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Corylus Colurna L. and Corylus Avellana L.) Using Multiplexed Nuclear Microsatellite Markers

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Corylus Colurna L. and Corylus Avellana L.) Using Multiplexed Nuclear Microsatellite Markers Ann. For. Res. 62(2): 173-182, 2019 ANNALS OF FOREST RESEARCH DOI: 10.15287/afr.2019.1709 www.afrjournal.org Molecular differentiation of Turkish and Common hazels (Corylus colurna L. and Corylus avellana L.) using multiplexed nuclear microsatellite markers Barbara Fussi1§, Darius Kavaliauskas1, Muhidin Šeho1 Fussi B., Kavaliauskas D., Šeho M.. Molecular differentiation of Turkish and Com- mon hazels (Corylus colurna L. and Corylus avellana L.) using multiplexed nucle- ar microsatellite markers. Ann. For. Res. 62(2): 173-182. Abstract. Corylus colurna is considered as important tree species under climate change for dry and warm conditions in Central Europe and was overused because of its valuable wood. Therefore Turkish hazel is now present only in small isolated populations and is protected under IUCN. Genetic conservation of this tree species plays a key role in future sustainable forest development. Turkish hazel co-occurs with Common hazel (C. avellana) in its whole distribution area and may form hy- brids. To differentiate between the pure species and their hybrids, cross-species amplifying markers are required. In this study we have evaluated existing simple sequence repeat (SSR) markers using altogether 128 samples of C. avellana and C. colurna. Fifteen nuclear SSRs have generated easy to-score alleles in the two species and 13 of them were highly polymorphic. For all 15 markers the mean al- lele number, average observed heterozygosity, genetic diversity and polymorphism information index were high. The two most polymorphic SSRs were L1.10 and CaT-B501 with 19 and 16 alleles, respectively. Structure analysis proved the dif- ferentiation of the two species C. avellana and C. colurna. No hybridization was detected in the analysed populations. Results also indicated that C. colurna from Balkan Peninsula and Asia Minor belong to separate groups. Our study presents highly polymorphic, easy to score, ready to use SSR-multiplexes, which can be applied in population genetics and gene conservation studies. Keywords: SSR, multiplex PCR, polymorphism, species differentiation, molecular markers, hazelnut Authors. 1Bavarian Office for Forest Genetics (AWG), Forstamtsplatz 1, 83317 Teisendorf, Germany. §Corresponding author: Barbara Fussi ([email protected]) Manuscript received November 21, 2018; revised December 20, 2019; accepted December 28, 2019; online first December 31, 2019. Introduction egy to increase the stability of forest stands is given through establishing mixed forests. By Climate change affects all forest ecosystems supplementing the existing spectrum of tree and goods that forests provide. A suitable strat- species in Central Europe, the risk can be min- 173 Ann. For. Res. 62(2): 173-182, 2019 Research article imized. Therefore we examine tree species two hazelnuts (C. colurna L. and C. avellana from the Mediterranean region for its drought L.) hybrids can be formed rarely (Alexandrov resistance and their suitability to be cultivat- 1995; Erdogan & Mehlenbacher 2000, Jahn ed in Germany. Turkish hazel is considered as 1930). Jahn (1930) described some differenc- a valuable tree species under climate change es between three possible hybrids between C. with high adaption potential to drought and colurna and C. avellana in botanical gardens frost (Šeho et al. 2019). Turkish hazel (C. in Hann. Münden, Göttingen and Jena (Ger- colurna L.) and Common hazel (C. avellana L.) many). However, he was not absolutely certain belong to the family of birch plants (Betulace- that these trees are natural hybrids. ae), with the subfamily Corylaceae which has In Germany, Common hazel is intensively four genera. The genus Corylus has 12 species traded and planted, and thus is of central inter- distributed in the temperate zones of Eurasia est from a nature conservancy point of view. and North America (Mitchell 1979, Fitschen Leinemann et al. (2013) studied Common ha- 1994, Alteheld 1996, Erdogan & Mehlenbach- zel using chloroplast markers (cpSSRs), iso- er 2000a). The genus is diploid (2n=2x=22), zymes and AFLPs with the aim to assess the and contains monoecious and wind-pollinated within and between population variation of 20 shrubs and trees (Bassil et al. 2013). Within natural populations from Central Europe. Sig- section Corylus, three subsections are tra- nificant differences between populations were ditionally recognized. C. colurna L. and C. found. Like other plant species, the distribution avellana belong to different subsections (Sub- of Common hazel is strongly affected by post- section Colurnae and Phyllochlamys, respec- glacial recolonization (Huntley & Birks 1983, tively) (Erdogan & Mehlenbacher 2000). Huntley 1990, Palmé & Vendramin 2002). A C. colurna is naturally distributed on the clear geographical structure was found on the Balkan Peninsula, Asia Minor, the Caucasus European scale separating Italy and the Bal- and Afghanistan (Temel et al. 2017, Šeho & kans from the rest of Europe (Palmé & Ven- Huber 2018, Šeho et al. 2019). By supple- dramin 2002). Together with pollen data the menting the existing spectrum of broad-leaved possible origin of Central European Common tree species with Turkish hazel in Central Eu- hazel would be France expanding into most rope, the risks raised by changing climate can of Europe and a local expansion in Italy and be minimized. To recommend suitable prove- the Balkans. C. avellana and C. colurna share nances for Central Europe research on genetic one haplotype indicating common ancestry or and phenotypic diversity between and within hybridization between the two species. For C. C. colurna populations is necessary. Turkish colurna there is a knowledge gap about poten- hazel can reach an age of 400 years, DBH of tial refugia or recolonization routes after the 60 cm (in some cases up to 170 cm) and height last glacial maximum. of 30 m depending on the growth conditions For further cultivation of Turkish hazel in (Alexandrov 1995, Alteheld 1996, Schmidt Central Europe it is important that the inva- 2003, Richter 2013, Šeho & Huber 2018, Šeho siveness of this tree species through hybrid- et al. 2019). In contrast, Common hazel is ization will be evaluated. Artificial crossing growing as a shrub. These two hazel species experiments between C. colurna x C. avellana have different morphology of leaves and fruits. L. yielded in the formation of few fruit clus- Turkish hazel has 17-20 mm long nuts ripen- ters with C. avellana as the male parent, but ing in 5 to 6 pods surrounded by deeply di- failed to set full seeds. The reciprocal cross, C. vided lobes. Nuts are smaller than those of C. avellana x C. colurna, set very few nuts only avellana. Leaves of Common hazel are slight- in one of three breeding years (Erdogan & Me- ly smaller and lighter green and the bark is less hlenbacher 2000). Percentage of seed germina- grey than that of Turkish hazel. Between these tion was higher when C. avellana was pollen 174 Fussi et al. Molecular differentiation of Turkish and Common hazels ... donor and C. colurna female crossing partner. of nuclear microsatellite loci across species/ Seedling vigor, recorded as stem diameter, taxa or genera depends on various factors was highly variable among the interspecific such as species generation time, type of breed- crosses. In general, the cultivated hazelnut C. ing system, genome size, etc. (Barbara et al. avellana, and the tree hazel species C. colurna 2007, Ellis & Burke 2007). Lower success and C. chinensis yielded very vigorous inter- rate of SSR transferability was observed in specific hybrid seedlings (Erdogan & Mehlen- selfing species and species with high genome bacher 2000). However, the crossability of C. size such as tree species and has therefor to be colurna and C. avellana was under discussion evaluated case-by-case (Barbara et al. 2007). in the 1960ies (Kasapligil 1963, 1964). Gel- Compared to “anonymous” SSRs EST-SSRs latly (1966) reported some hybrids and named are easier to be transferred between species es- them trazels, derived from open-pollination of pecially among plants (Ellis & Burke 2007 and C. colurna trees, morphologically indicating references therein). The transferability of SSR true hybrids (varieties called ‘Morrisoka’ and markers allows their usage in related species ‘Faroka’). Erdogan & Mehlenbacher (2000) for fingerprinting, mapping, and marker-assist- indicate that crossability between these two ed breeding (Ellis & Burke 2007). Examples species is complicated but possible. In their include the high transferability rate of SSRs study C. colurna x C. avellana resulted in in several tree species from apple (Malus do- cluster set every year, but with high proportion mestica) to European pear (Pyrus communis) of empty seeds and only few viable seedlings. (Pierantoni et al. 2004), from peach (Prunus Breeders need to perform large numbers of persica) to related Prunus species (Cipriani pollinations to obtain a few hybrid seedlings et al. 1999), and within oaks (from Quercus (Erdogan & Mehlenbacher 2000). Bassil et al. petraea to Quercus robur, Steinkellner et al. (2013) reported about hybridization support- 1997). ed by nuclear and chloroplast microsatellite The goal of this study was to establish high data, where C. colurna - C. avellana hybrids quality sets of molecular markers (i.e. ready formed a distinct group, while accessions of C. to use multiplexes of nSSRs) for both species colurna were placed in one cluster with two C. C. colurna and C. avellana and use them to avellana cultivars from southern Italy (‘Tonda investigate
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