Elimination of pollen contamination in Scots pine seed orchards: Is it possible?

Tomas FundaEMG postdoc seminar, Nov 6, 2013

Foto: Skogforsk

Tree Improvement scheme

www.for.gov.bc.ca/hre/forgen

Phenotypic selection

www.for.gov.bc.ca/hre/forgen

Tree Improvement scheme

Evaluation of genetic quality

www.for.gov.bc.ca/hre/forgenwww.for.gov.bc.ca/hre/forgen

Tree Improvement scheme

Seed orchard establishment

www.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

© Google

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

© Google

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

© Google