Trees Structure and Function Genetic diversity of chestnut tree in relation to susceptibility to leaf miner (Cameraria ohridella Deschka & Dimič

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Some species of the genus Aesculus are yearly severely infested by horse chestnut leaf miner (Cameraria ohridella). The larvae mine the leaves and under appropriate conditions can damage up to 100% of the leaf area. In this study, we assessed the

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           1 3 Trees Structure and Function ISSN 0931-1890 TreesDOI 10.1007/s00468-016-1506-2 Genetic diversity of chestnut tree in relationto susceptibility to leaf miner (  Camerariaohridella  Deschka & Dimič) Václav Bačovský, Tomáš Vyhnánek,Pavel Hanáček, Josef Mertelík & IvanaŠafránková           1 3 Your article is protected by copyright andall rights are held exclusively by Springer-Verlag Berlin Heidelberg. This e-offprint isfor personal use only and shall not be self-archived in electronic repositories. If you wishto self-archive your article, please use theaccepted manuscript version for posting onyour own website. You may further depositthe accepted manuscript version in anyrepository, provided it is only made publiclyavailable 12 months after official publicationor later and provided acknowledgement isgiven to the srcinal source of publicationand a link is inserted to the published articleon Springer's website. The link must beaccompanied by the following text: "The finalpublication is available at link.springer.com”.  ORIGINAL ARTICLE Genetic diversity of chestnut tree in relation to susceptibilityto leaf miner ( Cameraria ohridella  Deschka & Dimic ˇ) Va´clav Bac ˇovsky´ 1 • Toma´s ˇ Vyhna´nek 1 • Pavel Hana´c ˇek 1 • Josef Mertelı´k 2 • Ivana S ˇafra´nkova´ 3 Received: 16 March 2016/Accepted: 8 December 2016   Springer-Verlag Berlin Heidelberg 2016 Abstract  Key message  Species of   Aesculus  genus are divided intofive main sections according to their susceptibility andresistance to horse chestnut leaf miner using molecularmarkers.  Abstract   Some species of the genus  Aesculus  are yearlyseverely infested by horse chestnut leaf miner ( Camerariaohridella ). The larvae mine the leaves and under appro-priate conditions can damage up to 100% of the leaf area.In this study, we assessed the genetic diversity of eightspecies of horse chestnut and their genotypes which havevarying susceptibility to the leaf miner. Analysis of eightmicrosatellite loci showed high polymorphic informationcontent 0.45–0.77 (0.60 in average) and high geneticdiversity. For each locus, we found on average 5.50 alleles.During three vegetation periods, the leaf area damage of these genotypes was evaluated using the statistical softwareAssess 2.0. Different pressure of leaf miner and differentdevelopment at each locality was recorded, and in somecases less damage was found, in one case the lesser damagewas permanent. From the data, a pathological scale wasestablished and a dendogram of similarity was created.Based on SSR analysis, four groups of   Aesculus  trees weredistinguished according to their resistance or susceptibilityand five main section ?  Hybrid   were found. By cloning andanalysing the inner transcribed spacers, the ITS1 and ITS2,inner and inter species variability was examined. For thesequence of hybrid species  A.  9  carnea,  no compliancewas revealed in NBCI database. For this reason, the firstsequence of this genotype was obtained. Keywords  Aesculus    Resistance    Genetic diversity   SSR    Cloning    ITS Introduction  Aesculus  are monumental plants, mostly trees or shrubsspread over the northern hemisphere (Harris 2007). Speciesof   Aesculus  are widely used in forestry, pharmaceutics andhorticulture (Heinrich et al. 2005; Russel 2005), and can be found mainly in urban residential areas due to their highresistance to drought and smog. In the Czech Republic,they are also for seed production as a traditional source of food in game preserves (Mertelı´k et al. 2010). The size of the genus (number of species) and rich fossil records made  Aesculus  a good model for genetic (Kaneko et al. 1999;Thomas et al. 2008; Vyhna´nek et al. 2013) and phyloge-netic studies (Hardin 1957; Forest et al. 2001; Harris and Xiang 2009; Harris et al. 2009; Buerki et al. 2010), in which the genus is traditionally divided into five mainsections—  Aesculus ,  Macrothyrsus ,  Parryina ,  Calothyrsus Communicated by W. Osswald. Electronic supplementary material  The online version of thisarticle (doi:10.1007/s00468-016-1506-2) contains supplementarymaterial, which is available to authorized users. &  Va´clav Bacˇovsky´xbacovs@ibp.cz 1 Department of Plant Biology, Faculty of AgriScience,Mendel University in Brno, Zemeˇdeˇlska´ 1, 613 00 Brno,Czech Republic 2 Department of Phytoenergy and Biodiversity, The SilvaTarouca Research Institute for Landscape and OrnamentalGardening (VUKOZ), Kveˇtnove´ na´meˇstı´ 391,252 43 Pru˚honice, Czech Republic 3 Department of Crop Science, Breeding and Plant Medicine,Faculty of AgriScience, Mendel University in Brno,Zemeˇdeˇlska´ 1, 61300 Brno, Czech Republic  1 3 TreesDOI 10.1007/s00468-016-1506-2  and  Pavia  (Chanon 2005). These sections reflect the geo-graphic distribution of individual species and their simi-larity (Xiang et al. 1998).However, more detailed knowledge on individual spe-cies varieties is lacking and there is a dearth of informationon the natural behaviour of   Aesculus  trees under stressconditions or their susceptibility to phytophages andpathogenic agents. Despite a number of studies character-ising dangerous agents and their occurrence in  Aesculus (Tozlu and Demirci 2010; Sniesˇkien _ e et al. 2011; Zim-mermannova´-Pastircˇa´kova´ 2003; Steele et al. 2010; Sˇefrova´ 2007), few have focused on the host tree responseover the long term.In 2004, a new genotype  A. hippocastanum  L. Merte-lik06 (M06) was described. This individual has a high levelof resistant behaviour to the leaf miner  Cameraria ohri-della  Deschka & Dimicˇ (Mertelı´k et al. 2004). The resis-tance is probably caused by increased leaf levels of secondary metabolites (escin, chlorogenic acid, glycosidequercetin) (Nejezchlebova, not published). As a result of feeding on the leaves, the larvae of horse chestnut leaf miner die before completion their life cycle and the finaldamage to leaf lamina is significantly lower (about 50%compared to seedlings of horse chestnut without resistance)(Mertelı´k and Kloudova´ 2012).This study focuses on determining molecular markerswhich may be helpful in seeking new genotypes forresistance breeding to  C. ohridella  and identification of existing plants derived from M06.Simple sequence repeats (SSR) and internally tran-scribed spacers (ITS) are very variable loci with highdegree of polymorphism and both can vary among specieswithin one genus or among populations (White et al. 1990;Mishra et al. 2014; Ga´lova´ et al. 2015). For this reason, aset of SSRs and both ITS regions were used together withstatistical analysis to classify eight different species of   Aesculus  and their genotypes over regions of the CzechRepublic regions. Materials and methods DNA isolation Total genomic DNA was isolated from young leavesusing DNeasy  Plant Mini Kit (Qiagen, GE) andDNeasy  Plant Handbook (Qiagen, GE) was followed.Due to the larger amount of inhibitors present in leaves,trichomes were removed and the total quantity of planttissue was reduced to 0.50–0.80 g. An additional amountof P2 buffer (300  l L) was added and the first washingstep was repeated for better yield and higher purity. Thecentrifuge was set at 18,000 rpm. DNA concentrationand purity were assessed spectrophotometrically byPicopet 1.0 (Picodrop, UK). Plant material The individuals analysed in this study (Table 1) and theirlocation (Table S1) were chosen on the basis of observationof   C  .  ohridella  infestation and leaf area damage level. Theannual observation (the presence of the leaf miner larvae,atypical development of mining larvae, tree lower infes-tation within an area) from 2011 to 2013 was carried outduring autumn months. Trees with max. 50–60% of leaf area damage were chosen as plant material for collectionand for final statistical evaluation.  Aesculus  trees were selected from different locations tominimise the compounding impact of climate conditionand hyper infestation in any one area. The use of protec-tives was checked by personal communication with personsresponsible for maintenance of greenery in each location.The localisation of each individual growing under naturalconditions was checked by Global Positioning System(GPS) and (https://www.google.cz/maps).The  A. hippocastanum  trees with resistant behaviour tohorse chestnut leaf miner proven in conditions of naturaland artificial infestation, seedlings without resistance,  Aesculus turbinata, Aesculus glabra, Aesculus pavia, Aes-culus glabra  var.  arguta, Aesculus  9  carnea, Aesculusmarilandica  and  Aesculus parviflora  were planted inexperimental areas in The Silva Tarouca Research Institutefor Landscape and Ornamental Gardening Pru˚honice(VUKOZ). SSR amplification The microsatellite loci were amplified via PCR and eighthighly polymorphic combination primers (Table S2) wereused as referred to in Minami et al. (1998). Reaction PCRmixture was prepared as described in Vyhna´nek et al.(2009) and a DNA concentration 20–40 ng  l L - 1 was used.Profile reaction in T3 cycler (Biometra, GE) was set asfollowed—9 min of initiation denaturation at 94   C and7 min of final elongation at 72   C. For 25 9 cycles was seta 30 s denaturation at 94   C, primer annealing 30 s at52   C (AT3D6, AT5D2, AT6D2, AT6D8, AT7D8)/54   C(AT5D10)/55   C (AT6D12, AT7D1) and 60 s of extensionat 72   C. The products were separated in 8% non-dena-tured polyacrylamide (PAA) gels and stained by 0.2%AgNO 3 . SSR evaluation PCR products were evaluated as in Vyhna´nek et al. (2009)and transmitted into binary code (1/0) in Microsoft Excel. Trees  1 3  The dendrogram of similarity was calculated by theUnweight Pair Group Method with Arithmetic Mean(UPGMA) with Jaccard´s coefficient using the statisticalsoftware FreeTree 9.1 (Hampl et al. 2001). Final view datawere transferred by TreeView 1.6 (Page 1996). ITS amplification The concentration of the DNA template and isolationprocedure were the same as for SSR markers. The PCRmixture in a total volume 25.0  l L for ITS amplificationcontained 0.5 U  Taq  polymerase (Promega, USA),1 9  aliquot buffer, 0.1 mM of each dNTP (Promega, USA)and 0.3 M of each primer ITS5-F (5 0 -GGAAGGA-GAAGTCGTAACAAGG-3 0 ) as reported in Blattner andKadereit (1999), and ITS4 (5 0 -TCCTCCGCTTATTGA-TATGC-3 0 ) according to White et al. (1990) with 4ntexchange. The PCR conditions in T3 cycler comprised3 min initial denaturation at 94   C and final extension for10 min at 72   C before cooling. The denaturation at 94   Cfor 30 s, primer annealing at 55   C for 30 s, and an elon-gation at 72   C for 1 min were repeated 40 times. Cloning of PCR fragments After separation of PCR products on 1.5% agarose gel(TAE Buffer), DNA was isolated and purified using Invi-sorb Fragment CleanUp Kit (Stratec Molecular, GE),where the protocol was followed. The fragments wereligated into pGEM-T Vector System (Promega 2010) and Table 1  Species and plant materialSpecies Signification Infestation Additional information  Aesculushippocastanum AH1 A A24 (M06) seedling, resistant behaviourAH2 A A24 (M06) seedling, without resistanceAH3 A A24 (M06) seedling, without resistanceAH24 (M06) A Resistant behaviourAH4, AH5, AH26, AH28,AH29, AH31A Grafted plants (same donor), atypical mines, resistant behaviourAH30 A Without resistanceAH25 N Without resistance*AH27 F Atypical mines 2012/2013/2014, resistant behaviourAH13 F Lower infestation 2013AH14 F Lower infestation 2013AH15 F Lower infestation 2013AH16 F Lower infestation 2013AH17 F Lower infestation 2013AH18 F Heavy infestationAH19 FAH20 F Lower infestation 2013  Aesculus turbinata  ATA F Heavy infestation  Aesculus glabra  AGLA N Immune/resistant b, c  Aesculus pavia  APAV N Immune/resistant b, c  Aesculus glabra  var. arguta AAR N Immune/resistant b, c  Aesculus  9  carnea  ACAR R N Immune/resistant b ACAR N F Atypical mines, heavy damage of   G. aesculi , occurrence horse chestnutleaf miner, resistant behaviour  Aesculusmarilandica AMA N Immune  Aesculus parviflora  APAR N Immune a, b, c  A  evaluation under artificial infestation,  F   evaluation under natural infestation (field conditions),  N   no evaluation in natural or artificial conditions* No observation of   C. ohridella  on donor plant a Referred in Kenis et al. 2003 b Referred in Straw and Tilbury 2006 c Referred in D´Costa 2014Trees  1 3
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