Towards improvement of oil content in safflower (Carthamus tinctorius L.)

Indian Institute of Oilseeds Research (formerly DOR), Hyderabad

CO1 (1982)

Pro

du

ctio

n (

‘000

to

nn

es)

Source: IARI (2012)

“India imported Rs. 55,000 crore worth of vegetable oils in 2012-13”

India’s vegetable oil economy

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

Safflower, a multipurpose oilseed crop

Seed - cooking oil, bird seed

Petals - natural dyes (Carthamin) and medicinal use (herbal tea)

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 6.5Kanu (2011); Kostik et al. (2012)

Safflower oil is a good source of essential fatty acid (Linoleic acid, Omega-6)

Safflower is grown in dry land and limited irrigation conditions

Dharwad (Karnataka) Raipur (Jharkhand)

Safflower based intercropping systems

Safflower + Chickpea

Safflower + Linseed

Safflower + Wheat

Source: FAOSTAT (2012)

Country Area (ha) Production (tonnes)

Productivity (Kg/ha)

Mexico 1,72,866 2,57,451 1,489.3India 1,78,000 1,45,000 814.6Kazakhstan 2,43,600 1,27,210 522.2United States of America 64,790 81,390 1,256.2China, mainland 23,000 36,000 1,565.2

India loses its first place to Mexico in safflower production

State Area (ha) Production (tonnes)

Productivity (Kg/ha)

Maharashtra 1,05,000 58,000 552Karnataka 48,000 30,000 625Andhra Pradesh 11,000 9,100 825OthersOdisha, West Bengal, Jharkhand, Bihar, Chhattisgarh)

2,400 1400 580

Maharashtra state leads safflower production in India

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

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

Exotic varieties recorded higher oil content than Indian cultivars (ICRISAT Farm; Rabi 2012 -13)

Oil

co

nte

nt

(%)

Safflower Cultivars

Mexican

Indian

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)

Indian and Mexican safflower cultivars are genetically distinct

0 0.1

EC 755659EC 755660EC 755661

EC 755662EC 755663EC 755664

EC 755666

EC 755667

EC 755668

EC 755669

EC 755670EC 755671

EC 755672

EC 755673

EC 755674

EC 755675

EC 755676

EC 755677

EC 755678EC 755679

EC 755680

EC 755681

EC 755682EC 755683

EC 755684EC 755685EC 755686

EC 755687EC 755688

A-1 A-2A-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

SSF748PB 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 I5770

50

77

100

96

8158

Mex

ico

Indi

aNJ tree

Pair-wise Fst = 0.39639

Based on 33 polymorphic SSR loci

Kadirvel et al. (unpublished)

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%)

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

No

. of

F2

:3 p

rog

en

ies

Oil content (%)

NARI-57 (36%)

Centennial (42%)

Evaluation of F2:3 progenies for oil content

Quantitative inheritance

Evaluation of F2:3 progenies for seed yield

NARI57 x EC736516 (Centennial) cross – 265 F2 progenies

NARI-57 (49 g)

Centennial (25 g)

No

. o

f F

2 p

rog

enie

s

Seed yield per plant (g)

Ped

igre

e se

lect

ion

Evaluation of F3 families for oil yield

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

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

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

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.

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

1 2 3 42 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

Marker 1 LG Marker 2 LG r2ct448 T9 ct316 T1 0.1370ct594 T3 ct309 T2 0.8014ct32 T5 ct309 T2 0.1083ct32 T5 ct594 T3 0.1073

ct266 T5 ct137 T5 0.1156ct274 T7 ct137 T5 1.0000ct274 T7 ct266 T5 0.1156ct233 T5 ct32 T5 0.2321ct440 T5 ct32 T5 0.1913ct440 T5 ct233 T5 0.1188ct337 T6 ct32 T5 0.1198ct337 T6 ct233 T5 0.1225ct297 O3 ct266 T5 0.1071ct297 O3 ct518 T5 0.1828ct861 O4A ct518 T5 1.0000ct218 T11 ct331 T6 1.0000ct783 O1 ct15 T7 0.8647ct861 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)

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”

Prospects of candidate gene based allele mining for finding useful alleles contributing for high oil

content and quality in safflower

Kennedy pathway

Chloroplast

Pathways leading to synthesis of essential fatty acids

Hypothesis: “Superior alleles of candidate genes are dispersed across different genotypes in the germplasm collection”

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”

Marker-assisted backcrossing of high oleic trait in safflower

Ole

ic a

cid

co

nte

nt

(%)

Safflower Varieties

Variation for high oleic content in safflower varieties

“Genetics of high oleic trait appears to be simple and recessively inherited”

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

xRPBC2F1

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)

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

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”

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

India

Acknowledgements

Dr. N. MuktaMrs. B. Usha KiranDr. S. SenthilvelDr. K. AliveluMr. D. Ravi Mr. Ch. VeerrajuDr. K.S. VaraprasadAICRP-Safflower breeders

Funding sources:ICARMARICO Pvt. Ltd. Mumbai