2. 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

3. 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

4. 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

5. ABcABc aBCaBC aBCaBC ABcABc aBCaBC aBcaBc AbcAbc AbcAbc AbcAbc abCabC abCabC AbcAbc AbcAbc aBCaBC ABcABc abCabC aBcaBc AbcAbc Quantitative Trait Locus Mapping ABCABC ABCABC abcabc abcabc F1F1 F1F1 X ABCABC ABCABC abcabc abcabc Parent 1Parent 2 X HEIGHT GENOTYPE BBBbbb          BbBb Bb BB bb

6. 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

7. 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 )

8. Historical Reference Jermstad K.D. et al. 1998.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.

9. Maternal Grandmother (late flushing) Maternal Grandfather (early flushing) Paternal Grandmother (late flushing) Paternal Grandfather (early flushing) F 1 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, WA test site (n=408) Springfield, OR test site (n=408) 750 NDLEDL Moisture stress (MS) 10 15 20 MS NMS Twin Harbors, WA test site (n=224) (Jermstad et al. 2001a, 2001b) (WC750_FT10) (WC750_FT15) (WC750_FT20)(WC1500_FT15)(WC1500_FT20)NDL_NMSNDL_MSEDL_NMSEDL_MS Flushing temperature (FT) o C 3-generation pedigree and mapping populations Daylength (DL) Winter chill (WC) hours 1500 10 15 20 (WC1500_FT20) Growth cessation experiment (357< n <407)

10. 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

11. Fig. 2 Bud flush QTLS in Douglas-fir Verification pop. Detection pop. ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* wfl 1* ofl 1* gfl 9* gc 9* gfl 9* gc 9* gfl 9* gc 9* gh 9* gfl 9* gc 9* gfl 9* gc 9* gh 9* Jermstad et al 2003. Genetics 165: 1489-1506

12. 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.

13. 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)

14. Fig. 3 Cold-hardiness QTLS in Douglas-fir fch-s fch-n* fch-b* fch-s fch-s* sch-s* sch-b sch-s sch-n* sch-b* sch-n* sch-b* sch-n* sch-s* sch-b* sch-s* sch-n* sch-b* sch-n* Spring cold-hardinessFall cold-hardiness

15. Spring cold hardiness QTL only

16. Cold hardiness and bud flush QTL

17. 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

18. Cumulative Proportion of Variation Explained Cohort 1Cohort 2 Phenotypic 24.9% (H²=0.45) 15.2% (H²=0.29) Genotypic 55.0%52.4%

19. 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!

20. Spring cold hardiness QTL only

21. Candidate Genes: Targets for Association LGCandidate genes mapped in Douglas-fir Similar genes from other species AbbreviationGene productFunctionGene expression reference 3 1PtIFG_2006_a; PtIFG_2006_b CABBP2Chlorophyll a/b- binding protein type 2 Component of the photosynthetic light- harvesting complex Dubos et al. 2003 1estPpINR_RN01G08_b 4 DER1-likeUnknown protein with DER1- motif Degradation of misfolded proteins in the yeast endoplasmic reticulum Binh and Oono 1992 1estPaTUM_PA0006_a PmIFG_1592_a 40S-RPS240S ribosomal protein S2 Aids in protein synthesis as a structural component of ribosomes – 1estPpINR_RS01G05_a 4 ACRE146Avr9/Cf-9 rapidly elicited protein ACRE proteins are induced by fungal pathogens and other stresses – 1717 estPmIFG_102G09_c estPmIFG_102G09_b Alpha tubulin Major constituent of microtubules and cytoskeleton – 1estPpINR_AS01D10_b 4 TBEThiazole biosynthetic enzyme Biosynthesis of the thiamine precursor thiazole – 1PmIFG_1162_aUGTUridine diphosphate glycosyltransfe rases Transfer of glycosyl residues from activated nucleotide sugars to aglycones Fowler and Thomashow 2002