Genetic variation of salinity tolerance in Chinese natural bermudagrass (Cynodon dactylon (L.) Pers.) germplasm resources

Bermudagrass (Cynodon dactylon (L.) Pers.) is a widely used turfgrass in tropical, subtropical and warm-temperate regions in the world. It is a salinity-tolerant plant, but much genetic variation exists among its genotypes. Genetic diversity has been shown to exist from a morphologic level to a molecular level among Chinese natural bermudagrass germplasm resources; however, the extent of variation in their salinity tolerance has remained unclear. We conducted greenhouse hydroponic and sand culture experiments to evaluate the variation in salinity tolerance of natural bermudagrass germplasm resources obtained from their main areas of distribution in China and treated at a 33 dS m−1 salinity level for three weeks. Compared with non-saline controls, salinity stress significantly decreased shoot clipping weight (46.5% and 44.2%) and increased leaf firing (41.1% and 37.6%) in hydroponic and sand culture experiments, respectively, across all genotypes. However, significant genetic variations in relative leaf firing percentage (RLF) and relative shoot weight (RSW) were found among genotypes in both experiments, and their coefficients of variation ranged from 25.5% to 41.6%, indicating that considerable variation exists in the salinity tolerance of Chinese natural bermudagrass germplasm resources. Shoot Na+ concentrations increased under salinity stress for all genotypes in both experiments. However, significant genetic variations were also found in shoot Na+ concentrations. Salinity tolerance in bermudagrass genotypes was found to be significantly negatively correlated with shoot Na+ concentrations in both experiments. Using a cluster analysis of RLF and RSW data, all genotypes were classified into four groups with different salinity tolerances. The results of our evaluation indicate that there is much potential for improving salinity tolerance among Chinese natural bermudagrass cultivars.


Introduction
Saline soil naturally occurs extensively around the seashore and in arid and semi-arid regions . Using saline and non-potable water in turf landscape where there has been a shortage of fresh water and using de-icing salt on roads during the winter often results in the accumulation of high concentrations of ions in normal soils (Qian & Mecham 2005;Friell et al. 2012). High salinity is one of the most important environmental stresses impeding plant growth (Turhan et al. 2013;Yang et al. 2013). It severely reduces turf quality and aesthetic appearance (Chen et al. 2009;Tang et al. 2013aTang et al. , 2013b. Breeding salinity-tolerant turfgrass cultivars is a perfect approach for establishing highquality turf in highly saline soils (Lee et al. 2005;Marcum 2006).
Bermudagrass (Cynodon dactylon (L.) Pers.), which belongs to the Poaceae family, subfamily Chloridoideae, is a warm-season perennial turfgrass. It is widely used for turf establishment on golf courses, sports fields, residential lawns and public parks (Pompeiano et al. 2013). Bermudagrass grows naturally in a wide range of soil types, pH levels and soil water levels, which makes it one of the most widespread and common species in tropical, subtropical and warm-temperate regions (de Silva & Snaydon 1995;Taliaferro 2003). It has also been found in saline soil environments (Hameed et al. 2011). Significant genetic variation has been found in many featuresfrom a morphologic level to a molecular level (de Silva & Snaydon 1995;Wu et al. 2006a;Anderson et al. 2009;Gulsen et al. 2009;Zhou et al. 2013).
Bermudagrass is a miohalophyte and has been shown to be a salinity-tolerant turfgrass (Maas & Hoffman 1977;Worku & Chapman 1998), but genetic variation exists among its ecotypes (Marcum & Pessarakli 2006;Hameed et al. 2011;Hu et al. 2012). This indicates that, by means of breeding programmes, we can develop new cultivars with improved salinity tolerance. Increased leaf firing and reduced shoot growth often occur under salinity stress in bermudagrass (Marcum & Pessarakli 2006;Chen et al. 2009). The majority of salinity-tolerant bermudagrass genotypes have a higher rate of shoot growth and a lower percentage of leaf firing (or higher green leaf canopy area; Marcum & Pessarakli 2006). Therefore, relative shoot growth rate and leaf firing percentage are good indicators with which to evaluate relative salinity tolerance in bermudagrass.
Salinity stress has been found to increase shoot Na + concentration in bermudagrass (Dudeck et al. 1983;Chen et al. 2009). However, studies have shown that Na + accumulation in the shoots can be reduced by secretion through salt glands and by selective transport of K + ions over Na + ions from roots to leaves (Marcum 1999;Chen et al. 2009). Other studies have found that lower Na + concentration in the shoots in salinity-tolerant bermudagrass genotypes based on a limited number of accessions (Hameed et al. 2011;Hu et al. 2012). Marcum and Pessarakli (2006) confirmed that salinity tolerance was negatively correlated to shoot Na + concentration in 35 bermudagrass cultivars. However, this relationship was unclear in large-scale experiments with wild germplasm resources.
Bermudagrass is indigenous to, and widely distributed throughout, China: from Hainan Province (north latitude 18°14′, southern China) to northern Xinjiang Province (north latitude 44°42′, northern China) and from Shandong Province (east longitude 122°02′, eastern China) to western Xinjiang Province (east longitude 75°05′, western China). The climate and soil type of these habitats are very different (Liu et al. 1998). Some research has found large differences in morphology (e.g. rhizome characteristics, creeping characteristics, turf height, turf density, leaf length, leaf colour and seed yield; Liu et al. 2002Liu et al. , 2003Zheng et al. 2003a;Wu et al. 2006b;Wang et al. 2009a), electrophoretic isozymes (Zheng et al. 2003b) and chromosome numbers (Wu et al. 2006a) in Chinese natural bermudagrass germplasm. Genetic variation at a molecular level has also been found based on molecular marker technologies such as amplified fragment length polymorphism (Wu et al. 2006b), inter-simple sequence repeats (Li et al. 2011;Wang et al. 2013), simple sequence repeats (Wang et al. 2013) and sequence-related amplified polymorphism (Wang et al. 2009b). However, little information has been published regarding the salinity tolerance of Chinese natural bermudagrass germplasm.
Biodiversity is very important to plant breeders (Ulukan 2011). Wild bermudagrass from different ecological districts is an important source of biodiversity. The evaluation of salinity tolerance in these resources is a key determinant in breeding for improved salinity tolerance among bermudagrass cultivars. The objective of this study was to determine whether significant variation exists in salinity tolerance among Chinese natural bermudagrass germplasm resources from diverse geographical regions by measuring leaf firing and shoot growth responses following three weeks' exposure to high-salinity stress (NaCl, 33 dS m 1 ) and to examine the relationship between salinity tolerance and Na + concentrations in the shoots of these germplasm resources.

Hydroponic culture experiment
About 900 wild bermudagrass germplasm resources were collected from the main areas of distribution in China and 51 accessions were selected from this large sample pool as representative of diverse geographies for the materials in this experiment (Experiment 1; Table 1). The experiment was conducted from 21 June 2010 to 20 October 2010 in a solution culture system described by Qian et al. (2000) with small modifications. Uniform sprigs of each grass were planted into plastic cups (5 cm diameter × 5 cm depth) filled with coarse, acid-washed silica sand. The cup bottom was removed and covered with a nylon screen to hold sand and allow roots to grow through it. The cups were suspended by 2 cm thick polyvinyl chloride (PVC) sheets over plastic tanks containing 40 L of a constantly aerated nutrient solution (Hoagland & Snyder 1933). The nylon screen bottom of each cup was submersed 1.0 cm into the solution. Six tanks were used and each tank accommodated 51 cups; specifically, one cup represented one genotype. The experiment was conducted in greenhouses located at the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences (north latitude 32°02′, To ensure complete establishment, plants were grown for three months before the start of salt treatment. During this period, water lost from the nutrient solution was supplemented on another day using tap water to ensure the roots were constantly suspended in the solution, and the nutrient solution was adjusted at the same time to maintain a pH of about 6.0 using 1 mol L −1 hydrochloric acid solution. The culture solution was changed every week. Before the start of treatment, the shoots were clipped weekly to a 3 cm height and the roots at the base of the cups were clipped; all clippings were discarded. Three tanks were then subjected to NaCl treatment. To avoid salt shock, the salinity level was increased by about 4 dS m −1 daily for eight days, with a final salinity level of 33 dS m −1 . The turfgrasses were exposed to this salt concentration for three weeks. Another three tanks, used as controls, received no NaCl (1.25 dS m −1 ). After supplementation of any lost water, the solutions were monitored for salinity level using a conductivity metre (model YSI 85, YSI Incorporated, Yellow Springs, OH, USA), and adjusted if necessary. After the turfgrasses had reached the final salinity level, they were again clipped and the clippings discarded; they were not clipped again until the end of the experiment.
At the end of salt treatment, we estimated the relative leaf firing percentage (RLF), expressed as a percentage of total leaf surface firing in relation to control plants (Marcum et al. 1998), with 0% corresponding to no leaf firing and 100% corresponding to totally brown leaves. The shoots (clippings) were harvested at a height of 3 cm. The harvested plant materials were washed three times using double-distilled water (DD water), and then oven-dried at 80°C for 24 h; dried weights were measured and recorded. Relative shoot weight (RSW) was calculated from the following equation: clipping dry weight with salt treatment clipping dry weight with control Each dried shoot sample was grounded to a powder and approximately 20 mg was placed into a test tube containing 15 ml DD water and sealed. After extraction by boiling for 1 h and filtering, the Na + concentration of the extract was measured by Note: Means (n = 3) followed by the same letter in the same column are not significantly different (P < 0.05). CV, coefficients of variation; RLF, relative leaf firing percentage; RSW, relative shoot clipping weight.
flame photometry (Model FP6410; Shanghai Xinyi Instruments Inc., Shanghai, China) according to the method described by Matsushita and Matoh (1991). All ion concentrations were calculated on a tissue dry weight (DW) basis (mmol kg −1 DW).

Sand culture experiment
In this experiment (Experiment 2), 26 genotypes were randomly selected from Experiment 1 ( Table 2). Experiment 2 was conducted between 15 June 2011 and 23 October 2011 using PVC tubes with saline water irrigation, as described by Fu et al. (2005), with some modifications. Uniform sprigs of each grass were planted in PVC tubes (length 60 cm, diameter 10 cm) filled with fine river sand. A PVC cap with four 0.5-cm diameter holes was positioned at the bottom of each PVC tube for drainage. The plants were maintained in a greenhouse, with average maximum/ minimum temperatures of 30.1°C/22.2°C and a 13-h photoperiod. The PAR on a horizontal plane just above the canopy ranged from 1250 µmol m −2 s −1 to 1750 µmol m −2 s −1 , provided by sunlight.
The plants were grown in the PVC tubes for about three months, allowing the shoots and roots to establish before start of treatments. During this period, the tubes were watered every four days using tap water until the water drained freely from the holes at the bottom and were fertilized every eight days with compound fertilizer (9N-9P-7K) to provide 6 kg N ha −1 throughout the experiment. The turfgrasses were hand-clipped weekly to a height of 3 cm in this experiment.
Saline irrigation water was prepared by the addition of NaCl to tap water to obtain the desired salinity of 33 dS m −1 . The saline water, together with tap water as the control, was applied to all the turfgrasses: 600 ml per tube every four days. Excess water drained freely from the bottom of the tube to reduce salt accumulation in the sand. To avoid salt shock, salinity levels were gradually increased by increments of about 8 dS m −1 every two days after the start of the salt treatment. The turfgrasses were exposed to the final salt concentration for three weeks. During establishment, and during periods of increasing salinity, the clippings were discarded. Note: Means (n = 3) followed by the same letter in the same column are not significantly different (P < 0.05). CV, coefficients of variation; DW, dry weight; RLF, relative leaf firing percentage; RSW, relative shoot clipping weight.
After the final salinity had been reached, the turfgrasses were not clipped until the end of the experiment. At the end of the NaCl treatments, the RLF was estimated, and the RSW and shoot Na + concentrations were measured as described in Experiment 1.

Experimental design and statistical analysis
Experiment 1 followed a split-plot design with three replications, with salt treatment being the main plot and genotypes being the subplot. Experiment 2 followed a completely randomised design with three replications. All data were subjected to analysis of variance, Duncan's multiple range test and cluster analysis by SPSS 13.0 software (SPSS Institute, Cary, NC, USA).

Leaf firing
Salinity had an adverse effect on above-ground growth in all bermudagrass genotypes in both experiments (Tables 1 and 2). RLF increased under salinity stress, averaging 41.1% and 37.6% in the hydroponic culture system (HCS) and sand culture system (SCS), respectively, compared with control conditions, and the total coefficients of variations (CVs) among genotypes were 37.8% and 41.6%, respectively. The lowest RLF percentages were 10.0% and 8.3% and the highest were 68.3% and 65.0% in the HCS and SCS, respectively. The results indicated that the significant genetic variation in salinity tolerance existed among genotypes in both experiments, and the variation among Chinese natural bermudagrass germplasm resources was higher than the results reported by Marcum and Pessarakli (2006), who found that the percentage of green leaf canopy areaan indicator similar to RLFranged from 10% to 36% at a salinity level of 60 dS m −1 in 35 bermudagrass cultivars. The visual quality, or aesthetics, of turfgrasses has been shown to be more important than growth parameters (Marcum & Pessarakli 2010). Both leaf firing and green leaf canopy area have been widely used as evaluation indicators of salinity tolerance in turfgrasses (Qian et al. 2000Alshammary et al. 2004;Marcum & Pessarakli 2006;Chen et al. 2009). In terms of RLF, the most statistically significant group (i.e. the most salinity-tolerant genotypes) comprised of C416, C298, C737, C291 and C699 in the HCS and C291 and C720 in the SCS. The least statistically significant group (i.e. the most salinity-sensitive genotypes) comprised of C580, C177, C808, C089, C795, C653, C106, C572, C461 and C788 in the HCS and C801, C572 and C704 in the SCS (Tables 1 and 2).

Shoot growth
Shoot growths of all genotypes were inhibited after three weeks' exposure to 33 dS m −1 salinity level in both experiments (Tables 1 and 2). RSW averaged 46.5% and 44.2% in the HCS and SCS, respectively, under salinity stress compared with control conditions. Significant genetic variation was found in RSW, and the total CVs among all genotypes were 30.0% and 25.5% in the HCS and SCS, respectively.
The RSW of the turfgrasses indicates a regrowth ability and vigour under salinity stress, and is often used as an indicator in turfgrass salinity tolerance evaluation (Alshammary et al. 2004;Qian et al. 2004;Marcum & Pessarakli 2006). Although absolute growth has been used in some research (Lee et al. 2004a(Lee et al. , 2004b(Lee et al. , 2005, RSW under salinity stress compared with non-saline controls has been used in most studies (Alshammary et al. 2004;Marcum & Pessarakli 2006). In the present study, C737 and C180 were in the most statistically significant group, and C089, C580, C098, C799, C572, C653, C808, C795, C461, C177, C731, C106 and C704 were in the least statistically significant group in the HCS (Table 1). C291 had the highest RSW, and C580 and C704 had the lowest RSW among all genotypes in the SCS (Table 2).
Shoot Na + concentration and its relationship with salinity tolerance Salinity stress increased shoot Na + concentration in all bermudagrasses. Significant genetic variation was found among genotypes in both experiments (Tables 1 and 2). Across genotypes, shoot Na + concentration ranged from 287.70 mmol kg −1 DW to 692.62 mmol kg −1 DW in the HCS and from 380.67 mmol kg −1 DW to 673.86 mmol kg −1 DW in the SCS under salinity stress. Salinity tolerance (based on RLF and RSW) was found to be significantly negatively correlated with shoot Na + concentrations under salinity stress in both experiments (Table 3).
Plants suffer ion toxicity under salinity stress, but can increase salinity tolerance by Na + excretion from leaves (Munns & Tester 2008). Bermudagrass bears salt glands in the epidermis of the leaves, which can selectively secrete saline ions, such as Na + , and thus decrease shoot Na + concentrations (Worku & Chapman 1998). Marcum and Pessarakli (2006) confirmed that the salinity tolerance of bermudagrass was positively correlated with leaf salt gland Na + excretion rate. Chen et al. (2009) found that bermudagrass could reduce Na + accumulation in leaf not only through salt gland excretion, but also through selective transport of K + ions over Na + ions from roots to leaves. In this study, we proved that shoot Na + concentrations in natural bermudagrasses from China were negatively correlated with salinity tolerance (Table 3), which concurred with the findings of Marcum and Pessarakli (2006).

Cluster analysis
Cluster analysis can facilitate the evaluation of salinity tolerance among genotypes. Use of multivariate analysis for the evaluation of salinity tolerance is highly advantageous in that it allows for the simultaneous analysis of multiple parameters and increases accuracy in ranking the genotypes (Zeng et al. 2002). In the present study, we performed a cluster analysis of RLF and RSW data to classify the genotypes into distinct groups for each experiment (Table 4, Supplementary Figure S1, available online). Groups 1-4 in the hydroponic experiment and groups A-D in the sand culture experiment represent rankings of salinity tolerance from highest to lowest, and the difference between the groups is significant. In the hydroponic experiment, group 1 had 44% lower RLF and 33% higher RSW than group 4. Likewise, in the sand culture experiment, group A had 55% lower RLF and 40% higher RSW than group D. In the hydroponic experiment, shoot Na + concentrations under salinity stress conditions were not significantly different in the tolerant and moderately tolerant groups but were significantly lower than in the moderately sensitive and sensitive groups. Furthermore, in the sand culture experiment, shoot Na + concentrations were higher in the moderately tolerant group than in the tolerant group, and lower than in the sensitive group (Table 4). Ahmad et al. (2010) suggested that soil environmentas a selection pressureplays a key role in the evolution of salinity tolerance in plants. Francois (1988) found that bermudagrass genotypes from highly saline environments had higher salinity tolerance than those from normal soil. A positive correlation was observed between the percentages of shoot mortality in zoysiagrass clones treated with 6% NaCl and the rainfall of the regions where the clones were collected (Weng & Chen 2001). Because plants are subjected to ion injury and osmotic stress under salinity stress, plant ecotypes from saline or drought environments have stronger ion regulation and/or osmotic adjustment ability, which results in stronger salinity tolerance. In this research, certain genotypes from seashore regions, such as C291, C298, C737, C262, C173 and C802, and from inland arid regions, such as C699, C720, C638, C831 and C830, had higher salinity tolerance. However, genotypes from riverside, roadside and fields of humid regions, such as C580, C177, C795, C089, C653, C106, C572, C461, C731, C098, C799 and C801, had lower salinity tolerance. Based on the above comprehensive analysis, it can be deduced that certain genotypes, such as C291, have a higher salinity tolerance than others.

Relationship between the hydroponic and sand culture experiments
The hydroponic method is often used to evaluate salinity tolerance in many turfgrasses (Qian et al. 2000;Lee et al. 2005;Marcum & Pessarakli 2006). Some earlier studies found that the results of relative salinity tolerance evaluation were consistent whether the turfgrasses were grown in soil (or sand) and treated with saline water irrigation or whether they were grown in hydroponic conditions with the same salinity treatment (Alshammary et al. 2004), although ion accumulation occurred in the soil after multiple irrigations with saline water (Fu et al. 2005). In this study, a hydroponic experiment was conducted to evaluate the salinity tolerance of 51 bermudagrass genotypes and verified by a sand culture experiment. The results were significantly correlative based on four indexes (RLF, RSW, shoot Na + concentration in control and salinity stress) at a P < 0.01 level, and the correlation coefficients of each index were 0.653, 0.618, 0.766 and 0.794, respectively, between the hydroponic experiment and the sand culture experiment, which indicated Table 3. Simple correlation coefficients for RLF, RSW and shoot Na + concentrations in control (Na CK ) or under salinity stress (Na S ) in bermudagrasses grown in a hydroponic experiment or a sand culture experiment (within parentheses).