Elimination of pollen contamination in Scots pine seed orchards: Is it possible?
Tomas FundaEMG postdoc seminar, Nov 6, 2013
Foto: Skogforsk
Evaluation of genetic quality
www.for.gov.bc.ca/hre/forgenwww.for.gov.bc.ca/hre/forgen
Tree Improvement scheme
Seed orchardsArtificial populations of genetically superior treesMass production of improved seed
Assumptions• Random mating• No gene flow from outside
Genetic gain Parental breeding values Reproductive success Pollen contamination Inbreeding depression
Number of parents involved in mating (including contamination) Reproductive success Co-ancestry Inbreeding
Genetic diversity <http://www.gowanuslounge.com>
Foto: John Marshall
Seed orchard crops’ quality
Seed supply from seed orchards in Sweden
Overall 71%
Norway spruce 62%Scots pine 83%
Mature seed orchard
Newly established seed orchard
Site after preparation for new seed orchard
Foto: Kalamalka Forestry Centre, B.C.
Pollen contamination
Reduces genetic gainMay cause maladaptation of progenies
More than 50% pollen contamination detected in Swedish
seed orchards
www.psmicrographs.co.uk
Scots pine pollen grain
Reducing pollen contamination
Several methods tested pollen dilution zones buffer stands larger orchards supplemental pollination phenological isolation
• bloom delay (cooling by water mist)• different geographic region
Generally low success!
Foto: Kalamalka Forestry Centre, B.C.
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: SkogforskFoto: 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
Isolation tents – design
10 experimental units
6 tents (3 treatments × 2 replicates)
tent (no further treatment) tent + fan tent + supplemental pollination
4 controls
close to tents (centre)
at the edges
Foto: Skogforsk
Tent + fanFoto: Skogforsk
Tent + supplemental pollination
Provided by Skogforsk
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
Isolation tents – design
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
Foto: Skogforsk
pollen contaminationpollen leak
Foto: Skogforsk
Parentage determination
Seed collection & extraction
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
• 1408 seeds analyzed
• 9 microsatellite markers
• combined multilocus exclusion probability 0.9994
Experimental unit / Treatment
Contaminating 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 in tents
pollen contamination
××
Experimental unit / Treatment
Pollen leakcount
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 into tents
pollen leak
Experimental unit /
TreatmentPollen leak
countTotal
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 contamination Pollen leak
Experimental unit /
Treatment
Contamin. seeds count
Total analyzed
seeds
Pollen contam.
[%]
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
1-May 11-May 21-May 31-May 10-Jun 20-Jun 30-Jun0
100
200
300
400
500
ControlTunnel
Tem
pera
ture
sum
abo
ve +
5°C
Control
Tent
Heat sum accumulation tent vs. control
Tents closed
Tents open
Experimental unit / Treatment
Contaminating 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 / Treatment
Contaminating 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
Genetic diversity of seed crops
1.2 T2.2 T
1.3 T+F
2.3 T+F
1.4 T+P
2.4 T+P
1.1 C2.1 C
C_26 C
C_103 C
0
5
10
15
20
25
30
Colored: Effective number of fa-thers
White: Number of available fa-thers (census)
1.2 T2.2 T
1.3 T+F
2.3 T+F
1.4 T+P
2.4 T+P
1.1 C2.1 C
C_26 C
C_103 C
0
5
10
15
20
25
30
Colored: Effective number of fa-thers
White: Number of available fa-thers (census)
Genetic diversity of seed crops
Tent AC1006 AC4221 Z3029 Z4003 Z4022 Total SMP Total analyzed SMP success
1.4 T+P 9 14 15 23 21 82 146 56.22.4 T+P 23 20 12 17 29 101 160 63.1
Total 32 34 27 40 50 183 306 59.8
Foto: Skogforsk
Selfing
May lead to inbreeding depression
reduction in economic value of forests (timber production, …)
Fertilization of ovules by pollen from the same tree
Selfing
Lstiburek and El-Kassaby (2010) Minimum inbreeding seed orchard design
Factors affecting selfing
Distance between replicates of the same parent Number of parents Fecundity Parental representation Reproductive phenology …
SelfingTreatment Count of
selfed seedsAnalyzed
seeds Selfing [%]
1.2 T 15 140 10.7
2.2 T 43 124 34.7
1.3 T+F 31 143 21.7
2.3 T+F 51 150 34.0
Tents 140 557 25.11.4 T+P 14 146 9.6
2.4 T+P 12 160 7.5
Tents SMP 26 306 8.5All tents 166 863 19.2
1.1 C 2 142 1.4
2.1 C 2 146 1.4
C_26 C 12 129 9.3
C_103 C 6 128 4.7
Controls 22 545 4.0
2:3 T+F parentcomposition
Y3012
AC3056
Y4103
Z4032
AC3056
Y3012
AC3040
AC3015
×Z2081
AC3056
Y3014
AC3056
Foto: Skogforsk
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
Summary / Conclusion
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
Acknowledgements
Xiao-Ru Wang
Curt Almqvist Ulfstand Wennström Bengt Andersson
Jin PanBaosheng Wang
Takeshi Torimaru
Financial support
Föreningen Skogsträdsförädling
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
Summary / Conclusion
Foto: Skogforsk