Towards Expression Maps in

Chickpea

Günter Kahl, Carlos Molina, Ruth Jungmann, Ralf

Horres, Boulbaba L`Taief, Jörg Geistlinger, Dirk
Fischer, Peter Winter, Björn Rotter
University of Frankfurt & GenXPro Ltd
Frankfurt am Main, Germany
Chickpea
Genome Sizes of Legumes (1C, Mbp)
• Lablab niger 368
• Prosopis alba 416
• Lotus japonicus 466
• Medicago truncatula 454-526
• Lupinus albus 588
• Cicer arietinum 738
• Glycine max 1115
• Melilotus alba 1299
• Lotus cytisoides 1372
• Arachis sylvestris 1470
• Lens culinaris 4063
 Non-genic Space Genic Space
Satellites
• Minisatellites
Microsatellites
• Ribosomal RNA genes
• Pseudogenes • RNA-encoding genes

• Retropseudogenes • Regulatory genes

Retrotransposons Signal transduction genes

LINES • Housekeeping genes

• SINES Defense genes

Ty3 copia elements • Scavenger genes
DNA transposons • Membrane protein genes
• Centromeres
• Telomeres
• Spacer DNA
 Chromosome Model of Cicer arietinum

18S-5.8S-25S rDNA
5S rDNA
CaSat1
CaSat1/18S-5.8S-25S rDNA
CaSat2
CaRep
CaRep/ CaSat2
CaTy
CaDis
CaLin
CaEn/Spm
E Telomeres

A B C-G H
 Generation of Locus-specific Microsatellite
(STMS) Markers

Chickpea Cultivars
Abundance of simple sequence repeats (SSRs)
1........5.........10..........17
in the chickpea genome : MW
(Bp)

255

(TAA)n >> (GA)n > (CA)n

227

Development of STMS markers

Sequenced Microsatellite-Containing Clones: 425
191
Design of Flanking Primer Pairs: 310 (73 %)
PCR Products of Expected Size: 267 (62 %) (GA)n
STMS Marker Map of the Chickpea Genome
 Integrated Genetic Map of Chickpea:
Localization of Candidate R-Genes
LG 1 LG 2 LG LG LG 5 LG 6 LG 7 OPT12-1 LG 8
3 4 OPO04-1 ISSR8682
STMS13 R360-8-3 OPC11-2 STMS20
TR19 4.0 OPC15-1 OPA12-1 3.6
ECAMCAT01 STMS8 EAAMCTT02
OPU03-5 7.6 OPP15-2 11.
15.1 1.9 14.1 EAAMCTT06 15.3
21.4 2.9 OPC06-1 4.7 RGA-A 4 5.8 13.5
OPI06-3 OPP17-1 2.4 S-SAP E4-1 ISSR8231 ISSR889 S1E1
7.1 OPO04-2 8.6 3.6
17.8 5.0 OPP15-1 10.0 OPC20-1 EACMCAC08 2.3 ECAMCAG07 ISSR8843 1.8 OPT12-4 11.4
OPD05-1 4.7 1.0 6.1
8.2 OPJ13-2 2.1 OPC14-5 4.7 ECAMCAG06 23.6 OPA14-1 OPJ13-1 GAA46
OPU03-4 R360-8-1 7.0
TA113 OPD03-1 3.1 2.9 ECAMCAT13 7.0
15.9 OPA17-2 9.7 GA31 ECAMCAG05 7.5 ECAMCAG03
11.4 13.6 STMS11 10.6 STMS22
17.7 OPD03-4 OPC15-4 16.3 TS19 STMS6 GA24 OPG09-5
7.9 9.4 9.4 EACMCAC12 10.9 15.2 2.6 IGR3RX2A
ACONa OPC14-4 ISSR8561 GA47 6.4
9.6 0.6 OPAB09-5 13.3 EAAMCTA06 4.0 EAAMCTT08 ECAMCAT12 25.0
8.9 R260-07-2 2.2 3.9
5.8 ACONb OPU18 STMS15 1.0 EAAMCTA02 5.0 ISSR8591 2.2
TA130 OPU17-2
23.7 EAAMCTA07 8.5 0.6 7.0
OPD03-3 6.2 ISSR8262 TA14 1.0 OPC183 GA2 OPQ13-3 G6PD
7.6 TR44 4.8 ECAMCTA01 10.1 4.8
OPG09-1 7.3
STK11 14.4 EACMCAC07 EAAMCTA13 TS35 4.7 ACONc
5.8 EACMCAC01 1.6 STMS10 4.9 10.2
9.6 OPQ13-2 16.1 GA16 1.6 1.2 S-SAP DR22 EAAMCTT01 OPAB09-3 TS45
14.1 ECAMCTA04 STMS14 7.1 6.3
ISSR8481 8.6 REPT10 1.6 0.9
ECAMCTA11 ECMCAC03 15.5 ECAMCAG04 OPQ11
8.0 ISSR8841 10.1 3.6 ECAMCTA07 0.6 STMS28 2.9 10.6 REPT11 ECAMCAT08 9.6
S-SAP AR1 11.2 ECAMCTA02 1.2 TA135 3.3 ECAMCAG01 3.2 TR20 PGMb
11.0 STK 42Eh 5.6 ISSR8553 4.8 4.3 7.0
1.7 RGA-C
STK 24Ev 1.6 CS27 EAAMCTA08 TR2 ECAMCTA06 4.3 TA13 8.9 12.6
1.6 3.9 2.5 OPN06-2
2.9 ECAMCAT10 2.0
4.1 R260-09-1 2.7 EACMCAC04 0.8 TR31 ECAMCAT05 2.2
1.1 TA146 GA4 EAAMCTA11
2.8 Foc4 1.1 0.7 TA34 18. ECAMCAT02 TA72 8.5
10.2 TR43 1.5 EACMCAC10 1.9 0.7 OPC06-2
2.9 TA1 0.5 OPU17-1 3.3
ISSR8402 2.0 STMS4 6 ECAMCTA12 2.3 TA2 5.6 OPT12-2
17.5 24.1
2.6 TA8 1.0 OPP06-4 TR1 2.3 STMS23 GAA44 4.4
STMS26 5.9 OPP07-1
1.0 1.9 10.0 1.5 TR56 12.8 4.0 3.4
REPT3 2.4 OPP15-3 4.6 IGR5F1 2.2 TA21 3.8 TS72 TA5
2.1 AF0 OPP08 STK 25B 2.6 ECAMCAT06 TA78 2.9 EAAMCTA14
4.4 0.9 0.1
S-SAP AR4 8.5 6.5 ISSR8401 3.3 STMS19
1.8 ISSR858 2.3 OPM20X 1.7 R260-7-1 5.2 1.6 REPT8 6.2 ISSR8902 RGA-B 15.6
3.5 5.7 S-SAP AR5 11.1
6.1 OPU17-3 1.5 TA96 OPN06-4 4.5 ECAMCTA03 3.9 IGR3R1 TA3
2.3 REPT6 1.7 7.4 BIGR5F1A 3.6 IGR3X4A
20.3 R260-9-2 6.6 2.1 OPD03-2 14.1 EAAMCTT04 1.3 OPS13-2 IGR3R4 9.0
S-SAP AR6 S-SAP FF2.3 IGR3RX1A 6.0
EACMCAC06 5.3 12.9 S-SAP F4.1
OPC11-3 3.5 1.8 IGR3R3 1.8 OPD16-1 OPB08-1
R260-9-3 1.4 EAAMCTA12 1.0 AF2 3.3 TA28 9.5
7.9 12.7
2.7 ECAMCTA07 3.9 AF00 21.7 S-SAP BR 17.6 OPC14-2 STK86-D
TA203 BIGR5F5 4.3 S-SAP C41 9.3 TS62 4.9 RGA-D2a TS12
16.9 4.3 S-SAP E4.2 OPT12-5
0.5 Foc5 1.1
S-SAP DR23 IGR5F2 4.7 TAA58 1.9 OPB08-2
2.1 PGMa 0.8 TAA59 OPT13-3 0.8
ISSR888 1.1 TA27 TR7 10.4 13.4 0.6 BIGR5F2A
2.3
10.3 TA59 10.7
TA80
PGD6
C33
12.3 TA140 2.2 TS43 165.8 cM
17.7 3.4 CS27ASAP 9.7 TA18 OPT12-3 3.8 TS53
TA22 ISSR8903 0.8
0.1
ISSR8552 1.2 TS82 15.7 4.8 OPT18-2 TA179
S-SAP F4.4 4.7 TA176 7.2 GA13 22.4 3.1
13.8 7.1 TAA60 S-SAP DR21 ECAMCTA08 7.8 OPC20-2 3.7 TR59
IGR3R6 10.7 TR58 0.6 STMS7
2.9 ECAMCTA09 4.6
S-SAP AR2 14.5 TA64 IGR5F3 EAAMCTT03
2.2 TA194 19.2 ISSR807 19.7 REPT9 6.7 OPC10-1
1.1 TA71
5.1 ECAMCTA10 TA37 5.3 1.8 TAASH
3.0 EAAMCTA01 3.1 ECAMCAT11 ECAMCAT04 6.4 ECAMCAG10 4.0
7.3 8.8 OPN06-3 OPN06-5 4.3
OPA12-2 EACMCAC09 AF1
Kin12 7.2 0.6 OPL4-2 5.2 1.6
EACMCAC05 9.7 TA106 S-SAP F4.3 STMS24 3.6 TA39
8.7 Kin11 2.5 7.5 OPA12-3 5.9
LN211 3.5 GA9 0.6 TR26 TAA46 9.4 TR29
OPC11-2 7.7 21.7 OPG09-2 12.7 GAA42
4.2 OPQ11-1 7.4 ISSR8551 7.5 STMS5 2.0
13.5 EACMCAC02
20.5 3.5 OPS02-1 9.7 GA34 7.5 OPD06-1 TA180 ISSR842 2.0
2.6 OPD06-2 1.5 ECAMCTA05
6.9
TA110 STMS2 12.1 16.0 ISSR8112 15.5 EAAMCTA09
R360-8-2y OPP06-3 OPD03-5
ISSR830 248 cM ECAMCAT09
18.0 RGA-Ds 18.8
7.1 ECAMCAG11 ECAMCAT03
17.8 RGA-D TA76 239 cM OPG09
RGA-G GAA45
ISSR8801 ISSR8681 258 cM LG 9
12.0 ISSR864
C32 EAAMCTA10 266 cM LG 11 LG 10
11.8 32.5 EAAMCTA05 270 cM 6.6
OPA12-4
ISSR8842 EAAMCTA03 OPO04-4
5.2
4.4 S-SAP F4.2 EAAMCTA04 LG 12 OPA12-5
STMS12
15.8
GAA40 S-SAP FF2.4 LG 13 6.6
OPU04-3 18.0
11.2 EAAMCTT07 ECAMCAG02 OPU03-3
OPAB 09-1 11.2 STMS25 12.8 15.0 TS72 14.1
PGI 5.0 OPU03-2 8.7
12.4 S-SAP FF2.1 TA196 OPE32 2.7 OPQ13 OPA12-6
7.4 OPD05-2
S-SAP E4.3
10.6 OPC14-1
4.2 EAAMCTA15 5.0 cM 12.8 cM 24.3 cM 26.7 cM 36.5 cM
STMS21 23.5
4.8
S-SAP FF2.2
ISSR8661
20.9

3.6
4.2
ISSR810
ISSR8601 355 cM Classes of Molecular Markers (> 500)
4.1
GA11
EACMCAC11

383 cM
Co-Dominant: STMS Isozyme RGA cDNA
Dominant: AFLP SCAR S-SAP RAPD
Fusarium Resistance
Gene Clusters on
Linkage Group 2
Relating Linkage Groups to Chromosomes

PCR with LG-specific SSR markers

Chromosome sorting
A B C+D
C+D C C C
H
F+G
LG VII LG IV LG III+V

Flow cytometer
E
B
A LG VI LG I+ II LG VIII

C C C
E F+G H
 Integrated Genetic and Chromosome Map of Chickpea
LG 7 LG 4 LG 3 LG 5 LG 6 LG 1 LG 2 LG 8
OPT12-1
ISSR8682
STMS20 OPO04-1
R360-8-3 3.6 OPC11-2 STMS13 EAAMCTT0
STMS8 4.0 OPC15-1 OPA12-1 TR19
7.6 OPP15-2 OPU03-5 11.4 ECAMCAT01
EAAMCTT06 1.9 14.1 15.1 15.3
13.5
4.7 RGA-A 2.9 OPC06-1 5.8 S-SAP E4-1 21.4
3.6 ISSR889 OPP17-1 7.1 ISSR8231 OPI06-3 S1E1
OPT12-4 8.6 OPO04-2 2.4
2.3 11.4
1.8 EACMCAC08 10.0 OPC20-1 ECAMCAG07 ISSR8843 5.0 OPP15-1
6.1 OPJ13-1 4.7 OPC14-5 OPJ13-2 1.0 17.8 OPD05-1 GAA46
2.1 4.7 ECAMCAG06 23.6 OPA14-1 8.2 7.0
7.0 R360-8-1 3.1 OPU03-4 ECAMCAT13 TA113 OPD03-1 ECAMCAG0
GA31 15.9 OPA17-2 2.9 ECAMCAG05 7.5
10.6 STMS22 9.7 13.6 STMS11 11.4 OPG09-5
16.3 TS19 OPC15-4 STMS6 17.7
2.6 IGR3RX2A 9.4 9.4 15.2 GA24 OPD03-4
6.4 EACMCAC12 ISSR8561 GA47 ACONa 7.9 OPC14-4
ECAMCAT12 13.3 EAAMCTA06 OPAB09-5 10.9
4.0 0.6 25.0
3.9 2.2
EAAMCTT08 9.6 8.9 R260-07-2
OPU17-2 EAAMCTA02 STMS15 5.0 ISSR8591 2.2
TA130 ACONb OPU18
7.0 OPQ13-3 1.0 EAAMCTA07 23.7 0.6 5.8
1.0 TA14 8.5 OPC183 GA2 OPD03-3 6.2 ISSR8262 4.8 G6PD
TS35 4.8 ECAMCTA01 TR44 10.1 7.6 OPG09-1 7.3 ACONc
10.2 1.6 EACMCAC01 14.4 EACMCAC07 4.9 EAAMCTA13 STK11 4.7 TS45
OPAB09-3 STMS10 1.2 EAAMCTT01 OPQ13-2 5.8
6.3
ECAMCAG04 1.6 STMS14 14.1 ECAMCTA04 S-SAP DR22 7.1 9.6 16.1 GA16 OPQ11
15.5 0.9 ECMCAC03 ISSR8481 8.6 REPT10
1.6
STMS28 3.6 ECAMCTA07 2.9 ECAMCTA11 10.6 9.6
ECAMCAT08 0.6 REPT11 8.0 ISSR8841 10.1 S-SAP AR1 PGMb
1.7 TA135 11.2 ECAMCTA02 3.3 ECAMCAG01 3.2 TR20
RGA-C 1.2 4.8 7.0 ECAMCTA06 11.0 STK g 5.6 ISSR8553 12.6
8.9 OPN06-2 4.3 TR2 3.9 EAAMCTA08 4.3 TA13 1.6 STK Ev 1.6 CS27
2.5 TR31 EACMCAC04 ECAMCAT05 2.2 TA146 ECAMCAT10 2.0 EAAMCTA1
GA4 0.8
2.7
1.1
2.9
4.1 R260-09-1
8.5 OPC06-2 0.7 TA34 1.1 EACMCAC10 18.6 ECAMCAT02 TA72 2.8 TR43 Foc4
1.9 0.7 10.2 1.5
5.6 OPT12-2 STMS4 3.3 ISSR8402 ECAMCTA12 2.3 TA2 TA1 0.5 OPU17-1 24.1
5.9 2.0 17.5 2.9
OPP07-1 2.3 STMS23 TR1 GAA44 4.4 STMS26 2.6 REPT3 1.0 OPP06-4
3.4 TA5 1.5 TR56 10.0 12.8 4.0 1.9
4.6 IGR5F1 TA21 3.8 TS72 1.0 TA8 2.4 OPP15-3 EAAMCTA1
2.9 2.2 ECAMCAT06 TA78 2.1
3.3 STMS19 2.6 0.1 STK 2 8.5 6.5 ISSR8401 AF0 0.9 OPP08
S-SAP AR4 1.7 R260-7-1 REPT8 4.4 OPM20X
11.1 RGA-B 5.2 1.6 6.2 ISSR8902 1.8 ISSR858 2.3 15.6
IGR3R1 5.7 S-SAP AR5 3.5 OPN06-4 4.5 ECAMCTA03 3.9 6.1 OPU17-3 1.5 TA96 TA3
7.4 BIGR5F1A 1.7 OPD03-2 3.6 IGR3X4A 2.3 9.0
IGR3R4 2.1 EAAMCTT04 1.3 20.3 R260-9-2 6.6 RET6
12.9 14.1 IGR3RX1A S-SAP FF2.3 6.0 5.3 OPS13-2 S-SAP AR6 S-SAP F4.1
OPB08-1 1.8 1.8 EACMCAC06 OPC11-3 3.5
IGR3R3 1.0 AF2 9.5 OPD16-1 1.4 EAAMCTA12 12.7
7.9 STK86-D S-SAP BR 3.9 3.3 TA28 R260-9-3
RGA-D2a 21.7 AF00 9.3 TS62 17.6 OPC14-2 2.7 ECAMCTA07 TS12
4.9 S-SAP C41 4.3 S-SAP E4.2 OPT12-5 16.9 TA203 4.3 BIGR
1.9 OPB08-2 IGR5F2 1.1 S-SAP DR23 4.7 TAA58 0.5 Foc5
0.8 PGMa 2.1 0.8 OPT13-3
BIGR5F2A TR7 TAA59 ISSR888 1.1 TA27
0.6
2.2 TS43
10.4 PGD6 10.7
TA80 12.3 TA140 13.4 2.3
10.3 TA59 165.8 cM
3.8 C33 OPT12-3 17.7 CS27ASAP
TS53 9.7 TA22 0.8 TA18 3.4
0.1 TA179 ISSR8903 15.7 4.8 ISSR8552 1.2 TS82
3.1 7.2 GA13 TA176 OPT18-2 22.4 4.7
3.7 TR59 S-SAP DR21 OPC20-2 S-SAP F4.4 7.1 TAA60
ECAMCTA08 7.8 13.8
0.6 STMS7 14.5 S-SAP AR2 IGR5F3 IGR3R6 10.7 TR58
1.1 TA71 TA64 EAAMCTT03 ECAMCTA09 4.6
19.2 2.9 TA194

H
1.8 ISSR807 19.7 REPT9 6.7 OPC10-1 5.1 ECAMCTA10 2.2
TAASH 5.3 ECAMCAT04 6.4 ECAMCAG10 3.1 TA37
4.0 ECAMCAT11 3.0 EAAMCTA01 OPN06-3
1.6 AF1 4.3 OPN06-5 OPA12-2 EACMCAC09 7.3 8.8
0.6 OPL4-2 7.2 5.2 EACMCAC05 Kin12
3.6 TA39 TA106 OPA12-3 STMS24 9.7
9.4 7.5 S-SAP F4.3 2.5 5.9 8.7 Kin11
TR29 0.6 GA9 TAA46 LN211
12.7 GAA42 TR26 3.5 21.7 OPG09-2 OPC11-2 7.7
2.0 7.5 7.4 ISSR8551 OPQ11-1
EACMCAC02 STMS5 13.5 4.2
2.0 7.5 OPD06-1 GA34 TA180 ISSR842 20.5 3.5 OPS02-1
1.5 ECAMCTA05 9.7 2.6
OPD06-2 STMS2 16.0 ISSR8112 TA110
15.5 EAAMCTA09 12.1 R360-8-2y 6.9
ECAMCAT09 OPD03-5 OPP06-3
ECAMCAG11 18.8 ISSR830
ECAMCAT03 7.1 18.0 RGA-Ds
OPG09 TA76
GAA45 RGA-G
248 cM 17.8 RGA-D
ISSR8801 ISSR8681
258 cM
239 cM 266 cM
12.0 ISSR864
EAAMCTA10
270 cM C32
32.5 EAAMCTA05
11.8
ISSR8842 EAAMCTA03
5.2 EAAMCTA04
4.4 S-SAP F4.2
GAA40 S-SAP FF2.4
11.2 EAAMCTT07

A B C/D C/D E OPAB 09-1 11.2

PGI
12.4 S-SAP FF2.1
7.4
S-SAP E4.3 OPC14-1
10.6
4.2 EAAMCTA15
STMS21 23.5
4.8
S-SAP FF2.2
ISSR8661
20.9

Chromosomes 3.6
4.2
4.1
ISSR810
ISSR8601
GA11
EACMCAC11
355 cM

383 cM

F/G F/G
Medium-density genetic maps

High-density submaps

SNP Markers and Cross-genome Markers

 Targeting at SNPs:
Design of single nucleotide amplified polymorphism (SNAP) primers

Sequencing of PCR products of both parental lines after amplification with

Pfu DNA polymerase
Comparison of sequences, detection of SNPs
Development of SNAP-primers
Test for amplification

Example:
Forward primer:
Original primer-binding site in C. arietinum ICC 4958: 5´ act gta agt agc att cgt t t 3
´
Original primer-binding site in C. reticulatum PI 489777: 5´ act gta agt agc att cgt t c 3
´
SNAP-primer specific for the C. reticulatum allele: 5´ act gta agt agc att cggt c 3´

Reverse primer:
One of the original primers for the amplification of the fragment
or any primer in good distance for amplification
 Segregation of 3 SNAP markers from a
single amplification product in chickpea RILs

(bp) M P1 P2 RILs
1000

500

200 SNAP 124a

1000

500

200 SNAP 124b

1000

500

200 SNAP 124c

 Segregation of CAPS Markers in Chickpea RILs:

High-Throughput Microfluid Capillary Separation

PisPR 110-1 Mtmt 32 Mtmt 24

MCG: one sample/30sec; 3bp resolution, digital data output,

sample volume 100 nl, no gel preparation, CAPS and SNAPs
 Localization of cross-genome markers from pea and Mt on the
LG 1 genetic map of chickpea
STMS13

TA113
ACONa LG 2 LG 3 LG 4
ACONb STMS25
TR19 OPC11-2
TA196 Glucanase
OPQ13-2 PisPR 51 ISSR8231
Glucanase CuAO
STK g ISSR8841 ISSR8843
STK Ev TR43 PisPR 132
TA1/TA8 Cysteine
PRP10 PRP5 PisPR 24
Synthase PisPR 62
Chitinase II PisPR 82 STMS11/GA24
Ribonuclease T2 OPU18 RGA-A GAA47
ECAMCAC08 PisPR 32
FP-i STK11 GA16 TA130
PisPR 40 GA2
TA203 TR20
S-SAP AR1 PisPR 141 MDR TS19 TA13
foc4 TA96 G6P-T STMS10 TA146
S-SAP AR6 S-SAP AR4 STMS14 Catalase TA72
TA27 TA2
ISSR8552 foc5 TA59 S-SAP AR5 STMS28
TA194 Expansin TA135 STMS26
S-SAP F4.4 KAPP TR56 OPS13-2
Mtmt 10
PisPR 110 Kin12 TA37 S-SAP BR
Kin11 TA110 S-SAP C41 C33
OPC11-2 PGMa Transketolase
HRP
PGD6 GA13 OPT12-3
RGA-D
RGA-Ds PisGen 6 LAP
ISSR8801 EAAMCTT07 EAAMCTT03
C32 TA64
S-SAP F4.2 PisPR 38
S-SAP FF2.4 STMS24
PisPR 69
GDP Dis. GAA40 PGI TAA46
PisPR 124
Inhibitor S-SAP FF2.1 Glucanase ISSR842
PisPR 13
S-SAP E4.3 Ion Channel
Ion Channel TA122 OPL4-2
Regulator PisPR 42 S-SAP F4.3
Regulator TR26
S-SAP FF2.2 STMS21 Histone
ISSR8661 Mtmt 104
Aspartic H2A TA76
PisPR 59
Proteinase GAA45
GA11
Genic Markers
 Localization of resistance gene analogs and pathogenesis-
LG 1 related genes on the genetic map of chickpea
STMS13

TA113
ACONa LG 2 LG 3 LG 4
ACONb STMS25
TR19 OPC11-2
TA196 Glucanase
OPQ13-2 ISSR8231
Glucanase CuAO
STK g ISSR8841 ISSR8843
STK Ev TR43 PRP 10
TA1/TA8 Cysteine
PRP10 PRP5
Synthase
Chitinase II STMS11/GA24
Ribonuclease T2 OPU18 RGA-A GAA47
ECAMCAC08
FP-i STK11 GA16 TA130
GA2
TA203 TR20
S-SAP AR1 MDR TS19 TA13
TA96 G6P-T STMS10 TA146
S-SAP AR6 S-SAP AR4 STMS14 Catalase TA72
TA27
ISSR8552 foc4
foc5 TA59
TA194
S-SAP AR5
Expansin
STMS28
TA135
TA2
STMS26
S-SAP F4.4 KAPP TR56 OPS13-2
Pyruvate kinase
Kin12 TA37 S-SAP BR
Kin11 TA110 S-SAP C41 C33
OPC11-2 PGMa Transketolase
HRP
PGD6 GA13 OPT12-3
RGA-D
RGA-Ds LAP
ISSR8801 EAAMCTT07 EAAMCTT03
C32 TA64
S-SAP F4.2 S-SAP FF2.4 STMS24
GDP Dis. GAA40 PGI TAA46
Inhibitor S-SAP FF2.1 Glucanase ISSR842
S-SAP E4.3 Ion Channel
Ion Channel TA122 OPL4-2
Regulator S-SAP F4.3
Regulator TR26
S-SAP FF2.2 STMS21 Histone
ISSR8661
Aspartic H2A TA76
Proteinase GAA45
GA11
 LG 5 LG 6 LG 7 LG 8
OPT12-1 ECAMCTA11 EAAMCTT02
OPC15-1
S-SAP E4-1 SIE1
EAAMCTT06 GAA44
OPJ13-2
OPT12-4 ECAMCAG 03
Replication factor OPG09-5
Pectin OPC15-4 Proline-rich protein
Esterase STMS22
FDH TA21
HRP Protein P40
G6P-T S-SAP DR22
OPAB09-5 G6PD
Protein P40 STMS15 ACONc
TS35 TA28 TS 45
GTP-Binding TA14 TS62
Protein TR44 SAMS TAA58 PGMb
TA140 EAAMCTA 11
RGA-C ECAMCAT08 ECAMCTA02
TA18
Chalcone ISSR8402 EAAMCTA 14
OPC20-2
Isomerase STK 2 TR1 OPA12-3 TA 3
GDP Dis. GA4 PRP10 OPD03-2 S-SAP F4.1
Inhibitor S-SAP FF2.3 TR7 TA180 TS 12
OPT12-2 S-SAP E4.2 TA80
Pyruvate TA176
S-SAP DR23 ISSR8112
Kinase
TA5 S-SAP DR21 ISSR830
MDR STMS19 Plantacyanin
RGA-B TS43 S-SAP AR2 TA106
STK86-D TA179 GA9
TAASH
RGA-D2a
TA39
TR29
GA34
STMS2 Localization of resistance gene analogs
TS83
GAA42
ECAMCAT03 RGA-G GA26
GAA39
and pathogenesis-related genes on the
Pyruvate GA21
Kinase TR3 genetic map of chickpea
OPG09

LG 9 LG 10 LG 11 LG 12
OPA12-1 ISSR8253 AAT OPA12-5
Peroxidase MDR
ECAMCAG07 OPN06-1 STMS12 β -Amylase
STMS6 TS72 OPD05-2
LAP
OPQ13
OPC18-3
 Tools for Transcriptomics
• cDNA collection infantil
• Full-length (fl) cDNA collection non-existing
• Expressed sequence tag (EST) bank infantil
• Transcribed fragment collection non-existing

• cDNA arrays probed

• Fl-cDNA arrays non-existing
• EST arrays non-existing
• Exon arrays non-existing
• Antisense transcript arrays non-existing
• Oligonucleotide array probed
• Genomic array non-existing
 SuperSAGE

Nla III Eco P15I

AAAAAAAA Population of cDNAs
cDNA TTTTTTTT
26 bp tag
Generate Tags
Ligation of Tags to form 52 bp Di-
Tags Generate Di-Tags

Concatemers of 52 bp Di-
Tags Concatenate and
Clone Concatemers

Sequence Count Tags

Matsumura H, Reich S, Ito A, Saitoh H, Winter P, Kahl G, Reuter M, Krüger D, Terauchi R (2003)
SuperSAGE: A universal functional genomics tool for any eukaryotes. PNAS 100:15781-15723
 SuperSAGE of Chickpea Leaves
The ten most abundant tags

Tag sequence % of total Annotation (100% Match)

CATGTGTGCGAGTCAACGGGCGAGTA 6.4 26S rRNA gene (all plants)

CATGGGCCTATGCCACCAACTTTGTT 6.3 Chlor. a/b binding protein (C. arietinum)
CATGCAAGTGTGGCCACCAATTGGCA 5.2 Rubisco SSU (C. arietinum)
CATGCACAGGTTGATGGTTCAATGTA 4.3 Chlorophyll a/b binding protein (P. sativum)
CATGTGGTTGTAACATTCTCAATGTG 2.1 Rubisco activase (M. truncatula)
CATGAGCAGAAGAACCTGTATGTTGT 1.2 LHC chlor. a/b binding protein (P. sativum)
CATGTGCAGCTTGATCCACACTTGCA 1.1 Metallothionein (C. arietinum)
CATGGGGCACGTGGAATCCCGTGTGA 0.8 Hypothetical protein (M. truncatula)
CATGTTAGACAGCAGCACTAGCAGTT 0.6 Fructose P2 aldolase (M. truncatula)
CATGGCCCTATCCTCTTGTACTAGAC 0.5 Lipid transfer protein (C. arietinum)
28.6
 SuperSAGE: EcoP15 as Tagging Enzyme

• 26bp tags
• Accurate and quantitative expression analysis

Securely identify corresponding gene

CATGNNNNNNNNNNNNNNNNNNNNNN
26bp tag

Amplify cDNA fragment of corresponding gene

CATGNNNNNNNNNNNNNNNNNNNNNN

AAAAAAA

3‘-RACE PCR
 SuperSAGE: towards expression markers

150 specific SuperSAGE forward primers, chickpea leaf cDNAs

Product size range: 100 - 1150bp (mostly 150 - 600bp)

7% 1 fragment 7

24% 2 fragments 24

31% 3 fragments 31

24% 4 fragments 24

14
14% 5-6 fragments

0 10 20 30 40

3’-RACE with specific tag primers mostly produces 2-4 fragments

Gene families? Different alleles? Alternative splicing?

SuperSAGE tags: towards expression markers
Example:

SuperSAGE tag # 91 5‘-CATGGAGATGCACCTTTTGGAGAGCT-3‘

Forward primer design 5 ‘ATGGAGATGCACCTTTTGGAG-3‘
Reverse primer Anchored oligo(dT)
Amplification Two products, transcripts both encoding
carbonic anhydrase isoforms (EC 4.2.1.1)
Occurrence Leaf cDNA of Cicer arietinum and C.
reticulatum

BLASTing of SuperSAGE tag # 91:

>gi|46879218|gb|AC124951.19| Medicago truncatula clone mth2-7p23 >gi|

41688377|dbj|AP006692.1| Lotus corniculatus var. japonicus, chromosome 2
>gi|20672|emb|X52558.1|PSCAP Pea mRNA for carbonic anhydrase (EC 4.2.1.1)
Towards expression markers based on
SuperSAGE tags
Variation in transcripts encoding carbonic anhydrase
isoforms exploited for expression marker design in legumes
P.sativum ... CCTTTTGCAGAGCTATG ...
P.sativum ... CCTTTTGCAGAGCTATG ...
Cicer arietinum ... CCTTTTGGAGAGCTATG ... Alu1 site:
GA-CAPS
C.reticulatum ... CCTTTTGGAGAACTATG ...
marker
C.reticulatum ... CCTTTTGGAGAACTATG ...
******* *** *****

Leaf cDNA of Cicer arietinum and C. reticulatum

Amplification with SuperSAGE tag # 91 forward primer
Sequencing of three ~ 450 bp fragments
Alignment to Pisum sequences in GenBank: transcript for carbonic
anhydrase isoforms
Towards expression markers based on
SuperSAGE tags

Variations in transcripts encoding carbonic anhydrase

isoforms exploited for expression marker design in legumes

P.sativum TCTTGATTACTAAGTATTCTTCTCCCTAAAATGTCAA
P.sativum TCTTGATTACTAAGT-------------------CAA
Cicer arietinum TCT------CTAAGTATTCTCCTCCCTAAAATGTAMA
Cicer reticulatum TCT------CTAAGTA-------------------AA
Cicer reticulatum TCT------CTAAGTA-------------------AA

INDELs of 6 and 19bp downstream of tag #91

Localization of SuperSAGE tags from chickpea leaves on
the emerging physical map of M. truncatula

rDNA
More in 2007!
 3’-RACE primer design based on 26 bp
SuperSAGE tag information

Detection of SNPs and INDELs in

the 3‘-end of cDNAs

3‘ RACE forward primer PCR

&
oligo dT reverse primer

Identification of differentially expressed members of gene

families and alternatively spliced transcripts by SuperSAGE