Phenotypic and Genotypic Diversity for Drought Tolerance among and within Perennial Ryegrass Accessions

Perennial ryegrass (Lolium perenne L.) is a popular cool-season forage and turfgrass in temperate regions. Due to its self-incompatible and out-crossing nature, perennial ryegrass may show a high degree of heterozygosity. Perennial ryegrass generally is susceptible to drought stress, but variations of drought response of individual genotypes within a particular accession or cultivar are not well understood. The objective of this study was to characterize phenotypic diversity of drought tolerance within and among accessions in relation to genetic diversity in perennial ryegrass. Five individual genotypes from each of six accessions varying in origin and growth habits were subjected to drought stress in a greenhouse. Leaf wilting, plant height, chlorophyll fluorescence (Fv/Fm) and leaf water content (LWC) differed significantly among accessions as well as among genotypes within each accession under well-watered control and drought stress conditions. Fv/Fm was highly correlated with LWC under drought stress. Genetic diversity among and within accessions were identified by using previously characterized 23 simple sequence repeat markers. Across accessions, the mean major allele frequency, gene diversity, and heterozygosity values were 0.66, 0.43, and 0.66, respectively. Accessions with closer genetic distance generally had similar drought responses, while accessions with greater genetic distance showed distinct drought tolerance. Significant differences in drought tolerance among and within accessions, especially for individual genotypes within one accession, indicated that variations of drought response could be used for enhancing breeding programs and studying molecular mechanisms of stress tolerance in perennial ryegrass. Water deficit has become more of a problem for production of turf and forage grasses due to regional and localized drought and increasing demands for fresh water uses in other areas. The frequency and intensity of drought stress may also increase as a result of climate change, which could further impact growth and persistence of perennial grasses. In addition, cool-season perennial grass species normally require a large amount of water to maintain growth, and it can be challenging when water availability is limited and extensive irrigation is not practical. Therefore, improvement of drought tolerance of turf and forage grasses is becoming increasingly important to minimize effects of drought on the plants and to enhance water conservation. Phenotypic evaluation of drought response is crucial for selecting drought-tolerant materials for breeding programs. At whole plant and cellular levels, fundamental responses of the perennial grass plants to drought stress have been extensively studied (DaCosta and Huang, 2006; Jiang et al., 2009; Merewitz et al., 2010; Yu et al., 2013; Zhou et al., 2013). Some drought responses used for assessing plant stress tolerance include, but are not limited to, reduced growth, photosynthesis, LWC, Fv/Fm, increased electrolyte leakage, and water use efficiency in perennial forage and turfgrass species (DaCosta and Huang, 2006; Jiang et al., 2009; Merewitz et al., 2010; Shukla et al., 2015; Xu and Zhou, 2011; Yu et al., 2013). Through evaluating LWC and Fv/Fm, drought tolerance of 57Brachypodium distachyon accessions were differentiated, consistent with leaf wilting by visual observation (Luo et al., 2011). In a field study, large variations of LWC and Fv/Fm were found among 192 accessions of perennial ryegrass (Yu et al., 2013). Whole-plant responses to drought stress provide a basis for exploring genetic variation of drought tolerance. Perennial ryegrass is one of the most economically and environmentally important cool-season grass species. It is extensively used as a turf and forage grass around the world. Perennial ryegrass is primarily diploid (2n = 2x = 14), but a tetraploid cultivar (2n = 4x = 28) has been developed to improve forage quality and productivity (Nair, 2004) and to enhance turf management (Richardson et al., 2007). As a self-incompatible and outcrossing species (Cornish et al., 1979), perennial ryegrass shows a high degree of genetic diversity within the population. Genetic diversity of perennial ryegrass has been characterized based on morphological traits such as seedling vigor, leaf width and forage yield (Balfourier and Charmet, 1991; Casler, 1995), isozymes (Charmet et al., 1993; Fernando et al., 1997), and molecular markers (Posselt et al., 2006; Wang et al., 2009). Furthermore, molecular markers have been successfully used for cultivar identification (Momotaz et al., 2004), assignment of individual genotypes to specific cultivars (Wang et al., 2009), quantitative trait loci mapping of key agronomic traits and disease resistance (Brazauskas et al., 2013; Navakode et al., 2009), and associationmapping of drought, salt and freezing tolerance as well as spring regrowth (Aleli unas et al., 2015; Tang et al., 2013; Yu et al., 2013, 2015). High levels of genetic diversity were observedwithin turf-type cultivars (Kubik et al., 2001) or within foragetype populations (Wang et al., 2009). Phenotypic evaluation of drought response and exploration of physiological and molecular mechanisms of drought tolerance have been studied in perennial ryegrass (Huang et al., 2014; Jiang and Huang, 2001; Jiang et al., 2009; Liu and Jiang, 2010; Patel et al., 2014; Turner et al., 2012) and in turftype interspecific hybrids of meadow fescue (Festuca pratensis) with perennial ryegrass (Barnes et al., 2014). Most previous reports on drought tolerance used either seedlings from the mixed individuals (seeds) for a particular cultivar (Jiang et al., 2009; Turner et al., 2012) or seedlings from single seeds of different accessions (Liu and Jiang, 2010; Yu et al., 2013). The results did not show the stress response of individual genotypes within a cultivar or accession to drought stress conditions. Due to high level heterozygosity of perennial ryegrass and high genetic diversity within each cultivar (Kubik et al., 2001; Wang et al., 2009), it is speculated that each individual genotype (seeds) from one accessionmay differ in traits related to whole-plant drought tolerance. However, such research work has not been conducted in perennial ryegrass. Therefore, this experiment was designed to compare phenotypic variations of drought tolerance among and within accessions and to relate phenotypic responses to genetic diversity in perennial ryegrass accessions. Exploring variation of drought response among or within accessions is important for selecting appropriate plant materials for studying molecular response such as gene expression, conducting gene and trait association analysis, as well as for breeding purposes in perennial ryegrass. Materials and Methods Plant materials and growth conditions. Six accessions of diploid perennial ryegrasses were chosen for this study including Received for publication 30 Apr. 2015. Accepted for publication 9 June 2015. This research is supported by the Midwest Regional Turfgrass Foundation of Purdue University and One-Hundred Talents Program of Shanxi Province of China. Visiting student. Corresponding author. E-mail: yjiang@purdue. edu. 1148 HORTSCIENCE VOL. 50(8) AUGUST 2015 PI598453 (Wild, Romania) and PI577265 (wild, UK), PI403847 (cultivated, Canada), PI578760 (cultivated, United States), and PI197270 (cultivated, Finland) as well as PI204085 (unknown status, Cyprus) from the USDA National Plant Germplasm System at the Western Regional Plant Introduction Station in Pullman, WA. These six accessions were selected for the study because they vary in geographical origin, growth habits, and drought tolerance assessed by seedlings grown from single seeds (Yu et al., 2013). On 1 July 2013, 10 seeds of each accession were planted in different pots (9 cm deep and 10 cm in diameter) containing topsoil in a plant growth chamber at Purdue University in West Lafayette, IN. Our preliminary observation on simple sequence repeat (SSR) analysis revealed that five to six individual genotypes from one accession generally covered diversity of that accession. Therefore, 20 d after germination, five genotypes were randomly chosen for each accession with one genotype per pot. On 5 Sept. 2013, all pots were moved to a greenhouse. After 2 weeks, all genotypes from each accession were propagated with tillers to maintain genetic uniformity, with three pots for the well-watered control and three pots for drought treatment for each genotype. Each pot contained eight tillers and the same volume of soil. Plants were watered every 2 d and fertilized once a week with a soluble fertilizer (N–P2O5–K2O, 24– 8–16) (Scotts Inc., Marysville, OH) and micronutrients; they were cut once a week to the height of 6–7 cm, depending on the growth habits of each particular grass. During the growth period, the average air temperatures were 23 C/18 C (day/night) and average daily photosynthetically active radiation (PAR) intensity was 300 mmol·m·s in the greenhouse. All plants were well watered before initiation of drought stress. Drought treatment. Drought stress began on 19 Oct. 2013 (110 d after seeding) and ended on 24 Oct. 2013, lasting for 6 d. Drought stress was imposed by withholding water from the grasses until permanent wilting occurred to most of the plants (the leaves were no longer rehydrated at night and in the morning), particularly for susceptible accessions. All the plants were exposed to drought stress for the same amount of time. The control plants received regular irrigation during the treatment period. At the end of treatment, drought-stressed plants were rewatered to allow recovery for 5 d. During the time of stress and recovery, air temperatures and PAR intensity were similar to the conditions before drought stress. Whole-plant measurements. Leaf wilting was visually rated on a scale of 0 (no observable wilting), 1 (slightly wilted), 2 (moderately wilted) to 3 (severe