Determination of Genome Size, Chromosome Number, and Genetic Variation Using Inter-Simple Sequence Repeat Markers in Lotus Spp

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 species. 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 species. 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. The genetic similarity matrix was generated using the same photoperiod, similar to which was used at the the similarity coefficient of Nei and Li’s (1979) genetic initial planting stage. distance and was subsequently employed to perform 2016 Analysis of Genomic Variation in Lotus spp. 97 Reference* Tschechow andTschechow Kartaschowa 1932, Larsen 1955, Grant, 1965 Harney and Grant 1965 Sz.-Borsos etSz.-Borsos al. 1972 Grant 1965 ̶ Larsen 1955, Grant and Sidhu 1966 ̶ Larsen 1956, Grant 1965 Tschechow andTschechow Kartaschowa 1932, Grant and Sidhu 1966 ̶ ̶ Grant 1965 Ito et al. 2000 Ito et al. 2000 ̶ Harney and Grant 1964, 1965 ̶ Grant 1965 ̶ ̶ ̶ Grant and Sidhu 1966 Kawakami 1930, Harney and Grant 1964 Larsen 1954, 1955 ̶ ̶ Larsen 1955, Harney and Grant 1965 Grant 1965, Grant and Sidhu 1966 (Mbp) 1 - 0.44 (432) 0.44 0.64 (625) 0.64 0.44 (429) 0.44 0.46 (447) 0.46 0.47 (459) 0.47 0.45 (435) 0.45 0.49 (472) 0.49 0.31 (304) 0.46 (443) 0.46 0.71 (696) 0.71 0.49 (481) 0.49 0.39 (385) 0.39 (383) 0.40 (390) 0.40 (427) 0.44 0.46 (448) 0.46 0.35 (345) 0.57 (559) 0.45 (440) 0.45 0.36 (354) (408) 0.42 0.39 (379) (398) 0.41 0.33 (322) 0.38 (370) 0.39 (379) 0.28 (269) 0.28 (273) Cx accessions in this study. 0.00 0.03 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.02 0.01 0.00 0.02 0.00 0.00 SD 0.00 0.00 0.01 0.01 0.01 0.00 0.01 0.00 0.00 0.01 0.00 0.01 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 - C 0.44 0.31 0.46 0.88 1.28 0.39 0.39 0.47 0.40 0.35 0.89 0.71 0.49 0.46 0.49 0.36 0.44 0.39 0.57 0.81 0.45 0.33 0.42 0.38 0.39 0.28 0.28 0.46 ± 0.46 Nuclear content DNA (pg) x x x x x x x x x x x x x x x x x x x x x x x x x x x x 4 4 4 2 2 2 2 2 2 2 2 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Ploidy level ) 12 28 12 14 ̶ 24 ̶ 12 12 ̶ ̶ 12 12 12 ̶ 12 ̶ 14 ̶ ̶ ̶ 12 12 12 ̶ ̶ 14 14 Reported 12 28 12 14 14 24 12 12 12 24 14 12 12 12 14 12 14 12 14 14 24 12 12 12 14 14 14 14 Observed Chromosome numbern (2 Chromosome numbers and nuclear content DNA the of Lotus ̶ ̶ ̶ ̶ Turkey Japan Pakistan Hungary Slovakia China Canada Israel U.S.A. U.S.A. Japan Japan Morocco Israel U.S.A. U.S.A. U.S.A. Morocco Chile Greece Australia U.S.A. U.S.A. U.S.A. Distribution Table 1. Table ̶ ̶ PI206895 B-303 PI283617 PI308034 PI631901 PI236862 B-39 PI238336 W6 30912 W6 DLEG900459 B-129 MG-20 PI655736 NSL448193 PI258396 W6 32602 W6 W6 30230 W6 DLEG920102 PI631785 PI368908 PI229569 PI190349 PI215235 W6 30246 W6 PI196332 DLEG880039 Accession No. ssp. frondosus Species ssp. requienii var. carmeli var. scoparius *The references cited pertain to chromosome numbers only. L. angustissimus L. australis L. burttii L. conjugatus L. conjugatus L. corniculatus L. corniculatus L. denticulatus L. L. filicaulis L. halophilus L. heermannii L. humistratus L. japonicus L. japonicus L. maritimus L. pedunculatus L. peregrinus L. salsuginosus L. scoparius L. strigosus L. subbiflorus L. subpinnatus L. tenuis L. L. uliginosus L. unifoliolatus L. unifoliolatus var. unifoliolatus L. weilleri L. wrightii 98 H. Tanaka et al. Cytologia 81(1)

Fig. 1. Somatic chromosomes of Lotus species. (a) Lotus conjugates ssp. requienii (2n=14). (b) Lotus corniculatus ssp. frondosus (2n=12). (c) Lotus halophilus (2n=24). (d) Lotus heermannii (2n=14). (e) Lotus maritmus (2n=14). (f) Lotus peregrinus var. carmeli (2n=14). (g) Lotus scoparius var. scoparius (2n=14). (h) Lotus strigosus (2n=14). (i) Lotus subbiflorus (2n=24). (j) Lotus unifoliolatus (2n=14). (k) Lotus unifoliolatus var. unifoliolatus (2n=14). (l) Lotus salsuginosus (2n=12). Scale bars=10 µm. cluster analysis and dendrogram construction through the chromosome numbers of all of these species verified the neighbor-joining (NJ) method (Saitou and Nei 1987). with the previous reports (Kawakami 1930, Tschechow All computations were carried out using the statistic pro- and Kartaschowa 1932, Larsen 1954, 1955, 1956, Har- gram R version 3.1.1 (R Core Team 2014). ney and Grant 1964, 1965, Grant 1965, Grant and Sidhu The genotype and allelic frequency data were used to 1966, Sz.-Borsos et al. 1972), except for that of Lotus compute the genetic diversity indices, observed number salsuginosus which had 2n=12 (Fig. 1l), a value differ- of alleles (Na), expected number of alleles (Ne), Shannon ent from which was previously reported for this species index of genetic diversity (I), and Nei’s genetic diversity as 2n=14 (Grant 1965). (H) at the population level using POPGENE 1.32 (Yeh et al. 1997). Nuclear DNA content measurement The nuclear DNA content of all 28 accessions was Results and discussion estimated using FCM with the standard curve, 2C DNA content=-0.0304+0.0039×fluorescent intensity, calcu- Chromosome counting lated according to the method described by Kawasaki Table 1 shows the observed and reported chromosome and Murakami (2000). The estimated nuclear DNA con- numbers determined for the Lotus species examined in tents for the 28 Lotus accessions are shown in Table 1. this study. Lotus conjugatus ssp. requienii, Lotus cor- We used two parameters to estimate genome sizes: the niculatus ssp. frondosus, Lotus halophilus, Lotus heer- C value and the Cx value. The C value describes the mannii, Lotus maritmus, Lotus peregrinus var. carmeli, DNA content per holoploid (with chromosome number Lotus scoparius var. scoparius, Lotus strigosus, L. sub- n), which represents the complete chromosome comple- biflorus, Lotus unifoliolatus and Lotus unifoliolatus var. ment irrespective of the degree of generative polyploidy unifoliolatus, were counted for the first time in this study (Greilhuber et al. 2005). On the other hand, the Cx value (Fig. 1a–k). Based on an analysis of more than 10 nuclei, has been recently introduced to represent the DNA con- 2016 Analysis of Genomic Variation in Lotus spp. 99

Table 2. Details of selected ISSR primers, primer sequences, range of fragment size, total number of ampliﬁed and polymorphic bands, and per- centage of polymorphism obtained from the analysis of the 28 Lotus accessions.

Number of bands Primer code Primer sequence (5′–3′) Range of fragment size (bp) Polymorphism (%) Total Polymorphic

UBC807 AGA GAG AGA GAG AGA GT 153–499 33 33 100.0 UBC810 GAG AGA GAG AGA GAG AT 150–498 31 31 100.0 UBC811 GAG AGA GAG AGA GAG AC 154–500 31 31 100.0 UBC825 ACA CAC ACA CAC ACA CT 172–499 25 25 100.0 UBC834 AGA GAG AGA GAG AGA GYT 153–499 33 33 100.0 UBC835 AGA GAG AGA GAG AGA GYC 152–500 33 33 100.0 UBC836 AGA GAG AGA GAG AGA GYA 150–499 33 33 100.0 UBC840 GAG AGA GAG AGA GAG AYT 150–492 32 32 100.0 UBC841 GAG AGA GAG AGA GAG AYC 152–499 34 34 100.0 UBC846 CAC ACA CAC ACA CAC ART 161–500 29 29 100.0 UBC848 CAC ACA CAC ACA CAC ARG 151–500 36 36 100.0 UBC890 VHV GTG TGT GTG TGT GT 150–496 29 29 100.0

Total 379 379 100.0 Mean 31.58 31.58 100.0 tent per monoploid (with chromosome number x) leading 1979) with the NJ method, grouped 28 accessions into to a clearer characterization of genome sizes regardless four clusters, I, II, III and IV (Fig. 2). Cluster I includes of ploidy levels (Greilhuber et al. 2005, Akiyama et al. seven Lotus species comprising nine accessions with a 2008). The diploid L. unifoliolatus had an estimated Nei’s gene diversity (H) score of 0.3117 (Table 3). The 0.28±0.01 pg C-1, which was the smallest genome size range of genome sizes in Cluster I had a more narrow detected in this study. In contrast, the tetraploid L. distribution, from 397.6 to 472.1 Mbp, than the other australis showed a genome size of 1.28±0.03 pg C-1, clusters. Cluster II includes 10 diploid accessions of nine representing an approximate five-fold difference. The species with a large variety of origins. Cluster III with C value of the tetraploid species was larger than that of H=0.2482 and Cluster IV with H=0.2226 contain five the diploid species. When expressed as a Cx value, the and four accessions of different species, respectively. mean nuclear DNA content of Lotus species ranged from All members of Clusters III and IV originated from 0.28 pg Cx-1 in L. unifoliolatus to 0.71 pg Cx-1 in Lotus the North American continent. All of the accessions in wrightii. Leitch and Bennett (2004) reported that the Cluster IV are diploid with a same basic chromosome mean DNA content per monoploid tended to decrease number of x=7. with increasing ploidy level in many species. However, There are two types of Lotus that are distinct in their this observation did not hold in our study. distributions: the Old and New World species. The Old World species have a major center of diversity in the Microsatellite analysis Mediterranean and adjacent regions. In contrast, the The 12 selected primers of the UBC primer set were New World species have a center of diversity in Cali- used to generate bright and discernible amplified bands fornia and adjacent regions of the U.S.A. and Mexico and to assess phylogenetic relationships among 28 ac- (Degtjareva et al. 2003). According to a cladistic analy- cessions of Lotus. Eventually, a total of 379 reproduc- sis (Allan and Porter 2000, Allan et al. 2003, Arambarri ible and scorable amplification bands were generated 2000, Degtjereva et al. 2006, 2008), six accessions (L. that were all polymorphic. The number of amplifica- japonicus MG-20 and B-129, Lotus tenuis, L. burttii, tion bands generated by each primer varied from 25 L. filicaulis and L. corniculatus) in Cluster I are mem- (UBC825) to 36 (UBC848) with an average of 31 bands bers of the Lotus corniculatus group. L. corniculatus per primer. The size of the amplified bands ranged from ssp. frondosus, a diploid originating from China with 150 to 500 base pairs (Table 2). These results indicate a basic chromosome number of x=6, are also classi- that ISSR analysis with the selected primers is a suitable fied into Cluster I. This is probably the first time L. and powerful tool for species identification and for es- corniculatus ssp. frondosus has been used in a phylo- timation of the genetic diversity and close phylogenetic genetic study based on ISSR. This accession was clas- relationships among 28 accessions of Lotus that have a sified with L. corniculatus within this cluster; thus, we high level of polymorphism. assume that this accession has a strong relationship with other L. corniculatus species. Cluster II comprises the Genetic diversity and cluster analysis Tetragonobolus Scopoli (L. maritimus, Lotus conjugatus Genetic similarities obtained from the binary data and L. conjugatus ssp. requienii), the Lotus peduncu- were used to construct a phylogenetic tree. The tree, latus group (Lotus uliginosus and Lotus pedunculatus), based on a similarity coefficients index (Nei and Li and L. wrightii, Lotus subpinnatus, L. peregrinus var. 100 H. Tanaka et al. Cytologia 81(1)

Fig. 2. Phylogenetic tree based on neighbor joining method showing the relationship among the 28 Lotus accessions using the ISSR marker. (N refers to the New World species and O refers to the Old World species of Lotus.)

Table 3. Genetic diversity measures for four clusters of the 28 Lotus accessions.

Observed number of Effective number of Nei’s gene diversity Shannon’s information Cluster Population size alleles (Na) alleles (Ne) (He) index (I)

I 9 1.8522±0.36 1.5320±0.34 0.3117±0.17 0.4648±0.23 II 10 1.8760±0.33 1.5150±0.33 0.3062±0.16 0.4610±0.22 II 5 1.6332±0.48 1.4269±0.37 0.2482±0.20 0.3661±0.29 IV 4 1.5435±0.50 1.3863±0.38 0.2226±0.21 0.3253±0.30 carmeli, L. strigosus and Lotus angustissimus, all of ploidy level (diploid) and same basic chromosome num- which are diploid. Additionally, there is a subcluster that ber of x=7 with three other accessions in this cluster. contains L. angustissimus, L. conjugatus and L. con- Therefore, L. weilleri might have close genetic relation- jugatus ssp. requienii, all annual species that share the ships with the other accessions of Cluster IV. same origin, Eastern Europe. Therefore, we concluded L. subbiflorus, L. halophilus, and L. australis clus- that they have closely genetic relationships with each tered into different groups as reported by Degtjareva other. Cluster III includes five accessions, three of which et al. (2008). This discrepancy may be due to species are members of subgenus Acmispon, section Microlotus sampling and different analytical techniques, as was also (Lotus denticulatus, L. salsuginosus and Lotus humistra- reported by Degtjareva et al. (2008). Additional studies, tus); all three are New World species and share the same using both chloroplast and nuclear genome sequences ploidy level (diploid) with a basic chromosome number to reveal single nucleotide polymorphisms, are needed of x=7. The other two species are L. scoparius var. sco- to further evaluate the phylogenetic relationship among parius and L. australis, L. scoparius var. scoparius is Lotus species. also one of the New World accessions and was grouped into subgenus Syrmatium. Three accessions of Cluster Conclusion IV include two subgenera Acmispon (L. heermannii) and Syrmatium (L. unifoliolatus, L. unifoliolatus var. uni- In this study, we determined the chromosome number foliolatus), which are also New World species. Another and nuclear DNA content of the Lotus species and esti- Cluster IV member is Lotus weilleri that shares the same mated their molecular variability using ISSR. Informa- 2016 Analysis of Genomic Variation in Lotus spp. 101 tion on the nuclear genome size of 19 Lotus species is corniculatus group as determined by karyotype and cytophoto- available so far. Here, we have shown the ploidy level metric analyses. Can. J. Genet. Cytol. 15: 101–105. Degtjareva, G. V., Kramina, T. E., Sokoloff, D. D., Samigullin, T. H., and chromosome number of 11 species for the first time, Sandral, G. and Valiejo-Roman, C. M. 2008. New data on nrITS and our nuclear DNA content measurements represent phylogeny of Lotus (Leguminosae, Loteae). Wulfenia 15: 35–49. the first report for 20 out of 28 accessions in the genus Degtjareva, G. V., Kramina, T. E., Sokoloff, D. D., Samigullin, T. H., Lotus. The data indicate that genome size is species spe- Valiejo-Roman, C. M. and Antonov, A. S. 2006. Phylogeny of cific in Lotus and that there is no relationship between the genus Lotus (Leguminosae, Loteae): Evidence from nrITS sequences and morphology. Can. J. Bot. 84: 813–830. genome size and chromosome number or ploidy level. Degtjareva, G. V., Valiejo-Roman, C. M., Kramina, T. E., Mironov, E. Consequently, the results in this study suggested that M., Samigullin, T. H. and Sokoloff, D. D. 2003. Taxonomic and relative DNA contents are helpful in ascertaining the phylogenetic relationships between Old World and New World taxonomic status of the various species in Lotus. The members of the tribe Loteae (Leguminosae): New insights from biosystematic relationships within this genus can be fur- molecular and morphological data, with special emphasis on Or- nithopus. Wulfenia 10: 15–50. ther clarified in conjunction with other approaches. Diaz, P., Borsani, O. and Monza, J. 2005. Lotus-related species and The results of this study provide valuable information their agronomic importance. In: Márquez, A. J., Stougaard, J., about the genus and will support future genetic research Udvardi, M., Parniske, M., Spaink, H., Saalbach, G., Webb, J., towards improving the morphological, physiological and Chiurazzi, M. and Márquez, A. J. (eds.). Lotus japonicus Hand- agronomic traits of Lotus species. book. Springer, Berlin. pp. 25–37. Doležel, J. 1997. Application of flow cytometry for the study of plant genomes. J. Appl. Genet. 38: 285–302. Acknowledgements Doležel, J., Bartoš, J., Voglmayr, H. and Greilhuber, J. 2003. Nuclear DNA content and genome size of trout and human. Cytometry A We are grateful to GRIN and NBRP for providing 51: 127–128, author reply 129. us with the Lotus seeds. Doležel, J., Greilhuber, J. and Suda, J. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2: 2233–2244. References Ferreira, J. and Pedrosa-Harand, A. 2014. Lotus Cytogenetics. In: Tabata, S. and Stougaard, J. (eds.). The Lotus japonicus Genome. Akiyama, Y., Yamada-Akiyama, H., Yamanouchi, H., Takahara, M., Springer-Verlag, Berlin, Heidelberg. pp. 9–20. Ebina, M., Takamizo, T., Sugita, S. and Nakagawa, H. 2008. Fukui, K. 2006. Enzymatic maceration/air-drying method. In: Fukui, Estimation of genome size and physical mapping of ribosomal K., Mukai, Y. and Taniguchi, K. (eds.). Chromosome. Methods DNA in diploid and tetraploid guineagrass (Panicum maximum for the Plant Chromosome Research. Yokendo, Tokyo. pp. 6–7. Jacq.). Grassl. Sci. 54: 89–97. Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M., Akiyama, Y., Yamamoto, Y., Ohmido, N., Ohshima, M. and Fukui, Sharma, D. P. and Firoozabady, E. 1983. Rapid flow cytometric K. 2001. Estimation of the nuclear DNA content of strawberries analysis of the cell cycle in intact plant tissues. Science 220: (Fragaria spp.) compared with Arabidopsis thaliana by using 1049–1051. dual-step flow cytometry. Cytologia 66: 431–436. Grant, W. F. 1965. A chromosome atlas and interspecific hybridiza- Allan, G. J. and Porter, J. M. 2000. Tribal delimination and phylo- tion index for the genus Lotus (Leguminosae). Can. J. Genet. genetic relationship of Loteae and Coronilleae (Faboideae: Cytol. 7: 457–471. Fabaceae) with special reference to Lotus: Evidence from nucle- Grant, W. F. 1986. The cytogenetics of Lotus (Leguminosae). J. Nat. ar ribosomal ITS sequences. Am. J. Bot. 87: 1871–1881. Hist. 20: 1461–1465. Allan, G. J., Zimmer, E. A., Wagner, W. L. and Sokoloff, D. D. 2003. Grant, W. F. and Sidhu, B. S. 1967. Basic chromosome number, cyto- Molecular phylogenetic analyses of tribe Loteae (Leguminosae): genetic glucoside variation, and geographic distribution of Lotus Implications for classification and biogeography. In: Klitgaard, species. Can. J. Bot. 45: 639–647. B. B. and Bruneau, A. (eds.). Advances in Legume Systematics, Greilhuber, J., Doležel, J., Lysak, M. A. and Bennett, M. D. 2005. The Part 10, Higher Level Systematics. Royal Botanic Garden, Kew. origin, evolution and proposed stabilization of the terms ‘genome pp. 371–399. size’ and ‘C-value’ to describe nuclear DNA contents. Ann. Bot. Angulo, M. D. and Real, M. C. 1977. A new basic chromosome num- 95: 255–260. ber in the genus Lotus. Can. J. Bot. 55: 1848–1850. Grime, J. P. and Mowforth, M. A. 1982. Variation in genome size: An Arambarri, A. M. 2000. A cladistic analysis of the New World species ecological interpretation. Nature 299: 151–153. of Lotus L. (Fabaceae, Loteae). Cladistics 16: 283–297. Harney, P. M. and Grant, W. F. 1964. The cytogenetics of Lotus (Le- Bennett, M. D. and Leitch, I. J. 2005. Nuclear DNA amounts in angio- gunimosae). VII. Segregation and recombination of chemical sperms: Progress, problems and prospects. Ann. Bot. 95: 45–90. constituents in interspecific hybrids between species closely re- Bennett, M. D., Leitch, I. J., Price, H. J. and Johnston, J. S. 2003. lated to L. corniculatus L. Can. J. Genet. Cytol. 6: 140–146. Comparisons with Caenorhabditis (~100 Mb) and Drosophila Harney, P. M. and Grant, W. F. 1965. A polygonal presentation of (~157 Mb) using flow cytometry show genome size in arabidopsis chromatographic investigation on the phenolic content of certain to be ~157 Mb and thus ~25% larger than the Arabidopsis Ge- species of Lotus. Can. J. Genet. Cytol. 7: 40–51. nome Initiative estimate of ~125 Mb. Ann. Bot. 91: 547–557. Ito, M., Miyamoto, J., Mori, Y., Fujimoto, S., Uchiumi, T., Abe, M., Bennett, M. D. and Smith, J. B. 1976. Nuclear DNA amounts in Suzuki, A., Tabata, S. and Fukui, K. 2000. Genome and chromo- angiosperms. Philos. Trans. R. Soc. Lond. B Biol. Sci. 274: some dimensions of Lotus japonicus. J. Plant Res. 113: 435–442. 227–274. Kawakami, J. 1930. Chromosome numbers in Leguminosae. Bot. Borsos, O. S. 1973. Cytophotometric studies on the DNA content of Mag. Tokyo 44: 319–328. diploid Lotus species. Acta Bot. Hung. 18: 49–58. Kawasaki, S. and Murakami, Y. 2000. Genome analysis of Lotus Cheng, R. L. and Grant, W. F. 1973. Species relationships in the Lotus japonicus. J. Plant Res. 113: 497–506. 102 H. Tanaka et al. Cytologia 81(1)

Larsen, K. 1954. Cytotaxonomical studies in Lotus. I. Lotus Sato, S., Nakamura, Y., Kaneko, T., Asamizu, E., Kato, T., Nakao, M., corniculatus L. sens. lat. Bot. Tidsskr. 51: 205–211. Sasamoto, S., Watanabe, A., Ono, A., Kawashima, K., Fujishiro, Larsen, K. 1955. Cytotaxonomical studies in Lotus. II. Somatic chro- T., Katoh, M., Kohara, M., Kishida, Y., Minami, C., Nakayama, mosome and chromosome numbers. Bot. Tidsskr. 52: 8–17. S., Nakazaki, N., Shimizu, Y., Shinpo, S., Takahashi, C., Wada, Larsen, K. 1956. Cytotaxonomical studies in Lotus. III. Some new T., Yamada, M., Ohmido, N., Hayashi, M., Fukui, K., Baba, T., chromosome numbers. Bot. Tidsskr. 53: 49–56. Nakamichi, T., Mori, H. and Tabata, S. 2008. Genome structure Leitch, I. J. and Bennett, M. D. 2004. Genome downsizing in poly- of the Legume, Lotus japonicus. DNA Res. 15: 227–239. ploid plants. Biol. J. Linn. Soc. Lond. 82: 651–663. Swanson, E. B., Somers, D. A. and Tomes, D. T. 1990. III.5 Birdsfoot Nagaoka, T. and Ogihara, Y. 1997. Applicability of inter-simple se- Trefoil (Lotus corniculatus L.). In: Bajaj, Y. P. S. (ed.). Biotech- quence repeat polymorphisms in wheat for use as DNA markers nology in Agriculture and Forestry, Vol. 10 Legumes and Oil- in comparison to RFLP and RAPD markers. Theor. Appl. Genet. seed Crops I. Springer-Verlag, Berlin, Heidelberg. pp. 323–338. 94: 597–602. Sz.-Borsos, O., Somaroo, B. H. and Grant, W. F. 1972. A new diploid Nei, M. and Li, W. 1979. Mathematical model for studying genetic species of Lotus (Leguminosae) in Pakistan. Can. J. Bot. 50: variation in terms of restriction endonucleases. Proc. Natl. Acad. 1865–1870. Sci. U.S.A. 76: 5269–5273. Tschechow, W. and Kartaschowa, N. 1932. Karyologisch-systematische Ohmido, N., Kijima, K., Akiyama, Y., de Jong, J. H. and Fukui, K. Untersuchung der tribus Loteae und Phaseoleae Unterfom. 2000. Quantiﬁcation of total genomic DNA and selected re- Papilionatae. Cytologia 3: 221–249. petitive sequences reveals concurrent changes in different DNA Tsumura, Y., Ohba, K. and Strauss, S. H. 1996. Diversity and inheri- families in indica and japonica rice. Mol. Genet. Genomics 263: tance of inter-simple sequence repeat polymorphisms in Doug- 388–394. las-ﬁr (Pseudotsuga menziesii) and sugi (Cryptomeria japonica). Qian, W., Ge, S. and Hong, D. Y. 2001. Genetic variation within and Theor. Appl. Genet. 92: 40–45. among populations of a wild rice Oryza granulata from China Vriet, C., Smith, A. M. and Wang, T. L. 2014. Root starch reserves are detected by RAPD and ISSR markers. Theor. Appl. Genet. 102: necessary for vigorous re-growth following cutting back in Lotus 440–449. japonicus. PLoS ONE 9: e87333. R Core Team (2014). R: A language and environment for statistical Yeh, F. C., Yang, R. C., Boyle, T. B. J., Ye, Z. H. and Mao, J. X. 1997. computing. R foundation for statistical computing, Vienna. POPGENE: The user-friendly shareware for population genetic Saitou, N. and Nei, M. 1987. The neighbor-joining method: A new analysis. Molecular Biology and Biotechnology Centre, Univer- method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: sity of Alberta, Alberta, Canada. 406–425.