Chromosome Research 12: 93–102, 2004. # 2004 Kluwer Academic Publishers. Printed in the Netherlands
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Flow karyotypes and chromosomal DNA contents of genus Triticum species and rye (Secale cereale) Jai-Heon Lee1*, Youzhi Ma2, Toshiyuki Wako3, Lian Cheng Li2, Kee-Young Kim1, Seong-Whan Park1, Susumu Uchiyama4 & Kiichi Fukui4 1 Department of Plant Biotechnology, Dong-A University, Busan 604-714, South Korea; E-mail:
[email protected]; 2Institutes of Crop Breeding and Cultivation, China Academy of Agriculture Sciences, Beijing, China; 3Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba 305-8602, Japan; 4Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Osaka, Japan *Correspondence
Key words: flow cytometry, genome size, image analysis, Secale, Triticum, wheat Abbreviations: CHIAS: chromosome image analyzing system, PI: propidium iodide
Abstract The flow cytometry and chromosome imaging method were jointly used for analyzing genome content and chromosomal DNA content of hexaploid wheat (AABBDD), hexaploid triticale (AABBRR), tetraploid wheat (AABB), and AA, BB, DD genome donors and RR genome rye. Their genome sizes were 34.4 pg, 40.9 pg, 26.2 pg, 12.1 pg, 13.7 pg, 10.5 pg, and 16.9 pg, respectively. The 2C nuclear DNA content of BB genome donor with 13.7 pg was the highest value among the other genome donors, AA or DD. The genome content of tetraploid wheat, unlike hexaploid wheat or hexaploid triticale, was larger than the sum of the genomes of AA and BB genome donors. The DNA content of each chromosome ranged from 1.22 pg in DD genome donor to 2.61 pg in rye. Each chromosome peak was divided into three to four groups. Only one chromosome was included in the highest chromosomal DNA peak in hexaploid wheat, tetraploid wheat, DD genome donor and rye but two chromosomes in AA, BB genome donors, and hexaploid triticale. Correlation between 2C nuclear DNA content and chromosome density volume was the highest value compared with the other chromosomal parameters of chromosome area, or chromosome length.
Introduction Bread wheat (Triticum aestivum L.) is a hexaploid containing three different homeologous genomes, AA, BB, and DD. Each genome has seven pairs of chromosomes (2n ¼ 6x ¼ 42). The total genome size (2C) is 2 16 billion base pairs, 34.6 pg (Bennett & Smith 1976). Hexaploid
wheat originated from the hybridization of three different species with genomes of AA, BB, or DD approximately 8000 years ago (Feldman et al. 1995). The hybridization between T. urartu (AA) and T. searsii (BB) resulted in tetraploid wheat, T. dicoccoides (AABB). This tetraploid wheat then hybridized with T. tauschii (DD) to produce the hexaploid wheat (AABBDD) (Kimber & Sears
94 1987). The domesticated diploid wheat, T. monococcum (AA), which closely relates to the A genome donor, T. urartu, has been used as a model for the A genome of hexaploid wheat (Wicker et al. 2001). After polyploidization, rapid genomic changes occurred across the whole genome, such as non-Mendelian genome reorganization (Soltis & Soltis 1995) including elimination or modification of chromosome- and genomespecific sequences, especially low-copy non-coding sequences (Liu et al. 1998), and chromosomal rearrangements including inversions, duplications, deletions, and reciprocal or nonreciprocal translocations (Bonierbale et al. 1988; Brubaker et al. 1999). To date, many researchers have reported genomic evolution with information about specific genes or DNA sequences (Liu et al. 1998; review in Wendel, 2000; Feuillet et al. 2001; review in Rieseberg, 2001; Wicker et al. 2001), or about the chromosomal location of specific DNA sequences (Kimber & Sears 1987; Jiang & Gill 1994; Friebe & Gill 1996). From the practical perspective, the analysis of genome content and chromosome karyotyping can give additional information that helps in understanding genomic evolution in polyploid plants. Flow cytometry has been used for analysis of DNA contents of genomes and speci¢c chromosomes (Lee et al. 2002), and chromosomal karyotyping (Dolezel & Lucretti 1995; Lee et al. 1996, 1997, 1998, 1999, 2000; Lysak et al. 1999; Vrana et al. 2000). In addition, £ow cytometry can be used for the isolation of speci¢c chromosomes for the construction of chromosome-speci¢c libraries and for gene cloning from chromosomal-speci¢c regions of a complex genome (Wang et al. 1992; Macas et al. 1993; McCormick et al. 1993; Arumuganathan et al. 1994). Recently, image analysis methods have been introduced in plant chromosome research (Fukui 1986; Fukui & Iijima 1991). It has been shown that the genome size data obtained by image analysis methods are comparable to the data by £ow sorting (Uozu et al. 1997). They showed that the length parameter shows the best correlation with the genome size in rice. These image analysis methods can provide qualitative and quantitative data for chromosome research. In the present study, we set out (1) to compare genome contents of hexaploid wheat (AABBDD
J.-H. Lee et al. genome), hexaploid triticale (AABBRR genome), tetraploid wheat (AABB genome), and A, B, D genome donor and R genome rye, (2) to identify positive or negative changes in genome size during polyploidization, (3) to analyze chromosomal DNA contents and chromosome types in £ow karyotypes and to evaluate their potential for sorting of individual chromosomes. We also analyzed the correlations among DNA contents measured by £ow cytometry, and chromosome areas, chromosome density volumes and chromosome lengths by imaging methods.
Materials and methods Plant materials Eight different species, kindly provided by Dr B. S. Gill (Kansas State University, USA) and Dr P. S. Baenziger (University of NebraskaLincoln, USA), were used for our experiments; Wichita (T. aestivum, AABBDD genome, 2n ¼ 6x ¼ 42), T. dicoccoides (AABB genome, 2n ¼ 4x ¼ 28), T. monococcum (AA genome donor, 2n ¼ 14), T. searsii (BB genome donor, 2n ¼ 14), T. tauschii (DD genome donor, 2n ¼ 14), S. cereale (RR genome, 2n ¼ 14), and Newcale (AABBRR genome, 2n ¼ 6x ¼ 42) (references in Kimber and Sears, 1987).
Cell-cycle synchronization A slightly modified procedure for synchronization of cell cycle has been used (Lee et al. 1996). Briefly, seedlings (about 1-cm long primary roots) of Wichita and Newcale were incubated in Hoagland solution (Sigma) containing 1.25 mM hydroxyurea in the dark at room temperature for 18 h. The seedlings were rinsed in distilled water, and incubated in hydroxyurea-free Hoagland solution for 2 h. The seedlings were treated with 1 mM Trifluralin (a gift from DowElanco, Midland, MI, USA) for 4 h. The other plant materials, T. dicoccoides, T. monococcum, T. searsii, T. tauschii, and S. cereale rye, were treated with the same method as above except the duration of hydroxyurea treatment for 14 h, and Trifluralin treatment for 3 h. After accumulation of
Flow karyotypes and chromosomal DNA contents of genus metaphase chromosomes, seedlings were transferred into ice-cold water for overnight. The roots were cut 1 cm from the root tip, and fixed with 2% para-formaldehyde in Tris buffer (10 mM Tris, 10 mM Na2EDTA, 100 mM NaCl, 0.1% Triton X-100, pH 7.5) for 20 min (Dolezel et al. 1992). Flow-cytometric analysis Suspensions of nuclei were prepared as described earlier (Lee et al. 1997), and analyzed for DNA content [relative propidium iodide(PI) fluorescent intensity] using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Approximately 2000 G1 nuclei per line were analyzed. Analyses were repeated twice for each nuclear suspension. Chicken red blood cell nuclei (2.33 pg/2C) and barley cell nuclei (NE86954, 10.24 pg/2C, Lee et al. 1997) were used as internal standards. Metaphase chromosome suspensions were prepared (Lee et al. 1996) in chromosome isolation bu¡er (50 mM KCl, 10 mM MgSO4, 5 mM K2HPO4, 5 mM Hepes, 2 mM dithiothreitol, 0.2% Triton X-100, 25 mg/ml PI). Twenty root tips were used to prepare one chromosome suspension sample. Chromosomes were isolated from the terminal 1.5 mm of the root tip by using Polytron PT1300D homogenizer (Kinematica AG, Littau, Switzerland) at 9000 rpm for 15 sec (Dolezel et al. 1999). The chromosome suspension was ¢ltered through a 50-mm nylon mesh. Chromosomes stained with PI were analyzed using FACScan £ow cytometer. Fluorescence pulse area was measured in all cases. At least 2000 particles were analyzed to generate each £ow karyotype. To eliminate cell debris with similar £uorescence intensity with chromosomes, the gate window was set on a dot plot of FL1-H versus forward light scatter (FSC). Histograms of relative £uorescence intensity of gated particles were acquired on histograms of FL2 PI £uorescent pulse area (FL2-A, linear scale, 1024 channels), and of FL3 PI £uorescent pulse height (FL3-H, logarithmic scale, 4 decades of log channels). The data were collected, stored, and analyzed with the CellQuest software (Becton Dickinson). Numbers of expected chromosome types in each chromosome peak were calculated by the relative
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frequency of total events of each chromosome peak in linear £ow karyotypes. The DNA content of individual chromosome peaks was calculated based on relative PI £uorescence intensity of individual chromosome peaks, and G1 nuclei. Each peak revealed in the £ow karyotypes was sorted and the content of the peak was analyzed under the £uorescence microscope for identi¢cation of clumps, chromosomes, chromatids, or cell debris as described previously (Lee et al. 1996). The instrument settings were adjusted to place the peak of the largest chromosome of hexaploid wheat, Wichita, at channel 600. The same instrument settings were used for analyses of chromosomes isolated from other materials. Image analysis Chromosome images were photographed by a microscope with a 100X objective lens. The photographs were converted to grayscale images. Image analysis was done on NIH image software with some of CHIAS III macro programs (http://133.1.131.81/Eudejas/chias3/ chias3.html, Kato & Fukui, 1998). The chromosomal regions were determined from the images interactively. Remaining noise was erased manually and the images were then normalized. Chromosomal area, mean gray value of the area and chromosome length were measured. Chromosome length was measured with segmented line tool on the NIH image software. Density volume was calculated multiplying chromosomal area by mean gray value. The NIH Image software is written by Wayne Rasband at the US National Institute of Health and available from the Internet by anonymous http from zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 Port Royal Rd, Springfield, VA 22161, part number PB93504868.
Results and discussion Metaphase chromosomes of Triticum species and Secale cereale are shown in Figure 1. T. aestivum (AABBDD genome wheat) (Figure 1A) and Triticale, (AABBRR genome) (Figure 1B) have 2n ¼ 6x ¼ 42 chromosomes respectively. T. monococcum (AA genome donor) (Figure 1C),
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Figure 1. Metaphase chromosomes of genus Triticum species and Secale cereale rye. (A) Wichita (T. aestivum, ABD genome, 2n ¼ 42); (B) Newcale (Triticale, ABR genome, 2n ¼ 42); (C) T. monococcum (A genome donor, 2n ¼ 14); (D) T. searsii (B genome donor, 2n ¼ 14); (E) T. tauschii (D genome donor, 2n ¼ 14); (F) T. dicoccoides (AB genome, 2n ¼ 28); (G) Elbon(S. cereale, R genome, 2n ¼ 14). Scale bar ¼ 10 mm.
T. searsii (BB genome donor) (Figure 1D), T. tauschii (DD genome donor) (Figure 1E), and S. cereale (RR genome) (Figure 1G) have each 2n ¼ 14 chromosomes. T. dicoccodes (AABB genome) (Figure 1F) has 2n ¼ 4x ¼ 28 chromosomes. Genome sizes of Triticum species and Secale cereale are listed in Table 1. The DNA content of 2C nuclear was measured for each line. Three nuclear suspensions of each line were analyzed twice. The nuclear DNA content of T. searsii (BB genome donor, 13.7 pg) was larger than that of the other genome donors, AA (12.1 pg) or DD
(10.5 pg). In Chinese Spring (AABBDD genome), however, the content of interphase nuclear DNA of AA genome (12.3 pg) was a little larger than that of BB genome (12.2 pg), or DD genome (10.1 pg) (Lee et al. 1997). In wheat, recent studies (Feldman et al. 1997; for review, see Wendel, 2000; Feuillet et al. 2001) have indicated that genome rearrangements such as sequence duplication, deletions, and insertions occur rapidly after polyploidy formation. The 2C DNA contents of Wichita (AABBDD), or Newcale (AABBRR) were less than the sum of 2C DNA contents of
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Table 1. Genome size of genus Triticum species and Secale cereale rye.
Species T. aestivum (cv. Wichita) T. dicoccoides T. monococcum T. searsii T. tauschii S. cereale (c v. Elbon) Triticale (cv. Newcale)
Chromosome number (2n)
Ploidy level
Genome (genome donor)1
Mean DNA content (pg/2C)SD
Genome Size (1C) (Mbp)2
42 28 14 14 14 14 42
6 4 2 2 2 2 6
AABBDD AABB (AA) (BB) (DD) RR AABBRR
34.40.4 26.20.4 12.10.3 13.70.3 10.50.3 16.80.3 40.90.8
1.6 104 1.2 104 5.6 103 6.3 103 4.9 103 7.8 103 1.9 104
1 References in Kimber & Sears (1987). 2The 1C genome size (1.6 104 Mbp) of Chinese Spring wheat (Bennett & Smith, 1976) was used as a reference. The chicken red blood cell DNA (2.33 pg/2C) and barley DNA (NE86954, 10.24 pg/2C, Lee et al. 1997) were used as the internal control. The means were calculated from six duplicates per line.
AA, BB, and DD genome donor, or AA, BB, and RR genome donor, respectively. Interestingly, T. dicoccoides (AABB) had a little higher genome than the sum of the 2C DNA content of AA and BB genome donor. These interesting results might be derived from the di¡erent evolutionary process of AA, BB, or DD genomes, di¡erent parental backgrounds of hexaploid wheat in coding and non-coding sequences, or elimination of genomic or chromosome-speci¢c sequences during the polyploid formation (review in Rieseberg, 2001). In this study, root tip cells of six di¡erent species of Triticum and Secale cereale were synchronized e⁄ciently by optimizing the concentration and time with hydroxyurea and microtubule inhibitor tri£uralin. In wheat (Wichita, AABBDD genome) and triticale (Newcale, AABBRR genome), the high mitotic indexes (about 60%) were achieved in treatment with 1.25 mM hydroxyurea for 18 h, incubation in hydroxyurea-free Hoagland solution for 2 h, and treatment with 1 mM tri£uralin for 4 h. Material from T. dicoccoides, T. monococcum, T. searsii, T. tauschii, and S. cereale, also showed high mitotic indexes about 60% in treatment with 1.25 mM hydroxyurea for 14 h, incubation in hydroxyurea-free Hoagland solution for 2 h, and 1 mM tri£uralin for 3 h. After accumulation of metaphase chromosomes, overnight ice-cold water treatment improved chromosome condensation and spreading of cells. In Figure 2, the £ow karyotypes of Wichita (T. aestivum, AABBDD genome, 2n ¼ 6x ¼ 42) (Figure 2A), T. dicoccoides (AABB genome, 2n ¼ 4x ¼ 28) (Figure 2B), T. monococcum (AA genome donor, 2n ¼ 14) (Figure 2C), T. searsii
(BB genome donor, 2n ¼ 14) (Figure 2D), T. tauschii (DD genome donor, 2n ¼ 14) (Figure 2E), S. cereale (RR genome, 2n ¼ 14) (Figure 2F), and Newcale (AABBRR genome, 2n ¼ 6x ¼ 42) (Figure 2G) provide information for the number of chromosomal peaks, the chromosomal DNA content of each peak, and di¡erent chromosomal types. Each £ow peak was sorted and analyzed for identi¢cation of contents such as nuclei, clumps, chromosomes, chromatids, and cell debris using a £uorescent microscope. Three to four chromosome peaks in each line ranged from channel 350 for the smallest chromosome of D genome donor to channel 600 for the largest chromosome of R genome (rye) and ABR triticale. The 21 pairs of hexaploid wheat chromosomes (2n ¼ 6x ¼ 42) were classi¢ed into four groups (one peak with 3B chromosome, and three composite chromosome peaks) based on relative PI £uorescent intensity (Lee et al. 1997; Vrana et al. 2000). The £ow karyotypes of AB genome, and A, B, and D genome donor showed two large peaks and one small peak. The £ow karyotypes of R genome and ABR triticale showed one large peak and two small peaks, and one large peak and three small peaks, respectively. The DNA contents of individual chromosome peaks based on £uorescence intensity from 6 di¡erent species of Triticum and Secale cereale are shown in Table 2. The DNA content of each chromosome ranged from 1.22 pg to 2.61 pg. In wheat (Wichita, AABBDD genome) and Triticale (Newcale, AABBRR genome), four chromosome peaks revealed from 1.24 pg to 2.12 pg, and from 1.62 pg to 2.60 pg, respectively. The DD genome donor
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Figure 2. Observed flow karyotypes based on relative fluorescent intensity of propidium iodide. (A) Wichita (T. aestivum, ABD genome); (B) T. dicoccoides (AB genome); (C) T. monococcum (A genome donor); (D) T. searsii (B genome donor); (E) T. tauschii (D genome donor); (F) Elbon (S. cereale, R genome); (G) Newcale (Triticale, ABR genome) (references in Kimber & Sears 1987). Left-hand side pictures are linear scale flow karyotypes and right-hand side pictures are log scale flow karyotypes.
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Table 2. DNA content of individual chromosome peaks based on fluorescence intensity from different genus Triticum species and Secale cereale rye. AABBDD2 FL chr peak1 1 2 3 4 Total
DNA (pg)3
Chr. no.4
2.120.02 1 1.800.07 9 1.570.06 7 1.240.05 4 34.28 21
AABB DNA (pg)
AA Chr. no.
2.240.04 1 1.910.08 10 1.580.04 3 26.09
14
DNA (pg)
BB Chr. no.
DNA (pg)
2.010.03 2 1.860.03 1 1.580.05 4 12.20
7
DD Chr. no.
2.220.06 2 2.050.05 1 1.780.07 4 13.63
7
DNA (pg)
RR Chr. no.
1.870.06 1 1.510.07 3 1.220.05 3 10.06
7
DNA (pg)
AABBRR Chr. no.
2.610.04 1 2.400.08 5 2.160.05 1 16.75
7
DNA (pg)
Chr. no.
2.600.08 2 2.300.08 4 1.910.09 10 1.620.07 5 41.57 21
1 Chromosome peaks of flow karyotypes were described by numerical designation. 2Genome of species; AABBDD (T. aestivum cv.Wichita); AABB (T. dicoccoides); AA (T. monococcum); BB (T. searsii); DD (T. tauschii); RR (S. cereale cv. Elbon); AABBRR (Triticale cv. Newcale). 32C DNA content of each chromosome peak. 4Number of expected chromosomes were calculated by the relative frequency of total events of each chromosome peak in linear flow karyotypes. Note: Total DNA content based on PI fluorescence intensity in metaphase chromosomes may be different from that of interphase nuclei due to chromatin coiling and fluorochrome binding difference. The formaldehyde fixation may change the accessibility of DNA to PI.
showed the smallest chromosome peaks compared to the other genomes ranged from 1.22 pg to 1.87 pg. In rye (RR genome), the largest chromosome peaks ranged from 2.16 pg to 2.61 pg. Interestingly, in AABB genome there was only one chromosome in the largest DNA fraction of 2.241 pg even though there were two chromosomes in this range in BB genome donor. In AABBRR genome, however, DNA content of two chromosomes was 2.60 pg, but only one chromosome from AA, BB, or RR genome donor/genome had DNA content of this range. It has recently been shown that genome evolution in polyploids is associated with the elimination of low copy DNA sequences and/or chromosomal rearrangement between homoeologous chromosomes (review in Wendel, 2000; review in Rieseberg, 2001; Ozkan et al. 2001). Lee et al. (2002) reported that total DNA content in metaphase chromosomes was less than that of interphase nuclei due to chromatin coiling and di¡erences in £uorochrome binding. Unlike in maize which has di¡erent sets of knobs with a total 23 locations on 15 out of 20 arms (McClintock et al. 1981), total DNA content of each 6 di¡erent species of Triticum and Secale cereale in metaphase chromosomes and interphase nuclei was similar. Chromosome images on an 8 bit gray scale were analyzed on NIH image software with CHIAS III macro program (Kato and Fukui
1998). A part of the image of metaphase spread is shown in Figure 3A. Chromosomal area was completely covered in red color after setting appropriate threshold using ‘Set Density Slice’ command on CHIA III macro program (Figure 3B). ‘Erase Background’ eliminates background noises that were not covered in red color and replaces chromosomal area covered with red color. Some remaining noises were deleted by manual operation, and then only chromosomes were left in the image (Figure 3C). The original chromosome image has gray value range from about 100 to 255 and the range is di¡erent between the images. The ‘normalization’ command changes the gray value range of the chromosome image from about 100^255 to 1^254. The contrast was then enhanced and the gray value range became the same between the images, so comparison between di¡erent images could be achieved (Figure 3D). LUT was used for easy identi¢cation of the midrib of each chromosome. Appropriate colors were arranged corresponding to the gray value, and original gray value was not changed after applying LUT (Figure 3E). A line was drawn on the chromosomal midrib from one end to the other end by segmented line tool (Figure 3F). There are three tools for drawing a line, ‘Segmented line’ makes the most exact length. The length of the line was then measured for all chromosomes in one image of the metaphase
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Figure 3. Representative steps for measurement of chromosomes. (A) Original gray image obtained from a normally exposed photograph; (B) Set threshold by ‘Density Slice’ command; (C) Extracted chromosomes; (D) Normalized chromosome image; (E) The gray image was artificially colored by the look-up table; (F) Segmented line was drawn interactively on the chromosome axis, then the length of the line was measured.
spread, and total chromosome length was calculated. Chromosomal area was chosen by density slice command (as in Figure 3B). The chromosomal area and mean gray value were then measured. Density volume was calculated by multiplying the area and the mean gray value. In Table 3, correlations among 2C nuclear DNA content, metaphase chromosome DNA content, chromosome area, chromosome density volume, and total chromosome length are shown. Nuclear 2C DNA content was used as standard
for calculation of correlation values among the parameters. Density volume was calculated by multiplying chromosome area with the mean gray value of chromosome area. Chromosome density volume showed the highest correlation of 0.9968 with nuclear 2C DNA content among the other parameters measured by image analysis methods. The nuclear 2C DNA content had the lowest correlation of 0.9844 with the chromosome area. AABBDD genome had 366 mm2 of chromosome area, 42738 units of density volume, and 229 mm of
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Table 3. Correlations among 2C nuclear DNA content, metaphase chromosome DNA content, chromosome area, chromosome density volume, and total chromosome length.
Nuclear 2C DNA content3 Metaphase chromosome DNA content4 Chromosome area5 Chromosome density volume Total chromosome length
AABBDD1
AABB
AA
BB
DD
RR
AABBRR
Correlation2
34.4 34.2 366.1 42737 229.4
26.2 26.1 321.8 31636 194.3
12.1 12.2 141.2 16508 100.2
13.7 13.6 120.0 15972 79.5
10.5 10.1 121.6 13281 70.1
16.8 16.8 150.1 18884 101.4
40.9 41.6 496.3 51784 306.2
1.0000 0.9997 0.9844 0.9968 0.9869
1
Genome of species; AABBDD (T. aestivum cv.Wichita); AABB (T. dicoccoides); AA (T. monococcum); BB (T. searsii); DD (T. tauschii); RR (S. cereale cv. Elbon); AABBRR (Triticale cv. Newcale). 2Correlation was calculated between 2C nuclear DNA content vs. metaphase chromosome DNA content, chromosome area, chromosome density volume, or total chromosome length. 32C nuclear DNA content was taken from Table 1. 4Metaphase chromosome DNA content was taken from Table 2. 5Chromosome area, chromosome density volume, and total chromosome length were measured using ‘Image analysis program’ (http://133.1.131.81/Eudejas/chias3/chias3.html, Kato & Fukui, 1999).
chromosome length. These values were smaller than the sum of AABB and DD genomes or the sum of three diploid species, AA, BB, and DD genome. AABBRR genome had 496 mm2 of chromosome area, 51784 units of density volume, and 306 mm of chromosome length. These values were larger than the values of AABBDD genome and smaller than the sum of AABB and RR genome or three diploid species, AA, BB, and RR genome. AABB genome had 322 mm2 of chromosome area, 31636 units of density volume and 194 mm of chromosome length. Chromosome area and length were larger than the sum of two diploid species, AA and BB genomes. Chromosome areas of diploid species varied from 120 to 150 mm2. The BB and DD genomes have about 120 mm2 of chromosome area. The BB genome has, however, larger density volume and chromosome length than that of the DD genome. The density volume of the BB genome is 15972 units and the DD genome is 13281 units. Chromosome length of the BB genome, 5.68 mm is longer than that of the DD genome, 5.01 mm. The AA and RR genomes with similar mean chromosome length have longer mean chromosome length than that of BB and DD genomes. There were 7.16 mm for AA and 7.24 mm for RR genome, respectively. RR genome has larger chromosome area and density volume than those of AA genome. It is concluded that the 2C nuclear DNA content can be used for analysis of evolutionary process of polyploids. In general during the polyploid formation, the 2C DNA content is decreased. Chromosome density volume measured by the
chromosome imaging analysis is a more reliable parameter for measuring 2C nuclear DNA content than the other two parameters such as chromosome length or chromosome area. The di¡erent results from those obtained in rice (Uozu et al. 1997) are presumably due to the di¡erent types of chromosomes (Fukui 1986).
Acknowledgments This research was partially supported by the grant from Dong-A University, and by the grant number CG2122 from Crop Functional Genomics Center of 21C New Frontier Project in Korea. We thank professors T. Yoshida and T. Seki at the Center for Biotechnology of Osaka University in Japan for providing a collaborative research to us in flow cytometry for chromosome analysis. We are grateful to Dr. Dolezel for constructive discussion and comment.
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