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RESEARCH

Diversity of Response to Drought in a Collection of Lines of Medicago truncatula, M. ciliaris, and M. polymorpha Mounawer Badri,* Ghazoua Toumi, Saoussen Mahfoudh, Kamel Hessini, Meriem Abdelguerfi-Laouar, Aissa Abdelguerfi, Mohamed Elarbi Aouani, Chedly Abdelly, and Naceur Djébali

ABSTRACT In this study, we investigated variation for tolerance to water deficit in 47 lines of Medicago truncatula Gaertn., M. ciliaris (L.) All., and M. polymorpha L. collected from different ecogeographic regions in Tunisia. Plants were cultivated in the greenhouse under 100 and 30% of field capacity and they were harvested at the flowering stage. Nineteen morphophysiological parameters were measured for each species. Our results showed that variation of parameters were explained by the effects of species, line within species, treatment, species × treatment interaction, and treatment × line within species interaction. Drought treatment explained the most variance for the measured parameters. Of the 19 traits, 12 showed significant differences for tolerance to water deficit between M. truncatula, M. ciliaris, and M. polymorpha. M. ciliaris was the latest-flowering and it exhibits the highest biomass under both treatments. High broad-sense heritabilities (H2) were noted for most of parameters under control treatment and drought stress. Positive correlations were found between transpiration rate (E) and aerial and root fresh weights, and between photosynthetic rate (A) and stomatal conductance (gs), and between relative water content and number of nodules. Studied lines formed five groups based on drought response indices (DRI). A first group is formed by 23 most tolerant lines, a second group and a fourth group are constituted by 17 moderately affected lines, and a third group and a fifth group have 8 sensitive lines. Tolerant lines of three species can be good candidates in future breeding programs.

M. Badri, G. Toumi, S. Mahfoudh, K. Hessini, C. Abdelly, Laboratory of Extremophile Plants, Centre of Biotechnology of Borj Cedria, B.P. 901, Hammam-Lif 2050, Tunisia; M. Abdelguerfi-Laouar and A. Abdelguerfi, Ecole Nationale Supérieure Agronomique d’Alger, Algeria; ME Aouani, Centre of Biotechnology of Borj Cedria, B.P. 901, Hammam-Lif 2050, Tunisia; N. Djébali, Laboratory of Bioactive Substances, Centre of Biotechnology of Borj Cedria, B.P. 901, HammamLif 2050, Tunisia. Received 12 Apr. 2016. Accepted 22 June 2016. Assigned to Associate Editor Joseph Robins. *Corresponding author ([email protected]). Abbreviations: ADW, aerial dry weight; AFW, aerial fresh weight; AWC, aerial water content; DRI, drought response index; FLOR, days from emergence to first flower; LR, length of roots; LS, length of stems; NA, number of axes; NbNOD, number of nodules; NL, number of leaves; RDW, root dry weight; RFW, root fresh weight; RtWC, root water content; RWC, root water content; WUEi, instantaneous water use efficiency.

T

he genus Medicago is one of most important genera of forage plants (Reid et al., 1989). Small and Jomphe (1989) reported that it contains 85 species of which two are shrubby (M. arborea and M. strasseri), 20 are perennial herbaceous, and 63 are annual predominantly autogamous. The agricultural role of Medicago annual species (medics) was recognized from the 1930s when Trumble and Donald (1938) recommended the culture of M. truncatula on calcareous soils of southwestern Australia. In North Africa, livestock suffers from a chronic shortage in food fodder, especially during dry periods in the summer and autumn. Annual Medicago are mainly used as pasture species in North African extensive farming systems, especially to improve the low quality of natural pastures. They are best grazed by sheep, but can be grazed by cattle as well (Van Heerden, 2013). Medic pastures can be used for hay production; it was found that medics hay has the same nutrient value than that of good quality lucerne

Published in Crop Sci. 56:1–8 (2016). doi: 10.2135/cropsci2016.04.0224 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. crop science, vol. 56, november– december 2016

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Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

hay (Denney et al., 1979). Among these species, M. truncatula, M. ciliaris and M. polymorpha had different geographic distributions in Tunisia. In contrast to M. truncatula that is omnipresent in all bioclimates (Badri et al., 2007), M. ciliaris is located in humid to lower semiarid climates (Badri et al., 2008) and M. polymorpha spreads from humid to higher arid climates (Badri et al., 2016). Medicago truncatula was emerged as a model legume because of the many advantages it offers (Barker et al., 1990). It is diploid (2n = 16), self-pollinating, it has a low DNA content per haploid genome (450–500 Mbp), it is suitable for cultivation in the laboratory, and it has a short generation time (3–4 mo). Medicago ciliaris is selfpollinating and diploid (2n = 16). It could be intercrossed readily with M. intertexta and also with M. muricoleptis (Lesins and Lesins, 1979). Medicago ciliaris growing in Tunisian soils is preferentially nodulated by Sinorhizobium medicae (Zribi et al., 2007) and was reported as the most-tolerant species to salt stress (Ben Salah et al., 2009). In Tunisia, it grows spontaneously on heavy soils with clay and/or high salinity such as Sebkha edges. Medicago polymorpha is autogamous and diploid (2n = 14) (Lesins and Lesins, 1979). It forms a symbiotic relationship with Sinorhizobium medicae (Dourado et al., 2009). Observations of M. polymorpha at natural sites show that it is well adapted to neutral and slightly acid soils (Reid et al., 1989). A large number of lines within the three species with high genetic variability were found (Badri et al., 2007, 2008, 2016; Arraouadi et al., 2009; Lazrek et al., 2009) which constitutes a material of choice for selection and creation of suitable varieties well adapted to various environments. The response of plants to environmental stress depends on a number of factors related to genotypes and type of stress (Bita and Gerats, 2013). The changes and disturbances caused by water deficit on plant physiology affect reduction of leaf area for growing leaves, leaf curling and development of a waxy cuticle. An increase in the root/ aboveground biomass was observed in some species (Joslin et al., 2000; Slama et al., 2007; Badri et al., 2010) up to certain levels of stress. Energy generation mechanisms are also affected: the decrease of CO2 fixation consecutive to stomatal closure, and in cases of most severe stress, impaired functioning of photosystems resulting in a reduction of photosynthesis (Ashraf and Harris, 2013). This study aims to analyze the responses to water deficit in a collection of 47 lines of M. truncatula, M. ciliaris and M. polymorpha for the selection of the most tolerant genotypes to propagate in regions suffering from water scarcity. Harvest of plants was performed at flowering stage and several parameters of vegetative growth and photosynthesis were measured.

MATERIALS AND METHODS Plant Material and Experimental Conditions Forty-seven lines from Tunisian populations of M. truncatula, M. ciliaris and M. polymorpha were used in this study on drought 2

tolerance (Fig. 1; Table 1). Inbred lines were developed by singleseed descent until the F3 or F4 generation under greenhouse conditions. Seeds were scarified using sandpaper and were sown into black pots (17 cm diam.; 13 cm depth) of two liters filled with soil from Centre of Biotechnology of Borj Cedria and compost of sphagnum (2:1). Plants were cultivated in the greenhouse in well-watered (100% of filed capacity) and water deficit (30% field capacity) conditions. Plants were irrigated with distilled water. A reduction in the water supply was applied after 21 d from the date of germination of seeds. Pots were weighed every 2 d and lost weight was replaced with distilled water. Three replicates per line and per treatment were used. Plants were organized into a randomized complete blocks design and plants were harvested at the formation of the first green pod.

Morphologic and Photosynthetic Parameters Nineteen parameters were measured for studied lines, including days from emergence to first flower (FLOR, d), length of stems (LS, cm), length of roots (LR, cm), number of leaves (NL), number of axes (NA), aerial fresh weight (AFW, g), aerial dry weight (ADW, g), aerial water content (AWC, %), root fresh weight (RFW, g), root dry weight (RDW, g), root water content (RtWC, %), root dry weight and aerial dry

Fig. 1. Map of Tunisia with the location of natural populations of Medicago truncatula, M. ciliaris, and M. polymorpha from which studied lines were collected. 1, Enfidha; 2, Jelma; 4, Deguache; 6, Thala; 7, El Kef; 8, Soliman; 9, Bulla Regia; 11, Mateur; 14, Tataouine.

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Region

M. truncatula

M. ciliaris

M. polymorpha

Soliman

TN8.21 TN8.15 TN8.5 TN9.5 TN9.4 TN9.3 TN1.5 TN1.3 TN1.17 TN7.17 TN7.19 TN7.2 TN2.21 TN2.12 TN2.15 TN6.18 TN6.22 TN6.23 TN14.9 TN14.12 TN14.2 TN4.13 TN4.14 TN4.18 -

TNC8.7 TNC8.5 TNC8.9 TNC10.11 TNC10.6 TNC10.2 TNC1.3 TNC1.8 TNC1.3 -

-

TNP8.7 TNP8.14 TNP8.15 TNP9.6 TNP9.14 TNP9.29 TNP1.1 TNP1.11 TNP1.23 TNP7.9 TNP7.13 TNP7.22 -

-

-

-

-

-

-

TNC11.11 TNC11.2 TNC11.5 -

TNP11.10 TNP11.22 TNP11.27 -

Bulla Regia

Enfidha

El Kef

Jelma

Thala

Tataouine

Deguache

Mateur

Reference lines

Jemalong F83005.5 DZA315 DZA45

Analysis of the effects of species, treatment, line within species, species × treatment interaction, and treatment × line within species interaction on measured traits was performed using Proc GLM in SPSS version 16 (2007 Rel 1600 SPSS Inc., Chicago, IL). Comparison of means for measured traits for species and lines within species was performed using Duncan test at 5%. Estimation of variance components within and among lines was performed using Proc VARCOMP in SPSS software. Variance between replicates of the same genotype is the environmental variance, while variance between genotypes is the genetic variance. Broad-sense heritability (H2) of measured parameters was estimated as the ratio of genetic variance on the sum of genetic and environmental variances (Badri et al., 2011). Phenotypic correlations between measured traits under control treatment and drought stress were estimated by computing the Pearson correlation coefficient (r) using Proc Correlate in SPSS software. Hierarchical classification of studied lines was performed with Ward’s method using the Euclidean distance of dissimilarity based on mean DRI of lines. A Discriminant Analysis test was performed on DRI values for the groups of lines. Given that the group 5 was constituted by only one line and based on the dissimilarity level, the three lines TN2.12, TN14.2 and TNC10.6 were considered in the same cluster for Discriminant Analysis. The cluster and Discriminant Analyses tests were performed using the XLSTAT (Addinsoft) software version 7.5.

RESULTS Morphophysiological Variation

weight ratio (RDW/ADW), relative water content (RWC), number of nodules (NbNOD), water potential (Yw), net CO2 photosynthetic assimilation rate (A), transpiration rate (E), stomatal conductance (gs), and instantaneous water use efficiency (WUEi) as the ratio A/E. At harvest, plants were divided into leaves, stems and roots. Aerial and root water contents were estimated as follows: AWC (%) = 100 (AFW – ADW/AFW), where AFW is the aerial fresh weight, and ADW is the aerial dry weight. The relative water content (RWC) was calculated as follows: RWC (%) = 100 [(LFW – LDW) / (LTW – LDW)], where LFW is the leaf fresh weight, the leaf dry weight is LDW and LTW is the leaf turgid weight. The dry weight of the different organs was estimated after drying in an oven at 70°C for 48 h. The leaf water potential (Yw) was measured on the sixth leaf at dawn by means of the pressure chamber (Model 1000 Pressure Chamber Instrument, PMS Instrument Co., Albany, OR). The photosynthetic parameters were measured on the sixth leaf at dawn using a CO2 and H 2O infrared gas analyzer (Li-Cor 6200, Li-Cor Biosciences, Lincoln, NE). Measurements were done between 10:00 am and 12:00 pm on the leaves of plants cultivated under well-watered and drought stress treatments as described by Hessini et al. (2013). The drought response index (DRI) was estimated as the ratio of values of measured traits for plants under water deficit on those obtained in control treatment (Badri et al., 2011).


Data Analyses

Results from ANOVA showed that variation of morphophysiological parameters was explained by the effects of species, line within species, treatment, species × treatment interaction, and treatment × line within species interaction (Table 2). Water treatment explained most of the variation of the measured traits. All measured traits showed significant variation among lines of three species under control treatment and drought stress (Supplemental Tables S1 and S2). In addition, significant differences were also recorded for drought response indices (DRI) in studied lines (Supplemental Table S3). Of the 19 parameters, 12, 14, and 12 traits showed significant variation among studied species under control treatment, water deficit and for drought response (Supplemental Tables S2 and S4). In control treatment, M. ciliaris was the latest-flowering and it exhibited the highest number of leaves, AFW, NbNOD, and Yw while the largest values of RDW and the ratio RDW/ADW were registered for M. polymorpha. Furthermore, there was no significant difference for means of gs, A, E, and WUEi among species. On the other hand, the lowest reductions for FLOR, RDW, the RDW/ADW ratio, RWC and WUEi were found for M. ciliaris under water deficit while it showed the largest reductions for RtWC, NbNOD, Yw and E (Table 3). Higher to moderate heritability values were found for measured parameters for the three species (Supplemental

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Table 1. List of used lines of Medicago truncatula, M. ciliaris and M. polymorpha.

Table 2. Contribution of species, line within species, treatment, species × treatment interaction, and treatment × line within species interaction to the total variance of measured traits for lines of Medicago truncatula, M. ciliaris and M. polymorpha.


Species

Line(Species) F P

Treatment

Species × Treatment F P

Treatment × Line(Species) F P

Trait†

F‡

P

F

P

FLOR LS LR NL NA AFW ADW AWC RFW RDW RtWC

3228.00 23.57 3.42 15.53 95.07 14.04 17.24 3.67 34.17 59.24 6.95

0.000 0.000 0.035 0.000 0.000 0.000 0.000 0.027 0.000 0.000 0.001

468.08 7.31 4.12 9.51 49.76 9.18 13.41 7.22 7.28 9.76 3.05

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

4500.00 1302.00 18.01 1076.00 865.07 914.20 512.86 4743.00 646.12 25.75 3789.00

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

27.23 3.59 9.90 3.79 40.61 9.59 4.68 4.95 18.29 19.20 48.23

0.000 0.029 0.000 0.025 0.000 0.000 0.010 0.008 0.000 0.000 0.000

23.67 4.40 4.06 5.64 56.44 6.71 6.53 4.68 4.79 3.28 3.90

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

111.06 4.26 14.88

0.000 0.016 0.000

6.95 6.74 12.96

0.000 0.000 0.000

533.84 2056.00 319.37

0.000 0.000 0.000

12.25 8.16 21.99

0.000 0.000 0.000

2.45 3.46 7.89

0.000 0.000 0.000

125.72 16.04 4.53

0.000 0.000 0.013

21.88 15.50 6.71

0.000 0.000 0.000

2631.00 1201.00 952.59

0.000 0.000 0.000

21.21 3.63 3.29

0.000 0.030 0.041

5.10 9.34 3.09

0.000 0.000 0.000

4.09 5.36

0.020 0.006

6.90 4.32

0.000 0.000

470.23 103.50

0.000 0.000

0.45 6.31

0.637 0.003

2.37 2.12

0.001 0.003

RDW/ADW RWC NbNOD Yw A E gs WUEi

† Trait abbreviations and units of measurement: Days from emergence to first flower (FLOR, d), length of stems (LS, cm), length of roots (LR, cm), number of leaves (NL), number of axes (NA), aerial fresh weight (AFW, g), aerial dry weight (ADW, g), aerial water content (AWC, %), root fresh weight (RFW, g), root dry weight (RDW, g), root water content (RtWC, %), root dry weight and aerial dry weight ratio (RDW/ADW), root water content (RWC), number of nodules (NbNOD), water potential (Yw, MPa), photosynthetic rate (A, mmol CO2 m -2s -1), transpiration rate (E, mmol H2O m -2s -1), stomatal conductance (gs), and instantaneous water use efficiency (WUEi, [mol CO2 m -2s -1/mmol H2O m -2s -1]). ‡ F is the coefficient of Snedecor-Fisher with significance at P £ 0.05.

Table 3. Mean values of drought responses indices (DRI) for Medicago truncatula (Mtr), M. ciliaris (Mcil) and M. polymorpha (Mpoly) cultivated under 30% of field capacity (FC). FLOR†

LS

LR

NL

NA

AFW

ADW

Mtr Mcil Mpoly F† P†

80.53 ± 5.21 88.26 ± 12.64 78.57 ± 5.53 14.54 0.000

29.46 ± 12.36 31.65 ± 12.30 38.24 ± 12.67 5.22 0.007

85.06 ± 27.01 96.42 ± 39.52 103.00 ± 26.81 4.67 0.011

31.03 ± 13.03 32.17 ± 12.87 30.58 ± 13.93 0.15 0.858

79.69 ± 96.72 43.77 ± 14.61 58.33 ± 16.37 3.28 0.041

25.74 ± 13.56 20.42 ± 8.65 21.81 ± 14.69 2.15 0.121

56.38 ± 28.41 49.36 ± 20.00 44.09 ± 25.39 2.60 0.078

AWC

RFW

RDW

RtWC

RDW/ADW


61.13 ± 8.54 59.85 ± 7.47 63.24 ± 8.71 1.40 0.251

26.52 ± 18.40 21.81 ± 12.49 26.54 ± 13.50 1.08 0.344

98.05 ± 51.62 108.50 ± 50.33 72.29 ± 41.63 4.90 0.009

50.22 ± 7.42 39.73 ± 12.66 56.42 ± 16.73 17.10 0.000

180.43 ± 64.44 218.47 ± 56.14 163.17 ± 38.69 8.27 0.000

Yw

A

E

gs

WUEi


231.99 ± 50.33 190.67 ± 42.45 219.10 ± 33.76 9.21 0.000

45.02 ± 11.86 41.70 ± 14.16 42.83 ± 7.93 0.74 0.481

28.57 ± 9.97 18.40 ± 7.67 24.31 ± 12.75 7.85 0.001

39.23 ± 16.41 39.50 ± 18.01 40.38 ± 15.47 0.04 0.959

173.55 ± 71.62 240.39 ± 94.48 214.00 ± 92.19 5.61 0.005

RWC 66.39 ± 7.32 72.45 ± 7.17 70.33 ± 7.76 7.67 0.001

NbNOD 50.00 ± 38.24 32.97 ± 30.77 51.36 ± 28.71 3.23 0.043

† Trait abbreviations and units of measurement: Days from emergence to first flower (FLOR, d), length of stems (LS, cm), length of roots (LR, cm), number of leaves (NL), number of axes (NA), aerial fresh weight (AFW, g), aerial dry weight (ADW, g), aerial water content (AWC, %), root fresh weight (RFW, g), root dry weight (RDW, g), root water content (RtWC, %), root dry weight and aerial dry weight ratio (RDW/ADW), root water content (RWC), number of nodules (NbNOD), water potential (Yw, MPa), photosynthetic rate (A, mmol CO2 m -2s -1), transpiration rate (E, mmol H2O m -2s -1), stomatal conductance (gs), and instantaneous water use efficiency (WUEi, [mol CO2 m -2s -1/mmol H2O m -2s -1]). ‡ F is the coefficient of Snedecor-Fisher with significance at P £ 0.05.

Table S5). Moderate heritabilities (0.20 < H2 < 0.40) for most traits were registered for M. ciliaris under water deficit treatment. Among 171 possible correlations between parameters, 28 for M. truncatula, 29 for M. ciliaris, 27 for M. polymorpha 4

were significant (Supplemental Table S6). Twenty-three of 28 correlations, 28 of 29 correlations, and 23 of 27 were positively correlated for M. truncatula, M. ciliaris and M. polymorpha, respectively.

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Cluster Analysis The lines of three species were clustered into five groups (Fig. 2). A first group was formed by 23 lines including 12 of M. truncatula, 7 of M. ciliaris and 4 of M. polymorpha. A second group was constituted by 15 lines with 10 of M. truncatula and 5 of M. ciliaris. A third group includes 1 line of M. truncatula, a fourth group has 1 line of M. truncatula and 1 of M. ciliaris, and a fifth group was composed by 1 line of M. truncatula, 3 lines of M. ciliaris and 2 lines of M. polymorpha.

The identification of the main traits involved in classification of lines was performed based on Discriminant Analysis. Length of stems, NL, ADW, RDW, RDW/ ADW, Yw, E and WUEi are the determinant characters for classification of lines into five groups (Table 4). Lines of first group were most tolerant for LS and RDW while lines of second and fourth group were moderately affected for ADW and NL, and lines of third and fifth groups were most sensitive for NL and ADW.

DISCUSSION From an agronomic point of view, drought tolerance is the ability of the plant to grow and produce satisfactory yields in areas prone to episodic water deficits (Turner, 1996). Drought tolerance is a strategy that allows the plant to maintain its physiological functions despite a deterioration of its water status. In this study, variation of morphophysiological traits was mainly explained by treatment factor

Fig. 2. Dendrogram of the lines of Medicago truncatula, M. ciliaris and M. polymorpha clustered based on Euclidean distances of dissimilarity matrix with the Ward’s method. TN denotes lines of M. truncatula (Mtr), TNC denotes lines of M. ciliaris (Mcil) and TNP denotes lines of M. polymorpha (Mpoly). crop science, vol. 56, november– december 2016

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The NbNOD was positively correlated with AFW, ADW, RDW, A and gs for M. truncatula while it was correlated with LS, NL, AFW, RDW and RWC for M. ciliaris. Furthermore, the NbNOD was positively correlated with WUEi for M. polymorpha. Comparison of correlations between traits in three species showed 7, 13, and 11 specific correlations for M. truncatula, M. ciliaris, and M. polymorpha, respectively.

Table 4. Means of drought response indices (DRI) for classes of lines of Medicago truncatula, M. ciliaris and M. polymorpha in 30% of field capacity. Class


Variable

l†

F†

FLOR‡ LS LR NL NA AFW ADW AWC RFW RDW RtWC

0.96 0.69 0.97 0.70 0.94 0.72 0.72 0.91 0.77 0.61 0.84

0.55 6.49 0.52 6.20 0.90 5.50 5.62 1.37 4.30 8.98 2.65

RDW/ADW RWC NbNOD Yw

0.70 0.95 0.93

A E gs WUEi

1

2

3

4

37.40a

24.59b

23.02b

32.95ab

36.14a

27.03a

10.75b

29.54a

28.86a 63.34a

16.73a 39.53ab

15.98a 31.88b

19.55a 43.18ab

32.28a 118.22a

18.17a 59.65b

18.53a 78.41ab

21.54a 92.17ab

6.01 0.81 1.04

191.82bc

152.52c

255.83a

216.01ab

0.002 0.498 0.386

0.62 0.89 0.79

8.86 1.80 3.86

193.98c

248.60ab

278.03a

219.83bc

25.90a

26.87a

21.77b

17.56c

0.000 0.161 0.016

0.95 0.47

0.68 16.25

175.17c

192.11c

236.19b

301.60a

p-value 0.652 0.001 0.674 0.001 0.45 0.003 0.002 0.264 0.010 0.0001 0.061

0.566 0.0001

† l is Lambda of Wilks; F is the coefficient of Snedecor-Fisher with significance at P £ 0.05. ‡ Trait abbreviations and units of measurement: Days from emergence to first flower (FLOR, d), length of stems (LS, cm), length of roots (LR, cm), number of leaves (NL), number of axes (NA), aerial fresh weight (AFW, g), aerial dry weight (ADW, g), aerial water content (AWC, %), root fresh weight (RFW, g), root dry weight (RDW, g), root water content (RtWC, %), root dry weight and aerial dry weight ratio (RDW/ADW), root water content (RWC), number of nodules (NbNOD), water potential (Yw, MPa), photosynthetic rate (A, mmol CO2 m -2s -1), transpiration rate (E, mmol H2O m -2s -1), stomatal conductance (gs), and instantaneous water use efficiency (WUEi, [mol CO2 m -2s -1/mmol H2O m -2s -1]).

for three species, indicating that measured characteristics were significantly affected by drought stress. All measured parameters showed significant variation of response to water deficit for lines while only 12 of 19 characters exhibited significant difference among species. The highest values of AFW registered for M. ciliaris under the control treatment and drought stress suggest that lines from this species could be used as a forage resource for livestock in Tunisia in irrigated and dry areas. Annual medic pastures can produce 6 to 10 tons of dry matter per hectare in exceptional conditions (Kotzé, 1999). The largest reduction for RDW was registered for M. polymorpha while lowest reductions for FLOR and RDW were for M. ciliaris. The rapid phenological development with early flowering allows the plant to avoid dry periods. This strategy is applied to crop systems and has led to shifting the sowing date and/or select the earliest varieties to avoid water deficits at the end of the cycle. The optimization of water absorption is related to a complex set of morphological traits of roots: mass and volume, branching, depth (Ramanjulu and Bartels, 2002). Many plants adapted to arid areas control only very little their water loss through transpiration, but have very deep roots that can extract water from the soil. Root growth under dry conditions can be maintained by the osmotic adjustment which limits loss of turgor potential (Turner, 1986).The physiological activity of the leaves, under water deficit, and more particularly the photosynthesis is affected (Poormohammad Kiani et al., 2007). Reduction of photosynthesis due to the decrease of leaf water potential is 6

assumed to depend on both (i) the closing of stomata, with a consequent decrease in conductance to CO2 diffusion, and (ii) a biochemical limitation chloroplast to fix CO2 (Graam and Boyer, 1990), possibly associated with the regeneration of RuBP limiting ribulose bisphosphate, the substrate of the Calvin’s cycle (Gimenez et al., 1992). Our data showed that lowest reductions for transpiration and water use effeciency under drought stress were found for M. ciliaris. Among the mechanisms to reduce transpiration, reducing leaf area and the decrease in stomatal conductance play a decisive role. Regulation of stomatal conductance is the major mechanism involved at short term to reduce water loss: leaf water potential will be maintained even longer than stomatal closure early. This can occur at various leaf water potential as a function of genotype (Mojayad, 1993) and stage of development (Morizet and Merrien, 1990). Regulating stomatal conductance depends on leaf water potential, and humidity of air in field (Turner, 1996). Low conductance is usually proposed as a favorable adaptation to drought stress (Turner, 1982, 1986). If stomatal closure is not complete, due to the difference between the coefficients of diffusion of water and CO2 in the leaves, transpiration is smaller than the net assimilation: the water use efficiency is increased. Higher to moderate heritabilities values were found for measured traits for three species, indicating that much of their variation is under a genetic control. Thus, these traits can be used as good descriptors for genetic determinism for tolerance to water deficit for M. truncatula, M.

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Acknowledgments The authors thank Ken Moriuchi (UC Davis) for helpful comments on the manuscript, and Fethia Zribi for photosynthesis measurements. We are grateful to Asma Mahjoub for useful discussion. This work was financially supported by Tunisian-Algerian bilateral project (2013–2015), and the Tunisian Ministry of Higher Education and Scientific Research (LR10 CBBC 02).

Supplemental Materials Available Supplemental Tables S1 to S6 provide the following information. Supplemental Tables S1, S2, S3, and S4: Means of measured traits for lines and species grown under control treatment and drought stress. Supplemental Table S5: Genetic and environmental variances, and heritabilities for measured traits in lines of the three species grown under control treatment and crop science, vol. 56, november– december 2016

drought stress. Supplemental Table S6: Correlations between drought response indices (DRI) of traits measured in lines of the three species grown under water deficit.

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ciliaris, and M. polymorpha. Accordingly, Badri et al. (2015) reported that most of morphological traits exhibited high H 2 for populations of M. truncatula under well-watered and water deficit conditions. In the current study, most of correlations between traits for three species were positive, suggesting that most of measured characters were similarly affected by drought stress. Furthermore, specific correlations between traits were noted for three species, suggesting that relationship between characters was affected by species effect. Similarly, Arraouadi et al. (2011) showed most important correlations between measured traits for Tunisian and reference lines of M. truncatula under salt stress. In the same sense, Badri et al. (2015) demonstrated that relationship between the parameters for populations of M. truncatula was dependent on the treatment effect. The NbNOD was positively correlated with ADW and RDW for M. truncatula and M. ciliaris, indicating that most tolerant plants under water deficit were more able to form maximum number of nodules. Furthermore, the nodulation of plants was positively associated with photosynthetic assimilation and water use efficiency in M. ciliaris and M. polymorpha. Constitutive lines of three species were clustered into five groups. Lines of first group were most tolerant for LS and RDW while those of third and fifth groups were most sensitive for NL and ADW. Our results showed that there was no segregation of lines into classes according to species factor. Overall, lines of three species showed a large drought tolerance variation. Five clusters of lines differing in their sensitivities to water deficit were identified, with 23 tolerant, 17 moderately tolerant, and 8 susceptible lines. The tolerant lines were less affected for LS and RDW than the susceptible. A further study is needed to validate the behavior of the tolerant lines in the field to select the most drought-tolerant lines to cope with water scarcity in arid and semiarid regions. The evaluation of these lines in the field based on agronomic traits should also take into account the variability for some characteristics related to nutritive value.


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