Over the past few years, a major Quantitative Trait Locus (QTL) controlling
submergence tolerance was mapped to a 6.5 cM region of chromosome 9. It was
cloned, sequenced and annotated (Vanavichit et al., 2001). Due to the
physiological response mechanisms, submergence primarily reduced plant elongation
and delayed leaf senescence, plants can survive and recover from submergence
stress (Timothy and Eufrocino, 1996; Ito et al., 1999). Genetic linkage
between submergence tolerance to both suppressed elongation and delayed leaf
senescence was clearly shown by the QTL mapping analysis involving several segregating
recombinant inbred lines (Toojinda et al., 2003). Both traits were coincidentally
mapped between two marker loci, S10709 and RB0783 on chromosome 9, where their
candidate genes were identified.
Submergence tolerance is characterized by complex phenotype-associated traits. The tolerant plant can be generalized as the ability to survive and continue growing after several days in submerged conditions (Adkins et al., 1990). The phenotypic benefits provided the tolerant rice with the ability to survive and, also, recover, during the flooding period. The submergence-tolerant plants affected by several morphological adaptations decreased the chlorosis of tissues and reduced the shoot elongation on plant growth in order to save carbohydrates and energy for maintenance processes including protective antioxidant systems.
According to a different mapping population, a isogenic line (ISL) was developed by four advanced backcrosses to Khao Dok Mali 105 (KDML105), which was susceptible to submergence stress. The donor of submergence tolerance is a doubled haploid line (DH206) derived from a cross between FR13A and CT6241-7-1-2-2. This ISL of the BC4F7 generation is genetically 94% identical to KDML105 but carries the 6.5 cM of the heterozygous segment at position 102.5 to 109 cM on chromosome 9 (Siangliw et al., 2003).
In this report, we present the associated data between the phenotypic response under flooding and the genotypic alleles of the ISLs containing three Gene Targeted Markers (GTMs), which are localized on 36 kb region within the 6.5 cM of SubQTL9. These markers were identified based on their correlation with individual trait components. In addition, the understanding of the mechanisms used by rice to tolerate submergence stress involved in GTMs compared with individual traits related to submergence stress might open new avenues to the genetic improvement of rice.
MATERIALS AND METHODS
A set of near isogenic lines, ISL-132, was used for this study. The ISLs
population had been developed from BC4F7 by backcrossing an introgression specific
region of chromosome 9. In order to fine map the Sub1 region, DH206, derived
from a cross between FR13A and CT6241-7-1-2-2, was crossed and successively
backcrossed with KDML105 (high quality and submergence intolerance) which resulted
in BC4F8 of ISL-132 differing in the 36 kb of SubQTL9 region which carried
three genes encoding ethylene responsive element binding protein (ERFs) as Sub1A,
OsEREBP1 (Sub1C) and OsEREBP2 (Sub1B) under the
genetic background of KDML105.
Fresh young leaves were collected and ground in a 1.5 mL tube with liquid
nitrogen. Genomic DNA was isolated using the CTAB (acetylmethylammonium bromide)-NaCl
method (Roger and Bendich, 1994). The DNA pellets were re-dissolved (at a concentration
of 50 ng μL-1) in 50 μL of water for PCR-based polymorphism
Characterization of ISL132 of BC4F8
Three pair of primers (Table 1) for Gene Targeted Markers
(GTMs) were used to cover the 36 kb region on the long arm of chromosome 9,
to which major Sub1QTL had previous been mapped (Toojinda et al.,
2003). All polymorphic primers were developed based on the genomic sequence
from GenBank accession number AC90056 and DQ011607. Genotypic screening to maintain
a tolerance allele (FR13A) at major SubQTL9, as well as selection against
an intolerance allele (KDML105), was determined by using the GTMs.
Evaluation of Phenotypic Change under Submergence Stress in ISLs-132 of
The evaluated for several phenotypic adaptations to survival were analyzed
under complete submergence using a dark plastic tank, in August 2004. Individual
plants of ISLs-132 population were grown completely submerged for 20 days at
3 weeks after germination. The water level was maintained at 60 cm above the
tallest plants to prevent leaf tips from emerging into the air.
The measurements of a complex phenotype-associated trait were explained with five major traits responsible for submergence stress. The data of Plant Elongation (PE), Total Shoot Elongation (TSE), Relative Shoot Elongation (RSE) and Leaf Senescence (LS) at 0, 2, 4 and 20 days of submergence was collected. In these experiments, all trait evaluation methods were performed as reported by Siangliw et al. (2003) and Toojinda et al. (2003).
The ANOVA and regression based software (STATGRAPHICS 2.1) was used for
detecting significant correlations between the response traits and submergence
stress and relationship between markers and traits.
DNA Sequencing and Promoter Analysis
DNA sequences of PCR products from specific primers for the promoter regions
of Sub1A, OsEREBP1 (Sub1C) and OsEREBP2 (Sub1B)
were characterized using an ABI PrismTM Dye Terminator Cycle Sequencing
Ready Reaction Kit with Ampli Taq DNA polymerase for fluorescent sequencing
Molecular Characterization of 36 kb SubQTL9 region in ISLs
The use of Isogenic Lines (ISLs) is a powerful tool in genetics approach
to examine the physiological processes linked to a predicted gene while other
regions are similar. The region was represented on a part of the genomic sequence
of a Nipponbare PAC clone (GenBank accession number AC090056) and DQ011607 from
Indica rice. The genotypic screening to maintain a tolerance allele (DH206)
at major SubQTL9, as well as selection against an intolerance allele
(KDML105), was done in three groups as ISLs-132CC (12 plants), ISLs-132Cc (12
plants) and ISLs-132cc (12 plants), respectively To classify a submergence trait
haplotype of ISL-132-Sub1 of BC4F8 population compared to FR13 A (a tolerant
line) and KDML105 (an intolerant line), an interval segment of major SubQTL9
region, designated as the C region, was determined by effective PCR screening
markers related to submergence tolerance trait as reported by Siangliw et
al. (2003), Ruanjaichon et al. (2004) and Fukao et al. (2006)
Using Sub1A marker, no length polymorphism was observed among rice plants. Therefore DNA sequencing analysis was carried out to detect the point mutation among these identical PCR products. The result showed that the nucleotide sequence of exon 1 contained two SNP at the nucleotide position of 556 (T to C) and 677 (A to G). Consequently, a tolerant plant, FR13A, has the bases T and A similar to ISL-132CC whereas ISL-132cc and KDML105 have the bases C and G (Fig. 1).
In addition we did not find polymorphism with OsEREBP2 marker between
ISL-132cc and KDML105. On the other hand, FR13A showed identical allele from
ISL-132CC. Finally, OsEREBP1 marker which was developed from a gene encoding
ethylene responsive element binding protein showed a 25 nucleotides Insertion-Deletion
at 3UTR in FR13A as well as in ISL-132CC. But ISL-132cc and KDML105 were
not found (Fig. 2). According to 3 molecular markers results,
we found that the allele pattern of Sub1A, OsEREBP1 and OsEREBP2
from ISL-132CC is similar to FR13A (Fig. 3).
||Sequence analysis of Sub1A coding region between ISL-132CC
and ISL-132 compared to FR13 A (a tolerant line) and KDML105 (an intolerant
|| Effective DNA markers in the SubQTL9 were used for
|AIndel = Insertion-Deletion, B SNP =
Single nucleotide polymorphism
These genomic patterns might be associated with the tolerant symptom when
the plant is under water. Besides, the understanding of the mechanisms used
by rice to tolerate submergence stress involved in 4 candidate gene markers
compared with individual traits related to submergence stress might open new
avenues to the genetic improvement of rice.
To survey other regions around the major QTL for submergence tolerance in ISL-132 population, many types of molecular markers reviewed by Sianglew et al. (2003) were used for screening. The results showed that the interval segment of major SubQTL9 region was represented as a genotypic sequence of ISL-132 population as similar as KDML105 (data not shown). Furthermore, SSR genotyping with 12 chromosomes was used for quality control of isogenic line compared to KDML105 (recipient). We found that genome of ISL-132CC has 90% convergence with KDML105 using 42 SSR loci. The selected recombinant event with in the C region could potentially be used for examination of plant adaptation traits. The result could be treated as single Mendelian factors that are likely due to a single locus.
In addition, rice gene-chip expression array was applied for analysis of Single
Feature Polymorphism (SFP) in whole genome (606,000 loci). Using Significant
Analysis of Microarray (SAM) at 15% FDR, ISL showed 0.02% divergence with KDML105
(data not shown).
||Sequence analysis of Sub1C coding region and 3UTR
between ISL-132CC and ISL-132cc compared to FR13 A (a tolerant line) and
KDML105 (an intolerant line)
||Comparison of allele composition on SubQTL chr9 region
related to plant adaptation and survival ability under flash flooding condition.
Allele detection is based on PCR screening with effective markers including
sequence analysis. Submergence tolerance trait was observed by evaluation
of plant responses to submergence stress as plant elongation and survival
The identified SFP of 118 loci (101 genes) dispersed in each chromosome which
the major and minor QTL segments were mapped. The selected recombinant event
with SubQTL9 region could potentially be used for expression analysis.
Evidences Support 36 kb Region Containing Genes Responsible for Submergence
The efficiency of survival and recovery after submergence is strongly related
to the ability to limit leaf elongation and to stay green under water. Under
submergence conditions mechanisms of plant adaptation have played important
roles in leaf elongation and senescence. Toojinda et al. (2003) reported
that the complex phenotype-associated traits such as leaf senescence, percent
plant survival, suppression elongation and so on were coincidently mapped at
SubQTL9 for submergence tolerance identified as a major QTL. The main
finding in this study is the characterization of the ISL-132 of BC4F8 in response
to submergence stress. These were classified into three haplotypes which exhibited
differences in plant adaptation under water.
PE was calculated as the plant height (cm) for 20 days under submergence stress. The measurement was taken as the distance from the soil surface to the tip of the longest leaf. The results showed that the development of ISL-132CC and ISL-132Cc have similarity in average plant height of 38.3 and 38.9 cm for 20 days under flooding. However the development of ISL132-cc has the highest level of average plant height at 62.7 cm under flooding (Fig. 4A).
The TSE during flooding was used as an indicator of the increment in shoot height and calculated as the average difference in shoot height before and after flooding. A set of three genotypic classes were characterized under flooding for 20 days into two different types of TSE. The individual plants of the homozygous donor (DH206), ISLs132CC and the heterozygous class, ISLs-132Cc have average incremental height of 5.4 and 7.1 cm, respectively. But ISLs-132cc, the individuals carrying homozygous alleles of KDML105 has the highest level of average incremental height at 31.2 cm under flooding (Fig. 4B).
The incremental height of plant shoot elongation in ISL-132 was separated into two parts of leaf tissue under submergence conditions. The acceleration of leaf extension combined the elongation of both leaf sheath and leaf blade. The development of ISL132-cc showed the highest level of leaf sheath and leaf blade growth in shoot elongation when compared to ISL132-CC and ISL132-Cc (Fig. 5).
The impact of submergence stress on plant growth was compared to growth under normal conditions. RSE was used to investigate the relationship between the reduction and induction of plant growth during 20 days submergence stress. The results showed that both ISLs-132CC and ISLs-132CC showed 73.6 and 74.8% of shoot elongation compared to 100% under normal growth conditions. However, the ISLs-132cc shoots elongated 120.5% more than plant growth under normal conditions (Fig. 4C).
LS is characterized by the presence of yellowing in leaves which resulted from chloroplast damage. Submergence stress affected the ability of rice to retain its green leaf coloring area. Submergence tolerant rice is able to stay green longer than intolerant lines when flooded. After 20 days under water, each individual line was defined at the base, middle and tip of one leaf. Using LS-SPAD, the results showed that ISLs-132CC, ISLs-132Cc and ISLs-132cc were defined for average chlorophyll content of 23.8, 23.9 and 15.4 score unit at 20 day flooding, respectively (Fig. 4D). The interaction between ISL132-CC: ISL132-Cc and ISL132-cc across time is highly significant (p<0.01) with chlorophyll content. The results suggest that the long-term flooding has major effects for senescence among ISL132 classes.
In all experiments, the interaction between ISLs-132CC and ISLs-132Cc is not significant (p<0.01) across time. But the development of ISL132-cc interacts to NILs-132CC: ISLs-132Cc across time showed high significance (p<0.01) with all obtained data in responding traits of PE, TSE and RSE under submergence stress. The result suggested that the region of 36 kb consists of a tolerant donor allele which affected submergence tolerance traits as a dominant allele. Moreover, the responding traits under flooding were controlled by a smaller region in ISLs of BC4F8. A highly correlated regression analysis was observed with Sub1A, OsEREBP1 and OsEREBP2 markers that contributed a high percentage of phenotypic changes in TSE and LS.
Relationship Between Genotypic Allele and Time-Course
The data from phenotypic changes among traits responded to submergence stress
were observed. The analysis of variance (ANOVA) showed that the genotypic effect
(G) and time-course (T) was the significant source of variation. Genotype main
effects (G) from the development of ISL132-CC, ISL132-Cc and ISL132-cc were
significantly (p<0.01) different among genotypes varying across time (Table
||Shows the relationships between genotype and plant adaptation
traits, A) plant elongation (PE); B) total shoot elongation (TSE); C) relative
shoot elongation (RSE); D) Chlorophyll content, at 2, 4 and 20 days under
||Submergence-induced leaf sheath elongation and leaf blade
elongation were determined with three genotypic classes of ISL-132. At the
start of the submergence, the treatment plants used were 21 day seedlings
and were submerged for 20 days
||Analysis of variance for Total Shoot Elongation (TSE), Plant
Elongation (PE), Relative Shoot Elongation (RSE) and Leaf senescence (LS)
of the three genotyping classes of isogenic lines submerged for 20 days
|The data shows mean square value and the significance record
of main and interaction effects of time, **Significant at 1% level; *Significant
at 5% level, ns: Non-significant Plants were treated under water
for 0, 2, 4 and 20 days in a plastic aquarium tank, PE, Plant Elongation;
TSE, Total Shoot Elongation; RSE, Relative Shoot Elongation; LS, Leaf Senescence
The ISL132-CC and ISL132-Cc interaction showed no differences in traits such
as PE, TSE, RSE and LS at 1% level. The interaction between ISL132-cc and ISL132-CC:
ISL132-Cc was highly significant (p<0.01) in PE, TSE and RSE and not significant
in LS. The results suggested that the time-course was the main effect for leaf
damage by development of leaf senescence.
||Correlation matrix of submergence response traits in rice
obtained in 36 individuals ISLs-132 of BC4F8 population from the submergence-tolerant
(DH206) and submergence-intolerant (KDML105)
|Plant were treated under water for 20 days in a plastic aquarium
tank, %PS, percent plant survival; TSE, total shoot elongation; RSE, relative
shoot elongation; LS, leaf senescence. p-values below 0.05 indicate statistically
significant non-zero correlations at the 95% confidence level
Traits Response to Flooding Correlation
Strong phenotypic correlations among traits responsive to submergence stress
were observed in the ISLs-132 population (Table 3). A high
correlation of the PS% was found among PE, TSE, RSE and LS traits. The PE, TSE
and RSE showed high correlation (-0.78*, -0.75* and -0.34*) and have a negative
correlation with the PS%. The LS measured by the SPAD-502 chlorophyll meter
presented high positive (0.71*) correlation with percentage plant survival,
whereas leaf senescence had negative correlation with PE, TSE and RSE.
In all experiments two classes, ISLs-132CC and ISLs-132Cc, having less TSE under flooding, mostly exhibited a higher level of PS% and LS. However, the ISLs-132cc showing high TSE and low LS affected the PS% and scored as 0% after being de-submerged for 7 days.
The efficiency of survival and recovery after submergence is strongly related to the ability to limit leaf elongation and to stay green under water. Under submergence conditions mechanisms of plant adaptation have played important roles in leaf elongation and senescence. Toojinda et al. (2003) reported that the complex phenotype-associated traits such as leaf senescence, percent plant survival, suppression elongation and so on were coincidently mapped at SubQTL9 for submergence tolerance identified as a major QTL. The main finding in this study is the characterization of the ISLs-132 of BC4F8 in response to submergence stress. These were classified into three genotypic classes which exhibited differences in plant adaptation under water.
The isogenic lines are the powerful tool in the genetic approach and the examination of the physiological processes linked to a predicted gene because of the unrelated variations which reached out from the target trait. This successful approach has been used in various plants such as maize (Dorweiler et al., 1993), tomato (Alpert and Tanksley, 1996 and rice (Yamamoto et al., 1998). In the study, we have used ISL-132 of BC4F8 in comparisons between three genotypic classes. Each plant contained a segment in the same region which different in each class (DH206 segment for ISLs-CC, KDML105 segment for ISLs-cc and heterozygous for ISLs-Cc). This study showed that the ISLs-CC and ISL-CC obtained a high percentage plant survival with gene targeted markers (GTMs). The result of this study suggested that the region of 36kb consists of a tolerance donor allele which affected submergence tolerance traits as a dominant allele. Moreover the responding traits under flooding were controlled by a smaller region in ISLs of BC4F8. A highly significant statistic of regression analysis was observed with gene targeted markers (GTMs), Sub1A, OsEREBP1 and OsEREBP2, which contributed a high percentage of phenotypic changes in TSE and LS. Using SSR genotyping, BC4F8 is only 94% similar to KDML105 with 6% of its alleles being from DH206, the effects of plant adaptation under flooding are major caused by different allelic at the interval region of 36 kb containing three predicted gene. The new developing ISLs for one gene at one locus and their function can be inferred by gene cloning and several computer analysis tools.
The results from physiological adaptation revealed a strong pattern of shoot elongation among three genotypic classes explained as a validation data of TSE and RSE during flooding. The higher level of TSE of the ISLs-132cc class affected the accumulated elongation in at least two parts of the leaf extension. Most parts of plant elongation were observed at leaf sheath and leaf blade. However, only ISLs132-CC and ISLs132-Cc showed significant physiological adaptation in leaf blade elongation. Recent finding reports revealed that the acceleration of elongation under flooding conditions combines the elongation of both leaf sheath (Mazaredo and Vergara, 1982) and leaf blade (Jackson et al., 1987). Slow leaf sheath elongation or suppressing elongation at leaf sheath of individuals ISLs132-CC and ISLs132-Cc might be an effective role with genotypic classes.
The plant adaptation linking fast elongation underwater and susceptibility to flooding was not positive in the past experiments (Yamada, 1959; Mazaredo and Vergara, 1982). Plant growth on rapid elongation of the three genotypic classes in response to submergence stress was obtained for a few days. The susceptible homozygous ISLs-132cc showed the prolongation of plant growth on shoot elongation whereas the plant growth was suppressed in both ISLs-132CC and ISLs-132Cc. However the plant growth did not differ in control plants of each class. The relationship between susceptible and resistance to fast elongation underwater was recently reported by Toojinda et al. (2003) and Siangliw et al. (2003). Plant growth compared results between flooded and normal conditions revealed that the ability of plant growth of ISLs-132CC and ISLs-132Cc was inhibited when compared to control plants. The effect of plant growth on rapid shoot elongation is highly negatively correlated (r = -0.34*) with the survival data after flooding for 20 days. The relationship between plant growth on shoot elongation and survival using five rice cultivars, which has been confirmed using the IRRI gene Bank database on 903 cultivars, was reported by Setter and Laureles (1996). Vartapetian and Jackson (1997) revealed that the plant actively growing during submergence is much more susceptibility than the slowly growing plant in submergence response. The advantage of plants in suppression elongation might be regulated by their genotypic allele to maintain the energy source for recovering plants after de-submergence.
Evidence of leaf damage by the development of leaf senescence in underwater conditions was revealed in Arabidopsis and maize (Zhang et al., 2000; Subbaiah and Sachs, 2003). In rice, one of the best indicators to show recovery from submergence is leaf senescence promoted under flooding stress (Jackson and Ram, 2003). The ability to stay green or leaf senescence underwater is similar to responsiveness to survival and the ISLs population were screened for their reaction to survival. The senescence leaf was obtained for intolerance homozygous of ISLs-cc but the tolerance homozygous, ISLs-CC and heterozygous, ISLs-Cc stayed green during flooding for 20 days. ANOVA analysis of LS data showed high significance among genotypes which vary across time. However, no significant LS data was shown among the three genotypic classes. The results suggested that the time-course was the main effect for leaf damage by development of leaf senescence. The ability to stay green in the leaf area showed positive correlation with the percent plant survival but negative correlation with plant shoot elongation. The phenotypic association between the low level of leaf senescence and high plant survival were shown to be highly positive (Toojinda et al., 2003). Moreover a strong negative correlation between elongation and survival was found amongst four difference cultivars (Singh et al., 2001). The leaf senescence and tolerance score were indeed linked genetically to plant elongation during submergence of the rice plant. The rapid plant elongation and leaf senescence being negatively correlated to plant survival in rice was parallel to the responses given by exogenous ethylene (Jackson et al., 1987). All of these data suggested plant elongation and leaf senescence was directly linked to the physiological adaptation and plant survival after submergence.
The results can classify the ISLs-132 classes, ISLs-CC and ISLs-Cc representing a tolerance segment in 16.7 kb strongly related to the ability to limit leaf elongation, to stay green and to survive including recuperatation after submergence. The ISLs-132 classes having homozygous segment of tolerance donor of ISLs-CC or heterozygous of ISLs-Cc was differentially expressed on phenotypic changes under water with ISLs-cc which contained the intolerant segment. The physiological adaptations related directly to the survival ability. Although it is reasonable to conclude that ISL-classes consist of a genotypic allele of a donor, DH206, the range 36 kb was related to the tolerance trait under submergence on plant shoot elongation and leaf senescence. In addition, cloning of genes related to submergence tolerance might open new avenues for genetic improvement of rice crops. Finally, the ISLs-132 containing a tolerance segment of 36 kb associated with submergence tolerance in rice should be very useful to promote the breeding process in submergence stress-tolerant rice.
The authors would like to express their sincere thanks to the Rockefeller Foundation and Rice Gene Discovery Unit, National Center for Genetic Engineering and Biotechnology and DNA Technology Laboratory, National Center for Genetic Engineering and Biotechnology, for their support in this research.