Introduction
Northern red oak is one of the most important North American lumber species. It ranges from the peninsula of Nova Scotia to Minnesota and south to eastern Oklahoma, Alabama, and North Carolina (Sander, Reference Sander1990). By now, northern red oak has become widespread in European forests with notable populations in Germany (Schmitz, Reference Schmitz2014), and various other countries (Nicolescu et al., Reference Nicolescu, Vor, Mason, Bastien, Brus and Henin2020).
Previous studies identified northern red oak haplotypes using PCR-RFLP (Aldrich et al., Reference Aldrich, Parker, Michler and Romero-Severson2003; Magni et al., Reference Magni, Ducousso, Caron, Petit and Kremer2005; Feng et al., Reference Feng, Sun and Romero-Severson2008; Birchenko et al., Reference Birchenko, Feng and Romero-Severson2009; Pettenkofer et al., Reference Pettenkofer, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2020) and cpSSR markers (Zhang et al., Reference Zhang, Hipp and Gailing2015; Pettenkofer et al., Reference Pettenkofer, Burkardt, Ammer, Vor, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2019; Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020), which were originally developed for angiosperms (Weising and Gardner, Reference Weising and Gardner1999) or white oak species (Deguilloux et al., Reference Deguilloux, Dumolin-Lapègue, Gielly, Grivet and Petit2002; Zhang et al., Reference Zhang, Hipp and Gailing2015; Pettenkofer et al., Reference Pettenkofer, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2020). Chloroplast markers are commonly used for the certification of reproductive material due to their maternal inheritance (via seeds) and strong phylogeographic signature (Petit et al., Reference Petit, Csaikl, Bordács, Burg, Coart, Cottrell, van Dam, Deans, Dumolin-Lapègue, Fineschi, Finkeldey, Gillies, Glaz, Goicoechea, Jensen, König, Lowe, Madsen, Mátyás, Munro, Olalde, Pemonge, Popescu, Slade, Tabbener, Taurchini, de Vries, Ziegenhagen and Kremer2002; Chmielewski et al., Reference Chmielewski, Meyza, Chybicki, Dzialuk, Litkowiec and Burczyk2015). Here, we introduce fifteen novel and easy-to-score polymorphic cpSSRs for Q. rubra chloroplast haplotype identification. We also tested the transferability of those primers to the closely related species Q. ellipsoidalis E. J. Hill and the white oak species Q. robur L. and Q. petraea (Matt.) Liebl. (section Quercus).
Experimental
Chloroplast genome
The Northern red oak chloroplast genome was first completely sequenced in 2014 (Alexander and Woeste, Reference Alexander and Woeste2014) and again in 2019 (Pang et al., Reference Pang, Liu, Wu, Yuan, Li, Dong, Liu, An, Su and Li2019). Chloroplast genome sequences of additional oak species from several subsections were also included in the sequence alignment (online Supplementary Table S1). All sequences were downloaded from the NCBI genomic database (Sayers et al., Reference Sayers, Beck, Brister, Bolton, Canese, Comeau, Funk, Ketter, Kim, Kimchi, Kitts, Kuznetsov, Lathrop, Lu, McGarvey, Madden, Murphy, O'Leary, Phan, Schneider, Thibaud-Niessen, Trawick, Pruitt and Ostell2020) in November 2021.
Plant material and haplotypes
Northern red oak DNA was obtained from previously conducted red oak studies with known haplotypes based on cpSSR and cpCAPS (PCR-RFLP) analyses (Pettenkofer et al., Reference Pettenkofer, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2020; online Supplementary Table S2), Q. ellipsoidalis DNA from a previous study (Lind and Gailing, Reference Lind and Gailing2013), Q. robur samples with known chloroplast haplotypes (Burger et al., Reference Burger, Müller, Rogge and Gailing2021 and unpublished data), and sessile oak samples from three geographic regions were used for transferability tests.
Motif detection and primer design
A red oak chloroplast genome (Alexander and Woeste, Reference Alexander and Woeste2014) was browsed for repetitive motives (minimum six repetitions for mononucleotide, and three for longer repeat motives) using the tandem repeats finder online application (Benson, Reference Benson1999). An alignment of Quercus chloroplast genome sequences was performed using the Qiagen CLC Genomics Workbench (Hilden, Germany). All detected candidate loci were visually verified for the variability of the potential SSR, revealing 44 promising candidates. Primer design was done using the NCBI Primer blast online application (Ye et al., Reference Ye, Coulouris, Zaretskaya, Cutcutache, Rozen and Madden2012) with all available chloroplast reference genomes of Quercus rubra (taxid:3512). Target fragment size range was set from 70 to 500 bp, melting temperature (T m) between 57 and 63°C with differences ⩽3°C between both primers and at least two mismatches to unintended targets within the last five base pairs of the 3′ end. All primer pairs were modified with an appendix (Forward primer: 5′-CACGACGTTGTAAACGAC-3′; reverse primer: 5′-GTTTCTT-3′). This enabled the use of HEX (Sigma Aldrich; St. Louis, MO, USA) or 6-FAM™ (Sigma Aldrich; St. Louis, MO, USA) labelled universal M13 primers for economic reasons (Schuelke, Reference Schuelke2000).
Primer testing
For primer testing, PCRs were conducted following the touchdown PCR protocol and reagent mix of Götz et al. (Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020) in volumes of 14 μl, each using 1 μl of genomic DNA (5–10 ng), 1 μl labelled M13 forward primer (5 μM), 0.2 μl of the designed forward primer (5 μM), and 0.5 μl reverse primer (5 μM). Primer pairs producing distinguishable fragment sizes were multiplexed (Table 1). Electrophoretic separation for allele scoring of 1:150 diluted PCR products was done using an ABI Genetic Analyzer 3130xl (Applied Biosystems, Foster City, USA) with the GeneScan™ 500 ROX™ internal size standard (Applied Biosystems, Foster City, USA) using GeneMapper V 3.7 (Applied Biosystems, Foster City, USA).
Table 1. Primer sequences and fragment size range for allele binning of novel polymorphic primer pairs
Primer sequences of monomorphic markers are reported in online Supplementary Table S3.
a Including M13 primer appendix. Fragment sizes will be shorter if labelled primers without M13 tail (online Supplementary Table S3) are used.
b Additional white oak allele: 188.
c Comparatively poor amplification during PCR.
Initial tests were conducted for eight samples of four different chloroplast haplotypes (Pettenkofer et al., Reference Pettenkofer, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2020). Polymorphic markers were subsequently multiplexed in 80 northern red oak samples of seven different haplotypes (Table 2, online Supplementary Table S2), and eight white oak samples with two and three previously defined haplotypes, respectively (online Supplementary Tables S2 and S4). Three of the fifteen polymorphic markers (QRcp01, QRcp07, QRcp17) exhibited various alleles within previously defined widely distributed red oak chloroplast haplotypes A.1 and A.3 (Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020), revealing sub-haplotypes A.1_1, A.1_2, A.3_1, A.3_2, and A.3_3 (Table 2, online Supplementary Table S4).
Table 2. Origin and novel haplotypes (HN) of northern red oak populations of previously defined haplotypes (HP, following Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020)
Alleles of each haplotype are reported in online Supplementary Table S4.
1 Götz et al. (Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020), 2 Pettenkofer et al. (Reference Pettenkofer, Burkardt, Ammer, Vor, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2019), 3 Liesebach and Schneck (Reference Liesebach and Schneck2011), 4 Lind-Riehl et al. (Reference Lind-Riehl, Sullivan and Gailing2014), 5 Lind and Gailing (Reference Lind and Gailing2013).
a Taken from a German provenance trial.
Discussion
Since our novel primer pairs enabled the distinction of additional haplotypes within two common northern red oak haplotypes of the native range, they provide a powerful tool for further differentiation between northern red oak chloroplast haplotypes. Reproductive material for the establishment of forest plantations, such as for northern red oak, is increasingly demanded due to climate change. However, certified plantations within Europe are rather limited (Steiner, Reference Steiner2012). German red oak probably originated from a limited geographic range within the species' natural distribution (Pettenkofer et al., Reference Pettenkofer, Burkardt, Ammer, Vor, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2019; Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020). Additional chloroplast markers will be valuable tools to identify novel and rare haplotypes with a restricted native range as a prerequisite to narrow down the geographic origin of reproductive material and potential admixture. German red oak population haplotypes, for example, consist predominantly of haplotype A.1 (Pettenkofer et al., Reference Pettenkofer, Burkardt, Ammer, Vor, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2019; Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020). Our sample test set included samples with haplotype A.1 from different states of Germany (Brandenburg), Canada (Ontario), and the USA (Wisconsin, Tennessee). Interestingly, we observed an association between populations´ geographic origin and novel sub-haplotypes. Sub-haplotype A.1_1 was observed in all individuals from Germany and Ontario. Wisconsin and Tennessee populations, on the other hand, consisted of samples of sub-haplotype A.1_2. This result provides further evidence for the capability of our marker set to narrow down the origin of European red oak and supports the assumption of a geographically restricted origin of German Q. rubra stands (Pettenkofer et al., Reference Pettenkofer, Burkardt, Ammer, Vor, Finkeldey, Müller, Krutovsky, Vornam, Leinemann and Gailing2019; Götz et al., Reference Götz, Krutovsky, Leinemann, Müller, Rajora and Gailing2020).
All fifteen polymorphic red oak markers amplified in the closely related species Q. ellipsoidalis, but were monomorphic in the tested samples. Additional alleles might be revealed in future Q. ellipsoidalis investigations. All primer pairs amplified in both white oak species and ten primer pairs were polymorphic (Table 1, online Supplementary Table S3). We observed comparable variation within white oak and red oak samples (32 versus 31 total alleles for all polymorphic markers), even though white oak samples were underrepresented (16 versus 80 samples). This result supports the observation of higher variation in white oak haplotypes as compared to red oaks (Birchenko et al., Reference Birchenko, Feng and Romero-Severson2009). However, we observed no differentiation within previously defined white oak haplotypes (online Supplementary Fig. S3). Our new cpSSR markers could be used as a fast and effective method to define chloroplast haplotypes in North American red oaks as well as in European white oaks and might potentially differentiate white oak haplotypes in larger datasets.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262122000156.
Acknowledgements
We thank Alexandra Dolynska for technical support and Katrin Burger for the haplotype definition of pedunculate oak samples.
Financial support
Federal Ministry of Food and Agriculture (BMEL) through Fachagentur Nachwachsende Rohstoffe. Reference number: 22023314
Open access
