Molecular Breeding in Minor Oilseed Crops: Opportunities ... - icrisat

Molecular Breeding in Minor Oilseed Crops:
Opportunities and Challenges
V. Dinesh Kumar
Directorate of Oilseeds Research,
Rajendranagar, Hyderabad, India
2 nd National Workshop on “Marker Assisted Selection for Crop Improvement”
27-29 October 2010, ICRISAT, Patancheru, India

Outline of the presentation..
‣ Some basic statistics
‣ Importance of the minor oilseed crops
‣ Genomic resources available
‣ An update on molecular markers used so far
‣ where we stand with respect to MAS – Indian scenario
‣ Challenges and road ahead in each crop
‣ Some open issues..

Crop-wise % area to total oilseeds area and
% production to total production in India
GN RM SB Sun Ses Lin Saf Cas Niger
Area 22.4 22.8 34.5 6.6 6.6 1.5 1.1 3.1 1.4
Prod 25.9 26 35.7 4.2 2.3 0.61 0.7 4.2 0.42
Six crops’ share - 20.3 % area and 12.43 % production,
thus relegated to minor oilseeds’ status
But, they are being cultivated by poor farmers, on marginal lands.
traditionally grown, each with a specific niche area

India’s position with respect to it’s share compared
to world (2008-09)
Crop
Area % of
world (rank)
Production %
of world (rank)
Sunflower 8 (IV) 3.1 (X)
Safflower 50 (I) 37 (I)
Sesame 23 (I) 18.5 (I)
Castor 58 (I) 71 (I)
Linseed 23 (II) 7.4 (IV)
This clearly indicates the importance of these crops
We are the world leaders in production and thus should be the first to
adopt the recent techniques in these crops
Ironically, least information (of MAS) is available in these crops

Present scenario..
Productivity (kg/ha) in India and the world
There is a huge gap between what we harvest and
the potential of the crop

Sunflower
• Oil rich in linoleic acid - used for culinary purposes, in preparation of
vanaspati and in the manufacture of soaps and cosmetics
• Being rich in PUFA, considered a healthy oil
• Oil cake can be used as animal feed
Safflower
• Oil is rich in polyunsaturated fatty acid – linolenic acid
• Cultivated for both oil (healthy) and the petals –
for pharmaceutical and dye purpose
• The cake, particularly from decorticated seed, is used as a
concentrated cattle feed, and that from undecorticated seed
is sometimes used as a manure.

Sesame
• Rich source of edible oil (46 to 52 per cent).
• Oil has excellent keeping quality - cooking and pickling oil
• It is also used for anointing the body, for manufacturing perfumed oils and for
medicinal purposes.
• Sesamum-cake is a rich source of protein, carbohydrates and mineral nutrients,
such as calcium and phosphorus.
• The cake is edible and is eaten avidly by working classes.
• Oil rich in ergosterols and antioxidants

Castor
• Very unique oil – 90% ricinoleic acid thus most pure fatty acid found
– the only oil with a hydroxyl group
• Earns - about 1000 crores for the National Exchequer
• Perennial tree crop that is drought hardy
• Oil used as a lubricant in high-speed engines and aeroplanes,
in the manufacture of soaps, transparent paper, printing-inks,
varnishes, linoleum and plasticizers - over 150 uses
• It is also used for medicinal and lighting purposes.
• Value added castor oil is very expensive – more than 20 fold increase in value
(for 4 th order molecules)
• De-oiled cake can be used as manure and nematicide – NOT USED AS ANIMAL FEED

Linseed
• The oil content of the seed varies from 33 to 47 per cent.
• Now the oil is promoted as a healthy oil due to high levels of
alpha linolenic acid - anticholesterol
• Oil is an excellent drying oil used in manufacturing paint and
varnishes, oilcloth, waterproof fabrics and linoleum and as an
edible oil in some areas.
• Linseed-cake is a very good manure and animal feed.
• Linseed straw produces fibre of good quality. Linseed is used in
making paper and plastics.

Genomic resources in the selected oilseed crops
Crop Botanical name Family Nucleotide
sequences
ESTs
Protein
db
Genom
e size
(Mbp)
Chromosome
number
Sunflower
Safflower
Sesame
Castor**
Linseed
Helianthus
annuus L.
Carthamus
tinctorius L.
Sesamum
indicum L.
Ricinus
cummunis L.
Linum
usitatissimum L.
Asteraceae 12148 134343 6320 3000 2n=2x=34
Asteraceae 511 41588 154 1350 2n=2x=24
Pedaliaceae 332 3771 103 948 2n=2x=26
Euphorbiaceae 83468 63398 63078 400 2n=2x=20
Linaceae 4794 12604 1862 686 2n=2x=30
* as on 26-10-2010 at NCBI website (http://www.ncbi.nlm.nih.gov)
**castor 4X draft genome sequence available now

Molecular markers used in the crops
Crop Markers Used for
Sunflower
Safflower
Sesame
RAPD, ISSR, AFLP, EST-
SSR, SSR, InDels, SNP
RAPD, ISSR, AFLP, EST-
SSR, SSR, SNP
RAPD, ISSR, AFLP, EST-
SSR, SSR
Diversity, Phylogenetic, map
development, tagging and
mapping qualitative and
quantitative traits, MAS
Diversity, Phylogenetic, map
development, tagging and
mapping qualitative traits
Diversity, tagging of
qualitative traits
Castor RAPD, SSR, EST-SSR, SNP Diversity
Linseed RAPD, AFLP, SSR, EST-SSR Diversity, tagging

Sunflower
• All key components (saturated map, trait-marker association,
fine mapped loci, mapped QTLs) for MAS available
• Well saturated reference map developed with anonymous, gene-targeted
and perfect markers available
• A well characterized wild species’ gene-pool for introgression available
- already SNPs and their LD decay for wild populations available
• Well characterized resistance gene islands for introgression
• TILLING platform available
• Time and resources ripe for exploitation of MAS in crop improvement
SND, Alternaria, downey mildew and powdery mildew tolerance –
immediate task

Tagging and mapping in Sunflower
Trait Gene Markers used Closest
genetic
distance (cM)
Downy
mildew
Pl1
--
Pl5/ Pl8
Pl5/Pl8
--
--
Pl13
Orobanche Or 5
Or3
Sclerotinia
midstalk-rot
White rot
and black
stem
Chlorotic
mottle virus
Phoma basal
stem and
root necrosis
QTL
--
--
RFLP, CAPS
IFLP, NBS-LRR, SSR
TIR-NBS-LRR
SSR
STS
EST
TRAP, SSR
SSR, AFLP
RFLP, SSR
SSR
SSR
SSR
--
2.4
--
--
--
--
0.9
5.6
--
--
--
--
Reference
Gedil et al., 2001
Slabaugh et al., 2003
Radwan et al., 2003
Duble et al., 2004
Radwan et al., 2004
Bouzidi et al., 2007
Sujatha et al., 2009
Tang et al., 2003
Perez-Vich et al., 2004
Micic et al., 2004
Micic et al., 2005a
Micic et al., 2005b
QTL RFLP, SSR -- Bert et al., 2004
Rcmo-1 SSR 4.0 Lenardon et al., 2005
QTL SSR, AFLP -- Abou et al., 2006

Tagging and mapping in Sunflower…
Trait Gene Markers used
Rust
(Puccinia
helianthi)
R1 and
R(Adv)
Closest
genetic
distance (cM)
Reference
RAPD -- Lawson et al., 1996
Oil content QTL AFLP, SSR 20.9 Mokrani et al., 2002
Gammatocopherol
content
High stearic
acid content
Seed
hypodermis
color
Tph2 SSR, INDEL 3.6 María et al., 2006
Es1, Es2
and Es3
SSR 3.9 Perez-Vich et al., 2006
Hyp RFLP -- Leon et al., 1996
Seed-quality QTL SSR -- Ebrahimi et al., 2008
Selfpollination
and seed
dormancy
Restoring
pollen fertility
Nuclear
male-sterility
QTL SSR -- Gandhi et al., 2005
Rf1 RAPD, SCAR, AFLP 0.3 Horn et al., 2003
ms9
Rf (4)
SSR, TRAP
SSR and RFLPderived
STS-markers
1.2
0.9
Chen et al., 2006
Feng and Jan, 2008

Tagging and mapping in Sunflower…
Trait Gene Markers used
Plant height or
flowering dates
Lemon ray flower
color
Branching and
pericarp pigment
Closest
genetic
distance (cM)
Reference
QTL SSR -- Bert et al., 2003
Yf, Yf1 SSR, EST 2.3, 1.5 Yue et al., 2008
Hyp SSR -- Tang et al., 2006
Apical branching b (1) TRAP, RFLP 2.5 Rojas-Burros et al., 2008
Early domestication QTL SSR -- Wills and Burke, 2007
Chlorophyll
fluorescence
Chlorophyll
deficiency
Photosynthesis and
water status
Plant water status
and osmotic
adjustment
Drought or salinity
stress
Chilling and salt
stresses
QTL SSR -- Poormohammad et al., 2008
Yl SSR, TRAP 4.2 Yue et al., 2009
QTL AFLP 6.0 Herve et al., 2001
QTL AFLP, SSR 3.7 Poormohammad et al., 2007
QTL SSR -- Xianan and Baird, 2003
-- EST -- Paula et al., 2008

Validation at individual plants

Sujatha et al., 2009. TAG 119:795-803

Use of SNP – characterization of inbred lines
(Fusari et al., 2008, BMC Plant Biology 8:7)
• A set of 28 candidate genes related to biotic and abiotic stresses
studied in 19 sunflower inbred lines.
• A total of 14,348 bp of sequence alignment analyzed per individual (1
SNP/ 69 nucleotides and 38 indels identified)
• The number of haplotypes per region ranged from 1 to 9
(mean = 3.54 ± 1.88).
• Two putative gene pools identified (G1 and G2), with a large
proportion of the inbred lines being assigned to G1
• LD for G1 decayed more rapidly (r2 = 0.48 at 643 bp) than the LD
trend line for the entire set of 19 individuals
(r2 = 0.64 for the same distance)
This work could facilitate association mapping with lower marker
densities than those usually reported for other plant species

MAS as applied in breeding programmes of sunflower
• Much of the work in private sector – for downey mildew resistance,
high oleic content, herbicide tolerance (simple traits)
• Complex traits like Sclerotinia, Phoma and Phomopsis being taken up
• Candidate gene based MAS being followed for transferring resistance genes
from wild species (both in public and private research)
• AB-QTL adopted for transferring QTLs for transferring agronomic traits
(for increased oil content through introgression from wild)
• Transfer of restorer genes using ‘perfect markers’
• Gene pyramiding against stem rot
• In general, very limited application of MAS for complex traits – factors like
diagnostic gene-based assays, high-throughput cost effective assays,
methods to accurately define QTLs, TILLING and association mapping
being addressed now.

Safflower
• So far markers have been used for either diversity studies or
for tagging some qualitative traits (male sterility and high
linolenic acid)
• SNPs identified in only a small target region – again used for phylogenetic
studies
• Very recently, a skeletal map (with only 116 EST-markers) has been developed
- need for saturated map
• About 800 EST-SSRs have been developed –
but so far used only for diversity studies
• At DOR, the developed markers are being used for tagging wilt resistance
genes from different wild species sources
Immediate targets for MAS – resistance against many isolates of wilt
- Alternaria tolerance

Sesame
• So far, studies restricted to diversity studies using anonymous markers
• Recently, EST-SSRs (about 50) developed
• No molecular map developed
• Closed capsule trait has been tagged using AFLP
• Determinate growth habit (Dt) gene tagged using RAPD and ISSR
Exploitation of the variability in sesame germplasm using association
mapping should be a priority area
Mapping of genes for capsules per plant, seeds per capsule and earliness
- with an emphasis to increase the yield
Developing the molecular map is an essential activity to harness
benefits of MAS in future

Castor
• Marker work restricted to only assessment of diversity
• These studies have indicated very low genetic diversity in the world collection
- monotypic species – so only primary gene pool to be exploited
• So far mainly anonymous markers have been used for diversity study
• We have developed 519 class I EST-SSR primers and they are being used
in tagging wilt resistance gene(s)
• Recently complete genome sequence published
(Nature Biotech., August 22, 2010)
Immediate target traits – reduced ricin and RCA content – complex genomics
- Botrytis tolerance (devastating disease with 80% loss)
- sex expression problem
- heterotic pools for increased yields

Linseed
Mianly diversity studies so far – with anonymous markers
Very recently, 610 EST-SSR primer pairs developed in Canada
Immediate targets
• Development of good markers and a molecular map
• Bud fly tolerance
• Development of dual type genotypes for both fibre and oil

Where we stand – with respect to MAS
Crop Strength Weakness Opportunity Traits of
importance
Sunflower
Safflower
Sesame
Castor
Linseed
Saturated Maps,
international,
characterized
genepool
Skeletal map,
Markers (recently)
Germplasm
Germplasm
Some genomic
resources
Genomic
resources,
GENOME
SEQUENCED
Germplasm
Some genomic
resources
Germplasm
Still MAS not
much adopted
for QTLs
Very small
group working
on markers
No map,
Very small
group working
on markers
No map,
Small group,
Less
variability,
monotypic
No map,
Small group
MAS could be
adopted for
several traits
Saturated maps,
Use of MAS,
Germplasm
Saturated maps,
Use of MAS,
Germplasm
Maps and MAS
Germplasm
MAPS and MAS
SND, Alternaria
Yield plateau
Alternaria, wilt
Capsule
shattering
‘Ricin’ toxin
Botrytis ,
capsule borer
Sex expression
Bud fly,
Alternaria

Major challenges – across all the crops (except sunflower)
• Mostly grown on marginal soils without irrigation – so drought tolerant cultivars
the need of the hour
• Non availability of validated markers with high PIC values – scope for DArT
• Non availability of molecular maps
• Very small group is working on these crops – barely any on markers
• Not much of work on MAS in India – especially people with active
crop improvement activities
• Need for trait based mapping populations
• Capacity building – critical trained mass not available to take up the MAS
• Plant breeders – to be empowered better to take part in the marker work
• Exploitation of the germplasm available – association mapping !!!

Thank you

Crop-wise Area, production and yield of oilseeds in the world
(2008-09)
Crop
Area
‘000 ha
Production
‘000 tons
Yield
kg/ha
Groundnut 24590 38201 1554
Rapeseed and
2384 58364 1883
mustard
Soybean 96870 230953 2384
Sesame 7534 3603 478
Sunflower 25024 35643 1424
Safflower 691 615 890
Linseed 2437 2200 903
Castor 1525 1581 1037
Total 189658 371160 1957