Mapping of Quantitative Trait Loci Controlling Adaptive Traits in coastal Douglas-fir.

1
Mapping of Quantitative Trait Loci Controlling
Adaptive Traits in coastal Douglas-fir.

• Cold-Hardiness QTL Verification and Candidate
  Gene Mapping
• N.C. Wheeler, K.D. Jermstad, K.V. Krutovsky, S.N.
  Aitken, G.T. Howe,
• J. Krakowski, and D.B. Neale

2
Major Messages

• QTL Maps can be very informative
• Size does matter (for predicting number, effects,
  and location of QTL)
• Practice is good (verification has value)
• QTL studies may guide candidate gene
  prioritization

3
QTL Studies Are Informative and Useful

• Complex trait dissection / genetic architecture
• Number of QTL influencing a trait
• Size of the QTL effects (PVE)
• Location of the QTL (gross)
• Parental contribution of allelic effects
• QTL by environment/site interaction effects
• Provide a foundation for MAS
• Provide a framework for positional selection of
  candidate genes

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Quantitative Trait Locus Mapping
A B C
A B C
a b c
a b c
X
Parent 1
Parent 2
A B C
A B C
a b c
a b c
X
F1
F1
B b
5
QTL Study Requirements

• An appropriate population
• Pedigreed, large, replicated
• Appropriate markers
• Co-dominant, multi-allelic, fully informative
• Framework map with complete genome coverage
• Good phenotypes
• Analytical tools
• The problem is, most studies have failed to meet
  all requirements well, and are seldom repeated
  esp the population

6
A Case for Verification

• To assess the robustness of QTL it is necessary
  to verify them in time, space, and/ or genetic
  background.
• Definition the repeated detection, at a similar
  position on the genetic map, of a QTL controlling
  a trait under more than one set of experimental
  conditions (Brown et al. 2003. Genetics
  1641537-1546)

7
Historical Reference

• Jermstad K.D. et al. 1998. A sex-averaged
  genetic linkage map in coastal Douglas-fir
  (Pseudotsuga menziesii (Mirb.) Franco var
  menziesii) based on RFLP and RAPD markers.
  Theor. Appl. Genet. 97 797-802.
• Jermstad K.D. et al. 2001a. Mapping of
  quantitative trait loci controlling adaptive
  traits in coastal Douglas-fir. I. Spring bud
  flush. Theor. Appl. Genet. 102 1142-1151.
• Jermstad K.D. et al.. 2001b. Mapping of
  quantitative trait loci controlling adaptive
  traits in coastal Douglas-fir. II. Spring and
  fall cold-hardiness. Theor. Appl. Genet.
  102 1152-1158.
• Jermstad K.D. et al. 2003. Mapping of
  quantitative trait loci controlling adaptive
  traits in coastal Douglas-fir. III. QTL by
  environment interactions. Genetics 165 1489-1506.

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3-generation pedigree and mapping populations
Maternal Grandfather (early flushing)
Maternal Grandmother (late flushing)
Paternal Grandmother (late flushing)
Paternal Grandfather (early flushing)
F1 Parent
F1 Parent
(1991)
(1994)
clonally replicated progeny linkage map
(Jermstad et al. 1998)
clonally replicated progeny
Growth cessation experiment (357lt n lt407)
Bud flush experiment (n429)
Field Experiment
Turner,OR test site (n78) (Jermstad et al.
2001a)
Twin Harbors, WA test site (n224) (Jermstad et
al. 2001a, 2001b)
Springfield, OR test site (n408)
Longview, WA test site (n408)
Winter chill (WC) hours
Daylength (DL)
750
1500
NDL
EDL
Flushing temperature (FT) oC
Moisture stress (MS)
10 15 20
MS NMS
MS NMS
10 15 20
NDL_MS
EDL_MS
NDL_NMS
EDL_NMS
(WC750_FT15)
(WC750_FT20)
(WC1500_FT15)
(WC1500_FT20)
(WC1500_FT20)
(WC750_FT10)
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The Other Requirements

• Markers and genome coverage
• 74 evenly spaced, highly informative RFLP
  markers
• Map length of 900 cM, density every 12 cM
• Phenotypes
• Spring cold hardiness (1997, 2003)
• Bud flush etc (annually 96-2001)
• Analytical Tools
• Haley-Knott multiple marker interval mapping
  approach scanned LG at 5 cM intervals, 1 and 2
  QTL models

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Fig. 2 Bud flush QTLS in Douglas-fir
Jermstad et al 2003. Genetics 165 1489-1506
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Cold Hardiness in Douglas-fir

• Genetics of cold hardiness well documented.
• Traditional quantitative and genecological tests
• Freeze testing
• Fall and spring cold hardiness controlled by
  different genes
• Spring ch under stronger genetic control
• Deacclimation synchronized in all tissues (buds,
  needles, shoots)
• QTL studies support all these findings.

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Cold Hardiness Evaluation

• Cohort 1
• Shoot tips (4) from each of 2 ramets in each of 2
  field blocks
• Frozen in a temperature controlled chamber,
  multiple test temps
• Evaluated using visual assessment of tissue
  necrosis (3 tissues)
• Cohort 2
• Single shoot tip from each of 2 ramets in each of
  2 blocks
• 20 diced needles, frozen in controlled chamber,
  multiple test temps.
• Evaluated using electrolytic conductivity
  (needles only)

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Fig. 3 Cold-hardiness QTLS in Douglas-fir
14
Spring cold hardiness QTL only
15
Cold hardiness and bud flush QTL
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Validation?

• Of eight unique spring needle cold hardiness QTL
  in Cohort 2, four co-located with QTL in Cohort 1
• Two of the eight on new LGs, others on LGs with
  QTL in other locations.
• Thus, all genomic regions containing QTL for sch
  were verified, even given
• Different cohorts, 5 years apart
• Different test sites, 6 years apart
• Different methods of detection

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Cumulative Proportion of Variation Explained
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What has QTL mapping taught us

• Virtually all traits tested are controlled by a
  finite number of detectable genes (QTL), with
  known genomic positions (kind of)
• In Douglas-fir, the majority of Sch_QTL are
  repeated in time (yr to yr) and space
  (environment). Bud flush same
• Most QTL explain 2-10 of the phenotypic
  variation of a trait (a few notable exceptions)
• Family size is very important (gt250 desirable),
  as is clonal replication. More trees, more QTL
  with smaller effect, and smaller CI.
• But, we still do not know what the relevant genes
  are!

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Spring cold hardiness QTL only
20
Candidate Genes Targets for Association