Miscanthus Andersson is under development as a biomass crop and has been characterized by a wide range of markers including amplified fragment length polymorphism (AFLP; Hodkinson et al., 2002), restriction fragment length polymorphism (RFLP; Hernández et al., 2001), inter-simple sequence repeat (ISSR) PCR, and DNA sequences of nuclear and chloroplast regions generated using conventional (Hodkinson et al., 2002) and next-generation approaches including RNAseq and genotyping by sequencing (GBS; Ma et al., 2012). Simple sequence repeat (SSR) markers from maize and Brachypodium distachyon (L.) P. Beauv. (Hernández et al., 2001; Zhao et al., 2011) have been successfully applied to Miscanthus, and chloroplast SSRs have been developed by De Cesare et al. (2010).
Some nuclear SSR markers have also been developed, such as those for M. sinensis Andersson, M. floridulus (Labill.) Warb. (Ho et al., 2011), and several other Miscanthus species (Zhou et al., 2011). However, there is a need to develop additional SSR markers for Miscanthus as the total number of available markers is limited. There is also a need to test these markers on a range of species, especially M. sacchariflorus (Maxim.) Hack., M. sinensis, and M. ×giganteus Greef & Deuter ex Hodk. & Renvoize as these comprise the main species of germplasm collections. SSRs developed from Saccharum officinarum L. expressed sequence tags (ESTs) have been recently used by Kim et al. (2012) to generate genetic maps of M. sacchariflorus and M. sinensis with genome coverage of 72.7% and 84.9%, respectively. The numbers of linkage groups found for the two maps (40 for M. sacchariflorus and 23 for M. sinensis) were higher than the basic chromosome number for Miscanthus (x = 19). Additional markers, such as those generated in this study, will be required to make more saturated maps, especially from noncoding regions that are underrepresented in current maps. Recently, single-nucleotide polymorphism (SNP) markers generated using GBS markers have been used for high-resolution mapping and identified all 19 linkage groups in M. sinensis (Ma et al., 2012).
METHODS AND RESULTS
DNA samples were either freshly extracted or obtained from the DNA bank at Trinity College, Dublin. Fresh leaves were frozen in liquid nitrogen and ground manually to a fine powder. Total genomic DNA was extracted following a modified cetyltrimethylammonium bromide (CTAB) method (Hodkinson et al., 2007). Total genomic DNA from the M. sinensis clone SW217 was used by ATG Genetics (Vancouver, British Columbia, Canada) to build a nuclear microsatellite–enriched library. After digestion with multiple 4-cutter restriction enzymes, enrichment for SSRs containing fragments was obtained through biotinylated TCn, TGn, and GATAn simple sequence motifs. The selected fragments were cloned into the EcoRI site of the plasmid pUC19 and screened for positive clones using 32P-labeled TCn, CAn, and GATAn simple sequence motifs. Two 96-well microtiter plates containing single positive bacterial colonies, one selected for the presence of dinucleotide repeats and the second for the presence of tetranucleotide repeats, were produced. The 192 clones were sequenced by AGOWA GmbH (Berlin, Germany), and SSRs were identified in the clones using ‘find microsat Win32’ (Salamin, unpublished). All 192 clones contained SSRs (96 dinucleotides and 96 tetranucleotides). Eighty primer pairs were designed equally among these sets using Primer3 software (Rozen and Skaletsky, 2000; http://frodo.wi.mit.edu/primer3/) and tested with PCR. Selection of the final sample of 29 primers was based on clarity of product on an agarose gel. Primer details and GenBank numbers are provided in Table 1.
Characteristics of 29 primer pairs developed for microsatellite genotyping.
Twenty-nine primer sets provided reliable amplification, and 19 of these were selected to have a mixture of di- and tetranucleotide SSRs. A template DNA volume of 1 µL (40 ng·µL-1) was amplified with an initial denaturation of 5 min at 95°C followed by 35 cycles each with a denaturation of 1 min at 95°C, 1 min at a primer-specific annealing temperature (Table 1), and an extension of 1 min at 72°C, followed by a final extension at 72°C for 10 min. The reaction mixture (final volume) contained 1× reaction buffer containing 2 mM MgSO4, 0.125 µM dNTPs, 0.25 µM of each primer, and 0.5 U of Taq DNA polymerase (New England BioLabs, Herts, United Kingdom). Five different fluorescent dyes were used for primer labeling to allow multiplexing, in pools (Table 1). A polyA treatment at 65°C was applied for 30 min to the PCR products. Undiluted PCR products were then sized using an ABI 3130xl automated DNA sequencer (Applied Biosystems, Carlsbad, California, USA) and the resulting peaks were scored with GeneMapper version 4.0 software (Applied Biosystems). All 29 primer pairs produced good amplification on eight test genotypes of M. sacchariflorus, M. sinensis, and M. ×giganteus, but 11 loci were not consistently amplified across our entire collection and were discarded from further analyses. Our final analysis therefore included 19 SSR markers. Allele number, size range, expected heterozygosity (He), and polymorphism information content (PIC) were calculated using PIC Calculator Extra ( http://www.genomics.liv.ac.uk/animal/pic.html). He and PIC values were only calculated for M. sacchariflorus, M. sinensis, and M. ×giganteus because of sample size (Table 2).
Genetic properties of the newly developed markers for three Miscanthus species.a
Cross-amplification of the newly developed microsatellites of Miscanthus.a
Polymorphism at 19 microsatellite loci was studied in a collection of 166 individual grasses (Appendix 1), mostly belonging to the species M. sinensis, M. sacchariflorus, and M. ×giganteus. Fourteen individuals belonging to closely related genera were also included. All markers revealed considerable length polymorphism, with the number of alleles ranging from 13 to 44 per locus, with an average of 27.5 (Table 3). The loci amplified included a tetranucleotide repetition in nine cases and a dinucleotide repetition in the remaining 10. No major difference was observed between di- and tetranucleotide microsatellite loci in their ability to detect variation. Thirteen out of 19 primer pairs showed cross-amplification in non-Miscanthus species (Table 3). Average allele number was higher than the value of 12 found by Hernández et al. (2001) in a previous study using SSRs from maize. The higher number of clones used in our study (166 against 16 clones) and the introduction of species other than M. sinensis, M. sacchariflorus, and M. ×giganteus could account for the difference in allele number.
PIC and He values varied considerably among species (Table 2) and were the highest (0.88 and 0.89, respectively) for M. sinensis, 0.48 and 0.54 for M. sacchariflorus, and the lowest (0.33 and 0.41) in M. ×giganteus. The PIC value of M. sinensis (0.88) was consistent with the value of 0.83 in Hernández et al. (2001), both are higher than the average PIC value recently found by Zhao et al. (2011) in a study examining transferability of 49 microsatellite markers from Brachypodium distachyon to M. sinensis.
In the past few years, the first nuclear microsatellite markers for Miscanthus have been developed (Hung et al., 2009; Ho et al., 2011; Zhou et al., 2011). Both studies from Zhao et al. (2011) on transferability from Brachypodium P. Beauv. and from Hung et al. (2009) on nine new microsatellite loci specific for Miscanthus, were limited to M. sinensis, thus explaining the low level of polymorphism found compared to the markers in this study. Zhou et al. (2011) extended the test for their 14 newly developed markers to M. floridulus, M. lutarioriparius L. Liu ex S. L. Chen & Renvoize, and M. sacchariflorus, increasing the average number of alleles found to 16.1 and the PIC value to 0.76. A different approach was used by Ho et al. (2011) to develop 12 new SSR primer pairs for Miscanthus. They designed primers based on genic microsatellite loci (EST-SSRs) obtained through transcriptome sequencing and detected an average of 7.9 alleles per locus when tested on M. floridulus and M. sinensis.
The newly developed primers presented here were found to cross-amplify not only within Miscanthus species but also in other members of the Saccharinae, Andropogoneae, and Paniceae. They amplified DNA in Zea L. (Tripsacinae), Sorghum Moench (Sorghinae), Cymbopogon Spreng. (Andropogoninae), and Pennisetum Rich. (Paniceae). The primers are of high value for characterization of Miscanthus species and can be applied to other closely related genera including Saccharum L.
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 The study was funded by the National Development Plan of Ireland through Teagasc core funding. M.d.C. was financed under the Teagasc Walsh Fellowship Ph.D. Scheme. We thank TCD Botanic Gardens Dublin; Svalöf Weibull, Sweden; Royal Botanic Gardens Kew, United Kingdom; and the University of Hohenheim, Germany, for sharing their Miscanthus resources.