Evaluation of seedling emergence and relative DNA content under dry soil conditions of wild Festuca arundinacea

O R I G I N A L A R T I C L E

Evaluation of seedling emergence and relative DNA content under dry soil conditions of wild Festuca arundinacea

populations collected in Iran

Iman Rohollahi^1,2, Nayer Azam Khoshkholghsima³, Toshihiko Yamada⁴, Mohsen Kafi², Yoichiro Hoshino⁴, Abdolmajid Liaghat⁵and Ali Ashraf Jafari⁶

1 Department of Horticulture Science, Shahed University, Tehran, Iran

2 Department of Horticulture Science, College of Agriculture and Natural Resource, University of Tehran, Iran 3 Agriculture Biotechnology Research Institute of Iran (ABRII), Iran

4 Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Japan

5 Department of Irrigation and Reclamation, College of Agriculture and Natural Resource, University of Tehran, Iran 6 Research Institute of Forests and Rangelands, Tehran, Iran

Keywords

Drought tolerance; final germination; relative DNA content; turfgrass; wild populations.

Correspondence

Nayer Azam Khoshkholghsima, Agriculture Biotechnology Research Institute of Iran (ABRII), P.O. Box 31535–1897, Karaj, Iran.

Email: ksima@abrii.ac.ir Received 25 March 2014;

accepted 19 September 2014.

doi: 10.1111/grs.12074

Abstract

Tall fescue (Festuca arundinaceaSchreb.) is an important forage and turf grass species adapted to cold, arid and semiarid environments. Germination and seedling establishment ofF. arundinaceacultivars is constrained by the low soil moisture found in these regions. Genome size was found to positively correlate with latitude of origin, suggesting that structural changes in DNA may play a role in environmental adaptation. We investigated the effects of low soil mois- ture on the germination and early establishment and their correlation with rel- ative DNA content using 14 wild F. arundinacea populations collected from various regions in Iran and two commercial turf cultivars. The populations were evaluated under 100% ( 0.03 matric potential [MPa]), 80% ( 0.2), 60%

( 0.6) and 40% ( 1.4) field capacity (FC) conditions. Seed germination and root and leaf growth decreased under reduced soil water content. Cluster analy- sis revealed that the populations fell into four groups. Populations in cluster I showed the greatest tolerance to low soil moisture and cluster IV was the least tolerant. ‘Isfahan’ and ‘Gonabad’ populations had the best final germination and longer leaf and root length at 40% FC. Studies to identify indices that were involved in drought resistance revealed that final germination, leaf length and seedling vigor index were most important evaluating indicators forF. arundin- acea populations. Flow cytometric relative DNA content of the wild popula- tions was negatively correlated with germination, leaf length and seedling vigor index. The drought tolerance populations in cluster I could be potentially use- ful germplasm for a breeding program to develop superior cultivars for arid and semiarid regions.

Introduction

The genus Festuca L. is one of the largest in the family Gramineae whose members are widely adapted to a vari- ety of eco geographical regions (Yamada 2011). Tall fes- cue (Festuca arundinacea Schreb.) is the most economically important forage and turfgrass species in the genus and is grown throughout temperate regions of

the world (Saha et al. 2005). According to Bor (1970), F. arundinaceais one of the perennial grasses that are dis- tributed in cold and dry regions of central, northeastern and northwestern Iran. Investigation of germination and seedling performance under conditions of low soil mois- ture is an efficient process to screen genotypes of cool- season grasses for drought resistance (Gazanchian et al.

2006). Gazanchian et al. (2006) evaluated only three

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F. arundinacea entries and determined that none of them would emerge from soils at 25% field capacity (FC).

Traits associated with germination and seedling establish- ment of cool-season grasses under water stress conditions are rapid germination (Larsen and Bibby 2004), enhanced seedling vigor (Mut and Akay 2010), early root initiation and rapid root extension (Johnson and Asay 1993).

Gregory (2005) reported positive correlations between plant genome size and capacity for growth under cooler conditions. Several studies have shown positive (Minelli et al.1996), negative (Ceccarelliet al.1993) or no signifi- cant (Knight and Ackerly 2002) correlation between gen- ome size and generation time or relative growth rate. It is known that large genome species tend to have lower spe- cific leaf area (typically smaller, thicker leaves), compared to small genome species, which have wider range of spe- cific leaf area (Grime et al. 1997; Knight et al. 2005).

Smarda and Bures (2006) confirmed the relationship between 4,6-diamidino-2-phenylindole (DAPI) and propi- dium iodide (PI) staining in Festuca plants on the basis of measurements made in different seasons. DAPI could be used for estimating the absolute DNA content in Fest- uca. Ceccarelliet al. (1992, 1993) examined the collected F. arundinaceaepopulations’ seeds from the Italian penin- sula that germinated on water paper in Petri dishes under sterile conditions. Ceccarelli et al. (1993) has reported a significant negative correlation between the seed germina- tion and leaf length with genome size of F. arundinacea populations.

There is a lack of knowledge on water requirements for germination and subsequent establishment in soil condi- tions and the correlation between traits of seedling emer- gence under dry soil conditions and genome size in wild F. arundinacea populations. Here we examine how drought stress and genome size influence the germination and seedling development in Iranian wild F. arundinacea populations to identify drought tolerance indexes and good indicators during seed germination stage.

Materials and methods

Sixteen entries were evaluated in the present study. Seeds of 14 wildF. arundinaceapopulations were collected from different cold, arid and semiarid regions of Iran by the Rangelands and Forestry Research Institute (stored in 20°C for 1 year) (Table 1). In addition, two commercial cultivars (‘Bravado’ and ‘Barleroy’) from Barenbrug Hold- ing B.V. in the Netherlands were used as controls (Table 1). Each entry was assayed at four levels; 40, 60, 80 and 100% of field soil moisture capacity (FC) condi- tions (approximately 1.4, 0.6, 0.2 and 0.03 matric potentials [MPa], respectively) in greenhouse study.

Single factorial experiment was carried out based on a

completely randomized design with four replicates. Signif- icant difference for all data between soil moisture treat- ment and entries were determined using Proc GLM function (SAS Institute 2003). Means between soil water treatments and entries were separated by least significant difference.

Soil volumetric water content was determined by weighing the pots during the experimental period for each treatment. Germination and seedling establishment were monitored for 20 days. Germination was recorded at the emergence of the leaf above the soil surface of the seedlings in each pot. Days to first germination (T0) and days required to reach 50% germination (T50) were recorded as described by Usberti and Valio (1997). The leaf length and the maximum root length for each emerged seedling were measured for each pot at the end of the experiment (20 days after planting). Leaves and roots of each seedling were separated, oven dried (75°C for 48 h), and weighed. In this study mean daily germina- tion (MDG) and germination rate (GR) were calculated as described by Maguire (1962) and Hunteret al.(1984), respectively. Seedling vigor index (SVI) was calculated (Abdul-Baki and Anderson 1973) by multiplying the per- centage of germination for each accession by the mean length (cm) of the seedling (root plus leaf). Sensitivity index (SI) for leaf dry weight was determined on the basis of Fernandez and Reynolds (2000).

Relative DNA content using flow cytometry Relative DNA content of each entry was estimated by using DAPI stained flow cytometry (Partec PA; Partec GmbH, Munster, Germany) (Mishiba et al. 2000). DNA content of leaves of Hordeum vulgare cv. ‘Sultan’

(2n= 2x=14) were used as the internal standard.

Totally, 48 plants from 16 entries were examined. For each sample, three individuals were analyzed. The Relative DNA content was estimated according to following for- mula: Relative DNA content=(Festuca sp. G0/G1 peak mean/reference standard G0/G1 peak mean)9nuclear DNA content of reference standard (Hordeum vulgarecv.

‘Sultan’: 10.9 pg) (Buitendijk et al. 1997; Loureiro et al.

2007).

Results

Germination traits and seedling growth

Significant differences between soil moisture levels and genotypes were observed for all the evaluated traits. While all of the populations displayed some germination at 40%

FC, the averages for the ‘Quchan’ and ‘Yasuj’ populations were low (Table 2). The highest final germination (100%)

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Evaluation ofFestucain germination I. Rohollahiet al.

was exhibited by the ‘Isfahan’ population, 95% for ‘Da- ran’ and 90% for ‘Barleroy’ at 40% FC (Table 2). Also

‘Isfahan’ and ‘Daran’ showed the highest final germina- tion (100%) at 60% FC. Final germination for the

‘Quchan,’ ‘Yasuj,’ ‘Borujen’ and ‘Kamyaran’ accessions was 6.7, 13.3, 28.3 and 35.0% at 40% FC, respectively (Table 2). Soil water content at 100% FC did not enhance final germination for all populations. For instance, F. arundinacea collected from Isfahan, Yazdabad, Semi- rom and Ardabil had highest final germination, at 80%

FC (Table 2). Whereas some accessions obtained data of T50 at 40% FC, the low soil water content delayed T50 germination by 4 days across all populations in compari- son to 100% FC (see Supporting information, Table S1).

Whereas most populations achieved 50% or greater final germination at 40% FC, the low soil water content delayed days reached 50% germination compared with germination at 100% FC between 0 and 14 days depend- ing on the accession (Table 2). The ‘Isfahan’ population showed the maximum MDG (5.0) and GR (12.22) at 40% FC (Table 2). At 40% FC, GR in ‘Daran’ (9.7), ‘San- andaj’ (8.4) and ‘Gonabad’ (7.3) were higher than all oth- ers after the ‘Isfahan’ population, on the other, the lowest GR was obtained from ‘Quchan’ (0.1) and ‘Yasuj’ (0.6) (Table 2). GR and MDG were 6.4 and 4.5 in 40% FC for

‘Barleroy’ (Table 2). Overall means of SVI at 100, 80, 60 and 40% FC were 18.28, 16.84, 13.23 and 7.81, respec- tively (see Supporting information, Table S1). At 100%

FC the highest SVI was observed for ‘Isfahan’ and ‘Semi-

rom’ (22.3 and 22.6, respectively) on the other, at 60%

FC, ‘Isfahan’ and ‘Daran’ showed the maximum SVI (Table 3). At 40% FC, the highest SVI measurement occurred for the ‘Isfahan’ and ‘Gonabad’ populations and the lowest for ‘Quchan’ and ‘Yasuj’ (Table 3). Under 40%

FC SVI values decreased by 70 and 51% in ‘Barvado’ and

‘Barleroy’ from 100% FC, respectively (Table 3). SI at 40% FC for the ‘Tiran,’ ‘Gonabad’ and ‘Isfahan’ popula- tions showed the lowest reduction and highest stability in leaf dry weight (Table 3).

For all populations, the leaf and root length of seed- lings decreased significantly by more than 50% and 36%, respectively, between 100 and 40% FC (see Supporting information, Table S1). Although all of the entries emerged at 40% FC, the final germination averages for some entries were low (Table 2). Under 40% FC, ‘Isfahan’

had the greatest leaf (8.8 cm) and root length (6.4 cm), in comparison with other genotypes, otherwise ‘Yasuj’

had the least leaf (1.6 cm) and root (1.9 cm) growth (Table 3). Leaf length and root length decreased by 52 and 21%, respectively, in ‘Bravado’ while it decreased by 51 and 28% in ‘Barleroy’ in 40% FC compared to 100%

FC (Table 3).

Relative DNA content

Except one diploid population (‘Brojen’), all entries were hexaploid, but more research is needed to confirm this finding (Table 2) because ‘Brojen’ appeared as tall fescue

Table 1 Descriptions of collection sites and seed characteristics of 14 Iranian wildFestuca arundinaceapopulations and two turfgrass cultivars used in the present study

Entry number

Collection site

Relative DNA contentstandard deviation

Maximum difference (%)‡

Average temperature (°C)

Average precipitation (mm/year)

1000 seeds weight (g)

1 Sanandaj 18.180.113^e§ 0.88 13.3 467.6 1.81

2 Gonabad 17.910.022^gh 0.17 16.6 143.6 2.23

3 Isfahan 17.870.013^hi 0.10 14.6 117.1 2.38

4 Semirom 17.880.048^hi 0.38 11.5 305.4 2.49

5 Borujen 6.530.102^j 3.72 9.9 258.5 1.97

6 Kamyaran 18.890.071^a 0.53 13.0 466.6 1.88

7 Mashhad 18.170.105^e 0.82 14.6 254.4 2.10

8 Ardabil 17.820.007ⁱ 0.06 8.6 289.3 2.36

9 Karaj Dam 18.300.028^d 0.22 14.2 264.7 2.36

10 Yasuj 18.290.025^d 0.19 14.4 891.4 2.39

11 Barleroy† 18.030.058^f 0.45 – – 2.76

12 Bravado† 18.370.038^c 0.29 – – 2.77

13 Quchan 18.490.110^b 0.85 12.1 339.3 2.44

14 Tiran 17.980.099^fg 0.78 16.1 116.5 2.42

15 Daran 18.000.118^fg 0.93 10.0 341.3 3.19

16 Yazdabad 17.980.006^fg 0.04 16.2 109.9 2.91

†F. arundinaceacultivars as control.‡Maximum difference in relative DNA content found between two samples expressed in the percentage as 1009(larger/smaller content 1) (Smarda 2006).§Means within column followed by same letters were not significantly different based on the LSD test atP=0.01.

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Table 2 Effects of soil moisture content on final germination mean daily germination, germination rate, days to 50% germination of 16Festuca arundinaceaentries in seedling stage

Entries Cluster† RDC

G (%) MDG GR T50

100%‡ 80% 60% 40% 100% 80% 60% 40% 100% 80% 60% 40% 100% 80% 60% 40%

Sanandaj I 18.18^e§ 96.6^abc 100.0^a 98.3^ab 85.0^ab 4.8^abc 5.0^a 4.9^ab 4.3^ab 14.3^abc 14.3^bc 12.8^ab 8.4^bc 5.3^fg 6.0^f 6.0^f 7.6^fg Gonabad I 17.91^gh 93.3^abc 96.7^ab 93.3^bc 88.3^a 4.6^abc 4.8^ab 4.7^bc 4.4^a 12.2^b–e 12.2^de 10.3^cde 7.2^cd 6.0^ef 6.0^f 7.0^ef 9.3^e Isfahan I 17.87^hi 100.0^a 100.0^a 100.0^a 100.0^a 5.0^a 5.0^a 5.0^a 5.0^a 15.5^a 15.1^b 14.0^a 12.2^a 5.0^g 5.0^g 6.0^f 6.6^g Semirom II 17.88^hi 98.3^ab 98.3^ab 85.0^cde 55.0^cd 4.9^ab 4.9^ab 4.3^cde 2.7^cd 13.7^a–d 13.3^cd 8.7^ef 3.9^fgh 5.0^g 5.0^g 7.0^ef 15.0^b Borujen III 6.53^j 58.3^e 53.3^f 48.3^h 28.3^fg 2.9^e 2.7^f 2.4^h 1.4^fg 6.0^fg 3.5ⁱ 4.3^ij 1.6^ijk 9.0^b 10.0^bc 11.6^b – Kamyaran III 18.89^a 75.0^d 80.0^d 78.3^e 35.0^ef 3.7^d 4.0^d 3.9^e 1.8^ef 8.0^f 8.2^h 6.0^hi 1.2^ijk 8.0^c 8.3^c 9.0^cd – Mashhad II 18.17^e 96.6^abc 93.3^ab 93.3^bc 70.0^bc 4.8^abc 4.7^ab 4.7^bc 3.5^bc 13.0^a–d 11.8^e 10.7^cde 5.0^d–g 6.0^ef 6.0^f 6.6^fq 12.6^c Ardabil II 17.82ⁱ 78.3^d 90.0^bc 81.7^de 60.0^cd 3.9^d 4.5^bc 4.1^de 3.0^cd 12.4^b–e 13.3^cd 9.8^def 4.9^efg 5.0^g 5.0^g 6.6^f 11.0^d Karaj II 18.30^d 90.0^bc 83.3^cd 85.0^cde 65.0^cd 4.5^bc 4.2^cd 4.3^cde 3.3^cd 10.7^e–h 9.4^g 8.1^fg 4.3^e–h 6.3^de 7.3^d 8.3^de 14.0^bc Yasuj VI 18.29^d 93.3^abc 83.3^cd 51.7^gh 13.3^gh 4.7^abc 4.2^cd 2.6^gh 0.7^gh 11.2^de 9.4^gh 5.0^ij 0.6^gh 7.0^d 7.0^de 10.3^bc – Barvado I 18.03^f 96.6^bc 96.7^ab 98.3^ab 50.0^de 4.8^abc 4.8^ab 4.9^ab 2.5^de 15.5^a 18.2^a 11.6^bcd 2.5^jk 5.0^g 3.6^h 6.3^f 19.0^a Barleroy II 18.37^c 96.6^abc 100.0^a 96.7^ab 90.0^a 4.8^abc 5.0^a 4.8^ab 4.5^a 12.8^b–e 19.0^a 12.3^abc 6.4^hij 5.0^g 4.0^h 5.6^f 8.3^ef Quchan VI 18.49^b 56.7^e 65.0^e 63.3^f 6.7^h 2.8^e 3.3^e 3.2^f 0.3^h 3.9^g 4.4ⁱ 3.3^j 0.1^k 15.3^a 15.3^a 18.6^a – Tiran II 17.98^fg 88.3^c 95.0^ab 90.0^bcd 63.3^cd 4.4^c 4.8^ab 4.5^cd 3.2^cd 11.9^cde 11.2^ef 8.0^fgh 2.9^ghi 6.0^ef 6.3^ef 8.3^de 18.6^a Daran I 18.00^fg 98.3^ab 95.0^ab 100.0^a 95.0^a 4.9^ab 4.8^ab 5.0^a 4.8^a 14.5^ab 14.1^bc 12.0^abc 9.7^b 5.0^g 5.6^fg 6.6^f 5.0^efg Yazdabad II 17.98^fg 78.3^d 81.6^cd 58.3^fg 65.0^d 3.9^d 4.1^cd 2.9^fg 3.3^cd 11.2^de 9.9^fg 6.0^ghi 5.2^ghi 6.0^ef 6.0^f 11.0^b 13.3^c

†Classification was conducted on the basis of Ward’s method.‡100%, 80%, 60% and 40%, field soil moisture capacity (FC) 100% ( 0.03 matric potential [MPa]), 80% ( 0.2 MPa), 60%

( 0.6 MPa) and 40% ( 1.4 MPa).§Means within column followed by same letters were not significantly different based on the LSD test atP=0.01. G, germination percentage (%); GR, germi- nation rate; MDG, mean daily germination; RDC, relative DNA content (Mean); T50, days required to reach 50% germination.

©2015JapaneseSocietyofGrasslandScience,GrasslandScience4 EvaluationofFestucaingerminationI.Rohollahietal.

Table 3 Effects of soil moisture content on leaf and root length, seedling vigor index leaf dry weight and sensitivity indices at 40% field capacity of 16Festuca arundinaceaentries in seedling stage

Entries Cluster† RDC

LL(cm) RL (cm) SVI LD (mg)

100%‡ 80% 60% 40% 100% 80% 60% 40% 100% 80% 60% 40% 100% 80% 60% 40%

SI40

%§

Sanandaj I 18.18^e– 10.6^gh 9.8^g 7.9e 8.0^abcd 7.8^cde 7.6^def 6.5^bcd 5.9^a-d 17.8^def 17.3^cd 14.2^d-g 11.8^bc 11.2^fg 8.6^fg 6.1^ghi 5.8^abc 0.53 Gonabad I 17.91^gh 14.4^bc 12.7^ab 10.2^bc 8.6^ab 8.9^ab 8.5^abc 7.0^b 6.1^abc 21.7^ab 20.5^a 16.1^bcd 13.0^ab 13.0^def 9.9^def 8.5^ef 6.4^ab 0.50 Isfahan I 17.87^hi 13.4^bcd 12.0^cd 11.3^a 8.8^a 8.9^ab 7.7^cde 6.8^bc 6.4^a 22.3^ab 19.7^ab 18.0^a 15.2^a 13.8^def 9.7^ef 9.0^def 6.9^a 0.50 Semirom II 17.88^hi 14.2^bc 12.7^ab 10.6^abc 8.2^abc 8.8^abc 8.6^ab 6.7^bc 6.4^a 22.6^a 20.9^a 14.7^c-f 8.0^def 16.9^abc 13.6^b 8.5^ef 5.6^a-d 0.34 Borujen III 6.53^j 11.8^efg 10.9^f 7.6^e 3.9^h 7.2^e 6.8^fg 6.5^bcd 5.6^bcd 11.1^h 9.5^g 6.9ⁱ 2.7^gh 8.7^gh 6.5^hi 5.5ⁱ 3.0^ef 0.35 Kamyaran III 18.89^a 10.0^h 9.9^efg 8.9^d 4.6^h 9.0^ab 7.6^de 6.8^bc 5.2^de 14.3^g 14.0^f 12.4^g 3.7^g 8.6^gh 6.3^hi 5.9^hi 2.9^ef 0.37 Mashhad II 18.17^e 13.0^cde 11.7^d 10.6^abc 7.4^cde 8.2^a-d 8.0^a-d 6.9^bc 5.4^cd 20.9^abc 18.4^bcd 16.3^abc 9.0^de 14.4^cde 11.0^de 7.8^fg 5.0^cd 0.34 Ardabil II 17.82ⁱ 16.0^a 12.8^a 9.9^cd 7.2^de 9.3^a 8.8^a 8.2^a 6.0^abc 19.7^bcd 19.4^ab 14.8^b-e 8.0^def 14.7^cde 12.7^bc 10.9^bc 5.2^bcd 0.38 Karaj II 18.30^d 13.6^bcd 12.2^bcd 10.9^ab 6.2^g 8.7^abc 8.0^bcd 6.7^bc 5.2^de 20.1^abc 16.8^cd 15.0^b-e 7.4^ef 14.1^cde 11.4^cd 8.7^ef 4.3^de 0.30 Yasuj VI 18.29^d 11.8^efg 10.1^g 7.9^e 1.6ⁱ 8.5^a-d 7.8^cde 6.7^bc 1.9^f 19.0^cde 14.9^ef 7.6^hi 0.5^hi 11.8^ef 7.8^gh 5.9^hi 1.9^fg 0.09 Barvado I 18.03^f 14.5^b 11.6^de 9.7^cd 7.0^efg 8.4^a-d 7.1^efg 6.3^cd 6.0^abc 22.2^ab 18.0^bcd 15.6^bcd 6.5^f 17.9^a 13.0^b 12.0^ab 6.4^ab 0.38 Barleroy II 18.37^c 12.5^def 11.3^fe 7.5^e 6.3^fg 8.3^a-d 7.3^d-g 6.1^d 4.6^e 20.1^a-d 18.5^bc 13.1^efg 9.8^cde 17.4^ab 15.4^a 12.8^a 6.3^abc 0.34 Quchan VI 18.49^b 8.0ⁱ 7.3^h 6.3^f 2.3ⁱ 7.6^de 6.6^g 6.4^bcd 2.3^f 8.8^h 9.0^g 8.0^hi 0.0ⁱ 7.8^h 5.6ⁱ 4.9ⁱ 1.5^g 0.19 Tiran II 17.98^fg 10.8^gh 10.0^g 7.9^e 7.1^ef 7.8^cde 7.4^def 6.5^bcd 5.3^cde 16.4^efg 16.5^e 12.9^fg 7.9^def 12.9^def 8.9^fg 7.5^fgh 6.2^abc 0.49 Daran I 18.00^fg 12.5^def 12.9^a 10.0^bc 7.2^de 7.9^b-e 7.7^cde 6.6^bcd 5.8^a-d 20.0^a-d 19.5^ab 16.7^ab 12.3^b 14.3^cde 12.8^bc 10.8^bcd 6.6^a 0.40 Yazdabad II 17.98^fg 11.4^fgh 12.6^abc 9.0^d 7.8^bcde 8.2^b-e 7.8^b-e 6.9^b 6.1^ab 16.4^efg 16.5^e 9.4^h 7.9^def 14.7^bcd 14.1ab 9.7^cde 6.4^ab 0.46

†Classification was conducted on the basis of Ward’s method (see Figure 2). ‡100 and 40%, field soil moisture capacity (FC) 100% ( 0.03 matric potential [MPa]) and 40% ( 1.4 MPa).

§SI 40%, Sensitivity indices at 40% FC.–Means within column followed by same letters were not significantly different based on the LSD test atP= 0.01. RDC, relative DNA content (Mean); LL, leaf length (cm); RL, root length (cm); SVI, seedling vigor index and LD, leaf dry weight (mg).

©2015JapaneseSocietyofGrasslandScience,GrasslandScience5 I.Rohollahietal.EvaluationofFestucaingermination

morphologically. Many significant correlations were also found to occur between the relative DNA content and particular phenotypic traits of the plants (Table 4). Our

results indicate the significant negative correlation between overall germination percentage ( 0.56, P<0.05), leaf length ( 0.71, P<0.01) and SVI ( 0.65,

Table 4 Correlation matrix for 10 measured traits on 16Festuca arundinaceaentries at 100%, 80%, 60% and 40% field soil moisture capacity (FC) levels

Soil water content Traits

Correlation matrix

G MDG GR LL RL T0 LD RD SVI

100% FC† G 1

MDG 0.99** 1

GR 0.84** 0.86** 1

LL 0.41^ns 0.44^ns 0.47^ns 1

RL 0.51* 0.51* 0.26^ns 0.70** 1

T0 0.69** 0.73** 0.94** 0.54* 0.21^ns 1

LD 0.55* 0.56* 0.71** 0.74** 0.41^ns 0.70** 1

RD 0.26^ns 0.26^ns 0.31* 0.20^ns 0.05^ns 0.38** 0.68** 1

SVI 0.89** 0.90** 0.78** 0.76** 0.75** 0.70** 0.76** 0.14^ns 1

80% FC G 1

MDG 0.99** 1

GR 0.92** 0.92** 1

LL 0.50* 0.50* 0.62** 1

RL 0.38^ns 0.38^ns 0.37^ns 0.62** 1

T0 0.79** 0.79** 0.94** 0.65** 0.32^ns 1

LD 0.71** 0.71** 0.78** 0.71** 0.38^ns 0.80** 1

RD 0.11^ns 0.11^ns 0.14^ns 0.17^ns 0.16^ns 0.02^ns 0.49^ns 1

SVI 0.91** 0.91** 0.89** 0.80** 0.61* 0.81** 0.68** 0.04^ns 1

60% FC G 1

MDG 0.99** 1

GR 0.86** 0.85** 1

LL 0.43^ns 0.43^ns 0.47^ns 1

RL 0.51* 0.51* 0.26^ns 0.70** 1

T0 0.72** 0.72** 0.94** 0.54* 0.21^ns 1

LD 0.56* 0.56* 0.71** 0.74** 0.42^ns 0.70** 1

RD 0.09^ns 0.09^ns 0.05^ns 0.31** 0.07^ns 0.66** 0.55* 1

SVI 0.90** 0.90** 0.78** 0.75** 0.75** 0.70** 0.47* 0.23^ns 1

40% FC G 1

MDG 0.99** 1

GR 0.92** 0.92** 1

LL 0.85** 0.85** 0.74** 1

RL 0.67** 0.67** 0.57* 0.88** 1

T0 0.85** 0.85** 0.76** 0.81** 0.68** 1

LD 0.88** 0.88** 0.75** 0.92** 0.77** 0.86** 1

RD 0.12^ns 0.12^ns 0.008^ns 0.26^ns 0.19^ns 0.23^ns 0.41^ns 1

SVI 0.97** 0.97** 0.94** 0.90** 0.72** 0.84** 0.88** 0.09^ns 1

Overall G 1

MDG 0.99** 1

GR 0.94** 0.94** 1

LL 0.71** 0.71** 0.70** 1

RL 0.50^ns 0.50^ns 0.48^ns 0.89** 1

T0 0.87** 0.87** 0.93** 0.80** 0.58* 1

LD 0.65** 0.66** 0.73** 0.74** 0.49^ns 0.85** 1

RD 0.02^ns 0.02^ns 0.07^ns 0.02^ns 0.18^ns 0.26^ns 0.55* 1

SVI 0.93** 0.93** 0.90** 0.90** 0.72** 0.89** 0.70** 0.10^ns 1

RDC 0.56* 0.56* 0.56* 0.71** 0.55* 0.63* 0.60** 0.21^ns 0.65**

Significance at*P<0.05;**P<0.01 levels;^nsnot significant.†Field soil moisture capacity (FC). G, germination percentage; GR, germination rate;

LD, leaf dry weight; LL, leaf length; MDG, mean daily germination; RD, root dry weight; RDC, relative DNA content (Within 15 hexaploid entries);

RL: root length; SVI, seedling vigor index; T0, days to germination initiation;.

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Evaluation ofFestucain germination I. Rohollahiet al.

P<0.01) with relative DNA content (Table 4) within hexaploid entries.

Correlation

High correlation coefficients were observed between traits, principally at 40% FC (Table 4). There was an inverse relationship between the final germination and T0 at FC ( 0.69, P<0.01) and 40% FC ( 0.85, P<0.01). A direct correlation was found between SVI and G at FC (0.89, P<0.01) and 40% FC (0.97, P<0.01). Moreover, under 40% FC, high correlations occurred between leaf length and G, MDG, GR, as com- pared with FC, 80 and 60% FC (Table 4). With decreas- ing soil water content, associations between characters relatively increased, and species with high vigor emerged earlier and showed further final germination under low soil water content.

Cluster analysis

Cluster analysis grouped the 16 entries into to four clus- ters (Figure 1). All entries in cluster I obtained T50 and

population ‘Isfahan’ showed 100% final germination at 40% FC (Table 2). Highest leaf and root lengths were obtained from ‘Gonabad’ (8.6, 6.1 cm) and ‘Isfahan’

(8.8, 6.4 cm) in cluster I. The entries in cluster II had lower final germination, GR, leaf length, root length and SVI in comparison to those in cluster I. Entries in Clus- ter III, ‘Kamyaran’ and ‘Borujen’ (2x) also had low ger- mination in 40% FC (35 and 28.3%) and they could not obtain T50. In cluster IV, two populations that were collected from ‘Quchan’ and ‘Yasuj’ had lowest final ger- mination, SVI, low growth potential and stability response to low soil water content (Tables 2 and 3).

The highest T0 (15 days) was obtained from ‘Quchan.’

These results suggest critical and significant traits (final germination and SVI) for the evaluation of drought tol- erance F. arundinacea populations that showed good correlation with relative DNA content under drought stress (Table 4).

Discussion

It is well known that rapid and complete germination are important during the establishment of grass species (Lar- sen and Bibby 2004). Our study demonstrated that F. arundinacea population needs a minimum of 40% FC for germination and subsequent seedling establishment.

The findings of the current study are consistent with those of Gazanchian et al. (2006), who found that F. arundinacea entries need more than 25% FC for ger- mination. However, far too little attention has been paid to F. arundinaceain the Gazanchian et al.(2006) investi- gation.

There were substantially genetic differences in germina- tion and seedling development under conditions of drought stress among the different wild populations of F. arundinacea collected at different locations within Iran.

Cluster I populations had more rapid and greater final germination and greater stability for root growth under low soil water content. Especially, the ‘Isfahan’ population had the greatest final germination in low soil water ( 1.4 MPa). ‘Isfahan’ was collected from a low precipita- tion area. Our results for populations in cluster I sup- ported the idea that early root initiation and rapid root extension can help seedlings to avoid low soil water con- ditions (Johnson and Asay 1993). ‘Quchan’ and ‘Yasuj’

entries in cluster IV, with low final germination, delayed germination and low seedling vigor index at 40% FC (0.03), were the most sensitive entries at germination and emergence under low soil water content. In our study, the effect of low soil water content was more severe on leaf than on root length. As a result, root growth or SVI is probably a better indicator of establishment and sur- vival than leaf growth in F. arundinacea under drought

Figure 1 Classifications of 14 Iranian wildFestuca arundinaceapopu- lations and two turf cultivars based on six traits (final germination per- centage, leaf length, root length, leaf dry weigh, root dry weigh and seedling vigor index) at seedling emergence stage using Ward’s clus- ter analysis (Ward 1963) with Euclidian distance. Entry names are defined in Table 1.

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I. Rohollahiet al. Evaluation ofFestucain germination

conditions (Mut and Akay 2010). A high correlation coef- ficient between SVI and the other traits in our work, par- ticularly at 40% FC, indicated that it could be used as a selection criterion for improving low soil water tolerance at germination and emergence in cool season grasses. In cool-season grass species, quick germination depends on factors related with genotypes, germination environment and seed characters (Palazzo and Gurdarshan 1997; Ga- zanchianet al.2006; Mut and Akay 2010). Moreover, it is well known that rapid and complete germination are important during the establishment of grass species (Lar- sen and Bibby 2004; Mut and Akay 2010).

The ‘Isfahan’ population derived from a dry region, which had the greatest final germination in low soil water, and had the smallest relative DNA content. The relative DNA content changes by up to 6% among the populations of tall fescue studies. Differences of 5–10%

were enough to determine changes in nucleotype expres- sion (Price 1988). Our study showed that change in the basic amount of relative DNA content within hexaploid F. arundinacea populations might have an influence on Festuca establishment by affecting seed germination and growth rates during the early developmental stage under drought stress. Similar to our results on negative corre- lation between final germination, leaf length and gen- ome size has also been observed by Ceccarelli et al.

(1992, 1993) in F. arundinacea populations. Negative correlation between leaf length, leaf width and genome size of the populations has also been demonstrated by Caceres et al. (1998) in Dasypyrum villosum and by Grime et al. (1997) in 43 British plants. In this study, we found that other germination traits like SVI and GR have also had significant negative correlations with rela- tive DNA content.

In conclusion, our work revealed significant variation among populations of F. arundinacea from different eco- logical regions of Iran in response to low soil water con- tent. Low soil moisture content at 40% FC decreased the final emergency, SVI and seedling characters such as leaf and root length, especially in susceptible entries in com- parison with tolerant genotypes. Populations that were collected from dry regions ‘Isfahan’ also had the smallest DNA content, best germination, growth and SVI under drought conditions. Populations ‘Quchan’ and ‘Yasuj’

showed the worst final germination and SVI. Studies on identification indices of drought resistance indicated that final germination, leaf length, root length and SVI were most important and better indicators of drought tolerant F. arundinacea population with appropriate genome con- tent. Some wild populations could be valuable germplasm resources for the development ofF. arundinacea cultivars with improved establishment under conditions of reduced soil moisture.

Acknowledgements

This research was supported by Iran National Science Foundation Projects (90001304).

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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Means of measured germination and seedling characteristics of 16 Festuca arundinacea entries at four soil moisture levels.

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