Transcript (i) N.K. Singh

Pigeonpea Genomics Initiative
Nagendra K. Singh
NRC on Plant Biotechnology
Indian Agricultural Research Institute, New Delhi-110012
Status of Plant Genome
Sequencing
•Sequencing of 16 Plants in Progress
(10 completed + 6 about to be finished)
Arabidopsis, Rice, Poplar, Medicago, Sorghum, Papaya,
Cassava, Cucumber, Tomato, Potato, Maize, Soybean,
Citrus, Grape, Banana, Wheat
•Arabidopsis and Rice with high quality
BAC by BAC sequence data
International Rice Genome Sequencing Project
INDIAN INITIATIVE FOR RICE GENOME SEQUENCING
INTERNATIONAL TOMATO GENOME SEQENCING CONSORTIUM
Based on Song-Bin Chang’s Ph. D. Thesis 2004
Indo-US AKI Pigeonpea
Genomics Initiative
from Orphan Legume
to Draft Genome Sequence
Productivity (hg/ha) World-Food grains
30000
20000
Cereals
Cereals,Total +
15000
Pulses
Pulses,Total +
10000
5000
0
1961
1963
1965
1967
1969
1971
1973
1975
1977
1979
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
2001
2003
2005
2007
Productivity (hg/ha)
25000
Year
FAOSTAT, 2010
Pigeonpea

Pigeonpea (Cajanus cajan (L.) Millsp.) belongs to
family Fabaceae with chromosome no. 2n=22 and genome
size of 853 Mbp

A major source of protein to about 20% of the world population (Thu et al., 2003).

An abundant source of minerals and vitamins (Saxena et al., 2002).

Most versatile food legume with diversified uses such as food, feed, fodder and fuel.

It is hardy, widely adaptable crop with better tolerance to drought and high
temperature.

Plays an important role in sustaining soil productivity by fixing atmospheric nitrogen.
Area, Production and Productivity
Year
Area
(Mha)
Production
(MT)
Productivity
(Kg/ha)
World
2008
4.90
4.22
861
India
2008
3.72
3.07
825
(FAOSTAT 2010)
o
India produces about 75% of the global output of pigeonpea.
o
Very low average productivity (800 kg/ha) as compared to it’s
potential (2000 kg/ha) (Ali and Kumar, 2005).
Constraints to High Productivity

Growing traditional landraces on large area

Non-availability of quality seeds of improved varieties

Inferior plant type with low harvest index

Long crop duration (5-9 months)

Wilt, SMD, Water logging, Pod borer

Poor agronomic practices
First meeting of Pigeonpea Consortium on
10th Nov 2006 at NRCPB, New Delhi
Objectives:
1.100,000 ESTs and genic-SSR
/SNP markers
2. Genomic SSR markers
3. Mutant lines and mapping
populations as resource for
gene discovery
4.High density molecular linkage
map as a reference map
5.Markers and genes for
important agronomic traits
6. Pigeonpea genome informatics
platform
7. Sequencing gene-rich BAC
clones of pigeonpea
Impact of Indo-US AKI: Pigeonpea Genomics Initiative
Impact of Indo-US AKI: Pigeonpea Genomics Initiative
Sequencing of
Pigeonpea Genome
1st Draft of Pigeonpea Genome Sequence
Submitted to NCBI GenBank, July 2011
Pigeonpea Genome- Repeat Elements
Pigeonpea Genome- Gene Content
Pigeonpea Genome- Comparison with Soybean
152 homologs of genes for abiotic stress tolerance
•
•
•
•
•
•
56 genes for heat shock proteins (HSP),
32 genes for glutathione-S-transferase (GST),
28 genes for trehalose-6-phosphate synthase (TPS),
8 genes for glutamine synthase (GS),
7 genes for water channel protein aquaporins
several transcription factors e.g. DREB, NAC and MYB genes
Pigeonpea Genome- Comparison with Soybean
Circular Map
of Synteny
between
11 Pigeonpea
20 Soybean
Chromosomes
Based on
512 Single
Copy Genes
Pigeonpea Genome-miRNA
Pigeonpea Genome- Improved Assembly
Pigeonpea Genome- Improved Assembly
Pigeonpea Genome- Improved Assembly
Pigeonpea
GenomeDevelopment of
Genic-SSR and
SNP markers by
mRNA Sequencing
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
150bp
100bp
50bp
Agarose gel (4.0 %) showing allelic variation
among 30 genotypes of pigeonpea and related
wild species with genic-SSR marker ASSR-277
Asha
GTR 9
HDMO4-1
H2004-1
JA 4
PCMF 39-1
PCMF 43-7
GT288A
PS 971
PS 956
Pusa 9
Kudarat
ICPA 2089A
ICPR 2438
C. cajan cultivars
PCMF 40
UPAS 120
Ia1
TTB 7
Pusa Dwarf
Bahar
Maruti
Ia
GTR 11
R. aurea
C. platycarpus(1)
Ia2
Ib
C. platycarpus(2)
C. cajanifolius
IIa
II
C. lineatus
C. scricea
R. bracteata
C. albicans
IIb
Similarity coefficient
Dendrogram showing phylogenetic relationship of 30 genotypes of
Cajanus cajan and related wild species based on 20 genic-SSR markers
Wild species
Pusa 992
I
Pigeonpea Genome- High density linkage map based
on 366 genic-SNP and 24 genic-SSR markers
Pigeonpea Genome- SSR Mining
Pigeonpea Genome- HASSR Markers
M
1
2
3
4
5
6
7
8
1
3
4
5
6
7
8
1
2
HASSR-128
HASSR-127
300
200
2
3
4
5
6 7
8
HASSR-129
100
M 1
2
3
4
5
6
HASSR-283
300
200
100
7
8
1
2
3
4
5
HASSR-284
6
7
8
1
2
3
4
5
6
HASSR-285
7
8
Application of Genic SSR/SNP
Markers in QTL Mapping
Options for Enhancing Pigeonpea Productivity
1. Hybrids
2. Ideotype
Ideotype: Set of features delineating the shape, size, canopy
and external structure of the plant
•Plant height
•Number of primary and secondary branches
•Number and length of internodes
•Size, shape and position of leaves and reproductive organs
Application of Genic SSR/SNP
Markers in QTL Mapping
1.Development of molecular linkage map
of pigeonpea
2.Mapping of genes/QTLs for traits
involved in plant ideotype and maturity
Outline of work
Pusa Dwarf/ HDM04-1
Cross
F1
Marker polymorphism
F2
Genotyping
F2:3
Linkage map
Phenotyping
QTL mapping
Plant Material
Mapping Population: ♀ Pusa Dwarf X ♂ HDM04-1
Trait
Pusa Dwarf
HDM04-1
Plant height (cm)
88
118
No. of primary
branches/plant
20
5
No. of pods/plant
120
24
Days to Flowering
106
65
Days to Maturity
158
116
Growth habit
Determinate
Indeterminate
Pusa Dwarf
HDM04-1
Markers
A. Genic-SSR
•
Total 927 genic SSR markers, 772 developed from 454 TSA
contigs and 155 from Sanger ESTs under Indo-US AKI
project were used.
B. Genomic-SSR
• 45 genomic SSR markers from literature ( Odeny et al.,
2007, 2009)
• Additional 40 SSR markers were designed from public BAC
end sequence database at NCBI BatchPrimer 3 software
(You et al., 2008)
C. Intron length Polymorphism (ILP) Markers
A total of 60 ILP primers were designed using Medicago genome
as subject species genome by ConservedPrimers 2.0 software .
(http://rye.pw.usda.gov/ConservedPrimers/index.html)
D. Single Nucleotide Polymorphism Assay:
 Two pools of RNA from varieties namely Asha and UPAS120 were
sequenced by 454-FLX sequencing and TSA contigs were used for in
silico SNP identification (Indo- US AKI project).
 SNPs were identified by aligning 15,511 common large TSA contigs
between the two varieties.
 1536-plex and 768-plex Illumina GoldenGate assays were designed
and latter was used for genotyping of F2 population
GoldenGate Genotyping OPA
Segregation analysis:
 All markers were tested for goodness of fit by chi-square test.
Linkage analysis:
 Linkage analysis of segregating markers was done by Mapdisto software
(http://mapdisto.free.fr/MapDisto/) at LOD = 3
QTL analysis:
 Statistical analysis of Phenotypic data was performed SPSS software
version 10.0
 QTL analysis was done by QTL Network software version 2.1 (Yang
et al. 2008)
Markers Used for Parental Polymorphism Survey
Marker type
No.
tested
No.
amplified
Polymorphic
No (%)
Marker Source/
Reference
Genic-SSR
(EST- 454 seq.)
772
583 (75.5%)
28 (4.8%)
Indo-US AKI,
NRCPB
Genic-SSR
(EST-Sanger seq.)
155
31 (20%)
0
Indo-US AKI,
NRCPB
GENOMIC-SSR
(Genomic library)
45
32 (71.1%)
0
Odeny et al., (2007,
2009)
GENOMIC-SSR
(BAC end sequences)
40
27(67.7%)
0
NCBI GSS database
ILP
(454 TSA contigs)
60
51(85%)
0
NPTC, NRCPB
TOTAL
1072
724 (67.5%)
28
Polymorphism survey with genic-SSR markers
1 2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
M A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B M
500
400
300
200
100
Parental polymorphism survey with ASSR markers (1-24), 21-ASSR1486 (polymorphic)
L-100bp DNA ladder, A-Pusa Dwarf, B-HDM04-1
Genotyping of F2 with SSR Markers
L
1 2 3
4
5
6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 P1 P2
500
300
200
100
Genotyping of F2 population with ASSR8 in 4% metaphor agarose gel
L- 100bp DNA ladder, 1-22 - F2 genotypes, P1- Pusa Dwarf, P2- HDM04-1
Parental Polymorphism survey on PAGE
ASSR 66
L 1 2
77
1 2
95
1 2
148
205
206
1 2 1 2 1 2
247
1 2
277 281 286 300 304 317 363
390
L 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
250bp
200bp
150bp
Parental polymorphism survey for ASSR markers in 8% PAGE
L-50bp DNA ladder, 1- Pusa Dwarf, 2-HDM04-1
Genotyping on PAGE
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 P1 P2
300
200
100
Genotyping of ASSR206 on 8% polyacrylamide gels.
M-100bp ladder, P1- Pusa Dwarf, P2- HDM04-1, 1-22 F2 genotypes
F2 Progeny
Homogeneous F3 Families
Segregating F3 Families
F3 Recombinants
Phenotyping of F2 and F2:3 (20 plants/lines)
1. Plant height
2. Number of primary branches per plant
3. Number of pods per plant
4. Days to flowering
5. Days to maturity
6. Number of secondary branches
7. Pod bearing length
8. No. of seeds per pod
9. Growth habit (determinate/indetrminate)
Frequency Distribution of Plant Height and No. of Primary Branches
40
40
35
35
30
30
25
25
20
P1
Frequency
Frequency
Plant height F2
P2
15
P1
20
15
10
10
5
5
0
Plant height F3 families
P2
0
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
50
cm
No. of primary branches F2
66
74
82
90
98 106 114 122 130 138 146 154 162
cm
No. of primary branches F3 families
35
50
45
30
P2
40
25
35
20
P2
Frequency
Frequency
58
P1
15
30
25
20
15
10
10
5
P1
5
0
0
2
4
6
8
10
12
14
16
18
20
Branch numbers
22
24
26
28
30
4
5.2
6.4
7.6
8.8
10 11.2 12.4 13.6 14.8 16 17.2 18.4
Branch numbers
Frequency Distribution for No. of Pods
No. of pods F2
35
No. of pods F3 families
60
P2
30
50
25
Frequency
Frequency
40
20
15
30
20
10
P2
P1
10
5
0
P1
0
0
20
40
60
80 100 120 140 160 180 200 220 240 260 280
Pod numbers
0
15
30
45
60
75
90
105 120 135 150 165 180
Pod numbers
Frequency Distribution for Days to Flowering and Days to Maturity
Days to flowering F3 families
40
40
35
35
30
30
Frequency
Frequency
Days to flowering F2
25
20
15
P2
P1
25
20
15
10
10
5
5
0
0
45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109
50
55
60
Days
40
P1
P2
65
70
75
80
85
90
95 100 105 110 115 120
Days
Days to maturity F2
P2
Days to maturity F3 families
60
35
50
25
20
P1
15
Frequency
Frequency
30
40
30
20
10
P2
P1
10
5
0
0
80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160
110 118 126 134 142 150 158 166 174 182 190 198 206 214
Days
Days
Descriptive Statistics of Five Traits for the Parents and
Mapping Populations of Pusa Dwarf and HDM04-1
Trait
Pusa
HDM0
Dwarf
4-1
F2
F3
Range
Mean
SD
CV
Range
Mean
SD
CV
Plant height
85
120
49-190
130.4
31.2
23.9
59-160
112.12 22.67
20.22
No. of pri. branches
18
7
2-29
12.6
5.65
44.86
4-16.6
9.88
1.95
19.75
No. of pods
180
24
6-279
74.3
57.7
77.7
6.6-170.9
58.4
23.9
41.0
Days to flowering
90
65
51-99
74.75
10.50
14.05
59-116
90.95
11.17
12.28
Days to maturity
150
120
85-158
120.13
19.01
15.82
110.43-208
140.09 15.31
10.93
QTL Map for All Traits
QTL Map for All Traits
QTLs with additive and dominance epistatic effects for number of primary
branches per plant in F2:3 population from Pusa Dwarf/HDM04-1
▬ Interaction between QTLs with epistatic and main effects.
●
QTLs with only additive effect
▀ QTLs with only dominant effect
▀ QTLs with no dominance effect
Summary and Conclusions
 Draft of 511 Mb of pigeonpea genome sequence assembled, 47,004 genes, 437
HASSR markers
 Deep coverage TSA assembly of 43,324 genes, 550 genic-SSR and 2,304 genic-SNP
GoldenGate assays
 Intra-species reference map of 366 genic-SNP and SSR markers
 1,363 markers screened to find 135 polymorphic (9.9%) markersbetween Pusa
Dwarf and HDM04-1 (28 SSR and 107 SNP)
 Linkage map of 136 loci, 1056.82 cM, average interval 7.77 cM.
 2 QTLs for plant height, qPH3, qPH5 ( 28.2, 28.1% PEV)
 3 QTLs for primary branches, qPB3, qPB5, qPB9 (23.4, 11.1, 2.6% PEV)
 2 QTLs for number of pods, qPD3, qPD5, (16.4, 18.7% PEV)
 2 QTLs for days to flowering, qFL3, qFL5 (52.3, 8.8% PEV)
 3 QTLs for days to maturity, qMT3.1, qMT3.2, qMT5 (23.4, 4.4,15.6% PEV)
 Significant epistasis of qPB3 with qPB5 and qPB9 (3.5% PEV).
 Co-located of QTLs in two genomic regions on LG3 and LG5 with pleiotropic effect
 Useful for MAS of semi-dwarf short duration pigeonpea varieties
Acknowledgements
• ICAR for funding support under IndoUS AKI and NPTC Projects
• Doug Cook, UC Davis, Chris Town,
JCVI and Rajeev Varshney, ICRISAT
for quality files of BAC end sequences
Thank You