National seminar on Challenges in Innovative approaches in crop improvement

1. Towards improvement of oil content in
safflower (Carthamus tinctorius L.)
Indian Institute of Oilseeds Research
(formerly DOR), Hyderabad
CO1 (1982)

2. Production(‘000tonnes)
Source: IARI (2012)
“India imported Rs. 55,000 crore worth of
vegetable oils in 2012-13”
India’s vegetable oil economy

3.  Rapeseed/Mustard (Brassica spp.)
 Soybean (Glycine max)
 Groundnut (Arachis hypogaea)
 Sesame (Sesamum indicum)
 Castor (Ricinus communis)
 Sunflower (Helianthus annuus)
 Safflower (Carthamus tinctorius)
 Linseed (Linum usitatissimum)
 Niger (Guizotia abyssinica)
Safflower is a traditional oilseed crop of India,
known since prehistoric times, but
underutilized

4. Safflower, a multipurpose oilseed crop
 Seed - cooking oil, bird seed
 Petals - natural dyes (Carthamin) and
medicinal use (herbal tea)

5. Type of Oil Mean P/S Index
SFA (%) MUFA (%) PUFA (%)
Coconut 90.5 8.8 0.5 0.005
Linseed 9.65 22.1 68 7.05
Palm oil 76 22.5 1.25 0.016
Olive 14.35 78.4 7.0 0.49
Soybean 13.5 28.5 57.5 4.26
Sunflower 8.8 31.5 59.5 6.76
Groundnut 19.2 58.5 20 1.04
Safflower 7.2 16.6 76 10.55
~70 ~30
Sesamum 13 46 39 6.5
Kanu (2011); Kostik et al. (2012)
Safflower oil is a good source of essential fatty acid
(Linoleic acid, Omega-6)

6. Safflower is grown in dry land and
limited irrigation conditions
Dharwad (Karnataka) Raipur (Jharkhand)

7. Safflower based intercropping systems
Safflower + Chickpea
Safflower + Linseed
Safflower + Wheat

8. Source: FAOSTAT (2012)
Country Area (ha) Production
(tonnes)
Productivity
(Kg/ha)
Mexico 1,72,866 2,57,451 1,489.3
India 1,78,000 1,45,000 814.6
Kazakhstan 2,43,600 1,27,210 522.2
United States of America 64,790 81,390 1,256.2
China, mainland 23,000 36,000 1,565.2
India loses
its first
place to
Mexico in
safflower
production

9. State Area (ha) Production
(tonnes)
Productivity
(Kg/ha)
Maharashtra 1,05,000 58,000 552
Karnataka 48,000 30,000 625
Andhra Pradesh 11,000 9,100 825
Others
Odisha, West
Bengal, Jharkhand,
Bihar,
Chhattisgarh)
2,400 1400 580
Maharashtra state leads safflower
production in India

10. Seed yield
(Kg/ha)
Oil content
(%)
Oil yield (Kg/ha)
1000 30 1000 x 0.30 = 300
1000 40 1000 x 0.40 = 400
1500 30 1500 x 0.30 = 450
1500 40 1500 x 0.40 = 600
“Simultaneous improvements in seed yield
and oil content can double the oil yield
potential in safflower”
Need for improving oil yield potential of
Indian safflower cultivars

11. Country Oil content (%) in
popular cultivars
Reference
USA 31.5 – 46.6 Bergman and Kandel (2013)
Mexico 37.4 – 41.9 Lope Montoya Coronado
(2008)
India 28 - 30 AICRP-Safflower reports
Australia 33 - 42 GRDC (2010)
Argentina 43.4 Baümler et al (2014)
Canada 30.4 – 33.6 Mundel et al. (2004)
Turkey 24.5 – 28.5 Coşge et al. (2007)
Scope to improve oil content of Indian
safflower cultivars

12. Exotic varieties recorded higher oil content than
Indian cultivars (ICRISAT Farm; Rabi 2012 -13)Oilcontent(%)
Safflower Cultivars
Mexican
Indian

13. Evaluation of exotic varieties for seed yield
• No. of exotic varieties: 25
• Checks: 3 (A1, Bhima,
NARI57)
• Design: RBD with 3 reps
• Bed size: 5 rows of 5 m
length
• Spacing: 45 cm x 20 cm
Seed yield/plant (g)
Mean of three replications (No. of plants ranged from 15 to 72/entry/rep)

14. Indian and Mexican safflower cultivars
are genetically distinct
0 0.1
EC 755659
EC 755660EC 755661
EC 755662
EC 755663
EC 755664
EC 755666
EC 755667
EC 755668
EC 755669
EC 755670
EC 755671
EC 755672
EC 755673
EC 755674
EC 755675
EC 755676
EC 755677
EC 755678
EC 755679
EC 755680
EC 755681
EC 755682
EC 755683
EC 755684
EC 755685EC 755686
EC 755687
EC 755688
A-1
A-2
A-300
N IR A
N AR I-6
N AR I-38
N AR I-52
N AR I-N H -1
N AR I-H -15
N AR I-H -23
AK S207
PK V PIN K
B H IM A
PH U LE K U SU M AG IR N A
TAR A
SSF658
SSF708
SSF733
SSF748
PB N S-12
PB N S-40
SH AR D A
JSF-1
JSF-7
JSI-97
JSI-99
JSI-73
R VS113
N AR I57
70
50
77
100
96
81
58
Mexico
India
NJ tree
Pair-wise Fst =
0.39639
Based on 33
polymorphic
SSR loci
Kadirvel et al. (unpublished)

15. Development of breeding populations
Elite varieties Donors Target trait
A1 EC755684 (S-719) High oil (38%)
Bhima EC755660 (S-334) High oil (40%)
PBNS12 EC755664 (CW-99) High oil (37%)
NARI57 EC755671 (CCC-B4) High oil (40%)
EC755673 (Humaya-65) High oil (41%)
EC755675 (Aceitera) High oil (41%)
EC736487 High oil (45%)
EC736498 High oil (40%)
EC736501 High oil (40%)
EC736516 (Centennial) High oil (42%)

16. Development of breeding populations
A x B C x D
F1 F1x
E x F G x H
F1 F1x
x x x x x x x x x
x x x x x x x x x
x x x x x x x x x
x x x x x x x x x
x x x x x x x x x
x x x x x x x x x
Intercrosses
x x x x x x x x x
x x x x x x x x x
x x x x x x x x x
Single crosses:
high seed yield
x high oil
Double crosses
Four-way crosses
Single seed
descent (SSD) Multi-parent Advanced
Generation Intercross (MAGIC)
population for breeding and
genetics research

17. No.ofF2:3progenies
Oil content (%)
NARI-57
(36%)
Centennial
(42%)
Evaluation of F2:3 progenies for oil content
Quantitative inheritance

18. Evaluation of F2:3 progenies for seed yield
NARI57 x EC736516 (Centennial) cross – 265 F2 progenies
NARI-57 (49 g)
Centennial (25 g)
No.ofF2progenies
Seed yield per plant (g)
Pedigreeselection

19. Evaluation of F3 families for oil yield

20. Strategies for increasing oil
content in safflower
 Negative relationship between hull
content and oil content
 By reducing the hull content, oil
content has been increased from
42% to 50% in USA
Professor Paul F.
Knowles, UC-Davis

21. Marker development and
genotyping applications:
SSR and SNP
Mapping of
genes and QTLs
Linkage mapping
•Develop bi-parental
mapping populations
(RILs/DHLs)
•Construct genetic linkage
maps
•Marker-trait association
through statistical tools
Association mapping
Genome-wide association
analysis
•Develop genotype panels – germplasm/
MAGIC/ NAM populations
•High throughput genotyping using
genome wide SNPs
•Phenotype the panels
•Marker-trait association
Candidate gene
based allele mining
•Develop genotype panel
•Resequence candidate
genes
•Phenotype the panel
•Association of SNP
haplotypes with oil content
Validation and fine-mapping of QTLs
Marker-assisted selection
•Marker-assisted backcrossing (MABC)
•Marker-assisted pedigree selection (MAPS)
•Marker-assisted recurrent selection (MARS)
•Genome-wide selection
Integrate in mainstream
classical breeding
programme for cultivar
development
Germplasm
evaluation
Trait mapping and marker-assisted selection

22. Species Populations Markers and
maps
No. of
QTLs
Phenotypic
variance (%)
Reference
Arabidopsis RIL SSRs, CAPS
and SSLPs
4 4.5-16.7 Hobbs et al. (2004)
Brassica
napus
DH SSR 17 - Zhang et al. 2005
DH SSR 15 - Zhang et al. 2006
DH SSR 14 1.7-13.4 Delourme et al.
(2006)
DH SSR, SNP,
InDel, IFLP
12 9.15 - 24.56 Sun et al. (2012)
DH SSRs, SRAPs,
STSs and
IFLP
63 2.64–17.88 Wang et al. (2013)
Soybean RIL 94 3 9.4-15 Panthee et al. (2005)
RIL SSRs 11 4-11 Eskandari et al.
2013a; 2013b
Sunflower F2:3 RFLP 8 Leon et al., 2003
“Strong epistatic and QTL x Environment
interactions reported”
Experience of QTL mapping for oil content in crops

23.  Association mapping may be an
effective strategy for mapping oil
content considering its highly
polygenic nature.
 Phenotypic diversity, population
structure and linkage disequilibrium in
a germplasm panel are critical
information for conducting association
mapping reliably.

24. Towards germplasm panel for association
mapping of oil content
Usha Kiran et al. (unpublished)
Genetic diversity and
relatedness in the core
subset of 148 safflower
accessions based on
50 SSR loci
 No. of SSR alleles ranged from 1 to
15 with an average of 3.1 per locus
 Gene diversity ranged from 0.000-
0.866 with an average of 0.271
 Polymorphism information content
values of SSR primer-pairs ranged
from 0.000-0.853 with an average of
0.244

25. 1 2 3 4
2 0.41
3 0.095 0.4603
4 0.4015 0.3001 0.4864
5 0.0859 0.1843 0.084 0.1802
STRUCTURE revealed four populations
(52%) and admixture (48%) group in the
sub-core accessions
Pair-wise Fst values
Source of
variation
d.f. Sum of squares Variance
components
Percentage
of variation
Among
populations
4 199.462 0.90168 Va 15.01
Within
populations
291 1485.399 5.10447 Vb 84.99
Total 295 1684.861 6.00614
Population structure in the core subset

26. Marker 1 LG Marker 2 LG r2
ct448 T9 ct316 T1 0.1370
ct594 T3 ct309 T2 0.8014
ct32 T5 ct309 T2 0.1083
ct32 T5 ct594 T3 0.1073
ct266 T5 ct137 T5 0.1156
ct274 T7 ct137 T5 1.0000
ct274 T7 ct266 T5 0.1156
ct233 T5 ct32 T5 0.2321
ct440 T5 ct32 T5 0.1913
ct440 T5 ct233 T5 0.1188
ct337 T6 ct32 T5 0.1198
ct337 T6 ct233 T5 0.1225
ct297 O3 ct266 T5 0.1071
ct297 O3 ct518 T5 0.1828
ct861 O4A ct518 T5 1.0000
ct218 T11 ct331 T6 1.0000
ct783 O1 ct15 T7 0.8647
ct861 O4A ct297 O3 0.1828
Linkage disequilibrium among SSR loci
 About 1.4% of SSR locus
pair were in significant
LD
 LD preserved by linkage
is essential for
association analysis
 High level of LD between
unlinked loci would lead
to spurious association
 LD due to population
structure and
relatedness need to be
corrected before using
the germplasm panel for
association analysis
Usha Kiran et al. (unpublished)

27. Genomic resources are limited in safflower
Usha Kiran et al. (unpublished)
 About 1000 SSR markers have
been developed so far in safflower.
We are designing more SSR
markers
 No high resolution genetic linkage
map is available
 SSR polymorphism is very low
 Need to design SNP markers
“We have initiated de novo sequencing of safflower
genome through Next Generation Sequencing
(NGS) to facilitate resequencing of a pool of
promising genotypes and designing genome wide
SNP markers for trait mapping purposes”

28. Prospects of candidate gene based allele mining
for finding useful alleles contributing for high oil
content and quality in safflower
Kennedy pathway
Chloroplast

29. Pathways leading to synthesis of essential
fatty acids
Hypothesis: “Superior alleles of candidate genes are
dispersed across different genotypes in the
germplasm collection”

30. Market for high oleic oils is expanding
 Concern about health implications of trans fat in foods
 Food industry is looking for frying oil with low linolenic
(omega-3) and adequate quantity of linoleic acid (omega-
6)
 Need to improve the oxidative stability of commodity oils
 Its extraordinary high stability can maintain preferred
flavours in storage, extend shelf life, reduce packaging
and enable new applications
 High oleic is the naturally available option for high stable
oil
“US food markets have already
shifted to high oleic oil”

31. Marker-assisted backcrossing of high oleic
trait in safflower
Oleicacidcontent(%)
Safflower Varieties
Variation for high oleic
content in safflower
varieties
“Genetics of high oleic trait appears to be simple
and recessively inherited”

32. Why marker-assisted selection for high oleic trait?
Recurrent
parent (RP)
(Elite variety)
(OlOl)
High oleic
donor (olol)
x
F1 (Olol)xRP
BC1F1 (Olol)
x x x x x
BC1F2 familiesxRP
BC2F1
x x x x x
Year 1
Year 2
Year 3
Year 4
Recurrent
parent (RP)
(Elite variety)
(OlOl)
High oleic
donor (olol)
x
F1 (Olol)xRP
BC1F1
x x x x x
xRP
BC2F1
x x x x x
Year 1
Year 2
Year 3
“MAS would save one generation
and resources needed for a
backcrossing programme”
(olol)
(Olol)
(Olol)
(Olol)

33. A single base mutation in fatty acid desaturase-2
(FAD2-1) is known to cause high oleic accumulation
in safflower seed (Guan et al. 2012)
We have detected
the same
mutation in our
high oleic
sources

34. Designing and validation of genotypic assay for
marker assisted selection for high oleic trait
Low oleic (<30%) High oleic (70-80%)
“Conversion of
mega safflower
varieties to high
oleic if food
industry
promotes”

35. Safflower is an opportunity crop - drought
tolerant and high value oil – needs revival in
India

36. Acknowledgements
Dr. N. Mukta
Mrs. B. Usha Kiran
Dr. S. Senthilvel
Dr. K. Alivelu
Mr. D. Ravi
Mr. Ch. Veerraju
Dr. K.S. Varaprasad
AICRP-Safflower breeders
Funding sources:
ICAR
MARICO Pvt. Ltd. Mumbai