EUCARPIA Cereals Section Meeting Lleida, November 2006

Materials and Methods225 barley accessions (SBCC and 66 cultivars) were genotyped with 73 markers, distributed along the

barley genome (Figure 1). Population structure was evaluated with the software Structure (Falush et al., 2003). For association analysis, marker bands with frequencies in between 5% and 95% were used. To assess the association of each maker locus with traits, we fitted multiple regression models of the response trait on the set of allele indicator variables, taking or not into account population structure. To this end, four variables describing the probability of membership of each genotype to each of the four genetic groups, as reported by Kraakman et al. (2006), were included in the model. Multiple testing wasaddressed using the Bonferroni correction.

Materials and Methods225 barley accessions (SBCC and 66 cultivars) were genotyped with 73 markers, distributed along the

barley genome (Figure 1). Population structure was evaluated with the software Structure (Falush et al., 2003). For association analysis, marker bands with frequencies in between 5% and 95% were used. To assess the association of each maker locus with traits, we fitted multiple regression models of the response trait on the set of allele indicator variables, taking or not into account population structure. To this end, four variables describing the probability of membership of each genotype to each of the four genetic groups, as reported by Kraakman et al. (2006), were included in the model. Multiple testing wasaddressed using the Bonferroni correction.

Results and Discussion•When population structure was not considered, a large number of marker-trait associations were detected (311 for 30 traits). Many of these associations could be false positives. This figure was reduced to 65 when population structure (Table 1) was included in the model, a similar reduction as observed by Thornsberry et al. (2001). •For the trait days to heading, 12 associations were detected and 8 of them were close to QTL reported in the literature. The marker Bmag223, that is closely linked to the gene srh for rachilla hair length (Costa et al., 2001), showed a very high association with length of rachilla hairs. In other cases (as for leaf rust), associations found had no correspondence in the literature. The loss in statistical power and the uncertainty about the significance of associations due to multiple testing in AM seem counterbalanced by the increase in the scope of genetic diversity analyzed, and thepermanent nature of the populations and data analyzed. In view of these results, the authors are optimistic about the feasibility of AM in barley, increasing the number of markers, and about thevalue of the SBCC as an appropriate resource for gene and allele discovery. References:

Costa JM, Corey A, Hayes PM, Jobert C, Kleinhofs A, Kopisch-Obusch A, Kramer SF, Kudrna D, Li M, Riera-Lizarazu O, Sato K,Szücs P, Toojinda T, Vales MI, Wolfe RI (2001) Theor Appl Genet 103, 415-424.

Falush D, Stephens M, Pritchard JK (2003) Genetics 164, 1567–1587.Kraakman ATW, Martínez F, Mussiraliev B, van Eeuwijk FA, Niks RE (2006) Mol Breeding 17, 41-58.Thornsberry JM, Goodman MM, Doebley J, Kresovich S, Nielsen D, Buckler IV ES. (2001) Nat Genet 28, 286-289.

Results and Discussion•When population structure was not considered, a large number of marker-trait associations were detected (311 for 30 traits). Many of these associations could be false positives. This figure was reduced to 65 when population structure (Table 1) was included in the model, a similar reduction as observed by Thornsberry et al. (2001). •For the trait days to heading, 12 associations were detected and 8 of them were close to QTL reported in the literature. The marker Bmag223, that is closely linked to the gene srh for rachilla hair length (Costa et al., 2001), showed a very high association with length of rachilla hairs. In other cases (as for leaf rust), associations found had no correspondence in the literature. The loss in statistical power and the uncertainty about the significance of associations due to multiple testing in AM seem counterbalanced by the increase in the scope of genetic diversity analyzed, and thepermanent nature of the populations and data analyzed. In view of these results, the authors are optimistic about the feasibility of AM in barley, increasing the number of markers, and about thevalue of the SBCC as an appropriate resource for gene and allele discovery. References:

Costa JM, Corey A, Hayes PM, Jobert C, Kleinhofs A, Kopisch-Obusch A, Kramer SF, Kudrna D, Li M, Riera-Lizarazu O, Sato K,Szücs P, Toojinda T, Vales MI, Wolfe RI (2001) Theor Appl Genet 103, 415-424.

Falush D, Stephens M, Pritchard JK (2003) Genetics 164, 1567–1587.Kraakman ATW, Martínez F, Mussiraliev B, van Eeuwijk FA, Niks RE (2006) Mol Breeding 17, 41-58.Thornsberry JM, Goodman MM, Doebley J, Kresovich S, Nielsen D, Buckler IV ES. (2001) Nat Genet 28, 286-289.

Bin Grain yield Heading date 1000 kernel weight Leaf rust

Length of rachilla

hairs Genes

1 2 6 6 12

2 2 6 1

3 5 1 1

4 5 9 21 1 19 D Rph2

5 5 1 11 9 8 15 55 18 19

6 5 1 24 9 19

7 5 1

8 5 8 8 5 18

9 1 10 4 55 S 50 R srh. Eam5

10 1 10 13 23 6

11 1 23 2 4 6 8 9 19 22 33 35 7 14 22 20 37 E Vrn-H1. Rph9/12

12 1 23 8 7 1

13 30 2 14

14 20

15 20

DAK133ABC483MWG920.1AABG316ABG705AAdh6ABG395Ctig7136Ltp1Bmac096Bmac113Bmag337HvHEMH1EBmac970NRG045AWG530AleHvLEUABC324ABC302Ctig7358BCD926srhBmag223RSB001AABG473HvDHN007MWG514ABG003BHvBM5AHv635P2.4WG908MWG877BE456118AABG496Bmag222ABG391ABG390ABG463Bmag113CMWG602A

5H

Locus Allele Size (bp) No. accessions Heading date Length of rachilla hairs

1 125 12 117.3 d 1.4 cd 2 127 16 120.9 ab 1.9 a 3 155 17 120.3 b 1.1 e 4 157 35 119.4 bc 1.2 e

5 159 10 119.6 abcd 1.2 de

6 165 9 121.6 a 1.8 ab

Bmag223

7 169 11 118.2 cd 1.7 bc

Locus Allele Size (bp) No. accessions Length of rachilla hairs

1 181 65 1.7 a 2 191 15 1.2 c 3 193 22 1.5 b 4 195 20 1.5 b

Bmac113

5 197 10 1.5 ab

Locus Allele Size (bp) No. accessions Leaf rust Hairy seath 1 127 19 7.5 ab 1.3 ab 2 129 61 7.5 a 1.5 a 3 143 13 5.7 c 1.4 a 4 145 20 6.6 bc 1.4 a 5 147 28 6.8 b 1.1 b

Bmag337

6 149 8 6.4 abc 1.5 a

Locus Allele Size (bp) No. accessions

Grain yield (q.ha-1)

1000 kernel weight (g)

Leaf rust

Lemma type

Row number

1 4850 92 25.9 a 41.4 a 7.2 a 2.7 a 5.0 a 2 1200 50 25.7 a 42.6 a 5.7 b 2.6 a 5.0 a 3 150 7 24.9 a 38.2 b 7.8 a 2.2 b 4.0 b

HvBM5A

4 null 7 20.2 b 36.8 b 6.5 ab 1.7 c 4.0 b

Bin Grain yield Heading date Lemma type

Genes

1

2 3 8 23

3 21 3 8

4 3

5 5 21 3 8

6 5 21 1 10 15

7 23 1 10 31

8 31 29 6 10

9 31 2 29 9

10 31 11 29 6 7

11 31 11 3 24

12 1 11 3 24 2 9 33 35 O P Vrn-H2 Hln

13 8 3 24 8

MWG634JS103.3MWG077HvM040Ole1CDO542HvOLECDO122BCD402Bhvknox3BCD808BDhn6ABC303CDO795ABG484HvM003Tef2GBM1020Bmag353bBE54ABmag384BCD453BABG472ABG319AKFP221EBmac701HvMLOH1AMWG652BABG397HvBTAI3ABG319CHshHvM067HvZCCTBmy1HvBAMYHdAMYBABG601

4H

Locus Allele Size (bp) No. accessions Grain yield (q.ha-1)

Spike length (cm)

1 133 8 24.6 ab 3.5 abc 2 135 47 24.1 b 3.3 bc 3 137 11 23.9 bc 3.8 a 4 147 16 23.0 c 3.6 ab

EBmac701

5 149 24 25.5 a 3.1 c

Locus Allele Size (bp) No. accessions Row number 1 1400 144 4.0 b HvZCCT 2 nulo 12 5.0 a

Locus Allele Size (bp) No. accessions Heading date 1 127 15 122.2 a 2 129 13 119.8 bc 3 131 83 119.4 c

HvBAMY

4 132 43 120.9 ab

Locus Allele Size (bp) No. accessions Stem pigmentation

1 186 18 1.5 bc 2 189 40 1.4 c 3 190 43 1.6 ab 4 192 16 1.7 ab

HdAMYB

5 211 12 1.8 a

Bin Heading date Leaf rust Spike length Genes1

1 37 52 A Rph4

2 2 38 1

3 2 55

4 4 55

5 4

6

7 5 8 16

8 5

9 8 9

10 3 4 7 19 42 44

11 3 4 7 19 42 44 eam8

12 4

13

14 5 7 36 G eam8

BCD1434Hor5MWG938Act8AMWG837BABC165CABA004Bmac399GBM1007BCD098ABG053Ica1GBM1042JS074HvM020HvALAATBmag211Pcr2GBM1051HvGLUENDGlb1Bmac032ABC160scssr10477DAK123BMWG518PSR330Bmac144ABmag382MWG706AKFP170HvHVA1BCD1930BlpABC261MWG2028WMC1E8ABG387A

1H

Locus Allele Size (bp) No. accessions Leaf rust Hairy sheath Spike

density Spike length

(cm) 1 109 9 7.6 a 1.2 c 4.0 bc 3.3 ab 2 127 13 7.2 a 1.2 c 3.9 c 3.6 a 3 131 9 5.7 bc 1.7 a 4.2 bc 3.8 a 4 137 16 5.7 c 1.6 ab 5.4 a 2.9 b 5 141 23 7.0 a 1.4 bc 4.9 ab 3.6 a

Bmac399

6 151 8 7.1 ab 1.2 c 4.2 bc 3.6 ab

Locus Allele Size (bp) No. accessions Heading date Lodging

1 217 13 120.1 ab 4.7 bc 2 227 14 119.4 bc 5.0 bc 3 229 20 119.5 b 4.5 c 4 231 29 118.6 bc 5.1 bc 5 233 12 119.2 bc 6.2 a 6 235 11 117.7 c 5.6 ab 7 237 14 119.7 bc 5.1 bc

Bmac032

8 239 13 122.0 a 4.5 c

Locus Allele Size (bp) No. accessions Row number 1 126 10 5.0 a 2 134 129 5.0 a scssr10477 3 nulo 17 4.0 b

Bin Grain yield Heading date 1000 kernel weight Screening Leaf rust Row

number Glume &

glume awn

Lemma type Genes

1 6 49

2 1 6

3 3 29 30 8 3 12 2 21 6 12 B Rph1

4 3 29 30 16 30 3 12 4 9 13 15 16 17 18 17 22 30 13 12 Ppd-H1

5 2 29 22 1 2 17 22 17 13

6 1 29 22 10 19 17 22 1 14 17 21 13 20 53 54 C rph16

7 20 29 22 10 17 5 19 18 20 32 40 17 22 18 21 32 17 21 18

8 8 29 21 1 10 8 5 19 27 12 26 39 51 H 17 1 18 15 17 21 I Eam6, eog

9 8 30 26 3 13 8 9 18 10 17 26 17 5 18 13 24 25 30 13 F J Gth, vrs1

10 3 5 3 13 ant2

11 3 5 3 17 Pau

12 3 3 31 25

13 21 28 1 8 15 2 28 13

14 2 2 48

15 2 1 7 7 16 25 45 48 12

MWG844AABG058Bmac134ABG703BChs1BMWG878AHVM036ABG318MWG858ABG358scssr07759PoxMWG663Bgg60CDO537ABC468EBmac684Bmag378EBmac640Bmac093Bmac132HvBKASIABC451His3CMWG865vrs1MWG699Bmag125MWG503ABG072KsuD22ABC252HvM054ABC165zeo1

MWG844CCDO036B

2H

Locus Allele Size (bp) No. accessions Heading date Screening (%)

1 130 68 119.5 b 3.7 cd2 138 9 123.3 a 9.4 a 3 148 11 120.3 b 4.3 bcd 4 156 9 116.8 c 5.3 bc 5 158 11 115.7 c 3.4 cd 6 162 8 120.1 b 6.7 b 7 166 9 120.4 b 4.2 cd 8 168 12 119.7 b 2.7 d

Bmac134

9 172 10 120.1 b 4.0 cd

Locus Allele Size (bp) No. accessions Row number 1 110 57 5.0 a2 114 61 5.0 a 3 122 8 4.0 b

HvM036

4 134 16 5.0 a

Locus Allele Size (bp) No. accessions Grain yield (q.ha-1) Headingdate Lemma type

1 195 7 23.0 b 122.0 a 2.2 b2 213 38 24.6 a 118.3 b 2.6 a scssr07759 3 221 106 23.2 b 120.2 a 2.5 a

Locus Haplotype No. accessions

1000 kernel weight (g)

Glume & glume awn

Length of rachilla hairs

Row number

A 62 39.3 a 2.1 a 1.2 c 5.0 aD 85 37.7 b 1.8 b 1.3 b 5.0 a MWG699 K 9 42.2 a 1.9 ab 1.9 a 3.0 b

Locus Allele Size (bp) No. accessions Screening (%)

1 134 27 5.5 a2 136 91 5.7 a 3 138 13 3.6 b

Bmag125

4 140 21 3.1 b Locus Allele Size (bp) No. accessions Length of rachilla hairs Row number

1 149 27 1.7 a 5.0 a2 157 91 1.4 b 5.0 a 3 159 13 1.6 a 5.0 a

HvM054

4 161 21 1.6 a 4.0 b

Locus Allele Size (bp) No. accessions Heading date

1 217 12 121.3 a 2 219 103 120.1 a HvLTPPB 3 221 41 117.3 b

Locus Allele Size (bp) No. accessions Lodging 1 164 23 4.5 c 2 170 12 6.1 a 3 172 18 4.2 c 4 182 25 5.5 ab 5 196 16 5.3 ab 6 198 8 5.1 bc

Bmag006

7 200 22 4.3 c

Locus Allele Size (bp) No. accessions Stem pigmentation 1 149 22 1.7 ab 2 153 9 1.7 abc 3 155 36 1.9 a 4 157 11 1.4 cd 5 159 10 1.8 ab 6 161 18 1.3 d

Bmag013

7 163 18 1.5 bcd

Locus Allele Size (bp) No. accessions Row number 1 169 44 5.0 a 2 173 10 4.0 b Hv13GEIII 3 175 101 5.0 a

Locus Allele Size (bp) No. accessions Heading date

1 247 43 118.6 b 2 251 27 120.2 a HvM062 3 255 83 120.8 a

Bin Heading date Lodging Stem pigmentation Genes

1

2 8

3

4 5

5 5

6 5 8 3 15 3 20

7 8

8 8

9 3

10

11 15 1 2 7 9 18 19 20 40 Z sdw1

12 15 41

13 4 8 41

14 4

15 9

16 9 46 N M ant28, eam10

ABG070BCD907ABG655AJS195FHvLTPPBABC171AABG321ABA303MWG798BABG460BCD1532BCD706almABG396Bmag136Bmac067Bmag006Bmac209ABC325MWG571BABC176ABG377Bmag225ABG453Act8CCDO345ABG499CDO113BHis4BABG004Bmag013ABC161MWG883Hv13GEIIIABC174HvM062ABC166Bmac029ABC172

3H

Locus Allele Size (bp) No. accessions Glume and glume awn

1 150 19 1.8 ab 2 158 16 2.3 a 3 160 13 2.0 a 4 162 37 2.1 a

Bmag500

5 164 17 1.6 b

Locus Allele Size (bp) No. accessions Grain yield (q. ha-1)1 172 11 24.7 a 2 176 12 22.4 b 3 178 120 24.8 a

Bmag009

4 180 10 24.8 a

Locus Allele Size (bp) No. accessions Hairy sheath 1 172 58 1.4 b

EBmac806 2 nulo 82 1.6 a

Locus Allele Size (bp) No. accessions Spike length (cm) 1 176 15 3.3 c 2 218 9 3.1 c 3 222 20 3.5 bc 4 224 8 3.5 abc 5 226 14 3.4 c 6 228 21 3.9 a 7 230 17 3.8 ab

Bmac040

8 232 13 3.3 c

Locus Allele Size (bp) No. accessions Heading date

1 138 7 117.4 b2 140 53 120.5 a3 142 55 118.6 b

Bmac018

4 144 40 118.4 b

Bin Grain yield Heading date Genes

1 3

2 3

3

4

5 27 eam7

6 11 29 24 47 9 1 10 43 27 T eam7

7 11 29 3 26 9 1

8 11 29 9

9 2 1 2 17 18 20

10

11

12 9

13 9 lax-c

14 9 dsp9

MWG620Bmac316

ABG378

Bmag500MWG625AMWG602BDD1.1CJS10AGBM1021ABG387BGBM1068Ldh1HvM031Bmag173Bmac018Bmag009ABG474ABG388EBmac806MWG820ABC170Nar7GBM1008MWG934Tef1DAK148GBM1022Bmac040ABC807AABG713MWG514MWG798A

6H

Locus Allele Size (bp) No. accessions Heading date

Glume and glume awn

1 185 31 118.6 a 2.2 a 2 187 62 118.5 a 1.8 b Bmag3693 189 53 117.0 b 1.8 b

Locus Allele Size (bp) No. accessions Spike length (cm)1 194 18 3.3 bc 2 198 13 3.5 ab 3 200 15 3.1 c 4 202 31 3.6 a 5 248 10 3.6 ab 6 252 12 3.7 a

HvM004

7 256 11 3.4 abc

Locus Allele Size (bp) No. accessions Spike density Spike length (cm)1 230 10 4.3 bc 3.5 ab 2 232 8 4.6 b 3.8 ab 3 234 21 4.3 bc 3.4 bc 4 236 48 4.0 bc 3.6 ab 5 238 8 4.7 ab 3.0 c 6 244 9 5.8 a 3.2 bc 7 262 9 4.5 b 3.4 abc 8 264 21 4.3 bc 3.5 ab

Bmag120

9 268 8 3.3 c 3.9 a

Bin Heading date Spike length Genes

1 2 W brh1

2 3 4 9 6 17 20

3 3 10 9 34 39

4 3 10 9 4 5

5 6 10 31 4 5 8 16 28

6 8 10 31 55 X dsp1

7 3 10 36 23 16

8 3 14 36 16

9 3 14

10

11 8 9

12 8 9 31 Y ibi3

ABG704ABG320Bmag206Bmag007HvM004WaxyABC151AAW982580CDO475MWG089ABG380ABC158ksuA1AHvSS1ABC255ABC165DHvCMAABG701MWG808MWG2031Bmac064nudBmag369Amy2Iks2ABC1024RZ242Bmag120WG380BABC310Ris44ABC305ABG461AWG380AGBM1065Bmac156KFP255HvGLB2ThA1Bmag135

7H

Genome-wide Association in BarleyYahiaoui S1, AM Casas1, MA Moralejo2, B Medina1, MP Gracia1, FJ Ciudad3, P Codesal3, JL Molina-Cano2, JM Lasa1, E Igartua1

1Department of Genetics and Plant Production, Aula Dei Experimental Station, CSIC, PO Box 202, E-50080 Zaragoza, Spain2Centre UdL-IRTA, Lleida, Av. Rovira-Roure 191, E-25198, Lleida, Spain3ITA, Instituto Tecnológico Agrario, Junta de Castilla y León, PO Box 172, E-47071 Valladolid, Spain

ssociation mapping (AM) deserves investigation as another method for QTL detection in crops. We testedthe feasibility of carrying out association analysis between several agronomic and morphological traits in a

set of landrace-derived inbred lines of the Spanish Barley Core Collection (SBCC). A subpopulation structure was detected, and taken into account in the analyses, which reduced the number of significant marker-trait associations by 5-fold. Some associations found were coherent with findings in biparental populations.

ssociation mapping (AM) deserves investigation as another method for QTL detection in crops. We testedthe feasibility of carrying out association analysis between several agronomic and morphological traits in a

set of landrace-derived inbred lines of the Spanish Barley Core Collection (SBCC). A subpopulation structure was detected, and taken into account in the analyses, which reduced the number of significant marker-trait associations by 5-fold. Some associations found were coherent with findings in biparental populations.

AA

Figure 1. Description of significant marker-trait associations found in 159 Spanishinbred lines, with 73 markers. Alleles and effects are shown on the righthand sidetables; genes and QTL found in 55 populations for the same traits are shown at thelefthand side tables. List of genes and populations are presented in the addendum. Shown only significant associations in a multilocus model for each trait.

Figure 1. Description of significant marker-trait associations found in 159 Spanishinbred lines, with 73 markers. Alleles and effects are shown on the righthand sidetables; genes and QTL found in 55 populations for the same traits are shown at thelefthand side tables. List of genes and populations are presented in the addendum. Shown only significant associations in a multilocus model for each trait.

Table 1. Distribution of genotypes in four Model-based populations.

Populations inferred with StructureGermplasm groups Number of entries Pop. 1 Pop. 2 Pop. 3 Pop. 4

Spanish 6-row 152 18 0 49 85 Spanish 2-row 11 0 9 1 1 Reference 6-row 33 31 2 0 0 Reference 2-row 29 6 23 0 0

Acknowledgments: This study was funded by INIA project RTA01-088-C3, including ERDF funding.