Assessment of morphological and anatomical variability in Triticum species, Aegilops species, interspecific and intergeneric hybrids

Wheat species and wild relatives offer promising resources for wheat improvement and research in the current period of the genetic narrowing of modern wheat cultivars. The present study was performed to evaluate the morphological and anatomical traits of 20 diverse genotypes including Triticum and Aegilops species with intergeneric and interspecific wheat hybrids, which were compared with modern bread and durum wheat cultivars locally adapted to rainfed and irrigated conditions. The study showed that stomata density and size ranged from 55.3 to 108.6 stomata/mm and 401.4 to 1296 μm, respectively, in the selected genotypes. Moving tetraploid to hexaploid genotypes, increased chromosome numbers yielded lower densities of large stomata in wheat species and hybrids. In this regard, the stomatal patterns of two hexaploid wheat hybrids and a wheat species including ‘Agrotriticum’, ‘Aegilotriticum’, and T. compactum, were of low density and large size stomata compared to T. durum cv. ‘Kunduru 1149’ with high density and small size stomata. Interestingly, the wild progenitor of the bread wheat D genome, Ae. tauschii, had a high density of the smallest stomata among the studied genotypes. The study further indicated that morphological parameters decreased under rainfed conditions compared to those under irrigated conditions, with levels varying among the genotypes. The rainfed flag leaf area and 1000-grain weight varied from 0.9 to 23.7 cm and from 7.3 to 61.9 g, respectively under rainfed conditions, while they ranged from 1.2 to 35.7 cm and 11.5 to 69.9 g under irrigated conditions. The flag leaf area had a significant and strong association with 1000-grain weight under rainfed (r= 0.79) and irrigated (r = 0.77) conditions. T. turanicum and T. polonicum were characterized by the significantly highest 1000-grain weight in both rainfed and irrigated conditions. This study suggests that these wheat species with high 1000-grain weight might have promising alleles to be transferred into durum wheat to increase grain yield.


Introduction
Wheat is a staple source of nutrients for about 40% of the population worldwide (Giraldo et al., 2019). In the wheat evolution process, wild tetraploid wheat, Triticum dicoccoides, was produced from hybridization between diploid wild wheat Triticum urartu (genome AA) and most likely Aegilops speltoides (genome BB) Peng et al., 2011). Cultivated emmer wheat (T. dicoccum) was formed by plant selection of wild emmer and then evolved into the free-threshing ears of T. turgidum, T. polonicum, T. turanicum, and T. carthlicum by natural mutation (Peng et al., 2011). Modern hexaploid bread wheat evolved through polyploidization between T. turgidum ssp. durum (genome AABB) and Aegilops tauschii (genome DD) 10.000 years ago (Feldman et al., 1995).
Ancient wheat species, landraces, wheat wild relatives, and wheat hybrids offer biotic and abiotic stress tolerance, high biochemical and micronutrient contents, and quality in the improvement of new cultivars (Mathre et al., 1985;Cakmak et al., 2010;Arzani and Ashraf, 2017;Li et al., 2018;Ullah et al., 2018;Kishii, 2019). Wild wheat relatives and different wheat species have also been used as donors of drought tolerance in water deficit conditions (Peng et al., 2013;Ikanović et al., 2014;Suneja et al., 2019). Particularly in rainfed farming, drought can lead to severe losses (Tigkas and Tsakiris, 2015). Water deficit may affect agriculture through limiting plant productivity by inducing stomatal closure and thereby reducing photosynthesis and growth (Németh et al., 2002;Bibi et al., 2012). Photosynthesis occurring in tissues through gas exchange has been described to be closely linked with stomata density and size (Chandra and Das, 2000;Buckley, 2005;Zwieniecki et al., 2016). These characteristics of stomata on the upper and lower sides of the leaf considerably alter the gas exchange rate between inner and outer layers (Kardiman and Raebild, 2018). Low density and large stomata could guarantee a proper photosynthetic rate and low stomatal conductance, implying that they would beneficially contribute to plants under conditions of increased CO2 and decreased water availability in the future (Yin et al., 2020). Also, in rice, stomata size has been proved to be related to grain yield, suggesting utilization possibilities for the improvement of yield in breeding programs (Limochi and Eskandari, 2013). Understanding how stomatal behaviour plays a considerable role in growth of different wheat species, hybrids, and wild wheat relatives is therefore important.
Considering the use of genetic resources, the present study aimed to determine morphological and anatomical traits and their relationships in many Triticum and Aegilops species together with their hybrids.

Experimental conditions
A field study was conducted in a randomized complete block design with three replications under rainfed and irrigated conditions in 2019 at Sarayönü Vocational School, Selçuk University, Konya, Turkey. A panel of 20 genotypes comprising different wheat species, Aegilops species, landraces, modern wheat cultivars, intergeneric and interspecific wheat hybrids was selected for the study (Table 1). T. aestivum cultivars ('Karahan 99' and 'Konya 2002') and T. durum cultivars ('Kunduru 1149' and 'Meram 2002 were used as control cultivars. Each one of locally adaptive modern cultivars represented an adverse genetic background with adaptations to rainfed and irrigated conditions, respectively. The soil taken from the field (0-40 cm) and used in the experiment was clay-loam with low organic matter (1.60%) and high levels of CaCO3 (31.9%) and Ca (6008 mg/kg). EC was 0.61 mmhos/cm. Soil pH was 7.76 and no salinity problem (0.02%) was observed. It was low in P2O5 (44.1 kg/ha) and Mn (6.17 mg/kg). K2O (1128 kg/ha), Zn (0.74 mg/kg), and Cu (2.4 mg/kg) were found to be adequate. The soil was high in Mg (591.7 mg/kg) with a moderate level of Fe (3.9 mg/kg).
According to the meteorological data for the growing season regarding the long-term (1928-2018) and average annual rainfall (2019), the average rainfall values in the months of March, April, May, June, and July were very close to 136.9 mm in the long term and 92.8 mm in 2019. The relative humidity values were 53.8% and 48.2%, respectively, and the average temperature values were 15.2 °C and 17.2 °C, respectively.

Field experiment and measurements
The sowings were made in 5-cm rows with 20-cm spaces between rows. At sowing, DAP fertilizer (18% N, 46% P2O5) was top-dressed to plots at 130 kg ha -1 . In the tillering stage, ammonium nitrate (33% N) was applied by broadcasting. In total, 75 kg ha -1 nitrogen was used under rainfed conditions and 120 kg ha -1 was used under irrigated conditions. Weeds were controlled with a chemical herbicide. The plots under irrigated conditions were irrigated two times in the stem elongation and booting stages. Flag leaves were excised from plants ( Figure 3). Six flag leaf areas (FLA) per replication were measured using ImageJ software (https://imagej.nih.gov/ij). Harvesting was performed at the stage of full grain maturity (GS 92). Morphological parameters such as plant height per main stem, number of fertile tillers per plant, spike length per main spike, and number of spikelet's per main spike were determined for 10 plants per replication. 1000grain weight (TGW) was determined by weighing dehulled seeds (4 × 100) per replication.

Stomatal anatomy analysis
Microscopic slides pasted with a layer of super glue were pressed on the adaxial side of a fully expanded flag leaf selected from plants grown under irrigated condition in the pre-anthesis growth stage. Approximately 60 seconds later, the slides were taken off from the leaves and the epidermal tissues were peeled off from the leaf and attached on the slides. In total, three replicates were performed in each treatment. Stomata were imaged using Nikon Eclipse E400 light microscope equipped with DS-5M digital camera head and DS-L1 camera control unit (Nikon, Japan). The microscopic area was 0.8 mm 2 using a microscope resolution of 1280 × 960 pixel (96 dpi). Image analysis was performed using ImageJ software (https://imagej.nih.gov/ij/). Twenty-four stomata widths and lengths per replicate were measured for the stomatal size (area) ([stoma width × stomata length × π]/4). Stomatal density was calculated as number of stomata in six microscopic fields per replicate (Cortan et al., 2017) (Figure 1).

Results
In this study, significant variations were observed within and between genotypes in terms of values for morphological and anatomical traits of Triticum and Aegilops species and their hybrids (P < 0.0001).
Stomatal anatomy Stomata exhibited a diverse range of length, width, size, and density across different wheat and Aegilops species with hybrids. Stomata length ranged from 28 to 51.6 µm, stomata width from 18.3 to 32 µm, stomata size from 401.4 to 1296 µm 2 , and stomata density from 55.3 to 108.6 stomata/mm 2 (Figure 2). In the study, the hexaploid and tetraploid genotypes were discriminated precisely in terms of stomata density. The maximum stomatal length and width were observed in the 'Aegilotriticum' in wheat species and hybrids. Conversely, T. dicoccoides and T. durum cv. 'Kunduru 1149' had the lowest stomatal length and width, respectively. In general, the hexaploid wheat genotypes and hybrids possessed a higher stomata size compared to tetraploid wheat genotypes. In Aegilops species, Ae. tauschii, a unique genitor of bread wheat, indicated the lowest stomatal length and width ( Figure 2). However, the highest values were obtained from Ae. brachyathera.  (Tables 2 and 3) and was generally greater in plants in the irrigated group than the rainfed group.
Aegilops species had shorter plant height than wheat species and hybrids. The spike length ranged from 3.5 to 12.1 cm under rainfed condition and from 3.6 to 12.6 cm under irrigated condition (Tables 2 and 3). The average values in both conditions were relatively close for spike length and spikelet number, as well.
Significant variations in tiller number were also observed among genotypes in both growth conditions. The tiller number varied from 5.2 and 42.3 under rainfed condition and 7.4 and 33.7 under irrigated condition (Tables 2 and 3). Aegilops species and T. ispahanicum showed higher tillering capacity in both conditions than other wheat species and hybrids. The FLA ranged from 0.9 to 23.7 cm 2 under rainfed conditions and 1.2 to 35.7 cm 2 under irrigated conditions (Tables 2 and 3). There was a significant and strong relationship between FLA and TGW in rainfed (r 2 = 0.79) and irrigated (r 2 = 0.77) conditions (Figure 4).
The FLA under rainfed conditions was lower than that under irrigated conditions. Similarly, TGW values were reduced under rainfed conditions compared to irrigated conditions. The TGW varied widely among genotypes, ranging from 7.3 to 61.9 g under rainfed conditions and from 11.5 to 69.9 g under irrigated  (Tables 2 and 3). It was further observed that Aegilops species had lower TGWs than Triticum species and hybrids.   . Relationship between/within stomatal and morphological traits of the studied genotypes "*" indicates P < 0.05, "**" for P <0.01, and "***" P <0.0001

Discussion
Stomata size and density vary depending on genetic factors and environmental conditions; the size ranges from 10 and 80 μm in length with densities between 5 and 1000 mm -2 (Hetherington and Woodward, 2003). The present study showed that stomata density in two bread wheat cultivars ranged from 69.5 to 70.8 mm -2 (Figure 2). Similarly, the findings resulting from the current study were in agreement with those of Shahinnia et al. (2016) who found that adaxial stomata density in two bread wheat cultivars varied from 61 to 71 mm -2 . Mohammady et al. (2007) observed that stomatal traits varied from 44.8 to 51.9 µm of stomata length, 24.5 to 31.1 µm of stomata width. They also revealed that stomata density differed from 56.2 to 81.5 mm -2 in tetraploid and from 45.6 to 64.5 mm -2 in hexaploid wheat accessions.
The density of stomata on the leaves is a highly heritable characteristic (Hofmann and Dobrenz, 1983;Schoppach et al., 2016). Hexaploid genotypes proved to have lower stomata density compared to tetraploid wheat genotypes. In this study, T. durum cv. 'Kunduru 1149' had a high density of small stomata compared to 'Agrotriticum', 'Aegilotriticum', and T. compactum with low densities of large stomata (Figure 1). These results were similar to the findings reported by Doheny-Adams et al. (2012), who concluded that smaller stomata were usually found in higher densities. Researchers have recently demonstrated pivotal results regarding appropriate stomata traits of plants. Reduced stomatal density has been proved to improve drought tolerance and water-use efficiency in bread wheat, rice, and wood plants (Caine et al., 2019;Dunn et al., 2019;Yin et al., 2020).
In the present study, the hexaploid genotypes with low stomatal density may contribute to the improvement of new drought-tolerant cultivars in environments with reduced water availability. Regarding photosynthesis and yield-related stomata traits, leaves with a low density of large stomata have shown lower photosynthetic rates (Drake et al., 2013). It was also suggested that high-yielding rice cultivars possessed a higher stomatal density and slightly shorter stomatal length (Ohsumi et al., 2007). In the present study, tetraploid wheat species had high stomata density, short length, and small size. There are differences in the previous studies about ideal stomata traits in plants, suggesting a high density of small stomata for improved yield and photosynthesis but a low density of large stomata for drought tolerance and water-use efficiency. Hence, further work is needed to elucidate associations between stomatal traits and other physiomorphological, biochemical, and genetic traits under different environmental conditions. Water deficit has been shown to lead to severe reductions in most morphological traits. It was described, for example, that precipitation was the key indicator for wheat yield evaluation (Tigkas and Tsakiris, 2015).
Grain yield can be enhanced by 60% to 100% with 200 to 300 mm of irrigation (Zhang et al., 1999).
Meanwhile, in the current study, significant reductions under rainfed conditions compared to the irrigated condition were observed in morphological parameters, such as 9.4% for plant height, 19.7% for tillering, and 33.5% for FLA. The FLA is one of the key determinants underlying wheat grain yield, particularly under drought (Biswal and Kohli, 2013). Ideal FLA is therefore significant for sustaining optimal yield in water deficit conditions (Quarrie et al., 1999). As wheat cultivars that have adapted to irrigated conditions, the FLAs of T. T. ispahanicum, T. carthlicum, and T. dicoccoides had lower FLA values in both growth conditions. Also, 'Aegilotriticum', T. carthlicum, 'Elytritilops', and T. ispahanicum were the most impacted genotypes by water deficit of 48.5% to 50% As expected, Aegilops species had inherently lower leaf areas than wheat species and hybrids. Among the Aegilops species, a progenitor of bread wheat, Ae. tauschii, was the least affected species.
This study showed that T. ispahanicum and T. dicoccoides were the most tillering species in both growth conditions among the studied genotypes. Longnecker et al. (1993) presented similar findings, indicating that tillering was dependent on genotypic and climatic factors. Another previous study reported that the tillering potential of bread wheat was greater than that of durum wheat (Pinthus, 1969). In the present study, there were no clear discriminations in terms of tillering between durum and bread wheat genotypes or tetraploid and hexaploid wheat genotypes. Indeed, T. polonicum, T. turgidum, T. aestivum cv. 'Konya 2002', and T. durum cv. 'Karakılçık' had less tillering capacity than other genotypes in both growth conditions.
Evaluating the spike length and spikelet number, genotypes under rainfed conditions had trends like those seen under irrigated conditions. Genotypes with high values maintained notably greater values in both conditions. The Aegilops species 'Ae. ventricosa' had higher values regarding plant height, spike length, and spikelet number, while 'Ae. geniculata' had lower values.
TGW was shown to be one of the significant indicators determining grain yield . Previous studies indicated that TGW was significantly and strongly correlated with grain yield (Huang et al., 2020;Öztürk et al., 2020). A close look at the data in Tables 1 and 2 reveals that T. turanicum and T. polonicum had distinguishably higher TGW than the modern wheat cultivars and other studied genotypes in both rainfed and irrigated conditions. Consistent with the findings of the current study, Grausbruger et al. (2005) indicated that T. turanicum, known as Khorasan wheat, often had TGW values of greater than even 60 g. T. turanicum was suggested to be a natural hybrid between T. polonicum (Polish wheat) and durum wheat (Kuckuck, 1959). The caryopsis structure of 'T. turanicum' has similarities with T. polonicum, T. ispahanicum (Ispahan emmer), and T. carthlicum (Persian wheat) (Kosina, 1995). Also, T. turanicum was proposed to have wide genetic diversity with multiple agronomic adaptive traits (Lannucci and Codianni, 2019). Regarding T. polonicum, TGW can reach up to 80 g (Wang et al., 2002). In the present study, the TGW of T. polonicum in rainfed and irrigated conditions averaged 52.6 g and 59.1 g, respectively. The TGW of T. turgidum was close to that of modern durum wheat cultivars and higher than modern bread wheat cultivars in both growth conditions. T. ispahanicum possessed TGW values between those of modern bread and durum wheat cultivars in both rainfed and irrigated conditions. Other wheat species and hybrids had lower TGWs than modern wheat cultivars. Morphological parameters may be altered in response to water availability in the soil. It has been reported that wheat needs 450-650 mm of rainfall annually for optimal development (Zargar and Zargar, 2018). However, this desired rainfall may not occur in a timely manner in the wheat growth stages. Seasonal rainfall irregularity might further exacerbate the adverse effects on wheat growth under rainfed conditions. In the present study, the average TGW was reduced by 11.7% under rainfed conditions compared to the irrigated condition. T. carthlicum and T. dicoccoides maintained their TGWs under rainfed conditions.
In contrast, T. aestivum cv. 'Karahan 99', 'Elytritilops', and 'Aegilotriticum' were the most affected genotypes in terms of TGW under rainfed conditions compared to the irrigated condition among the wheat species and hybrids (Tables 2 and 3). In Aegilops species, the TGW was influenced most under rainfed conditions, excluding 'Ae. ventricosa'.

Conclusions
Wheat species, wild relatives, and hybrids have recently attracted researchers' interest based on the genetic diversity in the gene pool. The present study has characterized the variability in stomatal and morphological traits of the studied genotypes. The variations among wheat species, wild relatives, and hybrids were about 2-fold for stomata density and 3.2-fold for stomata size. It was shown that 'Aegilotriticum', 'Agrotriticum', T. compactum, and Ae. tauschii particularly offered differential stomatal characteristics. 'Ae. tauschii' had a high density of small stomata, while, conversely, T. compactum, 'Aegilotriticum', and 'Agrotriticum', a hexaploid wheat species and two man-made hybrids, possessed low densities of large stomata. Considering the value of genetic variability for the improvement of wheat in rainfed and irrigated conditions, the best-performing genotypes of T. turanicum and T. polonicum had considerably high TGW compared to adapted bread wheat cultivars, durum wheat cultivars, and other species. These genotypes with superior characteristics might be evaluated for hybridization studies to transfer valuable genes into wheat.