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

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

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

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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

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 164:1537-1546)

Historical Reference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.

Maternal Grandmother(late flushing)

Maternal Grandfather

(early flushing)

Paternal Grandmother(late flushing)

Paternal Grandfather

(early flushing)

F1 Parent F1 Parent

(1991) (1994)

clonally replicated progeny linkage map (Jermstad et al. 1998)

Turner,OR test site (n=78)(Jermstad et al.

2001a)

clonally replicated progeny

Bud flush experiment(n=429)

Field Experiment

Longview, WAtest site (n=408)

Springfield, ORtest site (n=408)

750 NDL EDL

Moisture stress (MS)

10 15 20 MS NMS MS NMS

Twin Harbors, WAtest site (n=224)(Jermstad et al. 2001a, 2001b)

(WC

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(WC

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Flushing temperature (FT) oC

3-generation pedigree and mapping populations3-generation pedigree and mapping populations

Daylength (DL) Winter chill (WC) hours

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10 15 20(WC

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)

Growth cessation experiment

(357< n <407)

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

Fig. 2 Bud flush QTLS in Douglas-fir

Verification pop.Detection pop.

LG17 LG16

LG15 LG14LG13 LG12

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11

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wlt 6*

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ofl 8*wfl 8*

wfl 8*

wlt 7ofl 8*

ofl 8

wfl 8*

ofl 8

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wlt 5*wlt 5*

otr 6wtr 6*

otr 6wtr 6*

wlt 7*

wtr 7*

Jermstad et al 2003. Genetics 165: 1489-1506

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.

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)

Fig. 3 Cold-hardiness QTLS in Douglas-fir

fch-s

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fch-n*

fch-b*

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Spring cold-hardiness Fall cold-hardiness

LG17 LG16

LG15 LG14LG13 LG12

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11

Spring cold hardiness QTL only

Cold hardiness and bud flush QTL

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

Cumulative Proportion of Variation Explained

Cohort 1 Cohort 2

Phenotypic 24.9%

(H²=0.45)

15.2%

(H²=0.29)

Genotypic 55.0% 52.4%

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 (>250 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!

Spring cold hardiness QTL only

Candidate Genes: Targets for AssociationLG Candidate genes mapped

in Douglas-firSimilar genes from other species

Abbreviation Gene product Function Gene expression reference 3

1 PtIFG_2006_a;PtIFG_2006_b

CABBP2 Chlorophyll a/b-binding protein type 2

Component of the photosynthetic light-harvesting complex

Dubos et al. 2003

1 estPpINR_RN01G08_b 4 DER1-like Unknown protein with DER1-motif

Degradation of misfolded proteins in the yeast endoplasmic reticulum

Binh and Oono 1992

1 estPaTUM_PA0006_a PmIFG_1592_a

40S-RPS2 40S ribosomal protein S2

Aids in protein synthesis as a structural component of ribosomes

–

1 estPpINR_RS01G05_a 4 ACRE146 Avr9/Cf-9 rapidly elicited protein

ACRE proteins are induced by fungal pathogens and other stresses

–

17

estPmIFG_102G09_cestPmIFG_102G09_b

Alpha tubulin

Alpha tubulin Major constituent of microtubules and cytoskeleton

–

1 estPpINR_AS01D10_b 4 TBE Thiazole biosynthetic enzyme

Biosynthesis of the thiamine precursor thiazole

–

1 PmIFG_1162_a UGT Uridine diphosphate glycosyltransferases

Transfer of glycosyl residues from activated nucleotide sugars to aglycones

Fowler and Thomashow 2002