The stages of physical mapping of Triticum aestivum cv CS

chromosome 5B

Sergeeva E.M.1, Timonova E.M.1, Bildanova L.L.1, Koltunova M.K.1,

Nesterov M.A.1, Magni F.2, Dolezel J.3, Faris J.4,

Sourdille P.5, Feuillet C.5, Salina E.A.1

1Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia2Institute of Applied Genomics, Udine, Italy 3Institute of Experimental Botany, Olomouc, Czech Republic4USDA-ARS Cereal Crops Research Unit, Fargo, USA5UMR INRA-UBP Genetics, Diversity and Ecophysiology of Cereals, Clermont-Ferrand, France

(Sorrells et al., 2003) 

5B chromosome

5BL 580 Mbp

5BS 290 Mbp

Steps of 5BS and 5BL physical mapping

In progress

In plans

Finished

2010

2011

2012

BAC library of 5BS (J. Dolezel, IEB)

Cytogenetic mapping of 5BS-deletion lines, introgressive lines

BAC library replication

Construction BAC library of 5BL in the end 2013 (J. Dolezel, IEB)

2013

2D POOLING

454 sequencing of 5.5% 5BS and 8.8% of 5BL, developing ISBP markers

ISBP and SSR mapping on genetic map and 5BS pool screening

Fingerprinting and preliminary MTP

BAC-end sequencing for non anchored contigs

Contigs anchored on maps

2012

2014

Steps of 5BS and 5BL physical mapping

In progress

In plans

Finished

2010

2011

2012

BAC library of 5BS (J. Dolezel, IEB)

Cytogenetic mapping of 5BS-deletion lines, introgressive lines

BAC library replication

Construction BAC library of 5BL in the end 2013 (J. Dolezel, IEB)

2013

2D POOLING

454 sequencing of 5.5% 5BS and 8.8% of 5BL, developing ISBP markers

ISBP and SSR mapping on genetic map and 5BS pool screening

Fingerprinting and preliminary MTP

BAC-end sequencing for non anchored contigs

Contigs anchored on maps

2012

2014

5BS = 290 Mbp

39695 reads16183252 bpmean = 475 bpN50 = 490 bp6% of total arm length

110793 reads 45307076 bpmean = 466 bp  N50 = 484 bp8.8% of total arm length

454 sequencing of chromosome 5B and development of ISBP markers

5BL = 580 Mbp

Analysis of 454-sequences. Identification of repetitive sequences i) no long GAA/GGA tracts and 454‐ reads consisting of GAA/GGAii) no telomeric repeat (TTTAGGG) sequence tracts

Content of different Transposable Element families

compared the 5BS and 5BL 454-data with 454-sequencing data on 5AS and 5AL (Vitulo et al., 2011) i) differing content of centromere associated (Li et al., 2013) Cereba

and Quinta retrotransposons between short and long armsii) the prevalence of retrotransposons Fatima and Sakura and DNA-

transposon Jorge in 5B chromosome comparing to 5A

Li B, Choulet F, Heng Y, et al. Plant J. 2013;73(6):952-65. Vitulo N, Albiero A, Forcato C et al PLoS One (2011) 6(10):e26421.

Mapping 454-reads to rice and Brachypodium chromosomes

BLASTn31029 reads matched with Brachypodium and 29952 matched with rice.

Results are in agreement with published data on EST mapping of wheat 5B chromosome to the genomes of these grasses (Linkiewicz, 2004, The International Brachypodium Initiative, 2010).

We assigned the mapped 454-reads to Gene Ontology annotations (http://www.geneontology.org/). Our results showed the similar pattern with chromosome 5A (Vitulo et al., 2011).

Functional assignment of 5B 454-reads

ISBP development and bioinformatical analysis

Comparison of ISBP marked 454‐reads with bread wheat DNA sequences from databases.The matching of 454‐reads of 5B chromosome with the bread wheat genomic sequences (E‐value  < 1e‐50).

5BS: Total analyzed1302154 reads matched (12%)From them 6 5BS-specific

5BL:Total analyzed 3923421 reads matched (11%)From them 96 5BL-specific

No data on chromosomal localization (89)

Mapping of SSR and ISBP markers on 5BPopulation – F2 from cross Chinese Spring and the disomic substitution line CS-5B dicoccoides CS –Dic5BxCSPlants – 366 crossing

ISBP marker AOON0_1

CS –cultivar Chinese Spring, CS-dic – disomic chromosome substitution line in which the 5B chromosome of CS was replaced by the 5B chromosome from T.dicoccoides, F1 – F1 plants from crossing CS×CS-dic, DT5BL – di-telosomic line for the 5B long arm, N5BT5A – nulli-tetrasomic line, F2 – F2 population from crossing CS×CS-dic.

62% of SSR and 7.5% of ISBP revealedpolymorphism between CS and CS‐5Bdic

SSR markers are more polymorphic between chromosomes 5B of CS and T.dicoccoides

AC931_2 BYRXJ_1 AML1U_2

Xwmc118

Xgwm814

Xgwm540

Xgwm0234b Xwmc740Xbarc340

5BS8-0.56-0.675BS1-0.67-0.71

Xwmc430

Xbarc243

Xwmc415Xgpw7346

Xbarc16

Xgwm271

Xbarc216

Xbarc232

Xbarc156Xwmc235

Xmag532

Xwmc376Xbarc4

5BS6-0.81-1.0

5BS4-0.43-0.56

5BS3-0.41-0.42

5BL11-0.59-0.66

5BL18-0.66-0.79

5BS5-0.71-0.81

5BS2-0.42-0.43

5BL16- 0.79-1.0

5BL1-0.55-0.59

Xmag705Xwmc149 Xgpw4098

Xgwm0234а

Xwmc363

Xgwm159

Xgpw4362

cpf341 (Skr)

Xgpw4463

Xbarc88

Xgpw358Xgpw8015

Xbarc21

Xbarc331

Xgwm213

Xgwm499

Xbarc74 (Ne1)

Xgpw3022

X1051/3 (Ph1)

TaCBFIIIc-B10

Xwmc289

Xgwm408

Xgwm497

Xpsp3037Xgpw3247

Vrn-B1

С-5BL1-0.55

C-5BS3-0.41

Xgwm118

Xwmc160

Xbarc59

Chi-B1

Xbarc308 Xwmc640

Xgwm605Xgpw5238

Xwmc682

Xksum69

Xwm133

Xcfe204 Xcfe229

С

cpf 4726 BSS74_1 AY4TI_1 AWE4U_1 A0Y4Y_1 BW7KR_1 A5AJD_1 AJ1NA_1 A2Q6Q_1 BG1R0_1 BJRIW_1 AJ5KT_1 AUW9J_1 APXYP_1 BOCLC_1 ABL9P_1 AD2SI_1 BQTB6_1 A2Z1J_2 AOL4L_1 AP9RD_1 B3SIZ_2 BLYF6_1 AKNFS_1BUW2W_3 BUN0O_1 A2Q6Q_1 A0S5G_1 BJRIW_1 ACXWN_1 AUHIM_1 A7ICL_1 BGOTC_2 AOON0_1

AC0Z2_1 AC0Z2_2 AV8ST_1 AE7GL_1 AIARO_1 BOU1R_2 APG9K_1 AID3Q_2 ASGRZ_3 A70JW_1 AB79U_2 AF0QM_1 APHKH_1 ANDOT_1 AVGCI_1 AG158_1 AWRK2_1 BA6UH_1 AFJ2U_1 B3CV6_1 AN9TG_1 AOY8Z_1 AWBUE_1 A70JW_1 AOOR7_1 AWBUE_2 AKOEF_1 AEIZG_1 A1X2X_1 AH0MV_2 A67VT_1

ATIVH_3 A17I8_1 AQGIL_1 A17I8_1 AMK1V_1 ABVHI_1 BEOR3_1

A5PS7_2 AIZ3E_1 A7T7G_2 BLWEB_2 ARLT4_1 AZUNB_1 BL2IX_1 AUHIM_2 APHFQ_2 AV49Y_1 ACRLC_1 ANJOM_1 ANGMC_1 AG4ZP_1 AM6WT_1 A7G3Q_1 AUAZX_3 BM1LN_3 A59RN_1 AI3A5_1 B2EE4_2 A27AD_1 AFWPE_1 A2EPP_1 ADZ88_1

AZNVV_1 AJYP0_2 AWE0Y_1 BQ1RV_1 A5ZC0_2 BT16D_3 A9463_1 AKEU0_2 A3O3X_1 APUAW_1 BAMR9_1 ALPC5_1 A7K8R_1 AEIBU_2 BHD3N_1 AAZQP_1 A1BXO_1 AKEU0_2 AC4L4_1 AYMJZ_1 A9463_1 AEIBU_2 AM6WT_1

B1K5O_1 AY4TI_1 A8FW8_1 AWOUG_1 ANWO3_2 BOYEL_1 ALSM8_1 AR693_1 A4B4U_1 A1VUC_1 AQ8AO_1 AEIBU_1 A525N_1 ABCWO_1 BSAI1_3 AQ8SF_1 A4YBI_1 ADV6O_1 A94EL_1 B2QFP_1 ATLVD_1 ADCY1_3 ADDI1_1 BRN68_1 BF9C0_1 BFGR6_1

AG4ZP_1 ASJWC_1 BOBR2_1 BMW9G_1 AKNFS_1 AGL35_1 AZEBG_1 AHXK3_2 BXNGH_1 BYYLW_2 AKJ8Z_1 BLPMC_1 BJFJH_1 AZSAJ_1 AT41B_1 ATC1D_2 A6S5N_1 BDDB7_1 AQY49_2AVC42_2 AOZCL_2 AK6D4_1

5B 5BS

Deletion map of wheat chromosome 5B

51 SSR markersAnd 6 genes on 5B

ISBP markers on 5BS

Deletion lines CS Introgressive lines - Saratovskaya 29, Novosibirskaya 29, Rodina

Introgressive region from wild species

Markers – 51 Genes - 7

L5366-180 (1) L21-4 (2) L73/00i (2)L842 (3)

Xgpw3247

Xgwm1043 Xgwm777Xgwm408 Xgwm271Xwmc289

Xgwm0213

Xgwm0371Xgwm639

Xgwm1246a Chi-B1Xgwm1072b Xgwm1016Xgwm0814_(LrTt2)Xgwm0880 Xgwm1257

Xgwm1043Xgwm777

Xgwm0213

Xgwm639Xgwm0371

Xgwm408Vrn-B1 Vrn-B1

Xgwm0604 Xgwm1246a Chi-B1Xgwm1072b Xgwm1016Xgwm0814 _(LrTt2) Xgwm0880 Xgwm1257 Xwmc235 Xwmc430 Xbarc156 Xbarc308 Xwmc640 Xgwm605 Xgpw5238 Xbarc232

Xgwm0213

Xgwm0371(3)Xgwm639 (3)

Vrn-B1 Xgwm408

Chi-B1 Xgwm0814 Xwmc235 Xwmc430 Xbarc156 Xbarc308 Xwmc640 Xgwm605 Xgpw5238 Xbarc232Xcfe204 Xcfe 229

Chi-B1

Xgpw3247 Vrn-B1 Xgwm408Xwmc289

Xgwm271Xwmc415

*TaCBFIVd22-B22

5BL-15BL-11

5BL-18

5BL-16Xwmc160 Xwmc118 Xbarc59Xgwm118

Xbarc243Xgpw7346Xpsp3037Xgwm497

Xbarc21

*TaCBFIIIc-B10

5BS-6

5BS-55BS-1

5BS-85BS-4

5BS-35BS-2

Xwmc363Xgpw4362

Xwmc682

Xgwm159 Xwmc376Xgwm540 Xbarc4

Xgwm234 Xwmc740 Xbarc216 Xbarc340Xmag705 Xbarc16 Xgpw4098 Xwmc149, SKr(4)

1. Timonova et al., 2012; 2. Adonina et al., 2012; 3. Leonova et al., 2011, 4. Alfres et al., 2009

Xksum69 Xbarc88Xgpw8015 Xgpw0358 Xgwm133 Xgwm213 Xbarc74_(Ne1) Xgpw4463 Xgpw3022 Xwmc759 Xmag532 Xbarc331 Xgwm499X1051I3_(Ph1)

Deletion map of wheat chromosome 5B

Xcfb341Xgwm234

Xbarc216Xbarc340Xgwm540Xbarc4Xwmc376Xbarc88Xgpw8015Xgpw358Xgwm133Xgwm335Xwmc759Xgwm639Xwmc415Xgpw3247Xgwm271

Xbarc232Xgpw4483Xwmc235Xgpw5238Xwmc118Xwmc430Xbarc59

4,1

9,43,43,41,20,91,31,20,71,92,85,34,71,58,5

2,0

18,2

1,53,24,72,44,52,4

5BBLYF6_1Xcfb341Xcfp4727Xgwm234AXMZZ_1BLWEB_2ACN7Q_1 ASJ96_1A2Z1J_2BYRXJ_1AUHIM_2B2QMF_1A5AJD_1AH0PA_1AUHIM_1AB79U_2A1X2X_1AC0Z2_2ANDOT_1AM6WT_1Xbarc340AEIZG_1APG9K_1 A67VT_1A5C5U_1AF0QM_1AOOR7_1Xgwm540Xbarc4AH0MV_2Xwmc376AKOEF_1BT16D_3BRN68_1BFTLX_1A1VUC_1AKJ8Z_1AD81G_1 AWLK5_1BCT8G_1 ADY4B_1BT08L_1 AVC42_2

1,70,44,62,82,01,60,93,11,71,50,60,40,81,90,91,01,80,70,20,60,90,70,81,02,01,30,90,20,11,00,80,50,30,50,52,22,0

5BS

1,4

AN9TG_1

0,8

Xbarc88

С

С

The genetic map for chromosome 5B was constructed with the help of the MAPMAKER v. 3.0b using the Kosambi mapping function at LOD 3.00

6 SSR markers and 39 ISBP markers24 SSR markers

cM cM

Genetic map of wheat chromosome 5B

Fingerprinting and processing of data

BAC‐library of 5BS chromosome (length 290 Mb) (IEB, Olomouc, Chezh Republic) J.Dolezel)

TaaCsp5BShA – 114 plates, 43776 clones, main insert size 122 Kb, coverage 15,8

Fingerprinting and processing(IGA, Udine, Italy)

● BAC‐DNA minipreprs● Hydrolysis and SNaPShot labelling● Gel electrophoresis ABI3730● GeneMapper processing

SizingQuality passed34463 clones (78,7%)

● FPB processing● GenoProfiler removing controls and contaminated clones 

Contaminated  279 clones (0,9%) + 13 removed controls

FPB sizes 32575 clones (5,5% no passed)

FPC Gel Entries – 32283 clones

FPC assembly statistics

FPC Gel Entries 32283 clones

FPC Bands 3315498

Bands/clone 102,7

5BS BAC-library 2D-pooling

Stage 1.114 plates

Stage 2. 15 96‐well plates

Stage 3.1,5 96‐well plates

1 well = 2 columns of matrix plate

1 WELL = 1 MATRIX PLATE

BAC library PCR-screening

Xcfb341Xgwm234

Xbarc216Xbarc340Xgwm540Xbarc4Xwmc376Xbarc88Xgpw8015Xgpw358Xgwm133Xgwm335Xwmc759Xgwm639Xwmc415Xgpw3247Xgwm271

Xbarc232Xgpw4483Xwmc235Xgpw5238Xwmc118Xwmc430Xbarc59

4,1

9,43,43,41,20,91,31,20,71,92,85,34,71,58,5

2,0

18,2

1,53,24,72,44,52,4

5BBLYF6_1Xcfb341Xcfp4727Xgwm234AXMZZ_1BLWEB_2ACN7Q_1 ASJ96_1A2Z1J_2BYRXJ_1AUHIM_2B2QMF_1A5AJD_1AH0PA_1AUHIM_1AB79U_2A1X2X_1AC0Z2_2ANDOT_1AM6WT_1Xbarc340AEIZG_1APG9K_1 A67VT_1A5C5U_1AF0QM_1AOOR7_1Xgwm540Xbarc4AH0MV_2Xwmc376AKOEF_1BT16D_3BRN68_1BFTLX_1A1VUC_1AKJ8Z_1AD81G_1 AWLK5_1BCT8G_1 ADY4B_1BT08L_1 AVC42_2

1,70,44,62,82,01,60,93,11,71,50,60,40,81,90,91,01,80,70,20,60,90,70,81,02,01,30,90,20,11,00,80,50,30,50,52,22,0

5BS

1,4

AN9TG_1

0,8

Xbarc88

С

С

24 SSR markers

MAPMAKER v. 3.0b using the Kosambi mapping function at LOD 3.00. Common markers located are boldfaced and colored. C – centromere. Connections between common markers are indicated by dotted lines.

6 SSR markers and

39 ISBP markers mapped,

9 ISBP and 1 SSR used for

anchoring

Genetic map of 5BS

Conclusions

- The physical mapping of 5BS is in progress:1) for saturation of genetic map we developed new 88 SSR

markers on the base of 5BS 454-sequences,2) for include more ISBP markers we will use Real Time PCR and

melting curve analysis 3) for optimization of BAC-library screening we will add to the

PCR/agarose analysis the Real Time PCR4) BAC End Sequencing of non-anchored BAC-contigs, and their

mapping

- To finish the physical map of 5BS to the March of 2014- To start construction of physical map of 5BL from the April 2014

Plans

Participants of 5B project

Institute of Cytology and Genetics SB RAS, Novosibirsk, RussiaSalina E.A. organization and coordination of work.

Sergeeva E.M. work with BAC library, fingerprinting, construction of physical map

Timonova E.M. construction of genetic and cytogenetic map

Bildanova L.L. ISBP PCR-testing

Koltunova M.K. development of markers, bioinformatical data analysis

Nesterov M.A. BAC library screening

Institute of Applied Genomics, Udine, Italy Magni F., fingerprinting, FP analysis

Institute of Experimental Botany, Olomouc, Czech RepublicDolezel J. construction of BAC library

USDA-ARS Cereal Crops Research Unit, Fargo, USAFaris J., development of F1 hybrids for CSXCS-5B dic

UMR INRA-UBP Genetics, Diversity and Ecophysiology of Cereals, Clermont-Ferrand, FranceSourdille P., Feuillet C., Poncet C. 454- and ISBP data

AcknowledgementInstitute of Cytology and Genetics SB RAS, Novosibirsk, RussiaAfonnikov D.A. for help in bioinformatical data analysis

Department of Plant Pathology, Kansas State University, USAGill B.S. for seeds of 5B chromosome deletion lines and CS-5B-dic