Introduction
The genus Triticum is an allopolyploid complex that is extremely important in agriculture. Triticum dicoccum (emmer wheat), Triticum dicoccoides and Triticum durum are within the AABB-genome emmer lineage, while Triticum timopheevii, Triticum araraticum and hexaploid Triticum zhukovskyi (AAAmAmGG) are within the AAGG-genome timopheevii lineage; the AAGG genome was assigned to T. timopheevii due to chromosome pair mismatching with emmer wheat (AABB) (Lilienfeld and Kihara, Reference Lilienfeld and Kihara1934; Love, Reference Love1941; Sachs, Reference Sachs1953; Wagenaar, Reference Wagenaar1966). Distinguishing individuals with AAGG from those with AABB more simply has been achieved using the morphology of hairy leaves in the timopheevii lineage (AAGG). However, checking hairy leaves and chromosome pairing is often complicated and actually impracticable for partial or sterile specimens, such as seed remains from archaeological sites.
Boscato et al. (Reference Boscato, Carioni, Brandolini, Sadori and Rottoli2008), in their quest to identify T. timopheevii from charred fossil remains, designed ribosomal internal transcribed spacer primers discriminating T. timopheevii and T. dicoccum but were unsuccessful in retrieving ancient DNA. We also applied this approach to modern timopheevii, but in vain. Golovnina et al. (Reference Golovnina, Glushkov, Blinov, Mayorov, Adkison and Goncharov2007) studied phylogenetic relationships in Triticum. In their alignment data (p. 206), we emphasized a single-nucleotide polymorphism (SNP) in the chloroplast matK region, which discerns timopheevii from emmer. Species-specific multiplex PCR is one of the most frequently used assay, as it is easy to perform, quick and cost-effective (Dubey et al., Reference Dubey, Meganathan and Haque2009; Mendonca et al., Reference Mendonca, Hashimoto, De-Franco, Porto-Foresti, Gadig, Oliveira and Foresti2010). Against this background, in the present study, we established a multiplex PCR marker to distinguish timopheevii from emmer and confirmed its accuracy using retained genetic resources.
Experimental
Plant samples
Five tetraploid taxa, T. durum, T. dicoccum, T. dicoccoides, T. timopheevii and T. araraticum, and one hexaploid species, T. zhukovskyi, were examined. All 199 accessions used in this study were from the collection of KOMUGI, Kyoto University (see Table 1; Kawahara, Reference Kawahara1997, Reference Kawahara1998), and represented nearly the entire geographical ranges of each species. Extraction of genomic DNA using cetyltrimethylammonium bromide was conducted in accordance with Escaravage et al. (Reference Escaravage, Questiau, Pornon, Boche and Taberlet1998).
Agarose gel electrophoresis of amplified multiplex PCR products. The bands below represent the matK amplicon, which differentiates AAGG (positive band) and AABB (negative) species. The upper bands represent rbcL, which is a positive control for confirming the validity of the experiment.
Plant materials of Triticum and results of PCR bands presented in this study
Species names in Triticum and Aegilops are often written as per van Slagen's criteria; this study, however, used the names based on the catalogue list of Kyoto University (Kawahara, Reference Kawahara1998). Detailed passport data for each accession can be found at: http://www.shigen.lab.nig.ac.jp/wheat/komugi/top/top.jsp. Underlined and double underlined accession no. represent multiplex PCR result in Fig. 1. Double underlined accessions no. were sequenced for confirmation for SNPs, and their nucleotide sequences were shown in online Supplementary Fig. S3.
Design of PCR primers
Sequences including an SNP site that varies between T. timopheevii and T. turgidum in the chloroplast matK region, which was first presented by Golovnina et al. (Reference Golovnina, Glushkov, Blinov, Mayorov, Adkison and Goncharov2007), were aligned using ClustalW (Thompson et al., Reference Thompson, Higgins and Gibson1994) (online Supplementary Fig. S1). Since Golovnina et al. did not search for polymorphisms within species, we examined whether this SNP is a fixed mutation within species. Initially, we designed the matK reverse primer using the T. timopheevii-specific site, which was located at the 3ʹ end of the primer (online Supplementary Fig. S1). The resulting PCR band was positive in both AAGG and AABB species. This means that a single mismatch primer would be inadequate for discriminating between these species. Therefore, a second artificial mismatch was introduced at the third base from the 3ʹ end of the reverse primer (matK_416R: 5ʹ-GAAAGAATCGCAATAAAGGT-3ʹ) in order to increase specificity (online Supplementary Fig. S1) (Bottema and Sommer, Reference Bottema and Sommer1993). The expected band size (203 bp) of the matK product was found in AAGG-genome species as well as two accessions of hexaploid T. zhukovskyi. A subset of accessions double underlined in Table 1 were sequenced for confirmation using the matK_233F primer (5ʹ-TTGTCCGAAAGAAAAAGAAA-3ʹ) as a forward primer and the matK_1534R primer as a reverse primer at an outboard to the SNP (online Supplementary Fig. S1).
An additional primer set for the rbcL gene was developed as a positive control amplicon, which enables a check of whether the PCR reaction was successful. A primer set (RUBISCO_834F: 5ʹ-AAATACTACTTTGGCTCATT-3ʹ, RUBISCO_1197R: 5ʹ-CACCAAATTGTAATACAGAA-3ʹ) was designed at conserved positions based on the alignment sequences from GenBank [No. LN626622 (T. timopheevii), LN626619 (T. aestivum) and KM352501 (T. turgidum)] (online Supplementary Fig. S2). The results showed that an approximately 363-bp band of the amplified rbcL product was detected in all PCR reactions.
Multiplex PCR amplification
The multiplex PCR reaction using a mixture of matK and rbcL primers was carried out as follows: 30 cycles of 45 s at 95°C for denaturation, 30 s at 47.5°C for annealing and 15 s at 68°C for polymerization (Taq DNA polymerase; New England Biolabs, Frankfurt am Main, Germany), with a final extension of 5 min at 68°C. The matK and rbcL primers were mixed at a ratio of 6:7. PCR products were observed using a 2% agarose gel, ethidium bromide and UV illumination. It was confirmed at least twice for each accession. As shown in Fig. 1, all of the AABB and AAGG species investigated could be distinguished (Table 1).
Discussion
Although there is still a risk of miss-identifying the species when we use only one SNP as a marker, the multiplex PCR marker presented here distinguished between AAGG from AABB species with complete accuracy. Multiplex PCR is rapid and affordable, allowing simultaneous detection of multiple loci, and thus, has been applied in various species, such as grapevine (Merdinoglu et al., Reference Merdinoglu, Butterlin, Bevilacqua, Chiquet, Adam-Blondon and Decroocq2005; Migliaro et al., Reference Migliaro, Morreale, Gardiman, Landolfo and Crespan2013), Chinese egret (Huang et al., Reference Huang, Zhou, Lin, Peng, Fang and Chen2012) and for the typing of high molecular weight alleles in wheat (Ma et al., Reference Ma, Zhang and Gale2003).
Our multiplex PCR marker should also be useful for the identification of ancient remains as well as modern genetic resources. The so-called ‘new-glume wheat’, featuring charred spikelets with a characteristic morphology as found at archaeological sites in Europe (Jones et al., Reference Jones, Valamoti and Charles2000; Kohler-Schneider, Reference Kohler-Schneider2003; Toulemonde et al., Reference Toulemonde, Durand, Berrio, Bonnaire, Daoulas and Wiethold2015), could be timopheevii, but its identity has remained unclear, mainly because its morphology is not exactly the same as that of modern cultivated timopheevii and its wild progenitor, T. araraticum. The process of domestication of timopheevii is mysterious due to the strong resemblance between charred seed remains and those of emmer.
Wild T. araraticum is distributed across large parts of West Asia, especially Iraq, east Turkey and Armenia (Zohary and Hopf, Reference Zohary and Hopf2000), but the genetic resources of this species have not been well collected, due to the inaccessibility of these areas to researchers. Recently, the situation in some areas has improved, and thus, archaeological and genetic resource investigations have been initiated or restarted (Gasparyan and Arimura, Reference Gasparyan, Arimura, Gasparyan and Arimura2014; Tanno et al., Reference Tanno, Kawahara and Takata2015, personal communication with M. Arimura). The multiplex PCR technique presented here is a novel approach for the rapid and easy identification of two important tetraploid wheat species.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262117000181.
Acknowledgements
The authors are grateful to S. Takenaka, Ryukoku University, and M. Arimura, Tokai University, for their advice. They also thank the two reviewers for their valuable comments. This study was supported by Grant-in-Aid for Scientific Research on Innovative Areas (Grant Number: JP 24101003) and Grant-in-Aid for Young Scientists (A) (Grant Number: JP 23682009). The authors would like to thank Enago (www.enago.jp) for the English language review.
Author Contribution Statement
K.-I.T. planned and coordinated the study, and wrote the paper. K.Y. planned and performed the experiment and analyses, and wrote the paper. A.T. and E.A. performed the experiment. K.K. analyzed the data. T.K. was responsible for plant materials.
