Biologia 68/4: 621—626, 2013 Section Botany DOI: 10.2478/s11756-013-0189-z Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra Konrad Celiński*, Ewa Maria Pawlaczyk, Aleksandra Wojnicka-Pó
ltorak, Ewa Chudzińska & Wies
law Prus-G
lowacki Adam Mickiewicz University, Department of Genetics, Umultowska 89, PL-61-614 Poznań, Poland; e-mail: celinski@amu.edu.pl Abstract: Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content. Key words: dwarf mountain pine; conservation genetics; genetic diversity; microsatellite transfer; SSR Introduction Microsatellites, also known as simple sequence repeats (SSR) or short tandem repeats (STR), are the smallest class of simple repetitive DNA sequences (Semagn et al. 2006). These repetitive DNA regions are present in both eukaryotic and prokaryotic genomes (Field & Wills 1998; Tóth et al. 2000). SSRs are composed of 1 to 6 nucleotide motifs repeated from 5 to 40 times (Oliveira et al. 2006; Selkoe & Toonen 2006). In contrast to the other part of genomes, microsatellite sequences are characterized by very high mutation rates, amounting to 10−2 –10−6 nucleotides per locus per generation (Sia et al. 2000; Zhang & Hewitt 2003). Microsatellites are commonly considered to be selectively neutral. However, many studies indicate, that both the number of repeats (Everett & Wood 2004; Moxon & Wills 1999) and their distribution in the genome are not completely random (Li et al. 2002; Morgante et al. 2002; Scotti et al. 2000). SSRs are widespread markers used in many fields of research, such as estimation of genetic diversity, gene flow analyses, gene mapping, individuals identification, parentage assignment, etc. (Christiakov et al. 2006; Oliveira et al. 2006). Microsatellite markers were used to describe genetic diversity and population differentiation among several conifer species, e.g. Picea abies (Scotti et al. 2006), Picea asperata (Wang et al. 2005), Pinus brutia and Pinus eldarica (Bucci et al. 1998), Pi* Corresponding author c 2013 Institute of Botany, Slovak Academy of Sciences
nus canariensis (Navascués et al. 2006), Pinus halepensis and Pinus pinaster (Gómez et al. 2005), Pinus heldreichii (Naydenov et al. 2005), Pinus strobus (Marquardt & Epperson 2004) or Pinus taeda (Al-Rabab‘ah & Williams 2002). Pinus mugo Turra is one of the most important conifer species of mountain ecosystems in Europe. In contrast to most of pine species, which are usually tall trees, Pinus mugo is rather a medium-sized shrub, reaching up to 3.5 m in height. In mountains it forms dense shrubs communities in subalpine regions above the timberline (1,400–2,700 m a.s.l.), which protect soil against erosion, immobilize snow patches and rocks, thus alleviating consequences of avalanches. According to Christensen (1987), the native range of this pine species occurance covers the mountains in Central (the Alps, the Sudeten) and South-Eastern Europe (the Tatras, the Carpathian Mountains and mountains of Balkan Peninsula). Some small isolated populations are also located in the central Italian Apennines (Richardson 1998). The International Union for Conservation of Nature (IUCN) placed Pinus mugo in 1998 on the Red List with the least concern status. To date studies conducted on Pinus mugo have concerned mostly its anatomical and morphological variability (Boratyńska et al. 2004; B˛aczkiewicz & PrusG
lowacki 2005), allozyme variation (Prus-G
lowacki & Szweykowski 1983; Slavov & Zhelev 2004), clonal struc- K. Celiński et al. 622 Fig. 1. Habitat of Pinus mugo (a) and a map showing locations of populations sampled in this study (b). Population codes are identified in Table 1. Table 1. The location of sampled populations of Pinus mugo. Code A B C D Location Tomanowa Prze
l˛ecz Zielony Staw Wielka Pańszczycka M
laka Żleb Żandarmerii Latitude (N) Longitude (E) Elevation (m) 49.2217 49.2303 49.2682 49.2104 19.9002 20.0009 20.0444 20.0771 1,590 1,680 1,270 1,345 ture (Prus-G
lowacki et al. 2005), taxonomic relationships between different taxa composing the Pinus mugo complex (Prus-G
lowacki et al. 1998; Lewandowski et al., 2000; Monteleone et al., 2006) as well as crossspecies hybridization processes (Wachowiak et al. 2006; Wachowiak et al. 2008; Kormutak et al. 2008; Kormutak et al. 2009). However, up to date no data have been available on the genetic diversity of Pinus mugo populations using nuclear microsatellite markers. Proper management, preservation and protection of each species require basic data on the level and current distribution patterns of genetic diversity in natural populations (Frankham et al. 2002). In this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. Successfully transferred markers were characterized in order to demonstrate their practical usefulness in further research. Material and methods Plant material One-year old pine needles were sampled from four natural populations of Pinus mugo located in the Tatra Mts in Southern Poland (Fig. 1. and Table 1). Within each population, 24 individuals were selected for needle collection with a minimum distance of 15 m between them to reduce the risk of clonality. The needles were stored at –20 ◦C until DNA isolation. Total genomic DNA was extracted from needles using a modified CTAB method (Doyle & Doyle 1990). Quality and quantity of the extracted DNA were measured with a NanodropTM ND-1000 spectrophotometer (ThermoScientific) and diluted to a final concentration of 20 ng/µL. DNA was stored at 4 ◦C until further analyzed. Microsatellite transfer Fifteen microsatellite markers originally developed for Pinus taeda (Auckland et al. 2002) and four markers, originally developed for Pinus sylvestris (Soranzo et al. 1998), were tested for their amplification in Pinus mugo (Table 2). From the 96 DNA samples collected, 16 were used to screen microsatellite markers and to confirm their polymorphism. The level of polymorphism of the SSR markers successfully transferred to Pinus mugo SSR markers was assessed using all 96 DNA samples. PCR was performed in a final volume of 10 µL, which contained: ∼50 ng of DNA template, 1 × complete reaction buffer, 0.2 mM dNTP, 0.2 µM each forward and reverse primer and 0.4U HiFiTaq polymerase (Novazym, Poznań, Poland). Amplification was carried out in a 2720 Thermal Cycler (Applied c ) at the following profile: initial denaturation Biosytems step (5 min at 94 ◦C), followed by 35 cycles of: 1 min at 94 ◦C, 1 min at annealing temperature calculated for each marker as Tm –5 ◦C, and 2 min at 72 ◦C, with a final elongation step of 10 min at 72 ◦C. PCR amplicons were initially assessed through electrophoresis in 2% agarose gels. Successful transfer of SSR marker to Pinus mugo was reported when a clear band of expected size and polymorphism was observed (class 1). Other observed results were classified as follows: class 2, monomorphic amplification product of the expected size; class 3, non-specific amplification, complex pattern or poor amplification; and class 4, no amplification. Polymorphism of successfully transferred loci was analyzed by allele sizing on an ABI 3130xl Genetic c ) using reverse primers laAnalyzer (Applied Biosystems belled with FAM, PET, NED and VIC fluorescent dyes (Apc ) and GeneScanTM 600LIZTM (Applied plied Biosystems c ) as an internal size standard. Individuals were Biosystems genotyped with the GeneMapper version 3.7 software (Apc ). plied Biosystems Characterization of microsatellite markers in Pinus mugo 623 Table 2. Transfer of 19 microsatellite loci developed for Pinus sylvestris and Pinus taeda (source species) to Pinus mugo (species of interest) and their allele sizes in each species. Transfer classes are: 1, polymorphic amplification product of expected size; 2, monomorphic amplification product of expected size; 3, non-specific amplification, complex pattern or poor amplification; 4, no amplification. Allele sizes (average) Source taxon Locus Result of transfer to P. mugo in P. mugo in source taxon Pinus sylvestris SPAC 11.4 SPAC 11.6 SPAC 12.5 SPAG 7.14 3 3 3 1 – – – 217 150 165 155 209 Pinus taeda PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX PtTX 4 3 2 2 3 4 3 4 3 4 1 1 3 3 3 – – 257 134 – – – – – – 208 278 – – – 337 122 260 155 223 280 335 338 332 182 224 305 167 127 214 2001 2003 3016 3018 3019 3027 3032 3068 3084 3107 4001 4011 4031 4177 4199 Sequencing of the microsatellite markers PCR products of three microsatellite markers successfully transferred to Pinus mugo (PtTX 4001; PtTX 4011; SPAG 7.14, see Results) were cloned using the pGEM
-T Easy Vector System (Promega). DNA sequencing was carried out using a BigDyeTM terminator, and analyzed with ABI 3130xl c ). Obtained seGenetic Analyzer (Applied Biosystems quences were aligned using MEGA tool version 3.1 (Kumar et al. 2004) and the MEGABLAST tool available on National Centre for Biotechnology Information (NCBI) website and finally deposited in GeneBank (NCBI). Genetic data analysis CERVUS version 3.0 (Kalinowski et al. 2007) was used to calculate the following parameters for each locus: number of alleles (NA ), frequency of frequent alleles (FFA), observed heterozygosity (HO ), expected heterozygosity (HE ), polymorphism information content (PIC) and probability of first parent exclusion (PE1). Deviation from the Hardy-Weinberg equilibrium (HWE), heterozygote excess and deficiency, and linkage disequilibrium (LD) were tested with GENEPOP version 4.0.10 (Raymond & Rousset 1995; Rousset 2008) and corrected for multiple comparisons using the Bonferroni approach. The presence of null alleles, stutter, and large allele dropout were assessed using MICROCHECKER (1,000 randomization; van Oosterhout et al. 2004). LOSITAN (50,000 simulations; Antao et al. 2008) was used to detect loci being potentially under selection. Results Microsatellite transfer rate The percentage of microsatellite markers transferred (classes 1 and 2 in Table 2) to Pinus mugo was moderately low (26%). Among satisfactorily amplified microsatellites in Pinus mugo there were four loci (PtTX 3016; PtTX 3018; PtTX 4001; PtTX 4011) which were originally developed for Pinus taeda and one locus (SPAG 7.14) originally developed for Pinus sylvestris. Out of the five microsatellites that were transferred to Pinus mugo (classes 1 and 2, Table 2), two PtTX 3016 and PtTX 3018 (40%) were monomorphic, with PCR products of 257 and 134bp, respectively. Under analyzed PCR conditions fourteen microsatellite loci were classified to classes 3 and 4 (nonspecific, complex patterns, poor amplifications or no amplifications) (Table 2). The length of microsatellites successfully transferred to Pinus mugo (PtTX 4001; PtTX 4011; SPAG 7.14) was similar to the length observed in the source species. Sequencing of microsatellite loci Obtained sequences of polymorphic loci (PtTX 4011; PtTX 4001; SPAG 7.14) in Pinus mugo confirmed that the analyzed DNA regions contain microsatellite repeats. There were no differences between repeat motifs detected in the source species and those observed in Pinus mugo. All the obtained sequences were deposited in the GeneBank nucleotide database (NCBI) under the following accession numbers: JQ995801, JQ995800 and JQ995802 (Table 3). Genetic polymorphism Three microsatellite loci successfully transferred to Pinus mugo differed in the observed levels of genetic polymorphism. The number of alleles (NA ) varied from 7 at PtTX 4001 to 24 at SPAG 7.14, with the average of 14.3 alleles per locus. The frequency of frequent alleles (FFA) also differed in the analyzed loci and ranged from 0.12 for the 190 bp allele at SPAG 7.14 to 0.53 for the 201 bp allele at PtTX 4001. Expected heterozygosity (HE ) and observed heterozygosity (HO ) ranged from K. Celiński et al. 624 Table 3. Accession numbers and population genetics parameters for loci PtTX 4001, PtTX 4011 and SPAG 7.14 in P. mugo. NA – number of alleles; FFA – frequency of frequent allele (length of allele in bp shown in parentheses); HE – expected heterozygosity; HO – observed heterozygosity; PIC – polymorphism information content; PE1 – average exclusion probability of first parent. Locus PtTX 4001 PtTX 4011 SPAG 7.14 Accesion no. NA FFA HE HO PIC PE1 JQ995801 JQ995800 JQ995802 7 12 24 0.53 (201) 0.22 (259) 0.12 (190) 0.635 0.861 0.939 0.604 0.563 0.813 0.579 0.841 0.930 0.225 0.556 0.765 Table 4. Linkage disequilibrium test (LD) between pairs of microsatellite loci in P. mugo. Locus pair PtTX 4001 & PtTX 4011 PtTX 4001 & SPAG 7.14 PtTX 4011 & SPAG 7.14 Chi2 df P-value 18.432111 Infinity 9.182199 8 8 8 0.018210 Highly sign. 0.327160 Table 5. Hardy-Weinberg equilibrium test (HWE) for microsatellite loci in P. mugo. Heterozygote deficit Heterozygote excess Locus PtTX 4001 PtTX 4011 SPAG 7.14 For all loci P-value S.E. 0.0955 0.0000 0.0000 0.0000 0.0044 0.0000 0.0000 0.0000 Switches (ave.) 18094.25 13114.00 3126.50 114444.92 0.635 and 0.604 at locus PtTX 4001 to 0.939 and 0.813 at locus SPAG 7.14, respectively. The value of polymorphism information content (PIC) in all the three loci was high (PIC > 0.5) and ranged from 0.579 to 0.930, with the total of 0.784. The average exclusion probability of the first parent (PE1) ranged from 0.225 to 0.765 (Table 3), with the total exclusionary power (first parent) 0.919 (data not shown). Linkage disequilibrium (LD) was found in two of the three analyzed pairs of loci (Table 4). Two loci (PtTX 4011 and SPAG 7.14) deviated from the HardyWeinberg equilibrium (HWE) (Table 5) and showed significant (P < 0.05) heterozygote excess (Table 5). MICROCHECKER indicated that null alleles may be present at SPAG 7.14 and PtTX 4001 loci. In addition, stuttering might have also resulted in scoring errors in those two microsatellite loci. There is no evidence for the presence of null alleles, large allele drop-out or stuttering at PtTX 4011. A comparison of four Pinus mugo populations from the Tatra Mts in Southern Poland, indicated that there is no evidence for non-neutrality of the transferred microsatellite loci, based on simulation tests using the FST -outlier detection method incorporated in LOSITAN. Discussion De novo development of polymorphic, selectively neutral and non-linked microsatellite markers for each species is both time- and cost-intensive. Therefore, transfer of microsatellites among related species seems to be a reasonable approach genetic diversity analysis. P-value S.E. 0.9045 1.0000 1.0000 1.0000 0.0044 0.0000 0.0000 0.0000 Switches (ave.) 18094.25 13114.00 3126.50 114444.92 In this study we investigated the possibility of microsatellite transfer from Pinus sylvestris and Pinus taeda to Pinus mugo. The transfer rate was moderately low (26%). Out of the total of 19 tested microsatellite markers, five gave clear and unambiguous amplification products, only three (60%) of which were polymorphic. Our results of transfer rates are similar to other cross-species amplification rates recorded for conifer species (Echt et al. 1999; Sheperd et al. 2002; González-Martínez et al. 2004). The rate of microsatellite markers successfully transferred between species depends directly on their divergence time. Kutil & Wiliams (2001) successfully transferred 100% of the tested microsatellite loci within the subsection Australes between P. taeda and P. palustris. Much lower transfer rates (29% and 32%) were observed between the subgenus Strobus and Pinus (Echt et al. 1999; Karhu et al. 2000, respectively) and between four Pinus species to Pinus pinaster (4%; Mariette et al. 2001). In our study the rate of successfully transferred microsatellite markers amounted to 26.6% from the subsection Australes to Pinus, and 25% within the subgenus Pinus. Due to the large and highly duplicated part of genomes, the transfer and development of new microsatellite markers for conifers, is a difficult task. For example, Soranzo et al. (1998) designed thirty-seven primer pairs, but only seven (20%) resulted in a single variable locus amplification in Pinus silvestris. Similar results have also been reported for Pinus strobus (Echt et al. 1996) and Picea abies (Pfeiffer et al. 1997). We found no differences in repeat motifs in microsatellite loci between the source species (Pinus Characterization of microsatellite markers in Pinus mugo sylvestris and Pinus taeda) and Pinus mugo (the species of interest). This means that these loci can be considered orthologous, because of their high sequence similarity among primer binding sites, flanking regions and repeat motifs (Kutil & Wiliams 2001). Three microsatellite loci successfully transferred to Pinus mugo were highly polymorphic. The number of alleles (NA ) in Pinus mugo was similar to those reported in the source species for loci PtTX 4001 and PtTX 4011 and even higher for SPAG 7.14 (Soranzo et al. 1998; González-Martínez et al. 2004). The differences in allele numbers may have resulted from the larger number of analyzed specimens (96) in our study than in the referred studies. The high value of polymorphism information content (PIC) indicates that the three microsatellite loci have sufficient polymorphism to be used for genetic studies in Pinus mugo. Linkage disequilibrium (LD) among pairs of loci found in our study connected with the presence of null alleles in some of the analyzed loci makes it difficult to use these microsatellite markers for genetic diversity studies in Pinus mugo. According to Chapuis & Estoup (2007) and Carlsson (2008), the presence of null alleles caused a small, but significant overestimation of both FST and genetic distance (GD). Therefore loci which are prone to provide null alleles should be used with caution and loci less prone to null alleles should always be preferred. Slatkin (2008) indicated several factors which can increase or strongly influence linkage disequilibrium, i.e. the Wahlund effect, bottleneck, genetic drift, natural selection or sampling method. We can not exclude the potential effect of any of these factors, except for natural selection because we found no evidence for non-neutrality in the microsatellite markers successfully transferred to Pinus mugo. Two of the three microsatellite loci were found to depart from the Hardy-Weinberg equilibrium (HWE). Further analyses indicated that heterozygote deficiency at these loci was responsible for this departure. Another possible explanation for the departure from HWE is connected with non-random mating, genetic bottlenecks or dramatic decline in spawning populations. One of the populations analyzed in this study (Wielka Pańszczycka M
laka) is located on a peatbog, surrounded by a spruce forest and almost completely isolated from other populations of Pinus mugo. We may not exclude bottlenecks in this population or non-random mating. There is also some evidence for preferably near-neighbour mating in shrubby Pinus mugo (Slavov & Zhelev 2004), which may also result in a departure from the Hardy-Weinberg equilibrium. According to our knowledge, this is the first study concerning the transfer of microsatellite markers to Pinus mugo and the use of these markers to analyze the level of genetic polymorphism in this species. We conclude that the cross-species amplification is an interesting alternative to the development of new microsatellites in conifer species. Considering the high level of polymorphism the described microsatellite loci provide a good and useful tool for studies on genetic diversity 625 in Pinus mugo. However, they should be used with caution in analyses, because some of them indicated linkage disequilibrium and presence of null alleles. Further studies are needed to transfer more microsatellite loci from other pine species or develop de novo new markers dedicated only to Pinus mugo. Acknowledgements This research was financially supported by the Ministry of Science and Higher Education of Poland (No. NN304060339). References Al-Rabab‘ah M.A. & Williams C.G. 2002. Population dynamics of Pinus taeda L. based on nuclear microsatellites. Forest Ecol. Manag. 163: 263–171. Antao T., Lopes A., Lopes R.J., Beja-Pereira A. & Luikart G. 2008. LOSITAN: a workbench to detect molecular adaptation based on a Fst-outlier method. BMC Bioinform. 9: 323 Auckland L.D., Bui T., Zhou Y., Shepherd M. & Williams C.G. 2002. Conifer microsatellite handbook. Texas A&M University, College Station TX, 57 pp. B˛aczkiewicz A. & Prus-G
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