characterization of commercial cultivars and ...

International Turfgrass Society Research Journal Volume 11, 2009

CHARACTERIZATION OF COMMERCIAL CULTIVARS AND NATURALIZED GENOTYPES OF STENOTAPHRUM SECUNDA TUM (WALTER) KUNTZE IN AUSTRALIA Donald S. Loch*, Matthew B. Roche, Jenny Sun-Yue, Vivi Arief, Ian H. Delacy and Christopher J. Lambrides ABSTRACT Stenotaphrum secundatum (Walter) Kuntze, known as "St Augustinegrass" in the USA and "buffalo grass" in Australia, is a widely used turfgrass species in subtropical and warm temperate regions of the world. Throughout its range, S. secundatum encompasses a great deal of genetic diversity, which can be exploited in future breeding programs. To understand better the range of genetic variation in Australia, morphological-agronomic classification and DNA profiling were used to characterize and group 17 commercial cultivars and 18 naturalized genotypes collected from across Australia. Historically, there have been two main sources of S. secundatum in Australia: one a reputedly sterile triploid race (the so-called Cape deme) from South Africa now represented by the Australian Common group naturalized in all Australian states; and the other a "normal" fertile diploid race naturalized north from Sydney along the NSW coast, which is referred to here as the Australian Commercial group because it has been the source of most of the new cultivars recently developed in Australia. Over the past 30 years, some US cultivars have also been introduced and commercialized; these are again "normal" fertile diploids, but from a group distinctly different from the Australian Commercial genotypes as shown by both DNA analysis and grouping based on 28 morphological-agronomic characteristics. The implications for future breeding within S. secundatum in Australia are discussed. Abbreviations: CTAB, cetyl tri-methyl ammonium bromide; dap, days after planting; ISSRs, Inter-Simple-Sequence-Repeats; L:W ratio, Length:Width ratio; M-A, morphologicalagronomic; PBR, Plant Breeder's Rights; PC, principal component; PP, Plant Patent; QLD/Qld, Queensland; NSW, New South Wales; VIC, Victoria; SA, South Australia; US, United States; WA, Western Australia. Keywords: Stenotaphrum secundatum, St Augustinegrass, DNA profiling, morphologicalagronomic classification, genetic variation, Australia D.S. Loch (formerly) Dep. of Primary Industries and Fisheries, Redlands Research Station, 26Delancey Street, Cleveland, Qld 4163, Australia (current address: 35 Hilltop Crescent, Alexandra Hills, Qld 4161, Australia); M.B. Roche, Dep. of Primary Industries and Fisheries, Redlands Research Station, 26 Delancey Street, Cleveland, Qld 4163, Australia; J. Sun-Yue, V. Arief, I.H. Delacy and C.J. Lambrides, School of Land, Crop and Food Sciences, University of Queensland, Brisbane, Qld 4072, Australia. * Corresponding Author: ([email protected]).

INTRODUCTION Stenotaphrum secundatum (Walter) Kuntze, known as "St Augustinegrass" in the USA and "buffalo grass" in Australia, is a warm-season turfgrass now widely used in subtropical and warm temperate regions of the world. The species occurs naturally as a seashore pioneer, and its inflorescences show adaptations for short-range dispersal by ocean currents. However, as Sauer (1972) observed, this does not account for its widely disjunct and apparently natural range, which would require a proven capacity for long-range sea dispersal. Busey (2003) summarizes hypotheses regarding the origin of S. secundatum: the first postulates an Old World origin along the coastlines and islands of the Indian Ocean and that it was brought to the New World by Europeans during the postColumbian era; an alternative hypothesis of a New World origin is consistent with the wide distribution and genetic diversity of S. secundatum in the Americas demonstrated by very early collections of the species prior to the 1800s; and the third hypothesis proposes an Old World origin and that the species was brought to the Americas by an early transoceanic dispersal predating the European voyages of discovery and colonization. The existing genetic variation in S. secundatum is not smoothly distributed across the species, but rather occurs as clusters of similar genotypes (Busey, 2003). These clusters have been variously designated as "Groups", "Races" (Busey et al., 1982; Busey, 1986), and "Demes" (Sauer, 1972). The so-called "normal" form of the species is as a fertile diploid with 2n=18 chromosomes (Busey, 2003). There are also polyploid types, most notably sterile triploids with 3n=27 chromosomes causing irregular meiosis (Long and Bashaw, 1961) as well as genotypes with c. 30-32 chromosomes (Busey, 2003) and tetraploids with 4n=36 chromosomes (Long and

Bashaw, 1961). Polymorphisms associated with the different chromosome types and clusters of similar genotypes have been demonstrated for a range of morphological and physiological/agronomic attributes, including disease, herbicide and drought resistance and shade tolerance (e.g. Atilano and Busey, 1983; Busey and Davis, 1991; Busey, 1993, 1996). In this paper, we report the results of pattern analysis studies on commercial cultivars and naturalized accessions of S. secundatum from Australia based on both morphological-agronomic data and DNA profiling. Our objective was to provide a logical framework within which to assess the results of associated studies of drought, shade, herbicide and wear tolerance, as well as regional adaptation (climate, soil), of these different genotypes. HISTORY OF STENOTAPHRUM SECUNDA TUM CULTIVAR DEVELOPMENT AND GENETIC VARIATION IN AUSTRALIA While the origins of S. secundatum elsewhere in the world may still be open to conjecture (Sauer, 1972; Busey, 2003), the sources of material present in Australia appear relatively clear. The species is regarded as an exotic one (Sharp and Simon, 2002), introduced to the country during the 1800s following European settlement. According to Sauer (1972), the "normal" fertile type was present in Australia by 1840. Subsequently, the sterile triploid Cape deme was imported during the 1840s as deck cargo on board the SS Buffalo, hence the species common name of buffalo grass (Aldous and Semos, 1999; P. McMaugh, personal communication, 2000). Sauer (1972) reports that the Cape deme was recorded near Sydney, New South Wales (NSW) by the mid-nineteenth century, in

South Australia (SA) by 1911, in Queensland (QLD) by 1917, in Western Australia (WA) by 1923, in Tasmania (TAS) by 1945, and in Victoria (VIC) by 1950. It was found in lawns and pastures, and also escaped to colonize seashore habitats. It became the "Common" buffalo grass in Australia, but Sauer's (1972) statement that it seemed to have completely replaced the "normal" race was premature with respect to his perceived demise of the latter.

grown to a limited extent in WA. This was followed in the 1990s by the "ST" series and by Palmetto®. A full list of Australian S. secundatum cultivars is presented in Table 1 in their approximate historic order of development, together with brief background details.

Stretching from Sydney along the NSW north coast is a variable (and apparently "normal") population of S. secundatum with its center of diversity arguably located in the lower Hunter Valley (P. McMaugh, personal communication, 2000). This has been the main source of new cultivars - the socalled "soft leaf' buffalo grasses - in Australia over the past 25 years or so. The first of these was 'Shademaster', which sets a high percentage of fertile seeds that will germinate readily (D.S. Loch, unpublished observations, 2000). Subsequent releases were developed from different seedlings found on turf farms and often attributed to Shademaster as a parent, or from superior plants identified from the diverse pool of genetic variation represented by the naturalized plant population. Despite claims by some breeders that the variant plants they discovered and developed arose through mutation, there is no evidence for this and a more plausible origin for these new cultivars is via normal sexual seed formation leading to genetically variable seedlings.

Three spaced-plant experiments were conducted on a fertile red volcanic (krasnozem) soil at Redlands Research Station (27°32'S lat., 153°15'E long., c. 25 masl) near Brisbane in southern Queensland, Australia. In each case, the 17 cultivars and accessions listed in Table 1 were arranged in a randomized block design with either 6 replicates (Experiment 1) or 3 replicates (Experiments 2 and 3). Each plot comprised 5 spaced plants, giving a total of 30 plants per cultivar in Experiment 1 and 15 plants in Experiments 2 and 3. Plants were spaced on a 1.5 x 1.5 m grid for experiments 1 and 2, but this was reduced to a spacing of 1.4 x 1.4 m for Experiment 3.

At the same time, cultivars developed in the United States of America (USA) or from US varieties were also imported into Australia, beginning in the late 1970s with Velvet™ which is still

MORPHOLOGICAL-AGRONOMIC CLASSIFICATION MATERIALS AND METHODS

In each experiment, plants were vegetatively propagated in the glasshouse (4 nodal cuttings per 60 x 60 mm pot) and, after 1-2 months, planted into a wellprepared seedbed in the field. Dates of planting for Experiments 1, 2 and 3 were 13 Feb. 2006, 25 Sept. 2006 and 1 May 2007, respectively. Slow release 18-10-9 fertiliser (275 kg ha"1) was applied at planting, together with oxadiazon {3-[2,4dichloro-5-(l-methylethoxy)phenyl]-5(1,1 -dimethyl-ethyl)-1,3,4-oxadiazol2(3H)-one} (Chipco Ronstar G®) broadcast at 150 kg ha"1 of 2% product for weed control.

Table 1. Genotypes used in spaced plant studies for morphological-agronomic characterisation of Stenotaphrum secundatum cultivars in Australia. Code

Cultivar

PBR application no. -

Utility patent application no.

Year granted

Trade Name

Comments

References

WA-1

Common

-

VEL

Velvet

SHM

Shademaster

ST85

ST-85

AccessionfromWA used by WA and QLD turf researchers as an T.C. Colmer, personal example of the Australian Common type ("Old Style" buffalo grass) communication, 2004 Califomian "Common" type introduced in the late 1970s and commercialized in P. McMaugh, personal WA, but not used in eastern states because of susceptibility to diseases communication, 2004 - . . . Commercialized c. 1985 as thefirst"soft-leaf buffalo grass cultivar; P. McMaugh, personal originatedfromnear Stockton in the lower Hunter Valley (NSW) communication, 2008 AU 199187058 1993 Selected dwarf seedling from a cross between ST-19 and 'Seville' Whiting (1993) (US PP4,097, 1977. Riordan et al., 1980)

ST26

ST-2691

-

AU 199228394

1997

ST-26

Selected seedling from a cross between ST-19 and'Seville' Whiting (1997a) (US PP4,097, 1977 Riordan et al., 1980) ST91 ST-1191 AU 199511474 1997 ST-91 Selected dwarf seedling from a cross between ST-19 and'Seville' Whiting (1997b) (US PP4,097, 1977 Riordan et al., 1980) SWL Sir Walter 1996/226 1998 Variant plant discovered among'Shademaster'on the breeder's turf McMaugh (1997) farm in the lower Hunter Valley (NSW) NSW-7 Shade Genotype originally selected near Coffs Harbour (NSW) in the late C.N. Beard, personal Invader 1980s; sold under trade mark no. 775835 granted in 1999 communication, 2008 PAL SSI00 1996/158 - 2002 Palmetto US cultivar based on a variant plant discovered adjacent to Scattini (1999) 'Bitterblue' and 'Floratam' (both sterile genotypes - Busey, 2003) on a Florida, USA, turf farm (US PP9,395, 1995) SAP B12 2002/342 - 2003 Sapphire Seedling derived from'Sir Walter' Paananen (2002); US PP16, 174 (2005) NSW-6 Coastal Genotype from the lower Hunter Valley (NSW); also sold as N.T. Hancey, Personal Shade "Grand Royal" communication, 2008 MAT Matilda 2004/078 2005 Seedling derivedfrom'Shademaster' on a Hawkesbury turf farm Paananen (2004) west of Sydney (NSW) SJM Sir James 2002/283 - 2005 Variant plant discovered among Common buffalo grass in the lower Loch and Roche (2004) Hunter Valley (NSW) MAR Marine 2005/033 - 2006 Seedling plant discovered adjacent to'ST-85','Shademaster'and Common Paananen (2005) buffalo grass on a Hawkesbuiy turf farm west of Sydney (NSW) NKL Ned Kelly 2005/298 - 2007 Variant plant discovered among Common buffalo grass in the lower Paananen (2006) Hunter Valley (NSW) KPR Kings Pride 2005/341 - 2007 Variant plant selected from a long-established buffalo grass lawn in McMaugh (2006) the lower Hunter Valley (NSW) TF01 TF01 2007/245 - 2008 Distinctive plant from the tidal reaches of the Bellinger River on the Roche and Loch (2007) north coast of NSW ST 135 ST-135 - . . . Selected dwarf seedling from a cross between ST-2691 and ST- H.F. Whiting, personal 1191; not commercialized communication, 2007 AMS TR 6-10 Amerishade US bred cultivar (US PP17, 095, 2006) trialled, but not yet comercialized, in Australia

Supplementary weed control was achieved by regular manual roguing of each experiment, and by spraying with fluroxypyr {[(4-amino-3,5-dichloro-6fluoro-2-pyri-dinyl)-oxy]acetic acid} (Starane 200®) at 500 ml ha 1 of 20% product for broadleaf weed control. Additional fertilizer (as urea) was applied to Experiment 1 on 20 Oct. 2006 at a rate of 100 kg N ha"1. All experiments were irrigated regularly as necessary to maintain unstressed growth. Measurements of lateral spread were made in all three experiments at approximately fortnightly intervals, beginning 4-8 weeks after planting. For each plant, mean diameter was determined from measurements of the widest diameter of spread taken from stolon tip to stolon tip across the centre of the plant (0° to 180°), the diameter at right angles to this (90 to 270°), and two intermediate measurements (45° to 225°, 135° to 315°) between the first two defining measurements. Comparisons of lateral spread in Table 2 show data for the final measurement date in each experiment. In Experiment 1, morphological measurements (2 per plant) were made on stolon attributes (13-27 July 2006), and on flowering culm and inflorescence attributes (28 Nov. - 14 Dec. 2006). Stolons of S. secundatum produce compound nodes as described by Bogdan (1952) for a wide range of warm-season grasses; each compound node in S. secundatum is subtended by 2 leaves. Measurements were taken from the fourth visible stolon internode and the outer leaf at the fourth visible node by which time cellular expansion in the stolon tip region virtually complete; these was considerations were balanced by the increasing likelihood of damage to stolon

leaves on older nodes. The solid inflorescences of S. secundatum are false spikes in which the panicle branches are contracted and usually reduced to l(-3) spikelets partially embedded in one face or the sides of the flattened corky rachis (Sharp and Simon, 2002; Busey, 2003). Additional measurements included height of the unmown sward (6 Nov. 2006) and inflorescence density (15 Dec. 2006). All data were analysed through GenStat Release 8.1 for Windows using standard Analysis of Variance procedures, which also generated Fisher's protected Least Significant Differences (LSDs) for comparison of treatment means. An examination of the error mean squares observed for analyses of each trait in the three experiments indicated that it was appropriate to combine the data for pattern analysis. Pattern analysis of the morphological-agronomic (M-A) data generated from the 17 genotypes was performed using a two-stage process in R version 2.7.1 (R Development Core Team, 2008). Firstly, a similarity matrix was generated by calculating Dice coefficients for all pairwise combinations of genotypes. The genotypes were then clustered into groups using the Incremental Sums of Squares (ISS) method. RESULTS There was significant variation among the 17 genotypes for each of the 23 morphological attributes measured (Table 2). There was also significant variation in the numbers of inflorescences produced per unit area, in height of the unmown swards, and in the rate of lateral spread (which was repeated three times and showed good consistency across different seasonal conditions).

Table 2. Morphological and agronomic measurements on 17 genotypes of Stenotaphrum secundatum available in Australia. See Table 1 for details of the abbreviated codes used to identify the different cultivars or accessions studied. Cultivar/Accession L§D SHM SJM SWL ST 26 ST 85 ST 91 ST 135 TF01 VEL WA-t AM) 051 Stolon: " ' Internode length (mm) SIL 50.9 60.7 50.1 55.2 60.7 42.3 52.9 43.7 55.3 64.7 47 1 47 1 31 9 29 1 65 0 32 6 51 6 3 3 Internode diameter (mm) SID 3.04 2.90 2.97 2.77 2.96 2.87 2.87 3.04 2.79 2.82 3 10 2 19 1 91 1 94 2 72 2 43 3 37 0 14 Leaf sheath length (mm) SLSL 17.2 19.6 17.7 17.0 18.7 18.9 17.4 16.2 21.0 21.3 17.0 12 3 10 3 10 3 18 2 15 8 19 9 o'85 Leafblade length (mm) SLL 13.4 21.4 15.8 19.6 20.0 18.0 14.2 17.0 23.5 23.9 14.5 12.1 10 1 11 0 167 140 2o's 12 Leaf blade width (mm) SLW 5.63 7.01 6.42 6.65 6.61 6.46 5.81 6.30 6.86 7.36 5.76 4.86 3.82 4.00 6 47 5 48 6 56 0 29 Leafblade L:W ratio SLWR 2.34 3.05 2.48 2.97 2.99 2.78 2.44 2.72 3.42 3.24 2.52 2.49 2 70 2 76 2 58 2 56 3 17 OH Branches at node 2 B2N 1.32 1.40 1.40 1.83 1.50 1.28 1.10 1.73 1.35 1.12 0.95 1.10 0.88 0.62 L18 L17 1 42 025 Flowering culm (tiller): — Flag leaf sheath length (mm) FLSL 53.4 41.1 46.9 50.3 43.2 58.0 48.5 38.3 46.1 42 5 54 2 47 7 34 1 32 0 43 2 42 1 48 5 3 2 Flag leafblade length (mm) FLL 31.3 24.4 33.8 33.4 27.9 40.5 28.7 25.6 31.4 30.2 32.2 46 3 21 5 212 28 2 25 3 30 4 5 2 Flag leafblade width (mm) FLW 6.49 5.81 6.26 6.67 6.42 6.63 6.14 5.31 6.13 6.68 6.16 6.62 5.04 4.63 6.25 5.63 6 01 0 47 Flag leafblade L:W ratio FLWR 4.66 4.22 5.46 4.99 4.21 5.88 4.60 4.85 5.11 4.41 5.24 6.83 4 28 4 09 4 43 4 39 4 94 0 69 Tiller leaf sheath length (mm) TLSL 40.1 34.6 31.0 39.9 36.6 36.9 38.5 26.2 31.8 33.0 38 5 35 2 25 2 211 36 2 30 7 42 1 3 3 Tiller leafblade width (mm) TLW 7.82 7.10 6.11 6.61 7.37 6.78 7.39 5.80 6.93 7.19 7.23 7.11 5.61 5.33 6.75 6.54 7 20 0 45 Tiller leafblade L:W ratio TLWR 9.66 9.62 10.94 13.41 10.56 8.83 9.66 8.26 10.61 10.67 10.54 11.20 8.84 7 28 1208 8 86 11 91 1 46 Internode length (mm) TIL 50.9 42.3 43.9 42.2 41.8 42.0 47.0 29.9 31.4 35.9 36.6 40 5 20 9 18 1 37 2 40 5 43 3 5 9 Internode diameter (mm) TIP 1.76 1.70 1.53 1.55 1.52 1.79 1.80 1.71 1.43 1.73 2.03 1.52 1.52 1.76 1.48 1.71 2 04 0 12 Inflorescence: ' ~ — Peduncle length (mm) PL 104.0 60.2 77.5 77.8 67.3 93.9 93.6 74.9 73.1 69.8 83.2 80.6 49.4 52 0 55 2 71 3 101 4 8 6 Peduncle width (mm) PW 1.36 1.63 1.32 1.44 1.36 1.59 1.44 1.62 1.32 1.64 1.52 1.16 1.25 1.26 1 37 1 50 1 60 0 10 Inflorescence length (mm) IL 93.8 81.5 65.8 86.3 90.3 89.6 88.6 74.5 89.1 86.0 84.8 77 2 64 7 56 1 78 2 79 7 83 4 4 5 Inflorescence minimum width (mm) IMNW 2.15 2.47 2.19 2.34 2.28 2.48 2.16 2.41 2.32 2.49 2 36 2 06 2 05 2 00 2 17 2 30 2 40 0 12 Inflorescence maximum width (mm) IMXW 4.54 4.55 4.30 4.32 4.47 4.82 4.61 4.43 4.10 4 76 4 64 4 09 3 97 4 19 4 15 4 44 Vl7 0 23 No. of inflorescences per ' ' ' flowering tiller lPT 2.9 2.7 2.5 2.4 2.8 2.7 2.8 2.4 2.4 2.7 2.8 2.3 2.3 2.3 2.6 2 8 2 6 02 Agronomic measurements: ~ " Inflorescences m"2 305 dapt Attribute

Code

AMS

KPR MAR MAT

__ PAL

NKL

SAP

Experiment 1 ID Unmown sward height 266 dap (cm)

906.9

395.1

348.6

511.8

357.6

406.5

961.6

183.7

150.2

440.0

486.5

706.1

380.4

315.1

376.3

1069 '

111 0

-Experiment 1 SH Plant diameter 87 dap (cm) -

29.7

34.8

26.8

37.7

31.0

19.9

30.4

21.6

27.5

35.8

24.5

13.9

16.6

3.2

35.9

10.3

28.5

107.4

160.0

159.6

106.1

121.2

129.7

122.2

142.0

100.0

Experiment 1 PD1 Plant diameter 85 dap (cm) -

111.1

167.4

Experiment 2 PD2 Plant diameter 154 dap (cm) -

155.5

170.9

134.2

155.4

185.3

Experiment 3 PD3 t dap = days after planting.

139.5

167.5

125.9

126.0

160.1

113.8 97.3

157.2 131.4

187.5 114.3

132.1 112.4

68.9

37.9

32 8 1745 57 4 ' ' '

179.1

129.6

110.5

79.2

69.9

192.7

69 2

179.6

125.3

106.2

78.5

68.8

199.1 —

75.4

94 8

143 8 46 14 1

1390

13 6

126.1

16.3

Group

1

2

3

4

5

Figure 1. Dendrogram for Australian Stenotaphrum secundatum eultivars and accessions derived from pattern analysis of morphological-agronomic data. See Table 1 for the genotypic identification codes used.

Figure 2. Biplots showing genotype (red) and attribute (black) relationships for the three main principal components, which collectively account for 76.45% of the total variation in the pattern analysis of morphologicalagronomic data presented in Fig. 1. For example, in the biplot on the left, genotypes ST91 and ST 135 were near the mean for SLWR and below average for all other attributes. See Tables 1 and 2, respectively, for the codes used to identify genotypes and attributes.

These genotypes ranged from dwarf, slow-spreading plants with short internodes and fine stems and stolons to more robust genotypes that spread rapidly with longer internodes and thicker stems and stolons (e.g. 'Sir Walter', 'Kings Pride', 'Ned Kelly', 'TF01'). The dwarf genotypes also produced shorter, thinner inflorescences and peduncles. Leaf length and shape differed markedly between stolons and flowering tillers, and comparative measurements of these attributes among cultivars also differed between stolons and flowering tillers. Among the more robust genotypes, for example, 'Matilda' and 'TF01' had long, relatively narrow leaves on flowering tillers while the Common WA-1 had long, relatively broad leaves; but on stolons, 'Sir James' and 'Sir Walter' produced long, relatively narrow leaves while 'Matilda' had shorter, broader leaves and the Common WA-1 had shorter, narrower leaves. The dendrogram produced by morphological-agronomic classification of the data in Table 2 showed 5 main groups (Fig. 1). The first of these comprised the two slow-growing, fine-textured, dwarf genotypes, 'ST-91' and ST-135, developed from US germplasm. The second group included most of the Australian-derived cultivars: 'Ned Kelly', 'Kings Pride', 'Sir Walter' and 'TF01' in one sub-group; and 'Sir James', 'Marine' and 'Matilda' in the other. 'ST-85' (developed from US germplasm) sits alone in Group 4. However, there is some overlap between Australian and US genotypes in Groups 3 and 5, though the biplots of the 3 main principal components (PCs) in Fig. 2 indicate that, for example, Velvet and 'Shademaster' in Group 3 and WA-1 with Palmetto and 'ST26' in Group 5 are not as closely similar as their inclusion within these discrete groups may suggest.

DNA PROFILING MATERIALS AND METHODS A further 15 naturalized accessions of S. secundatum were added to the 17 genotypes listed in Table 1 for DNA profiling. With the exception of 2 variegated genotypes (designated VAR-1 and VAR-2), these were designated and numbered according to the Australian state of origin: NSW (6 accessions), QLD (4 accessions), VIC (1 accession), TAS (1 accession), and SA (1 accession). One accession of the sole Australian native species, Stenotaphrum micranthum, was also included and coded MIC. This species occurs on islands off the Queensland coast, but is not found on mainland Australia (Sharp and Simon, 2002). DNA was isolated from 0.2-0.3g fresh leaves ground in liquid N and homogenised in 1.2 ml of CTAB (cetyl trimethyl ammonium bromide) buffer for 2 hrs at 65°C with shaking every 15 mins. The samples were then centrifuged and the supernatant poured off and extracted twice with an equal volume (700 jn/) of chloroform:isoamyl (24:1). DNA was precipitated using 0.6V isopropanol and washed twice with 70% ethanol. DNA pellets were dried and resuspended in 100 jlx/ TE buffer and incubated with RNAse. InterSimple-Sequence-Repeat (ISSR) markers were generated by PCR using 15 |Ltl reactions containing 67 mM Tris HC1 (pH 8.8), 16.6 mM (NH 4 ) 2 S0 4 , 0.45% (v/v) Triton-X-100, 200 |ng/ml gelatine, 1.0 mM MgCl2, 1.2 mM dNTPs, 0.8 ¿iM primer, 20 ng genomic DNA and 0.9 unit of Taq polymerase. Reactions were placed in a Geneworks thermal cycler programmed for 36 cycles of 40 s at 94°C for denaturation, 1 min at 54-57.8°C for annealing and 2 min at 72°C for primer extension. A 5 min denatureation step at 94°C prior to the commencement of the first cycle and a 15 min

extension step at 72°C after the last cycle were included. Three fluorescently labelled primers were used i.e. VIC (GA)9C, 6FAM (AG)9C, NED (GA)9T. Amplification products were separated by capillary electrophoresis using an ABI 3130 genotyper and visualised using Genemapper software. The dominant markers generated were used to produce a dendrogram using the same methods of pattern analysis as described above for the M-A data. RESULTS The dendrogram produced by DNA profiling grouped the 32 commercial cultivars and naturalized accessions of S. secundatum tested into 3 main groups, with S. micranthum showing substantially greater differences from those genotypes (Fig. 3). These groupings can be described as follows. 1. Australian Common Group: 9 naturalized accessions from QLD, NSW, VIC, TAS and WA. 2. Australian Commercial Group: 9 commercial cultivars plus 4 naturalized accessions collected from Windsor and the

lower Hunter Valley through to Caloundra in southern Queensland. The variegated form, VAR-2, which originated in the lower Hunter Valley, alsofitsinto this group. 3. American Commercial Group: 7 cultivars from the US or from US germplasm, plus one naturalized accession from SA. Variegated foliage is not an attribute uniquely associated with a particular genotypic group, but could arise in any or all of these. While VAR-1 (origin unknown) was split off on its own strand between the Australian Common and Australian Commercial groups in the dendrogram in Fig. 3, it would appear to be more closely associated with the Australian Common group based on the Similarity Coefficient; these details need to be checked in future work. VAR-2, on the other hand, falls clearly within the Australian Commercial group. DISCUSSION The results of our DNA analysis confirm Sauer's (1972) statement that early introductions of S. secundatum to Australia

Figure 3. Dendrogram for Australian Stenotaphrum secundatum samples based on D N A analysis. See Table 1 and text for the identification codes used for cultivars and accessions tested.

came from two basic genotypic groups: the sterile triploid Cape deme (which was the basis of the widely used and naturalized Australian Common group during the late 19th century and for much of the 20th century) and a "normal" fertile diploid group. Although reputedly sterile (Sauer, 1972), the Australian Common group does show limited genetic variation (Fig. 3), as described by Busey (2003) for comparable material in the USA. Interestingly, the NSW Common genotypes tested showed greater genetic diversity than did the remaining Common genotypes across the rest of Australia (Fig. 3), perhaps suggesting that multiple imports of the Cape deme through Sydney in the mid-nineteenth century were localized with regard to their subsequent distribution rather than being spread widely across the country. However, contrary to Sauer's (1972) conclusion, the "normal" group survived (and apparently thrived) in coastal areas north of Sydney through to about the mid-North Coast of NSW. Whether or not this group extends into southern QLD is still subject to conjecture and on-going investigations into the origin of QLD-3, the only example of the "normal" group so far collected from southern QLD. Selection of superior variant plants from the "normal" group has been the major contributor to the development of new S. secundatum cultivars in Australia since the mid-1980s (see Table 1 and Fig. 3), hence our naming it the Australian Commercial group. Elsewhere in VIC, TAS, WA and southern QLD, the dominant naturalized forms would appear to be from the Australian Common group, although more intensive sampling of the naturalized populations is required before this could be confirmed. Clearly, in Australia, there is not the same degree of genetic diversity among S. secundatum as described by Busey (2003) for the USA. However, as markets for S. secundatum have grown accompanied by the development of new cultivars, US cultivars

have also been introduced and released in Australia. These were also derived from Busey's (2003) "normal" type, but show distinctly different DNA profiles when compared with Australian-derived "normal" types (Fig. 2). This is supported by our M-A classification (Fig. 1), which shows the Australian Commercial and the American Commercial genotypes forming two distinct, but overlapping, groups as a result of differences in their morphology and agronomic development. To this could also be added additional differences, for example, in stigma colour: the Australian genotypes thus far seen all produce reddishpurple stigmas, while at least some of the US genotypes have white stigmas (e.g. Velvet, Palmetto, Amerishade) as described by Busey (2003). There is also evidence from our results (e.g. the accession SA-1 from Moonta, SA) that the American Commercial group is now beginning to enter the naturalized germplasm in Australia (as could also happen with the Australian Commercial group now that planting material is being distributed extensively around the country). Earlier work by Kim (2005), using 18 ISSR primers but with a smaller group of 10 S. secundatum genotypes and applying Nearest Neighbour grouping techniques, produced the same 3 broad groups with the same individual members as in the present studies. To date, the "breeding" of new S. secundatum cultivars in Australia has been restricted to the haphazard (one-off) selection of superior plants found among naturalized material or as contaminant seedlings on turf farms. In future, a more scientific and methodical approach should see deliberate crosses made, not just among the Australian Commercial genotypes but between these and American Commercial genotypes, in an attempt to exploit the best characteristics of both groups, particularly as more information is gathered regarding

regional adaptation and needs and key attributes like disease and herbicide tolerance in these different environments across Australia.

Bogdan, A.V. 1952. Observations on stoloniferous grasses in Kenya. J. East Afr. Nat. Hist. Soc. 20(2):71-76. Busey,

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