DOI: 10.5061/dryad.g79cnp5n2
title: SSR and cpDNA marker dataset genetic integrity of M.sylvestris in Saxony, Germany

description: Malus sylvestris (Mill.) is the only indigenous wild apple species in
 Central Europe. Agriculture, forestry and urbanization increasingly
 endanger Malus sylvestris natural habitats. In addition, the risks of
 cross-hybridization associated with increase in the cultivation of the
 domesticated apple Malus ×domestica (Borkh.), threatens the genetic
 integrity of M. sylvestris. The present study investigated the number of
 hybrids, genetic diversity and genetic structure of 292 putative M.
 sylvestris that originate from five different natural M. sylvestris
 populations in Saxony, Germany. All samples were genetically analyzed
 using nine nuclear microsatellite markers (ncSSR) and four maternally
 inherited chloroplast markers (cpDNA) along with 56 apple cultivars
 commonly cultivated in Saxony. Eighty-seven percent of the wild apple
 accessions were identified as pure M. sylvestris. The cpDNA analysis
 showed six private haplotypes for M. sylvestris, whereas three haplotypes
 were present in M. sylvestris and M. ×domestica. The analysis of molecular
 variance (AMOVA) resulted in a moderate (ncSSR) and great (cpDNA)
 variation among pure M. sylvestris and M. ×domestica individuals
 indicating a low gene flow between both species. The genetic diversity
 within the pure M. sylvestris populations was high with a weak genetic
 structure between the M. sylvestris populations indicating an unrestricted
 genetic exchange between these M. sylvestris populations. The clear
 distinguishing of M. sylvestris and M. ×domestica confirms our expectation
 of the existence of pure M. sylvestris accessions in this area and
 supports the argument for the implementation of preservation measures to
 protect the M. sylvestris populations in Saxony. 
description: <strong>Sampling</strong> Two
 hundred and ninety-two potential <i>M. sylvestris</i> trees
 sampled from five different areas within Saxony, Germany formed the basis
 of this study. The mapped trees were mainly found in sparse forests, along
 forest edges, stonewalls or other open landscape structures. The selection
 of trees was based on several morphological characters described by Wagner
 (1996) including fruit diameter &lt; 3.5 cm, no fruit over color,
 absence of hairiness in the leaf lower surface, for example. In addition,
 56 apple cultivars (<i>M. ×domestica</i> Borkh.)<i>
 </i>often cultivated in Germany were included as control genotypes.
 Leaf material of these 56 apple cultivars was provided by the Fruit
 genebank of the Institute of Breeding Research on Fruit Crops of the
 Julius Kühn-Institute (JKI) at Dresden-Pillnitz. Fresh leaf materials were
 collected in 2 ml tubes and dried using silica gel according to a modified
 protocol of (Chase & Hills, 1991). Leaf materials were stored at
 room temperature until DNA isolation. LGC Genomics (Berlin, Germany)
 performed DNA isolation and quantification. All samples were diluted to 10
 ng/ µl. <strong>Genetic
 analysis</strong> Nine ncSSR primer pairs
 (CH01H10, CH04C07, CH01H01, Hi02C07, CH01F03b, GD147, CH01F02, CH02C09,
 GD12) developed for <i>M. ×domestica</i> (Liebhard et al.,
 2002) were combined in three multiplexes with three primers per multiplex.
 . Four cpDNA primer pairs located at the <i>matK_dup </i>(NCBI
 GenBank: AF309231), <i>rps16</i> intron (NCBI GenBank:
 JQ391664) region and the intergenic spacer region
 <i>rps16_trnQ</i> (NCBI GenBank: AB604694) and
 <i>rpl2_trnH (</i>NCBI<i> </i>GenBank: JQ392141),
 respectively, were developed based on <i>Malus </i>sequence
 data obtained from the NCBI database (www.ncbi.nlm.nih.gov). Multiplex PCR
 was performed using the Type-It kit (Qiagen, Germany) according to the
 manufacturer’s protocol with three microsatellites per PCR in a total
 volume of 10 µl. PCR fragments were analyzed on a CEQ 8000XL Genetic
 Analyzer System (Beckman Coulter, Germany), for which forward primers were
 labelled with BMN-5, BMN-6 and DY751 (Biomers, Germany). PCR products were
 then diluted 1:100  using sterile water and 2 µl of this dilution was
 mixed with 29.9 µl of sample loading solution (Beckman Coulter) and 0.1 µl
 of size standard. <strong>Data
 analysis</strong> First, samples were grouped into
 pure <i>M. sylvestris</i>, <i>M. ×domestica</i> or
 hybrids using the model-based clustering method with STRUCTURE software
 version 2.3.4. (Pritchard, Stephens, & Donnelly, 2000). In order
 to improve the accuracy of the inference, the analysis was performed with
 prior information on the population (POPINFO model) in which each
 individual was defined either as ‘<i>M. sylvestris</i>’ or
 ‘<i>M. ×domestica</i>’. To accurately classify the
 individuals, 5 runs with <i>K </i>= 2 was performed with the
 program. The parameters were 50,000 burn-in periods and 50,000 Markov
 Chain Monte Carlo repetitions using the admixture model with correlated
 allele models. All <i>M. sylvestris</i> individuals with a
 probability of lower than 90 % that the assigned population is right were
 classified as hybrid. The probability of membership to the <i>M.
 ×domestica</i> genepool for the 56 reference <i>M.
 ×domestica</i> cultivars was &gt; 90 % indicating almost a zero
 chance of introgression from <i>M. sylvestris</i>. 
   Secondly, the
 number of natural <i>M. sylvestris</i> populations
 (<i>K</i>) were estimated with STRUCTURE. For this, we run the
 program again including all pure <i>M. sylvestris</i>
 accessions with <i>K</i> varying from 2 to 5 with 5 runs for
 each <i>K</i> value without POPINFO model. The remaining
 parameters were described as above. STRUCTURE HARVESTER (Earl &
 Vonholdt, 2012) was used for detecting the most likely value for
 <i>K</i> based on Evanno’s ΔK method (Evanno, Regnaut,
 & Goudet, 2005). Statistical
 analysis The mean number of alleles by
 locus (<i>N<sub>a</sub></i>), effective number of
 alleles (<i>N<sub>e</sub></i>), observed
 heterozygosity (<i>H<sub>o</sub></i>), expected
 heterozygosity (<i>H<sub>e</sub></i>) and number
 of private alleles (<i>PA</i>) were calculated for each ncSSR
 loci within the pure <i>M. sylvestris</i> individuals and the
 apple cultivars using the software GENALEX ver. 6.5 (Peakall &
 Smouse, 2006, 2012). Additionally, the allele frequencies of the
 chloroplast DNA markers were compared among the <i>M. sylvestris
 </i>individuals and the apple cultivars. An ‘Analysis of Molecular
 Variance’ (AMOVA) was performed based on nuclear and chloroplast marker
 data. The molecular variance (<i>ɸ</i><sub>PT,
 </sub>an analogue of <i>F</i><sub>st</sub>)
 and the migration rate (<i>N<sub>m</sub></i>) was
 calculated among the pure <i>M. sylvestris</i> genotypes and
 the apple cultivars as well as among the different <i>M.
 sylvestris</i> populations using GENALEX ver. 6.5. The correspondence
 (<i>rxy</i>) between geographic and genetic distance was
 performed by Mantel test with statistical testing by 9,999 permutations
 using the software GENALEX ver. 6.5 (Mantel, 1967).
description: Missing genetic data are indicated with -9.