1. Association mapping in forage crop grasses Leif Skøt

2. Potential for high-resolution mapping Evaluation of many alleles Complements linkage mapping Control for population structure Association mapping in Lolium perenne

3. Traits Water soluble carbohydrates Ruminant productivity Abiotic stress tolerance (drought, cold) Flowering time Plant development Forage quality traits Seed production

4. Turner et al. New Phytol. 2005 Fructose and Glucose Alkaline Invertase locus map to QTL for Glucose and Fructose

5. Invertases: 1. Acid (vacuolar and Cell wall) 2. Alkaline Vacoule Sucrose Glucose Fructose Glucose Fructose Glucose Fructose Acid Invertase Fructan Fructosyl- transferase Alkaline Invertase Sucrose Acid Invertase Chloroplast / Amyloplast Starch -substrates for respiration -biosynthesis of primary and secondary compounds - regulation of gene expression by sugars Cytosol -sugar storage -osmoregulation -response to cold stress Cell Wall

6. Effect of sucrose accumulation on gene expression Depleted in soluble carbohydrate Plants - low light short photoperiod Excised Leaves Sucrose solution Sorbitol solution (osmotic Control) Dark Water 24h Actin Suc W Sor Alk Inv.

7. TLC - Water Soluble Carbohydrates 1 2 3 4 5 Photoperiod Sucrose Alkaline invertase expression along the developing leaf 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 Ct 1 2 3 4 5

8. SNPs in Alkaline Invertase Locus of Lolium perenne LG 6 1 kb M-L D-N SNPNo Indel6 Syn13 Non-syn8 Non-coding65 Total92

9. LD in candidate genes Alkaline Invertase

10. LD in Alkaline invertase locus

11. Pop Inferred clusters 123456789 1 0.0050.000 0.9890.005 2 0.0000.0010.0000.0010.0100.0010.9820.0050.000 3 0.0010.0030.0000.9710.0150.0090.000 0.001 4 0.0080.9460.0000.0060.0130.0010.000 0.027 5 0.0030.0420.0000.0050.000 0.0010.0000.950 6 0.0040.0010.9890.0000.0030.0010.0000.0020.000 7 0.9760.0140.0010.0000.0010.000 0.0070.000 8 0.0090.0230.0070.0080.9130.0170.007 0.011 9 0.0020.0010.0070.0010.0110.9750.0020.0010.000 Population structure 70 AFLP markers

12. SNPWSCNDMD 040504050405 202111 3821211 492111 805111 825112 953111 10531112 19541 19701121 221411 2283121 26473112 487911 5259111 535911 SNP0405 202 382 492 805 825 953 1053 1954 1970 2214 2283 26470.05 4879 5259 5359 Association analysis Alkaline Invertase and WSC Linear mixed model (WSC) ANOVA

13. E38M4710 0 6 10 12 19 23 25 26 27 28 30 33 34 35 38 41 42 43 45 46 53 55 56 58 59 69 85 103 INV1:2 R2869 B6102b CDO545 OSW LpF2 LpF1 LpF3 C764 LpF4 ACA_CTA_254ACA_CTA_321 E40M5909 RZ144S2539 ACA_CAC_225 E39M4910 E37M5601 GSY60.1 CDO385.1 PSR690 E41M5710 RZ952 C390 LtemCOa LtemCOb E39M5802 E41M5701 ACA_CAC_246 ACA_CTA_204 ACP CAT ACA_CTA_254 ACA_CTA_321 C764 S2539 ACA_CAC_225 0 10203040 LOD AFLP markers associated with flowering time in Lolium perenne Fructan epistatic QTL

14. Properties of LpCO gene Synteny with rice HD1 locus Homologous to CONSTANS Complements Arabidopsis co-2 mutant (Martin et al. 2004, PMB 56: 159) Upregulated in response to LD Map to major QTL for flowering time

15. N-I CAA[C/A]A V-M HD1 Peroxidase SNPs in HD1 locus in Lolium perenne LG7 1 kb SNPNo Indel0 Syn2 Non-syn2 Non-coding8 Total12

16. LD in candidate genes HD1

17. Association analysis HD1 locus and flowering time SNPFlowering timeWSC 04050405 320 513 1475 2118 2389 4443<0.05<0.001 4717 5443 Linear mixed model (pops random effect)

18. SNPBa9955Ba10113Ba10158Ba10278Ba10284Ba10732Ba10870Ba11304Ba12945 040504050405040504050405040504050405 320 513*** 1475**** 2118***** 2389* 4443************ 4717 5443******* Association analysis HD1 locus and flowering time ANOVA * P<0.05; ** P<0.01; *** P<0.001

19. N-I CAA[C/A]A V-M HD1 Peroxidase SNP 4443 in HD1 locus 1 kb CAACA Consensus recognition sequence for RAV1/AP2…

20. Acknowledgements IGER Janet Humphreys Mervyn Humphreys Joe Gallagher Danny Thorogood Ian Armstead Sue Heywood Mark Hirst Ruth Sanderson Lesley Turner University of Birmingham Zewei Luo Mike Kearsey