Genetic analysis of salt tolerance at seedling and ... · Genetic analysis of salt tolerance at seedling and reproductive stages in rice (Oryza sativa) REZAMOHAMMADI 1,3,MERLYNS

Genetic analysis of salt tolerance at seedling and reproductive stages in rice(Oryza sativa)R E Z A M O H A M M A D I

1,3, M E R L Y N S . M E N D I O R O2, G E N A L E E N Q . D I A Z

2, G L E N N B . G R E G O R I O1 and

R A K E S H K . S I N G H1,4

1Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila,Philippines; 2Genetics and Molecular Biology Division, Institute of Biological Sciences, University of the Philippines, Los Ba~nos,Laguna 4031, Philippines; 3Present address: Agricultural Biotechnology Research Institute of Iran (ABRII), North-West and West region,Tabriz, Iran; 4Corresponding author, E-mail: [email protected]

With 2 figures and 9 tables

Received July 16, 2013/Accepted June 20, 2014Communicated by Y. Saranga

AbstractSalinity is a major abiotic stress that limits rice production across riceareas as high-yielding modern rice varieties are generally sensitive to saltstress. The study was conducted to deduce heritability and combiningability estimates of rice for various morphological and physiologicaltraits using a 7 9 7 full-diallel-cross analysis at seedling and reproduc-tive stages. The salinity stress treatment was 12 dS m�1 at the seedlingstage and 8 dS m�1 at the reproductive stage. Diallel analysis revealedhigh h2bs for salinity tolerance scores and shoot height, moderate for shootdry weight and root dry weight and low for Na+ and K+ concentrationsand K+/Na+ ratio. The low-to-moderate narrow-sense heritability fornumber of panicles, number of fertile spikelets, grain weight, spikelet fer-tility and K+/Na+ ratio suggests a large breeding population and delayedselection for tolerance until later generations. Significant maternal effectsindicate that selection of the female parent is very important for desiredtrait development. The results of this study confirmed that salinity toler-ance at the seedling and reproductive stages is regulated by a differentset of genes that could be pyramided using different donors to enhancethe level of tolerance.

Key words: rice (Oryza sativa) — salinity stress — combiningability — heritability — reproductive stage — salt tolerance

Salinity is one of the serious environmental stresses that limitthe productivity of agricultural crops, which are mostly sensitiveto the presence of high concentrations of salts in the soil (Flow-ers and Yeo 1995). A soil is considered saline if the electricalconductivity of its saturated extract (ECe) is above 4 dS m�1

(US Salinity Laboratory Staff 1954). More than 800 million haof land throughout the world is salt-affected by either salinity(397 million ha) or the associated condition of sodicity (434 mil-lion ha) (Munns and Tester 2008). In Asia alone, 21.5 millionha is salt-affected, of which 12 million ha is saline and 9.5 mil-lion ha is alkaline/sodic. The mechanism of salinity tolerance isstill not very clear, but tolerant plants use a combination ofmechanisms to overcome salinity stress. Salinity stress inhibitsplant growth through many interruptions, such as osmoticeffects; excessive uptake of toxic ions (Na+ and Cl�); inabilityof partitioning at the organ, tissue and cell level; and poor regu-lation of antioxidants with impaired signal pathways (Munns2002, Singh and Flowers 2010).More than half of the world’s population depends on rice

(Oryza sativa L.) as a staple crop; in Asia, rice supplies 30–80%of the daily calories consumed (Narciso and Hossain 2002). Riceis a relatively salt-sensitive crop with threshold salinity of3 dS m�1, above which yield loss occurs (Maas and Hoffmann1977). Maas and Grattan (1999) indicated that rice yields

decrease by 12% for every unit (dS m�1) increase in ECe above3 dS m�1.Rice is relatively tolerant of salt stress during germination,

active tillering and grain filling and is sensitive during the earlyseedling and reproductive stages [panicle initiation (PI), anthesisand fertilization] (Zeng et al. 2001, Singh et al. 2008, Singh andFlowers 2010).The effect of salinity on rice depends on the amount of

salinity, duration of exposure, cultivar, crop growth stage,water regime, soil physical properties, temperature and solarradiation (Neue et al. 1998, Ali et al. 2013). Symptoms ofsalinity stress at the seedling stage include whitened tips ofaffected leaves, chlorotic patches on some leaves, plant stun-ting, reduced tillering, patchy field growth and, in severe cases,plant death.Salinity at the reproductive stage significantly reduces spikelet

number per panicle, tiller number, fertility, panicle length (PL)and the number of primary branches per panicle (Heenan et al.1988, Cui et al. 1995, Khatun et al. 1995, Zeng and Shannon2000, Zeng et al. 2002, Singh et al. 2010). The number of spik-elets per panicle was found to be the most sensitive yield com-ponent. This component is determined at the early reproductivestage, around PI (Counce et al. 2000). The loss in spikelet num-ber per plant was most significant when the stress was imposedbefore PI or between PI and booting stage (~16 days after PI).This loss of potential spikelets is attributed to the degenerationof primary and secondary branches and flower primordia (Zenget al. 2001). Yeo and Flowers (1986) found that during repro-ductive-stage development, tolerant genotypes tended to excludesalt, thus resulting in less salt concentration in flag leaves anddeveloping panicles, which resulted in higher grain yield. Raoet al. (2008) reported a grain yield reduction by 27%, 46% and50% at an ECe of 8 dS m�1 in tolerant, semitolerant and sensi-tive rice cultivars, respectively. The reproductive stage is crucialas it ultimately determines grain yield; however, the importanceof the seedling stage cannot be ignored as it affects crop estab-lishment. Hence, pyramiding of contributing traits at both stagesis needed for developing resilient salt-tolerant cultivars (Moradiet al. 2003, Singh et al. 2010).The diallel cross has proved to be of considerable value to

plant breeders in making decisions concerning the type of breed-ing system to use and in selecting breeding materials that showthe greatest promise. It gives information on the genetic structureof populations under study and on the genetic mechanism(s)controlling various traits (Hayman 1954, Griffing 1956).

Plant Breeding, 133, 548–559 (2014) doi:10.1111/pbr.12210© 2014 Blackwell Verlag GmbH

Salt tolerance, a complex characteristic that changes with plantdevelopment stages, is controlled by a number of genes or groupof genes and involves a number of component traits that arelikely to be quantitative in nature (Flowers and Yeo 1995).Genetic studies indicated that both additive and dominant effectsare important in the inheritance of almost all characteristics asso-ciated with salt tolerance (Mishra et al. 1990, Gregorio and Se-nadhira 1993, Chauhan 2007). At the seedling stage under salineconditions, characteristics associated with salinity tolerance suchas shoot height (SH), low Na+ and high K+ concentrations in theshoots and large dry weight of shoots and roots showed highlysignificant additive effects as heritability in these characteristicsis high. Characteristics at the maturing stage such as plant height(PH) and yield per plant showed highly significant additiveeffects, suggesting the greater importance of additive gene actionin the inheritance of these characteristics (Moeljopawiro andIkehashi 1981, Akbar et al. 1986, Mishra et al. 1990). Yeo et al.(1990) demonstrated genetic diversity for sodium uptake andcommented that salt tolerance persists within modern ricecultivars.The study of Gregorio and Senadhira (1993), using a nine-par-

ent complete diallel analysis of Na+/K+ ratio in shoots, showedthat both additive and dominance gene effects govern salinitytolerance (low Na+/K+ ratio). Environmental effects were signifi-cantly large, and narrow-sense heritability was low (19%). Thecombining ability study showed the significant effects of bothgeneral combining ability (GCA) and specific combining ability(SCA) in the inheritance of salinity tolerance in rice. Moderatelytolerant parents (e.g. IR4595-4-1-13 and IR98884-54-3-1E-P1)were the best combiners for salt-tolerant progeny. The presenceof reciprocal effects among crosses necessitates the use of high-yielding susceptible parents as males in a hybridization pro-gramme. Garcia et al. (1997) reported heritabilities of about 0.4–0.5 for two segregating breeding lines for Na+, K+ and Na+/K+.Chauhan (2007) also observed low estimates of narrow-senseheritability for yield per plant (0.20) in normal environments, til-lers per plant (0.27) in moderate sodic environments, 100-seedweight (0.3) in high sodic environments, tillers per plant (0.2) inmoderate saline environments and 100-seed weight (0.22) in ahigh-saline environment. Mahmood et al. (2002, 2004) reportedthat non-additive gene effects were more evident for panicle fer-tility, days to maturity, shoot dry weight (SDW), paddy yield,Na+ and Na+/K+ ratio, but both additive and dominance effectswere important for PH, PL and shoot Na+ and K+ concentra-tions.A huge number of studies were conducted to decipher the dif-

ferent physiological and genetic factors associated with salinitytolerance during the early seedling stage, but little attention hasbeen devoted to understanding the genetics of salinity toleranceduring the reproductive stage. Hardly any published studies onsalinity tolerance at the reproductive stage in the last 20 yearsincluded the grain yield trait as well (Flowers 2004). The fewreports on reproductive-stage tolerance are mainly because of

difficulties in phenotyping in terms of both technical feasibilityand time consumed for screening. Therefore, it is imperative tohave sound knowledge of the genetic mechanisms underlyingboth seedling- and reproductive-stage salinity tolerance to speedup breeding programmes for the development of cultivars thatare tolerant of both growth stages. This study was conducted toestimate heritability, GCA, SCA and reciprocal effects amongseven rice parental lines in both the seedling and reproductivestages under control and salinity stress conditions, to have anidea about the genetic mechanisms of salinity tolerance at bothcrucial growth stages using a diverse set of genetic materials indifferent cross-combinations. A complete diallel was used todeduce the role of maternal effects for salinity tolerance.

Materials and MethodsPlant materials: The seven diverse parental lines involved in a fulldiallel cross, including reciprocals, were the seedling-stage-susceptiblePusa Basmati 1 and SADRI (IRGC acc. 32329), moderately tolerantIR64, seedling-stage-tolerant FL478 (IR66946-3R-178-1-1) and thereproductive-stage-tolerant varieties CSR28, IR4630-22-2-5-1-3 andIR70023-4B-R-12-3-1, which were selected based on IRRI salinityscreening experiments (unpublished data). Seeds of the seven parentswere sown in wooden seeding boxes at three dates with 10-day intervalsbetween successive sowings. Twenty-one-day-old seedlings weretransplanted in the concrete hybridization blocks at IRRI. All crosseswere made following the crossing procedures described by Jenningset al. (1979). The resulting 42 single crosses from the full diallel crossand seven parents were assessed in greenhouse conditions for seedling-stage salinity tolerance and in field conditions for reproductive-stagesalinity tolerance.

Studied parameters: The study included different parameters such as ascore based on the IRRI-modified standard evaluation system (SES)(Gregorio et al. 1997), SH (cm) and root and SDW (g), Na+ and K+

uptake (mmol/g dwt) and K+/Na+ ratio for the seedling-stage screeningas they are the most important ones for discriminating between tolerantand sensitive genotypes. The SES is a scoring of the plants in a rangeof 1–9 based on morphological manifestation/stress symptoms understress conditions where a lower score of SES indicates tolerant and ahigher score a sensitive reaction (Table 1). However, for thereproductive-stage screening, we included traits such as days toflowering (DFL), PH (cm), PL (cm), panicles per plant, fertile spikelets(FrSp) (%), straw dry weight per plant (StDW) (g), grain weight (GW)per plant (g), 1000-grain weight (TGW) (g) and K+/Na+ ratio for theflag leaf that were critical for distinguishing tolerant from sensitivegenotypes.

Screening for salinity tolerance at the seedling stage: After breakingdormancy, seeds of 42 F1s and seven parents were placed in Petri disheswith moistened filter papers and incubated at 30°C for 48 h to germinate.Pregerminated seeds were sown (two seeds per hole) on a styrofoamsheet having 100 holes with a nylon net bottom in the two sets, one forthe control and the other for salinity stress. The sheets were left to floaton a Simple Nutrient Addition Program (SNAP) nutrient solution. It is abuffer-based nutrient solution (SNAP A and SNAP B) developed by the

Table 1: Modified standard evaluation system (SES) score of visual salt injury at seedling stage in rice (Gregorio et al. 1997)

Score Observation Tolerance

1 Normal growth, no leaf symptoms Highly tolerant3 Nearly normal growth, but leaf tips or few leaves whitish and rolled Tolerant5 Growth severely retarded; most leaves rolled; only a few are elongating Moderately tolerant7 Complete cessation of growth; most leaves dry; some plants dying Susceptible9 Almost all plants dead or dying Highly susceptible

Genetic analysis of salt tolerance 549

Institute of Plant Breeding (IPB), Los Ba~nos (Santos and Ocampo 2005).It is good for one month without replacement and maintenance of dailypH. The nutrient solution pH was checked every week for any deviationfrom 5 (adjusted by adding either 1 N NaOH or HCl). The seedlingswere grown in the phytotron facilities at IRRI that are maintained at 29/21°C day/night temperature and at a minimum relative humidity of 70%.Irradiance inside the glasshouse was maintained at at least 80% ofincident solar radiation. The experiment was conducted in a randomizedcomplete block design (RCBD) in three replications with 10 plants perexperimental unit. After 4 days, the seedlings of the salinity setunderwent salinization (EC = 12 dS m�1) by dissolving NaCl in thenutrient solution. Fourteen days after salinization, salt stress symptomswere evaluated using the modified SES as per Gregorio et al. (1997) toquantify the visual symptoms of salt toxicity (Table 1). This scoringdistinguishes the susceptible from the tolerant and moderately tolerantgenotypes. Data recording and shoot sampling for Na+ and K+

concentrations were performed 14 days after salinization, in both thecontrol and salinized sets, when susceptible parents were severelyaffected by salt stress (Figure S1). For Na+ and K+ concentration analysis,the whole shoot was separated from the root and washed twice in tapwater and twice in deionized water and bagged and then oven-dried for3 days at 70°C. Dried samples were finely ground, and 10 mg of powderfrom each sample was used for Na+ and K+ analysis. Na+ and K+ wereextracted in 100-mM acetic acid at 90°C inside the hot-water bath for atleast 2 h. The Na+ and K+ concentrations were determined by a flamephotometer (Model 420; Sherwood, Cambridge, UK).

Screening for salinity tolerance at the reproductive stage: Theexperiment was conducted at IRRI in an artificially salinized field inconcrete blocks (8 m 9 4.8 m and 0.5 m depth). The 21-day-oldseedlings of 42 F1s and seven parents were transplanted in the controland saline concrete blocks using a RCBD with three replications (FigureS2). Single plants were transplanted per hill. Seven plants perexperimental unit with a distance of 20 cm were maintained for each ofthe seven parents and 42 F1s.

Saline water solution was prepared by dissolving technical grade salt(NaCl) in water to salinize the soil by irrigation of saline water in salineblocks. Salinity stress in the saline blocks was imposed initially by irri-gating the saline water of ECe 3–4 dS m�1 during transplantation at theseedling stage, which increased to ECe 6–8 dS m�1 after the active til-lering stage and was maintained until maturity. This salinity stress levelat the reproductive stage is optimum to distinguish between sensitive andtolerant genotypes. More than this stress level results in a lot of sterilityamong the tolerant genotypes as well, whereas less salinity poses diffi-culty in distinguishing between tolerant and sensitive genotypes. Atmaturity, data were recorded on five plants in each of the entries of theparents and F1s in both the control and saline blocks. The flag leaf ofthe main tiller was used for Na+ and K+ concentration analysis using thesame methodology as for shoot samples at the seedling stage.

Diallel statistical analysis: Data were obtained from the 42 F1s andseven parents under control and saline conditions, and combiningabilities as well as heritabilities were estimated according to Griffing’s

method 1 model 1 (Griffing 1956) using WINDOSTAT (version 8.5) (www.windostat.org). GCA and SCA analyses were carried out according tothe procedure outlined by Griffing (1956), using method I (full set ofdiallel crosses, including reciprocals) as a fixed model. Broad-senseheritability was calculated as additive variance + dominance variance/(dominance variance + additive variance + error variance/reps.). Narrow-sense heritability was calculated as additive variance/(dominancevariance + additive variance + error variance/reps.). The GCA/SCAvariance ratios were calculated to determine the relative importance ofthe effects of GCA and SCA (Baker 1978).

ResultsThe analysis of the data showed a significantly differentresponse among the parents and F1 progeny for the evaluatedtraits under control and salt stress conditions at the seedling andreproductive stages. By comparison of trait means under controland stressed conditions, a considerable effect due to salinitystress was observed in all traits evaluated at both stages(Tables 2 and 3). Majority of the traits at the seedling stagewere normally distributed, except for the distribution of ShDW,which was close to normal distribution (Table 2 and Fig. 1). Forthe reproductive stage, the frequency distribution of all studiedtraits under salinity stress and most traits under control condi-tions, except for the distribution of PnN, StDW and GYLDunder control conditions, was normal distribution (Table 3 andFig. 2).

Responses to salinity at the seedling stage

Analysis of variance for combining ability was performed on alldata obtained from the parents and F1s for seven characteristicsunder normal environments and salinity stress at the seedling stage.The mean squares of GCA effects were significant for all traits(P ≤ 1%) under normal environments and salinity stress (Table 4).The mean squares of SCA effects were significant for SES, SH,SDW and root dry weight (RDW) (P ≤ 1%), but SCA effects werenot significant for Na+ and K+ uptake and K+/Na+ under normaland saline conditions. Reciprocal effects were significant for alltraits at 1% probability except for SDW, RDW, K+ uptake undernormal conditions and K+ uptake under saline conditions, indicat-ing the significant role of maternal effects (Table 4). General andSCA effects for all studied traits under normal conditions andsalinity stress at the seedling stage are presented as follows.

Standard evaluation system (SES) score for salinity toleranceAll seven parents had significant GCA effects for SES undersalinity stress except for IR64, and it was positive for IR4630,PB1 and SADRI but negative for FL478, CSR28 and SAL729.

Table 2: Mean values, range and skewness of studied traits of seven parents and 42 F1 progeny grown under normal and salt stress conditions at seed-ling stage in phytotron

Trait

Range Mean Skewness1

Normal Saline Normal Saline Normal Saline

SES score – 1–7 – 4.34 – �0.40Shoot height (cm) 41–75 22–50 55.80 35.52 0.67 0.34Shoot DW (g/plant) 0.08–0.49 0.03–0.25 0.18 0.09 1.40 1.13Root DW (g/plant) 0.01–0.07 0.00–0.06 0.03 0.02 1.03 0.73Shoot Na+ (mmol/g dwt) 0.05–0.22 0.18–0.91 0.11 0.42 0.77 0.97Shoot K+ (mmol/g dwt) 0.65–1.15 0.40–1.00 0.94 0.74 �0.31 �0.32Shoot K+-Na+ ratio 4.52–17.42 0.46–4.40 9.41 2.02 0.49 0.49

1A variable is reasonably close to normal if its skewness has values between �1.0 and +1.0.

550 R. MOHAMMAD I , M. S. MEND IORO , G. Q. DIAZ e t a l .

FL478 had the lowest GCA with desirable negative effects forSES; thus, it could be a good combiner as a parent for SES.PB1 had the highest GCA with undesirable positive effectsamong the parents for SES as it is the most susceptible parentunder salinity stress at the seedling stage (Table 2). In the F1hybrids, PB1 9 IR4630 had the lowest desirable negative SCAeffects, followed by FL478 9 IR64 and SADRI 9 FL478(Table 6). The GCA/SCA ratio was 0.58, suggesting incomplete

dominance and the involvement of additive and non-additivegene action for this trait.

Shoot heightFor SH, the effects of GCA under normal environments werepositive for SADRI; on the other hand, IR4630, FL478 andIR64 had negative effects (Table 5). Under salinity stress, theeffects of GCA for SH were significant for all parents except

Table 3: Mean values, range and skewness of yield components of seven parents and 42 F1 progeny grown under normal conditions and salt stress atreproductive stage in field conditions

Trait

Range Mean Skewness1

Normal Saline Normal Saline Normal Saline

Days to flowering 65–100 70–106 81.5 86.5 0.48 0.67Plant height (cm) 113.7–240.7 89.7–201.0 160.7 139.2 0.92 0.57Number of panicles 6.7–40.3 5.7–34.3 14.1 15.1 2.10 0.80Panicle length (cm) 24.0–40.3 21.3–37.3 30.2 28.4 0.42 0.41Straw dry weight (g/plant) 20.9–150.4 8.9–148.2 53.6 42.5 1.38 1.04Number of fertile spikelets 204–4045 7.3–2552.3 1252.0 918.8 1.10 0.49Grain yield (g/plant) 3.7–110.2 0.2–74.1 32.4 21.0 1.30 0.80Spikelet fertility % 19.1–91.7 0.43–89.5 62.0 51.7 �0.57 �0.571000-grain weight (g) 15.0–32.3 13.1–29.0 25.5 22.1 �0.81 �0.49Flag leaf K+-Na+ ratio 1.9–13.4 0.4–9.7 6.1 2.9 0.61 0.81

1A variable is reasonably close to normal if its skewness has values between �1.0 and +1.0.

Sh-Na+-N Sh-K+-N

Sh-Na+-S Sh-K+-S

Sh-K+/Na+-N Sh-K+/Na+-S

25

20

15

10

5

0

0

05.00 7.50 10.00 12.50 15.00 17.50 0.00 1.00 2.00 3.00 4.00 5.00

5

15

10

10

20

20

0

5

15

10

20

30

00.40 0.60 0.80 1.00

10

20

30

00.60 0.70 0.80 0.90 1.00 1.10 1.20

10

20

Frequency

Frequency

Frequency

Frequency

Frequency

Frequency

30

0.00

0.00 0.20 0.40 0.60 0.80 1.00

0.05 0.10 0.15 0.20 0.25

Fig. 1: Frequency distribution ofshoot Na+ and K+ concentrations(mmol/g dwt) and K+/Na+ ratio ofseven parents and 42 F1 progeniesin three replications grown undercontrol (N) and salt stress (S)conditions at seedling stage inphytotron


FL478 and SAL729. The effects were positive for SADRI andCSR28 (Table 5). The largest positive SCA effects were in theSADRI 9 IR64 cross under salinity stress (Table 6). The maxi-mum SCA with negative effects was measured forSADRI 9 CSR28. The GCA/SCA ratio was 0.39. This sup-ported the involvement of non-additive gene action for this traitunder salinity stress (Table 5).

Shoot dry weightFor SDW under normal conditions, IR4630, PB1 and SADRIhad significant GCA effects. Under normal conditions, SADRIhad the highest positive GCA effects for SDW (Table 5).SADRI was found to be the best general combiner under normalconditions. However, under salinity stress, GCA effects werepositive only for FL478, CSR28, IR64 and SAL729; FL478 hadthe highest and most significant GCA effects. Under salinitystress, maximum positive SCA effects were observed forIR64 9 FL478, followed by PB1 9 IR4630. These crosses per-formed better than their reciprocals (Table 6), indicating thepresence of reciprocal effects. The lower GCA/SCA ratio (0.16)supported the involvement of non-additive effects for this trait.

Root dry weightFor RDW under normal conditions, GCA effects were significantonly for PB1 and SADRI but positive for SADRI and negative

for PB1 (Table 5). SADRI was the best general combiner undernormal conditions. Highly desirable positive GCA effects forRDW were measured for FL478 and CSR28. The highest nega-tive value was for PB1 (P ≤ 0.001) (Table 5). The largest posi-tive value of SCA was estimated for IR64 9 FL478, followedby PB1 9 IR4630 (Table 6). The lower GCA/SCA ratio (0.18)for RDW supported the involvement of non-additive effects forthis trait.

Na+ uptakeFor Na+ uptake (mmol/g dwt) under normal conditions, GCAeffects were significant for PB1 in a positive trend and forCSR28 in a negative trend, whereas the effects were not signifi-cant for the rest of the parents (Table 5). Undesirable positiveGCA effects for Na+ uptake under saline conditions were mea-sured for PB1, but SAL729 had a desirable negative GCA value(P ≤ 0.05). Results showed that FL478 was not a good generalcombiner for Na+ exclusion (Table 5). The highly desirable neg-ative value of SCA was estimated for SAL729 9 SADRI, whichwas a cross between a tolerant and susceptible parent. The larg-est positive values of SCA for Na+ uptake were estimated for itsreciprocal SADRI 9 SAL729, indicating the presence of mater-nal effects (Table 6). The GCA/SCA ratio was 0.70. This sup-ported the involvement of additive and non-additive gene actionfor this trait.

K+ uptakeFor K+ uptake (mmol/g dwt) of the shoot under normal condi-tions, GCA effects were significant and positive for PB1 andCSR28 but negative for SADRI. For K+ under salinity stress,GCA effects were significant for FL478, PB1 and CSR28, butnot significant for IR4630, IR64, SAL729 and SADRI. BothFL478 and CSR28 parents were the best combiners, exhibitingvery highly desirable positive GCA effects for K+.IR4630 9 SADRI had the highest positive SCA values amongthe crosses, but when SADRI was a female parent in theSADRI 9 SAL729 cross, it had the largest negative SCA valueof all the crosses. The GCA/SCA ratio was 1.5. This supportedthe involvement of additive gene action, which is a fixable com-ponent of inheritance (Table 5).

K+/Na+ ratioFor K+/Na+ ratio under normal conditions, GCA effects weresignificant for PB1, CSR28, IR64 and SADRI only. GCA effectswere positive for CSR28, but negative for PB1, IR64 andSADRI. For K+/Na+ ratio under salinity stress, GCA effects weresignificant and positive for FL478 and SAL729 but negative forPB1. Thus, FL478 and SAL729 were the best combiners withdesirable positive GCA effects for K+/Na+ ratio. Gregorio andSenadhira (1993) suggested that maintaining a good K+/Na+ bal-ance in the shoot by Na+ exclusion and high K+ uptake is a typi-cal characteristic of salinity tolerance in rice. The largest positivevalues of SCA for K+/Na+ ratio were estimated forIR4630 9 SADRI, followed by SAL729 9 SADRI. The largestnegative effect of SCA was for FL478 9 CSR28, the crossbetween two tolerant parents. The high GCA/SCA ratio (0.87)supported the involvement of both additive and non-additivegene action for this trait (Table 5).

Responses to salinity at the reproductive stage

Analysis of variance for combining ability was performed on alldata obtained from the parents and F1s for nine characteristics

FL K+/Na+-S

FL K+/Na+-N25(a)

(b)

20

15

10

5

0

25

20

15

10Frequency

Frequency

5

00.00 2.00 4.00 6.00 8.00 10.00

0.00 5.00 7.50 10.00 12.502.50

Fig. 2: Frequency distribution of flag leaf K+/Na+ ratio of seven parentsand 42 F1 progenies in three replications grown under (a) control (N)and (b) salt stress (S) at reproductive stage in field conditions


Table

4:Analysisof

variance

fortestsof

thecombining

ability

ofallstudiedtraitsin

a79

7diallelcrossof

rice

(Oryza

sativaL.)at

theseedlin

gstageundernorm

alandsalin

ized

conditionsat

EC12

dSm

�1

Source

ofvariation

df

Meansquares

SESScore

SHSD

WRDW

Na+

K+

ShK-N

a

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

e

GCA

64.39

**28

4.42

**94

.31*

*0.00

5**

0.00

142**

0.0000

8**

0.00

010**

0.002*

*0.0272

**0.021*

*0.016*

*11

.11*

*0.77

**SC

A21

0.66

**15

.25*

*19

.74*

*0.00

2*0.00

072**

0.0000

7**

0.00

005**

0.000ns

0.0066

ns0.002ns

0.003ns

2.38

ns0.18

nsReciprocal

210.28

**6.10**

7.76

**0.00

1ns

0.00

044**

0.0000

3ns

0.00

003**

0.001*

*0.0103

**0.002ns

0.005ns

5.70

**0.41

**Error

960.13

2.88

3.01

0.00

10.00

018

0.0000

20.00

001

0.000

0.0043

0.002

0.004

1.82

0.17

EC,electrical

conductiv

ity;GCA,generalcombining

ability;RDW,root

dryweight;SC

A,specific

combining

ability;SD

W,shootdryweight;SE

S,standard

evaluatio

nsystem

;SH

,shootheight.

*,**

,ns,sign

ificantat

the0.05

and0.01

prob

ability

levelandno

tsign

ificant,respectiv

ely.

Table

5:General

combining

ability

(GCA)effectsof

thesevenrice

parental

lines

(Oryza

sativaL.)andheritabilityestim

ates

forallstudiedtraits

attheseedlin

gstageundernorm

alandsalin

ized

conditionsat

EC12

dSm

�1

Parent

SESScore

SHSD

WRDW

Na+

K+

ShK-N

a

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

eNormal

Salin

e

IR46

300.49**

*�5

.31***

�3.73*

**�0

.025

**�0

.012**

*�0

.002

ns�0

.002*

�0.006

ns�0

.002

ns0.01

1ns

�0.013

ns0.46

ns�0

.055

nsFL

478

�1.03***

�1.19**

0.74

ns0.01

4ns

0.014*

**0.001ns

0.003*

**0.006ns

�0.027

ns0.01

2ns

0.042*

*�0

.25ns

0.201*

PB1

0.59**

*�0

.03ns

�1.35*

*�0

.019

*�0

.014**

*�0

.003**

�0.004**

*0.020*

**0.092*

**0.04

1***

�0.047**

�0.96**

�0.443**

*CSR

28�0

.24**

�0.74ns

1.41

**0.00

4ns

0.005ns

0.001ns

0.003*

*�0

.017**

*�0

.013

ns0.02

6*0.044*

*1.64**

*0.128ns

IR64

0.14

ns�1

.60***

�1.73*

**�0

.012

ns0.002ns

�0.001

ns0.001ns

0.003ns

�0.025

ns�0

.004

ns�0

.012

ns�0

.56*

0.041ns

SAL729

�0.24**

�0.55ns

0.29

ns0.00

9ns

0.006ns

0.002ns

0.000ns

�0.006

ns�0

.039*

�0.007

ns0.014ns

0.35

ns0.245*

SADRI

0.30**

9.43**

*4.36

***

0.02

9***

�0.001

ns0.003*

*�0

.001

ns�0

.001

ns0.013ns

�0.079

***

�0.027

ns�0

.67*

�0.116

nsh2 ns

0.45

0.71

0.37

0.24

0.17

0.11

0.19

0.32

0.25

0.49

0.31

0.23

0.21

h2 bs0.85

0.92

0.85

0.60

0.69

0.63

0.71

0.48

0.43

0.64

0.42

0.42

0.34

GCA

/SC

A0.58

1.63

0.39

0.33

0.16

0.10

0.18

1.05

0.70

1.68

1.51

0.61

0.87

EC,electrical

conductiv

ity;RDW,root

dryweight;SC

A,specific

combining

ability;SD

W,shootdryweight;SE

S,standard

evaluatio

nsystem

;SH

,shootheight.

*,**,**

*,ns,significantat

the0.05,0.01

and0.001probability

levelandnotsignificant,respectiv

ely.


under control and stress conditions at the reproductive stage. Themean squares of the effects of GCA and SCA were significantfor all traits (P ≤ 1%). This indicated that both additive and dom-inance genetic variances were important in the measured charac-teristics (Table 7). Reciprocal effects suggested that maternalinfluence was significant for all traits under control and stressconditions except for TGW under control conditions and numberof panicles, PL and K+ uptake under stress conditions. Thisshowed that the maternal effect made an important contributionin these traits.

Number of DFLFor DFL under control and stress conditions, all seven parentshad significant positive GCA effects for IR4630, PB1, CSR28and SAL729, but negative effects for FL478, IR64 and SADRI.SAL729 had the highest GCA with positive effects for DFL, butSADRI and FL478 had the highest GCA with negative effectsamong the parents. These can be good combiners as parents forearly maturity under salinity stress. The largest negative effect

among F1 crosses was measured for SAL729 9 FL478 (Table9). The GCA/SCA ratio was 1.99. This shows that GCA vari-ance was much higher than SCA variance, which clearly indi-cates the preponderance of additive effects for this trait.

Plant heightAll parents had significant GCA effects for PH except IR4630under control conditions. Effects were positive for CSR28,SADRI and SAL729; on the other hand, FL478, PB1 andIR64 had negative GCA effects (Table 8). All parents had sig-nificant GCA effects for PH except CSR28 under saline con-ditions. As in normal conditions, SADRI had the highestGCA with positive effects for PH under a saline environment.FL478 and IR64 had the highest GCA with negative effectsamong the parents. SADRI 9 CSR28 had the largest positiveSCA effect. The GCA/SCA ratio was 0.96. This shows thatGCA and SCA variances are almost the same. This supportedthe involvement of additive and non-additive gene action forthis trait.

Table 6: SCA effects of selected F1s and reciprocals (Rec.) for all measured traits of a 7 9 7 diallel cross at the seedling stage of rice under salineconditions at EC 12 dS m�1

Cross1

Trait

Rec.

Trait

SES SH SDW RDW K-Na SES SH SDW RDW K-Na

1 9 2 0.33 0.50 �0.006 �0.001 0.393 2 9 1 1.20 1.64 �0.021 �0.007 �0.2941 9 3 �0.67 0.67 0.019 0.003 0.058 3 9 1 �1.09 4.90 0.032 0.009 �0.0511 9 7 �0.14 0.33 0.011 0.004 0.990 7 9 1 �0.14 �1.82 0.000 0.001 0.5342 9 3 0.83 �0.17 �0.021 �0.005 �0.120 3 9 2 0.27 �1.08 0.000 0.000 �0.1962 9 4 0.67 0.83 0.008 0.002 �0.462 4 9 2 0.27 �2.51 �0.011 �0.002 0.1322 9 5 �0.78 �0.50 �0.030 �0.008 �0.240 5 9 2 �0.78 0.97 0.046 0.010 0.1902 9 7 0.17 2.83 0.008 0.000 0.568 7 9 2 �0.78 4.21 0.007 0.003 0.0194 9 7 �0.17 3.83 0.020 0.005 0.085 7 9 4 �0.61 �2.79 0.013 0.005 �0.2015 9 6 �0.33 0.33 �0.006 �0.002 �0.535 6 9 5 0.10 �1.77 �0.001 0.000 0.3685 9 7 �0.17 �1.00 �0.010 �0.002 0.078 7 9 5 �0.61 5.85 0.022 0.006 �0.0846 9 7 �0.50 5.17 0.026 0.004 0.837 7 9 6 0.10 �0.67 �0.004 �0.002 �0.236

EC, electrical conductivity; RDW, root dry weight; SCA, specific combining ability; SDW, shoot dry weight; SES, standard evaluation system; SH,shoot height.1Parent 1 = IR4630-22-2-5-1-3 (IR4630), Parent 2 = IR66946-3R-178-1-1 (FL478), Parent 3 = Pusa Basmati 1 (PB1), Parent 4 = CSR28, Parent5 = IR64, Parent 6 = IR70023-4B-R-12-3-1 (SAL729), Parent 7 = SADRI (IRGC acc. 32329).

Table 7: Analysis of variance for tests of the combining ability of all studied traits in a 7 9 7 diallel cross of rice (Oryza sativa L.) at the reproduc-tive stage under normal and salinized field conditions

Source ofvariation df

Mean squares

DFL PH PN PL StDW

Normal Saline Normal Saline Normal Saline Normal Saline Normal Saline

GCA 6 357.68** 315.9** 2945.94** 3499.1** 33.54** 49.7** 25.02** 55.2** 1814.68** 2357.1**SCA 21 12.28** 11.9** 232.55** 271.5** 15.11** 20.9** 8.45** 6.8** 423.88** 520.3**Reciprocal 21 4.19** 4.86** 51.43** 22.7* 10.84** 6.9 ns 2.00** 0.72 ns 181.72** 102.3*Error 96 0.57 0.65 25.55 12.3 4.12 5.1 0.96 0.98 73.25 53.0

FrSp GW SpFr% TGWFL K-Na

Normal Saline Normal Saline Normal Saline Normal Saline Saline

GCA 6 111846 ns 468715** 153.96** 335.28** 617.53** 1308.9** 38.58** 35.8** 8.61**SCA 21 375117** 216000** 296.95** 137.92** 272.18** 193.8** 6.91** 4.1** 1.72**Reciprocal 21 179825** 123061** 128.98** 78.82** 63.59** 135.6** 1.42 ns 3.1** 1.08**Error 96 52680 44208 39.22 23.63 28.33 50.4 0.935 1.5 0.45

DFL, days to flowering; FrSp, fertile spikelets; GCA, general combining ability; GW, grain weight; PH, plant height; PL, panicle length; PN, numberof panicles per plant; SCA, specific combining ability; SpFr, spikelet fertility; StDW, straw dry weight per plant; TGW, 1000-grain weight.*, **, ns, significant at the 0.05 and 0.01 probability level and not significant, respectively.


Number of panicles per plantFor number of panicles per plant (PN) under control conditions,the GCA effect was significantly positive for IR64 and SADRI,

but negative for IR4630, FL478 and CSR28. The largest nega-tive GCA effect for PN was measured for FL478 (Table 8).Unlike in control conditions, the largest positive GCA effect forPN was measured for SADRI, which was significantly different

Table 8: General combining ability (GCA) effects of the seven rice parental lines (Oryza sativa L.) and heritability estimates for all studied traits atthe reproductive stage under normal and salinized field conditions

Parent

DFL PH PN PL StDW

Normal Saline Normal Saline Normal Saline Normal Saline Normal Saline

IR4630 3.54*** 2.86*** �1.97 ns �3.06*** �1.15* �0.66 ns �0.55* �0.40 ns 1.01 ns �1.33 nsFL478 �3.92*** �4.54*** �12.12*** �15.35** �1.86*** �3.16*** �2.13*** �2.28 *** �12.81*** �14.13***PB1 2.73*** 1.12*** �4.21** �3.44*** �0.24 ns 0.15 ns 1.31*** 0.78 ** �1.92 ns �3.71*CSR28 2.30*** 2.48*** 3.50** �0.88 ns �1.31* �0.85 ns �0.10 ns �0.62 * �4.39* �2.26 nsIR64 �5.03*** �3.83*** �17.13*** �14.77*** 1.97*** 0.65 ns �0.91*** �1.99 *** �10.72*** �12.54***SAL729 6.80*** 7.38*** 4.15** 6.08*** 0.76 ns 0.91 ns 0.72** 1.01*** 8.97*** 11.70***SADRI �6.42*** �5.47*** 27.77*** 31.42*** 1.83*** 2.98*** 1.67*** 3.51 *** 19.85*** 22.27***h2ns 0.78 0.76 0.55 0.64 0.19 0.23 0.28 0.54 0.34 0.38h2bs 0.96 0.95 0.95 0.98 0.67 0.79 0.88 0.94 0.82 0.91GCA / SCA 2.18 1.99 0.68 0.96 0.19 0.20 0.23 0.67 0.35 0.35

FrSp GW SpFr% TGWFL K-Na

Normal Saline Normal Saline Normal Saline Normal Saline Saline

IR4630 �130.19* �117.73* �3.06 ns �2.41* �0.24 ns �0.65 ns 0.49* 0.26 ns 0.17 nsFL478 81.95 ns �48.40 ns 3.51* 0.32 ns 8.50*** 9.83*** 1.54*** 1.89*** �0.19 nsPB1 �8.67 ns 18.43 ns �2.93 ns �1.49 ns �4.27** �2.99 ns �2.44*** �2.02*** �0.91***CSR28 10.33 ns �25.04 ns 1.72 ns �0.39 ns 2.20 ns �2.09 ns 1.52*** 0.11 ns 0.89***IR64 118.09* 147.43** 2.28 ns 2.95* 7.53*** 11.02*** �0.28 ns 0.15 ns �1.12***SAL729 �97.33 ns �271.64*** �4.39** �7.43*** �9.89*** �17.92*** �2.03*** �2.13*** 0.75***SADRI 25.81 ns 296.95*** 2.87 ns 8.45*** �3.83** 2.82 ns 1.20*** 1.74*** 0.40*h2ns 0.02 0.19 0.05 0.21 0.23 0.43 0.43 0.50 0.36h2bs 0.74 0.74 0.76 0.76 0.88 0.78 0.91 0.76 0.76GCA / SCA 0.01 0.18 0.03 0.20 0.17 0.63 0.45 0.95 0.46

FrSp, fertile spikelets; GW, grain weight; PH, plant height; PL, panicle length; PN, number of panicles per plant; SCA, specific combining ability;SpFr, spikelet fertility; StDW, straw dry weight per plant; TGW, 1000-grain weight.*, **, ***, ns, significant at the 0.05, 0.01 and 0.001 probability level and not significant, respectively.

Table 9: SCA effects of selected F1s and reciprocals for all measured traits of a 7 9 7 diallel cross of rice (Oryza sativa L.) at the reproductive stageunder salinized field conditions

Cross1

Trait

Rec.

Trait

DFL PH PN PL StDW DFL PH PN PL StDW

1 9 7 1.88 �4.43 2.83 0.43 8.57 7 9 1 1.23 10.15 1.40 1.54 17.432 9 6 �1.00 �3.55 1.83 0.57 13.18 6 9 2 �4.38 �11.78 �2.03 �2.51 �10.762 9 7 �0.33 �1.10 �1.00 �1.03 2.55 7 9 2 2.47 11.01 4.40 1.15 17.153 9 4 3.83 6.82 0.50 0.12 16.72 4 9 3 �0.98 3.78 2.42 1.17 10.523 9 6 �3.00 �5.95 3.00 �0.77 1.25 6 9 3 3.95 4.15 3.16 0.86 11.033 9 7 1.50 �3.53 �0.33 �0.22 �7.07 7 9 3 �0.69 2.20 �1.57 2.65 �12.594 9 7 2.50 4.12 �4.67 �0.43 �6.63 7 9 4 0.62 16.42 3.09 �0.32 9.736 9 7 �0.33 �2.45 1.50 �0.17 �9.95 7 9 6 �1.60 7.46 �12.07 �6.68 27.98

Trait

Rec.

Trait

FrSp GW SpFr TGW K+/Na+ FrSp GW SpFr TGW K+/Na+

1 9 3 569.7 13.31 19.78 2.78 �1.13 3 9 1 7.19 0.34 �7.24 �0.99 1.161 9 7 �65.5 �2.45 �8.82 �1.18 0.27 7 9 1 �265.2 �6.33 �17.22 0.97 �0.102 9 7 �424.7 �11.48 �6.17 �0.08 �1.10 7 9 2 470.6 13.05 �0.04 0.71 1.174 9 5 �106.2 �3.26 0.02 �0.57 �0.63 5 9 4 449.3 10.79 11.15 0.72 0.114 9 6 176.7 3.63 2.57 1.40 �0.26 6 9 4 125.9 1.47 6.83 �1.78 �1.914 9 7 �511.7 �15.93 �7.49 �2.55 0.46 7 9 4 378.6 10.70 1.11 0.04 �0.34

FrSp, fertile spikelets; GW, grain weight; PH, plant height; PL, panicle length; PN, number of panicles per plant; SCA, specific combining ability;SpFr, spikelet fertility; StDW, straw dry weight per plant; TGW, 1000-grain weight.1Parent 1 = IR4630-22-2-5-1-3 (IR4630), Parent 2 = IR66946-3R-178-1-1 (FL478), Parent 3 = Pusa Basmati 1 (PB1), Parent 4 = CSR28, Parent5 = IR64, Parent 6 = IR70023-4B-R-12-3-1 (SAL729), Parent 7 = SADRI (IRGC acc. 32329).


from the other parents (P ≤ 0.001). SADRI 9 FL478 had thehighest positive SCA effect for PN, followed by IR64 9 CSR28(Table 9). A lower GCA/SCA ratio (0.20) for PN supported theinvolvement of non-additive effects for this trait.

Panicle lengthFor PL under control conditions, GCA effects were significantlypositive for PB1, SAL729 and SADRI. Under control conditions,SADRI and PB1 were the best general combiners for PL. FL478had the largest negative GCA effect for this trait (Table 8).Under stress conditions, GCA effects were significantly positivefor PB1, SADRI and SAL729. Similar to normal conditions,FL478 had the largest negative GCA effect for this trait(Table 8). SADRI 9 SAL729 had the largest negative SCAeffect. The GCA/SCA ratio was 0.67, which supported theinvolvement of additive and non-additive gene action for thistrait.

Straw dry weight per plantFor SDW, GCA effects were significant and positive for SADRIand SAL729, whereas they were negative for FL478, CSR28and IR64. For SDW, SADRI was the best general combiner.FL478 had the largest negative GCA effects for this trait(Table 8). Under saline conditions, negative GCA effects wereobserved for FL478, PB1 and IR64. Maximum negative SCAeffects were observed for SADRI 9 PB1 and SAL729 9 FL478(Table 9). A lower GCA/SCA ratio (0.35) supported the involve-ment of non-additive effects for this trait under stress conditions.

Number of FrSpFor FrSp under normal conditions, GCA effects were significantand positive for IR64 and negative for IR4630. Under salineconditions, GCA effects were significant for IR4630, IR64,SAL729 and SADRI only. SADRI had the highest positive GCAvalue for number of FrSp, and SAL729 had the highest negativeGCA value (Table 8). IR4630 9 PB1 had the highest positiveSCA effects, and CSR28 9 SADRI had the highest negativeSCA effects (Table 9). The GCA/SCA ratio (0.18) indicated thatnon-additive gene action was predominant.

Grain weightFor GW under control conditions, GCA effects were positiveand significant only for FL478. However, under stress condi-tions, GCA effects were significantly positive for IR64 andSADRI (Table 8). Under stress conditions, highly desirable posi-tive GCA effects for GW were measured for SADRI, which wassignificantly different from the other parents (P ≤ 0.001). Understress conditions, SADRI was the best general combiner forGW. The largest positive values of SCA were estimated forIR4630 9 PB1, followed by SADRI 9 FL478, IR64 9 CSR28and SADRI 9 CSR28. SADRI 9 FL478 and SADRI 9 CSR28performed better than their reciprocals (Table 9), indicating thatusing SADRI as a female parent will produce the best combina-tions. This indicated the presence of reciprocal effects amongcrosses. The lower GCA/SCA ratio (0.19) for GW supported theinvolvement of non-additive effects for this trait.

Spikelet fertilityFor spikelet fertility (SpFr) under control conditions, GCAeffects were significant for all parents except IR4630 andCSR28. Under stress conditions, GCA effects were significantfor FL478, IR64 and SAL729 but were not significant for

IR4630, PB1, CSR28 and SADRI. As in control conditions, thelargest positive GCA effects for SpFr were measured for IR64and FL478 and high, undesirable negative GCA effects weremeasured for SAL729 (Table 8). IR4630 9 PB1 had the highestpositive SCA effect, whereas SADRI 9 IR4630 had the highestnegative SCA effect. The GCA/SCA ratio was 0.63, which sup-ported the involvement of additive and non-additive gene actionfor SpFr.

1000-grain weightAll parents had significant GCA effects except IR64 for TGWunder control conditions. FL478, CSR28 and SADRI were thebest combiners for TGW (Table 8). Under stress conditions,FL478 and SADRI were the best combiners. IR4630 9 PB1 hadthe highest positive SCA effect and CSR28 9 SADRI had thehighest negative SCA effect for TGW. The GCA/SCA ratio was0.95, which shows that GCA and SCA variances are almost thesame. This supported the equal involvement of additive and non-additive gene action for this trait.

Flag leaf K+/Na+ ratioFor flag leaf K+/Na+ ratio, GCA effects were significant for allparents under stress conditions except for IR4630 and FL478.GCA effects were positive for CSR28, SAL729 and SADRI.Negative GCA effects were observed for PB1 and IR64(Table 8). For K+/Na+ ratio, the highest positive values of SCAwere estimated for SADRI 9 FL478, followed byPB1 9 IR4630. The ratio of GCA/SCA was 0.46, which indi-cated that non-additive gene action was dominant for this trait.

Heritability estimates

The broad-sense (h2bs) and narrow-sense (h2ns) heritability esti-mates for all seven traits studied at the seedling stage are listedin Table 5. The broad-sense heritability was high for salinityscore (saline = 0.85) and SH (normal = 0.92, saline = 0.85),moderate for SDW (normal = 0.60, saline = 0.69) and RDW(normal = 0.63, saline = 0.70) and low for Na+ (normal = 0.48,saline = 0.43), K+ (normal = 0.64, saline = 0.42) and K+/Na+

ratio (normal = 0.42, saline = 0.34). High broad-sense heritabil-ity indicated the involvement of both additive and non-additivevariances and the little influence of environmental effects,whereas low broad-sense heritability indicated much influence ofenvironmental effects. Narrow-sense heritability was low for alltraits except for SH under normal conditions (h2ns = 0.71). Nar-row-sense heritability is important for breeding programmesbecause it estimates the relative importance of the additive por-tion of genetic variance that can be transmitted to the next gener-ation.Broad-sense (h2bs) and narrow-sense (h2ns) heritabilities were

estimated for all the studied characteristics at the reproductivestage and are listed in Table 8. The traits with high narrow-senseheritability values can be the most reliable traits for selection inbreeding programmes for salinity tolerance.

DiscussionResults of frequency distribution analysis showed that the mostmeasured traits, especially Na+ and K+ concentrations, exhibitcontinuous normal distribution; thus, the quality of the data wasacceptable for genetic analysis. Diallel studies in rice havereported the preponderance of both additive and non-additive


gene action for most of the traits associated with salt tolerance(Narayanan and Sree Rangasamy 1990, Gregorio and Senadhira1993, Gregorio et al. 2002, Chauhan 2007). Based on the com-bining ability analysis, it is possible to deduce whether the par-ents could be carefully selected for the improvement of aspecific trait as GCA relates to the additive genetic variance thatis heritable over generations (Gu et al. 1998). Significant GCAeffects were found among the parents for the different traitsunder salinity stress at the seedling stage, and FL478 was foundto be the best general combiner for salinity score, SDW and K+

uptake, and a good combiner for Na+ uptake and K+/Na+ ratio.It could be used for improving salinity tolerance at the seedlingstage. CSR28 also had the highest positive GCA values for K+

uptake and hence could also be used to improve the K+ uptakeof breeding lines to develop better and more tolerant rice varie-ties. FL478 and CSR28 were the best general combiners forRDW. A high positive GCA value for SDW, K+ uptake and K+/Na+ ratio means that the parental line has high potential to gen-erate superior offspring for salinity tolerance.In this study, a higher GCA/SCA ratio for traits such as K+

uptake and K+/Na+ ratio also supported the preponderance ofadditive effects for these traits. A low GCA/SCA ratio for SH(saline), SDW and RDW exhibited greater relevance of non-additive gene actions that govern these traits. In F1 hybrids, thehighest desirable negative SCA effects of salinity score were forPB1 9 IR4630, followed by FL478 9 IR64 andSADRI 9 FL478, which are probably contributed by extremelysalt-tolerant genotypes such as IR4630 and FL478. The highestpositive values of SCA for K+/Na+ ratio were estimated forIR4630 9 SADRI, followed by SAL729 9 SADRI, probablybecause of the combination of highly salt-tolerant parents suchas IR4630 and SAL729 with a common parent SADRI, which isquite tolerant of salinity stress at the reproductive stage but verysensitive at the seedling stage. The best combinations had sus-ceptible parents crossed to either moderate or tolerant parents.Similar findings were also reported earlier in a 7 9 7 diallelstudy on phosphorus deficiency (Chaubey et al. 1994). The pres-ence of reciprocal effects among crosses necessitates the use oftolerant parents as females in hybridization programmes, and thematernal effect was substantiated by earlier reports (Gregorioand Senadhira 1993, Tsukahara et al. 1995). This implies thatFL478, CSR28 and SAL 729 could be involved as female par-ents in the development of improved varieties for seedling-stagesalinity tolerance.Estimates for GCA under salinity stress at the reproductive

stage revealed that, among the parents, SADRI was the best gen-eral combiner for GW and an average to good combiner formost yield component characteristics except percent SpFr.SADRI, FL478 and IR64 had high negative GCA values fornumber of DFL, and they can be the best general combiners forearly heading and maturity. Significant reciprocal effects showedthat the maternal effect made an important contribution to thesetraits, especially for SDW and RDW under saline conditions(Table 4). There seems to be a quite significant associationbetween GCA and parental tolerance (Gu et al. 1998); therefore,SADRI could be the best candidate variety to be used forimproving GW and yield components under salinity stress at thereproductive stage.Among the F1 hybrids, SADRI 9 CSR28 and

SADRI 9 FL478 had the highest GW with 55.68 and 54.29 g,respectively. Normally, SCA effects do not contribute tangiblyto the improvement of self-fertilizing crops, except when com-mercial exploitation of heterosis is feasible (Menon and Sharma

1997). SCA represents dominance and epistatic interaction,which can be related to heterosis. However, in self-pollinatedcrops such as rice, the additive 9 additive type of interactioncomponent is fixable in later generations. Effects of general andSCA are an important indicator of the potential of parental linesto generate superior breeding populations. This has been reportedin other crops such as sorghum as well (Igartua et al. 1994). Asmall combining ability effect indicates poor ability to transfergenetic superiority to hybrids.In the reproductive-stage study, the higher GCA/SCA ratio for

DFL and PH supported the involvement of additive effects forthese traits. The high estimate of narrow-sense heritability furthersupported the involvement of additive effects. High GCA effectsare mostly because of the effects of additive genes or the effectsof additive 9 additive interaction (Griffing 1956, Chaubey et al.1994). With this, breeders can use the good general combinersin specific breeding programmes for improving GW under salin-ity stress (Gravois 1992). Both additive and non-additive geneswere found to control the expression of PL and SpFr. A highGCA/SCA ratio means that additive gene action is predominant,but low GCA/SCA supports non-additive gene action.Broad-sense heritability values were high for salinity score

and SH, moderate for SDW and RDW and low for Na+, K+

uptake and K+/Na+ ratio for seedling-stage tolerance, and highfor DFL, PH, PL and straw dry weight at the reproductive stage.This indicated the involvement of both additive and non-additivevariances and the little influence of environmental effects onthese traits. If the heritability of the trait is low to moderate, thebreeding population must be large and selection for tolerancemust be performed in later generations under salt stress in con-trolled conditions (Gregorio and Senadhira 1993, Singh et al.2010).Narrow-sense heritability values were low for all the traits

except SH under normal conditions. The lower narrow-sense her-itability values indicate the importance of non-additive geneaction in controlling these traits. Gregorio and Senadhira (1993)also observed low to moderate heritability for traits in the seed-ling stage under salt stress in rice. Reports on diallel analysis inrice under salinity stress have indicated significant additive anddominant genetic effects in many studies (Akbar et al. 1986,Mishra et al. 1990, Narayanan and Sree Rangasamy 1990). Thehigh narrow-sense heritability values for DFL, PH, PL and TGWindicated the predominance of additive gene action for thesetraits, which can be the most reliable criteria for selection inbreeding programmes for salinity tolerance. Mahmood et al.(2002), in combining ability studies in rice under saline soil con-ditions, reported high additive effects for PH, PL and number ofproductive tillers/plant but more non-additive gene effects forpanicle fertility, days to maturity, SDW, paddy yield, Na+ andK+ concentrations of the shoot and Na+/K+ ratio, which is inconcordance with our study for most of the traits. Many otherdiallel studies in rice substantiated the results about the abun-dance of both additive and dominant genetic effects that contrib-uted significantly to the total heritable variation for traits such asdays to 50% flowering, productive tillers per plant, PL, panicleweight, filled grains per panicle, SpFr, TGW, Na+ and K+ con-centrations, Na+/K+ ratio, harvest index and GW per plant (Kal-aiyarasi et al. 2002, Mahmood et al. 2004, Deepa Sankar et al.2008). Additive genetic effects were important for number ofproductive tillers and number of primary branches per panicle,whereas dominant genetic effects were important for days tomaturity. The higher narrow-sense heritability indicates the pres-ence of additive gene action. In this case, selection in the early


generation may be fruitful. Falconer and Mackay (1996) statedthat the lower narrow-sense heritability was caused by low addi-tive effects and high dominant gene action. Heritability estimatescan be used to determine selection procedures in breeding pro-grammes.Direct selection under stress for grain yield has also been

reported as one of the best approaches to breed stress-tolerantrice varieties for salinity or drought stress (Mishra 1996, Munnset al. 2006, Venuprasad et al. 2007, Kumar et al. 2008), butselection for the component traits to breed a stress-tolerant ricevariety has also been proposed (Yeo et al. 1990) and long used(Singh et al. 2008, 2010, Serraj et al. 2009). However, morpho-logical traits such as the number of grains per panicle, SpFr, PH,number of fertile tillers and flowering time in comparison withnon-stress are good indicators associated with salt tolerance inrice (Singh and Flowers 2010). These observations demonstratedthe value of diallel analysis in choosing parents for the improve-ment of salinity tolerance in rice.One of the most important indications of this study is the role

of different genes for seedling- and reproductive-stage responses.Significantly low GCA values belonged to FL478 for the nega-tive SES score for seedling-stage salinity tolerance. A lower SESscore denotes salinity tolerance at the seedling stage, and FL478is a well-known seedling-stage salinity-tolerant check with ausual SES score of 1–3 until 10–12 dS m�1. The same FL478,which is highly tolerant at the seedling stage, has negative ornon-significant GCA values for yield-associated traits such aspanicles per plant, PL and FrSp that directly relate to salinity tol-erance at the reproductive stage. FL478 is a sensitive rice varietyfor reproductive-stage tolerance, and its non-significant or lowGCA for panicles per plant, PL and FrSp clearly substantiatesour field observations about its reproductive-stage sensitivity forsalinity stress. Contrary to this, SADRI, which has significantGCA values for GW and yield-associated traits, is quite tolerantof salt stress at the reproductive stage. These results clearlyreveal and confirm that salinity tolerance at the seedling andreproductive stages is not regulated by the same set of genes butis regulated independently by a different set of genes that couldbe pyramided using pertinent donors to enhance the level oftolerance. For this approach, Mohammadi et al. (2013) used anF2 population of SADRI/FL478 cross to identify reproductive-stage-specific QTLs for salinity tolerance, and they reported 35QTLs for yield components under salinity stress (Mohammadiet al. 2013). Selection for GW under stress conditions could bethe best approach to evaluate the tolerance of rice varieties.

Acknowledgements

The authors thank A.G. Sajise, N.A. Vispo and J.C. Amas for technicalassistance in hybridization and salinity screening, and the Head of thePlant Breeding, Genetics, and Biotechnology Division, IRRI for permit-ting us to conduct this research.

ReferencesAkbar, M., G. S. Khush, and D. Hilleris-Lambers, 1986: Genetics of SaltTolerance in Rice. Proceedings of the International Rice GeneticsSymposium, 399—409. IRRI, Los Ba~nos, Philippines.

Ali, S., R. K. Gautam, R. Mahajan, S. L. Krishnamurthy, S. K. Sharma,R. K. Singh, and A. M. Ismail, 2013: Stress indices and selectabletraits in SALTOL QTL introgressed rice genotypes for reproductivestage tolerance to sodicity and salinity stresses. Field Crops Res. 154,65—73.

Baker, R. J., 1978: Issues in diallel analysis. Crop Sci. 18, 533—536.

Chaubey, C. N., D. Senadhira, and G. B. Gregorio, 1994: Genetic analy-sis of tolerance for phosphorous deficiency in rice (Oryza sativa L).Theor. Appl. Genet. 89, 313—317.

Chauhan, M. S., 2007: Genetic studies on salt tolerance in rice (Oryzasativa L.), 212. Dr. B.R. Ambedkar University, Agra, UP, India.

Counce, P. A., T. C. Keisling, and A. J. Mitchell, 2000: A uniform,objective, and adaptive system for expressing rice development. CropSci. 40, 436—443.

Cui, H., Y. Takeoka, and T. Wada, 1995: Effect of sodium chloride onthe panicle and spikelet morphogenesis in rice. Jpn. J. Crop Sci. 64,593—600.

Deepa Sankar, P., N. Subbaraman, and S. L. Narayanan, 2008: Heterosis,combining ability and gene action studies in TGMS based rice hybridsunder normal and salt affected environments. Indian J. Agric. Res. 42,177—182.

Falconer, D. S., and T. F. C. Mackay, 1996: Introduction to QuantitativeGenetics, 4th edn. Longman, Harlow, Essex, UK.

Flowers, T. J., 2004: Improving crop salt tolerance. J. Exp. Bot. 55,307—319.

Flowers, T. J., and A. R. Yeo, 1995: Breeding for salinity resistance incrop plants-where next? Aust. J. Plant Physiol. 22, 875—884.

Garcia, A., C. A. Rizzo, J. UdDin, S. L. Bartos, D. Senadhira, T. J.Flowers, and A. R. Yeo, 1997: Sodium and potassium transport to thexylem are inherited independently in rice, and the mechanism ofsodium: potassium selectivity differs between rice and wheat. PlantCell Environ. 20, 1167—1174.

Gravois, K. A., 1992: Genetic effects determining rice grain weight andgrain density. Euphytica 64, 161—165.

Gregorio, G. B., and D. Senadhira, 1993: Genetic analysis of salinity tol-erance in rice (Oryza sativa L.). Theor. Appl. Genet. 8, 333—338.

Gregorio, G. B., D. Senadhira, and R. D. Mendoza, 1997: ScreeningRice for Salinity Tolerance. IRRI Discussion paper Series No.22. Inter-national Rice Research Institute, Los Ba~nos, Laguna, Philippines.

Gregorio, G. B., D. Senadhira, R. D. Mendoza, N. L. Manigbas, J. P.Roxas, and C. Q. Guerta, 2002: Progress in breeding for salinity toler-ance and associated abiotic stresses in rice. Field Crop Res. 76, 91—101.

Griffing, B., 1956: Concepts of general and specific combining ability inrelation to diallel crossing system. Aust. J. Biol. Sci. 9, 436—493.

Gu, X., X. Yan, S. Zheng, and Y. Lu, 1998: Diallel cross analysis of salttolerance in rice seedlings. J. Southern China Agric. Univ. 19, 31—35.

Hayman, B. I., 1954: The theory and analysis of diallel analysis. Genet-ics 39, 251—271.

Heenan, D. P., L. G. Lewin, and D. W. McCaffery, 1988: Salinity toler-ance in rice varieties at different growth stages. Aust. J. Exp. Agric.28, 343—349.

Igartua, E., M. P. Gracia, and J. M. Lasa, 1994: Characterization andgenetic control of germination-emergence responses of grain sorghumto salinity. Euphytica 76, 185—193.

Jennings, P. R., W. R. Coffman, and H. E. Kauffman, 1979: RiceImprovement. International Rice Research Institute, Los Ba~nos, Philip-pines.

Kalaiyarasi, R., G. A. Palanisamy, and P. Vaidyanathan, 2002: Thepotentials and scope of utilizing TGMS lines in inter-subspeciescrosses of rice (Oryza sativa L.). J. Genet. Breed. 56, 137—143.

Khatun, S., C. A. Rizzo, and T. J. Flowers, 1995: Genotypic variation inthe effect of salinity on fertility in rice. Plant Soil 173, 239—250.

Kumar, A., J. Bernier, S. Verulkar, H. R. Lafitte, and G. N. Atlin, 2008:Breeding for drought tolerance: direct selection for yield, response toselection and use of drought-tolerant donors in upland and lowland-adapted populations. Field Crop Res. 107, 221—231.

Maas, E. V., and S. R. Grattan, 1999: Crop yields as affected by salinity.In: R. W. Skaggs, and J. van Schilfgaarde (eds), Agricultural Drain-age, Agron Monogr 38, 55—108. ASA, CSSA, SSA, Madison, WI.

Maas, E. V., and G. J. Hoffmann, 1977: Crop salt tolerance-currentassessment. Proc. Am. Soc. Civil Eng., J. Irrig. Drainage Div. 103,115—134.

Mahmood, T., G. Shabbir, M. Sarfraz, M. Sadiq, M. K. Bhatti, M. S.Mehdi, M. Jemil, and J. Hassan, 2002: Combining ability study in rice


(Oryza sativa L.) under salinized soil condition. Asian J. Plant Sci. 1,88—90.

Mahmood, T., M. Turner, F. L. Stoddard, and M. A. Javed, 2004:Genetic analysis of quantitative traits in rice (Oryza sativa L.) exposedto salinity. Aust. J. Agric. Res. 55, 1173—1181.

Menon, U., and S. N. Sharma, 1997: Genetics of yield determining fac-tors in spring wheat over environments. Indian J. Genet. 57, 301—306.

Mishra, B., 1996: Highlights of Research on Crops and Varieties for SaltAffected Soils, 28. Central Soil Salinity Research Institute (CSSRI),Karnal, India.

Mishra, B., M. Akbar, and D. V. Seshu, 1990: Genetic studies on salin-ity tolerance in rice towards better productivity in salt-affected soils.Proceedings of the Paper Presented at the Rice Research Seminar, July12, 1990. IRRI, Los Ba~nos, Philippines.

Moeljopawiro, S., and H. Ikehashi, 1981: Inheritance of salt tolerance inrice. Euphytica 30, 291—300.

Mohammadi, R., M. S. Mendioro, G. Q. Diaz, G. B. Gregorio, and R.K. Singh, 2013: Mapping quantitative trait loci associated to yield andyield components under reproductive stage salinity stress in rice(Oryza sativa L.). J. Genet. 92, 433—443.

Moradi, F., A. M. Ismail, G. B. Gregorio, and J. A. Egdane, 2003: Salinitytolerance of rice during reproductive development and association withtolerance at the seedling stage. Indian J. Plant Physiol. 8, 105—116.

Munns, R., 2002: Comparative physiology of salt and water stress. PlantCell Environ. 25, 239—250.

Munns, R., and M. Tester, 2008: Mechanisms of salinity tolerance.Annu. Rev. Plant Biol. 59, 651—681.

Munns, R., R. A. James, and A. Lauchli, 2006: Approaches to increasingthe salt tolerance of wheat and other cereals. J. Exp. Bot. 57, 1025—1043.

Narayanan, K. K., and S. R. Sree Rangasamy, 1990: Genetic analysis forsalt tolerance in rice. In: IRRI (ed.), Rice Genetics II, 167—173. IRRI,Los Ba~nos, Laguna, Philippines.

Narciso, J., and M. Hossain, 2002: World Rice Statistics. IRRI, LosBa~nos, Philippines.

Neue, H. U., C. Quijano, D. Senadhira, and T. Setter, 1998: Strategiesfor dealing with micronutrient disorders and salinity in lowland ricesystems. Field Crops Res. 56, 139—155.

Rao, P. S., B. Mishra, S. R. Gupta, and A. Rathore, 2008: Reproductivestage tolerance to salinity and alkalinity stresses in rice genotypes.Plant Breed. 127, 256—261.

Santos, P. J. A., and E. T. Ocampo, 2005: SNAP hydroponics: develop-ment & potential for urban vegetable production. Philippine J. CropSci. 30, 3—11.

Serraj, R., A. Kumar, K. L. McNally, I. Slamet-Loedin, R. Bruskiewich,R. Mauleon, J. Cairns, and R. J. Hijmans, 2009: Improvement of

drought resistance in rice. In: D. Sparks (ed.), Advances in Agronomy,Vol. 103, 41—99. Academic Press, Burlington, MA.

Singh, R. K., and T. J. Flowers, 2010: The physiology and molecularbiology of the effects of salinity on rice. In: M. Pessarakli (ed.), Hand-book of Plant and Crop Stress, 3rd edn, 901—942. Taylor and Francis,Boca Raton, Florida.

Singh, R. K., G. B. Gregorio, and A. M. Ismail, 2008: Breeding ricevarieties with tolerance to salt stress. J. Indian Soc. Coastal Agric.Res. 26, 16—21.

Singh, R. K., E. D. Redo~na, and L. Refuerzo, 2010: Varietal improve-ment for abiotic stress tolerance in crop plants: special reference tosalinity in rice. In: A. Pareek, S. K. Sopory, H. J. Bohnert, andGovindjee (eds), Abiotic Stress Adaptation in Plants: Physiological,Molecular and Genomic Foundation, 387—415. Springer, Dordrecht,the Netherlands.

Tsukahara, M., T. Hirosawa, E. Nagai, H. Kato, R. Ikeda, and K. Maruy-ama, 1995: Genetic analysis of plant regeneration ability in cell sus-pension cultures of rice (Oryza sativa L). Breed. Sci. 45, 425—428.

United States Salinity Laboratory Staff, 1954: Diagnosis and improve-ment of saline and alkali soils. US Department of Agriculture, Agricul-tural Handbook No. 60. US Government Printer, Washington, DC.

Venuprasad, R., H. R. Lafitte, and G. N. Atlin, 2007: Response to directselection for grain yield under drought stress. Crop Sci. 47, 285—293.

Yeo, A. R., and T. J. Flowers, 1986: Salinity resistance in rice (Oryzasativa L.) and a pyramiding approach to breeding varieties for salinesoils. Aust. J. Plant Physiol. 13, 161—173.

Yeo, A. R., M. E. Yeo, S. A. Flowers, and T. J. Flowers, 1990: Screen-ing of rice (Oryza sativa L.) genotypes for physiological characterscontributing to salinity resistance, and their relationship to overall per-formance. Theor. Appl. Genet. 79, 377—384.

Zeng, L., and M. C. Shannon, 2000: Salinity effects on the seedlinggrowth and yield components of rice. Crop Sci. 40, 996—1003.

Zeng, L., M. C. Shannon, and S. M. Lesch, 2001: Timing of salinitystress affects rice growth and yield components. Agric. Water Manage.48, 191—206.

Zeng, L., M. C. Shannon, and C. M. Grieve, 2002: Evaluation of salt tol-erance in rice genotypes by multiple agronomic parameters. Euphytica127, 235—245.

Supporting InformationAdditional Supporting Information may be found in the online version ofthis article:Figure S1. Performance of parental lines of rice (Oryza sativa L.).Figure S2. The reproductive stage experiment was conducted at IRRI.


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Genetic analysis of salt tolerance at seedling and ... · Genetic analysis of salt tolerance at seedling and reproductive stages in rice (Oryza sativa) REZAMOHAMMADI 1,3,MERLYNS