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Journal of Food, Agriculture & Environment Vol.10 (3&4): 875-878. 2012

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Combining ability and inbreeding depression of physiological parameters in F3 population of wheat under drought stress
Mohammad Reza Naroui Rad 1, 2 *, Mihdzar Abdul Kadir 1, Mohd Rafii Yusop 3, Hawa Z. E. Jaafar 4 and Mahmood Danaee 1
Department of Agriculture Technology, Faculty of Agriculture, University Putra Malaysia (UPM), 43400 UPM Serdang, Malaysia. 2 Agriculture and Natural Resources Research Center of Sistan, Iran. 3 Institute of Tropical Agriculture, 4 Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, 43400 Serdang, Malaysia. *e-mail: narouirad@gmail.com
1

Received 23 June 2012, accepted 1 October 2012.

Abstract
The study was conducted with the objective of identifying parents to be used in a breeding program to develop lines with well performance under drought stress. Combining ability, inbreeding depression and inheritance of membrane stability index, stomatal conductance, relative water content and chlorophyll content were investigated in bread wheat obtained from half-diallel crossings among eight parental lines in third generation. Cultivars with names of Irena/Babax//Pastor, S-78-11, Tajan, Chamran, Moghan3, Hamoon, Veery/Nacozari and Hirmand have different performance for this traits. Eight parental genotypes and their resulting 28 F3s were grown in three replications of randomised complete block design. Drought stress was performed with irrigation at 75% soil moisture depletion, the data collected were subjected to analysis of variance and combining abilities were carried out according to Griffings method 2, model 1. General combining ability and specific combining ability effects were significant for traits membrane stability index, stomatal conductance, relative water content and chlorophyll content; however, the cultivar Chamran was introduced as a good combiner based on general combining ability and also the line Irena/Babax//Pastor for membrane stability index. The most inbreeding depression was recorded for chlorophyll content. Key words: Physiological parameters, combining ability, drought stress, inbreeding depression.

Introduction The best option for crop production, yield improvement and yield stability under soil water stress condition is to develop drought tolerant varieties. A physiological approach would be the most attractive way to develop new wheat varieties rapidly, but breeding for specific, water stress tolerance need a deeper understanding of the yield determining traits like physiological characters. This is where the knowledge of crop responses to water stress may be best put to use 1. One of the most complex designs that has been used extensively for the genetic analysis of quantitative characters such as drought is the diallel cross. The diallel cross designs are frequently used in plant breeding research to obtain information about genetic properties of parental lines or estimates of general and specific combining abilities and heritability 2 . The physiological characters play an important role in grain yield, stomata play an important role in regulating plant water stress, and relative water content varies from one species to another, and is influenced by the environmental conditions under which a plant is grown 3. In general, breeding for drought tolerance involves combining good yield potential in the absence of the stress and the selection of high heritable traits that provide drought stress tolerance 4. The concept of combining ability refers to the capacity or ability of a genotype to transmit superior performance to its crosses. The value of an inbred line depends on its ability to produce superior hybrids in combination with other inbreds combining ability analysis helps to evaluate inbreds in terms of their genetic value 5. In addition, diallel cross provides early

information on the genetic behavior of attributes in the generation6. Several methods have been proposed for the genetic analysis of data from a diallel cross. The approaches of Griffing 7 and Hayman 8 are statistically similar in their analyses of variance, the Griffings 6,7 general and specific combining abilities are mathematically identical to Haymans 8 additive and dominance components. They differ, however, in the genetic assumptions, information and interpretation. Therefore, this study was conducted (1) to estimate the combining ability of selected lines and cultivars for tolerance to drought using physiological traits as indirect indicators and (2) to determine the inbreeding depression of related traits after three generations. Materials and Methods Plant material: Eight bread wheat cultivars were used as parents (Table 1). Parental genotypes were derived from Seed and Plant Improvement Institute, Karaj, Iran, and tolerance status was determined from preliminary and advance experiments in this institute. Crosses for a half-diallel among these wheats were made in the Agriculture and Natural Resources Research Center of Sistan, Iran. F3 hybrids and their parents were planted under shelter in pots. Trial: In the season (201011), the 28 F3 hybrids and their eight parents were sown in plastic pots filled with a soil mixture containing soil/sand/organic matter in a ratio of 1:1:1 in Experiment Farm of University Putra Malaysia. Four seeds were sown in each
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Table 1. Genotype name and pedigree.

No 1 2 3 4 5 6 7 8 Pedigree/Name Irena/Babax//Pastor S-78-11 Tajan Chamran Hamoon Moghan3 Veery/Nacozari Hirmand Tolerance status Tolerant Tolerant Susceptible Tolerant Semi-tolerant Susceptible Tolerant Semi-tolerant

pot. The pots were irrigated after 75% depletion of the soil water. Each pot was filled with 3 kg of air-dried soil and soil field capacity was calculated on the soil dry weight basis. Water stress treatments were 75% of moisture depletion of field capacity by weight. The pots were weighed at two days intervals to compensate the water loss by evapotranspiration and irrigation was performed after 75% depletion of field capacity of soil, and genotypes were arranged as a completely randomised block design. The chlorophyll content was measured 3 times. Measurements were made on the flag leaf on two seedlings per pot, with a chlorophyll meter, (SPAD-502, Soil Plant Analysis Development (SPAD) Section, Minolta Camera Co,Osaka, Japan). Three readings were taken along the middle section of the leaf, and the mean was used for analysis and values were expressed as SPAD units. Stomatal conductance was measured using an IRGA (Infra Red Gas Analyzer, LCA-4, Analytical Development Corporation, UK) on the abaxial surface of the mid portion of flag leaf between 10:00 am and 14:00 pm at grain filling stage. Leaf membrane stability index (MSI) was determined according to the method of modified by some researcher 14, 15. Leaf discs (100 mg) were thoroughly washed in running tap water followed by washing with double distilled water, thereafter the discs were heated in 10 ml of double distilled water at 40oC for 30 min. Electrical conductivity (C1) was recorded by EC (electrical conductivity) meter. Table 2. Analysis of variances for F3 generation (mean squares). Subsequently the same samples were placed in a Stomatal Membrane Relative boiling water bath (100oC) for 10 min and their S.O.V DF conductance stability index water content electrical conductivity was also recorded (C2). The Replication 2 145.8 32.9 0.009 MSI was calculated as: Genotype 35 6524.05** 14.23** 137.56**
Error 70 76.83 19.26 4.22

Results and Discussion Combining ability analysis: Analysis of variance revealed significant differences among parents and hybrids for physiological traits, indicating the different responses of genotypes to water stress condition and possible selection of drought tolerant genotypes under water deficit (Table 2). Analysis of variance for stomatal conductance, membrane stability index, relative water content and chlorophyll content revealed significant mean squares of GCA and SCA for all the traits indicating the importance of additive and dominance genetic effects and variance of GCA for all traits except relative water content were more than variance of SCA and this indicates more additive control than non-additive effects for this traits (Table 3). Knowledge about genetic mechanism, involved in the expression of yield-related traits, is helpful in developing superior genotypes. The information about the relative contribution of components of variation, i.e. additive, non-additive and epistasis, is essential for effective plantimprovement exercise 9. Among various techniques, genetic analysis formulated by Griffing 6,7, provides a workable approach, to evaluate newly developed cultivars for their parental usefulness and to assess the gene action involved in various attributes, so as to design an efficient breeding plan, for further genetic upgrading of the existing material. On the basis of GCA estimation effects it can be concluded that for each physiological parameter different parents were considered as good general combiners indicating genetic diversity (Table 4). Chamran, Hamoon and Veery with significant negative and significant GCA effects were good combiners for stomatal conductance under drought stress. The best general combination with positive and significant effects was Irena/Babax//Pastor and S-78-11 for membrane stability index, Irena/Babax//Pastor and S-78-11 for relative water content and cultivar Chamran for chlorophyll content. Farshadfar et al. 10 did

Clorophyll content 18.77 60.27** 8.71

Membrane stability index (MSI) = [1 - (C1/ C2)] 100. Leaf relative water content (RWC) was estimated according to the method of Ekanayake et al. 16. Leaf material was weighed (2 leaves) to determine fresh weight and placed in distilled water at +4C for 19 h, and turgid weight was recorded. Finally, the samples were dried in an oven at 65-70C for 48 h and dry weights were recorded. RWC was calculated as: RWC= [(fresh weight- dry weight)/ (turgid weight- dry weight)] x100 Statistical analysis: Data were subjected to analysis of variance. Data obtained from the 28 hybrids of F3 and eight parents were subjected to analysis by Griffings method 2, model 1. To investigate relative performance of parent lines and crosses ANOVA was performed on variables by SAS 17.

** Significant at 1% level.

Table 3. Analysis of variance (mean squares) for combining ability in F3 generation.

Source of variation Replication GCA SCA Error DF 2 7 28 70 Stomatal conductance 124.25 32258.4** 7000.2** 133.1 Membrane stability index 34.29 93.44** 87.51** 22.59 Relative water content 0.45 146.95** 157.12** 4.28 Chlorophyll content 25.83 295.8** 28.23** 9.09

**Significant at 1% level.

Table 4. Values of general combining ability (GCA).

Line/Cultivar Irena/Babax//Pastor S-78-11 Tajan Chamran Hamoon Moghan3 Veery/Nacozari Hirmand SE (gi)
** Significant at 1% level.

Stomatal conductance 63.8** 19.4** 35.3** -54.7** -31.3** -28.1** -35.1** 30** 3.41

Membrane stability index 4.79** 1.85 -0.82 -2.15 -0.71 -1.60 -0.10 -1.26 1.40

Relative water content 1.72** 1.72** -3.28** 5.33** -2.33** -1.33** -2.17** 0.33 0.61

Clorophyll content 0.32 0.49 -2.3** 8.8** -4.4** -3.2** 0.10 0.26 0.88

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not obtain any genotypic variability for relative water content in first generation of wheat under drought stress. The best specific combinations (Table 5) for improvement of stomatal conductance, membrane stability index, relative water content and chlorophyll content were VeeryHirmand, IrenaMoghan3, S-78-11Moghan3 and Moghan3Hirmand crosses, respectively, indicating that parents of these crosses are genetically diverse. Crosses S-781Hirmand, Chamran Veery/Nacozari, Irena/Babax// PastorMoghan3 and Irena/Babax//PastorTajan produced the most significant positive heterosis for chlorophyll content, chlorophyll a, b and total chlorophyll, respectively11. This could be due to either epistatic or maternal effects, also GCA and SCA were highly significant (P0.01) for chlorophyll content, chlorophyll a and b and total chlorophyll (Table 3). Table 5. Values of specific combining ability (SCA).
Hybrids Irena S-78-11 Irena Tajan Irena Chamran IrenaHamoon IrenaMoghan3 IrenaVeery IrenaHirmand S-78-11Tajan S-78-11Chamran S-78-11Hamoon S-78-11Moghan3 S-78-11Veery S-78-11Hirmand TajanChamran TajanHamoon TajanMoghan3 TajanVeery TajanHirmand ChamranHamoon ChamranMoghan3 ChamranVeery ChamranHirmand HamoonMoghan3 HamoonVeery HamoonHirmand Moghan3Veery Moghan3Hirmand VeeryHirmand SE.sij Stomatal conductance 51.49** -30.7** -61** 86.9** -47.5** -23.9** 24.8* -38.9** 11.7 23.3* -25.1* 14.1 -36.7** -30.1** 18.4 -3.1 35.2** 49** 12.8 4.7 26.6* 35.1** -22.6* -30.06** -88.9** 27.44** 66.2** -49.5** 10.46 Membrane stability index -8.8** -0.82 1.52 5.07 8.29* -2.87 -2.37 -1.87 -2.54 7.68* -3.43 6.47* 2.57 2.13 -2.98 -3.76 3.74 3.57 -6.32 7.24* -5.60 3.57 -3.87 -1.37 1.79 2.18 -6.65* -2.48 3.30 Relative water content -8.73** 1.60 0.99 -7 6.3** 11.8** -5** 2.94 -5.01** 6.33** 13.3** -6.5** -2.3 -4.6* -1.67 -3 -3.8* 8.6** 4.7* 0.7 -5.4** 8.7** -5.29** 6.21** -3.2 -3.7* -8.2** 1.5 1.87 Chlorophyll content 3.09 0.25 1.03 3.03 0.81 -3.19 -5.02 2.09 -0.13 -3.13 1.64 -1.02 -2.52 1.37 -2.63 -4.86 1.81 1.98 2.81 -1.41 -2.08 -1.58 -3.41 2.25 1.09 1.70 5.53* 0.53 2.7

Table 6. Inbreeding depression for traits in F3 generation.

Hybrids Irena S-78-11 Irena Tajan Irena Chamran IrenaHamoon IrenaMoghan3 IrenaVeery IrenaHirmand S-78-11Tajan S-78-11Chamran S-78-11Hamoon S-78-11Moghan3 S-78-11Veery S-78-11Hirmand TajanChamran TajanHamoon TajanMoghan3 TajanVeery TajanHirmand ChamranHamoon ChamranMoghan3 ChamranVeery ChamranHirmand HamoonMoghan3 HamoonVeery HamoonHirmand Moghan3Veery Moghan3Hirmand VeeryHirmand Stomatal conductance -0.32 2.01 0.30 3.64 0.96 2.27 4.61 2.68 2.65 3.07 2.43 2.75 3.82 1.98 2.28 4.38 2.68 2.81 2.98 4.50 7.47 4.70 12.73 7.98 1.67 2.55 3.26 6.76 Membrane stability index 6.67 4.05 1.62 3.49 -0.57 0.00 4.98 4.48 1.65 6.25 0.50 -1.91 3.08 0.52 4.06 5.18 2.10 2.30 -2.33 3.67 4.60 4.19 8.51 -4.05 2.02 -2.15 -6.04 5.31 Relative water content 1.52 -0.51 0.00 5.56 0.00 0.55 1.02 -1.13 5.71 4.23 5.09 9.71 7.80 1.83 7.03 5.13 6.03 6.16 9.02 7.51 3.23 5.46 5.36 7.06 6.71 7.48 6.85 5.34 Chlorophyll content 5.51 4.37 3.58 -3.25 -5.74 -6.21 -4.94 1.84 4.02 -3.85 -1.31 12.78 6.16 10.13 8.44 9.65 9.89 11.86 11.45 5.61 -0.21 11.05 8.53 12.56 6.83 7.86 11.56 11.06

Conclusions For stomatal conductance, cell membrane stability, relative water content general combining ability (GCA) and specific combining ability (SCA) were highly significant, but variance for GCA had a more value than SCA except relative water content, indicating the importance of additive effects of genes compared to interaction effects (dominance and epistasis). The line Chamran had a high negative GCA value for stomatal conductance and a good performance under drought stress. Also, cultivar Irena/Babax// Pastor had high positive value for cell membrane stability index, also this line had a high GCA value for relative water content, and cultivar Chamran had the best performance for GCA effect among genotypes for chlorophyll content. Therefore, these lines and cultivars were recommended to put into the breeding program. Acknowledgement The authors gratefully acknowledge Dr Mihdzar Abdul Kadir for his contribution. References
Desalegn, D., Bedada, G., Zewdie, A. and Gelalcha, S .2001. Drought tolerance of some bread wheat genotypes in Ethiopia. Afr. Crop Sci. J. 9(2):385-392. 2 Iqbal, M., Navabi, A., Salmon, D. F., Yang, R. C., Murdoch, B. M., Moore, S. S. and Spaner, D. 2007. Genetic analysis of flowering and maturity time in high latitude spring wheat. Euphytica 154(1-2):207218. 3 Munir, F. 1997. Genetic Control of Some Metric Traits Determining Grain Yield in Bread Wheat. M.Sc. (Hons.) Agri, thesis. Dept. Pl. Br. Genet., Univ., Agric., Faisalabad, Pakistan, pp. 68-94. 4 Jones, H. G. 2007. Monitoring plant and soil water status: Established and novel methods revisited and their relevance to studies of drought
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*and ** Significant at 5% and 1% statistical levels.

Inbreeding depression: With some exceptions, inbreeding reduces offspring fitness in essentially all naturally outcrossing plants and to a lesser extent in selfing species. The negative effects of mating between relatives have been noticed for many centuries. The negative effects of inbreeding have since been observed in both outcrossing and selfing species for a variety of traits with consequences for offspring fitness 12, 13. Totally more inbreeding depression in this experiment was obtained for chlorophyll content than other traits. Crosses HamoonMoghan3, HamoonVeery, S-78-11Veery and S-78-11Hirmand had the most inbreeding depression for stomatal conductance, membrane stability index, relative water content and chlorophyll content, respectively (Table 6).

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tolerance. J. Exp. Bot. 58:119130. Singh, P. and Narayanan, S. S .1993. Biometrical Techniques in Plant breeding. Kalayani Publishers, New Delhi, India, pp.45-73. 6 Griffing, B. 1956a. A generalized treatment of the use of diallel crosses in quantitative inheritance. Heredity 10:31-50. 7 Griffing, B. 1956b. Concept of general and specific combining ability in relation to diallel crossing systems. Aust. J. Biol Sci. 9:463-493. 8 Hayman, B. I . 1954. The analysis of variance of diallel tables. Biometrics 10:235-244. 9 Azhar, F. M. and Ajmal, S. U. 1999. Diallel analysis of oil content in seed of Gossypium hirsutum L. J. Genet. and Breed. 53:19-23. 10 Farshadfar, E., Rasoli, V. and Farshadfar, M. 2011. Inheritance of drought tolerance indicators in bread wheat (Triticum aestivum L.) using a diallel technique. Aust. J. Crop Sci. 5(7):870-878. 11 Naroui Rad, M. R., Abdul Kadir, M. and Yusop, M. R. 2012. Genetic behaviour for plant capacity to produce chlorophyll in wheat (Triticum aestivum) under drought stress. Aust. J. Crop Sci. 6(3):415-420. 12 Charlesworth, D. and Charlesworth, B. 1987. Inbreeding depression and its evolutionary consequences. Ann. Rev. Ecol. and Sys. 18:237268. 13 Keller, L. F. and Waller, D. M. 2002. Inbreeding effects in wild populations. Trends Ecol. and Evol. 17(5):230-241. 14 Premchandra, G., Saneoka, H. and Ogata, S. 1990. Cell membrane stability, an indicator of drought tolerance as affected by applied nitrogen in soybean. J. Agric. Sci. 115:63-66. 15 Sairam, R . 1994. Effect of moisture stress on physiological activities of two contrasting wheat genotypes. J. Exp. Biol. 32:594-597. 16 Ekanayake, I., De Datta, S. and Steponkus, P. 1993. Effect of water decit stress on diffusive resistance, transpiration, and spikelet desiccation of rice. Annals of Botany 72:73-80. 17 SAS 1988. SAS/STAT Users Guide, Release 6.03. SAS Institute, Cary, NC, pp.145-213.
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