Physiological response , molecular analysis and water use efficiency of maize ( Zea mays L . ) hybrids grown under various irrigation regimes

Aksum University, Shire Campus, P. O. Box. 314, Shire, Ethiopia. Department of Agronomy, University of Agricultural Sciences, Dharwad-580 005, India. Department of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad-580 005, India. Department of Crop Physiology, University of Agricultural Sciences, Dharwad-580 005, India. Department of Soil Science, University of Agricultural Sciences, Dharwad-580 005, India.


INTRODUCTION
Maize (Zea mays L.) a miracle crop, is grown over a wide range of climatic conditions in semi arid and sub-tropics of Indian continent.Besides, it is a water demanding crop; higher grain yields can be achieved when water and nutrients are not limiting.Occurrence of drought is unpredictable as it can occur at any stage of the crop.However, maize is very sensitive to water and other environmental stresses in the period one week before flowering to two weeks after flowering (Grant et al., 1989;Pandey et al., 2000;Cakir, 2004).Drought during this period result in easily measured increase in the anthesis-silking interval (ASI) as the silk emergence is delayed (Zaidi et al., 2007).Further, the water stress occurring at different crop developmental stages could potentially limit biomass accumulation and consequently reduce grain yield of the maize crop.
Throughout the tropics, periodic drought caused by uncertain and ill distributed rainfall and soils with low water holding capacities cause sizeable reduction in maize yield.In India, majority of maize is grown under irrigated conditions and most farmers in south India cultivate maize under rainfed condition also.Significant yield losses in maize from drought are expected to increase with global climate change as temperature rise and rainfall distribution changes in key traditional areas.There is a need to identify suitable management techniques in maize which can withstand water stress situations.Most of the maize grown in the irrigated areas of the Navalgund and Nargund taluks of Dharwad district, Karnataka, India suffers from such water shortages at key developmental stages.
The hypothesis of the study was that under water limited conditions, an early maturing maize hybrid would be a better alternative crop in the area of study.In this context, a field experiment was performed to compare response of maize hybrids of different maturity to varying irrigation schedules in the same location and under the same crop management.Crop development, soil water extraction pattern, biomass and grain yield; and molecular diversity were characterized for maize hybrids.The objectives of this study were: i) to compare agronomic and physiological responses of maize hybrids of different maturity groups to irrigation scheduling; and characterize their molecular diversity and (ii) to quantify the relative yield contribution of maize hybrids and the variations in their water use efficiency (WUE).

Site description
The field experiment was conducted at Water Management Research Center (WMRC), Belvatagi, University of Agricultural Sciences, Dharwad in Malaprabha Command Area, Karnataka, India during winter season 2010.The experimental site is located in the northern agroclimatic zone (zone-3) of Karnataka at latitude of 15°16' N, and longitude of 75°23' E with an altitude of 579 m above sea level.The soil of the experimental site was analyzed for its physico-chemical properties (Table 1).
The meteorological data gathered during the experimental period are presented in Figure 1.The experimental crop received a very less amount of rainfall (101 mm) during the growing period, only in the month of November.Mean maximum temperature ranged from 31.70 (November) to 37.5°C (March) while the mean minimum temperature ranged from 11.1 (January) to 21.87°C (November).There was an uneven seasonal rainfall distribution coupled with 20.81 mm mean growing season evaporation.The percent relative humidity also declined from November (63.18%) to March (48.96%).Thus, due to frequent drying of top soil (six inches), irrigations were provided based on irrigation water/cumulative pan evaporation (IW/CPE) ratio.The higher temperatures during March resulted in higher evaporation of 6.26 mm which exceeded previous three years average by 0.76 mm (data not shown).

Experimental design and treatments
The experiment was laid out in a split plot design with three replicates using a net plot size of 3.0 x 5.6 m for biometric observations.The maize plants were accommodated in 0.6 m inter-row spacing with 0.2 m intra-row spacing between the plants.Irrigation schedules and maize hybrids were randomized in main and subplots, respectively.The treatment combinations comprised four irrigation schedules [I 1 = 0.4 IW/CPE, I 2 = 0.6 IW/CPE, I 3 = 0.8 IW/CPE, and I 4 = irrigations at critical growth stages of maize that is (i) at knee-high stage (V 5 or 35 DAE), (ii) anthesis stage (VT or 65 DAE) and (iii) grain development (R 4 or 90 DAE)]; and three maize hybrids [H 1 =PEEHM5 (extra early), H 2 = PEHM2 (early) and H 3 = 900 M Gold (full season)].

Characteristics of maize hybrids used in the study
PEEHM5 and PEHM2 (extra-early and extra maturing hybrids) were released from IARI, India and are recommended for cultivation in Karnataka state.900 M gold is a full season single cross hybrid of Monsonto Ltd.

Crop husbandry
Maize hybrids were sown on 2 nd November 2010 by marking and opening of shallow furrows at 0.6 m apart and seeds were dibbled uniformly at 0.2 m interval in furrows using a seed rate of 25 kg ha - 1 .Nitrogen, phosphorus and potash were applied at 150, 75 and 37.5 kg ha -1 , respectively to all the plots.Entire doses of P 2 O 5 and K 2 O were applied at planting, while N was applied in three splits that is ⅓ each at the time of sowing, at vegetative stage and before flower initiation stage.The experimental plot was maintained weed free throughout the growth period using pre-emergence *Corresponding author.E-mail: bnakumar@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviations: WUE, Water-use efficiency; RWC, relative water content; SPAD, soil plant analysis development system; IW/CPE, irrigation water/cumulative pan evaporation ratio; DAE, days after emergence; RAPD, random amplified polymorphic DNA.application of pendimethalin 30 EC at 1.0 kg a.i ha -1 followed by manual weeding.Irrigation was applied manually to a depth of 60 mm.The scheduling of irrigation was done based on progressive total of evaporation, after attaining the pre-determined values of cumuli-tive pan evaporation (CPE) (Prihar et al., 1974).Thus, CPE values for different IW:CPE ratios viz., 0.4,0.6 and 0.8 at a constant depth of 60 mm irrigation water (IW) were calculated to be 150, 100 and 75 mm, respectively.The total water use, depth of irrigation water and the number of irrigations provided are presented in Table 2.

Gas exchange measurements and soil plant analysis development system (SPAD) chlorophyll meter values
Leaf gas exchange parameters photosynthesis (P n ), stomatal conductance (g s ), and internal CO 2 concentration (C i ) were measured in the top fully expanded leaf at anthesis stage using a portable infra-red gas analysis system (LI-6400 LICOR, Nebraska, Lincoln, USA) under uniform light conditions.The readings were taken after at an ambient CO 2 concentration of 380 ppm.Three measurements per leaf were taken for each genotype x irrigation combination in each replicate; a total of 48 readings were taken at each time.Gas exchange measurement was taken on a day with sufficient sunlight and no artificial light source was used for illumination.
Chlorophyll content was determined non-destructively using a SPAD-502 meter (Minolta, Japan), on third fully expanded leaf from the top at 60 DAE (V 12 ) and 90 DAE (R 4 ) by clamping the SPAD sensor over the leaf lamina.In each plant, five readings were recorded from single leaf.

Soil moisture measurements
Soil moisture was measured gravimetrically before and after irrigation at grand growth, anthesis and at physiological maturity in soil layers: 0 to 15, 15 to 30 and 30 to 45 cm.Soil samples were taken from each plot at about 15 cm away from the crop line.The soil moisture measurements were used to calculate consumptive use and moisture extraction pattern.

Molecular analysis and genomic DNA extraction
Genomic DNA was extracted by cetyltrimethyl ammonium bromide (CTAB) extraction procedure (Doyle and Doyle, 1987).Fresh leaf samples of 1 g were ground to powder in liquid nitrogen and transferred to a 1.5 ml centrifuge tube to which 1 ml of pre-heated (60°C) extraction buffer was added.The extraction buffer consisted of 2% CTAB (w/v), NaCl (4 M), Tris HCl (pH 8.0 1 M) and PVP (0.1%), mercapto ethanol 1% (v/v), RNAse A (2 mg/ml), chloroform: iso-amyl alcohol (24:1) (v/v), ethanol (70%) and TE buffer (Tris HCl, 10 mM (pH 8.0), and 1 mM EDTA (pH 8.0) were the additional solutions required.The samples containing tubes were incubated at 65°C in circulating water bath for 15-20 min.An equal volume of chloroform: iso-amyl alcohol (24:1) was added and mixed for about 5 min.Samples were centrifuged at 12000 rpm for 15 min and the supernatant was decanted and transferred to a fresh tube.
The PCR programme included an initial denaturation step at 95°C for 5 min followed by 39 cycles with 94°C for 1 min for DNA denaturation, annealing at 31.6°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 8 min were carried out.The amplified DNA fragments were electrophoretically separated on 1.4% agarose gel in 1X Tris-acetate-EDTA (TAE) buffer (for each liters of stock contains 4.84 g of Tris base, 1.14 ml of glacial acetic acid and 2 ml of 0.5 M EDTA) and stained with ethidium bromide (10 mg/ml).Thirty five 10-mer primers randomly selected were used in RAPD analysis.A 250 bp DNA ladder (Bangalore Genie) was used as a marker with molecular size of 5000,4500,4000,3500,3250,3000,2750,2500,2250,2000,1750,1500,1250,1000,750, 500 and 250 bp.20 µl of sample was loaded onto each well and amplified DNA was separated with 70 V constant current for 3 h.The amplified pattern was visualized on a UV trans illuminator and photographed.

Data collection and analysis
Observations on number of days to 50% anthesis, days to 50% silking, anthesis -silking interval, cob length, number of grains per row, above ground biomass, 1000-seed weight, grain yield, harvest index and soil moisture extraction pattern were recorded.Above ground biomass was determined using five plants per plot and the samples were oven dried to a constant weight at 80°C.Antheis silking interval (ASI) was computed as the difference between silking and anthesis dates (Kuchanur et al., 2013).Relative water content (RWC) was measured to determine the plant water status of leaf discs sampled from the third leaf from the top adopting the procedure given by Barrs and Weatherly (1962) as: RWC (%) = (Fresh weight-oven dry weight) / (Turgid weight-oven dry weight) x 100 The water-use efficiency (kg ha -1 mm -1 ) was estimated in terms of grain yield as the ratio between grain yield (kg ha -1 ) and total consumptive use of water (mm).
The data collected were analysed using analysis of variance (ANOVA) and Fisher's LSD test to determine the significant differences at P<0.05 levels between treatment means.All statistical analyses, except for the molecular analyses were performed with MSTATc (Russel, 1986).

RAPD data analysis and scoring
For RAPD data analysis, the bands with same molecular weight and mobility were treated as identical fragments.RAPD products were scored for presence or absence of each amplicon evaluated.Only those bands that could be unequally scored across all the samples were included in the analysis.Pair wise similarity matrices were generated using Jaccard's coefficient of similarity.Data matrices were prepared in which the presence of a band was coded as 1, whereas the absence as 0. The data matrices were analyzed by the SIMQUAL program of NTSYSpc© (version 2.02j) (Rolf, 1998).
Dendrogram of the similarity coefficients was performed using unweighted pair group method of arithmetic means (UPGMA) through the programme, Popgene Version 1.31 (Microsoft windows based Freeware for population genetic analysis).

Physiological responses of maize hybrids to irrigation scheduling
The comparisons of means of irrigation levels, maize hybrids and their interactions are shown in Tables 3 and  4. The above ground biomass (AGB) at harvest was significantly highest in I 3 (259.56g plant -1 ) compared to I 1 (164.96g plant -1 ).Averaging across irrigation levels, H 3 produced significantly higher AGB (247.64;P<0.05) over other hybrids.Among interaction effects, H 3 produced more AGB in I 3 compared to other treatments (Table 3).Effect of different irrigation regimes and maize hybrids as well as their combined effect on days to reach 50% anthesis was significant (Table 3).Water stressed regimes I 1 and I 2 resulted in more number of days to reach 50% anthesis.Among hybrids, H 3 took 66.75 days to reach 50% anthesis (P<0.05) over other hybrids.The combined effect also showed a similar trend.A same trend was found for number of days to reach 50% silking.Averaging across all hybrids, the interval between anthesis and silking (ASI) was significantly influenced by irrigation schedules.I 1 extended the ASI (8.4 days) while the least was recorded in I 3 (4.11days; P<0.05).The effects of hybrids and their combined effects was not significant (Table 3).The cob length, number of grains, 100 seed weight and HI were significantly more in I 1 (14.97 cm, 22.40 g, 28.2 g and 46.22%, respectively) (P<0.05)compared to other irrigation schedules.A similar trend was noticed for these yield components in H 3 (14.44cm, 34.14 g, 27.5 g and 51.71%, respectively) (P<0.05;Table 3).

Grain yield
Averaging across all the maize hybrids, the yield of I 3 was significantly higher by about 3019 kg ha -1 than that of I 1 (Table 3).The yield differences were significant (P<0.05) at all the irrigation levels.The hybrid 900 M Gold (H 3 ) produced higher grain yields in the range 2143 to 2733 kg ha -1 than other hybrids.Among interaction effects, H 3 produced higher grain yields in all the irrigation levels and yield increase ranged from 1077 to 2774 kg ha -1 , while for H 1 , it was in the range of 810 to 3423 kg ha -1 ; and for H 2 in the range of 446 to 2860 kg ha -1 .

Gas exchange measurements
Irrigation levels did not show significant differences for P n , g s , and C i (P>0.05).Among hybrids, H 1 had the highest C i (P<0.05) compared to other hybrids.There were significant interaction effects between irrigation levels and maize hybrids for P n being significantly highest for H 1 at I 4 (P<0.05)(Table 4).

SPAD chlorophyll meter readings
Averaging across hybrids, I 3 recorded maximum SPAD value both at anthesis and grain filling stages (P<0.05).Among hybrids, H 3 recorded the maximum values (P>0.05).There was a trend in response of hybrids to irrigation levels with respect to SPAD values being higher in H 3 at all the irrigation levels (P>0.05)(Table 4).

Relative water content (RWC)
Significantly higher RWC was recorded at anthesis stage in I 3 which was 8.8% higher than I 1 (P<0.05).

Water use efficiency (WUE)
I 4 had significantly the highest value of 23.80 kg ha -1 mm (P<0.05).Providing water at higher frequency, I 3 resulted in decrease in WUE by 4.5 kg ha -1 mm.Over all, the WUE for maize hybrids was in the range 17.25-26.16kg ha -1 mm.There were significant interactions between irrigation levels and maize hybrids being highest with H 3 at I 1 (30.12 kg ha -1 mm) but was at par with H 3 at I 4 (29.75 kg ha -1 mm) (Table 3).

Soil moisture extraction pattern
At sowing, the soil profile was close to field capacity in all the plots.The depletion of soil moisture was higher in the top soil layer due to delayed irrigation (I 1 ) both at vegetative stage and anthesis stage.On the contrary, lowest depletion was found at all the soil depths in I 3 .The interaction effects were not significant (P>0.05)(Data not shown).

RAPD analysis
Random amplified polymorphic DNA analysis of three maize hybrids on 34 primers produced a total of 351 amplified fragments, 202 of which were polymorphic, and the percentage of polymorphism was 57.55.These amplified fragments ranged in size from 250 to 5000 bp (Figure 2).On average, 10.32 bands were amplified per primer and 5.94 were polymorphic (Table 6).Jaccard's coefficient of similarity ranged from 0.70 to 0.74.A highest genetic diversity was observed between H 1 and H 3 (0.73).The dendrogram revealed two distinct clusters; H 1 and H 2 clustered distinctly away from H 3 .

Effect of irrigation scheduling on physiological responses of maize hybrids
Results of this study show higher grain yield with 0.8 IW/CPE (I 3 ) on account of higher cob length, cob girth, number of grains per row, and 1000-seed weight.The significant increases in these yield components were due to beneficial effect of sufficient moisture available in the soil.This result is in conformity with the findings of Farshad et al. (2008) who showed that missing single irrigation at any of the growth stages in maize significantly decreases grain yield.Scheduling irrigation at critical stages of growth also significantly improved grain yield than providing more irrigation in I 3 .The moisture stress encountered in I 1 resulted in more number of days to reach 50% anthesis, days to reach 50% silking and thus widening the interval between anthesis and silking.Continuous stress due to low frequency of irrigation in I 1 also prolonged the days to reach 50% silking by about eight days.In  (2013) reported that in maize, moisture stress increased significantly the days required for 50% anthesis, 50% silking and ASI.In this study, the association of ASI with grain yield was significantly negative (r= -0.73; P<0.05) (Table 5).Monneveux et al. (2006) reported in two drought tolerant populations viz.DTP1 and DTP2 that significant yield gains in the populations were associated with a significant increase in number of cobs per plant and grains per ear and significant reductions in ASI.Further, it is evident in the literature that the shortening of ASI is associated with high grain yield under drought (Edmeades et al., 2000;Moser et al., 2006).The increase in grain yield of H 3 was about 32.30% over H 1 and 26.23% over H 2 .This might be due to genetic and morphological characteristics of maize hybrids exploiting climatic maxima at important growth stages.In the present study, all the yield traits have contributed for yield increment with significant positive correlations for 1000seed weight (r= 0.81); cob length (r= 0.83); harvest index (r=0.82);SPAD values (r=0.79) and WUE (r=0.61)(P<0.05)(Table 5).H 3 exhibited a higher HI which might be due to a genetically strong source-sink relation resulting in higher yields.A higher total biomass production in well watered situations has been reported by Moser et al. (2006).Among interaction effects, H 3 performed equally well in all the irrigation levels.The grain yield ranged from 4582 to 7442 kg ha -1 in H 2 and from 3527 to 6950 kg ha -1 .In this study, lesser number of irrigations resulted in reduction in grain number and 100 grain weight.This is in agreement with the findings of Moser et al. (2006) which showed a decrease in kernel number and 1000-kernel weight resulted in lower grain yields due to water shortage in maize.
The RWC decreased significantly (P<0.05) with increasing moisture stress.I 3 had higher RWC by about 82.53% compared to I 1 (75.24%).This may be attributed to better availability of soil moisture in the crop root zone.The earlier findings in maize revealed that, water potential and RWC (Chen et al., 1990) and relative water content (Schlemmer et al., 2005;Kuchanur et al., 2013)   declined under low water conditions.The chlorophyll concentration is a measure of functional stay green (Barker et al., 2005).The chlorophyll content as measured by SPAD values decreased under water stress but it was more drastic under I 1 .A higher photosynthetic rate was found in I 4 being highest with H 1 (16.04 µ mole m -2 s -1 ).The other parameters of stomatal aperture traits were not significant.Moderate stress did not significantly change the relative water content (RWC).Severe stress at silking stage did significantly decrease the leaf RWC and increase leaf  relative conductivity (Li-Ping et al., 2006).A highest WUE was observed at I 4 (23.80 kg ha -1 mm) than I 1 (21.25 kg ha -1 mm).The increased water application resulted in increase in crop water use without a corresponding increase of yield which was reported by Kar and Verma (2005).Providing irrigation at critical stages that is I 4 resulted in better grain yield and WUE over I 2 on account of optimum number of irrigations.This is in agreement with the findings of Jiotode et al. (2002) which revealed better WUE with irrigation at critical growth stages of maize.While Maqsood et al. (2012) reported that providing six irrigations at different growth stages of maize along with higher N rates up to 200 kg ha -1 has increased maize grain yield.WUE values for rainfed maize have been reported in the literature in the ranges 11.4 to 14.4 kg ha -1 mm -1 (Meena et al., 2009); 9.3 to 13.8 kg ha -1 mm -1 (El-Tantawy et al., 2007); and 11.0 to 18.0 kg ha -1 mm -1 (Tijani et al., 2008).
Despite providing a highest amount of water (420 mm) in I 3 , the WUE was significantly low (P<0.05)which could be attributed to a greater use of water with relatively lesser increase in yield.Trooijen et al. (1999) found greater WUE of maize with limited irrigation, but full irrigation of maize was more profitable than limited irrigation.During vegetative growth stages, soil moisture depletion from different soil layers varied with irrigation levels.Moisture extraction was more from the top layer (0-15 cm) irrespective of irrigation levels.However, it was more at I 1 coupled with moisture loss through evaporation from soil.Similar trends were found during anthesis stage except in I 3 wherein depletion of moisture from 15 to 30 cm depth was seen.Maximum moisture extraction from deeper layers may be due to stress as a result of less number of irrigation which encourages more rootgrowth into deeper profiles.It is also reported in other study that drought sensitive inbred maize lines despite having deeper rooting markedly reduced WUE on account of inefficient photosynthesis (Hund et al., 2008).
Based on the water extraction patterns, the lack of significant differences in water extraction at depth among maize hybrids and their interaction with water levels was mainly due to a well developed root system by maize.The relationship between yield and irrigation is affected by factors such as climate, soil properties and irrigation practices (Tolk and Howell, 2003) and determining the level of irrigation needed to optimize profits can be complex and depends on both biophysical and economic factors (English et al., 2002;Payero et al., 2008).

Conclusion
The study focuses to bring interrelations between different maturity group hybrids and their WUE through several physiological, molecular and agronomic analyses.Our findings show that providing four irrigations at critical growth stages followed by either three or five irrigations seem to have higher WUE.This approach could save water up to 29% with slight reduction of grain yield by 12% over providing full irrigation.Further, WUE was increased to a maximum of 23.80 kg ha -1 mm with decreased frequency of irrigation at critical growth stages.The RAPD analysis revealed that extra-early and early maturity hybrids clustered significantly differently from full season hybrid.This diversity may be of use in crop improvement to introgress certain traits from full season hybrid into early maturity groups for enhanced productivity while emphasizing on the reduction in number of days to reach physiological maturity.To integrate differential responses during phenological stages, it is suggested that field research be combined with thoroughly calibrated and validated crop-water productivity models to further improve strategies obtained from field experiments.In areas where water supply is going to be a constraint in future, farmers must choose varieties and irrigation strategies to ensure sustainable production.Under these situations per day productivity of crop both based on crop duration and on water use need to be given due consideration with a view to save water resources as well as to have temporal benefit so that farmers will have an option to choose additional crop for inclusion in the double cropping system.This perhaps is the only alternative that net returns can be maximized, especially for the farmers in tail-end regions of canal areas.

Figure 1 .
Figure 1.Daily values of rainfall (mm), evaporation (mm), maximum and minimum temperatures (°C) for the period between November, 2010 to March, 2011 (cropping season) at the experimental site.

Table 2 .
Total water used (TWU), depth of irrigation water (DIW) and WUE under different irrigation levels.

Table 3 .
Yield and yield components of maize (Zea mays L) under different irrigation levels.

Table 4 .
Relative water content (RWC), SPAD values and stomatal aperture traits of maize (Zea mays L) under different irrigation levels.

Table 5 .
Associations of growth and yield traits of maize (Zea mays L) with grain yield under different irrigation levels.

Table 6 .
Total number of amplicons, number of polymorphic bands and per cent polymorphism of maize hybrids.