Simplified way of applicability of introgression breeding for submergence tolerance in rice with special emphasis on physiology of submergence tolerance

1. INTROGRESSION BREEDING FOR
SUBMERGENCE TOLERANCE IN
RICE
GEETANJALI BARUAH
JRF & Ph.D. Scholar
Assam Agricultural University,
Jorhat-13, Assam

2. Flooding is one of the most important environmental stresses
worldwide
Flash flooding adversely affects at least 16 % of the rice lands of the
world (Khush, 1984)
In the rainfed lowland areas of eastern India, submergence is the third
most important limitation to rice production (Widawsky and O'Toole,
1990)
Complete submergence due to frequent flooding adversely affects
plant growth and yield
The problem

3. Rice is the only crop plant adapted to aquatic environments
because of its well-developed aerenchyma tissues
However, complete submergence due to frequent flooding can
adversely affect plant growth and yield
Two types of flooding cause damages to rice:
 Flash flooding
 Deepwater flooding
Submergence tolerance is required in rainfed areas
The problem

5. Carbohydrate concentration
• A strong positive correlation
• Influenced by growth conditions before submergence
• Level of carbohydrates remaining after submergence is more critical
Alcoholic Fermentation (AF)
• Major metabolic adaptation
• ATP produced by this process is very small (5%)
Experimental observations (Fukao et al. 2006):
(1) Enzymes of AF often increase under flooding
(2) Hypoxia pretreatment increased tolerance
(3) Mutants lacking ADH die more quickly
(4) Rates of AF are related to the tolerance
(5) High sugar supply improved survival
PHYSIOLOGY OF SUBMERGENCE
TOLERANCE

6. Stem elongation
• A strong negative correlation
• Sensitive cultivars survived on application of a gibberellins biosynthesis
inhibitor, paclobutrazol
• Addition of GA reduced survival of submergence tolerant lines
Aerenchyma Formation
PHYSIOLOGY OF SUBMERGENCE
TOLERANCE
Fig. 2: Diffrences in formation of lysigenous aerenchyma and patterns of radial O2 loss (ROL)
in rice roots under drained soil conditions & waterlogged soil conditions, Nishiuchi et al., 2012

7. Post Submergence Events
• High light intensity and higher oxygen levels
• Generation of reactive oxygen species and toxic oxidative products as
acetaldehyde
• Two mechanisms:
Presence of natural antioxidants: ascorbate, α-tocopherol, carotenoids,
glutathione etc.
Presence of antioxidant enzyme systems: superoxide dismutase,
catalase, peroxidase etc.
Role of Ethylene generated during submergence
• Submergence-intolerant cultivars usually showed increased levels of leaf
chlorosis
• Ethylene produced during submergence trigger leaf senescence
• This was proved by using an ethylene inhibitor, 1-methyl cyclopropene (MCP).
PHYSIOLOGY OF SUBMERGENCE
TOLERANCE

8. Two distinct strategies of
growth controls –
 Quiescence strategy
(Colmer and Voesenek 2009)
Escape strategy
(Bailey-Serres and Voesenek
2008; Colmer and Voesenek
2009)
 Both strategies depend on
ethylene- responsive
transcription factors
Fig. 3: Strategies of adaptation to excess water stresses in the
form of submergence or waterlogging, Nishiuchi et al., 2012

9. Xu et al. (2006) discovered SUB 1
locus contains-
SUB 1 A, SUB 1 B & SUB 1 C
All encode ethylene responsive factors
Upregulated under submergence
But only SUB 1 A confers flash flood
tolerance
Reason:
 Restricted shoot elongation
 Supresses expression of α-amylase &
sucrose synthase and regulates alcohol
fermentation (Fukao et al., 2006)
 Enhances expression of SLR1 & SLRL1
(Fukao et al., 2008) and genes involved
in ABA-mediated accimation
 Reduces accumulation of ROSFig.4: Schematic representation of
Sub 1 locus (Fukao et al. 2006, Xu et
al. 2006)
Fig.4: Schematic representation of
Sub 1 locus (Fukao et al. 2006, Xu et
al. 2006)
Sub 1 gene cluster and how it works

10. Introgression is the movement of a gene from donor to recipient
parent by the repeated backcrossing of an F1 hybrid with one of its
parent.
Purposeful introgression is a long-term process
WILD RELATIVE
CULTIVAR

11. (1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
Overview of ‘Marker Genotyping’Overview of ‘Marker Genotyping’

12. MAB has several advantages over conventional backcrossing:
Effective selection of target loci
Minimize linkage drag
Accelerated recovery of recurrent parent
 Plants with desirable genes/QTLs are selected and alleles can be
‘fixed’ in the homozygous state
plants with undesirable gene combinations can be discarded
1 2 3 4
Target
locus
1 2 3 4
RECOMBINANT
SELECTION
1 2 3 4
BACKGROUND
SELECTION
TARGET LOCUS
SELECTION
FOREGROUND
SELECTION
BACKGROUND SELECTION
Marker-assisted backcrossing (MAB)

13. Selection for target gene
or QTL
Useful for traits that are
difficult to evaluate
Also useful for recessive
genes
1 2 3 4
Target
locus
TARGET LOCUS SELECTION
FOREGROUND SELECTION

14. Use of flanking markers to select
recombinants between the target
locus and flanking marker
Linkage drag is minimized
Require large population sizes
--depends on distance of flanking
markers from target locus
RECOMBINANT SELECTION
1 2 3 4

15. Use of unlinked markers to
select against donor
Accelerates the recovery
of the recurrent parent
genome
Savings of 2, 3 or even 4
backcross generations may
be possible
1 2 3 4
BACKGROUND SELECTION

16. Conventional backcrossing
Marker-assisted backcrossing
F1 BC1
c
BC2
c
BC3 BC10 BC20
F1
c
BC1 BC2
TARGET
GENE
TARGET
GENE
Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection:
new tools and strategies. Trends Plant Sci. 3, 236-239.
Markers can be used to greatly minimize the amount
of donor genome

18. Donors: Two breeding lines derived from FR13A i.e. IR49830 and IR40931
Recipient parents:
Samba Mahsuri and CR1009 from India
IR64 from the Philippines (IRRI)
Thadokkham 1 (TDK1) from Laos
BR11 from Bangladesh
Strategy employed to transfer the tolerant Sub1 allele into the mega
varieties
 closely flanking markers used for recombinant selection to reduce the target
introgression size
 background markers used to select for recurrent parent alleles
The fully converted Sub1 lines were selected at the BC2F2 or BC3F2
generation

19. Sowing
14-21 days old
Submerge for 14 days
10 to 21 days
Desubmerge
Scoring

20. Molecular Markers developed in the Sub1 gene cluster

21. Fig.: Graphical genotypes of the IR64-Sub1 BC & BR11-Sub1 A recombinant
plants with RT-PCR analysis

22.  All Sub1 varieties had significantly higher survival rates compared
with the original recipient parents
 IR 64- Sub 1, TDK1-Sub1 and CR1009-Sub1 showed the same high
level of tolerance
 Whereas BR11-Sub1 was slightly less tolerant
 Samba Mahsuri-Sub1 was the least tolerant among all the Sub1 lines
 The heterozygous plants of F1 hybrids of IR64/IR64-Sub1 were
significantly less tolerant than the plants homozygous for the tolerant
allele
Findings:

23.  During submergence increased ethylene levels trigger accumulation
of Sub 1 A transcripts
 During submergence, transcription of both Sub 1 A & Sub 1 C is
strongly up regulated and down regulated upon desubmergence
 Sub 1 A down regulates Sub 1 C (Xu et al.,2006)

25. More submergence tolerant
No negative side effect in terms of yield
and grain quality when grown under
control Conditions
Starch and soluble carbohydrate levels
declined more slowly
mRNA levels coding for α-amylases and
sucrose synthases were lower
Pyruvate decarboxylase (PDC) and
alcohol dehydrogenase (ADH) activity
was increased
Ethylene production was lower
Transcription of expansin genes was
suppressed
Fig.: Sub 1 confers tolerance to M2O2, a
submergence intolerant japonica rice
variety
Findings

26. Sub1A is confirmed as the primary contributor to tolerance, while
Sub1C alleles do not seem important
Lack of dominance of Sub1 suggests that the Sub1A-1 allele should
be carried by both parents for developing tolerant rice hybrids
Sub1 could be solution for providing a substantial enhancement in
the level of tolerance of sensitive mega varieties
With the identification of physiological traits, DNA markers and
genes associated with submergence tolerance the prospects for
breeding suitable rice cultivars for rainfed lowlands have been
improved
Summary &
conclusion

27. THANK YOU
FOR YOUR ATTENTION