Analysis of Genetic Diversity in Crocuses with Carpathian Basin Origin Using Aflp-Markers

Acta Biologica Hungarica 61 (Suppl.), pp. 149–155 (2010) DOI: 10.1556/ABiol.61.2010.Suppl.15

ANALYSIS OF GENETIC DIVERSITY IN CROCUSES WITH CARPATHIAN BASIN ORIGIN USING AFLP-MARKERS

G. SURÁNYI,* C. MÁTHÉ, ÁGNES MOSOLYGÓ, G. BORBÉLY and G. VASAS

Department of Botany, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary

(Received: August 9, 2010; accepted: October 10, 2010)

Crocus taxonomy has until now been based primarily on morphology, taking chromosome numbers into consideration. The genetics and genome structure of the genus, the relationships and diversity within the genus are not well known. Amplified fragment length polymorphism (AFLP) is a whole genome approach to study genetic variation that is gaining in popularity for lower-level systematics. The present study employed the AFLP technique for analyzing relationships among taxa of the Crocus genus (particularly the Crocus vernus aggregate) with Carpathian Basin origin. The molecular variance obtained was based on amplification, separation and detection of EcoRI and Tru1I double-digested Crocus spp. genomic DNAs. Our results confirm the relatedness of C. tommasinianus, C. vittatus and C. heuffelianus at the Verni series of the Crocus genus. C. banaticus is taxonomically isolated as the sole member of the subgenus Crociris based on unique morphological features, but the difference is not convincing from AFLP data. The second interesting AFLP analysis result is the position of C. scepusiensis which separated it from the Crocus vernus aggregate.

Keywords: AFLP-markers – Crocus banaticus – Crocus scepusiensis – Crocus vernus aggregate – molecular taxonomy

INTRODUCTION

Crocus vernus Hill, with the two subspecies C. vernus subsp. vernus and C. vernus subsp. albiﬂorus, is a taxon distributed throughout Middle-Southern Europe. The species is morphologically variable and presents a range of karyotypes at the diploid and polyploid levels; all accessions have large genomes and very large chromosomes. Crocus taxonomy has until now been based primarily on morphology, taking chromosome numbers into consideration [8]. Little is known about the genetics and genome structure of the genus and about the relationships, and diversity within the genus [3–5]. Two subspecies of Crocus vernus Hill are recognized: C. vernus Hill ssp. albiﬂorus (Schultes) Asch. & Graeb., a small white, purple-striped or purple type growing mostly at high altitudes in the Alps, the Pyrenees, and in Sicily {2n = 8}; and

* Corresponding author; e-mail: [email protected]

C. vernus ssp. vernus, a somewhat larger and more richly coloured type of the Carpathians, the former Yugoslavia and central Italy {2n = 8, 10, 12, 16, 18, 20, 22} [3, 8]. Many different names have been used for these plants (and some still are used), but the overlapping variation in morphology and lack of correlation between chromosome number and morphology hinder resolving uncertainties regarding the real taxonomical division of the Crocus vernus aggregate. C. vernus is a good model for the investigation of chromosome evolution in wild species: the diploid number based on only four pairs of large chromosomes limits problems of chromosome identification [11]. The variation in chromosome number and wide geographical range make it likely that multiple evolutionary events have taken place [3, 6]. Frello and Heslop-Harrison investigated the physical organization of some major tandemly-organized repetitive DNA sequences using in situ hybridization along the chromosomes of different C. vernus accessions [3, 4]. The present study attempts to contribute DNA polymorphism-based molecular biological approach to the theme by applying AFLP method [14]. The present taxonomy and classification of the Crocus genus based on Mathew’s former and recent works [8, 9, 13] is as follows: The genus Crocus 1. Subgenus Crocus A) Section Crocus / Type species: C. sativus L. Series Verni Mathew / Type species: C. vernus Hill. Series Scardici Mathew / Type species: C. scardicus Kos. Series Versicolores Mathew / Type species: C. versicolor Ker-Gawl. Series Longiflori Mathew / Type species: C. longiflorus Raf. Series Kotschyani Mathew / Type species: C. kotschyanus Koch. Series Crocus / Type species: C. sativus L. B) Section Nudiscapus Mathew / Type species: C. reticulatus Stev. ex Adams Series reticulati Mathew / Type species: C. reticulatus Stev. ex Adams Series Biflori Mathew / Type species: C. biflorus Mill. Series Orientales Mathew / Type species: C. korolkovii Regel ex Maw. Series Flavi Mathew / Type species: C. flavus Weston Series Aleppici Mathew / Type species: C. aleppicus Baker Series Carpetani Mathew / Type species: C. carpetanus Boiss. & Reut. Series Intertexti (Maw) Mathew / Type species: C. fleischeri Gay Series Speciosi Mathew / Type species: C. speciosus M. Bieb. Series Laevigati Mathew / Type species: C. laevigatus Bory & Chaub. 2. Subgenus Crociris (Schur) Mathew / Type species: C. banaticus Gay The species comprising series Verni: C. vernus Hill, C. tommasinianus Herb., C. heuffelianus Herb., C. scepusiensis Rehmann et Wol., C. vittatus Schloss. et Vukit.

Ampliﬁed fragment length polymorphism (AFLP) is a whole genome approach to study genetic variation that is gaining in popularity for lower-level systematics. The strengths of AFLP include high levels of polymorphism and the ability to sample

Acta Biologica Hungarica 61, 2010 Genetic diversity in crocuses with AFLP 151 randomly across the genome. The technique appears to be least successful where species are closely related and cross-fertile. We employed the ampliﬁed fragment length polymorphism technique for analyzing relationship among taxa of the Crocus genus (particularly the Crocus vernus aggregate) with Carpathian Basin origin. The desired goal of this study was to make our AFLP method suitable for standardized measurement of the variation occurring within and between Crocus spp. populations. The molecular characterization of crocuses of Carpathian Basin origin is part of the CROCUSBANK European Consortial Research Project.

MATERIALS AND METHODS

Sample collection

Samples of the Crocus and Crociris subgenera [8] included C. heuffelianus, C. tommasinianus, C. scepusiensis, C. vittatus and C. banaticus. All above-mentioned taxa were collected between 2006 and 2009 in Hungary and the Carpathian Basin (Table 1). The plant materials applied for DNA extraction were from both the origi- nally collected plant samples (various populations) and their ﬁeld-cultivated or tissue- cultured vegetative derivates (shoots, corms; Botanical Garden of University of Debrecen). Table 1 Origin of collected Crocus samples used in molecular characterization Geocoordinate Geocoordinate Species Country County Locality (N) (E) Crocus heuffelianus Romania Jud. Bihor Betﬁa 46°57'50.3" 22°01'07.4" Crocus heuffelianus Romania Jud. Bistrica- Borsa 47°34'01.5" 25°01'01.0" Năsăud Crocus heuffelianus Romania Jud. Bihor Finis 46°37'43.3" 22°18'02.3" Crocus scepusiensis Slovakia NA Ždiar 49°14'47.0" 20°18'37.6" Crocus tommasinianus Hungary Tolna Gyulaj 46°31'55.9" 18°16'56.6" Crocus vittatus Hungary Veszprém Kup 47°13'53" 17°25'08" Crocus sativus L. Spain Ledana

DNA puriﬁcation and AFLP analysis

DNA from all plant samples was puriﬁed using modiﬁed phenolic extraction [1] or using TRI Reagent Solution (Molecular Research Center, USA) for extraction. The exception was the reference saffron DNA which was a lyophilized DNA sample from Spain (Ledana). All DNA samples were stored at –18 °C until use. The AFLP method was adapted from Vos et al. [14] using separated restriction and ligation of DNA samples. For this method EcoRI and TrulI (isoschisomer of MseI)

Acta Biologica Hungarica 61, 2010 152 G. SURÁNYI et al. restriction endonuclease enzymes, R+ buffer, T4 DNA ligase, T4 DNA ligase buffer and Taq DNA polymerase and buffer (all from Fermentas, Lithuania) were used. EcoRI and TrulI adapters and primers were synthesized by Biocenter Ltd (Szeged, Hungary). Enzymatic digestion was carried out in 30 μl volumes. A 200 ng total genomic DNA sample was digested with 3 μl (30 U) EcoRI restriction enzyme in R+ buffer. The reactions were incubated at 37 °C for 4 or 20 hours, then 2 μl (2 U) TrulI restriction enzyme was added and incubated at 65 °C for 1 or 2 hours. Ligation was carried out in a 40 μl total volume adding 10–30 μl double-digested DNA, 1–1 μl (10 μM) EcoRI and 1–1 μl (100 μM) Tru1I adapter lower and upper strands, 2 μl T4 DNA ligase (2 U), 4 μl 10× T4 DNA ligase buffer. The incubation took place overnight at 20 °C.

Fig. 1. Separation of AFLP fragments from double-digested Crocus DNA samples (TruI1/1 h and EcoRI/ 20 h) on polyacrylamide gel (5–10% linear gradient; the gel was silver stained). The order of samples: (1) Molecular mass standard, (2) C. sativus, (3) C. scepusiensis, (4) C. banaticus, (5) C. vittatus, (6) C. tommasinianus, (7) C. heuffelianus (Finis)

Acta Biologica Hungarica 61, 2010 Genetic diversity in crocuses with AFLP 153

Fig. 2. Genetic distances between C. sativus (Ledana, Spain) and wild Crocus spp. entries from Carpathian Basin. The dendrograms based on AFLP-marker analysis: A – Type1, B – Type2, C – Type3

The DNA amplification was based on PCR method, which was simplified by using only two AFLP primers, ACG and CAA with three extension bases for EcoRI and Tru1I, respectively. There was no preselective amplification. PCR reactions were carried out in 20 μl total volume adding 4–6 μl ligated DNA template. Thermocycler conditions were: 94 °C for 2 min, followed by 35 cycles 94 °C for 30 s, 52 °C for 1 min, 72 °C for 1 min; followed by 72 °C for 4 min.

Acta Biologica Hungarica 61, 2010 154 G. SURÁNYI et al.

Gel and statistical analysis of ampliﬁed DNA samples

The products of DNA ampliﬁcation were loaded on 6% or 5–10% linear gradient polyacrylamide gels [6] in TBE (Tris-borate EDTA) electrophoresis buffer using 18×20 cm gel apparatus. Gels were developed by applying silver staining [12], then digitalized and analyzed with UVITEC Imaging System and UVIBANDMAP Analysis Software (UVITEC, Cambridge). The presented dendrograms (Fig. 2A, B, C) are based on calculated values of genetic distances; for the calculation we used the Nei’s indices [10] which show the measure of average differences between isolates.

RESULTS The molecular variance obtained was based on ampliﬁcation, separation and detection of EcoRI and Tru1I double-digested Crocus spp. genomic DNAs (Fig. 1). The compared Crocus taxa were collected from the Carpathian Basin (Table 1). Our results conﬁrm the relatedness of C. tommasinianus, C. vittatus and C. heuffelianus at the Verni series (Fig. 2A, B, C). C. scepusiensis is also known to belong to the Verni aggregate. However, our approach shows its separation from other species belonging to this series (Fig. 2A, B, C). The investigated C. scepusiensis entries are the closest relatives of the C. sativus accession {Ledana, Spain} (Fig. 2A and B). The separation of C. banaticus (Crociris subgenus) entries from the Crocus spp. accessions belonging to the Crocus subgenus is not convincing (Fig. 2A, B, C).

DISCUSSION The analysis of AFLP markers resulted in three (Fig. 2A, B, C) more or less different types of phylogenetic trees for the investigated crocuses. Explanation of our genetic variability analysis results (based on Nei’s index calculations) is not complete because of a very limited number of investigated Crocus spp. entries (less than five isolates of each collected wild Crocus taxa). The results of Frello and Heslop-Harrison indicate a general high variability of certain repetitive DNA sequences within the C. vernus aggregate and show differences to the classical taxonomy of the genus Crocus [3, 4]. Recent data of the Frello and Heslop-Harrison laboratories [3, 4] – concerning in situ hybridization of given repetitive sequences in Crocus genus – fit our AFLP results. C. banaticus is taxonomically isolated as the sole member of the subgenus Crociris based on unique morphological features. However, the subset of four of the seven repetitive sequences included in its genome does not separate it from species in the other subgenus Crocus. In contrast, six of the seven repetitive sequences are not detected in C. korolkowii, placed in the subgenus Crocus. Furthermore, the subdivision of the subgenus Crocus into two sections is not reflected in the distribution of the sequences [4]. Due to the results concerning C. scepusiensis and C. banaticus, we will need more samples from wild Crocus species of the Carpathian Basin and various C. sativus

Acta Biologica Hungarica 61, 2010 Genetic diversity in crocuses with AFLP 155 accessions (from Crocusbank Collection) as well, in order to establish genetic variation occurring in different populations. It is worth mentioning that C. heuffelianus, a taxon formerly believed to be closely related to C. scepusiensis is highly variable in its chromosome number and morphology [2]. However, there is an interesting conclusion of this study that AFLP analysis does not conﬁrm that C. scepusiensis belongs to the C. vernus aggregate. We have shown discrepancies concerning its taxonomical position (Fig. 2A, B, C). This might be caused by possible high cytogenetic and DNA polymorphism within this taxon, but the conﬁrmation of this statement needs further study.

ACKNOWLEDGEMENTS

We are especially grateful to A. Molnár V. and G. Sramkó for collecting invaluable ﬁeld plant material for our study, and also to Á. Albert, B. A. Lukács, O. Horváth, R. Kish, A. Molnár, M. Toldi for assisting to the collections.

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