DOI: 10.5061/dryad.6wwpzgmv4
title: ISSR analysis of Tulipa suaveolens (Liliaceae) populations in the European part of the species range

description: Tulipa suaveolens Roth is a decorative bulbous, polycarpic species in the
 Liliaceae family. Currently, the species populations are declining due to
 the degradation of natural steppes. In the present article, we evaluated
 the genetic diversity of 216 specimens from 22 natural T. suaveolens
 localities in the European part of the species range using 10 inter simple
 sequence repeat (ISSR) markers. The polymerase chain reaction (PCR)
 yielded 250 unambiguous and reproducible polymorphic bands with a mean
 polymorphism information content (PIC) value of 0.27. Neither the
 principal component analysis nor the clustering method split the dataset.
 However, the Bayesian model-based STRUCTURE analysis detected two genetic
 clusters of T. suaveolens. The analysis of the biogeographical
 distributional pattern of the revealed genetic groups confirmed our
 hypothesis that the evolution and distribution of T. suaveolens were
 likely affected by the Early Khvalynian transgression of the Caspian Sea
 and the following Enotayevsk regression. Furthermore, this hypothesis was
 supported by the results of the NEW HYBRIDS analysis identifying the
 specimens from the populations located outside the Early Khvalynian
 flooding limit as pure parents, while the specimens of the populations
 within the flooding limit were classified as backcrosses and second
 generation hybrids.
description: The
 samples were collected from 22 natural <i>T. suaveolens</i>
 localities in the Astrakhan, Volgograd, Orenburg, Rostov, Samara and
 Saratov provinces, the Krasnodar region, the Republics of Kalmykia and
 Dagestan as well as from the Crimea and Western Kazakhstan (Table 1). Ten
 plants were selected from each locality except the population no. 22.
 Leaves were dried in silica gel and put in plastic zip-bags.
 DNA was
 extracted using the NucleoSpin® Plant II kit (MACHEREY-NAGEL, Germany)
 according to the manufacturer’s protocol. DNA purity (absorbance ratio
 A260/A280) and quantity (absorbance at 260 nm) were measured by the Qubit
 fluorometer (Thermo Fisher Scientific, Carlsbad, CA). The samples were
 normalized at 10 ng/μl for the ISSR analysis following the Qubit
 concentrations. The polymerase
 chain reaction (PCR) was carried out in the Mastercycler gradient
 amplifier (Eppendorf, Germany) with 10 ISSR primers from the “Sintol”
 company (Moscow, Russian Federation) that were pre-selected for
 <i>T. suaveolens </i>(Kashin et al. 2016; Kritskaya et al.
 2019). The PCR was performed in 20 µl reaction volumes. The reaction
 mixture contained 4 µl ready-to-use PCR mix MaGMix (200 µM of each dNTP,
 1.5 mM MgCl<sub>2</sub>, 1.5 U SmarTaqDNA-polymerase and
 buffer; Dialat Ltd., Moscow, Russian Federation), 15 µl of deionized
 water, 3.4 pmol of each primer and 1 µl of template DNA. The PCR
 amplification regime was as follows: preliminary denaturation during 5
 minutes at 95° C, then 35 cycles of 30 seconds each at 95° C, 30 seconds
 at 44° C and 2 minutes at 72° C, then final elongation during 10 minutes
 at 72° C. DNA bands were visualized on 2% agarose gels<i>
 </i>(10 cm long and 15 cm wide) ran at 5V/cm for 2 h. Gel images
 were documented using the gel<i> </i>documentation system
 (Doc-print VX2, Germany). One Kb and 100 bp ladder were used<i>
 </i>as reference. For each
 specimen, two technical replicates with each ISSR primer were conducted
 (Gel replicability score ~ 95%). All DNA samples
 were amplified and produced clear, reproducible bands which were<i>
 </i>scored as present (1) or absent (0), using the CorelDRAW X8
 software.<i> </i>A binary matrix (1/0) containing 250
 polymorphic loci was generated and used for the subsequent<i>
 </i>data analysis for diversity and population structure.
 The
 informativeness of the ISSR markers was evaluated using polymorphism
 information content (PIC), resolving power (RP), mean resolving power
 (MRP), the marker index (MI) and the Shannon index (H’). PIC is the
 probability in detecting polymorphism by a primer: PIC =
 1−∑(<i>P<sub>i</sub></i>)<sup>2</sup>,
 where <i>P<sub>i</sub></i> is the frequency of the
 <i>i</i><sup>th</sup> allele (Sehgal et al. 2009).
 RP is the ability of each primer to detect the level of variation between
 individuals and was calculated according to Prevost and Wilkinson (1999):
 RP = ∑Ib, where Ib (band informativeness) takes the values of
 1–[2|0.5–<i>p</i>|], where <i>p</i> is the
 proportion of individuals containing the band. MI for each primer was
 calculated as a product of polymorphism information content and the
 effective multiplex ratio (MI = PIC × EMR (Varshney et al. 2007)). The
 Shannon index (H’) was calculated by the formula H’ =
 −<sub><i>i=1</i></sub><sup><i>R</i></sup><i>pi</i>ln<i>pi</i> (Shannon and Weaver 1949). Genetic diversity within each population was estimated through the percentage of polymorphic loci (P), the mean effective number of alleles (Ne), the mean Shannon Information Index (I), Nei’s gene diversity (h'), the overall Genetic differentiation (G<sub>ST</sub>), the Gene flow (N<sub>m</sub>) and the number of rare alleles/loci per population using the POPGene v 1.32 (Yeh 1997) software. The analysis of the obtained matrix was performed by the Neighbour Net method in the SPLITS TREE 4.6 software (Huson 1998). The principal component analysis (PCA) was carried out using R environment (R Core Team 2020) with RStudio (Version 1.2.5033) by running the scriptfile (see Appendix 1). The population structure analysis was conducted by the Bayes method in the STRUCTURE 2.3 software (Pritchard et al. 2000, Evanno et al. 2005, Jakobsson and Rosenberg 2007). The analysis was performed twice based on the genetic admixture model. The burn-in took 500,000 iterations; the subsequent building of the Markov chain took one million iterations for <i>K</i> (the hypothetical number of populations) from 1 to 10 with five-time repetition for each value of <i>K</i>. The obtained data were interpreted in the STRUCTURE HARVESTER software (Earl and VonHoldt 2012). Since we supposed that <i>T. suaveolens</i> populations were long isolated from one another due to the geological processes of the Late Pleistocene, we performed an additional NEW HYBRIDS 3.1.1 analysis (Anderson and Thompson 2002). The method evaluates the posterior probability of each specimen belonging to one of the probable hybrid classes and requires no preliminary knowledge about the parent classes (Anderson and Thompson 2002). NEW HYBRIDS is also used to reveal the restrictions of the gene flow between populations of the same species (Misiewicz and Fine 2014, Palm et al. 2019, Reid et al. 2019, Ortíz-Gamino et al. 2020) resulting from the physical barriers separating populations during the species distribution. All data were analyzed ten times from overdispersed starting values, over one million sweeps after a burn-in of 500,000 sweeps, following the recommendations of Anderson and Thompson (2002). The ArcGIS® 10.6 software was used to analyze the geographical distribution of the revealed genetic groups.