Genetics of gene expression in barley

1. Genetics of gene expression in barley Birmingham University: Elena Potokina Zewei Luo Mike J Kearsey SCRI(Dundee): Arnis Druka Robbie Waugh Material: Steptoe x Morex doubled haploid (DH) recombinant inbred lines Tool: 22,801 unigene Affymetrix Barley Chip • Genetical Genomics Approach: • Expression Data Analysis across parental lines and DH progeny • eQTL analysis (identification of cis- and trans-acting regulators) • Exploring the relationship between eQTL and mapped phenotypic traits

2. Seedlings 12 days after planting The length of the seedling leaves of any of the segregating lines was not significantly different at this developmental stage (data not shown) Morex Steptoe Golden Promise • 9 seeds per pot were planted in the vermiculate • all the seedlings from each pot were counted, measured and pooled for the RNA isolation 12 days after the planting

3. Material: Steptoe feed barley Morex malting barley x AA BB (3 replications) (3 replications) 35 Double Haploid lines(each in 1 replication) Morex at 10 cm seedling stage (12 days after planting). Leaves were collected separately from 9 seedlings. Totally: 41 hybridisations onto Affymetrix Barley1 Chip

4. 22801 contigs

5. Probe set structure: Probe Set: Contig8450_at Description: BEST BLASTX NR: 10/02/02 BAB89658.1 6e-43 (AP003262) hypothetical protein~similar to Arabidopsis thaliana chromosome 3, At3g02290 [Oryza sativa (japonica Probe Set: Contig8450_s_at Description: BEST BLASTX NR: 10/02/02 BAB89658.1 6e-43 (AP003262) hypothetical protein~similar to Arabidopsis thaliana chromosome 3, At3g02290 [Oryza sativa (japonica

6. Calculation of an expression value: Probe Level Analysis Method Expression value is calculated by takingthe difference between the PM and MM of every probe pair and averaging the differences over the probe set

7. Filtering of genes before eOTL analysis: from 22,801 to 13,720 P – “present”, A – “absent”, M – “marginal”

8. 22 801 contigs on the chip Filtering based on presence-absence score of parents 13 720 contigs 2 124 genes are differentially expressed (15.5%) 10 623 genes are constantly expressed (77.4%) 972 genes cannot be classified because of high standard error (7.1%) Difference in gene expression between parental genotypes ?

9. 562 genes are differentially expressed between Steptoe and Morex after Multiple Testing Correction

10. Steptoe DH73 DH74 Morex

11. S/M map for eQTLs: 34 lines, 220 markers - 7 chromosomes (RFLP and SNP markers) 3H 4H 2H … 1H

12. eQTL analysis criteria: 1) Composite Interval Mapping (CIM) , LOD > 3.0 2) Single Marker Analysis (SMA) p-value < 0.001 13 720 genes subjected to QTL analysis 1 609 genes with at least one significant QTL 13 720 genes subjected to eQTL analysis different QTLs = QTLs on different chromosomes

13. cis- or trans- regulation ? 50 genes mapped by SNP markers eQTL for Contig 10081_at cis- 72% Contig 10081_at mapped by SNP marker

14. cis- or trans- regulation ? 50 genes mapped by SNP markers eQTL for Contig 10984_at trans- 28% Contig 10984_at mapped by SNP marker

15. 261 eQTLs are assigned to MWG503 (2H) 121 eQTLs are assigned to MWG865 (2H) How many eQTLs are linked to each of 222 markers: hotspot on chromosome 2H ? Markers - Chromosomes

16. To perform genetical genomics approach we need: • Segregation population (DH lines, RILs, F2...) • Set of molecular markers polymorphic between parental lines • Information about alleles of the markers in each of progeny lines • (progeny lines genotypes) • Genetic map as a linear arrangement of the markers from • recombination values • RNA profiling data for each of progeny lines

17. Gene arrays are not just for measuring gene expression labeled mRNA labeled total genomic DNA Expression analysis Single Feature Polymorphism (SFP) detection

18. Val V1/n1 +V2/n2 How to define Single Feature Polymorphism (SFP markers) from RNA profiling data? Contig5061_at7: two groups from k-mean clustering Contig5061_at3: two groups from k-mean clustering Variance across all 40 chips Var Group1 + Var Group 2

19. Val V1/n1 +V2/n2 Contigs with putative SFPs are in the 90 percentile:

20. DHi = aa DHi = ? AA aa aa AA t-test: p=10-20 t-test: p=10-3 Pr { DHi AA AA, σAA ,aa,σaa} Pr > 0.90 genotyping success: 60% genotyping success: 100% Genotyping of DH lines across the population based on SFPs: (genotyping success: percentage of DH lines successfully genotyped)

21. Sequencing data were available for 129 probes to test the accuracy of SFPs prediction t-test: p=10-3 t-test: p=10-20 AA aa aa AA

22. Mapping of the predicted SFPs: 1012 • only one SFP/probe per contig was mapped • contigs predicting less than 31 DH line out of 34-DHset were removed 648 • 81 were removed from analysis by mapping software as identical 567 • 19 did not join to any of linkage group by LOD=3 and higher 548 SFPs/contigs successfully formed 7 linkage groups corresponding to 7 barley chromosomes

23. 0 Contig21640_at5 Contig2337_at10 12 Contig4887_s_at11 13 MWG837 15 Contig5857_at7 19 Contig5859_at7 21 Contig22516_at9 27 Contig21247_at7 0 MWG835A 33 HV05F11u_x_at5 Contig3217_s_at6 2 MWG938 35 Contig14013_s_at4 7 MWG036A 41 ABG500A 12 MWG837 43 Contig14742_x_at7 14 Hor1 49 Contig12283_at10 56 Contig6251_at3 22 ABA004 57 Contig2947_at10 HK04F07r_at5 59 Contig5576_at2 Contig10724_at11 30 BCD98 HK04G05r_at5 HVSMEg0015I15r2_at7 36 ABG053 60 Contig15811_at8 rbags10j11_s_at3 41 Ica1 Contig6997_at11 61 Contig17095_at8 48 ABG500A 64 HVSMEb0014F22f_s_a10 55 ABC164 67 HK05M22r_at9 57 WG789B 68 Contig3479_at3 61 ABR337 70 Contig4121_at3 65 Glb1 Contig12839_at1 Glb1 71 Contig14005_at9 73 ABC160 72 Contig10681_at6 73 Contig20245_at8 75 Contig12759_at10 86 ABG464 80 Contig20070_at9 93 His3B 81 HV_CEa0009C07r2_at10 95 ABC307A 83 Contig10249_at11 104 cMWG706A 88 Contig4450_at3 109 ABC257 90 Contig200_at5 114 cMWG733 91 Contig15928_at6 120 AtpbA 97 Contig22762_at8 122 ABG702 100 Contig7574_at9 126 ABC322B 102 Contig7898_at5 128 ABC261 103 Contig3246_s_at7 131 Cab2 105 Contig3532_at6 133 Aga7 108 Contig10829_at7 136 MWG912 115 cMWG706A 138 ABG387A 121 Contig9336_at4 122 Contig12420_at6 129 rbaal30e02_s_at3 133 Contig2449_at5 136 Contig14682_at8 Contig2951_at6 138 Contig4250_s_at6 139 ABG387A Contig5061_at7 Contig14709_at8 141 Contig17218_at7 143 Contig14590_at10 145 Contig6092_at5 150 Contig1079_x_at7 Comparison of SFP map and base RFLP map of barley: chromosome 1H RFLP map SFP map

24. Alignment of haplotypes from the SFP and SNP barley maps: chromosome 1H

25. 0 Contig9243_at5 0 Contig125_at9 0 Contig7699_at8 0 Contig1568_x_at7 0 HVSMEl0003E22r2_at10 6 Contig7295_s_at10 6 Contig12220_at3 2 HW01O15u_at7 13 Contig2400_at11 0 Contig21640_at5 8 Contig1300_x_at8 0 Contig11315_at9 7 Contig7522_at7 8 Contig4161_at1 14 HV_CEb0001A10f2_at7 2 Contig13877_at7 4 Contig10150_at7 15 Contig1579_s_at6 12 Contig4887_s_at11 16 Contig3947_s_at10 3 HVSMEi0018A05r2_x_5 9 Contig17530_at6 5 Contig11996_s_at10 Contig7698_at7 18 Contig7192_at8 24 Contig6315_s_at8 10 Contig11003_at1 13 MWG837 Contig10114_at9 HVSMEl0008P20r2_at9 8 6 Contig2659_at11 10 27 rbaak13h13_s_at8 12 ABG703B 20 MWG620 Contig8506_at7 15 Contig5857_at7 35 Contig9522_at11 Contig7951_at7 14 Contig19578_at9 7 Contig14594_at9 36 Contig7106_at9 14 Contig4769_at4 21 Contig2305_at10 17 Contig17510_s_at5 19 Contig5859_at7 8 Contig18822_at3 46 Contig13951_at2 18 Contig12308_at11 16 Contig7870_at5 10 Contig16774_at10 24 Contig24914_at2 48 Contig10757_at8 9 Contig3141_at7 19 rbah35f01_s_at4 21 Contig22516_at9 17 HVSMEl0023I10r2_at8 49 Contig6519_at2 21 Contig589_x_at6 25 Contig8716_at11 11 Contig11397_s_at11 19 Contig10968_at9 27 Contig21247_at7 19 Contig12833_at7 51 rbaal22m22_s_at7 23 Contig4823_at8 12 ABG320 52 Contig5083_at8 30 HVSMEn0020P13r2_x_7 24 Contig308_x_at4 20 EBro04_SQ003_B10_s6 33 HV05F11u_x_at5 54 Contig2893_at10 25 MWG077 Contig594_x_at7 17 Contig6752_s_at5 21 Contig11952_at8 33 Contig8091_at7 25 55 Contig5974_s_at10 35 Contig14013_s_at4 Contig19241_at11 21 Contig40_x_at3 23 Contig1164_at3 57 ABG705 35 EBem09_SQ002_H11_x5 26 Contig12089_at7 32 Contig5487_s_at11 41 ABG500A 59 Contig6996_at1 25 Contig11188_at11 22 Contig10425_at1 29 Contig327_s_at9 36 HVSMEg0004N17r2_x_4 60 Contig26251_at7 31 Contig61_s_at3 28 Contig1786_s_at6 43 Contig14742_x_at7 24 Contig570_s_at1 35 HV_CEa0009K11r2_at11 61 HVSMEi0008J09r2_x_11 40 HA16L09r_s_at8 33 ABG318 Contig5845_at4 28 Contig11509_at7 49 Contig12283_at10 62 Contig18379_at1 39 Contig2769_s_at8 39 Contig9300_at4 42 Contig19209_at1 33 Contig6681_at4 Contig3237_at3 29 Contig4028_x_at7 41 Contig25371_at10 56 Contig6251_at3 63 Contig8209_at6 Contig12753_at7 43 HVSMEb0010F06r2_at10 43 Contig9927_s_at8 30 Contig5588_at11 EBro03_SQ005_O21_s4 41 rbaal1i15_s_at9 45 Contig20626_at11 57 Contig2947_at10 34 MWG798B 45 HV_CEb0011B08r2_at9 31 Contig20455_at8 Contig16134_at11 47 Contig3579_x_at11 64 37 Contig19045_at6 HVSMEi0019L06r2_at9 59 Contig5576_at2 48 HV11O16u_at3 42 Contig12802_at8 33 Contig3024_at5 46 ABC152A 65 HY07G16u_at8 39 Contig21913_at5 HY10B05u_x_at2 HK04G05r_at5 34 Contig15204_s_at9 67 EBma03_SQ003_J21_s4 52 rbags22p06_s_at10 40 Contig6119_at10 49 Contig18594_at3 45 HW03O03u_at5 68 Contig10497_at1 38 Contig253_s_at5 60 Contig15811_at8 Contig6254_at6 Contig12614_at8 Contig10972_at4 53 Contig4666_at6 Contig5231_at11 50 Contig8346_at7 42 70 39 HA11J15u_s_at11 Contig6997_at11 HVSMEl0008I17r2_at5 Contig7176_at4 48 Contig7594_at7 51 Pox 60 Contig10126_at11 71 Contig18705_at4 41 ksuA1A 45 Contig9352_at5 53 Contig8914_at9 61 Contig17095_at8 72 HVSMEm0003A13r2_at5 61 Contig10125_s_at6 53 HC113H12_SK_at10 55 Contig4265_s_at9 42 Contig13249_at1 HV_CEa0003N18r2_at3 73 Contig5847_at2 64 HVSMEb0014F22f_s_a10 48 57 Contig25428_x_at2 Contig9408_at1 43 Contig18524_at5 Contig8330_at10 74 HVSMEg0008B24r2_at9 58 Contig7102_at5 55 Contig16020_at1 67 HK05M22r_at9 75 Contig13997_at11 49 Contig16307_at4 62 Contig1659_x_at11 44 Contig13250_at11 Contig9717_at7 59 76 HP01B09w_at9 68 Contig3479_at3 Contig19984_at11 51 Contig4997_s_at6 50 rbaal37j13_s_at9 Contig14824_at11 57 Contig4605_at2 77 Contig10883_at9 60 Contig26451_at5 54 Contig23811_at10 52 HV_CEb0010H02r2_at2 79 Contig9283_at9 70 Contig4121_at3 Contig14514_at8 Contig5948_at6 61 86 S78730_at3 65 Contig21442_at4 61 ABG484 Contig20540_at4 54 Contig861_at1 Contig12839_at1 87 ABC302 Contig23373_at1 67 Contig1430_at5 62 HVSMEi0003C20r2_at11 71 63 56 Contig3840_at7 88 Contig3054_at8 64 Contig19630_at10 63 Contig10535_at11 Contig14005_at9 Contig4320_at6 73 Contig2949_s_at9 89 Contig7450_at2 Contig18530_s_at9 Contig11000_at4 Contig13173_at10 57 76 Contig8408_at5 64 90 Contig19506_at3 72 Contig10681_at6 Contig4706_at4 Contig11524_at7 HVSMEh0089C16r2_at4 65 Contig5531_at5 91 Contig10407_at11 77 Contig18723_at9 65 rbags21b15_at9 73 Contig20245_at8 Contig18566_at10 93 HVSMEn0020J05f_x_a5 58 Contig11942_at11 66 Contig1559_at10 79 Contig2613_s_at2 64 66 Contig10310_at7 96 HU09F16u_s_at7 Contig12739_at2 75 Contig12759_at10 62 Contig2810_at7 67 Contig7293_at11 81 Contig3166_at1 97 EBro03_SQ004_E10_a9 68 rbaak37h22_at6 63 Contig5815_at5 65 Contig10706_at8 80 Contig20070_at9 104 Contig7020_at8 84 CDO113B 69 Contig11844_at9 Contig6993_at6 105 HV_CEb0012I09r2_x_11 65 Contig8756_at9 87 Contig18557_at3 66 Contig1042_x_at11 70 Contig17958_at4 81 HV_CEa0009C07r2_at10 106 Contig4267_at7 66 Contig8154_at3 72 Contig8511_at2 89 Contig30_at9 111 Contig24438_at1 69 Contig6770_at8 83 Contig10249_at11 71 Contig14898_at10 113 Contig12567_at7 91 Contig410_at2 67 Contig2163_at11 72 Contig12607_at10 71 HA04k13r_at8 73 Contig18240_at8 114 Contig24449_at10 88 Contig4450_at3 92 Contig14467_at2 68 Contig24926_at10 73 Contig9258_at11 116 Contig4384_at10 Contig13806_at11 93 Contig6460_at5 90 Contig200_at5 76 Contig14030_at8 69 Contig1661_x_at3 117 Contig5182_at9 73 74 Contig24154_at5 79 Contig24074_at1 Contig4906_at11 Contig15356_at10 119 Contig6939_at9 70 rbaal9p16_x_at1 91 Contig15928_at6 95 81 Contig2253_s_at4 121 Contig16074_at9 Contig10854_s_at11 81 Contig16261_at7 HV_CEb0007N06r2_at11 Contig742_at11 82 Contig8908_at10 97 Contig22762_at8 122 Contig13943_s_at3 71 74 96 Contig7199_at7 Contig6198_at7 Contig7183_at4 123 Contig16318_at1 Contig15923_at4 83 100 Contig7574_at9 Contig26114_at3 83 Contig5836_s_at4 97 Contig16567_at7 126 Contig8979_at11 72 Contig15402_at4 76 Contig590_at7 84 HV04J01r_at6 130 rbah38o04_s_at9 98 Contig7928_s_at10 102 Contig7898_at5 Contig13835_at11 73 Contig20066_at9 140 Contig6745_at4 90 HVSMEh0095L04f_s_a5 85 78 Contig4108_x_at9 ABG004 Contig5754_at9 HU04E13r_at9 103 Contig3246_s_at7 145 Contig14054_at5 74 Contig7248_at1 103 86 MWG503 Contig5753_at3 80 ksuD17 148 HVSMEm0007B06r2_s_2 92 Contig3205_s_at8 105 Contig3532_at6 87 Contig14108_at11 75 Contig2170_at5 151 Contig10941_at11 106 Contig18959_at10 85 Contig2885_at8 88 Contig8616_at4 Contig19601_at10 154 Contig9724_at11 Contig13416_at9 108 Contig10829_at7 108 Contig5075_at3 99 HY02C09u_x_at4 89 Contig4350_at6 76 157 Contig1181_x_at3 86 Contig5801_at10 HVSMEm0003G02r2_at11 92 Contig7630_at7 118 Contig21378_at9 158 EBro02_SQ008_M09_a7 115 cMWG706A 97 Contig15767_at10 Contig477_at6 77 Contig7278_at5 Contig21308_at6 119 Contig13892_at9 106 Contig7474_s_at3 165 87 121 Contig9336_at4 98 Contig19953_at8 Contig24410_at6 78 Contig8776_s_at10 120 Contig5646_at7 Contig8372_at7 102 Contig6795_at10 166 Contig6838_at9 122 Contig12420_at6 109 Contig9190_at3 121 Contig4700_at9 82 Contig14342_at10 104 Contig2809_at7 HB14F21r_at10 90 HV11N12u_at10 168 Contig10095_at8 108 Contig2046_at2 129 rbaal30e02_s_at3 Contig2129_x_at3 baal31g17_s_at8 Contig4300_at1 92 HVSMEa0009E14r2_at3 125 112 iHxk2 83 110 Contig10730_at11 169 Contig6095_at6 Contig17946_at6 Contig4374_s_at7 133 Contig2449_at5 111 Contig2044_at4 171 Contig2130_at1 96 HVSMEg0011O06r2_s_10 126 Contig3927_s_at11 117 ABC252 116 Contig10937_at5 172 Contig9707_at4 84 HVSMEm0001B03r2_at3 136 Contig14682_at8 97 Contig17043_at6 123 Contig6105_s_at1 129 Contig19223_at10 173 Contig14157_at5 86 Contig6283_s_at10 138 Contig4250_s_at6 125 Contig24523_at3 174 HVSMEf0003C10r2_at10 132 HM09F12r_s_at9 117 Contig5903_s_at11 98 Contig12824_at6 Contig13190_at7 130 Contig10785_at1 181 Contig15607_at8 94 139 ABG387A 135 Contig13352_at6 133 Contig12574_at10 190 Contig5252_at8 118 Amy1 Contig10950_at11 136 ABG319C 136 Contig13874_at6 138 Contig1433_at1 196 Contig17228_at11 Contig5061_at7 95 Contig7157_at11 126 Contig16766_at8 140 Contig5135_s_at10 199 Contig331_x_at3 141 137 Contig1159_at2 157 ASE1C 99 Contig1048_at6 Contig17218_at7 142 Contig11295_at11 200 Contig1003_at2 130 ABC170A Contig679_s_at1 140 Contig12390_at1 145 Contig17366_at7 203 Contig9042_x_at2 106 Contig10197_at8 143 Contig14590_at10 142 HVSMEb0010A16r2_at7 HV_CEb0009L11r2_at1 147 bBE54C 205 Contig8225_at6 158 Contig11126_at11 131 107 Contig3777_at8 149 Contig4801_at4 207 HV_CEa0001E19r2_at11 147 Contig1179_at8 Contig12587_at5 145 Contig6092_at5 Contig12030_at11 150 HVSMEg0016P19f_x_a3 209 Contig9470_at6 111 Contig4056_at6 ABA307A 167 155 ABC172 150 Contig1079_x_at7 132 HVSMEc0012C08r2_at10 151 Contig13815_at5 214 Contig14544_at4 113 Contig25162_at1 216 Contig8849_at11 156 HVSMEm0015J14r2_at5 133 HU11B09u_at11 115 ABG461 219 ABG463 150 Contig19427_at11 122 HS04A09u_s_at2 The SFP genetic map of barley

26. Summary We suggest a simple algorithm to detect SFPs from RNA profiling data of any segregation population combining homozygote progeny lines Number of detectedSFP markers is sufficient to create a high density genetic map for any unknown cross Construction of genetic map, measurement of gene expression and mapping of eQTL can be achieved within the same experiment

27. Thanks to : Mike Kearsey Zewei Luo Robbie Waugh Arnis Druka