Rice is a staple cereal of India cultivated in about 43.5 Mha area Affected by abiotic stresses like submergence Flash flood - plants are completely submerged for up to 2 weeks 30% (12–14 Mha) of the rice growing area affected Severe loss in rice productive with an average of only 0.5–0.8 t/ha

1. Genetics of Submergence Tolerance in Rice
Credit Seminar
Presented By :-
Jyoti Prakash Sahoo
Roll No. 14-AMJ-15
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2. Flood - The problem in rice cultivation…
• Rice is a staple cereal of India cultivated in about 43.5 Mha area
• Affected by abiotic stresses like submergence
• Flash flood - plants are completely submerged for up to 2 weeks
• 30% (12–14 Mha) of the rice growing area affected
• Severe loss in rice productive with an average of only 0.5–0.8 t/ha
Kuppar village in Jammu, India
India – Tamil Nadu Flood
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3. FR-13 A with undesirable trait
FR-13A Submerged for two weeks
 FR13A (Dhalputtia) from Odisha recognized for submergence tolerance
 Major tolerant source – SUBMERGENCE 1 (Sub1) on chromosome 9 identified for
managing flash flooding.
(Mackill et al. 1996)
Submergence tolerant cultivar…
Derived lines - IR40931, IR49830
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4. Discovery of SUB1 gene and the development of submergence
tolerant rice…
2003 - Jalashree, Jalkuwari and Plaban
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5. Mapping and molecular characterization of SUB1…
FR13A Cultivar
(IR40931-26)
(Tolerant indica derivative)
Nipponbare (M-202)
(Intolerant japonica cultivar)
X
F1
169 F2
624 RAPD primers used to screen the bulks
Five specific bands and were used to genotype whole F2 population
Sub1 gene mapped with RFLP marker on chromosome 9 using MAPMAKER
Xu and Mackill, 1996
Sub 1
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6. Journey of Sub1 QTL to gene…
• Major QTL on chromosome 9 mapped in 169 F2 population using RAPD and
RFLP. (Xu and Mackill, 1996)
• A high-resolution linkage mapping of Sub1 locus in 2950 F2 population using
AFLP and RFLP marker . (Xu et al., 2000)
• Map-based cloning & sequencing of Sub1 locus revels, Sub1A, Sub1B and
Sub1C genes (Xu et al., 2006)
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7. Genomic organization of Sub 1 QTL…
Fig. ERF gene structure and organization in tolerant indica (IR40931-26) and
intolerant japonica (Nipponbare).
Xu et.al (2006)
o The Sub1 region, flanked by the markers CR25K and SSR1A
o Encodes three ERF domain Sub1A, Sub1B and Sub1C
o SUB1A shares 87.7% and 77.2% sequence identity with SUB1B and SUB1C, respectively
15 bp
11 bp
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8. • Downregulation of Sub1C and upregulation of Alcohol dehydrogenase 1
(Adh1) indicating that Sub1A is a primary determinant of submergence
tolerance
Expression profiling of genes in indica & japonica cultivars…
Fig. Semi-quantitative RT–PCR assessment of gene transcript levels in shoot tissue
from tolerant (FR13A) and intolerant (Nipponbare) genotypes
Xu et.al (2006)
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9. Haplotypes of the Sub1 locus…
Line Phenotype Subspecies Sub1A
allele
Sub1B
allele
Sub1C
allele
FR13A Tolerant Indica A1 B-1 C-1
Goda Heenati Tolerant Indica A1 B-6 C-1
Kurkaruppan Tolerant Indica A1 B-3 C-1
IR64 Intolerant Indica A2 B-1 C-3
IR24, Swarna Intolerant Indica Absent B-8 C-6
IR50 Intolerant Indica Absent B-9 C-7
Nipponbare Intolerant Japonica Absent B-2 C-2
Liaogeng Intolerant Japonica Absent B-2 C-2
M-202 Intolerant Japonica Absent B-2 C-2
• Tolerance associated with the presence of Sub1 A-1 , Sub1 B-1,6,3 , Sub 1C-1 allele
Xu et.al (2006)
• A survey of Sub1 locus haplotypes in 17 indica and four japonica varieties identified two
Sub1A, nine Sub1B and seven Sub1C alleles on the basis of variation in AA sequence
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10. Flooding survival in rice with Sub1…
alpha-amylase
Sucrose synthase
Expansin A
Alcohol Dehydrogenase,
Pyruvate Decarboxylase
Perata et al., 2007
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11. • Effective method to introgress a single locus controlling a trait of interest while
retaining the essential characteristics of the recurrent parent.
Introgression Breeding of Sub1 mega varieties using Marker-assisted
backcrossing (MABC)…
• Plants selected having the
marker of the donor
parent at the target locus
• To maintain the target
locus in a heterozygous
state (one donor allele
and one RP allele)
• Selecting BC progeny
with the target gene
• Reduce the size of the
donor chromosome
segment
• Selecting BC progeny with
the greatest proportion
of RP genome using
markers unlinked to the
target locus
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12. Flow of work through MABC in rice for submergence tolerance…
High yielding but
Submergence
susceptible
Submergence
tolerance

13. Advantage of MABC over Conventional backcross breeding…
 Simpler method compared to phenotypic screening
 Save time and resources
 Selection at seedling stage possible for traits like grain quality
 Minimizing linkage drag in two or three backcross generations
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14. Submergence tolerant rice varieties released/ at advanced
stage of release in India…
Introgression of Sub1A1 is being carried out at RARS, AAU, Assam using Swarna-Sub1 as DP
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15. Case Study…
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16. Objectives…
• Development of mega varieties with Sub1 introgression
• Assess the performance of Sub1 in different genetic backgrounds
• Determine the roles of the Sub1A and Sub1C genes in conferring tolerance
• Assess the tolerance level in F1 heterozygous hybrids for the Sub1A-1 allele
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17. Developing mega varieties with Sub1 introgression…
To transfer the tolerant Sub1 allele into the mega varieties (MAB Strategy followed)
• Closely flanking markers used for recombinant selection to reduce the target
introgression size
• Background markers used to select for recurrent parent alleles
Donors :
Two breeding lines derived from
FR13A i.e. IR49830 and IR40931
Recipient parents (Mega Varieties) :
• Samba mahsuri and CR1009 from india
• IR64 from the philippines (IRRI)
• Thadokkham 1 (TDK1) from Laos
• BR11 from bangladesh
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18. Developing molecular markers within Sub 1 cluster…
Septiningsih et al., 2009
• Primer3 was used to design PCR primer
• BLAST used to find regions of similarity of markers for Sub1 target region
• CLUSTALW used for sequence alignments
Finally developed total 11 pairs of markers in Sub1 cluster
The fully converted sub1 lines were selected at the BC2F2 or BC3F2 generation
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19. Submergence screening performed in the greenhouse at IRRI…
Trays de-submerged
Sub1 lines along with parents and the susceptible check- IR42
planted into trays
Submerged when seedlings were 14–21 days old
When susceptible check IR42 varieties shown >50% damage
Survival of plants Scored after 10–21 days recovery
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20. Performance of mega varieties with submergence tolerance…
IRRI, Philippines
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21. Performance of Sub1A/Sub1C recombinant…
IR64 X IR40931
After screening 2240 BC1F2
IR64-Sub1 BC
(Sub1B and Sub1C present
but Sub1A still segregating)
BR11 X IR40931
After screening 2137 BC1F2
BR11-Sub1A
(Sub1A present but Sub1B
and Sub1C segregating)
Sub1A-2(IR64) Sub1B-1 and Sub1C-1(IR40931)
Sub1A-1(IR40931) Sub1B and Sub1C-3(BR11)
Septiningsih et al., 2009
Both allowed
to self pollinate
and genotyped
To select the homozygous recombinants
RM8300 & ART5
recombinant
markers used
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22. • Both trials were conducted in a submergence tank using RCBD with three and two replications
for trials 1 and 2, respectively. Plants were submerged after they were 14 d old and desubmerged
when most of IR42 were damage. Scoring was done after 21 d recovery.
Submergence screening of homozygous recombinants…
No significant
difference in
tolerance
having Sub1
locus from IR64
No significant
difference in
tolerance
having Sub1
locus from
IR40931
Intolerant
Phenotype
Tolerant
Phenotype
Septiningsih et al., 2009
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23. • Sub1C expression appeared to be independent of the Sub1A allele
• The expression of Sub1A-1 was slightly reduced in the presence of the Sub1C intolerant
allele, but it does not significantly reduce the level of tolerance
Expression profiling of recombinants & parental lines…
S= Submerged for 6 hour
C= Non submerged control
S – Susceptible, T - Tolerance
Recombinant
Septiningsih et al., 2009
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24. Performance of heterozygous Sub 1A lines…
• Heterozygotes were found to be less tolerant then homozygotes
• Sub1 locus does not show dominance and tolerance is closely associated with the
dosage of the Sub1A alleles
S = Susceptible, M – Moderately tolerant, T = Tolerant
Septiningsih et al., 2009
IR64 IR64–Sub1
X
F1
(compared with parents)
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• All mega varieties with Sub1 introgression had a significantly higher survival rate
than the original parents.
• An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not
significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to
be independent of the Sub1A allele
• Even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2,
plants remained intolerant.
• Survival rates and Sub1A expression were significantly lower in heterozygotes
compared with the homozygous tolerant parent.
Findings…

26. Conclusion…
 Sub1 provided a substantial enhancement in the level of tolerance of all the
susceptible mega varieties.
 Sub1A-1 is confirmed as the primary contributor to tolerance, while Sub1B & Sub 1 C
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.
 The marker-assisted introgression of the SUB1 has successfully improved
submergence tolerance in a wide range of mega-varieties
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27. Acknowledgement…
 Dr. M. K. Modi (Major advisor)
 My parents, respected teachers, my seniors and all my friends
 Department of Agricultural Biotechnology and AAU
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