ACTA BOT. CROAT. 78 (2), 2019 169 Acta Bot. Croat. 78 (2), 169–174, 2019 CODEN: ABCRA 25 DOI: 10.2478/botcro-2019-0012 ISSN 0365-0588 eISSN 1847-8476 Screening for drought resistance during germination of modern and old Iberian wheat cultivars Ivo Pavia1, Luís Rocha1, José Moutinho-Pereira1, José Lima-Brito1,2,3, Carlos Correia1* 1 CITAB – Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal 2 Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal 3 BioISI-UTAD – Biosystems & Integrative Sciences Institute-University of Tras-os-Montes and Alto Douro, Portugal Abstract – Climate change is expected to reduce water availability in several wheat (Triticum aestivum L.) grow- ing areas. Drought, during and immediately after sowing can compromise germination, seedling development, and ultimately crop yield. Our study’s objective was to investigate early drought stress resistance and evaluate genetic diversity of four bread wheat cultivars – three modern elite germplasm lines (‘Antequera’, ‘Jordão’ and ‘Roxo’) and an ancient Portuguese cultivar (‘Mestiço’) – at five water potentials, ranging from 0 to –1.5 MPa. Germination was annotated daily during eight days. Dry and fresh weights as well as root and shoot growth were measured. Genetic variability among cultivars was evaluated using start codon targeted (SCoT) markers. Overall, when water potential was lowered, germination rate and seedling growth were delayed. Final germina- tion percentages were only affected by the higher stress intensity. This study reinforces the importance of re- search into ancient wheat cultivars, such as ‘Mestiço’. This cultivar showed the highest genetic difference when compared with the remaining cultivars. ‘Mestiço’ presented the highest coleoptile size, high vigor index values, and the lowest variation in water content due to stress conditions. Therefore, it may be able to germinate in ex- tremly dry soil and be the least affected by drought stress. Still, due to the comparatively reduced yield of older wheat cultivars, stress tolerance should be researched within elite cultivars. Among these, Jordão revealed par- ticularly good germination and early biomass development characteristics across water potentials, presenting a likely choice for future studies under drought stress conditions. Keywords: ancient wheat cultivar, drought, genetic diversity, germination, Triticum aestivum * Corresponding author e-mail: ccorreia@utad.pt Introduction Wheat (Triticum spp.) is one of the most important sta- ple crops worldwide (Schillinger et al. 2008). Dryland wheat farming in Mediterranean climates is highly dependent on winter precipitation and overwinter water storage in the soil. Climate change may cause many dryland production areas to become even drier, compromising the germination, emer- gence and overall yield of rainfed bread wheat (Triticum aes- tivum L.) (Eitzinger et al. 2003, Singh et al. 2013). Seed germination is critical in the early stage of growth in most plants and may determine the success of crop pro- duction (Finch-Savage and Bassel 2016). Drought is an im- portant environmental factor that negatively affects seed germination and seedling growth in dryland soil (Singh et al. 2013, Khayatnezhad and Gholamin 2011). The effects of drought stress can be mimicked resorting to polyethylene glycol (PEG). PEG is a non-ionic water polymer that is not expected to penetrate the plant tissue rapidly, thus compro- mising water uptake (Almansouri et al. 2001). Genetic diversity analysis and fingerprinting in cereals have been successfully carried out by start codon targeted (SCoT) markers (Cabo et al. 2014, Pour-Aboughardareh et al. 2017). These are highly polymorphic functional mark- ers based on the short-conserved region in plant genes sur- rounding the ATG translation start codon (Collard and Mackill 2009). Ancient wheat cultivars present an important group of genetic resources and may present abiotic stress tol- erance characteristics (Martins-Lopes et al. 2009, Carvalho et al. 2010). Nonetheless, possible stress tolerance character- istics within elite crop germplasm should not be disregarded (Gilliham et al. 2017). Our objective was to quantify early drought stress re- sistance and evaluate genetic diversity of four winter wheat cultivars – three modern elite germplasm lines developed in the Iberian Peninsula (‘Antequera’, ‘Jordão’ and ‘Roxo’) and an ancient Portuguese bread wheat cultivar (‘Mestiço’). Short communication PAVIA I., ROCHA L., MOUTINHO-PEREIRA J., LIMA-BRITO J., CORREIA C. 170 ACTA BOT. CROAT. 78 (2), 2019 Materials and methods Seeds of durum wheat (Triticum durum Desf.) ‘Core’ and bread wheat (Triticum aestivum L.) ‘Antequera’, ‘Jordão’, ‘Roxo’ and ‘Mestiço’ were used in this study. Seeds of durum wheat and modern (elite) bread wheat ‘Antequera’, ‘Jordão’ and ‘Roxo’ were stored at the Plant Breeding Station, Elvas (Portugal). Seeds of ‘Mestiço’ were obtained from the Plant Germplasm Bank, at the Department of Genetics and Bio- technology, University of Tras-os-Montes and Alto Douro, Vila Real (Portugal). Seeds of T. aestivum were disinfected using a solution of sodium hypochlorite 1% for 3 min and then washed with distilled water. Twenty seeds per cultivar/treatment (in 3 rep- etitions) were germinated in 90 mm Petri dishes with filter paper moistened with polyethylene glycol solutions (PEG- 6000) of 0, –0.33, –0.75, –1 and –1.5 MPa water potential in the dark at 25 °C. Germination was annotated daily during eight days. PEG solutions were changed at the fourth day. Germinability (G), mean time of germination (MT), mean germination rate (MR) and synchrony of germination (Z) were calculated according to Ranal et al. (2009). Five days after seed germination, longest root and coleoptile growth as well as dry and fresh weight were measured in fifteen seed- lings per cultivar × water potential combination. Water con- tent was calculated based on these values. Vigor index (VI) was calculated based on coleoptile length and germinability according to Kumari et al. (2014). Logit transformation was performed in data expressed in proportion/percentage (G and water content) prior to statistical analysis; original val- ues are shown. ANOVA and Tukey post-hoc analysis were performed in IBM SPSS Statistics V23. DNA was extracted from bulks of seven seedlings from each cultivar using DNeasy Plant Mini-kit (Qiagen) accord- ing to the manufacturer’s instructions. DNA integrity was checked by electrophoresis on 1.5% agarose gels, quantified on Nanodrop ND-1000, and diluted to 30 ng μL–1. DNA amplification was performed with 8 SCoT primers (Fig. 1a) developed by Collard and Mackill (2009). Reaction mixture was made as reported by Cabo et al. (2014) and PCR condi- tions were as follows: initial denaturation of 3 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 2 min at 72 °C; final extension was of 5 min at 72 °C. Bands were scored as described by Cabo et al. (2014). Genetic simi- larity among cultivars was accessed by an unweighted pair group method using arithmetic averages (UPGMA) den- drogram of genetic similarity constructed with the software “NTSYS-pc, Numerical Taxonomy System” (Rohlf, 1998). Results and discussion Dynamics of seed germination Wuest and Lutcher (2012) reported critical water poten- tials for 75% germination of several dryland wheat cultivars to be between –1.1 and –1.6 MPa. By contrast, Singh et al. (2013) achieved 75% germination only at –0.75 MPa and no germination at –1.5 MPa in five commonly rainfed win- ter wheat cultivars. Here, although all cultivars’ germina- bility (G) decreased at –1.5 MPa, we did not observe com- plete germination inhibition. G of modern cultivars reached near 100% from 0 till –1 MPa, while that of Mestiço was around 80% (Tab. 1). The low G value registered in Mestiço might not reflect a characteristic of this cultivar. Mestiço was not stored at the same location as the remaining cultivars, which might have significantly influenced germination per- formance (Wuest and Lutcher, 2012). Germinability was on- ly affected when the higher stress level was applied (Tab. 1). At –1.5 MPa, overall G dropped to 64.2%. In Antequera and Jordão it dropped to 71.7 and 78.3%, respectively, while in Mestiço it fell to 58.3%. In Roxo it dropped steeply from 95% at –1 MPa till 48.3% at –1.5 MPa. Mean time of germination (MTG) steady increased with lower water potentials in all cultivars from near 1 till over 4 days (Tab. 1). Mean germi- nation rate (MR) was similar in all cultivars, with the excep- tion of Mestiço, and decreased with the decrease of the water potential (Tab. 1). Synchrony of germination (Z) was overall higher in Antequera and Jordão (Tab. 1). The observed delay in germination with the decrease of water potential is con- sistent with the findings of other authors (Almansouri et al. 2001, Singh et al. 2013, Kumari et al. 2014). Early biomass production Drought highly affected fresh weight, while dry weight variation was only observed between cultivars (Fig. 2a). At 0 MPa all cultivars showed similar water content, ranging from 84.67 till 89.45% (Fig. 2b). Mestiço showed the low- est variation in water percentage between 0 and –1.5 MPa, with 89.45 and 45.81% respectively. The remaining cultivars dropped to values lower than 40% at –1.5 MPa (Fig. 2b). Khayatnezhad and Gholamin (2011) found that the effect of PEG on leaf area of 10-day-old plants was greater than the effect on fresh weight, leading us to believe that Mestiço, which showed the lowest water percentage variation across water potentials (Fig. 2b), will also be the least affected by the future growing reduction due to drought stress. Drought stress decreased both root and shoot size (Fig. 3a). Field and laboratory experiments indicate that cultivars with long coleoptiles have superior emergence on deep seed- ing (Singh et al. 2013). The highest coleoptile growth was verified in Mestiço at 0 MPa, with an average of 8.22 cm. Coleoptile length coupled with the ability of seeds to germi- nate at low water potentials are important factors control- ling winter wheat seedling emergence (Singh et al. 2013). In contrast to the modern cultivars, Mestiço is a tall wheat vari- ety (Maçãs, B. and Coutinho, J., unpublished data). Modern varieties assuredly have reduced height (Rht) genes which have been associated with reduced coleoptile size (Ellis et al. 2004). This is a possible explanation for the reduced coleop- tile size in modern cultivars when compared with Mestiço. Vigor index (VI) that considers both coleoptile length and germinability may present an important clue regarding cul- tivars ability to withstand drought stress. VI values ranged from 10.47 till 786.67 (Fig. 3b). VI was influenced by both cultivars and drought stress, as reported by others (Kumari et al. 2014). The highest values were observed, for all cul- tivars, at 0 MPa and the lowest at –1.5 MPa. The highest DROUGHT RESISTANCE DURING GERMINATION OF IBERIAN WHEATS ACTA BOT. CROAT. 78 (2), 2019 171 averaged VI values across the different water potentials were observed in Mestiço and Jordão. Ancient wheat varieties as a source of genetic diversity Ancient bread wheat cultivars show a high genetic diver- sity (Carvalho et al. 2010) and interesting agronomic traits, such as aluminum resistance (Martins-Lopes et al. 2009). Although the limitations of bulk analysis are recognized, low intra-cultivar variability is expected and it allows assessment of genetic variation among cultivars. SCoT markers showed an average of 64.6% of polymorphic bands (Fig. 1a). The genetic profile of each of the studied cultivars, based on the DNA amplification with SCoT markers, is presented in Fig- ure 1b. Mestiço showed a higher genetic difference than the Fig. 1. Results of the genetic diversity analysis: (a) sequence, number of bands and polymorphism percentage of each start codon targeted (SCoT) marker, (b) genetic profile, based on DNA amplification with SCoT markers, of bread wheat (Triticum aestivum L.) ‘Antequera’, ‘Jordão’, ‘Roxo’ and ‘Mestiço’ and durum wheat (Triticum durum Desf.) ‘Core; and (c) unweighted pair group method with arithmetic mean dendrogram of genetic similarity of the studied wheat cultivars; MW – molecular weight in base pairs. PAVIA I., ROCHA L., MOUTINHO-PEREIRA J., LIMA-BRITO J., CORREIA C. 172 ACTA BOT. CROAT. 78 (2), 2019 elite cultivars (Fig. 1c). This was expected as Mestiço is an ancient Portuguese wheat cultivar, not subjected to a mod- ern breeding program. Such a distinction is also observed in the germination (Tab. 1) and growth responses (Figs. 2, 3). Among the studied cultivars, Mestiço may be the least affected by any future growing reduction due to drought stress (Khayatnezhad and Gholamin 2011). Introgression of alleles from ancient cultivars, such Mestiço, into mod- ern ones can be useful when breeding for suboptimal en- vironments, such as those prevalent in the Mediterranean Basin (Soriano et al. 2018). Furthermore, we propose fu- ture research into Rht genes able to maintain reduced height in modern cultivars without affecting early growth, such as Rht8 (Ellis et al. 2004). Screening for drought resistance using PEG assays Development of stress-tolerant varieties is a core objec- tive of many breeding programs. Still, success has been lim- ited due to lack of adequate screening techniques (Kumari et al. 2014). Due to the vast number of ancient cultivars stored in germplasm banks, most are still to be agronomically char- acterized. Nonetheless, the use of ancient wheat cultivars in breeding programs may be useful when breeding for sub- optimal environments such as those prevalent in the Medi- terranean Basin (Soriano et al. 2018). We believe that PEG experiments, such as the one described here, are suitable for screening drought tolerance prior to field trials. This screen- ing could significantly reduce the overall cost and manpower Tab. 1. Germination parameters in wheat cultivars on drought stress. Data are mean values ± SD, n = 60. G ‒ germination percentage; MT ‒ mean time of germination; MR ‒ mean germination rate; Z ‒ synchrony of germination. Different letter (a, b, c) correspond to statistical differences among results by the Tukey-test (p < 0.05) Cultivar (cv) G (%) MT (day) MR (day–1) Z Antequera 92.00 ± 11.46 a 2.48 ± 1.42 a,b 0.531 ± 0.271 a 0.656 ± 0.216 a Jordão 94.00 ± 9.30 a 2.28 ± 1.15 a 0.549 ± 0.260 a 0.683 ± 0.223 a Mestiço 80.67 ± 14.00 b 2.69 ± 0.98 b 0.419 ± 0.057 b 0.490 ± 0.159 a,b Roxo 88.67 ± 21.42 a 2.48 ± 1.33 a,b 0.519 ± 0.265 a 0.597 ± 0.247 b Water potential (W, in MPa) 0 95.83 ± 6.34 a 1.16 ± 0.23 a 0.884 ± 0.140 a 0.804 ± 0.209 a –0.33 97.08 ± 4.98 a 1.77 ± 0.37 b 0.586 ± 0.112 b 0.557 ± 0.123 b –0.75 95.83 ± 5.97 a 2.15 ± 0.19 c 0.469 ± 0.041 c 0.748 ± 0.171 a –1 91.25 ± 9.08 a 2.79 ± 0.31 d 0.363 ± 0.044 d 0.523 ± 0.202 b,c –1.5 64.17 ± 14.72 b 4.56 ± 0.40 e 0.221 ± 0.019 e 0.400 ± 0.113 c cv × W Antequera × 0 96.67 ± 5.77 1.05 ± 0.05 0.954 ± 0.045 0.904 ± 0.095 Antequera × –0.33 96.67 ± 5.77 1.59 ± 0.25 0.640 ± 0.112 0.523 ± 0.075 Antequera × –0.75 96.67 ± 2.89 1.98 ± 0.16 0.506 ± 0.040 0.838 ± 0.103 Antequera × –1 98.33 ± 2.89 2.86 ± 0.31 0.352 ± 0.037 0.554 ± 0.151 Antequera × –1.5 71.67 ± 5.77 4.93 ± 0.39 0.204 ± 0.016 0.459 ± 0.192 Jordão × 0 100.00 ± 0.00 1.03 ± 0.03 0.968 ± 0.027 0.933 ± 0.058 Jordão × –0.33 100.00 ± 0.00 1.53 ± 0.12 0.655 ± 0.052 0.472 ± 0.040 Jordão × –0.75 100.00 ± 0.00 2.10 ± 0.13 0.477 ± 0.029 0.835 ± 0.205 Jordão × –1 91.67 ± 7.64 2.49 ± 0.40 0.408 ± 0.062 0.718 ± 0.159 Jordão × –1.5 78.33 ± 2.89 4.23 ± 0.33 0.238 ± 0.019 0.459 ± 0.043 Mestiço × 0 86.67 ± 2.89 1.52 ± 0.13 0.660 ± 0.057 0.477 ± 0.051 Mestiço × –0.33 91.67 ± 5.77 2.26 ± 0.34 0.450 ± 0.072 0.632 ± 0.141 Mestiço × –0.75 86.67 ± 2.89 2.38 ± 0.11 0.420 ± 0.018 0.541 ± 0.125 Mestiço × –1 80.00 ± 8.66 3.00 ± 0.06 0.334 ± 0.007 0.437 ± 0.266 Mestiço × –1.5 58.33 ± 14.43 4.31 ± 0.02 0.232 ± 0.011 0.363 ± 0.071 Roxo × 0 100.00 ± 0.00 1.05 ± 0.05 0.954 ± 0.045 0.904 ± 0.095 Roxo × –0.33 100.00 ± 0.00 1.70 ± 0.28 0.599 ± 0.095 0.602 ± 0.177 Roxo × –0.75 100.00 ± 0.00 2.12 ± 0.10 0.473 ± 0.024 0.779 ± 0.050 Roxo × –1 95.00 ± 5.00 2.80 ± 0.22 0.359 ± 0.028 0.384 ± 0.084 Roxo × –1.5 48.33 ± 10.41 4.76 ± 0.25 0.211 ± 0.011 0.320 ± 0.064 p-values cv p < 0.001 p < 0.001 p < 0.001 p = 0.001 W p < 0.001 p < 0.001 p < 0.001 p < 0.001 cv × W p = 0.044 p = 0.003 p < 0.001 p = 0.006 DROUGHT RESISTANCE DURING GERMINATION OF IBERIAN WHEATS ACTA BOT. CROAT. 78 (2), 2019 173 Fig. 2. Mean values of (a) fresh and dry weigh, and (b) water content of 5-day old seedlings of four Iberian cultivars at five water potentials, n = 15. Results of two-way ANOVA showed p-values for cultivar (cv), water potential (W) and the interaction of both factors (cv × W) for each parameter. p-values for fresh weight: cv = 0.015, W < 0.001, cv × W = 0.275; p-values for dry weight; cv < 0.001, W = 0.047, cv × W = 0.933; p-values for water content: cv < 0.001, W < 0.001, cv × W = 0.049. Fig. 3. Mean values of (a) root and coleoptile length, and (b) vigor index of 5-day old seedlings of four Iberian cultivars at five water potentials, n = 15. Results of two-way ANOVA showed p-values for cultivar (cv), water potential (W) and the interaction of both factors (cv × W) for each parameter. p-values for root length: cv < 0.001, W < 0.001, cv × W < 0.001; p-values for coleoptile length: cv < 0.001, W < 0.001, cv × W < 0.001; p-values for vigor index: cv < 0.001, W < 0.001, cv × W = 0.003. PAVIA I., ROCHA L., MOUTINHO-PEREIRA J., LIMA-BRITO J., CORREIA C. 174 ACTA BOT. CROAT. 78 (2), 2019 required for field trials, as reported previously by others (Singh et al. 2013, Kumari et al. 2014, Gilliham et al. 2017). Conclusion This study reinforces the importance of research into an- cient wheat cultivars, such Mestiço. This cultivar showed the highest coleoptile size, high VI values, as well as the lowest variation in water content due to stress conditions. All these features lead us to believe it may be able to germinate in ex- tremely dry soil and may be the least affected by any future growing reduction due to drought stress. Still, to be relevant to breeders, research into improving stress tolerance should focus on elite cultivars. Paradoxically, breeding programmes have been traditionally focused on maximising yield, while disregarding abiotic stress tolerance. Among the modern cul- tivars, Jordão revealed particularly good germination charac- teristics, such G and Z as well as VI values across water po- tentials. This elite cultivar, which has shown high and stable yield in various environments (Coco, 2013), presents a like- ly choice for future studies under drought stress conditions. Acknowledgements We thank Eng. Coutinho (INIAV-Elvas) for supplying seeds of durum wheat ‘Core’ and bread wheat ‘Antequera’, ‘Jordão’ and ‘Roxo’ stored at the Plant Breeding Station at Elvas (Portugal). The authors IP (PD/BD/113611/2015) and LR (PD/BD/113612/2015) acknowledge their doctoral grants funded by the FCT (Portuguese Foundation for Science and Technology) under the Doctoral Program “Agricultural Pro- duction Chains – from fork to farm” (PD/00122/2012). This work was also supported by National Funds by FCT, under the project UID/AGR/04033/2019. References Almansouri, M., Kinet, J.M., Lutts, S., 2001: Effect of salt and os- motic stresses on germination in durum wheat (Triticum du- rum Desf.). Plant and Soil 231, 243–254. Cabo, S., Ferreira, L., Carvalho, A., Martins-Lopes, P., Martín, A., Lima-Brito, J., 2014: Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritor- deums and their respective parents. Journal of Applied Ge- netics 55, 307–312. Carvalho, A., Guedes-Pinto, H., Martins-Lopes, P., Lima-Brito, J., 2010: Genetic variability of Old Portuguese bread wheat cul- tivars assayed by IRAP and REMAP markers. 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