to the artificial regeneration programmes, and thus can ultimately be a major source of genetic material in future forests. Established seed orchards have produced seed for reforestation for a substantial n u m b e r of years. It is estimated that the productive life expectancy of a Douglas-fir seed orchard is about 20 years (Y.A. E1-Kassaby, personal


801

Table 2. Allelic frequencies for the 49 natural populations, breeding, production, and infusion populations Allele

Natural

Breeding

Production

Infusion

FEST2-1 FEST2-2 FEST2-3 IDH-1 IDH-2 IDH-3 IDH-5 IDH-7 PGMI-1 PGM1-2 PGM1-3 PGM1-5 PGM2-1 PGM2-2 PGM2-3 PGM2-4 PGI1 PGI2-1 PGI2-2 PGI2-3 PGI2-5 6PGI-1 6PG 1-2 6PG1-3 6PG2-1 6PG2-2 6PG2-3 GDH G6P-1 G6P-2 G6P-3 G6P-5 SKDI-1 SKD1-2 SKD1-3 MDHI-1 M D H 1-2 MDH1-3 MDH1-4 MDH2-1 MDH2-2 MDH2-3 MDH3-1 MDH3-2 MDH3-3 MDH3-5 LAP1-1 LAP1-3 LAP2-1 LAP2-3 ACOI-1 ACO1-3 ACO2-1 ACO2-2 ACO2-3 AATI-1 AAT1-2 AAT1-3 AAT2-1 AAT2-2 AAT2-3

0.978 0.021 0.001 0.801 0.095 0.044 0.057 0.003 0.879 0.055 0.066 0.001 0.901 0.073 0.026 0.0001 1.000 0.877 0.117 0.003 0.003 0.901 0.033 0.066 0.939 0.059 0.002 1.000 0.550 0.427 0.011 0.011 0.657 0.134 0.209 0.838 0.161 0.0001 0,0001 0.959 0.000 0.041 0.874 0.011 0.001 0.114 0.932 0.068 1.000 0.000 0.969 0.031 0.963 0.036 0.001 0.991 0.001 0.009 0.862 0.120 0.018

1,000 0,000 0,000 0.839 0.090 0.056 0.015 0.000 0.849 0.075 0.076 0.000 0.920 0.071 0.009 0.000 1.080 0.807 0.190 0.003 0.000 0.928 0.023 0.050 0.893 0.106 0.001 1.000 0.544 0.446 0.008 0.001 0.739 0.146 0.115 0.992 0.008 0.000 0.000 0.962 0.008 0.029 0.886 0.007 0.000 0.107 0.807 0.193 0.995 0.005 0.961 0.039 0.870 0.125 0.005 0.999 0.000 0.001 0.805 0.139 0.055

0.997 0.003 0.000 0.826 0.089 0.051 0.034 0.000 0.854 0.068 0,078 0.000 0,905 0,078 0.014 0.002 1.000 0.825 0.172 0.002 0.001 0.912 0.024 0.065 0.904 0.095 0.001 1.000 0.561 0.428 0.006 0.004 0.672 0.156 0.172 0.989 0.011 0.000 0.000 0.965 0.004 0.031 0.895 0.006 0.000 0.099 0.841 0.159 0.996 0.004 0.967 0.033 0.910 0.086 0.004 0.998 0.000 0,002 0.834 0.129 0.037

0.977 0.015 0.008 0.833 0.159 0.008 0.000 0.000 0.864 0.098 0.038 0.000 0.879 0.106 0.015 0.000 1.000 0.879 0.106 0.008 0.008 0.924 0.030 0.045 0.947 0,045 0,008 1,000 0,508 0.424 0.000 0.068 0.735 0.121 0.144 0.992 0.008 0.000 0.000 0.947 0.000 0.053 0.765 0.000 0.008 0.227 0.924 0.076 1.000 0.000 0.985 0.015 1.000 0.000 0.000 1.000 0.000 0.000 0.909 0.045 0.045

iPrivate alleles with frequency of 0.011, 0.010, and 0.010 were detected for PGM2-4, MDHI-3, and MDH1-4, respectively.


802 Table 3. Proportion of polymorphic loci (95% criterion) and number of alleles per Locus in the breeding, production, and infusion populations Population

PLP~5

N,,

Natural Breeding Production Infusion

0.53 0.65 0.65 0.65

2.14 2.50 2.65 2.30

experience). Early results from the breeding and testing of tree improvement activities lead to roguing thereby influencing seed orchards' genetic structure during their productive lifetime (Fig. 1). Table 5 gives the average allelic frequencies of the 49 NPs (EI-Kassaby and Ritland, 1996), 1GSOs before and after genetic roguing, and the 2GSOs. The number of polymorphic loci varied slightly among the NPs (17), 1GSOs before (18) and after (18) genetic roguing, and the 2GSOs (17), as in discussing Table 2, we classified alleles as common vs rare, and widespread vs local. Losses of alleles from the NPs to seed orchards were observed to occur as follows (see Table 5 and Fig. 2): (i) loss from all seed orchards: four rare and local alleles (FEST2-3, PGM1-5, MDH1-3, MDH3-3); two rare and private alleles (AAT1-2, MDH1-4). (ii) loss from 1GSOs before and after genetic roguing but recovered in 2GSOs: one rare and local allele (IDH-7). (iii) loss from 1GSOs due to genetic roguing: one rare and private allele (6PG2-3). (iv) loss from the 2GSOs: one rare and private allele (PGI2-3). In total, nine alleles were lost at some point, but only three alleles (in cases 2 and 4 above) are true allelic losses. In general, all lost alleles were rare and local in natural populations (Fig. 2). Gain of previously unobserved alleles occurred twice: the LAP2-3 allele was present in all seed Table 4. Expected heterozygosity H and genetic divergence D and their changes in the breeding, production, and infusion populations Population Natural populations b. Breeding C. Production d. Infusion Change b to a Change c to a Change d to a Change c to b Change d to b Change d to c a.

H 0.171 0.176 0.167 0.151 0.005 0.004 --0.020

sE 0.051 0.038 0.039 (I.041 0.021 0.007 0.017

D 0.042 0.061 0.032 0.059 0.019 0.010 (I.017 0.029 0.002 0.027

sE 0.012 0.025 0.015 0.026 0.021 0.012 0.(123 0.014 0.030 0.018


803

Table 5. Allele frequencies at polymorphic loci, averaged over the 49 natural populations, for first-generation seed orchards before and after genetic roguing, and second-generation seed orchards Allele FEST2-1 FEST2-2 FEST2-3 IDH-1 IDH-2 1DH-3 IDH-5 IDH-7 PGMI-1 PGM1-2 PGM1-3 PGM1-5 PGM2-1 PGM2-2 PGM2-3 PGM2-4 PGI1 PGI2-1 PGI2-2 PGI2-3 PGI2-5 6PGI-1 6PG1-2 6PG1-3 6PG2-1 6PG2-2 6PG2-3 GDH (36P-1 G6P-2 G6P-3 G6P-5 SKD1-1 SKD1-2 SKD1-3 MDH1-1 MDH1-2 MDH1-3 MDH1-4 MDH2-1 MDH2-2 MDH2-3 MDH3-1 MDH3-2 MDH3-3 MDH3-5 LAPI-1 LAP1-3 LAP2-1 LAP2-3 ACOI-1 ACO1-3 ACO2-1 ACO2-2 ACO2-3 AATI-1 AAT1-2 AAT1-3 AAT2-1 AAT2-2 AAT2-3

Natural populations 0.978 0.021 0.001 0.801 0.095 0.044 0.057 0.003 0.879 0.055 0,066 0,001 0.901 0.073 0.026 0.0001 1.000 0.877 0.117 0.003 0.003 0.901 0.033 0.066 0.939 0.059 0.002 1.000 0.550 0.427 0.011 0.011 0.657 0.134 0.209 0.838 0.161 0.0001 0.000~ 0.959 0.000 0.041 0.874 0.011 0.001 0.114 0.932 0.068 1.000 0.000 0.969 0.031 0.963 0.036 0.001 0.991 0.001 0.009 0.862 0.120 0.018

Firstgeneration

Secondgeneration

(before)

Firstgeneration (after)

0.998 0.002 0.000 0.839 0.087 0.052 0.021 0.000 0.860 0.065 0.075 0.000 0.914 0.071 0.014 0.001 1.000 0.801 0.196 0.003 0.001 0.914 0.027 0.059 0.898 0.101 0.001 1.000 0.562 0.429 0.005 0.005 0.693 0.138 0.169 0.990 0.010 0.000 0.000 0.969 0.003 0.028 0.879 0.007 0.000 0.115 0.824 0.176 0.993 0.007 0.959 0.041 0.910 0.084 0.006 0.999 0.000 0.001 0.828 0.131 0.041

0.997 0.003 0.000 0.836 0.088 0.048 0.029 0.000 0.844 0.067 0.090 0.000 0.905 0.079 0.015 0.001 1.000 0.790 0.206 0.003 0.001 0.915 0.025 0.059 0.899 0.100 0.000 1.000 0.552 0.438 0.005 0.005 0.665 0.156 0.179 0.992 0.008 0.000 0.000 0.968 0.003 0.030 0.870 0.009 0.000 0.121 0.842 0.158 0.993 0.007 0.958 0.042 0.933 0.062 0.005 0.999 0.000 0.001 0.842 0.121 0.037

0.986 0.014 0.000 0.819 0.084 0.037 0.058 0.002 0.855 0.060 0.079 0.000 0.925 0.060 0.011 0.002 1.000 0.800 0.194 0.000 0.000 0.920 0.031 0.040 0.932 0.060 0.002 1.000 0.527 0.460 0.008 0.005 0.633 0.142 0.225 0.987 0.012 0.000 0.000 0.928 0.000 0.072 0.856 0.000 0,000 0.144 0.908 0.092 0.998 0.002 0.963 0.037 0.971 0.010 0.019 1.080 0.000 0.000 0.865 0.112 0.023

1private alleles with frequency of 0.011, 0.010, and 0.010 were detected for PGM2-4, MDH1-3, and MDHI~, respectively.


804 0

o 25

~

~ eo

Natural populations First generation (before roguing) First generation (after roguing) Second generation

~ lO

6

Z

o 0.00

0.05

0.20

0.50

0.80

0.95

0.99

1.00

Frequency of allele (upper cutoff) Pigare 2. Distribution of allele frequencies at all isozyme loci, listed in eight frequency classes. Note the increased loss of rare alleles in later generations. orchards but not in any of the 49 NPs and MDH2-2 was detected in the 1GSOs but not present in either the NPs or the 2GSOs (Table 5 and Fig. 2). N a for the 49 NPs was 2.143 _+ 0.015, which differs significantly from that obtained from the studied 12 1GSOs before (2.279 +_0.032) and after genetic roguing (2.242 + 0.034) and the 2GSOs (2.250 _+ 0.020) (Table 6). This significant increase of N a between the NPs and the 1GSOs is attributed to the inclusion of parent trees not included in the original breeding and testing programme (see above). PLP95 in the NPs (52.6 _+ 1.0) differed significantly from that for the 1GSOs before (62.5 _+ 1.4) and after (60.4 _+ 1.7) roguing and did not differ from that observed for the 2GSOs (56.3 _+ 2.4) (Table 6). No significant differences were observed among the NPs and the seed orchards under the PLP~, criterion (Table 6). The three heterozygosity parameters studied (PLP, N,,, and H) did not show any extreme values when the observations were made at the individual orchard level. In general, all the heterozygosityparameters were higher than that observed for the 49 NPs as well as other studied Douglas-fir populations (Yeh and O'Malley, 1980; E1-Kassaby and Sziklai, 1982; Markel and Adams, 1987; Li and Adams, 1989: Moran and Adams, 1989). These results indicate that the genetic diversity in these seed orchard populations has been maintained. H did not differ significantly among the NPs (0.171 +_ 0.051), 1GSOs before (0.172 0.003) and after (0.173 + 0.001) genetic roguing and 2GSOs (0.163 _+ 0.010) (Table 7). H for the 1GSOs ranged from 0.161 to 0.188 (before genetic roguing), from 0.157 to 0.191 (after genetic roguing), and from 0.130 to 0.182 for the 2GSOs (Table 7). These figures indicate that the genetic diversity present in NPs was retained in all the seed orchards populations, and that the early stages of selection and breeding did not cause any erosion to the heterozygosity levels. There were no significant changes of H between (i) the NPs and 1GSOs before genetic roguing, (ii) 1GSOs before and after genetic roguing, and (iii) 1GSOs after genetic roguing and 2GSOs (Table 7). The average D values for the 49 NPs (0.042 +_0.012), 1GSOs before (0.039 +_0.017) and


805

Table 6. Proportion of polymorphic loci (95% criterion) and number of alleles per locus, in natural populations and seed orchard generations Population

PLP95

Na

A. 49 Natural populations Mean SE among populations

0.526 0.010

2.143 0.015

B. Seed orchards: first-generation before thinning Orchard 1 0.6 2.30 Orchard 2 0.7 2.30 Orchard 3 0.65 2.45 Orchard 4 0.65 2.25 Orchard 5 0.65 2.15 Orchard 6 0.65 2.10 Orchard 7 0.55 2.20 Orchard 8 0.60 2.25 Orchard 9 0.60 2.40 Orchard 10 0.55 2.30 Orchard 11 0.70 2.45 Orchard 12 0.60 2.20 Pooled orchard 0.625 2.279 SE of mean 0.014 0.032 C. Seed orchards: first-generation after thinning Orchard 1 0.55 2.20 Orchard 2 0.65 2.30 Orchard 3 0.65 2.45 Orchard 4 0.65 2.40 Orchard 5 0.65 2.10 Orchard 6 0.65 2.10 Orchard 7 0.55 2.15 Orchard 8 0.55 2.25 Orchard 9 0.55 2.30 Orchard 10 0.55 2.30 Orchard 11 0.70 2.25 Orchard 12 0.55 2.10 Pooled orchard 0.604 2.242 SE of mean 0.017 0.034 D. Seed orchards: second-generation Orchard 2a 0.50 Orchard 2b 0.55 Orchard 2c 0.60 Orchard 2d 0.60 Pooled orchard 0.563 SE of mean 0.024

2.25 2.20 2.30 2.25 2.25 0.020


806

Table 7. Heterozygosity H, genetic divergence D and their changes among generations in the seed orchards Population

H

SE

D

SE

0.051

0.042

0.012

0.039 0.038 0.036 0.037 0.033 0.032 0.035 0.036 0.035 0.034 {).034 0.034 0.035 {).0018

0.044 0.060 0.044 0.105 0.069 0.074 0.040 0.047 0.045 0.044 0.054 0.042 0.039 -0.003

0.016 0.023 0.019 0.047 0.026 {).027 0.018 0.020 0.017 0.018 0.024 0,017 0.017 0.014

0.174 0.186 0.175 0.191 0.177 0.159 0.162 0.167 0.169 0.17(I 0.183 0.157 0.173 0.001

0.040 0.039 0.036 0.039 0.036 0.031 0.036 0.036 0.036 0.036 0.036 {).036 0.036 0.003

0.047 0.058 0.046 0.050 0.086 0.079 0.040 0.049 0.039 0.045 0.059 0.045 0.033 --0.006 0.009

0.018 0.022 0.020 0.018 0.031 0.030 0.019 0.022 0.015 0.018 0.026 0.018 0.015 0.005 0.011

0.130 0.165 0.173 0.182 0.163 0.010

0.030 0.035 0.040 0.038 0.036 0.009

0.028 0.031 0.050 0.032 0.020 -0.013 --0.019 ......0.022

0.014 0.014 0.024 0.013 0.012 0.009 0.013 0.009

A. Natural populations

Average of 49 populations 0.171 B. First-generation before thinning

Orchard 1 Orchard 2 Orchard 3 Orchard 4 Orchard 5 Orchard 6 Orchard 7 Orchard 8 Orchard 9 Orchard 10 Orchard 11 Orchard 12 Pooled orchard Change A to B

0.176 0.188 0.175 0.186 0.169 0.161 0.163 0.168 0.169 0.170 0.178 0.161 0,172 0,0027

C. First-generation after thinning

Orchard 1 Orchard 2 Orchard 3 Orchard 4 Orchard 5 Orchard 6 Orchard 7 Orchard 8 Orchard 9 Orchard 10 Orchard 11 Orchard 12 Pooled orchard Change B to C Change A to C D. Second-generation Orchard 2a Orchard 2b Orchard 2c Orchard 2d Pooled orchard Change C to D Change B to D Change A to D


807

after (0.033 __- 0.015) genetic roguing, and 2GSOs (0.020 ___0.012) were not significantly different from each other, indicating lack of divergence (i.e. they are not genetically different from each other) (Table 7). The changes in the genetic divergence from the NPs to 1GSOs before and after genetic roguing were small and did not differ significantly from zero; however, a marginally significant change was observed between the NPs and the 2GSOs (Table 7). The observed significant change in genetic divergence between the NPs and the 2GSOs should be viewed as an early indication of changes that could develop due to the reduced number of parents in the advanced generation seed orchards.

Distribution of variation Brown (1978) compared the genetic organization of allozyme variation between natural plant populations and their domesticated cultivars, and found a considerable amount of reorganization. These differences were best illustrated when the Gsr values obtained for wild vs cultivated populations were compared. For example, the Gsr values for cultivated Phlox drummondii (Levin, 1976) and Raphanus sativus (Ellstrand and Marshall, 1985) were 0.78 and 0.31 while values of 0.29 and 0.15 were obtained for their respective wild populations. However, we must realize that these large differences occurred after many generations of selection. It is important to evaluate the effect of domestication on the reorganization of genetic variation in forest tree species. It is important also to evaluate the commonly-held notion that the among-population variation observed in the wild will not be maintained through seed orchard crops because it represents variation from few or single populations (E1-Kassaby and Namkoong, 1994). Recent studies on forest tree species have demonstrated that the levels of genetic diversity in seed orchards are similar to, or even higher than, those observed in natural populations (Miiller-Starck, 1987; Bergmann and Ruetz, 1991; Chaisurisri and E1-Kassaby, 1994; and the present study). The results from gene diversity analyses of the 49 NPs, the 1GSOs before and after genetic roguing, and the 2GSOs confirm this. The Gsr value for the 49 NPs was 0.045, typical of that reported for Douglas-fir and most coniferous trees (Mouna, 1989; E1-Kassaby, 1991; Hamrick et al., 1992). The Gsr values of the domesticated populations were much lower, being 0.010, 0.013, and 0.015 for the 1GSOs before and after roguing and the 2GSOs, repectively. These lower values indicate that the small (5%) amongpopulation variation observed among natural populations is not maintained through seed orchard crops. This reduction in among-population variation is expected because each domesticated population consists of pooled genetic material from natural populations. The small remaining Gsr values, on order 0.01, among the domesticated population is probably caused by the sampling of finite numbers of genotypes in the formation of seed orchards, which produces small but detectable gene frequency differences among seed orchards when sampled completely. The pattern of divergence of domesticated and natural populations can be further analysed by examining how these populations are projected onto the axes of principal components after a PC analysis of the matrix of genetic relationship. Figure 3 shows the projection of populations onto each of the first five dimensions of variation, which progressively accounted for 21%, 15%, 10%, 9%, and 8% of the total variation, respectively. Seed orchard populations tend to project into intermediate ranges, while

808


natural populations are projected over the full range, indicating a lack of divergence of domesticated populations, and a general tendency to be intermediate (Fig. 3). Conclusion

In a companion study, E1-Kassaby and Ritland (1996) characterized the genetic structure of natural Douglas-fir as complex with little structure in the pattern of genetic relationship among populations, and little correlation of relationship with physical distance. The species' wide range is continuous, with high outcrossing rates (E1-Kassaby et al., 1981: Shaw and Allard, 1982; Neale and Adams, 1985; Ritland and E1-Kassaby, 1985; Yeh and Morgan, 1987), gene flow (E1-Kassaby and Ritland, 1996), and lack of multilocus structures or linkage disequilibrium (Yeh and Morgan, 1987). The results from the present study demonstrate that Douglas-fir, unlike agricultural crop plants that have been domesticated for hundreds or thousands of generations, has not experienced any major reduction of genetic diversity due to phenotypic selection and breeding. The domesticated populations we studied are all in their early stages of domestication, so that high levels of heterozygosity are expected. Nevertheless, phenotypic selection and breeding programmes for this species in British Columbia should maintain effective gene resource management for this economically and ecologically important species. Proper breeding and testing were advocated as a viable gene conservation strategy (Namkoong, 1995). Furthermore, the dynamics of rare alleles in these domesticated populations needs further study, using different types of genetic markers. Isozymes are well known to encompass a very restricted portion of the total genome of an organism (see E1-Kassaby and Ritland, 1996), and in Douglas-fir domestication, cannot fully measure the losses of variation that occur in the initial stages of domestication. The ideal markers would lie adjacent to loci controlling physiologically and ecologically important characters, and lacking knowledge of the locations of these genes. would at least be numerous and highly polymorphic. Theoretically, the effect of random genetic drift on depleting the heterozygosity in seed orchard populations is slow, roughly equalling a rate of 1/(2Ne) per generation, for N,. is the effective population size (Savolainen and Yazdani, 1991; Savolainen and Karkkainen, 1992). In our study, the population size was ca 20(0500 individuals during each generation, so the expected change of heterozygosity is quite small, in the order of 0.1%, which is undetectable with our sample size of marker loci. This sampling of 200-500 individuals has a much larger impact on rare alleles. In a sample of N outbred, diploid individuals, the probability that an allele of frequency p is not found, e.g. is lost, is (l-p) 2N.This probability is plotted in Fig. 4 for the cases of N = 50, 250 and 500 over the range o f p from 0.0001 to 0.1. This figure shows that, generally, alleles of frequency 0.001 or less in nature have a greater than 50% chance of loss in our domesticated Douglas-fir populations, while alleles of frequency 0.01 or greater are almost certainly not lost. Our data do seem to indicate a loss of rare alleles, but considerably larger sample sizes of populations in nature are needed to firmly document the loss of rare alleles in the domesticated populations. Furthermore, the value of rare alleles, e.g. whether they represent mutations or are of adaptive significance, needs further investigation. Other factors as well have not received attention in the present study. The effect of random genetic drift on depleting the genetic variability in Douglas-fir orchards should be considered in concert with other factors such as differences in male reproductive success


8 ~ PC

1

°~

o~-,

,-~

fill

-1.00

-0.75

Natural populations First generation (before r o g u i n g ) First generation (after r o g u i n g )

IN

id

809

~ ~ ~--~ secondgeneration

dmll m,i)u,m, fl B

-0.50

-0.25

0.00

El

0.25

mB fl

0.50

0.75

~ 1.00

48P02 o, , 1!,)11,,

12

O__.r.. -1.00 or) tO

. m

.... -0.75

, . . . . . . . . . . . . . . . . . . . . . . . -0.50

-0.25

0.00

0.25

0.50

0.75

1.00

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

121 I48PC3

0 t~

6 Z

-1.00

-0.75

24iPC j1218 om!,H0 0

.......

-1.00

18

~-~-7-

-0.75

-0.50

. . . . . -0.25

0.00

m'~''

''~p . . . .

0.25

0.50

m. . . . 0.75

m' 1.00

PC5

6 0

....

~ ....

I ....

', ' ~ . . . . . .

-1.00-0.75-0.50-0.25

I '~' ' ' I . . . .

r-r-~'

; ~'

0.00 0 . 2 5 0 . 5 0 0 . 7 5 1.00

Projection onto P C axis Figure 3. Projections of orchard populations in the five dimensions of principle components, relative to natural populations (e.g. a full PCA was done with all 49 + 28 = 77 populations, and all projected on the axis). Note that orchard populations tend to fall into the intermediate ranges and the declining per cent of variation as well as scale increase across the first five dimensions.

810


1.0 ffl

0.8

\

0 o

0.6

. - , . .

0.4 2 0.2 0.0 10 .4

1

I

10 3

10 .2

10 1

Frequency p of allele in original population Figure 4. The probability that an allele of frequency p is not found, e.g. is lost, in a sample of N individuals. (Aspit et aL, 1989; Roberds et al., 1991; EI-Kassaby and Ritland, 1992; Nakamura and Wheeler, 1992), female reproductive success (EI-Kassaby et aL, 1989; Reynolds and E1-Kassaby, 1990; E1-Kassaby and Cook, 1994), as well as differences of reproductive phenology among the seed orchard parents (E1-Kassaby et al., 1988; Erickson and Adams, 1989; EI-Kassaby and Askew, 1991; Copes and Sniezko, 1991; E1-Kassaby and Davidson, 1991). These factors cause restricted mating among individuals, and thus increase the rate of random genetic drift and its effects on the depletion of the genetic variability. The effective management of genetic resources requires a thorough understanding of the biological dynamics of forest tree populations and the impact of environmental influences, natural and/or man made, on shaping present and future populations.

Acknowledgements

The authors thank S. Barnes, C. Cook, L. EI-Kassaby, D. MacLeod, and B. Newbery for technical assistance, D. Ashbee, C. Heaman, J. Woods, and C. Ying from the British Columbia Ministry of Forests' Research Branch and all the Coastal Douglas-fir seed orchards' managers for providing access to research materials. This work was supported in part by Pacific Forest Products Limited, a Science Council of British Columbia grant to Y.A.E. and a Natural Sciences and Engineering Research Council of Canada grant to K.R.

References

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811

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