© 2016 The Japan Mendel Society Cytologia 81(1): 95–102 Determination of Genome Size, Chromosome Number, and Genetic Variation Using Inter-Simple Sequence Repeat Markers in Lotus spp. Hidenori Tanaka, Awatsaya Chotekajorn, Sayumi Kai, Genki Ishigaki, Masatsugu Hashiguchi and Ryo Akashi* Faculty of Agriculture, University of Miyazaki, 1–1 Gakuen Kibanadai Nishi, Miyazaki 889–2192, Japan Received August 21, 2015; accepted December 15, 2015 Summary Lotus is a leguminous and cosmopolitan genus in the Loteae tribe consisting of more than 200 spe- cies. The number of chromosomes has been reported for many Lotus species; however, molecular studies have focused only on a few important species of this genus. The present study was conducted to estimate the genome size and ploidy levels of 28 Lotus accessions, and to identify their genetic diversity using inter-simple sequence repeat (ISSR) analysis. The chromosome number of 16 accessions agreed with previous reports (except for Lotus salsuginosus), while that in 11 accessions were reported here for the first time. The smallest nuclear DNA content was identified in the diploid, Lotus unifoliolatus (0.28±0.01 pg C-1). In contrast, the tetraploid, Lotus australis, had a genome size of 1.28±0.03 pg C-1, representing a five-fold difference in genome size among the Lotus spe- cies. When expressed as a per Cx value, Lotus species genome sizes ranged from 0.28 pg Cx-1 in L. unifoliolatus to 0.71 pg Cx-1 in Lotus wrightii, representing a 2.5-fold difference. There was no relationship between genome size and chromosome number or ploidy level; thus, genome size is species specific in the Lotus species. On ISSR analysis, a total of 379 fragments were generated with 12 primers, and all accessions were grouped into four clusters by phylogenetic analysis. The results of this investigation will be useful for plant breeders attempting to expand the genetic variation found in this species by crossbreeding using these resources. Key words Chromosome number, Flow cytometry, Genome size, Lotus. The genus Lotus (Leguminosae) is a cosmopolitan this group, only four species have been domesticated and group in the Loteae tribe, consisting of over 200 spe- improved by plant breeding for agricultural purposes so cies with a wide ecological habitat, including xerophytic far. More detailed studies on this genus could reveal its desert plants, alpine perennials, and saline- and alkaline- untapped agronomic potential (Ferreira and Pedrosa- tolerant species. The regional center of origin for Lotus Harand 2014). is probably the Mediterranean basin, where the greatest The Lotus genus has three basic chromosomes, chro- diversity of species occurs (Swanson et al. 1990). Lotus mosome number, 5, 6 and 7 (Angulo and Real 1977). species are either annuals or perennials, and their dis- Cytogenetic analysis of 108 Lotus species has been tribution is generally along the arid deserts or steppes, reported, revealing 25 tetraploids, 71 diploids, and 12 equatorial fully humid areas of the tropics, warm tem- with both diploid and tetraploid cytotypes (Grant 1986). perate fully humid subtropical areas, and humid snowy However, the Lotus taxonomy is more complicated with areas except for extremely cold regions (Vriet et al. yet undeciphered phylogenetic relationships between 2014). Lotus plants are generally used as forage, as orna- different species. Estimations of genome sizes may pro- mental plants, and as a cover crop to indicate and control vide useful information for the analysis of phylogenetic soil erosion, and they are used in bioremediation (Diaz relationships and contribute to an understanding of the et al. 2005). Lotus japonicus is used as a model species complexity of genomes (Doležel 1997). Genome size in many legume-related research studies, and its whole information can also serve as a criterion in the selection genome sequence is currently available (Sato et al. of breeding material for cross breeding. The genome 2008). In addition to their ability to grow in varied con- size is still unknown for about 98% of angiosperm spe- ditions, their ability to form nodules enables symbiotic cies (Bennett and Leitch 2005) and more studies are relationship with nitrogen-fixing bacteria, thus, contrib- needed in this area to better understand the genetic uting to the ecological balance. Despite the wide ecolog- relationship between important species. Even though ical habitats and the abundance of species belonging to the Lotus genus has many species, information on the genome size of Lotus species is available only for 20 of * Corresponding author, e-mail: [email protected] those (Borsos 1973, Cheng and Grant 1973, Bennett and DOI: 10.1508/cytologia.81.95 Smith 1976, Grime and Mowforth 1982, Ferreira and 96 H. Tanaka et al. Cytologia 81(1) Pedrosa-Harand 2014). Several techniques are avail- Chromosome preparation and counting able for the determination of genome information. Flow Chromosome preparation and counting were per- cytometry (FCM) is a powerful and accurate tool to formed using the modified method described by Fukui estimate cellular DNA content in plants (Galbraith et al. (2006). Chromosomes stained with 4′,6-diamino-2-phen- 1983, Ohmido et al. 2000, Akiyama et al. 2001). Sample ylindole (DAPI) were observed with a fluorescence preparation using this method usually requires only a microscope (AXIO Imager.M1, Carl Zeiss, Oberkochen, few minutes and expensive reagents are not generally Germany). The number of chromosome in the Lotus spe- needed (Doležel et al. 2007). cies was counted by using at least five nuclei per acces- Genetic diversity and phylogenetic relationships in sions. plants can be estimated using several techniques, such as restriction fragment length polymorphism (RFLP), Genome size estimation using flow cytometry random amplified polymorphic DNA (RAPD), amplified FCM was conducted to estimate the genome sizes of fragment length polymorphism (AFLP) and microsatel- 28 Lotus accessions according to the method of Doležel lites. The inter-simple sequence repeats (ISSR) are the et al. (2007). Approximately 10000 nuclei were analyzed regions that lie within the microsatellite repeats and of- per sample, and the measurement was replicated three fer great potential to determine intra-genomic and inter- times for each accessions. The holoploid genome size (C genomic diversity compared to other techniques. The value) of Lotus species was estimated according to the ISSR marker can be highly variable within a species, but calibration curve from a reference standard, which was reveals more reliable and reproducible bands when com- Arabidopsis thaliana ecotype Columbia, with a nuclear pared with RAPD, and is more simple and cost effective DNA content of 0.16 pg C-1 (Bennett et al. 2003). The than AFLP or sequence-related amplified polymorphism monoploid genome size (Cx-1) of all species was also (SRAP) (Tsumura et al. 1996, Nagaoka and Ogihara calculated as a mass value (pg) and number of Mbp 1997, Qian et al. 2001). (1 pg=978 Mbp, Doležel et al. 2003). In this study, we conducted a detailed cytological analysis in the Lotus species by determining the chro- DNA isolation and ISSR-PCR amplification mosome number, ploidy level and estimation of the Total genomic DNA was extracted from fresh young nuclear DNA content of these species using FCM. More- leaves of each accession using DNeasy Plant Mini Kit over, we classified and determined the genetic variation (Qiagen, Hilden, Germany) according to the manufac- within this genus using ISSR markers. The information turer’s instruction. ISSR primers used in this study were from this study will be useful for future breeding pro- synthesized according to the primer set published by the grams in the Lotus spp. University of British Columbia, Canada (UBC primer no. 9). A total of 98 primers were initially screened. Materials and methods From the preliminary screening, 24 primers that could amplify visible bands were selected for further exami- Plant material nation. Eventually, 12 of those primers yielded bright Twenty-eight Lotus accessions were used in this study and discernible bands and were used for the succeeding (Table 1). Lotus australis, Lotus corniculatus, Lotus analyses of all 28 accessions. PCR was performed in a filicaulis and Lotus subbiflorus were conserved in our 10 µL reaction volume containing 4.0 µL of 0.25 ng μL-1 laboratory. Lotus burttii and L. japonicus were provided DNA, 5.0 µL of 2×AmpliTaq Gold 360 Master Mix (Life by the Legume Base at the National BioResource Project Technologies, CA, U.S.A.) and 1.0 µL of 10 µM each of (NBRP), University of Miyazaki, Japan (http://www. primer with the following conditions: an initial 10 min legumebase.brc.miyazaki-u.ac.jp/) and another accessions denaturation at 95°C; followed by 45 cycles of 30 s at were provided by the Germplasm Resources Informa- 95°C, 45 s at 52°C, 2 min at 72°C; and a final elongation tion Network (GRIN) at the United States Department step of 10 min at 72°C. The amplification products were of Agriculture’s Agricultural Research Service in U.S.A. analyzed using MultiNA Microchip Electrophoresis Sys- (http://www.ars-grin.gov/). The seeds were sown in tem (Shimadzu Biotech, Kyoto, Japan) with the DNA- Golden peat bun (Sakata Seed Corporation, Kanagawa, 500 Kit. Japan) and grown in a phytotron for one month under a photoperiod of 16-h light and 8-h dark at 25 and 23°C, Genetic diversity and cluster analysis respectively. The seedlings were subsequently trans- The ISSR bands were scored using the binary scoring planted to vermiculite in 6-cm diameter pots and grown system based on the presence or absence of reproducible in a phytotron for one month, and were eventually re- polymorphic bands as one or zero, respectively, and only transplanted to 12-cm diameter pots in the greenhouse. discernible bands were scored and used for cluster anal- Light regiments in both subsequent phases maintained ysis.