University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Biochemistry -- Faculty Publications Biochemistry, Department of 2015 Genetic variation in Miscanthus X giganteus and the importance of estimating genetic distance thresholds for differentiating clones Katarzyna Glowacka University of Illinois at Urbana-Champaign, [email protected] Lindsay V. Clark University of Illinois at Urbana-Champaign Shivani Adhikari University of Illinois at Urbana-Champaign Junhua Peng Huazhong Agricultural University J. Ryan Stewart Brigham Young University FSeeollow next this page and for additional additional works authors at: https:/ /digitalcommons.unl.edu/biochemfacpub Part of the Biochemistry Commons, Biotechnology Commons, and the Other Biochemistry, Biophysics, and Structural Biology Commons Glowacka, Katarzyna; Clark, Lindsay V.; Adhikari, Shivani; Peng, Junhua; Stewart, J. Ryan; Nishiwaki, Aya; Yamada, Toshihiko; Jorgensen, Uffe; Hodkinson, Trevor R.; Gifford, Justin; Juvik, John A.; and Sacks, Erik J., "Genetic variation in Miscanthus X giganteus and the importance of estimating genetic distance thresholds for differentiating clones" (2015). Biochemistry -- Faculty Publications. 429. https://digitalcommons.unl.edu/biochemfacpub/429 This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Katarzyna Glowacka, Lindsay V. Clark, Shivani Adhikari, Junhua Peng, J. Ryan Stewart, Aya Nishiwaki, Toshihiko Yamada, Uffe Jorgensen, Trevor R. Hodkinson, Justin Gifford, John A. Juvik, and Erik J. Sacks This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ biochemfacpub/429 GCB Bioenergy (2015) 7, 386–404, doi: 10.1111/gcbb.12166 Genetic variation in Miscanthus 3 giganteus and the importance of estimating genetic distance thresholds for differentiating clones KATARZYNA GŁOWACKA1,2,LINDSAYV.CLARK1 , SHIVANI ADHIKARI1 , JUNHUA PENG3 , J. RYAN STEWART4 ,AYANISHIWAKI5 , TOSHIHIKO YAMADA6 , UFFE JØRGENSEN7 , TREVOR R. HODKINSON8 ,JUSTINGIFFORD1 , JOHN A. JUVIK1 and ERIK J. SACKS1 1Energy Biosciences Institute, University of Illinois, 1206 W. Gregory Dr., Urbana, IL 61801, USA, 2Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyn´ska 34, Poznan 60-479, Poland, 3College of Plant Science and Technology, Huazhong Agricultural University, No. 1, Shizishan Street, Wuhan, Hubei 430070, China, 4Plant & Wildlife Sciences, Brigham Young University, 167 WIDB, Provo, UT 84602, USA, 5Field Science Center, Faculty of Agriculture, University of Miyazaki, 1-1 Nishi Gakuenkibanadai, Miyazaki-shi Miyazaki-ken 889-2192, Japan, 6Field Science Center for Northern Biosphere, Hokkaido University, Kita 11 Nishi 10, Kita-ku, Sapporo, Hokkaido 060-0811, Japan, 7Dept. of Agroecology, Aarhus University, Blichers Alle 20, Postboks 50, Tjele DK-8830, Denmark, 8School of Natural Sciences, Trinity College Dublin, Dublin D2, Ireland Abstract Miscanthus 9 giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among leg- acy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type-specimen. A subset of accessions was also evaluated by restriction-site associated DNA sequencing (RAD-seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differ- ences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD-seq, the former is currently more cost-effective for determining if two accessions are genetically identical. We con- cluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In con- trast with the Mxg legacy cultivars, genetic similarity to the type-specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much-needed variation to growers. Keywords: genetic diversity, genotyping error, interspecific hybrids, Miscanthus sacchariflorus, Miscanthus sinensis, RAD-seq, SSR Received 7 September 2013 and accepted 19 October 2013 thus, all progeny derived from crossing Msa and Msi Introduction are by definition Mxg. In Japan, indigenous populations Miscanthus 9 giganteus (Mxg) is a nothospiecies, of tetraploid Msa and diploid Msi are common (Hirayo- derived from M. sacchariflorus (Msa) and M. sinensis shi et al., 1957; Adati & Shiotani, 1962; Nishiwaki et al., (Msi) (Hodkinson & Renvoize, 2001; Hodkinson et al., 2011). In southern Japan, sympatric populations of Msa 2002b). In this article, we adhere strictly to the Interna- and Msi infrequently produce interspecific triploid tional Code of Nomenclature for algae, fungi, and progeny (i.e. Mxg; Nishiwaki et al., 2011; Dwiyanti et al., plants (2012), especially with regard to nothospecies; 2013). Notably, one such Mxg was introduced from Yokohama Japan to Denmark in 1935 by Aksel Olsen Correspondence: Erik J. Sacks, tel. 217-333-9327, fax 217-244-3637, (Greef & Deuter, 1993; Linde-Laursen, 1993). During the e-mail: [email protected] last 20 years, Mxg has become an important feedstock 386 © 2013 John Wiley & Sons Ltd GENETIC VARIATION IN MISCANTHUS 9 GIGANTEUS 387 crop for the emerging bioenergy industries in Europe lineages that originated from a single genet. However, and the United States (Scurlock, 1999; Clifton-Brown technical replication enables estimation of genotyping et al., 2004; Heaton et al., 2008; Somerville et al., 2010). error rates, and subsequent consensus calling has pro- The USDA Biomass Crop Assistance Program (BCAP) ven to be a valuable approach for mitigating these alone supported the planting target of ~20 000 acres of errors (Zhang et al., 2006; Christelova et al., 2011). Mxg during 2011 and 2012 (K. Novak, personal commu- A key question about Mxg genetic diversity is: Are nication). Under the BCAP program, Mxg ‘Illinois’ was the legacy cultivars in North America derived from dif- the predominant cultivar planted, with a smaller area ferent and/or additional introductions (i.e. different planted to ‘Freedom’. genets) than the European genotype? Were multiple Numerous accessions of sterile triploid Mxg of genotypes of Mxg introduced or was there only one? To unknown provenance, which we call legacy cultivars, resolve the seemingly incongruous conclusions of Greef have been named in Europe and North America for et al. (1997) and Chouvarine et al. (2012), we tested the commercial sale and scientific research, but how much hypothesis, that Mxg in North America was derived genetic diversity is represented by these accessions is a from the European genotype, by estimating genetic sim- key question. All of the legacy Mxg in Europe and ilarity among a broad sample of accessions from North North America are cultivars that have been maintained America, and comparing them with previously studied by people in public and private gardens, and agricul- accessions from Europe, including the type-specimen tural fields; Mxg is neither native nor naturalized on for the species. Additionally, we assessed the potential these continents. Greef et al. (1997) evaluated 31 Euro- of new Mxg progeny, from planned interspecific crosses pean accessions of Mxg with amplified fragment length and collections in the wild (Japan), to increase genetic polymorphism (AFLP) markers but found only two dis- diversity of this newly important bioenergy crop. We tinct genotypes that were 98% similar, indicating a strik- also explored the limits of inference in modern molecu- ing lack of genetic diversity. Given error rates lar marker systems, such as simple sequence repeats commonly observed for AFLPs (Douhovnikoff & Dodd, (SSRs) and restriction-site associated DNA sequencing 2003; Bonin et al., 2004; Lasso, 2008), it is doubtful that (RAD-seq), for distinguishing different multilocus DNA the two Mxg groups observed by Greef et al. (1997) fingerprints in Miscanthus. were truly different genotypes, although distinguishing between genotyping error and mutation (i.e. a horticul- Materials and methods tural sport) is difficult (Pompanon et al., 2005; Cipriani et al., 2010). Such a lack of genetic diversity represents a Plant material significant risk to growers, as the emergence of a single virulent pest or disease could damage or destroy all We studied 85 Miscanthus accessions (Table 1), including 50 commercial production. If Mxg in North America was Mxg, 28 Msa, and seven Msi. Two sugarcane cultivars were obtained solely from Europe, then we would expect included as an out-group. We compared the Mxg type-speci- – genetic diversity to be similarly