towards understanding genetic basis of chapatti (indian flat bread) making quality
DESCRIPTION
International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012TRANSCRIPT
Towards understanding genetic basis of chapatti (Indian flat bread) making quality
M. GARG, S. ARORA, R.P. Singh*, A. SINGH, J.K. ROY
National Agri-Food Biotechnology Institute
*Deptt. Of Plant Breeding and Genetics
Punjab Agricultural University, Ludhiana.
Sample ID Character Hardness Pina-D1 Pinb-D1
Chapatti
Score
CHINESE SPRING Check 48.66 a a -
PBW 343 Check 86.26 b a 7.5
WH 291 Chapatti 60.81 b a 5
SONALIKA Chapatti 66.88 a b 6
C 306 Chapatti 94.64 b a 9
LOK 1 Chapatti 67.51 b a 7
Parameter/cultivar
C306 LOK1 WH291 SONALIKA
Chapatti score Very Good Good Poor Medium Poor
Grain hardness (g) Hard Med Soft Med hard Med Soft
Seed size Med Med Small Large
Color White Brown Brown Brown
Water absorption High Med Med Med
Protein content Med Med High Med
Gluten Medium Medium High Low
Starch gelatinisation Medium High Medium High
RVA Breakdown High High Low Medium
HMW-GS-ABD Null, 20, 2+12 2*,17+18,
2+12 2*,20, 2+12 2*,7+9, 2+12
Puroindolines (5DS) PinaD1b, PinbD1a
PinaD1b, PinbD1a
PinaD1b, PinbD1a
PinaD1a, PinbD1b
11
Affymetrix GeneChip® Wheat Genome Array
7DAA 14DAA 21DAA 28DAA 35DAA Fig. 1 Caryopsis at three developmental stages- 7, 14, 21, 28, & 35 days after
anthesis (DAA)
Expression profiling of 17,344 probe
sets at three developmental stages-
7DAA, 14DAA, 28DAA
RNA EXTRACTION
3’-IVT
Hybridization
Washing, Staining and scanning
File generation and Data analysis
AGCC-MAS5,RMA, GCRMA, PILER)
{GeneSpring, Array star,
Overview of experiment
Quality control of samples RIN calculation
Correlation coefficient (r)
Positive hybridization control
Summary
Data analysis of7DAA Good vs. Poor
61,290 (Total probe sets)
Normalized & filter by percentile
37,277 (Probe sets)
Statistical data analysis
2,989 (Probe sets)
Fold change analysis
59
1027 137
Functional annotation:
Up-regulating
Catalytic subunit activity
Nutrient reservoir activity
DNA binding activity
Translational initiation factor activity
Down regulation:
Catalytic subunit activity
Magnesium ion binding activity
GO ANALYSIS:
Two fold regulation:
Molecular function: 43.6%
Biological function: 34.0%
Cellular function: 22.6%
Contd….
Data analysis of28DAA Good vs.Poor
61,290 (Total probe sets)
Normalized & filter by percentile
33,083 (Probe sets)
Statistical data analysis
527 (Probe sets)
Fold change analysis
• 194 (113↑, 81↓)
• 72 (33↑, 26↓)
• 19 (9↑, 10↓)
Functional annotation:
Up-regulating
Response towards heat (HSP-16, 26)
Lipid binding proteins and its transport (LTP3)
Down regulation:
Catalase activity (Response towards oxidative stress)
Starch biosynthesis activity (WGBSSI)
GO ANALYSIS:
Two fold regulation: Molecular process: 33.3%
Biological process: 66.6%
Contd…
30% - storage proteins
30% -no prior information on gene
function. Rest - Transcription
factors, trypsin inhibitors,
metal binding, lipid transfer, cell
wall related proteins, etc.
photosynthesis, regulatory genes
Major genes – No prior
information on gene function.
Rest - cytochrome C heme
attached protein, peroxidases.
starch synthesis, proteases
1 2
3,4 5
23
S.
No.
Probe Set ID UniGen
e ID
(DAA) Fold
change
Function
1 Ta.24736.1.S1_at NA 7 +284.7 Predicted protein (Hordeum
vulgare )
2 Ta.27778.4.S1_x_at Ta.5420
6
7 +108.1 Pre-alpha-/beta-gliadin A-II
3 Ta.7158.1.S1_at Ta.7158 7 +105.9 Transcribed locus
4 Ta.6175.1.S1_at Ta.3546
3
7 +102.3 No significant similarity in non-
redundant (nr) protein
sequences database
5 Ta.24114.14.S1_x_
at
Ta.6588
1
7 +284.7 Triticum aestivum gamma-
gliadin gene
Differentially expressed genes (up and down)
At the three development stages among two good and two poor chapatti varieties Source-Dr. Joy K Roy
24
6 Ta.6984.1.A1_at Ta.5710
0
7 -58.0 Predicted protein & also
similar with PHD zinc finger
protein-like [Oryza sativa
Japonica Group]
7 TaAffx.104444.1.S1
_at
Ta.3578
4
7 -47.5 No significant similarity
8 Ta.23013.3.S1_s_a
t
NA 7 -31.5 B22EL8 [Hordeum vulgare]
9 Ta.14507.2.S1_at Ta.5418
6
7 -29.9 Predicted protein & also similar
with nucleotide-binding protein
1 [Zea mays]
10 Ta.4957.1.S1_at Ta.4957 7 -26.6 Triticum aestivum granule bound starch synthase I (GbssI)
11 Ta.23366.2.S1_at NA
28 +59.96
Triticum aestivum TaPrx-B mRNA for peroxidase, partial cds
12 Ta.28744.1.S1_at Ta.28744
28 +35.22
Transcribed locus, Triticum aestivum cDNA
13 Ta.14050.1.S1_at Ta.1405
0
28 +37.26
Hordeum vulgare subsp. vulgare mRNA for predicted protein
Source-Dr. Joy K Roy
25
14 TaAffx.91902.1.A1_at Ta.4769
5
28
+32.62 Transcribed locus, Triticum aestivum cDNA
15 Ta.23366.2.S1_x_at NA
28 +28.88
Triticum aestivum TaPrx-B mRNA for peroxidase, partial cds
16 Ta.2025.1.S1_at
Ta.2025
28 -1727.4
Transcribed locus_Hordeum
vulgare subsp. vulgare cDNA
clone: FLbaf53o13
17 Ta.24114.7.A1_at Ta.30989
28 -92.767 Triticum aestivum granule
bound starch synthase I (GbssI)
18 TaAffx.80118.1.S1_at
Ta.48558
28 -32.63 Transcribed locus
19 Ta.14489.1.S1_at
NA
28 -28.99
S.cereale acclimation
protein/Triticum aestivum cold-
responsive protein (Wlt10)
20 Ta.27445.1.S1_at Ta.27445
28 -28.62
Medicago sativa FtsH protease
(FtsH) mRNA/Triticum aestivum
clone wlp1c.pk007.f6:fis Source-Dr. Joy K Roy
26
zEEMAN et al. Biochem. J. (2007) 401 (13–28
DAY NIGHT
Nagai Y S et al. Plant Cell Physiol 2009;50:635-643 © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant
Physiologists. All rights reserved. For permissions, please email:
Tian Z et al. PNAS 2009;106:21760-21765 ©2009 by National Academy of Sciences
GBSS genes
GC rich 11 EXONS 10 INTRONS
GBSS A1 gene 2781bp
GBSS D1 gene 2862bp
GBSS B1 gene
2794bp
GBSS genes
GC rich 11 EXONS 10 INTRONS
Saito et al., JARQ 44 (2), 109–115 (2010)
Good starch swelling properties-
Good for Udon noodles
Low amylose content (27-28% compared to 29%
22-23% compared to 25%)
Amylopectin matrix in
starch granule can swell
better
38
Japanese Udon Indian Chapatti Softness
39
Null GBSS A1 gene Deletion mutation -19bp
2762bp (2781bp wild type)
Null GBSS A1 gene
Insertion mutation from Turkey
(Transposable element) 2854bp (2781bp wild type)
Transp.elemt
IR IR 12bp
Target site duplication 8bp
Null GBSS B1 gene Absence (2794bp wild type)
Null GBSS D1 gene Deletion mutation 2850bp (2862bp
wild type)
Yanagisawa et al EUPHYTICA, 121-3, 209-214
GBSS D1 gene
Allele GBSS-D1e
Nakamura et al. Plant physiology
Waxy
Waxy
Normal
Nakamura et al. Plant physiology
49 49
50
51
S.No Chapatti Quality
Cultivar GBSS 4A1 5+10/ 2+12
PINa-D1
1 Good PBW550 A 5+10 b
2 Good C306 A 2+12 b
3 Good LOK1 A 2+12 b
4 Good PBW175 A 2+12 b
5 Good HI1563 A 2+12 b
6 Good K8027 A 5+10 b
7 Good HD2888 A 2+12 b
8 Good HI1500 A 2+12 b
9 Good PBW175 A 2+12 b
52
10 HI977 A 5+10 b 11 HI1563 A 2+12 b 12 HD2987 A 5+10 b 13 HD2236 A 2+12 b 14 HD2385 A 2+12 b 15 WH416 A 2+12 b 16 JWS17 A 2+12 b 17 K8434 A 2+12 b 18 HD2307 A 2+12 b 19 HD2380 A 2+12 a
20 TAWA267 A 2+12 b
53
1 Poor PBW343 P 5+10 b
2 Poor PBW621 P 2+12 b
3 Poor SONALIKA P 2+12 a
4 Poor WH291 P 2+12 b
5 Soft IITR67 P 2+12 a
6 Soft NAPHAL P A a
7 HUW629 P 5+10 b
8 UAS305 P 5+10 b
9 DHT5 P 2+12 LIGHT b
10 CPAN4202 P 2+12 b
11 UTKALIKA P 5+10 b
12 DWR39 P 2+12 b
54
• Batey et al., 1997. Cereal Chem. 74(4):497–50
3 6 9 12 15
500
1000
1500
2000
Rapid visco
analyzer’s graph
Influenced of RVA breakdown on chapatti making quality
Variety Breakdown (cP)
Final Viscosity (cP)
Set back (cP)
Peak time (min)
Pasting temp (˚C)
C306 648.7 1580.7 828.3 5.7 69.7
LOK1 752.3 1821.0 842.0 5.9 85.2
WH291 374.3 1393.0 697.7 5.4 65.8
Sonalika 504.0 1923.0 935.7 6.0 87.7
Commercial
C306
Sonalika
Lok1
WH291
High RVA breakdown in good chapatti variety (C306, Lok1)
Low RVA breakdown in poor chapatti variety (WH291)
Medium RVA breakdown in poor chapatti variety (Sonalika)
•The analysis will be repeated on the above samples and to be conducted on a larger samples to calculate correlation between RVA breakdown and good chapatti
500 bp
250 bp
668 bp
778 bp GBSSB1
GBSSB1null
F1
BC1
BC2
MAs
BC3
1st Year
2nd Year MAS
BC3F2
BC4
58
59
60