Molecular Variation and Fingerprinting of Leucadendron Cultivars (Proteaceae) by ISSR Markers

Abstract

• Background and Aims There are more than 80 species of Leucadendron and most are used as cut flowers. Currently, more than 100 cultivars are used by industry and many of them are interspecific hybrids. The origin of most cultivars is unclear and their genetic diversity and relationships have not been studied. This investigation was carried out to evaluate the genetic variation and relationships among 30 Leucadendron cultivars.

• Methods ISSR markers were applied to determine the genetic variation and to discriminate Leucadendron cultivars. Sixty-four ISSR primers were screened and 25 primers were selected for their ability to produce clear and reproducible patterns of multiple bands.

• Key Results A total of 584 bands of 305–2400 bp were amplified, of which 97 % were polymorphic. A dendrogram generated using the Unweighted Pair Group Method with Arithmetic Average based on a distance measure of total character difference showed that the Leucadendron cultivars clustered into two main groups. Twenty-four of the 30 cultivars can be unequivocally differentiated, but identical profiles were observed for three cultivar pairs, ‘Katie's Blush’ and ‘Silvan Red’, ‘Highlights’ and ‘Maui Sunset’, and ‘Yellow Crest’ and ‘Yellow Devil’.

• Conclusions ISSR profiling is a powerful method for the identification and molecular classification of Leucadendron cultivars. A fingerprinting key was generated based on the banding patterns produced using two ISSR primers (UBC₈₅₆ and UBC₈₅₇). In addition cultivar-specific ISSR bands were obtained for 17 of the 30 Leucadendron cultivars tested.

INTRODUCTION

Leucadendron are Proteaceae that produce male and female flowers on separate plants. The flowers have distinct and colourful petal-like bracts (Vogts, 1982; Matthews, 2002) that, along with long stems and long-lasting foliage, make many Leucodendron species highly desirable cut flowers. There are 80 species of Leucadendron. Based on fruit characteristics, the species have been divided into two sections, Alatosperma and Leucadendron, with each section having several subsections (Williams, 1972).

There are more than 100 Leucadendron cultivars (International Proteaceae Register, 2002). The most widely grown cultivar, ‘Safari Sunset’, is grown in Australia, New Zealand, South Africa, Hawaii and Israel (Littlejohn and Robyn, 2000). Several commercial cultivars (Littlejohn et al., 1998; Sedgley and Yan, 2003) are the result of interspecific hybridization (Van den Berg and Brits, 1990; Littlejohn et al., 1995; Yan et al., 2001a, b).

There is limited knowledge regarding the genetic diversity and interspecies relationships in Leucadendron, limiting the efficiency of breeding programmes. Moreover, the parentage of Leucadendron cultivars is not always certain due to inadequate documentation. Some cultivars may have arisen from selection of seed populations, while others have arisen from hybridization of undocumented parents (Littlejohn and Robyn, 2000). Therefore, methods for the fast and accurate identification of Leucadendron species and cultivars would be of substantial benefit to the Leucadendron cut-flower and breeding industry.

Random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990) and ISSR markers (Zietkiewicz et al., 1994) are two molecular typing approaches that have been used to detect variation among plants. Each method has been used extensively to identify and determine relationships at the species and cultivar levels (Rajaseger et al., 1997; Raina et al., 2001; Martins et al., 2003). These methods are widely applicable because they are rapid, inexpensive, simple to perform, do not require prior knowledge of DNA sequence and require very little starting DNA template (Esselman et al., 1999).

The ISSR method has been reported to produce more complex marker patterns than the RAPD approach (Parsons et al., 1997; Chowdhury et al., 2002), which is advantageous when differentiating closely related cultivars. In addition, ISSR markers are more reproducible than RAPD markers (Goulão and Oliveira, 2001), because ISSR primers, designed to anneal to a microsatellite sequence, are longer than RAPD primers, allowing higher annealing temperatures to be used. ISSR analysis has been used for cultivar identification in numerous plant species, including rice (Joshi et al., 2000), apple (Goulão and Oliveira, 2001) and strawberry (Arnau et al., 2003).

This paper reports on the use of ISSR markers to differentiate Leucadendron cultivars, to determine the molecular relationships among the cultivars tested and to develop a fingerprinting key for Leucadendron.

MATERIALS AND METHODS

Plant materials

Leaf tissue for DNA extraction was collected from the 30 Leucadendron cultivars listed in Table 1.

Table 1.

Leucadendron cultivars used in this study

Cultivars	Parental origin	Source of collection	References
Buttercup	Leucadendron sp.	Amarilo Proteas§	IPR*
Corringle Gold	L. gandogeri Schinz ex Gand. × L. spissifolium (Salisb. ex Knight) I.Williams	Proteaflora	IPR, Matthews (2002)
Devil Blush	L. salignum Berg.	Proteaflora	Proteaflora†
Fire Glow	L. salignum Berg.	Proteaflora	IPR, Proteaflora
Gem	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Proteaflora	Matthews (2002)
Highlights	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Proteaflora	IPR
Inca Gold	L. laureolum (Lam.) Fourc. × L. salignum Berg. (yellow form)	Amarilo Proteas	Matthews (2002)
Jubilee Crown	L. laxum I.Williams hybrid	Amarilo Proteas	IPR, Littlejohn and Robyn (2000)
Katie's Blush	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Amarilo Proteas	Proteaflora
Maui Sunset	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Amarilo Proteas	IPR, Matthews (2002)
Pisa	L. floridum R.Br hybrid	Amarilo Proteas	IPR, Matthews (2002)
Pixie Red	L. salicifolium (Salisb.) I.Williams × L. procerum (Salisb. ex Knight) I.Williams	UWA	Sedgley and Yan (2003)
Pom Pom	L. discolor Buex ex Meisn.	Proteaflora	IPR
Red Devil	L. salignum Berg.	Proteaflora	IPR, Proteaflora
Red Centre	L. discolor Buex ex Meisn.	Proteaflora	Proteaflora
Safari Sunset	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Amarilo Proteas	Matthews (2002)
Safari Goldstrike	L. strobilinum Druce × L. laureolum (Lam.) Fourc.	Proteaflora	Matthews (2002)
Silvan Red	L. laureolum (Lam.) Fourc. × L. salignum Berg.	Proteaflora	IPR, Matthews (2002)
Spring Gold	L. gandogeri Schinz ex Gand.	Proteaflora	Proteaflora
Summer Sun	L. laureolum (Lam.) Fourc. × L. discolor Buex ex Meisn.	Proteaflora	Proteaflora
Tall Red	L. salignum Berg. × L. eucalyptifolium Buex ex Meisn.	Proteaflora	Proteaflora
Yellow Devil	L. salignum Berg.	Proteaflora	IPR, Proteaflora
Yellow Crest	L. salignum Berg.	Amarilo Proteas	IPR
436	L. coniferum Meisn. × L. discolor Buex ex Meisn.	UWA	UWA‡
772	L. floridum R.Br × L. discolor Buex ex Meisn.	UWA	UWA
802	L. floridum R.Br × L. gandogeri Schinz ex Gand.	UWA	UWA
868	L. floridum R.Br × L. procerum (Salisb. ex Knight) I.Williams	UWA	UWA
1386	L. uliginosum R.Br × L. discolor Buex ex Meisn.	UWA	UWA
1325	L. strobilinum Druce × L. procerum (Salisb. ex Knight) I.Williams	UWA	UWA
1424	L. uliginosum R.Br × L. salicifolium (Salisb.) I.Williams	UWA	UWA

^*International Proteaceae Register (http://www.nda.agric.za/docs/Protea2002/proteaceae_register.htm).

^†Proteaflora (http://www.proteaflora.com/).

^‡G. Yan, R. Sedgley and B. Croxford (unpubl. res.).

^§Hilltop Rise, Karnup, WA 6176.

DNA extraction and electrophoresis

Genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Clifton Hill, Victoria, Australia). Approximately 0·1 g of leaf material from one individual plant for each cultivar was ground to a fine powder in liquid nitrogen with a mortar and pestle before isolation of DNA according to the manufacturer's instructions. DNA was visualized by agarose gel electrophoresis followed by staining with ethidium bromide (Sambrook et al., 1989). Known amounts of lambda DNA (MBI, Fermentas, Hanover, MD, USA) were included on the gel to quantify the DNA.

ISSR amplification

ISSR primers (UBC set 9) were from the Biotechnology Laboratory, The University of British Columbia, Canada (Table 2). Optimal conditions for DNA amplification were empirically determined by testing different concentrations of genomic DNA (10, 15, 25 and 40 ng), MgCl₂ (1·5, 2, 2·5, 3·0 mm) and primers (0·15, 0·2, 0·3 and 0·4 μm). The optimal annealing temperature was found to vary according to the base composition of the primers (Table 2). PCR amplifications were performed in 25-μL reaction mixtures containing 10 ng DNA, 1·5 mm MgCl₂, 1× PCR buffer (10 mm KCl, 10 mm (NH₄)₂SO₄, 20 mm Tris–HCl, 2 mm MgSO₄, 0·1 % Triton X-100 pH 8·8), 0·3 μm primer, 200 μm of each dNTP (Promega, Annandale, NSW, Australia) and 1 unit of Taq DNA polymerase (New England BioLabs, Baverly, MA, USA). To reduce background amplification, 2 % (v/v) formamide was added to the reactions (Fang and Roose, 1997; Raina et al., 2001). Amplifications were carried out using a thermocycler (iCycler, Biorad, Regents Park, NSW, Australia) with an initial denaturation/activation step of 4 min at 95°C, followed by 45 cycles of 30 s at 94°C, 45 s at annealing temperature (Table 2) and 2 min extension at 72°C. A final extension for 10 min at 72°C was included. Optimal conditions were determined based on the resolvable PCR products generated by each primer. A negative control which contained all PCR components except DNA (replaced by water) was included in every experiment to test for DNA contamination of the reagents. PCR products were visualized using agarose gel electrophoresis stained with ethidium bromide (Sambrook et al., 1989).

Table 2.

ISSR primers used in this study and summary of ISSR markers from 30 Leucadendron cultivars

Primer (UBC)	Primer sequence*	Annealing temperature (°C)†	Fragment size range	Fraction polymorphic fragments‡
813	(CT)8T	50	430–1625	12/12
814	(CT)8A	50	385–1980	19/19
815	(CT)8G	52	395–1690	27/27
817	(CA)8A	52	595–1355	14/15
820	(GT)8T	52	575–2055	16/16
824	(TC)8G	50	595–2230	24/24
826	(AC)8C	52	500–1735	23/25
834	(AG)8YT	52	370–1490	34/34
836	(AG)8YA	52	325–1990	30/31
840	(GA)8YT	54	255–1645	35/35
843	(CT)8RA	54	350–1965	21/21
845	(CT)8RG	54	305–2100	18/18
848	(CA)8RG	55	450–1665	18/19
852	(CT)8RA	52	310–2195	17/18
855	(AC)8YT	55	310–1825	27/28
856	(AC)8YA	55	480–1710	14/14
857	(AC)8YG	54	250–2000	29/29
858	(TG)8RT	52	330–1845	21/21
859	(TG)8RC	55	300–1340	25/26
860	(TG)8RA	52	330–2400	17/18
889	DBD(AC)7	52	320–1800	26/27
890	VHV(GT)7	54	310–1575	36/37
891	HVH(TG)7	54	340–1495	27/27
868	(GAA)6	50	470–1675	30/30
873	(GACA)4	50	595–1355	10/13
				570/584

^*Single letter abbreviations for mixed-base positions: Y = (C, T), R = (A, G), B = (non A), D = (non C), V = (non T), H = (non G).

^†Determined empirically.

^‡Number of polymorphic fragments/number of fragments amplified.

Data analysis

Gels were photographed (Kodak Digital Science1D™, Eastman Kodak Co., Rochester, NY, USA) and the sizes of the fragments estimated (Digital Science 1D Image Analysis Software, Eastman Kodak Company, Rochester, USA). Each ISSR band was considered as a character and the presence or absence of the band was scored in binary code (present = 1, absent = 0). A data matrix was assembled and analysed using Phylogenetic Analysis Using Parsimony (PAUP; Swofford, 1998) and a pairwise distance matrix was generated based on total character differences. The genetic relatedness among the Leucadendron cultivars was analysed using Unweighted Pair Group Method with Arithmetic Average (UPGMA) based on distance measure of total character difference. Bootstrap analysis using UPGMA search with 1000 replicates was performed to obtain the confidence of branches of the tree.

RESULTS

Selection of primers and reproducibility

Initially, 64 ISSR primers were screened against genomic DNA from three Leucadendron cultivars (‘Katie's Blush’, ‘Gem’ and ‘Red Centre’) for their ability to amplify DNA fragments. Of the 64 primers, eight produced no distinct bands on a smeary background and 31 resulted in very faint bands upon a highly smeared background. The remaining 25 primers (Table 2) produced robust amplification patterns. As an example, the pattern obtained for each cultivar with primer UBC₈₅₇ is shown in Fig. 1. Within the set of 25 primers giving robust patterns, there were 23 di-nucleotide repeat primers, 20 primers with 3′ anchors and 3 primers with 5′ anchors (Table 2). The single tri- and tetra-nucleotide repeat primers were both unanchored.

Fig. 1.

ISSR marker profiles obtained from Leucadendron cultivars. ISSR amplification products were produced using primer UBC₈₅₇ and separated on 1·8 % agarose gel. The cultivars (Table 1) are shown across the top of the figure. The lanes containing 1-kb and 100-bp marker ladders (Promega, Annandale, NSW, Australia) are indicated, as are the sizes of selected marker bands. The bands marked with arrows are the cultivar-specific bands amplified (Table 3).

The reproducibility of the ISSR amplifications was assessed using selected primers (UBC₈₂₀, UBC₈₂₆, UBC₈₃₄, UBC₈₆₀, UBC₈₆₈, UBC₈₉₀ and UBC₈₉₁) with different DNA samples isolated independently from the same cultivar and amplified at different times. Under the optimized PCR conditions, the banding profiles were consistent among PCR experiments (data not shown).

ISSR diversity

The set of 25 ISSR primers showed multiband patterns in each cultivar and no band was detected in any negative control amplification. This primer set amplified a total of 584 bands from the 30 Leucadendron cultivars tested. Primer UBC₈₁₃ resulted in the smallest number of bands (12) and primer UBC₈₉₀ generated the largest number of bands (37). The average number of bands per primer was 23·4. Band size ranged from 305 bp (UBC₈₄₅) to 2·4 kb (UBC₈₆₀). Among the 30 Leucadendron cultivars, 570 (97·6 %) of the ISSR bands were polymorphic. The percentage of polymorphic fragments per primer was 76–100 % (Table 2). Identical ISSR profiles were obtained for three cultivar pairs: ‘Katie's Blush’ and ‘Silvan Red’, ‘Highlight’ and ‘Maui Sunset’, and ‘Yellow Crest’ and ‘Yellow Devil’.

Molecular relationship and fingerprinting of Leucadendron cultivars

UPGMA analysis based on total ISSR character difference was carried out to group the 30 Leucadendron cultivars. A dendrogram resulting from a cluster analysis of the distance matrix showed two main groups, designated A and B (Fig. 2). The UPGMA dendrogram showed a high confidence level. Bootstrap analysis using 1000 replicates showed that seven forks had 100 % bootstrap support and 21 of the 28 forks had greater than 50 % bootstrap support.

Fig. 2.

Dendrogram of 30 Leucadendron cultivars based on UPGMA analysis of ISSR polymorphisms. The Leucadendron cultivars (Table 1) clustered into groups (A and B) and subgroups (numbers on left). Numbers below the lines indicate bootstrap values (percentage of 1000 replicates). Bootstrap values greater than 50 % are shown.

Cultivar-specific ISSR bands were obtained for 17 of the 30 Leucadendron cultivars tested (Table 3). Using the ISSR data from primers UBC₈₅₆ and UBC₈₅₇, a fingerprinting key was generated (Fig. 3) that is able to distinguish 27 cultivars out of 30 cultivars.

Table 3.

Cultivar-specific bands revealed by ISSR fingerprinting for 17 Leucadendron cultivars (other cultivars listed in Table 1 cannot be characterized by the presence and/or absence of specific bands)

Cultivar	Characterized by the presence of ISSR markers*	Characterized by the absence of ISSR markers
Buttercup	UBC860–2460
Devil Blush	UBC840–420, UBC855–1420
Fire Glow	UBC855–465
Gem	UBC868–795
Inca Gold	UBC855–920
Jubilee Crown	UBC836–325, UBC845–305, UBC856–800, UBC857–350
Maui Sunset, Highlights	UBC815–1685
Pixie Red	UBC855–1000, UBC891–500
Red Devil	UBC814–805
Safari Goldstrike	UBC868–470
Safari Sunset	UBC836–590
Spring Gold	UBC848–1820	UBC855–540
Summer Sun	UBC859–825
Tall Red	UBC824–1260, UBC852–310, UBC860–1300
436	UBC857–870
1386	UBC857–450
1424	UBC891–525	UBC891–670

^*An ISSR primer (i.e. UBC₈₂₄) is followed by the size, in bp, of the cultivar-specific fragments.

Fig. 3.

ISSR marker fingerprinting key for Leucadendron cultivars generated from amplification products of primers UBC₈₅₆ and UBC₈₅₇. ISSR bands are designated by the primer name (i.e. UBC₈₅₆) followed by the fragment size, in bp. The presence (P) or absence (A) of bands allows 24 of the Leucadendron cultivars (Table 1) tested to be uniquely identified (bold text).

DISCUSSION

ISSR markers

The 64 primers, including di-, tri- and tetra-nucleotide repeat primers, tested in this study amplified DNA fragments from Leucadendron genomic DNA with different efficiencies. Several studies have reported that 5′-anchored ISSR primers generated informative bands (Charters et al., 1996; Matthews et al., 1999). However, in the present study, primers with 3′-anchors were more successful in amplifying specific bands than primers with 5′-anchors. Most non-anchored or 5′-anchored primers produced smeared patterns, with or without faint bands. Since primer specificity determinants are located within the first eight nucleotides at the 3′ end (Caetano-Anollés, 1994), anchoring primers at their 3′ ends will lower the number of sequences which have homology to the primers, thus producing distinct bands (Parsons et al., 1997). The success in amplifying specific bands also depends on the anchoring motif. For example, while primers (AG)₈T, (AG)₈C and (AG)₈G produced smeared profiles, the AG repeat primers anchored with YT or YA produced clear and distinct products.

The failure of several primers to give clear banding patterns may be because those primers require special amplification conditions, such as alternative chemical stabilizers or different annealing temperatures. The type of gel electrophoresis and staining method used can also influence the number of scorable bands and the level of polymorphism observed (Godwin et al., 1997, Wiesner and Wiesnerová, 2003). Both Charters et al. (1996) and Matthews et al. (1999) used polyacrylamide gels as a resolving medium and silver staining for visualization. Compared with agarose gels, polyacrylamide gels visualized by autoradiography of radiolabelled samples, or to some extent visualized by silver staining, were reported to give higher resolution (Godwin et al., 1997).

Fingerprinting of Leucadendron cultivars and cultivar-specific markers

Until now, identification of Leucadendron species has relied on morphological characters, especially of the fruit and seed. A fingerprinting key, based on the banding patterns of ISSR markers that can be used to identify Leucadendron cultivars, has now been generated (Fig. 3). The high level of polymorphism of the ISSR markers detected in Leucadendron facilitated the development of the DNA fingerprinting key. In fact, the fingerprinting key for Leucadendron cultivars could be developed using only two primers, UBC₈₅₆ and UBC₈₅₇ (Fig. 3). The use of a small number of primers is advantageous for reducing the time and cost of analysis. The ideal of using a single ISSR primer has been achieved for the differentiation of 30 strawberry cultivars (Arnau et al., 2003). The ISSR assay also identified cultivar-specific markers (Table 3) that have the potential of being used as diagnostic tools for cultivar identification, or that could be developed into cultivar-specific Sequence Characterized Amplified Region (SCAR) markers.

The Leucadendron fingerprinting key cannot differentiate the cultivar pairs ‘Highlights’ and ‘Maui Sunset’, ‘Katie's Blush’ and ‘Silvan Red’, and ‘Yellow Devil' and ’Yellow Crest’. The International Proteaceae Register (2002) lists ‘Highlight’ and ‘Maui Sunset’ as the same cultivar. The present findings certainly indicate that these two cultivars are closely related, but a more extensive analysis is required to assert with statistical certainty that they are identical. The International Proteaceae Register (2002) lists ‘Yellow Crest’ and ‘Yellow Devil’ as separate cultivars released by different companies, while some horticulturalists believe that ‘Yellow Crest’ is a former name of ‘Yellow Devil’ (P. Armitage, pers. comm.). The present results support the view that these cultivar names are synonyms. The ISSR analysis also failed to differentiate ‘Katie's Blush’ and ‘Silvan Red’. ‘Katie's Blush’ is a stable sport of ‘Silvan Red’ with variegated foliage (Matthews, 2002).

Relationships among Leucadendron cultivars

The dendrogram displaying the molecular relationships among the 30 Leucadendron cultivars tested separates them into two main groups. Cultivars in group A are either selections of L. salignum or L. salignum × L. laureolum hybrids (Matthews, 2002). Group B contains of a heterogeneous group of cultivars, but clustering is based on parental lineage. For example, the L. discolor cultivars ‘Red Centre’ and ‘Pom Pom’ group together with ‘436’ which has L. discolor as the male parent (Fig. 2).

Group A can be divided into three clear subgroups (A1, A2 and A3), with ‘Maui Sunset’ and ‘Highlights’ being distantly separated from the other members of group A (Fig. 2). Cultivars of L. salignum (subgroup A1) formed a sister group to the hybrid progeny of a L. salignum × L. laureolum cross (subgroup A2). ‘Tall Red’ which clustered with the L. salignum cultivars, is recorded as a L. salignum × L. eucalyptifolium hybrid (Proteaflora, 2004). The ISSR analysis showed that ‘Tall Red’ is the most distantly related cultivar in the L. salignum branch, supporting the view that this cultivar is a hybrid.

‘Maui Sunset’ is recorded as the result of a cross between L. laureolum × L. salignum (International Proteaceae Register, 2002; Matthews, 2002). However, plant morphology suggests that ‘Maui Sunset’ is actually a trihybrid of (L. laureolum × L. discolor) × L. salignum (B. Croxford, The University of Western Australia, pers. comm.). ‘Maui Sunset’ is morphologically very similar to hybrids between L. discolor and L. salignum, but has slightly longer bracts. In addition, at one growth stage, ‘Maui Sunset’/‘Highlights’ shows colour which is not displayed by L. discolor × L. salignum hybrids. The placement of ‘Maui Sunset’ on a separate branch from L. salignum and from the progeny of crosses between L. salignum and L. laureolum (Fig. 2) supports the view that ‘Maui Sunset’ is genetically dissimilar to L. laureolum × L. salignum hybrids and may be a trihybrid. Crossing L. laureolum × L. discolor results in plants that are morphologically similar to L. laureolum, which may explain the confusion in the parentage of ‘Maui Sunset’ (B. Croxford, University of Western Australia, pers. comm.).

In general, Leucadendron cultivars in group B cluster according to their reported pedigrees (Fig. 2 and Table 1). An exception is subgroup B5, where the reported parentage of the cultivars is broad. However, there is at least some uncertainty in the parentage of several of these cultivars. For example, ‘Pisa’ is only reported to be a L. floridum hybrid (Matthews, 2002), ‘Jubilee Crown’ has been only noted as a L. laxum hybrid (Littlejohn and Robyn 2000) and there is no clear information on the origin of ‘Buttercup’ (International Proteaceae Register, 2002).

This study further demonstrates that ISSR markers are a powerful tool for generating fingerprinting keys and have the potential to identify cultivar-specific markers for Leucadendron. The elucidation of the relationships among the 30 Leucadendron cultivars, the identification of species-specific ISSR markers and the generation of a fingerprinting key are important resources for the breeding and management of Leucadendron germplasm.