Journal of Applied Botany and Food Quality 88, 127 - 133 (2015), DOI:10.5073/JABFQ.2015.088.018 1Department of Plant Breeding & Genetics, University College of Agriculture, University of Sargodha, Pakistan 2 Department of Experimental Design and Bioinformatics, Warsaw University of Life Sciences, Warsaw, Poland 3 Department of Agronomy, Warsaw University of Life Sciences, Warsaw, Poland 4 International Potato Center (CIP), Lima, Peru Multitraits evaluation of Pakistani ecotypes of berseem clover (Trifolium alexandrinum L.) under full-irrigation and water restriction conditions Muhammad Mubashar Hussain1, Saeed Rauf1, Jakub Paderewski2, Ikram ulHaq1, Dorota Sienkiewicz-Paderewska3, Philippe Monneveux4 (Received January 20, 2015) Summary Berseem clover (Trifolium alexandrinum L.) is an important forage crop in Pakistan and many ecotypes are grown across the country. Its yield is however frequently affected by insufficient irrigation due to unavailability of water. In the present study, twenty Pakistani ecotypes of berseem clover have been evaluated in lysimeters under full irrigation and water restriction conditions. In the full irrigation treatment soil humidity was maintained at field capacity, while in the water restriction treatment water was only supplied after severe wilting and to maintain humidity in the deep profile of the soil. Assessed traits included forage yield, calculated as the sum of the biomass harvested at 70 and 110 DA days after emergence, and morpho-physiological traits. Significant effects of water restriction were noted on yield, leaf gas exchange parameters, canopy tempera- ture and osmotic adjustment. Most morpho-physiological traits had higher broad sense heritability than forage yield, both under full ir- rigation and water restriction conditions. Water restriction increased genetic and phenotypic variability and heritability of most traits under study. Under these conditions forage yield was positively as- sociated to leaf temperature and recovery rate index and, under full irrigation, to net photosynthetic rate, canopy depression temperature and leaf area. The possible use of these traits as indirect selection criteria in berseem clover breeding programs is discussed. Some ecotypes with favorable traits such as high forage yield potential, good adaptation to water restriction and aptitude to multiple harvest- ing have also been identified. Introduction Trifolium alexandrinum L. (berseem clover or Egyptian clover) is one of the most important leguminous forages in the Mediterrane- an region and the Middle-East (Sardana and narwal, 2000; El- BaBly, 2002; IannnuccI, 2002; dE SantIS et al., 2004). It con- tributes to soil fertility and improves soil physical characteristics (GravES et al., 1996) and its forage is superior to grasses in pro- tein and mineral contents (laGharI et al., 2000). Berseem clover originated in Syria and was introduced into Egypt in the 6th century (hannaway and larSon, 2004), India in the 19th century and Paki- stan, South Africa, the USA and Australia in the 20th century (hEuzé et al., 2014). It is grown in the semi-arid regions of the world un- der pure stand and crop mixture (MartInEllo and IannuccI, 1998; vaSIlakoGlou and dhIMa, 2008). Limitation of water supply is a major production constraint for this crop (IannuccI et al., 2000; lazarIdou and koutrouBaS, 2004). Studies concerning the evaluation of tolerance to water restriction are scarce in berseem clover. IannuccI et al. (2000) reported a sig- nificant reduction in total dry weight, plant height and proline con- tent due to water stress treatments. lazarIdou and tSIrIdIS (2004) noted a 75 % decrease in biomass, leaf area and transpiration rate as a result of water restriction. Evaluation and selection of berseem clover germplasm in a specific environment have generally con- cerned small sets of ecotypes and have been based on a single or a reduced number of traits (IannuccI et al., 2000; lazarIdou and koutrouBaS, 2004). The present experiment compared the yield of a large set of berseem clover ecotypes originated from different regions of Pakistan under full irrigation and water restriction conditions. As in the semi-arid environment and under limited irrigation the berseem plant has of- ten to rely on moisture stored in a deep profile of soil to maintain its growth, the water restriction treatment consisted in maintaining humidity only in the deep profile of the soil. The ability of the dif- ferent ecotypes to utilize the stored soil moisture content and main- tain its yield under these specific water restriction conditions was evaluated using a multiplicity of traits including canopy temperature (and its response to variation in air temperature), gas exchange para- meters, osmotic potential, chlorophyll and carotenoid contents, wa- ter use efficiency, recovery rate index and wilting rate index, as well as calculating a drought resistance index on the basis of comparisons between the two treatments. The genetic variability and heritability of the assessed traits and their relation with yield were also exa- mined, in order to identify potential criteria for the further selection of ecotypes under stored moisture stress. Materials and methods Plant material Twenty Pakistani berseem clover ecotypes were provided by the National Agriculture Research Council and the Forage Research Institute of Faisalabad, Pakistan. Out of them, 17 originated from Punjab, one from Balochistan, one from Khyber Pakhtunkhwa and one from Singh Provinces (Tab. 1). Experimental conditions The experiments were conducted in lysimeters of 60 cm diameter and 40 cm depth. Field conditions are known to exploit full yield potential of the ecotypes but make difficult to ensure uniform mois- ture storage and avoid confounding factors such as soil heteroge- neity or presence of multiple stress factors (arauS and caIrnS, 2014). Conversely, the use of lysimeters facilitates better control in the application of uniform treatments (MaSuka et al., 2012). Each lysimeter was filled with 50 kg of dry soil with an equal quantity of silt and loam. Soil fertility was increased by adding 5 % of or- ganic matter. The field capacity of the soil, measured using the gravi- metric method, was 14 % by weight. The seeds were inoculated be- fore sowing with 5 μL of rhizobium bacterial suspension (Rizobium leguminosarum bv. trifolii). The experiment was conducted in a com- pletely randomized design with three replications and two factors, i.e. ecotypes (20) and water regimes (2). The two contrasting water regimes consisted in a full irrigation and a water restriction regime. In the full irrigation regime, soil was flooded when the moisture level fell below the field capacity of the soil, while the water restriction regime was created by irrigating the crop when severe wilting (90 %) 128 M. Huassain, S. Rauf, J. Paderewski, I. ulHaq, D. Sienkiewicz-Paderewska, P. Monneveux Tab. 1: List of ecotypes of berseem clover (Trifolium alexandrium L.) used in the study Number Ecotype Origin Source 1. L-94 Punjab, Pakistan Forage Research Institute, Faisalabad, Pakistan 2. Anmol Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan 3. P-209 Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan 4. BerseemQueta Balochistan, Pakistan National Agriculture Research Council, Pakistan 5. Sandal Bad Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan 6. Agaiti Punjab, Pakistan Forage Research Institute Sargodha, Pakistan 7. SB-12 Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan 8. Berseem Peshawar KPK, Pakistan National Agriculture Research Council, Pakistan 9. Berseem Tandojam Sindh, Pakistan National Agriculture Research Council, Pakistan 10. Pachati Punjab, Pakistan Forage Research Institute Sargodha, Pakistan 11. Pak Berseem Punjab, Pakistan National Agriculture Research Council, Pakistan 12. Punjab Berseem Punjab, Pakistan National Agriculture Research Council, Pakistan 13. Chenab Punjab, Pakistan National Agriculture Research Council, Pakistan 14. Samarkand Punjab, Pakistan National Agriculture Research Council, Pakistan 15. L-48 Punjab, Pakistan National Agriculture Research Council, Pakistan 16. SK-1 Punjab, Pakistan National Agriculture Research Council, Pakistan 17. Super Berseem Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan 18. P-22 Punjab, Pakistan National Agriculture Research Council, Pakistan 19. SB-10 Punjab, Pakistan National Agriculture Research Council, Pakistan 20. Sandal bar Punjab, Pakistan Forage Research Institute Faisalabad, Pakistan was observed for each ecotype, through a 3 cm diameter pipe allow- ing storing the moisture in the lower profile of the soil (30-40 cm). The quantities of water supplied to each lysimeter were 16.5 L in the full-irrigation regime (11 irrigations) and 4.5 L in the water restric- tion regime (3 irrigations). The lysimeters were covered with a plas- tic tunnel to avoid chilling stress or damage due to frost during the night and maintain the optimum temperature for growth (25±3 °C) during the day. Relative humidity was around 40 % and photon flux density was 600 μmol m-2 s-1 during the peak photosynthesis hour. Air temperature and humidity in the tunnel were registered at regular intervals using a digital thermometer and a hygrometer. Thinning was carried out to keep the plant population close to 100 plants per lysimeter. During the entire crop growth cycle, weeds were removed manually and no herbicide or pesticide was applied. The different ecotypes were regularly evaluated for insect and dis- ease, and no attacks were identified during the entire crop cycle. Soil and plant trait measurements A soil sampler with a coring cylinder of 3.8 × 10 cm was used to col- lect soil samples at various depths (10-40 cm) in each lysimeter. The samples were quickly transported to the laboratory without exposure to air. The soil sample was oven-dried at 60 °C (ED-115, Binder, Tuttlingen, Germany) to determine soil moisture content at constant weight. All ecotypes were harvested at 70 and 110 DAE. For each harvest, fresh biomass (FB) was measured on a digital balance (GW 6202, Sartorius, Germany). Plants were then dried in a heating oven (ED- 115, Binder, Tuttlingen, Germany) at 70 °C for 72 hours to deter- mine dry biomass (DB). Forage yield was estimated as the sum of biomass of the two harvests (krEnzEr et al., 1992). Leaf area (LA) was measured on well expanded leaves at 70 days after emergence (DAE), using a leaf area meter (CI-202, Camas, USA). Canopy temperature was assessed at 60 DAE using a hand-held infrared thermometer (model IR-AHT, Chino Co., Tokyo, Japan) at a uniform height (1.5 m), angle (60°) and distance between the thermometer and the target. Measurements were made in windless conditions during the afternoon. Each measurement was repeated three times. The air temperature was subtracted from the canopy tem- perature to determine the canopy temperature depression (CTD). Net photosynthesis rate (PN), transpiration rate (Tr), leaf temperature and ambient air temperature were measured at 68 DAE on 10 days old leaves at the top of canopy around noon with a gas exchange apparatus (CI-340, Camas, USA). Each measurement was repeated three times. Leaf temperature depression (LTD) was measured by subtracting the temperature of ambient air around the leaf from the leaf temperature. Chlorophyll contents were measured using the acetone extraction method. Collected samples (0.5 g of fresh leaf) were dissolved in 15 ml of acetone. The leaf extract was centrifuged at 8000 RPM for 5 minutes and absorbance was assessed at 663 nm, 645 nm and 470 nm using a UV-Vis spectrophotometer (UV 2600, Schemadzo, Japan). Chlorophyll a (Chla), Chlorophyll b (Chlb) and carotenoids (Car) were calculated according to hIScox and ISraElStaM (1979) as Chla = 11.75A663 – 2.35A645, Chlb = 18.61A645 – 3.96A663 and Car = (1000A470 – 2.27 Chla – 81.4 Chlb)/227 withA663, A645 and A470 being the absorption values at 663, 645 and 470 nm, respectively. Osmotic adjustment (OA) was determined for each ecotype by sub- tracting the osmotic potential under full irrigation from the osmotic potential under water restriction, after irrigation and recuperation of full turgor, as described by BaBu et al. (1999). Osmotic potential was measured using a vapor pressure osmometer (VAPRO 5520, Wescor, Utah, USA). At 70 DAE and after irrigation leaf samples were col- lected in both treatments when 80 % of plants recovered from wilt- ing. The leaf samples were pressed in the Eppendorf tube to extract the cell sap which was centrifuged at 8000 RPM for five minutes to collect the supernatants. 10 μL of supernatants were used for the measurement of osmotic potential (in MPa) in both regimes. Evaluation of berseem germplasm against drought stress 129 The water used by the plant (WU) was estimated as the product of transpiration rate (Tr) by leaf area (lazarIdou and noItSakIS, 2003). Transpiration was asses on fully expanded leaves at the top of canopy with a hand-held photosynthesis system (CI-340 4845 NW, Camas, WA, USA) between 10:00 and 11:00 am. Plant water use efficiency (WUE) was calculated according to lazarIdou and koutrouBaS (2004) as the ratio of above-ground dry biomass to the water transpired by the plant. Drought resistance index was calcu- lated as DRI = (YS/YN) / (MS/MN) where YS was the biomass of an ecotype under stress (water restriction) conditions, YN the biomass of this ecotype under non-stress (full irrigation) conditions, MS the mean biomass yield of all ecotypes under stress and MN the mean yield of all ecotypes under non-stress conditions. A recovery rate index was calculated for each ecotype as RRI = [(R1/T1) + (R2/T2) + (R3/T3)] / R where R1 was the number of plants which recovered from wilting the first day after restoring water in the lower profile of the lysimeter, R2 the number of plants which recovered from wilt- ing during the second day, R3 the number of plants which recovered from wilting during the third day and R the final recovery after 3 days. T1, T2 and T3 had values of 1, 2 and 3, respectively. A wilt- ing rate index was also calculated for each ecotype as WRI = (W1/ T1+W2/T2+W3/T3) / % W where W1 was the number of plants show- ing symptoms of wilting 10 days after restoring the soil moisture content, W2 and W3 being the number of plants that showed symp- toms of wilting 2 and 3 days after the first measurement, respec- tively, and W the total number of wilted plants wilting after 3 days. T1, T2 and T3 had values of 1, 2 and 3, respectively. Statistical analyses The data were subjected to the analysis of variance using the CROP- STAT 7.2 software. Genotypic and phenotypic coefficients of va- riation were estimated as GCV % = √Genotypic variance/overall average of the ecotypes × 100 and PCV % = √Phenotypic variance/ overall average of the ecotypes × 100, respectively. PCV % and GCV % reflected the variation in the germplasm at phenotypic and genotypic levels. The phenotypic variation is the sum of genotypic variance (σ2g)+ environmental variance (σ2E). Genetic purity of each of the ecotypes (inbreeding coefficient = 1) was maintained for seve- ral generations by growing them in isolation. Therefore variation within ecotypes was considered environmental which was used to estimate overall σ2E. Variation between ecotypes was used to esti- mate the phenotypic variance (σ2p). Genotypic variance was estimat- ed by subtracting the environmental variance from the phenotypic variance. Broad sense heritability (h2) was calculated according to allard (1960) as h2= (σ2g/ σ2p) × 100. A stepwise selection procedure based on the Akaike information criterion for multiple regression models (vEnaBlES and rIplEy, 2002) was conducted to determine the traits associated with fresh and dry biomass. The plant trait values were used to describe mul- tiple regression models for fresh forage yield (sum of first and sec- ond harvest) and dry forage yield (sum of first and second harvest), both measured in the water restriction treatment. Calculation of re- gressions and stepwise selection were carried out using the ‘lm’ and ‘step’ procedures of the R software (R 2013), respectively. A genotype plus genotype by environment (GGE) analysis (yan and kanG, 2003) was carried out to analyze the fresh and dry biomass in the first and second harvests for the two treatments. Both fresh and dry biomasses were arranged into a two-way genotype-by-combina- tion of the harvest number and the water regime. A biplot analysis was carried out on traits showing positive influence on yield in order to select promising ecotypes. The traits were standardized before the analysis in accordance with different scales of the chosen variables. The biplot calculations were made using the ‘scale’ and ‘svd’ proce- dures of the R software (R 2013). Results As shown by the average pattern of water removal in different layers at various intervals (Tab. 2) moisture content was higher in upper layers in the full irrigation treatment, compared to the water restric- tion treatment. In this last one, moisture content was maintained higher in the lower layers than in the upper ones. Significant effects due to ecotypes, water regimes and interaction (ecotypes × water regimes) were noted for all traits, except carotene contents which showed insignificant differences due to water treatments (Tab. 3). Water restriction effects were particularly drastic on fresh forage yield (FFY), canopy temperature depression (CTD) and transpira- tion rate (Tr) which reduced 160, 79 and 61 %. Water restriction increased the phenotypic and genotypic coefficients of variation for all traits except leaf area. High phenotypic and genotypic co- efficients of variation were noted in both treatments except for leaf area. The broad sense heritability of FFY, leaf area (LA), chloro- phyll b (Chlb) and carotene content (CAR) was less under water restriction conditions. Most physiological traits had a higher herit- ability than forage yield. Osmotic adjustment (OA) had higher phe- notypic and genotypic coefficients of variation than drought resist- ance index (DRI), recovery rate index (RRI) and survival rate index (SRI) (Tab. 4). DRI and SRI had higher heritability than water use efficiency (WUE), OA and RRI. Tab. 2: Moisture gradients in soil layers of lysimeters under two regimes at 60 days after emergence of plants. Moisture content (%) Full irrigation Water restriction 24 h 48 h 72 h 24 h 48 h 72 h 10 17 14 10 5 5 4 20 14 10 8 7 6 6 30 10 8 7 10 9 7 40 5 6 7 15 12 10 Soil depth (cm) The biplot analysis realized on biomass retained 88 % of the vari- ation for fresh biomass (Fig. 1) and 80 % for dry biomass (Fig. 2). In both treatments, the evaluated ecotypes showed differential per- formance across the two harvests. Under full irrigation conditions, the highest fresh biomass was noted in ‘SB-10’ and ‘L-94’ for the first harvest and in ‘Berseem Tandojam’, ‘Pachati’ and ‘Agaiti’ for the second harvest. The highest dry biomass was noted in ‘SB-10’ and ‘L-94’ for the first harvest and in ‘SB-10’ and ‘Sandal bar’ for the second harvest. Under water restriction, the highest biomass was noted in ‘SB-12’, ‘Berseem Queta’ and ‘P-22’. The best fitted models for fresh biomass under water restriction and full irrigation conditions were respectively FB (fresh biomass) = 6.63 – 0.02 Chla + 0.8 LTD – 0.17PN + 3.18 DRI + 0.84 WUE + 0.03 RRI – 0.01 SRI (R2 = 0.4) and FB = 15.07 + 0.06 LA + 2.09 LTD (R2 = 0.1). The best fitted model explaining dry biomass under water restriction and full irrigation conditions were DB (dry bio- mass) = 1.560 + 0.26 LTD + 0.73 DRI + 0.21 WUE (R2 = 0.32) and DB = 2.45 + 0.01 LA + 0.13 PN – 1.02 E + 0.42 CTD (R2 = 0.47), respectively. The biplot analysis realized on the traits associated to yield under water restriction (ie, DRI, LTD, RRI and WUE) showed a negative association between WUE and LTD, and between DRI and RRI (Fig. 3). The ecotypes ‘Anmol’, ‘Agaiti’ and ‘SB-12’ had the high- est WUE (and the lowest LTD values). Conversely, the ecotypes ‘Super’ and ‘Chenab’, followed by ‘Samarkand’, ‘L-48’, ‘SK-1’, ‘P- 130 M. Huassain, S. Rauf, J. Paderewski, I. ulHaq, D. Sienkiewicz-Paderewska, P. Monneveux Tab. 3: Mean, analysis of variance, genotypic coefficient of variation (GCV%), phenotypic coefficient of variation (PCV%) and heritability (h2) for fresh forage yield (FFY), dry forage yield (DFY), canopy temperature depression (CTD), leaf temperature depression (LTD), net photosynthesis rate (PN) and transpiration rate (Tr) of berseemclover under full irrigation and water restriction conditions. Plant traits Mean Mean sum of squares‡ GCV% PCV% h2 S Full Water G T G × T Full Water Full Water Full Water irrigation restriction irrigation restriction irrigation restriction irrigation restriction FFY† (g) 26.85 10.31 21.67** 4104.15** 11.73** 22.65 23.52 43.22 46.33 0.52 0.51 DMY† (g) 4.79 2.98 0.88* 49.27** 84.63** 25.21 27.87 47.41 51.58 0.53 0.54 CTD (°C) 2.80 1.56 0.52* 50.70** 0.34* 21.94 42.03 28.91 52.57 0.76 0.80 LTD (°C) 2.20 1.51 0.52* 16.65** 1.26** 18.64 41.05 23.3 49.51 0.80 0.83 LA (cm2) 125.92 112.67 637.98** 4957.92** 381.39** 8.19 4.87 12.47 11.23 0.66 0.43 Chla (mg kg-1) 90.23 67.44 1637.60** 15258.72** 1510.28** 27.31 31.72 31.27 35.8 0.87 0.89 Chlb (mg kg-1) 17.41 26.92 112.56** 2582.44** 45.10* 23.02 27.19 28.02 35.31 0.82 0.77 Carotene 19.84 19.39 85.10** 19.14NS 128.42** 30.44 31.94 43.44 46.38 0.70 0.69 PN (μmolm-2 s-1) 8.01 5.61 70.78** 247.25** 17.95** 40.69 62.67 51.36 74.22 0.79 0.84 Tr (mmolm-2s-1) 1.74 1.08 0.34* 12.93** 0.25* 14.90 28.07 21.43 34.64 0.70 0.81 † FFY and DMY were sum over two harvests ‡ G = variation due to ecotypes with df = 19; T = water treatment df = 1 and G×T = ecotypes × water regimes with df = 19 ** P ≤ 0.01, *P ≤ 0.05 Fig. 1: GGE biplot of fresh biomass in the first (1) and second (2) cuts under water restriction (S) full irrigation (W) for twenty ecotypes of berseem clover (Trifolium alexandrinum L.). 22’, ‘SB-10’, ‘Sanadal bar’, ‘Pak berseem’ and ‘Punjab’ had high LTD and low WUE values. The highest DRI and lowest RRI val- ues were observed in ‘Berseem Peshawar’, ‘Agati’, ‘Sandal Bad’, ‘Super’, ‘Pachati’, ‘L-48’, ‘Berseem Tandojam’ and ‘Sanadal bar’. The ecotypes ‘L-94’, ‘SB-10’, ‘P-209’, ‘P-22’, ‘Anmol’ and ‘Pun- jab’ had the highest RRI and lowest DRI values. The biplot analysis realized on the traits associated to yield under full irrigation (ie, PN, LA and CTD) showed a positive association between PN and LA (Fig. 4). Those traits were poorly correlated to CTD. The ecotype ‘SB-10’ had high PN and LA values. ‘P-209’, ‘P-22’, ‘Sanadal Bar’, ‘Samarkand’ and ‘Pachati’ showed high PN values, while ‘Berseem Peshawar’, ‘Berseem Queta’, ‘Sandal Bad’, ‘Berseem Tandojam’, ‘L-48’, ‘L-94’, ‘Anmol’ and ‘Punjab’ had low PNvalues. The ecotypes ‘P-209’, ‘P-22’, ‘Sandal Bar’, ‘Samarkand’, ‘L-94’ and ‘SK-1’ had the highest LA while ‘Punjab’, ‘Berseem Pe- shawar’, ‘Agaiti’, ‘SB-12’ and ‘Chenab’ had the lowest. ‘Punjab’, ‘Chenab’, ‘Pak’, ‘Agaiti’, ‘SB-12’ and ‘Pachati’ had high CTD values. Contrastingly, the ecotypes ‘L-94’, ‘Sandal Bad’, ‘Anmol’, ‘Berseem Queta’, ‘Berseem Tandojam’,’P-209’ and ‘L-48’ had low CTD values. PN was not associated to CTD and LA. ‘Pachati’ and ‘Pak Berseem’ combined high CTD and PNvalues, while ‘P-209’, ‘P22’, ‘Sanadal Bar’, ‘Samarkand’ and ‘SK-1’ had high LA and PN values. The ecotypes ‘Berseem Peshawar’, ‘Berseem Queta’, ‘Ber- seem Tandojam’ and ‘L-48’ had lower CTD, PN and LA values. Discussion After having used yield as an exclusive breeding objective, many breeders progressively replaced this empirical approach by indirect Tab. 4: Mean, analysis of variance, genotypic coefficient of variation (σ2g), phenotypic coefficient of variation (σ2p) and broad sense heritability (h2) for osmotic adjustment (OA), drought resistance index (DRI), water use efficiency (WUE), recovery rate index (RRI) and survival rate index (SRI) under water restriction conditions. Plant traits Mean ecotypes σ2g σ2p h2 mean sum of square OA 0.23 0.14** 71.34 85.31 0.81 DRI 1.08 0.32** 36.18 37.40 0.94 WUE 1.43 1.01** 39.39 42.93 0.84 RRI 44.75 1136.01** 48.35 60.43 0.78 SRI 83.58 5195.23** 50.38 52.41 0.92 **P ≤ 0.01) Evaluation of berseem germplasm against drought stress 131 selection (JackSon et al., 1996), based on the selection for ‘sec- ondary traits’ or plant characteristics that provide additional infor- mation about how the plant performs under a given environment (lafIttE et al., 2003). Any trait to be used as a surrogate of yield in the evaluation or selection process has to be genetically variable, highly heritable, genetically associated with yield, easy, inexpensive and fast to observe or measure, non-destructive and stable over the measurement period (EdMEadES et al., 1997). In the present study, all measured traits had high genotypic and phenotypic coefficients of variation. Moreover, water restriction increased the variability of various traits as previously observed by IannuccI et al. (2000), Fig. 2: GGE biplot of dry biomass in the first (1) or second (2) cut under water restriction (S) andfull irrigation (W) for twenty ecotypes of berseem clover (Trifolium alexandrinum L.). Fig. 3: GGE biplot for standardized leaf temperature depression (LTD), drought resistance index (DRI), recovery rate index (RRI) and water use efficiency (WUE) for twenty ecotypes of berseem clover (Trifolium alexandrinum L.) under water restriction. LTD, DRI, RRI, and WUE were averaged across replications for each combination of Genotype-by-Trait. The first principal component axis (PC1) retains 41 % and the second 31 % of sum of squares. El-BaBly (2002) and lazarIdou and koutrouBaS (2004). Phy- siological traits had higher broad sense heritability than yield. Final- ly, an association was noted between forage yield and LTD and RRI under water restriction conditions, suggesting the use of these traits as indirect selection criteria for yield under stress in berseem clover. Under full irrigated conditions, yield was positively associated to PN, canopy temperature depression (CTD) and leaf area. Gas exchange measurements which provide simultaneous information about LTD and PN appears provide useful prediction of forage yield. The use of leaf gas exchange traits has been proposed for the selection of sunflower (kalyar et al., 2013). These measurements however re- quire expensive equipment and are slow, stable over the measure- ment period. The use of the recovery index consequently appears as a good option for the prediction of forage yield under water restric- tion. Similarly, under full irrigated conditions, leaf area and canopy temperature depression could represent good candidates for indirect selection. Leaf area is strongly reduced by water stress in berseem clover (MartInIEllo and tExEIra da SIlva, 2011) and is closely as- sociated to yield in these conditions (ahMEd, 2006). CTD is a highly integrating trait resulting from the effects of several biochemical and morpho-physiological features acting at the root, stomata, leaf, and canopy levels. It is useful mainly in hot and dry environments, with high vapor pressure deficits (tuBEroSa, 2012). In wheat, significant genetic gains in yield have been reported in response to direct selec- tion for CTD in these environments (BrEnnan et al., 2007). The mean sum of square (MSS) due to ecotypes × water regimes was high when compared with MSS due to ecotypes for traits such as FFY and DMY (Tab. 3), suggesting differential performance of ecotypes over water regimes (dESMaraIS et al., 2013). This was confirmed by the magnitude of genetic variation (GCV%) which in- creased under water restriction, particularly for CTD, LTD and PN, indicating that this treatment successfully discriminated the ecotypes for their tolerance. GGE biplot analysis of multiple harvests under water regimes showed that the biomass of the first cut is not always a good indica- tor of biomass of the second cut. Some ecotypes like ‘SB-10’ from Punjab, however, showed stable biomass across cuts. As the deve- lopment of high-yielding and stable ecotypes across multiple har- vests is a major breeding objective of forage breeding (chakroun et al., 1990; krEnzEr et al., 1992), these ecotypes should be recom- mended for cultivation. The relationships observed between traits under water restriction conditions suggest that ecotypes with a higher LTD may recover well from drought stress. LTD is an important index of drought avoidance, transpiration and root growth under drought stress. It has been noted earlier that this trait positively correlated with bio- mass in various forage grass species (acuna et al., 2011; MErEwItz et al., 2014). It also appears that simultaneous selection for LTD and DRI or for RRI and WUE might be difficult. However, simultaneous selection may be practiced to some extent for LTD and RRI or WUE and DRI. Under full irrigated conditions, simultaneous selection for PN and CTD or PN and LA may be possible for the selection of high- yielding ecotypes under irrigated conditions. Selection for low LA might lead to higher CTD. kalyar et al. (2013) also showed that a higher temperature depression was negatively related with leaf area in irrigated sunflower and suggested that reduced leaf area could par- ticipate in leaf cooling. A large variation has been reported in berseem clover for the adap- tation to multiple cuts (GravES et al., 1996; putnaM et al., 1999; roSS et al., 2003; ranJBar, 2007) and the knowledge of produc- tivity across cutting regimes is of primary importance in breeding programs (JuSkIw et al., 2000). Among the tested ecotypes, ‘SB-10’ (from Punjab) showed the highest productivity. This ecotype was also characterized a good aptitude for double harvesting in irrigated conditions. This aptitude that highly depends harvesting management 132 M. Huassain, S. Rauf, J. Paderewski, I. ulHaq, D. Sienkiewicz-Paderewska, P. Monneveux (aBdEl-Gawad, 1993; GEwEIfEl and raMMah 1990; IannuccI et al., 2000) should however be confirmed under in different dates of harvest. Under water restriction conditions, the highest forage yield was noted in ‘SB-12’ (Punjab), ‘Berseem Queta’ (Balochistan) and ‘P-22’ (Punjab). These ecotypes could be recommended to farmers according to their needs and demand and for further use as progeni- tors in breeding programs. References ABDEL-GAWAD, K.I., 1993: Effect of cutting management on forage, protein and seed yields of berseem clover (synthetic 79 var.). Zagazig J. Agric. Res. 20, 67-75. ACUÑA, H., INOSTROZA, L., TAPIA, G., 2011: Strategies for selecting drought tolerant germplasm in forage legume species. In: Mofizur Rahman, I.M, (ed.), Water Stress, 277-300. Intech. AHMED, M.A.S., 2006: Response to three methods of recurrent selection in a Khadawari bersim (T. alexandrinum L.) population. Alex. J. Agric. Res. 51, 13-23. ALLARD, R.W., 1960: Principles of Plant Breeding. Johan Wiley and Sons Inc, New York. ARAUS, J.L., CAIRNS, J.E., 2014: Field high-throughput phenotyping: the new crop breeding frontier. Trends Plant Sci. 19(1), 52-61. BABU, R.C., PATHAN, M.S., BLUM, A., NGUYEN, H.T., 1999: Comparison of measurement methods of osmotic adjustment in rice cultivars. Crop Sci. 39, 150-158. BRENNAN, J.P., CONDON, A.G., VAN GINKEL, M., REYNOLDS, M.P., 2007: An economic assessment of the use of physiological selection for stoma- tal aperture-related traits in the CIMMYT wheat breeding programme. J. Agric. Sci. 145, 187-194. CHAKROUN, M., TALIAFERRO, C.M., MCNEW, R.W., 1990: Genotype- environment interactions of bermudagrass forage yields. Crop Sci. 30, 49-53. DE SANTIS, G., IANNUCCI, A., DANTONE, D., CHIARAVALLE, E., 2004: Changes during growth in the nutritive value of components of berseem clover (Trifolium alexandrinum L.) under different cutting treatments in a Mediterranean region. Grass For. Sci. 59, 378-388. DES MARAIS, D.L., HERNANDEZ, K.M., JUENGER, T.E., 2013: Genotype- by-environment interaction and plasticity: exploring genomic responses of plants to the abiotic environment. Annual Rev. Ecol. Evol. Syst. 44, 5-29. EDMEADES, G.O., BOLAÑOS, J., CHAPMAN, S.C., 1997: Value of secondary traits in selecting for drought tolerance in tropical maize. In: Edmeades G.O., Bänziger, M., Mickelson, H.R., Peña-Valdivia, C.B. (eds.), Devel- oping drought and low-N tolerant maize, 222-234. CIMMYT, El Batan, Mexico. EL-BABLY, A.Z., 2002: Effect of irrigation and nutrition of copper and mo- lybdenum on Egyptian clover (Trifolium alexandrinum L.). Agron. J. 94, 1066-1070. GEWEIFEL, H.G.M., RAMMAH, A.M., 1990: Seed production of six Egyptian clover cultivars as influenced by cutting system and potassium fertiliza- tion. Zagazig J. Agric. Res. 17, 589-598. GRAVES, W.L., WILLIAMS, W.A., THOMSEN, C.D., 1996:Berseem clover: a winter annual forage for California agriculture. University of California, Division of Agriculture and Natural Resources, Publication no 21536. HANNAWAY, D.B., LARSON, C., 2004: Berseem clover (Trifolium alexandri- num L.). Oregon State University, Species Selection Information System http://forages.oregonstate.edu/php/fact_sheet_print_legume.php?Spec ID=196&use=Forage HEUZÉ, V., TRAN, G., BASTIANELLI, D., BOUDON, A., LEBAS, F., 2014: Ber- seem (Trifolium alexandrinum). Feedipedia.org. A programme by INRA, CIRAD, AFZ and FAO; http://www.feedipedia.org/node/248 (last ac- cessed March 30th, 2015). HISCOX, J.D., ISRAELSTAM, G.F., 1979: A method for the extraction of chlo- rophyll from leaf tissue without maceration. Canad. J. Bot. 57, 1332- 1334. IANNUCCI, A., RASCIO, A., RUSSO, M., DI FONZO, N., MARTINIELLO, P., 2000: Physiological responses to water stress following a conditioning period in berseem clover. Plant and Soil 223, 219-229. IANNUCCI, A., RUSSO, M., ARENA, L., DI FONZO, N., MARTINIELLO, P., 2002: Water deficit effects on osmotic adjustment and solute accumula- tion in leaves of annual clovers. Europ. J. Agron. 16, 111-122. JACKSON, P., ROBERTSON, M., COOPER, M., HAMMER, G., 1996: The role of physiological understanding in plant breeding: from a breeding perspec- tive. Field Crop. Res. 49, 11-39. JUSKIW, P.E., HELMS, J.H., SALMON, D.F. 2000: Forage yield and quality for monocrops and mixtures of small grain cereals. Crop Sci. 40, 138-147. KALYAR, T., RAUF, S., TEIXEIRA, DA SILVA, J.A., HAIDAR, S., IQBAL, Z. 2013: Utilization of leaf temperature for selection of leaf gas exchange traits for the induction of heat resistance in sunflower (Helianthus an- nuus L.). Photosynthetica 51, 419-428. KRENZER, E.G., THOMPSON, J.D., CARVER, B.F., 1992: Partitioning of geno- type × environment interactions of winter wheat forage yield. Crop Sci. 32, 1143-1147. LAFITTE, H.R., BLUM, A., ATLIN, G., 2003: Using secondary traits to help identify drought-tolerant genotypes. In: Fischer, K.S., Lafitte, R.H., Fukai, S., Atlin, G., Hardy, B. (eds.), Breeding rice for drought-prone environments, 37-48. IRRI, Los Baños, Philippines. LAGHARI, H.H., CHANNA, A.D., SOLANGI, A.A., SOOMRO, S.A., 2000: Com- parative digestibility of different cuts of berseem (Trifolium alexandri- num) in sheep. Pak. J. Biol. Sci. 3, 1938-1939. LAZARIDOU, M., KOUTROUBAS, S.D., 2004: Drought effect on water use ef- ficiency of berseem clover at various growth stages. In: New directions for a diverse planet: Proceedings of the 4th International Crop Science Congress Brisbane, Australia (Vol. 26). LAZARIDOU, M., TSIRIDIS, A., 2004: Soil water deficit effects on growth and physiology of berseem clover. In: Proceedings International Soil Con- gress (ISC), Natural Resource Management for Sustainable Develop- ment. Erzrum, Turkey, 17-23. Fig. 4: GGE biplot for standardized net photosynthesis rate (PN), leaf area (LA), and canopy temperature depression (CTD) under full irrigation for twenty ecotypes of berseem clover (Trifolium alexandrinum L.). The first principal component axis (PC1) retains 47 % and the second 36 % of sum of squares of averaged by replications trait-by-ecotypes combinations. Evaluation of berseem germplasm against drought stress 133 ing rate effects in oat-berseem clover intercrops. Canad. J. Plant Sci. 83, 769-778. SARDANA, V., NARWAL, S.S., 2000: Influence of time of sowing and last cut for fodder on the fodder and seed yields of Egyptian clover. J. Agric. Sci. 134, 285-291. TUBEROSA, R., 2012: Phenotyping for drought tolerance of crops in the ge- nomics era. Front. Phys. 3, 1-26. VASILAKOGLOU, I., DHIMA, K., 2008: Forage yield and competition indices of berseem clover intercropped with barley. Agron. J. 100, 1749-1756. VENABLES, W.N., RIPLEY, B.D., 2002: Modern applied statistics with S. Springer, New York, USA. YAN, W., KANG, M.S., 2003: GGE biplot analysis: a graphical tool for breed- ers, geneticists, and agronomists. CRC Press. Boca Raton, USA. Address of the authors: M. Huassain, S. Rauf, I. ulHaq, Department of Plant Breeding & Genetics, University College of Agriculture, University of Sargodha, Pakistan, 38000. J. Paderewski, Department of Experimental Design and Bioinformatics, Warsaw University of Life Sciences, Nowursynowska 159, 02-776 Warsaw, Poland. D. Sienkiewicz-Paderewska, Department of Agronomy, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland. P. Monneveux, International Potato Center (CIP), Avenida La Molina 1895, La Molina, Lima, Peru. LAZARIDOU, M., NOITSAKIS, B., 2003: The effect of water deficit on yield and water use efficiency of lucerne. In: Kirilov, A., Todorov, N., Katerov, I. (eds.), Optimal forage systems for animal production and the environ- ment, 344-347. Grassland Sci. Europ., Vol 8. MARTINIELLO, P., IANNUCCI, A., 1998: Genetic variability in herbage and seed yield in selected half-sib families of berseem clover, Trifolium alex- andrinum L. Plant Breed. 117, 559-562. MARTINIELLO, P., TEIXEIRA DA SILVA, J.A., 2011: Physiological and bio- agronomical aspects involved in growth and yield components of culti- vated forage species in Mediterranean environments: a review. Europ. J. Plant Sci. Biotech. 5, 64-98. MASUKA, B., ARAUS, J.L., DAS, B., SONDER, K., CAIRNS, J.E., 2012: Phe- notyping for abiotic stress tolerance in maize. J. Int. Plant Biol. 54, 238- 249. MEREWITZ, E., BELANGER, F., WARNKE, S., HUANG, B., BONOS, S., 2014: Quantitative trait loci associated with drought in creeping bentgrass. Crop Sci. 54, 2314-2324. PUTNAM, D.B., WILLIAMS, W.A., GRAVES, W.L., GIBBS, L., PETERSON, G., 1999: The potential of berseem clover varieties for California. In: Pro- ceeding of 29th California Alfalfa Symposium. University of California Coop. Extension, 108-119. R 2013: R, Project for Statistical Computing: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. RANJBAR, G.A., 2007: Forage and hay yield performance of different ber- seem clover (Trifolium alexandrinum L.) genotypes in Mazandaran con- ditions. Asian J. Plant Sci. 6, 1006-1011. ROSS, S.M., KING, J.R., O’DONAVAN, J.T., IZAURRALDE, R.C., 2003: Seed- © The Author(s) 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).