INTROGRESSION BREEDING FOR SUBMERGENCE TOLERANCE IN RICE

GEETANJALI BARUAH

JRF & Ph.D. Scholar

Assam Agricultural University,

Jorhat-13, Assam

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

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

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

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

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

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

Xu et al. (2006) discovered SUB 1 locus contains-

SUB 1 A, SUB 1 B & SUB 1 CAll 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

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

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Overview of ‘Marker Genotyping’Overview of ‘Marker Genotyping’

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)

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

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

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

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

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

Sowing

14-21 days old

Submerge for 14 days

10 to 21 days

Desubmerge

Scoring

Molecular Markers developed in the Sub1 gene cluster

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

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:

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)

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

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

THANK YOU FOR YOUR ATTENTION