Foto: Skogforsk
Tomas Funda
EMG postdoc seminar, Nov 6, 2013
Tree Improvement scheme
www.for.gov.bc.ca/hre/forgen
Tree Improvement scheme
 Phenotypic selection
www.for.gov.bc.ca/hre/forgen
Tree Improvement scheme
 Evaluation of genetic quality
www.for.gov.bc.ca/hre/forgen
www.for.gov.bc.ca/hre/forgen
Tree Improvement scheme
 Seed orchard establishment
www.for.gov.bc.ca/hre/forgen
Seed orchards
Artificial populations of genetically superior trees
Mass production of improved seed
Assumptions
• Random mating
• No gene flow from outside
Seed orchard crops’ quality
Genetic gain
 Parental breeding values
 Reproductive success
 Pollen contamination
 Inbreeding depression
<http://www.gowanuslounge.com>
Genetic diversity
 Number of parents involved in
mating (including contamination)
 Reproductive success
 Co-ancestry
 Inbreeding
Foto: John Marshall
Seed supply from seed orchards in Sweden
Overall 71%
Norway spruce 62%
Scots pine 83%
Newly established seed orchard
Site after preparation
for new seed orchard
Mature seed orchard
Pollen contamination
Reduces genetic gain
May cause maladaptation of progenies
www.psmicrographs.co.uk
Scots pine pollen grain
More than 50% pollen
contamination detected in Swedish
seed orchards
Foto: Kalamalka Forestry Centre, B.C.
Reducing pollen contamination
Several methods tested





pollen dilution zones
buffer stands
larger orchards
supplemental pollination
phenological isolation
Foto: Kalamalka Forestry Centre, B.C.
• bloom delay (cooling by water mist)
• different geographic region
Generally low success!
Foto: Western Forest Products Ltd. B.C.
Isolation tents
Scots pine seed orchard “Västerhus”
 ca 15 km east of Örnsköldsvik
 established 1991
 13.7 ha
 28 replicated parents
Västerhus seed orchard‘s location
Foto: Skogforsk
Foto: Skogforsk
Isolation tents
Scots pine seed orchard “Västerhus”
 ca 15 km east of Örnsköldsvik
 established 1991
 13.7 ha
 28 replicated parents
Target area for Västerhus seed
© Google
Isolation tents – design
10 experimental units
6 tents (3 treatments × 2 replicates)
tent (no further treatment)
tent + fan
tent + supplemental pollination
Foto: Skogforsk
Tent + fan
Foto: Skogforsk
Tent + supplemental pollination
4 controls
close to tents (centre)
at the edges
Provided by Skogforsk
Isolation tents – design
One ramet of 4 parents (AC3056, Y3012, Y3014 and Z2081)
sampled in each experimental unit
Extra pollen from 5 parents (AC1006, AC4221, Z3029, Z4003, Z4022)
applied via SMP in two tents
1:1 C
1:3 T+F
1:4 T+P
1:2 T
2:3 T+F
2:2 T
2:1 C
2:4 T+P
C_26 C
C_103 C
1 AC3056
14 Y3014
27 AC3056
40 Y4103
52 Y3012
64 AC3015
77 Y3014
90 AC3056
103 AC3040
131 Z2081
2 Z2081
15 Y3012
28 Z2081
41 AC3065
53 AC3056
65 AC3056
78
91 Z2081
104 Z2081
132 Y3012
3 Y3014
16 AC3056
29 Y3012
42 Z2081
54 Y4103
66 Y3012
79 AC3056
92 Y3014
105 AC3056
133 Y3014
4 AC1075
17
30 AC3056
43 Y3014
55 Z4032
67 Z2081
80
93
106 Y3014
134 Z2081
5 AC3056
18 AC3065
31 Z4032
44 Y3012
56 AC3056
68 Y3014
81 Z2081
94 Y3012
107 Y3012
135 AC3056
6 Y3014
19 AC3040
32 Y3014
45
57 Y3012
69 Y3012
82 Y3014
95 AC3040
108 AC3056
136 Y3012
7 Z2081
20 AC1075
33 AC3056
46 Z2081
58 AC3040
70 AC3056
83 Y3012
96 AC3015
109 Z2081
137 AC3040
8 AC3056
21 AC3056
34 Y3012
47 Y3014
59 AC3015
71 AC3056
84 AC3056
97 AC3056
110 Y4103
138 AC3065
9 Y3012
22 Z2081
35 Z2081
48 Y3012
60
72 AC1075
85 AC3040
98 Y3012
111 AC3056
139
10 AC3040
23 AC3040
36 Y3014
49 AC3056
61 Z2081
73 Y3014
86 Y3012
99 Z2081
112 Y3014
140 Y4103
11 AC3015
24 Y3012
37 Y3012
50 Z4032
62 AC3056
74 Z2081
87
100 Y4103
113 Z2081
141 AC1075
12 Y3012
25 AC3056
38
51 AC3040
63 Y3014
75 Z2081
88 AC3015
101 AC1075
114 AC3056
142 Z2081
13 AC3056
26 Z4032
39 Z2081
144 AC3056
76 Y3014
89 Z2081
102
115 Y3012
143 Y3012
-
Questions
Do the plastic tents effectively isolate trees from background pollen sources?
What is the effect of each treatment on genetic diversity of resulting seed crops?
Does the limited number of fathers in tents result in higher rates of self-fertilization?
selfing
pollen contamination
pollen leak
Foto: Skogforsk
Foto: Skogforsk
Parentage determination
Seed collection & extraction
• 1408 seeds analyzed
• 9 microsatellite markers
• combined multilocus
exclusion probability 0.9994
Germination
DNA isolation
Microsatellite genotyping
Simple paternity exclusion
Not complete match
with any candidate father
pollen contamination
Complete match
with a candidate father
within-orchard mating
Father the same as mother
self-fertilization
Father different from
mother – outcrossing
Pollen contamination in tents
Experimental unit Contaminating
/ Treatment
seeds count
Number of
analyzed seeds
Pollen
contamination [%]
1.2 T
0
140
0.0
2.2 T
0
124
0.0
1.3 T+F
0
143
0.0
2.3 T+F
0
150
0.0
1.4 T+P
0
146
0.0
2.4 T+P
0
160
0.0
Tents
0
863
0.0
1.1 C
6
142
4.2
2.1 C
6
146
4.1
C_26 C
8
129
6.2
C_103 C
8
128
6.3
Controls
28
545
5.1
× ×
pollen contamination
Pollen leak into tents
Experimental unit
/ Treatment
Pollen leak
count
Total
analyzed seeds
Pollen leak [%]
1.2 T
0
140
0.0
2.2 T
0
124
0.0
1.3 T+F
1
143
0.7
2.3 T+F
0
150
0.0
1.4 T+P
2
146
1.4
2.4 T+P
1
160
0.6
Tents
4
863
0.5
pollen leak
Pollen contamination
Experimental Contamin.
unit /
seeds
Treatment
count
Total
analyzed
seeds
Pollen
contam.
[%]
Pollen leak
Experimental
Total
Pollen leak
Pollen leak
unit /
analyzed
count
[%]
Treatment
seeds
1.2 T
0
140
0.0
1.2 T
0
140
0.0
2.2 T
0
124
0.0
2.2 T
0
124
0.0
1.3 T+F
0
143
0.0
1.3 T+F
1
143
0.7
2.3 T+F
0
150
0.0
2.3 T+F
0
150
0.0
1.4 T+P
0
146
0.0
1.4 T+P
2
146
1.4
2.4 T+P
0
160
0.0
2.4 T+P
1
160
0.6
Tents
0
863
0.0
Tents
4
863
0.5
1.1 C
6
142
4.2
2.1 C
6
146
4.1
C_26 C
8
129
6.2
C_103 C
8
128
6.3
28
545
5.1
Temperature sum above +5°C
Controls
Tents open
500
400
Control
Control
Tunnel
Tent
300
Tents closed
200
100
0
1-May
11-May 21-May 31-May
10-Jun
20-Jun
30-Jun
Heat sum accumulation tent vs. control
Pollen contamination in controls
Experimental unit Contaminating
/ Treatment
seeds count
Total analyzed
seeds
Pollen
contamination [%]
1.2 T
0
140
0.0
2.2 T
0
124
0.0
1.3 T+F
0
143
0.0
2.3 T+F
0
150
0.0
1.4 T+P
0
146
0.0
2.4 T+P
0
160
0.0
Tents
0
863
0.0
1.1 C
6
142
4.2
2.1 C
6
146
4.1
C_26 C
8
129
6.2
C_103 C
8
128
6.3
Controls
28
545
5.1
Pollen contamination in controls
Experimental unit Contaminating
/ Treatment
seeds count
Total analyzed
seeds
Pollen
contamination [%]
1.2 T
0
140
0.0
2.2 T
0
124
0.0
1.3 T+F
0
143
0.0
2.3 T+F
0
150
0.0
1.4 T+P
0
146
0.0
2.4 T+P
0
160
0.0
Tents
0
863
0.0
1.1 C
6
142
4.2
2.1 C
6
146
4.1
C_26 C
8
129
6.2
C_103 C
8
128
6.3
Controls
28
545
5.1
Genetic diversity of seed crops
30
25
Colored: Effective number of
fathers
20
White: Number of available
fathers (census)
15
10
5
0
Genetic diversity of seed crops
30
25
Colored: Effective number of
fathers
20
White: Number of available
fathers (census)
15
10
5
0
Foto: Skogforsk
Tent
AC1006 AC4221
Z3029
Z4003
Z4022
Total SMP
Total
SMP success
analyzed
146
56.2
1.4 T+P
9
14
15
23
21
82
2.4 T+P
23
20
12
17
29
101
160
63.1
Total
32
34
27
40
50
183
306
59.8
Selfing
Fertilization of ovules by pollen from the same tree
May lead to inbreeding depression 
reduction in economic value of forests (timber production, …)
Selfing
Factors affecting selfing
Distance between replicates of the same parent
Number of parents
Fecundity
Parental representation
Reproductive phenology
…
Lstiburek and El-Kassaby (2010)
Minimum inbreeding seed orchard design
Selfing
Treatment
Count of
selfed seeds
Analyzed
seeds
Selfing [%]
1.2 T
15
140
10.7
2.2 T
43
124
34.7
1.3 T+F
31
143
21.7
Y3012
2:3 T+F parent
composition
2.3 T+F
51
150
34.0
AC3056
Tents
140
557
25.1
Y4103
1.4 T+P
14
146
9.6
Z4032
2.4 T+P
12
160
7.5
Tents SMP
26
306
8.5
All tents
166
863
19.2
1.1 C
2
142
1.4
2.1 C
2
146
1.4
Z2081
C_26 C
12
129
9.3
AC3056
C_103 C
6
128
4.7
Y3014
545
4.0
AC3056
Controls
22
AC3056
Y3012
AC3040
AC3015
×
Foto: Skogforsk
Summary / Conclusion
 Too early to make a conclusion …
 The isolation tents fully eliminated pollen contamination from the studied
Scots pine seed orchard
 The effective number of father (Ne) was very low in the tents
 Selfing was too high in the tents
 Supplemental mass pollination (SMP) substantially improved seed crops
in terms of both Ne and selfing
© Google
Foto: Skogforsk
Next steps
 Seed crop from 2012 and possibly 2013
 Fine scale assessment of mating dynamics in the tents
 Cost-benefit evaluation of the tent isolation method
Foto: Anders Fries
Xiao-Ru Wang
Curt Almqvist
Ulfstand Wennström
Bengt Andersson
Jin Pan
Baosheng Wang
Takeshi Torimaru
Financial support
Föreningen Skogsträdsförädling
Summary / Conclusion
 Too early to make a conclusion …
 The isolation tents fully eliminated pollen contamination from the studied
Scots pine seed orchard
 The effective number of father (Ne) was very low in the tents
 Selfing was too high in the tents
 Supplemental mass pollination (SMP) substantially improved tent seed crops
in terms of both Ne and selfing
© Google
Foto: Skogforsk