Mentha Longifolia (L.) L.: a Model Species for Mint Genetic Research
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HORTSCIENCE 40(5):1225–1229. 2005. genome was reported as 1C = 385 Mbp (Ben- nett and Leitch, 2005), and in the range of 2C = 0.84 to 0.99 pg (Gobert et al., 2002), or 1C Mentha longifolia (L.) L.: A Model = 405 to 477 Mbp. The M. longifolia C value is relatively small among those of cultivated Species for Mint Genetic Research plants, being comparable to that of rice (C = 400 to 466 Mbp) and about half that of tomato K.J. Vining, Q. Zhang, A.O. Tucker, C. Smith, and T.M. Davis (C = 980 Mbp) (Bennett and Leitch, 2005). University of New Hampshire, Department of Plant Biology, 46 College Road, Phylogenetic analysis of Mentha indicates that Rudman Hall, Durham, NH 03824 M. longifolia is an ancestor of M. spicata, and may be the latter’s organelle genome source Abstract. Mentha longifolia, a wild relative of the polyploid, cultivated Mentha (mint) (Bunsawat et al., 2004). In turn, M. spicata is species, was evaluated as a potential model system for genetic research relevant to the a parent of M. ×gracilis and of M. ×piperita cultivated mints. Fourteen Mentha longifolia accessions maintained by the US Department (Tucker and Naczi, 2005; Tucker et al., 1991; of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Re- Tucker and Fairbrothers, 1990). Mentha ca- pository (NCGR), were highly diverse with respect to geographic origin, oil composition, nadensis is believed to have arisen as a hybrid verticillium wilt resistance, aspects of morphology, and molecular marker polymorphism. of M. longifolia and M. arvensis (Tucker and Accession CMEN 584 was the only carvone chemotype, while CMEN 682 was the only ac- Chambers, 2002). cession with high menthol content. Trans-piperitone oxide was the primary oil component We have examined a set of M. longifolia of accessions CMEN 17 and CMEN 18, while pulegone was most abundant in CMEN 20, accessions maintained by the NCGR, with CMEN 500, CMEN 501, and CMEN 585. Four accessions—CMEN 585, CMEN 17, CMEN particular attention to two traits of economic 501, and CMEN 81—were consistently resistant to verticillium wilt, while CMEN 584 relevance: oil composition and resistance to and CMEN 516 were highly susceptible. Pairwise similarity coeffi cients were calculated verticillium wilt, an important disease of pep- and a UPGMA (unweighted pair-group analysis) tree was constructed on the basis of 63 permint. This paper documents the phenotypic informative randomly amplifi ed polymorphic DNA (RAPD) marker bands. CMEN 585 and genetic diversity among these M. longifolia and CMEN 584 shared the greatest number of bands (16), and formed a distinct cluster accessions and reviews the features that make in the UPGMA tree. Seven pairs of accessions had no bands in common, emphasizing M. longifolia a potentially useful model species the high degree of molecular diversity represented by these accessions. The favorable for Mentha genetic and genomic research. features of diploid (2n = 2x = 24) genome constitution, comparatively small genome size (400 to 500 Mb), self-fertility, fecundity, and diversity with respect to economically rel- evant traits, contribute to M. longifolia’s potential usefulness as a model system for the Materials and Methods cultivated mints. As a perennial species amenable to vegetative propagation, M. longifolia’s Germplasm. Fourteen accessions initially spectrum of susceptibility/resistance to an important vascular wilt disease encourages identifi ed as M. longifolia, including 4 sub- its further evaluation as a system for broader studies of plant–microbe interactions and species, were obtained as rooted plants or disease resistance mechanisms. rhizomes from the NCGR. The USDA National Plant Germplasm System Plant Information The principal Mentha (Lamiaceae = mint genetically, and few genomic resources have (PI) numbers for each of these accessions, as family) species of commerce in the United been developed. Gene identifi cation in Mentha well as their chromosome numbers (if known) States are vegetatively propagated polyploids, has been limited to genes encoding enzymes and geographic origins, are listed in Table 1. making them diffi cult or intractable subjects for involved in essential oil biosynthesis. These Plants were maintained in a greenhouse at the transmission genetic analysis and conventional genes have been extensively characterized, University of New Hampshire in 22-cm pots, breeding. Native spearmint (Mentha ×villoso- and genetic manipulation of peppermint oil and were propagated vegetatively. Observations nervata Opiz) is triploid (2n = 3x = 36), although biosynthesis has been initiated (Mahmoud et of morphology were made by direct visual morphologically similar to fertile, tetraploid al., 2004; Burke et al., 2004; Mahmoud and examination and by light microscopy. spearmint (M. spicata L., 2n = 4x = 48). Scotch Croteau, 2001). Oil composition. Oils from whole fl owering spearmint (M. ×gracilis Sole) is heptaploid (2n A diverse and widely distributed Mentha plants were distilled with a neo-Clevenger of = 7x = 84). ‘Mitcham’ peppermint (M. ×piperita germplasm base has been documented (Tucker Moritz after Kaiser and Lang with the modifi ca- L.) is hexaploid (2n = 6x = 72) (Tucker and Naczi, and Naczi, 2005). As of December 2004, the tion of Hefendehl (Kaiser and Lang, 1951; von 2005; Tucker and Fairbrothers, 1990; Udo et al., NCGR in Corvallis, Ore., maintained 441 Rudloff, 1969). Mass spectra were recorded with 1962). Polyploidy increases composite genome Mentha accessions as vegetative clones and 52 a 5970 Hewlett-Packard Mass Selective detector size and allelic complexity, hampering structural as seed, representing 20 species and a diversity coupled to a HP 5890 GC using a HP 50 m × 0.2 and functional genomics studies, and may be of interspecifi c hybrids (GRIN). Twenty-one mm fused silica column coated with 0.33 mm accompanied by poor fertility. Not surprisingly, accessions are M. longifolia, and six are listed FFAP (crosslinked). The GC was operated under no genetic linkage maps have been constructed as M. longifolia × M. longifolia hybrids. In the following conditions: injector temperature for Mentha. Other than an extensive literature addition, of the 67 accessions listed as simply 250 °C; oven temperature programmed to 60 on the genetics of oil quality, both classical Mentha hybrid, 30 include M. longifolia in the °C held for 1 min to 115 °C at 2.5 °C per min, (Hefendehl and Murray, 1976; Hendriks et al., known or inferred pedigree. The USDA col- then to 210 °C at 1.0 °C per min and held for 1976) and molecular (Croteau and Gershen- lection of M. longifolia accessions represents 30 min; injection size 1mL (about 50% solution zon, 1994), few traits have been characterized a wide range of geographic, phenotypic, and in spectroscopy grade n-pentane) split 1:10. genetic diversity. The MSD EI was operated at electron impact Mentha longifolia has the widest natural source 70 eV, 250 °C. Identifi cations were made Received for publication 12 Dec. 2004. Accepted for geographic distribution of any Mentha species, by Kovats Indices and library searches of our publication 25 Mar. 2005. We gratefully acknowledge from western Europe to central Asia and in volatile oil library supplemented with those of Sara Olson for helping to develop the verticillium southern Africa. It may encompass 22 subspecies NBS, NIST, and Wiley. resistance screening protocol. We also thank Dennis (Tucker and Naczi, 2005). Almost all are diploid Verticillium resistance screening. Qualita- Johnson and Linda Ciuffetti for providing V. dahliae (2n = 2x = 24), but some tetraploid (2n = 4x = cultures, and J. Brent Loy and Rosanna Freyre for tive assessment of verticillium resistance in all helpful comments on the manuscript. This is Scientifi c 48) forms have also been described (Chambers 14 accessions was conducted with a wild-type Contribution 2267 from the New Hampshire Agricul- and Hummer, 1994). The sexual fertility of Verticillium dahliae strain provided by Dennis tural Experiment Station (NHAES). This research was the diploid, and even the tetraploid, forms has Johnson at Washington State University. Based supported in part by NHAES Project H433, and grants been documented (Fagbemi and Morton, 1982; on the outcome of these initial trials, a subset of from the Mint Industry Research Council. Murray, 1960). The size of the M. longifolia resistant and susceptible accessions was chosen HORTSCIENCE VOL. 40(5) AUGUST 2005 1225 AAugustBook.indbugustBook.indb 11225225 66/14/05/14/05 112:10:102:10:10 PPMM Table 1. Features of Mentha longifolia accessions used in this study. Accession numbers, status and geographic origin as listed on http://www.ars-grin.gov/cor/ mentha/meninfo.html. Accession Status 2nz Collected from CMEN 17 (PI 557755) Breeding material 24 Unknown European country CMEN 18 (PI 557756) Wild material 24 Netherlands CMEN 19 (PI 557757) Wild material 24 France CMEN 20 (PI 557770) Wild material 24 Syria CMEN 34 (PI 557758) Wild material ---y India CMEN 500 (PI 212313) Wild material 48 Afghanistan CMEN 501 (PI 212314) Cultivated material 48 Afghanistan CMEN 516 (PI 557760) Cultivated material --- Italy CMEN 584 (PI 557769) Uncertain improvement status 24 South Africa CMEN 585 (PI 557767) Uncertain improvement status 24 South Africa CMEN 592 (PI 557766) Wild material 24 Uzbekistan CMEN 635 (PI 557768) Wild material 24 Nepal CMEN 682 (PI617491) Cultivar: ‘Velvet’ --- Russia CMEN 81 (PI 557759) Probable hybrid of M. longifolia × M. spicata --- United States zChambers and Hummer (1994). yUndetermined chromosome number. for closer examination and for use as crossing were moved to the greenhouse under natural RAPD marker diversity analysis. Only parents for future genetic studies. The latter tri- light and ambient temperature. informative markers (bands that were present als differed from the initial assessments in that Observations were taken 8 weeks postinocula- in at least two accessions and absent in at least a quantitative rating scale was used, and a V. tion. For plants with dead primary stems, stem two) were included in the analysis.