Kudzu (Pueraria lobata Ohwi) karyotyping using FISH and Chromosome Image Analysis System IV

Chromosome Science 16: 17-21, 2013 Ohmido et al. 17 Regular Article Kudzu (Pueraria lobata Ohwi) karyotyping using FISH and Chromosome Image Analysis System IV Nobuko Ohmido, Akane Shimoura, Seiji Kato, Sachiko Isobe, and Satoshi Tabata Received: May 26, 2013 / Accepted: July 4, 2013 2013 by the Society of Chromosome Research Abstract In this study, kudzu (Pueraria lobata Ohwi), a new biomass plant widely grown in Japan, was investigated by chromosome karyotyping. Four strains of Japanese kudzu were examined, and all were found to contain 22 chromosomes. The 5S and 45S rdna genes, which contain fundamental repeat sequences used in karyotyping, were determined by fluorescence in situ hybridization (FISH). Our results found one 5S rdna locus and three 45S rdna loci on the chromosomes of all four strains, which suggest that Japanese kudzu is not greatly differentiated at the cytological level. A further 11 chromosomes were identified, and the rdna loci were mapped by chromosome image analysis system IV (CHIAS IV), following which kudzu chromosome karyotyping was performed. This is the first report of cytogenetic analysis of P. lobata, a Japanese kudzu. Keywords: Pueraria lobata, kudzu, FISH, CHIAS IV, karyotyping. Nobuko Ohmido (*) and Akane Shimoura Graduate School of Human Development and Environment, Kobe University, Kobe, 657-8501, Japan Tel: +81-78-803-7763 E-mail: ohmido@kobe-u.ac.jp Seiji Kato Yamanashi Prefectural Agritechnology Center, Kitamori, Yamanashi, 408-0003, Japan Sachiko Isobe and Satoshi Tabata Kazusa DNA Research Institute, Kisarazu, 292-0818, Japan Introduction Kudzu (Pueraria lobata Ohwi) is a perennial climbing plant that thrives in high temperatures and belongs to the Fabaceae family. It has a twining vine and broad trifoliate leaves (Tsugawa, 1986A). Kudzu is regarded as a harmful and invasive weed due to its vigorous flourishing habit (Fig. 1). It is distributed throughout Asia, including Japan, Korea, China, and other regions in Southeast Asia, as well as in North and South America. Kudzu can adapt to grow in the wide range of climates found in Japan as well as in other regions. It is reported that the line between Hokkaido Erimo Cape (42 31 N) and Rumoi-shi, Hokkaido (44 N) represents the northern limit of its spread in Japan (Tsugawa, 1986B). Kudzu is very productive and highly adaptable. It grows quickly, and as such, was introduced to the US as a control measure against soil erosion and a garden plant, and in 1876 it was introduced by China as a forage crop. However, kudzu grows wild in wasteland after cultivation and destroys the ecosystem, and it was placed on the Federal Noxious Weeds List in 1997 (Forseth and Innis 2004). More recently, kudzu has received renewed attention as a potential biomass plant. The root contains abundant starch that could be used as a food source, and there is evidence that it was used in East Asia as herbal medicine in the past. Recently, tissue culture for production of certified plant material to obtain herbal medicines like as isoflavonoids, are established (Thiem 2003). The fibers from the vine can also be used in the production of cloth (Tsugawa et al., 1986). As production of any biomass plant is influenced by both climate and soil conditions, genetic knowledge of kudzu is necessary for future breeding programs. Conventional kudzu research has been limited to ecological characteristics and environmental impact. The genes and chromosomes of kudzu have not been investigated extensively, and therefore the genetic differentiation of kudzu in Japan and those elsewhere is not clear. A previous study of the somatic chromosomes of P. lobata in US strains reported that they contained 22 chromosomes (Kumar and Hymowitz 1989). However no chromosome karyotyping was conducted. In this study, we initially confirmed the number of kudzu chromosomes of four strains growing in various regions of Japan; Kobe-Hyogo, Kawachinagano-Osaka, Kisarazu-Chiba and Senjojiki-Aomori. We then examined the loci of the 5S and 45S ribosomal RNA (rdna) genes,

18 Kudzu (Pueraria lobata Ohwi) karyotyping using FISH and Chromosome Image Analysis System IV Figure 1. External growth and morphology of Pueraria lobata, Japanese kudzu plants of the Kawachinagano strain. (A) Elongated leaf morphology. (B) Roots emerging from the node (C). which contain a basic repeat element, to compare kudzu strains by fluorescence in situ hybridization (FISH). We examined the cytological differences at the ploidy level of samples from several regions of Japan by counting the number of chromosomes using DAPI staining, and fluorescence signals from the rdna genes. We karyotyped chromosomes of the Kobe strain using the chromosome image analysis system IV (CHIAS IV) to investigate kudzu chromosomes at prometaphase. This represents the first study of the morphological structure of the kudzu chromosome. Materials and Methods Plant materials and root collection Kudzu (Pueraria lobata Ohwi) was used throughout this study. Plantlets were collected from Kobe city-hyogo prefecture (Kobe strain), Kisarazu-Chiba (Kazusa strain), Senjojiki-Aomori (Senjojiki strain), and Kawachinagano- Osaka (Kawachinagano strain). Plant cuttings were taken as 2-year old shoots with trifoliate leaves and a single node (Fig. 1C). Indolebutyric acid was used to encourage root formation at the node. Roots were grown from a plant node immersed in water for 7-10 days, which was aerated by an air bubbling device. Chromosome preparation Mitotic chromosome samples from root tips of the four strains were prepared for microscopic observation using an enzymatic maceration and air-drying method (Ohmido et al. 2001) with some modifications. Briefly, kudzu root tips were pretreated in 2 mm 8-hydroxyquinoline at room temperature for 4 h, fixed in ethanol and acetic acid (3:1, v/v), and stored at -20 C for up to several weeks until chromosome preparation. For chromosome preparation, the stored root tips were washed thoroughly in water, macerated in an enzyme solution containing 2.5% Pectolyase Y-23 (Seishin Pharmaceutical Co., Ltd, Chiba, Japan) and 1% Cellulase Onozuka RS (Yakult Honsha Co., Ltd, Tokyo, Japan), and incubated at 37 C for 20 min. After washing, the tips were crushed in 10 µl of ethanol: acetic acid (3:1) using fine forceps, and then air-dried on glass slides. DNA probes The 45S rdna gene, which contains a large number of tandem repeats originating from rice, was used as a probe for FISH analysis. The 5S rdna gene sequences were amplified by PCR using the following primers; 5 -cggtgcattagtgctggtat-3 and 5 -gatcccatcagaactcccca-3, which were based on the 5S rdna sequence (291 bp units) from red clover belonging to the Fabaceae (Kataoka et al. 2012), and kudzu genomic DNA was used as a template. The 5S rdna was PCR-labeled with digoxigenin-11- dutp (Roche), and the 45S rdna sequence was labeled using a Nick Translation Kit (Roche) with Cy3-dUTP (GE Healthcare Life Sciences) according to the method described by Ohmido et al. (2010). Fluorescence in situ hybridization (FISH) FISH using the two rdna genes was performed using chromosome spreads prepared according to the method of Ohmido et al. (2010). For immunocytochemical fluorescence detection, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated antidigoxigenin antibody (Roche) in 4 SSC at 37 C for 60 min in a damp, dark box. After washing with 2 SSC, the slides were further incubated with FITC-conjugated antisheep antibody (Vector Laboratories) in 4 SSC at 37 C for 60 min in a dark box to amplify the signal. After washing and air-drying, the chromosome samples were stained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector Laboratories). The slides were observed under a fluorescence microscope (BX50; Olympus) equipped with a high-sensitivity digital camera (SPOT-RT3, Seki Technotron Co.). The FISH images were captured using blue, green, and ultraviolet light excitation and emission filter sets. Digitally captured images were adjusted using IP Lab Spectrum, version 4.0.8 (Scanalytics Inc.) and Photoshop version 3 (Adobe Systems Inc.). Quantitative karyotype analysis using CHIAS IV After imaging, CHIAS IV (http://www2.kobe-u. ac.jp/~ohmido/index03.htm) was used to analyze the kudzu chromosomes as described by Kato et al. (2009) and Kataoka et al. (2012). Quantitative chromosome karyotypes were constructed based on the digitized intensity of the DAPI signals. The fluorescence signal images and original chromosome images for construction of the idiogram

Ohmido et al. 19 were captured as red-green-blue (RGB) images with 8-bit grey levels. The numerical density profiles representing the condensation patterns (CPs) along the mid-rib of each chromatid were measured using CHIAS IV. The uneven CPs appearing on chromosomes at the mitotic prometaphase stage were analyzed and used to develop a quantitative chromosome map and idiogram. Results Chromosome numbers in kudzu strains in Japan We prepared kudzu chromosome spreads and used FISH to investigate the two ribosomal RNA genes (Fig. 2). Four kudzu strains; Kobe, Kawachinagano, Kazusa, and Senjojiki, were used. These strains consistently contained 22 chromosomes as determined by DAPI staining, enabling us to conclude that Japanese kudzu contain 22 somatic chromosomes (Fig. 2A, 2C, 2E, 2G). Previously, somatic chromosome numbers of 20, 22, and 24 had been reported for members of this genus (Goldblatt, 1981). Furthermore, another two varieties of P. lobata originating from the US and seven varieties of P. phaseoloides originating from Asia and South Africa also contained 22 chromosomes (Kumar and Hymowitz, 1989). As this result was consistent, Japanese kudzu might be a diploid plant with 2n=22. Genetic variability of rdna loci among four Japanese kudzu strains We used double-color FISH to map the loci of the 5S and 45S rdna genes of four strains to confirm chromosomal arrangement. Previous studies of rice demonstrated that the 45S rdna varies due to genetic variation among species (Fukui et al. 1994, Shishido et al. 2000). It was thus appropriate to use two rdna loci to estimate genetic differences among the four strains of Japanese kudzu. Microscopic observation of the chromosomes after FISH revealed 5S rdna gene locus as two clear FITC fluorescence signals. 5S rdna (green) was detected two sites on 22 chromosomes of the long arm in the four strains (Fig. 2B, 2D, 2F, 2H). This indicated that one 5S rdna locus was detected in four strains of kudzu, suggesting diploidy. We also observed two major and four minor Cy3 fluorescence signals from 45S rdna (red) of the short arm of three pairs of chromosomes (Fig. 2B, 2D, 2F, 2H). Therefore, one pair of major loci and two pairs of minor 45S rdna loci exist in Japanese kudzu strains. The major 45S rdna locus was seen in the satellite regions, mostly on the short arms of chromosomes, at the nucleolus organizer region (NOR). All four strains contained an identical number of 45S and 5S rdna loci. We observed no other variability of the rdna loci among the four Japanese strains. Karyotyping of kudzu chromosomes The kudzu chromosomes were analyzed using CHIAS IV, and a chromosome ideogram was created (Fig. 3). The cytological features of the kudzu chromosomes were similar Figure 2. FISH images of 45S and 5S rdna on mitotic chromosomes (2n=22) of four Japanese kudzu strains. A, C, E and G show chromosome images, and B, D, F and H show FISH images mapping the 45S rdna (red) and 5S rdna (green). (A, B) Kobe strain, (C, D) Kawachinagano strain (E, F) Kazusa strain, (G, H) Senjojiki strain. 5S rdna sites are representing *. 45S rdna major and minor sites are representing arrows and arrowheads, respectively. Bar = 10 µm. Figure 3. Kudzu karyotyping of the Kobe strain with mapping of the 45S and 5S rdna genes by CHIAS IV. (A) FISH images of two rdna loci. (B) Pseudo-colored chromosomes demonstrating specific condensation patterns of individual chromosomes (C) Colorgrams (D) Kudzu ideogram. Red and green circles demonstrate 45S rdna and 5S rdna loci, respectively.

20 Kudzu (Pueraria lobata Ohwi) karyotyping using FISH and Chromosome Image Analysis System IV in all four strains. There was essentially no variability in size, shape and number of chromosomes among the strains. We used five well-spread prometaphase chromosomes of the Kobe strain to which FISH had been applied, to ascertain characteristics; i.e., identification of individual chromosomes, centromere region, and the positions of the 45S and 5S rdna sites (Fig. 3). Prometaphase chromosomes counterstained with DAPI showed prominent, uneven condensation patterns (CP) along the chromosome. The CPs of the chromosome were analyzed following the CHIAS IV procedures (Kato et al. 2009, Kataoka et al. 2012). Firstly, chromosome images were stained with DAPI, and fluorescence images were prepared for CHIAS IV (Fig. 3A). The chromosome-specific patterns of the CPs unique to each chromosome were clearly seen as pseudo-coloration (Fig. 3B). The density profiles of DAPI, Cy3, and FITC fluorescence along the chromatids were analyzed digitally. In total, 110 chromosomes of five spreads were analyzed by CHIAS IV. The map was based on mitotic prometaphase chromosomes, which ranged in length from 3.8 to 5.8 µm. Density profiles of the chromosome (DAPI, blue), and the characteristic chromosomal positions of the 45S rdna (Cy3, red) and 5S rdna (FITC, green) were assessed (Fig. 3C). Finally, a quantitative ideogram was constructed based on the chromosome condensation pattern and rdna mapping (Fig. 3D). Numerical data for arm length, arm ratio, total length, and rdna signal positions are shown in Table 1. Of the 11 pairs of chromosomes, four were large (chromosomes 1-4), four were ribosomal genes localized to the chromosome (chromosomes 5-8), and three were small (chromosomes 9-11) (Fig. 3D). Characteristic highly condensed heterochromatic regions were observed on the long arms of chromosomes 1-4, 7 and 9-11. The 45S and 5S rdna mapping by FISH was used as a landmark for kudzu chromosome karyotyping. 45S rdna loci were localized on the short arms of the medium-sized chromosomes (5, 6, 8); 5S rdna was localized close to the centromere region on the long arm of chromosome 7 (Fig. 3D). Table 1. Characteristics of the chromosomes of the Kobe strain of kudzu. Chromosome No. Relative length a) Arm ratio b) r DNA c) 1 10.59 1.51 2 10.02 1.21 3 9.98 1.26 4 9.97 1.39 5 9.91 1.51 45S 6 9.68 1.19 45S 7 9.04 1.84 5S 8 8.90 1.35 45S 9 7.58 1.76 10 7.32 1.22 11 7.01 1.41 a) Relative chromosome length (%). b) Short arm length/long arm length. c) Loci of 5S rdna or 45S rdna genes. Discussion Kudzu is a common plant in wide areas of wasteland and on hill slopes in Japan, Korea, China and other South Asian countries. Kudzu was introduced to the US to reduce soil erosion in the 19th century, and was placed on the Federal Noxious Weeds List in 1997 (Forseth and Innis 2004). The ecological impact of kudzu, as well as its taxonomy, cultivation, physiology, spread and use have been investigated (Tsugawa 1986A, 1986B). However, genetic and chromosomal data are not available. It has been reported that kudzu, Pueraira lobata and P. phaseoloides in the US, South Africa, South America and Southeast Asia contain 22 chromosomes (Kumar and Hymowitz, 1989). However, no chromosomal characterization information was provided. In this study, karyotyping of the chromosome was demonstrated using the somatic prometaphase stage, and showed a characteristic chromosomal CP. Moreover, we determined the locations of the 5S rdna and 45S rdna genes by FISH. Our results suggest that at the chromosomal level, there was no variability among the four strains studied, and all are diploid with n=11. The ideogram we report here is the first chromosome karyotype of the genus Pueraria. The fluorescence signals of the 5S and 45S rdna genes were detected at four loci on kudzu chromosomes. The intensity of the 45S rdna fluorescence signal demonstrated one major locus and two minor loci in all strains. 5S rdna loci were detected on interstitial regions of the long arms in four strains. 45S rdna were detected on the short arms of three chromosome pairs. This suggests no cytogenetic polymorphisms in all Japanese kudzu examined. In many of the observed chromosomes, the major fluorescent 45S rdna gene signal was located in NOR regions. This suggests that the 45S rdna gene is highly active in kudzu. The ideogram of the Kobe strain was based on chromosomes in the prometaphase stage. This enabled assessment of quantitative chromosome features, such as, relative length, arm ratio, and the loci of the 5S and 45S rdna genes (Fig. 3, Table 1). Relative lengths were 7.01-10.6%, and chromosomes 9-11 were relatively small. The arm ratio (short arm length/long arm length) ranged between 1.19-1.84. According to Levan et al. (1964) chromosome classification, which describes an arm ratio of 1.0-1.7 as a meta centromeric chromosome type, and an arm ratio of 1.7-3.0 as a submetacentromeric chromosome type, kudzu chromosomes 7 and 9 were sub-meta centromeric chromosome types, while all others were meta centromeric chromosome types (Fig. 3D). The nuclear DNA contents of kudzu are approximately 2.01 pg/2c, and 990 Mb/1C, as determined by flow cytometry (Sliwinska and Thiem, 2007). The kudzu genome is larger than those of other Fabaceae plants; e.g., Lotus japonicus (2n=12; 466 Mbp; Ohmido et al. 2010), and Trifolium pratense (2n=14; 440 Mb; Kataoka et al. 2012). This may be due to large amounts of heterochromatin on the internal regions of the long arms of the chromosomes, which stained strongly with DAPI (Fig. 2, 3A). We estimated that the average size of one chromosome is 88 Mb. This chromosome size is larger than those of other legume plants; e.g., soybean (2n=40, 1150 Mb; Ohmido et al. 2007, Chung et al. 1998). Statistical analysis of arm lengths is ranged from 2.8 to 4.8 µm in red clover. The length of kudzu prometaphase chromosome is ranged from 3.8 to 5.8 µm. The total chromosome length of Kudzu (54.8 µm) to red clover (26.4µm) is approximately two fold. The chromosome length is well representing the difference genome size between kudzu and red clover. Additionally, 8 of 11 Kudzu

Ohmido et al. 21 chromosomes have condensed heterochromatic regions on the long arm. The condensed regions might be contributing the much larger genome size. FISH mapping and chromosome karyotyping using CHIAS IV facilitated construction of a quantitative ideogram, which constitutes a comprehensive Fabaceae chromosome map of L. japonicus and T. pratense (Ohmido et al. 2010, Kataoka et al. 2012). T. pretense, among the varieties of allogamous red clover, contains rdna gene polymorphisms. In breeding varieties, chromosomal positions of the 45S rdna locus contained polymorphisms, whereas no such polymorphisms were detected in the rdna loci of the Japanese kudzu strains. These results support the hypothesis that overall, Japan kudzu are cytologically uniform. We found no genetic differences, neither in chromosome number nor in the loci of the 5S and 45S rdna genes, among the Japanese kudzu strains. However, Pappert et al. (2000) reported high genetic and clonal diversity of kudzu using allozyme analysis in the southeastern US. They concluded that US populations of P. lobata to contain many genotypes with relatively high levels of genetic differentiation among populations and the diversity resulted from multiple introductions to the US over an extended period of time. Thus investigation of the genetic diversity of kudzu worldwide might be important. Starch from kudzu is used as food materials and traditional pharmaceutical products for a long time in the oriental countries. Additionally, kudzu has potentially new usages such as the production of medicinal properties, fiber and ethanol productions. Recently Kudzu is to be used as a new biomass crop, further genetic research is essential. Then the genetic information referring these useful phenotypic genes would be researched and analyzed using the DNA maker mapping in the near future. This chromosome map of kudzu would become genetically fundamental data for physical mapping. On the other aspect, kudzu is known as a hazard plant to be exterminated and is placed on the list of the Federal Noxious Weeds in US. It might be a basic data to obtain an effective extermination method. As for these reasons, the basic chromosome karyotype and mapping of ribosomal DNA gene loci we report here is a vital resource for future kudzu genetic research. Euphytica 40: 221-226 Levan A, Fredga K, Avery A. Sandberg A (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220 Ohmido N, Fukui K, Kinoshita T (2010) Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH). Proceedings of the Japan Academy Series B 86:103-116 Ohmido N, Ishimaru A, Kato S, Sato S, Tabata S, Fukui K (2010) Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes. 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