Rice near-isogenic-lines (NILs) contrasting for grain yield under lowland drought stress

Field Crops Research 123 (2011) 38–46

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Rice near-isogenic-lines (NILs) contrasting for grain yield under lowland drought stress R. Venuprasad ∗,1 , S.M. Impa, R.P. Veeresh Gowda 2 , G.N. Atlin 3 , R. Serraj 4 International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 8 April 2011 Accepted 9 April 2011 Keywords: Rice Drought NILs Yield Roots

a b s t r a c t The development of near-isogenic-lines (NILs) is a very important tool for both genetic and physiological dissection of drought resistance in rice. Two pairs of NILs differing for grain yield under drought stress were isolated and characterized for yield, yield related traits, and several physiological traits in a range of contrasting environments. In replicated field trials both NIL pairs differed significantly for grain yield under drought stress but showed similar yield potential, phenology, and yield component traits under non-stress conditions. A polymorphism analysis study with 491 SSRs revealed that both NIL pairs are at least 96% genetically similar. These NILs show that small genetic differences can cause large difference in grain yield under drought stress in rice. In both pairs the drought-tolerant NILs showed a significantly higher assimilation rate at later stages both under stress and non-stress conditions. They also had a higher transpiration rate under non-stress condition. The most tolerant NIL (IR77298-14-1-2-B-10) had significantly higher transpiration rate and stomatal conductance in severe stress conditions. In one pair the tolerant NIL had constitutively deeper roots than the susceptible NIL. In the second pair, which had higher mean root length than the first pair, the tolerant NIL had more roots, greater root thickness, and greater root dry weight than the susceptible NIL. Deeper root length may allow tolerant NILs to extract more water at deeper soil layers. It is concluded that enhanced rooting depth is an important strategy for dehydration avoidance and rice adaptation to drought stress, but root architecture might not be the only mechanism causing the significant yield increase we observed in lowland drought stress environments. To further dissect the drought avoidance mechanisms in rice, analysis of root hydraulic properties may be necessary. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Drought is a major constraint to rice production in rainfed areas. Progress in breeding for high yielding and drought-tolerant (DT) varieties has been traditionally slow. Recently phenotypic screening methods have been developed that can reliably select DT lines based on yield under drought (Venuprasad et al., 2007a; Kumar et al., 2008; Venuprasad et al., 2008; Guan et al., 2010; Verulkar et al., 2010). These methods also led to the discovery that individual QTLs can have a large effect on yield under drought stress in both upland (qtl12.1; Bernier et al., 2007) and lowland conditions (DTY3.1 ; Venuprasad et al., 2009). The QTLs, DTY3.1 and qtl12.1,

∗ Corresponding author. Tel.: +234(02)7517472; fax: +44 208 7113786. E-mail address: [email protected] (R. Venuprasad). 1 Present address:Africa Rice Centre (AfricaRice), PMB5320, Ibadan, Nigeria. 2 Present address: Monsanto, Bangalore, India. 3 Present address: International Maize and Wheat Improvement Centre (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico. 4 Present address: International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria. 0378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2011.04.009

explained 31% and 51% of the variance for grain yield under stress respectively. Because many of the most important rice varieties grown in rainfed regions of Asia are highly susceptible to drought, the ability to improve them by introgressing one or a few QTL for DT from tolerant donors could have a large impact on the resilience and sustainability of rice production in drought-prone upper-toposequence fields. One of the most important cases of a small chromosomal region or regions having a major effect on drought tolerance was observed by Venuprasad et al. (2007b) in a family of backcross derivatives of the lowland rice mega-variety IR64, a line grown on millions of hectare world-wide that is valued for its quality, short duration, and field resistance to diseases and bests. It is, however, highly susceptible to drought (Verulkar et al., 2010). BC3 introgression lines developed using the tungroresistant lndian line Aday Sel were observed to differ significantly in yield under severe drought stress in both lowland and upland conditions; NIL IR77298-14-1-2 was found to be significantly more tolerant than the recurrent parent IR64, while IR77298-5-6 was highly susceptible. The two lines share at least 94% of their genetic material with each other, and are 92%

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identical in SSR marker genotype to IR64 (Venuprasad et al., 2007b). The mechanism by which large-effect QTL improve drought tolerance (DT) has not been unequivocally determined. Bernier et al. (2009) compared a set of near-flowering lines with and without the QTL qtl12.1; though large differences in yield under stress were observed they could not clearly establish the underlying physiology. Information on these causal mechanisms would help in planning breeding programs and in developing screens to accelerate the development and delivery of improved varieties (Serraj et al., 2011). Attempts to identify key drought-resistance traits and devise suitable screens that could be used in breeding have only been partly successful (Serraj et al., 2009). One limitation in such studies is that mostly genetically unrelated genotypes are used, which complicates the physiological dissection of drought resistance mechanisms. Use of closely related genetic materials such as near-isogeneic lines (NIL) should be useful in such attempts (Lafitte, 1999; Price and Courtois, 1999; Bernier et al., 2009). In this study we first report on the development of NILs within IR77298-14-1-2 and IR77298-5-6 that differ substantially in DT, but that share from 96.5% to 98.8% of 491 SSR markers. We compared the field performance of the NILs with respect to grain yield and yield components in a wide range of environments, and evaluated several physiological parameters in an effort to shed light on the mechanisms causing differences in field performance under drought stress in these NILs. 2. Materials and methods The study was conducted on the experiment station of the ˜ International Rice Research Institute (IRRI), Los Banos, Laguna, Philippines, in the dry seasons (DS; December to March) of 2006, 2007 and 2008, and the wet seasons (WS; June to September) of 2007 and 2008. IRRI is located at 14◦ 13 N latitude, 121◦ 15 E longitude, and at an elevation of about 21 m above mean sea level. The soil type is a Maahas clay loam, isohyperthermic mixed typic tropudalf. 2.1. Definition of upland, lowland, stress and non-stress environments In this article, the term lowland refers to field trials/experiments conducted under flooded, puddled, transplanted, and anaerobic conditions while upland refers to field trials/experiments conducted under direct-sown, non-puddled, non-flooded, and aerobic conditions in leveled fields. Lowland or upland trials conducted under irrigated conditions where no stress was imposed are referred to as non-stress trials. Dry season (DS) trials in which drought stress was artificially imposed during the reproductive stage are referred to as managed-stress environments. Rainfed trials in the wet season (WS), in which stress occurred naturally due to periods of low rainfall, are referred to as natural-stress environments. 2.2. Development of NILs Aday Sel is a tungro (virus disease) tolerant rice variety from India while IR64 is a popular high-yielding but tungro susceptible lowland rice variety developed at IRRI, Philippines. Tungro tolerance from Aday Sel was transferred to IR64 background through a backcross program at IRRI (Khush et al., 2004); IR77298-5-6 (a BC3 F2:3 line) and IR77298-14-1-2 (a BC3 F3:4 line) were two tungro tolerant sister lines identified in a pedigree breeding program from a BC3 generation cross (IR77298). IR77298-5-6 has been released in Philippines as Tubigan 6 (NSIC 140) as a replacement to IR64 in tungro affected regions. Since their development the above two lines

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were maintained as bulks for several generations and were fixed for most agro-morphological characters including days to flowering, height, biomass and yield potential. These lines were subsequently found to differ in yield under drought conditions; IR77298-14-1-2 was high yielding (tolerant) while IR77298-5-6 was poor yielding (susceptible) under drought, but under irrigated non-stress conditions they had similar yields (Venuprasad et al., 2007b). These two lines were highly similar genetically SSR based polymorphism revealed 94% similarity and coefficient of parentage (COP) was 0.9; further these two lines were 92% similar to the recurrent parent IR64 (Venuprasad et al., 2007b). Though these two lines were fixed for most characters, some within-line segregation for drought resistance was observed, presumably resulting from residual genetic heterogeneity within the lines. Thus, the opportunity existed to purify these lines and isolate NILs for drought resistance within each of these two lines. From each of these two lines thirty random plants were selected and out of them single-panicle-derived lines were produced. These sixty lines were evaluated in replicated trials in three conditions – upland drought stress, lowland drought stress and lowland non-stress conditions (Venuprasad et al., unpublished data). There was considerable genetic variation observed in the two stress trials but not in the non-stress trial. Based on consistency of yield in upland and lowland stress trials, a pair of contrasting lines was selected in each of the two families; these were lines IR77298-5-6-B-11 and IR77298-5-6-B-18 in the IR77298-5-6B family and lines IR77298-14-1-2-B-10 and IR77298-14-1-2-B-13 in the IR77298-14-1-2-B family. The IR77298-5-6-B-18 line had significantly higher yield than IR77298-5-6-B-11 line in stress trials but showed similar yield levels under non-stress conditions. Similarly, IR77298-14-1-2-B-10 had significantly higher yield than IR77298-14-1-2-B-13 under stress. These four NILs along with parents and checks were evaluated for yield and other morphophysiological traits in stress and non-stress conditions during DS 2007–DS 2008 in a total of twelve field experiments and a glass house experiment. 2.3. Phenotyping of NILs 2.3.1. Field experiments In total, 12 field experiments were conducted during 2007 and 2008 in a range of moisture conditions. They comprised three lowland non-stress trials (one each during DS 2007, DS 2008 and WS 2008), two lowland stress trials (one each during DS 2007 and DS 2008), two lowland natural stress (rainfed) trials (one each during the WS of 2007 and 2008), two upland non-stress trials (in WS 2007), two upland mild stress trials (one each in DS 2007 and DS 2008), and one upland severe stress trial (in DS 2008). All the experiments were laid out as alpha lattice designs and consisted of four to five replications with four-row plots. Plots were 2 m long in upland and 5 m long in lowland trials and were spaced at 0.25 m apart. 2.3.1.1. Management of lowland evaluation trials. In the lowland non-stress trials, seeds were sown in the nursery and 21-day old seedlings were transplanted to the main field. One seedling was transplanted per hill at a spacing of 10 cm between hills in a row. After transplanting, approximately 5 cm of standing water was maintained in the field until drainage before harvest. Inorganic NPK fertilizer was applied at the rate of 90–30–30 kg ha−1 . Weeds and insect pests were controlled chemically. Initial field establishment and management practices for lowland stress and natural stress trials were similar to the non-stress trials described above. In the natural-stress trials from 30 DAT until maturity no irrigation water was provided and the crop was solely dependent on rainfall (860–940 mm rainfall; 498–512 mm evapo-transpiration (ET)). In the stress trials, stress was imposed by draining water from the paddy at 30 days after transplanting

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(DAT) and withholding irrigation until the soil moisture tension reached −30 kPa at 20 cm depth. Fields were then re-irrigated by flash flooding, and drained again after approximately 24 h. This cycle was repeated until harvest. Severe leaf rolling was observed during each stress period. This protocol is routinely used at IRRI to screen for drought tolerance in lowland environments (for example – Venuprasad et al., 2007a, 2008). 2.3.1.2. Management of upland evaluation trials. In the upland trials, dry seed was direct-sown at a rate of 8 g m−2 into level, unpuddled, unflooded leveled upland fields. Inorganic PK and ZnSO4 fertilizers were each applied at the rate of 40 kg ha−1 as basal dose. N was applied in three splits at the rate of 30–40 kg ha−1 per split. Weeds were controlled by application of herbicide and by hand weeding. Insecticides were used to control insect pests when necessary. For the first four weeks after sowing (WAS) irrigation was applied by overhead sprinklers once in 3 days for 2–3 h. After the initial establishment the upland non-stress trials (conducted during WS) were maintained as rainfed. Due to frequent rainfall (940 mm total) and lower ET (512 mm total) throughout the season no stress symptoms were observed on the plants. In the upland mild stress experiments (conducted during DS) from after four weeks the trials were irrigated to field capacity every week until harvest. Approximately 40 mm of water was applied during each irrigation and a total of about 800 mm of water was added by sprinkler irrigation. However, due to higher ET (600–880 mm total) and low rainfall (130–355 mm total) throughout the DS, mild stress symptoms were observed on the plants prior to irrigation. The upland severe stress trial was managed similarly to the mild stress trial explained above but after seven weeks, irrigation was withheld until soil water tension reached about −30 kPa at 20 cm soil depth. Trials were then re-irrigated to field capacity. This stress cycle was then repeated until maturity. This irrigation regime resulted in stress levels that caused leaf rolling and tip burning on most entries at the end of each drying cycle. The repeated cycles of stress ensured that all entries experienced stress during the sensitive stage of 15 days before and after flowering. On average, this protocol has been found to reduce yields by 50–70% relative to fully irrigated controls (Venuprasad et al., 2007a). In this experiment a total of about 600 mm water was added by sprinkler irrigation. Rainfall and evapo-transpiration were 355 and 600 mm respectively. 2.3.2. Glasshouse experiment In WS 2007 an experiment was conducted in the glasshouse at IRRI to evaluate root traits under both control and stress conditions. The four selected NILs along with the parents and nine other lines from the same population were used. The lines were established in a nursery bed and 24-day old seedlings were transplanted in pipes measuring 1.05 m in length with diameter 0.18 m at the rate of two seedlings per pipe. Pipes contained compacted soil from the IRRI farm: first, the bottom 50 cm of each pipe was filled with soil and compacted to 40 cm, second, soil was added to fill the next 40 cm of pipe and compacted to about 30 cm (up to 70 cm filled), and finally, the next 30 cm was filled with soil and compacted to about 20 cm (totally 90 cm filled). The top 10 cm was covered with puddled soil from a typical lowland field. The bottom of the pipes was sealed; pipes in stress treatments were provided with a drainage hole. Both the stress and control treatments had four replications each arranged in a randomized complete block design. Two weeks after planting NPK was provided at the rate of 80–30–30 kg ha−1 . In the control pipes water was maintained at a depth of 2 cm throughout the experiment. In stress pipes a 2 cm water depth was maintained until a month after planting and subsequently stress was imposed; during stress treatment no water was added. In both control and stress treatments, plants were sam-

pled 34 days after stress imposition. At the time of sampling, all the entries in the control treatment had flowered and in the stress treatment all the entries exhibited severe leaf rolling. 2.4. Data collection and analysis 2.4.1. Yield and related traits In all the trials data on days to 50% flowering, plant height at maturity, grain yield, biomass, harvest index and number of panicles were recorded. Days to 50% flowering was recorded when the panicle was exserted in approximately 50% of the plants in a plot. Final plant height was measured as the distance from the ground to the panicle tip of three random plants from each plot. Grain from each plot was harvested, dried to a moisture content of about 14%, and weighed to obtain grain yield. For measurement of harvest index, all the plants in 0.5 linear meter of a row (in upland) or one linear meter of a row (in lowland) were harvested at ground level at maturity and dried to 0% moisture. From this sample, number of panicles, biomass weight, and grain weight data were obtained and harvest index was computed as the ratio of grain weight to total above-ground harvested biomass weight. 2.4.2. Physiological traits Physiological characterization was carried out in the lowland stress and non-stress trials conducted during DS 2008 (Section 2.3.1). The NILs were characterized for relative water content (RWC), and leaf gas exchange measurements (stomatal conductance (gs), assimilation rate (A), transpiration rate (Tr), and intrinsic leaf transpiration efficiency (TE)). Data were collected in three replications and on three random plants in each replication. Data on the above traits (except RWC) were recorded on two dates in control (64 and 83 DAS) and three dates in stress (78, 87, and 97 DAS). RWC measures the absolute amount of water required by the plant to reach artificial full saturation (Gonzalez and GonzalezVilar, 2001). RWC was measured under stress conditions on two dates (78 and 97 DAS). RWC was measured in first fully expanded leaves from the top during vegetative stage and in flag leaf during reproductive stage. The leaves were directly placed in a preweighed plastic centrifuge tube and placed on ice in a cooler. The tubes were taken to the laboratory and weighed immediately to determine initial fresh weight. The tubes were then filled with distilled water, recapped, and placed in dark 4 ◦ C room overnight. The next morning, leaves were blotted with paper towels and were weighed immediately to determine the fully turgid weight. Leaves were then dried at 70 ◦ C to constant weight for dry mass determination. The RWC was computed as per Slayter (1967). Leaf gas exchange measurements were measured using an open system portable infrared gas analyzer (LI-6400, LI-COR Biosciences), at a block temperature of 30 ◦ C with a PPFD of 1400 ␮mol m−2 s−1 under both stress and non-stress conditions. 2.4.3. Root traits Soon after sampling, the pipes containing soil and roots were soaked in water and the entire soil column was extracted carefully and divided into two parts: the top 30 cm as one part and the rest as another. Roots were separated from soil by carefully washing with a fine jet of water on a 1-mm screen until all soil washed through the screen. After cleaning, roots were stored in an alcohol solution (50% isopropanol) in a refrigerator at 4 ◦ C for later analyses. Data on maximum root length, root thickness, root volume, total root number, and root dry weight were recorded. Maximum root length was measured as the distance from base of the plant to the lowermost root tip. Number of primary roots at the base of the plants were counted and recorded as the total root number. To measure root thickness, eight random basal roots were sampled

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close to the base of the plant and thickness was measured using magnifying lens. Volume of the roots was recorded by water displacement method (Hemamalini et al., 2000). To obtain root weight data, the root samples were placed in a hot air oven (80 ◦ C) for 2 days and weighed after they attained constant weights. Root volume and root dry weight data for the 0–30 cm and >30 cm sections were obtained separately and summed up to obtained total root volume and total root dry weight data respectively. 2.4.4. Data analysis Statistical analysis was performed using SAS v8.2 (SAS Institute Inc., 2004). Mixed models were employed in which lines were treated as fixed whereas replicates and blocks within replicates (wherever applicable) as random. While the physiological traits and root traits data were analyzed trial-wise, combined analysis over trials for each evaluation environment was performed for grain yield and yield-related traits data. For the combined analysis trials within environments were also considered random. Least squares estimates of each trait on each line were obtained using the REML option of the SAS MIXED procedure (SAS Institute Inc., 2004). 2.5. Genotyping the NILs All molecular marker work was conducted in the Gene Array and Molecular Marker Analysis (GAMMA) Lab, Plant Breeding, Genetics and Biotechnology (PBGB) division, IRRI. Leaf samples of all lines were collected from field and were freeze-dried in a lyophilizer. Miniprep scale DNA extraction was done in deep-well-plates (Axygen scientific, California, USA) via a modified CTAB protocol using a GENO grinder. The quantity and quality of DNA was checked on 0.8% agarose gels and concentration was adjusted to ∼20 ␩g/␮L by comparing with lambda (␭) DNA standards. The four NILs along with IR64 were genotyped with a set of 491 SSR markers from the 500 rice microsatellite (RM) markers (ResGen, Invitrogen Corporation, Huntsville) described in Temnykh et al. (2001). PCR amplification was performed as described in Temnykh et al. (2001). PCR products were resolved on 8% nondenaturing polyacrylamide (PAGE) gels as described by Sambrook et al. (1989). The genetic distance between markers (in cM) was also assumed from Temnykh et al. (2001) study. GGT software (van Berloo, 2008) was used to obtain graphical genotypes of these lines. 3. Results 3.1. Genetic polymorphism between NILs The NILs of the IR7798-5-6-B family were polymorphic at only 6 loci (1.22%) while the NILs of the IR77298-14-1-2-B family were polymorphic at 17 loci (3.46%) (Figs. 1 and 2). Of the polymorphic loci in the IR77298-5-6-B NILs, at three loci, RM438 (chromosome 2, 58.4 cM), RM258 (chromosome 10, 70.8 cM) and RM304 (chromosome 10, 73.0 cM), the tolerant NIL had the donor alleles while the susceptible NIL had IR64 alleles; while at other three polymorphic loci i.e., RM31 (chrom.5, 118.8 cM), RM591 (chromosome 10, 118.3 cM), RM287 (chromosome 11, 68.6 cM), the susceptible NIL had the donor alleles while the tolerant NIL had IR64 alleles. Of the rest, 455 loci were monomorphic for IR64 alleles, 25 loci (∼5% of the total) were monomorphic for the donor alleles, and five loci were heterozygous. Similarly in the IR77298-14-1-2-B family, of the polymorphic loci, the tolerant NIL had donor alleles at 14 loci of which four were located on chromosome 2 (RM109, 0 cM; RM492, 53 cM; RM262, 70.2 cM; RM341, 82.7 cM), one on chromosome 4 (RM241, 106.2 cM), five on chromosome 8 (RM310, 57 cM; RM126, 57 cM; RM547, 58.1 cM; RM44, 60.9 cM; RM72, 60.9 cM), one on chromosome 10 (RM228, 96.3 cM), and three on chromosome 11 (RM209, 73.9 cM; RM229, 77.8 cM; RM21, 85.7 cM). At three loci,

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RM71, RM231, and RM25, located on chromosomes 2 (49.8 cM), 3 (15.7 cM), and 8 (52.2 cM) respectively, the susceptible NIL had the donor alleles. Of the remaining loci, 421 loci were monomorphic for the IR64 alleles, 43 loci were monomorphic for the donor alleles and 10 loci were heterozygous. 3.2. Grain yield and yield-related traits The yield of lines in evaluation environments is given in Table 1. Grain yield of all the four NILs was on par both under lowland and upland non-stress conditions. In all the stress trials the ‘tolerant’ NILs (IR77298-14-1-2-B-10 and IR77298-5-6-B-18) out-yielded the corresponding ‘susceptible’ NILs (IR77298-14-12-B-13 and IR77298-5-6-B-11 respectively) of the same family. These differences were significant in the lowland stress environment in both the families but in the rainfed lowland and upland mild stress environments the differences were significant only in the IR77298-14-1-2-B family. Under severe upland stress no significant difference between lines was observed. On average, the tolerant NILs out-yielded the susceptible NILs by about 15% in rainfed lowland, ∼44% in lowland stress and ∼93% in upland mild stress environments. Among these lines IR77298-14-1-2-B-10 was the most drought-tolerant as it consistently out-yielded all other lines in all the stress environments. Similarly, IR77298-5-6-B-11 was the most drought susceptible, while IR77298-14-1-2-B-13 and IR77298-5-6-B-18 are intermediate in drought resistance. No significant difference between yield of IR64, the recurrent parent, and that of the NILs was observed in the lowland non-stress environment. But in the lowland stress, rainfed and upland non-stress environments IR64 yields was either on par with or worse than the most susceptible NIL. In Fig. 3, where the line means are plotted against the trial means, it can be observed that NILs grain yield means decrease with trial mean due to increasing stress severity. However, the tolerant NILs show relatively lower yield reduction compared to the respective susceptible NILs in the severe stress environments. The superiority of the tolerant NILs is observed when the trials mean drop below 2.5 ton ha−1 . The NILs of the IR7798-14-1-2-B family showed significantly lower days to 50% flowering (DTF), by about 7 days on average, than the IR7798-5-6 family NILs (Table 2) and IR64 (data not shown) in most environments. In neither family did the contrasting NILs show any significant differences in DTF in any of the lowland environments or in the upland non-stress environments. However, in the upland stress environment in both the families the tolerant NILs had significantly lower DTF than the susceptible NILs by about 10 days. In the upland mild stress environment in the IR7798-14-1-2B family the tolerant NIL flowered about 10 days earlier than the susceptible NIL. The tolerant and susceptible NILs in both families had similar plant height, number of panicles, biomass and harvest index in lowland stress and non-stress environments in field conditions (Table 2). Only in a few cases in the upland environments did the tolerant NILs have significantly higher biomass and harvest index than the corresponding susceptible NILs. 3.3. Physiological parameters The tolerant NILs maintained relatively higher RWC compared to their respective susceptible NILs under stress at both stages, although these differences were only significant in the case of one pair of NILs (IR77298-14-1-2-B) at 97 DAS (Table 3). With the progression of stress application (78 to 97 DAS) the NILs showed a reduction in leaf RWC; on average, the susceptible NILs showed higher reduction trends compared to the tolerant NILs, although this difference might not be statistically significant. At 97 DAS, RWC

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Fig. 1. Graphical genotype depicting polymorphism on the 12 chromosomes between the two contrasting near isogenic lines (NILs) of the IR77298-5-6-B family. The chromosomal regions shaded in green and blue represent monomorphic regions for IR64 and donor alleles respectively. The regions shaded in red represent polymorphic regions while the yellow regions represent heterozygous regions. The name of the marker is given next to the chromosome, and the chromosome number indicated below respective chromosome. The scale (in cM) is given at the left of the figure (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Fig. 2. Graphical genotype depicting polymorphism on the 12 chromosomes between the two contrasting near-isogenic lines (NILs) of the IR77298-14-1-2-B family. The chromosomal regions shaded in green and blue represent monomorphic regions for IR64 and donor alleles respectively. The regions shaded in red represent polymorphic regions while the yellow regions represent heterozygous regions. The name of the marker is given next to the chromosome, and the chromosome number indicated below respective chromosome. The scale (in cM) is given at the left of the figure (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

was significantly higher in the tolerant than in the susceptible line of IR77298-14-1-2-B family. Under non-stress conditions there was an increase in stomatal conductance (gs), assimilation rate (A) and transpiration rate (Tr) with age of plants while under stress there was a slow increase

of these parameters initially and then a drastic reduction at later stages (Table 3). The NILs in IR77298-14-1-2-B family had almost similar gs under non-stress but under stress the tolerant NIL showed higher gs than the susceptible NIL, although the difference was significant only at 97 DAS. On the other hand the NILs in

Table 1 Combined analysis of grain yield (kg ha−1 ) of contrasting near-isogenic lines in different moisture regimes: IRRI DS 2007–WS 2008. Designation

Lowland environments Non-stress

IR77298-14-1-2-B-10 IR77298-14-1-2-B-13 IR77298-5-6-B-18 IR77298-5-6-B-11 IR64 #

3396 3035 2858 3724 2431

± ± ± ± ±

593 688 607 589 590

#

Upland environments

P

Stress

a a a a a

1744 1259 1164 774 695

± ± ± ± ±

172 212 172 172 172

P

Rainfed

a b b c c

2483 1949 2234 2171 1530

± ± ± ± ±

566 566 566 566 566

P

Non-stress

a b ab ab c

2565 2540 2855 2542 1506

± ± ± ± ±

588 588 588 588 588

P

Mild stress

a a a a b

1733 754 1432 957 718

Genotypes with same letter are not significantly different. Duncan multiple range test was used to categorize genotypes.

± ± ± ± ±

585 585 585 585 587

P

Stress

a b ab ab c

485 177 151 33 87

± ± ± ± ±

P 78 78 77 78 77

a a a a a

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Fig. 3. Scatter plot diagram of grain yield of near-isogenic lines (NILs) and IR64 versus the trial means. The NILs of the IR77298-14-1-2-B and IR77298-5-6-B families are indicated as red and green lines respectively; solid and dotted lines represent drought tolerant and susceptible NILs respectively. The R2 of fit is given next to the respective line name in the legend (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Table 2 Yield-related traits of contrasting near-isogenic lines (NILs) in different moisture regimes: IRRI DS 2007–WS 2008. Trait

Environment

IR77298-14-1-2-B family Tolerant NIL

IR77298-5-6-B family

Susceptible NIL

Pdiff

Tolerant NIL

Susceptible NIL

Pdiff

Days to 50% flowering

Lowland non-stress Lowland stress Lowland rainfed Upland non-stress Upland mild stress Upland stress

80.73 84.2 70.27 74.04 78.77 84.18

± ± ± ± ± ±

2.41 2.75 9.03 2.59 2.24 2.27

80.44 83.27 69.98 75.78 87.61 93.96

± ± ± ± ± ±

2.57 2.86 9.03 2.59 2.24 2.27

ns ns ns ns ** **

86.52 90.43 76.24 80.98 88.45 94.47

± ± ± ± ± ±

2.4 2.75 9.03 2.59 2.24 2.26

88.41 90.93 75 80.9 91.75 104.36

± ± ± ± ± ±

2.4 2.75 9.03 2.59 2.24 2.27

ns ns ns ns ns **

Height (cm)

Lowland non-stress Lowland stress Lowland rainfed Upland non-stress Upland mild stress Upland stress

101.87 69.68 86.08 92.93 67.17 67.61

± ± ± ± ± ±

8.81 2.24 5.94 10.1 4.16 2.28

93.03 63.4 83.34 89.38 59.75 62.36

± ± ± ± ± ±

8.9 3.11 5.94 10.1 4.16 2.28

ns ns ns ns ns ns

95.97 65.63 81.27 91.3 63.75 65.02

± ± ± ± ± ±

8.8 2.24 5.94 10.1 4.16 2.28

94.7 60.54 78.57 87.99 64.51 61.36

± ± ± ± ± ±

8.8 2.24 5.94 10.1 4.16 2.28

ns ns ns ns ns ns

No. of panicles (m−2 )

Lowland non-stress Lowland stress Lowland rainfed Upland non-stress

750.07 701.32 705.16 774.4

± ± ± ±

107.76 61.93 97.31 52.75

740.96 757.48 688.29 788

± ± ± ±

119.28 85.65 97.31 52.75

ns ns ns ns

764.39 744.26 722.27 808

± ± ± ±

106.36 61.92 97.31 52.75

698.08 620.13 705.3 740

± ± ± ±

105.31 61.93 97.31 52.75

ns ns ns ns

Biomass (g m−2 )

Lowland non-stress Lowland stress Lowland rainfed Upland non-stress Upland mild stress Upland stress

3234.81 2027.85 1729.01 2873.6 1305.65 936

± ± ± ± ± ±

890 359.6 382.67 389.53 291.36 75.11

3432.15 1616.1 1544.57 2790.4 1130.31 844

± ± ± ± ± ±

889.37 419.16 382.67 389.53 291.36 75.11

ns ns ns ns ns ns

3169.37 1968.6 1634.57 2989.6 1381.11 1052

± ± ± ± ± ±

888.26 359.48 382.67 389.53 291.36 75.11

2966.56 1596.33 1442.85 2597.6 1449 854

± ± ± ± ± ±

887.38 359.6 383.87 389.53 291.37 75.11

ns ns ns * ns *

Harvest index

Lowland non-stress Lowland stress Lowland rainfed Upland non-stress Upland mild stress Upland stress

0.34 0.2 0.34 0.19 0.24 0.08

± ± ± ± ± ±

0.02 0.03 0.02 0.03 0.07 0.02

0.36 0.16 0.35 0.19 0.12 0.02

± ± ± ± ± ±

0.03 0.04 0.02 0.03 0.07 0.02

ns ns ns ns ** *

0.34 0.18 0.38 0.22 0.16 0.07

± ± ± ± ± ±

0.02 0.03 0.02 0.03 0.07 0.02

0.34 0.12 0.37 0.19 0.16 0.02

± ± ± ± ± ±

0.02 0.03 0.02 0.03 0.07 0.02

ns ns ns ns ns ns

*, ** Significant at P < 0.05 and 0.01 levels respectively; ns: non-significant.

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Table 3 Relative water content (RWC), stomatal conductance (gs; mol m−2 s−1 ), assimilation rate (A; ␮mol m−2 s−1 ), transpiration rate (Tr; mmol m−2 s−1 ), intrinsic leaf transpiration efficiency (A/T; ␮mol/mmol) of contrasting near-isogenic lines (NILs) under lowland drought stress and non-stress conditions: IRRI DS 2008. Trait

IR77298-14-1-2-B family Tolerant NIL

Stress RWC (78 DASa ) RWC (97 DAS) Non-stress gs (64 DAS) gs (83 DAS) Stress gs (78 DAS) gs (87 DAS) gs (97 DAS) Non-stress A (64 DAS) A (83 DAS) Stress A (78 DAS) A (87 DAS) A (97 DAS) Non-stress Tr (64 DAS) Tr (83 DAS) Stress Tr (78 DAS) Tr (87 DAS) Tr (97 DAS) Stress A/T (78 DAS) A/T (87 DAS) A/T (97 DAS)

IR77298-5-6-B family Susceptible NIL

Pdiff

Tolerant NIL

Susceptible NIL

Pdiff

0.76 ± 0.05 0.75 ± 0.01

0.70 ± 0.04 0.65 ± 0.03

ns *

0.78 ± 0.02 0.76 ± 0.01

0.76 ± 0.03 0.71 ± 0.05

ns ns

0.50 ± 0.03 0.98 ± 0.02

0.54 ± 0.02 1.04 ± 0.02

ns ns

0.48 ± 0.03 1.28 ± 0.02

0.43 ± 0.03 1.00 ± 0.01

ns ns

0.52 ± 0.10 0.79 ± 0.09 0.13 ± 0.01

0.42 ± 0.06 0.58 ± 0.08 0.09 ± 0.01

ns ns *

0.44 ± 0.03 0.72 ± 0.14 0.10 ± 0.00

0.43 ± 0.04 0.70 ± 0.11 0.09 ± 0.00

ns ns ns

25.2 ± 0.73 37.3 ± 1.07

25.60 ± 0.35 28.7 0 ± 0.87

ns *

23.60 ± 0.71 34.00 ± 0.86

23.40 ± 0.67 29.9 0 ± 0.86

ns *

30.18 ± 1.23 30.46 ± 1.60 12.06 ± 0.60

23.25 ± 1.13 27.33 ± 1.20 7.01 ± 0.40

* ns *

23.23 ± 0.44 27.13 ± 2.20 9.13 ± 0.02

21.60 ± 0.75 24.92 ± 1.60 6.40 ± 0.50

ns ns *

5.80 ± 0.26 11.5 ± 0.10

6.06 ± 0.04 10.4 ± 0.10

ns *

5.48 ± 0.35 12.30 ± 0.30

5.07 ± 0.33 9.70 ± 0.10

ns *

6.2 ± 0.99 9.39 ± 0.69 2.96 ± 0.09

5.38 ± 0.71 7.60 ± 0.69 2.33 ± 0.20

ns ns *

5.50 ± 0.22 8.32 ± 1.09 2.29 ± 0.01

5.75 ± 0.46 7.91 ± 0.94 2.04 ± 0.15

ns ns ns

4.17 ± 0.30 3.27 ± 0.10 4.07 ± 0.16

4.48 ± 0.40 3.69 ± 0.20 3.33 ± 0.37

ns ns Ns

4.22 ± 0.10 3.37 ± 0.20 4.01 ± 0.00

3.80 ± 0.20 3.27 ± 0.20 2.95 ± 0.37

ns ns *

* Significant at P < 0.05 level; ns: non-significant. a days after seeding.

IR77298-5-6-B family did not show any noticeable differences in gs both in non-stress and stress conditions. The drought tolerant NILs in both the families maintained significantly higher A and Tr than the respective susceptible NILs in non-stress (83 DAS) conditions. At later stages of stress (97 DAS), while the drought tolerant NILs in both the families showed significantly higher A values than the respective susceptible NILs, only the tolerant NIL in IR77298-141-2-B family exhibited significantly higher Tr than the respective susceptible NIL. The NILs of IR77298-14-1-2-B family did not differ significantly in leaf transpiration efficiency (TE; A/T ratio) but the tolerant NIL in IR77298-5-6-B family showed significantly higher TE than the respective susceptible NIL at later stages of stress (97 DAS; Table 3). 3.4. Root traits On average, relative to the non-stress control, in stress a large reduction in number of roots (54%), root volume (65%), and root dry weight (61%) was observed (Table 4). On the other hand, there was a large increase in maximum root length (64%) and slight increase in root thickness (3%) under stress relative to the non-stress. The tolerant NIL in the IR77298-14-1-2 family had a significantly higher number of roots, root thickness, and root dry weight than the susceptible NIL under stress but not under non-stress. In the IR77298-5-6 family the tolerant NIL exhibited significantly higher rooting depth than the susceptible NlL under non-stress conditions only. 4. Discussion In this study we report development of two pairs of nearisogenic lines (NILs) contrasting for yield under drought stress in two sister families (IR77298-5-6-B and IR77298-14-1-2-B) of a rice cross. The two sister families (IR77298-5-6-B and IR77298-14-1-

2-B) were themselves genetically very similar to one another (94% similar) and were also at least 92% similar to the recurrent parent IR64 (Venuprasad et al., 2007b). A genetic polymorphism study with 491 SSRs confirmed that the contrasting (tolerant and susceptible) lines in both the families are highly homogeneous; the two sublines derived from IR77298-5-6-B differed by less than 2% while the two sublines derived from IR77298-14-12-B differed by less than 4% of the genome (Figs. 1 and 2). Thus these lines are genetically very close to one another. The contrasting NILs were selected based on yield differences in severe stress environments in which stress levels reduced mean trial yield by over 70% (data not shown); in rice this level of stress is essential to distinguish lines based on drought resistance and not based on yield potential (Pantuwan et al., 2002; Venuprasad et al., 2007a). Good response to selection in these environments was observed in the subsequent evaluation environments. In both families, the tolerant and susceptible NILs had similar grain yield and days to 50% flowering in non-stress (Tables 1 and 2) but the tolerant NILs in both families always yielded higher than the corresponding susceptible NILs in stress environments. In addition, number of panicles, biomass and harvest index of the tolerant and susceptible NILs of a family were similar in non-stress conditions. The two lines selected in a family are almost homogeneous genetically and possess similar yield potential and phenology under non-stress, but under drought stress they show contrasting yield. Thus the two lines in a family can be considered as near-isogenic-lines for contrasting for grain yield under drought stress. To our knowledge these are the first thoroughly characterized NILs contrasting for grain yield under drought stress in rice. These NILs show that small genetic differences could bring about large difference in grain yield under drought stress in rice. To cope with drought stress, plants may adopt several strategies including changes in phenological and physiological traits. Among such drought resistance mechanisms, the ability of plant to

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45

Table 4 Root traits of contrasting near-isogenic lines (NILs) grown in PVC pipes in glasshouse under drought stress and control conditions: IRRI WS 2007. Trait

Condition

IR77298-14-1-2-B Tolerant NIL

IR77298-5-6-B Susceptible NIL

Pdiff

Tolerant NIL

Susceptible NIL

Pdiff

Maximum root length (cm)

Non-stress Stress

51.50 ± 3.71 90.00 ± 4.65

42.75 ± 3.71 79.75 ± 4.65

ns ns

50.25 ± 3.71 75.00 ± 4.65

39.00 ± 3.71 71.25 ± 4.65

* ns

No. of roots per plant (#)

Non-stress Stress

231.13 ± 29.0 115.50 ± 8.01

164.00 ± 29.0 94.38 ± 8.01

ns *

216.00 ± 29.0 112.75 ± 8.01

164.13 ± 29.0 110.88 ± 8.01

ns ns

Root thickness (mm)

Non-stress Stress

0.74 ± 0.05 1.05 ± 0.07

0.76 ± 0.05 0.85 ± 0.07

ns *

0.82 ± 0.05 0.67 ± 0.07

0.79 ± 0.05 0.74 ± 0.07

ns ns

Root volume (cm3 )

Non-stress Stress

6.03 ± 2.09 2.84 ± 0.51

3.73 ± 2.41 1.86 ± 0.51

ns ns

5.76 ± 2.09 1.88 ± 0.51

3.26 ± 2.09 1.76 ± 0.51

ns ns

Root dry weight (g)

Non-stress Stress

3.20 ± 0.68 1.44 ± 0.18

1.73 ± 0.78 0.82 ± 0.18

ns **

2.86 ± 0.68 0.98 ± 0.18

1.51 ± 0.68 1.14 ± 0.18

ns ns

*,** Significant at P < 0.05 and 0.01 level respectively; ns: non-significant.

accumulate more dry matter per unit of water transpired i.e., transpiration efficiency and the ability to harness water from deeper soil profiles by having better root systems are two important traits that directly influence yield under drought stress (Passioura, 1996; Gowda et al., 2011). The NILs were characterized for several physiological traits under lowland non-stress and stress conditions, where traits were recorded at regular time intervals which corresponded to early, mid and late stages of stress. Though in many cases the differences were not significant in this preliminary study, the following preliminary conclusions could be made by comparing across the two families. The tolerant NILs in both the families maintained significantly higher A and Tr than the respective susceptible NILs under non-stress (83 DAS) and under severe stress they also had significantly higher A (Table 3). In addition, under severe stress, the tolerant NIL of IR77298-14-1-2-B family maintained significantly higher RWC, gs and Tr than the susceptible NIL while the tolerant NIL of IR77298-5-6-B family maintained significantly higher TE than the susceptible NIL. The higher gs of IR77298-14-1-2-B10 may be due to its ability to maintain better plant water status and thereby cell turgidity. The higher transpiration efficiency in the other tolerant NIL (IR77298-5-6-B-18) is mainly due to its higher assimilation rate. IR77298-14-1-2-B-10 had higher gs, A, and Tr than the other lines under severe stress. This may explain the yield superiority of the IR77298-14-1-2-B-10 over other lines under stress (Table 1). Our findings confirm those of Centritto et al. (2009), who observed that rice lines with higher photosynthesis and stomatal conductance were more productive under stress than other lines. Deeper and thicker root system has been regarded as drought resistance traits in rice (Fukai and Cooper, 1995; Nguyen et al., 1997; Gowda et al., 2011). In our study, in neither family did the tolerant NILs have significantly deeper roots than the susceptible NILs under stress (Table 4). Root thickness did not differ in the IR772985-6-B family and differed only slightly in the other family, with the tolerant NIL having slightly thicker roots (P < 0.05) than the susceptible NIL. Constitutive differences in root length between the tolerant and the susceptible NIL were observed in the IR77298-56-B family only (Table 4). The most tolerant NIL, IR77298-14-1-2-B-10, had longer and more numerous, and thicker roots and a greater root volume and root dry weight than all other lines, however, the differences were small and not always significant. The tolerant NILs had higher transpiration rates than the susceptible NILs and therefore seem to have had access to more water, but there were no significant differences between the tolerant and susceptible NILs particularly for root length. Although clear and large differences in yield were observed in the field, differences in root traits were relatively minor. Thus through use of NILs we showed that large differences in root pheno-

type are not always the cause of significant yield differences among lines under drought stress in rice. This seems somehow contradicting the thinking that large differences in root architecture are needed to breed for drought resistance in rice (for example – Shen et al., 2001; Steele et al., 2006; Gowda et al., 2011). Similar observations were made by Bernier et al. (2009) where a large-effect QTL for yield under severe upland drought stress showed relatively small but significant difference in water uptake and only minor and usually non-significant differences for root morphological traits. Although the increase in water uptake reported by Bernier et al. (2009) was small (equivalent to 13 mm ha−1 ), it was estimated, based on model simulations in wheat, that every extra millimeter of water transpired could possibly lead to a yield gain of 20–60 kg ha−1 in well-managed disease-free cereal crops (Manschadi et al., 2006). It was therefore concluded that the effect of qtl12.1 on grain yield under stress could come mainly, if not entirely, from an increased plant water uptake (Bernier et al., 2009). These studies together indicate that there is a need to refine the existing root screening methodologies to observe these differences before using root traits in a trait-based approach to breed for dehydration avoidance improvement in rice (Serraj et al., 2009; Henry et al., 2011). Analysis of root hydraulic properties could be considered for further dissecting the drought avoidance mechanisms in rice. All these NILs were 85–92% similar to IR64 genetically and also appear very similar to IR64 in plant type, but they out-yield IR64 in both irrigated and stress environments. In comparison to yield of the standard check ‘Apo’, these lines performed well under both irrigated and stress in lowland but showed poor performance under upland stress, thus, we can conclude that these lines are unsuited for upland environments but can be considered as varieties for stress prone lowland environments. In fact one of the parental lines, IR77298-5-6, has already been released in Philippines as a lowland variety. Our earlier studies showed that this variety is highly drought susceptible (Venuprasad et al., 2007b), thus, IR77298-56-B-18, a drought tolerant selection from the susceptible parent can be immediately useful as a variety in drought prone lowland environments as it combines both high yield potential and drought resistance with the same duration as the released variety. Alternatively IR77298-14-1-2-B-10, the most drought tolerant NIL, can also be used as a variety in drought prone lowlands after suitable testing in target environments. IR64 is very widely grown as a short-duration lowland variety throughout South and SouthEast Asia, where it is often planted in upper-toposequence fields in rainfed environments subject to drought. It has been shown to be highly drought-susceptible (Verulkar et al., 2010), and therefore the availability of a highly similar line (IR77298-14-1-2-B-10) with significantly improved lowland drought tolerance is potentially of importance to millions of farmers in the region.

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On comparing regions of genetic polymorphism between tolerant and susceptible NILs, the regions around RM324-RM521 on chromosome 2 and RM72 on chromosome 8 appear interesting. A QTL affecting yield under lowland drought stress linked to RM324 was reported by Venuprasad et al. (2009) and the contrasting NILs in both the families have polymorphisms in this region (Fig. 1). The contrasting NILs in the IR77298-14-1-2-B family are also polymorphic in the region around RM72 (Fig. 2) and this region is also known to be important in drought tolerance (Kamoshita et al., 2008; Venuprasad et al., unpublished data). Saturation of these regions with more markers is needed. Further work including classical QTL analysis and gene expression is ongoing to identify the genes involved in stress response. Acknowledgements This work was supported by funds from Rockefeller and GCP. We thank Dr. A. Kumar, Tess Sta Cruz, Amante Modesto, Marc Esperitu for their help in conducting the field trials and the IRRI drought physiology technical staff for assistance in the physiology experiments. References Bernier, J., Serraj, R., Kumar, A., Venuprasad, R., Impa, S., Gowda, V., Owane, R., Spaner, D., Atlin, G., 2009. The large-effect drought-resistance QTL qtl12.1 increases water uptake in upland rice. Field Crop Res. 110, 139–146. Bernier, J., Kumar, A., Venuprasad, R., Spaner, D., Atlin, G.N., 2007. A large-effect QTL for grain yield under reproductive-stage drought stress in upland rice. Crop Sci. 47, 507–516. Centritto, M., Lauteri, M., Monteverdi, M.C., Serraj, R., 2009. Leaf gas exchange, carbon isotope discrimination and grain yield in contrasting rice genotypes subjected to water deficits during reproductive stage. J. Exp. Bot. 60, 2325–2339. Fukai, S., Cooper, M., 1995. Development of drought-resistant cultivars using physiomorphological traits in rice. Field Crops Res. 40, 67–86. Gonzalez, L., Gonzalez-Vilar, M., 2001. Determination of relative water content. In: Reigosa Roger, M.J. (Ed.), Handbook of Plant Ecophysoilogy Techniques. Springer, Netherlands. Gowda, V.R.P., Henry, A., Yamauchi, A., Shashidhar, H.E., Serraj, R., 2011. Root biology and genetic improvement of drought avoidance in rice. Field Crop Res. 122, 1–13. Guan, Y.S., Serraj, R., Liu, S.H., Xu, J.L., Ali, J., Wang, W.S., Venus, E., Zhu, L.H., Li, Z.K., 2010. Simultaneously improving yield under drought stress and nonstress conditions: a case study of rice (Oryza sativa L.). J. Exp. Bot. 61, 4145–4156. Hemamalini, G.S., Shashidhar, H.E., Hittalmani, S., 2000. Molecular marker assisted tagging of morphological and physiological traits under two contrasting moisture regimes at peak vegetative stage in rice (Oryza sativa L.). Euphytica 112, 69–78. Henry, A., Gowda, V.R.P., Torres, R.O., McNally, K.L., Serraj, R., 2011. Variation in root system architecture and drought response in rice (Oryza sativa): phenotyping of the OryzaSNP panel in rainfed lowland fields. Field Crop Res. 120, 205–214. Kamoshita, A., Babu, R.C., Boopathi, N.M., Fukai, S., 2008. Phenotypic and genotypic analysis of drought-resistance traits for development of rice cultivars adapted to rainfed environments. Field Crops Res. 109, 1–23. Khush, G.S., Angeles, E., Virk, P.S., Brar, D.S., 2004. Breeding rice for resistance to tungro virus at IRRI. SABRAO J. Breed. Genet. 36, 101–106.

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