Journal of Experimental Botany, Vol. 42, No. 238, pp. 659-665, May 1991 Variation in the DNA Content of Species

N. HAMMATT1, N. W. BLACKHALL and M. R. DAVEY2

Department of Botany, University of Nottingham, Nottingham NG72RD, UK

Received 1 October 1990 Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021

ABSTRACT Nuclei were isolated from cotyledons of a range of accessions from 14 species of Glycine. These were stained with ethidium bromide and the relative fluorescence for each genotype was measured by flow cytometry. The DNA content was estimated by comparison of relative fluorescence with that from nuclei from seedling leaves of Allium cepa, whose DNA content has been calculated previously by chemical assay. The 4C amounts for diploid Glycine ranged from 3-80 to 6-59 pg. Two groups of diploid species appeared from the analysis. The first consisted of species with amounts ranging from 3-80 to 516 pg and included G. canescens (AA), G. argyrea (A^), G. clandestina (A2A2), G. microphylla (BB), G. latifolia (B^,), G. tabacina 2/i = 40 (B2B2), G. tomentella 2/1 = 38 (EE) and 2n = 40 (DD), G. max and G. soja (GG), G. arenaria and G. latrobeana. A second group had higher DNA contents ranging from 5-27 to 6-59 pg, and consisted of G. curvata, G. cyrtoloba (CC), and G.falcata (FF). The polyploid species, G. tabacina 2/i = 80 (AABB, BBB^), G. tomentella 2/i = 78 and 2/i = 80 (AAEE and DDEE, respectively) contained amounts approximating to the sums of the respective parental diploid species thought to have given rise to these allotetraploids. Intraspecific variation was detected in the DNA content of G. canescens. Within the overall distribution of DNA amounts found in A genome species, each genome contained a range of DNA contents specific to that species. This phenomenon was also detected amongst B genome species.

Key words: Flow cytometry, genome size, soybean, nuclei, ethidium bromide.

INTRODUCTION The genus Glycine consists of two subgenera, with the between perennial Glycine species has derived from mor- eudiploid chromosome complement for ali Glycine species phological and biochemical evidence and, more recently, being 2n = 2x = 40. The first subgenus, Soja, contains the from studies of meiotic chromosome pairing in interspe- annual, cultivated soybean, G. max L. Merr., and its cific hybrids (Newell and Hymowitz, 1983; Grant, Grace, conspecific, twining progenitor, G. soja. The second sub- Brown, and Putievsky, 1984; Singh and Hymowitz, genus, Glycine, comprises 15 perennial species, some 1985a, b; Singh, Kollipara, and Hymowitz, 1987, 1988). diploid and some tetraploid, which are indigenous to Based on this information, Hymowitz and Singh (1988) , parts of South-East Asia, and several Pacific have assigned genome symbols to Glycine species, and island groups. Most perennial species produce a substan- these are given in Table 1. tial tap root and are prolific twiners (Fig. 1A), but the Variation in intraspecific DNA content of has rare G. latrobeana is a dwarf scrambling herb, similar to been found using microdensitometry, including studies by G.falcata (Fig. 1B). The latter is unique in producing Price, Chambers, and Riggs (1980) on Microseris doug- pods underground. The subgenus is characterized by the lasii, Miksche (1971) on Picea sitchensis and Watson production of white to deep purple chasmogamous (1987) with Bulbine species. In this technique, the amount flowers, inserted singly along an extended rachis (Fig. lc), of nuclear staining with Feulgen's reagent, as assessed by and single or clustered axillary, cleistogamous flowers a microdensitometer, is proportional to the DNA content (Fig. ID). of the nucleus (Bennett and Smith, 1976). Flow cytometry, Current understanding of the systematic relationships which is both more convenient and more efficient than

1 Present address: Horticulture Research International, East Mailing, West Mailing, Kent ME19 6BJ, UK. 2 To whom correspondence should be addressed. © Oxford University Press 1991 660 Hammatt et al.—DNA Amounts in Glycine Species microdensitometry, has also been used to assess the 1600, Canberra, ACT, Australia. Those with a PI prefix were amounts of DNA in plants. Nuclei are isolated and kindly donated by C. A. Newell, The Monsanto Co., 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA, and stained with a fluorochrome and the relative fluorescence, T. Hymowitz, Department of Agronomy, University of Illinois, which is emitted and measured with a flow cytometer, is Urbana, Illinois 61801, USA, who also provided seeds of related to the DNA content of each nucleus. Several accessions with a three figure number. Seeds of G. soja 3080 laboratories have reported on this technique, using differ- were supplied by AVRDC, P.O. Box 42, Shanua, Tainan 741, ent procedures to isolate and stain nuclei (Galbraith, Taiwan, and soybeans by W. Ellingson, AgriPro, Ames, Iowa, USA. Seeds of those species used as DNA standards (Triticum Harkins, Maddox, Ayres, Sharma, and Firoozabady, aestivum cv. Chinese Spring, Secale cereale cv. Petkus Spring, 1983; De Laat, Gohde, and Vogelzang, 1987; Ulrich, Hordeum vulgare cv. Sultan and Pisum sativum cv. Minerva Fritz, and Ulrich, 1988; Fahleson, Dixelius, Sundberg, Purple) were provided by the AFRC Institute of Science and Glimelius, 1988; Dolezel, Binarova, and Lucretti, Research, Trumpington, Cambridge, UK, while those of A Ilium 1989). Owing to uncertainty relating to the ability of flow cepa cv. Ailsa Craig were purchased locally (Stewarts Garden Supplies, George Street, Nottingham). cytometry to resolve intraspecific variation in DNA Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 amounts, Rayburn, Auger, Benzinger, and Hepburn (1989) confirmed, by this technique, intraspecific variation Seed production in Zea mays previously detected using procedures other Seeds of Glycine were scarified by removal of a small piece than flow cytometry. of the testa, and sown in Levington Universal Compost (Fisons Horticultural Division, Ipswich, UK), in 7-0 cm diameter, plastic As discussed by Hammatt, Jones, and Davey (1989), plant pots, and maintained at 27 °C on a glasshouse bench in the subgenus Glycine could be exploited as a potentially natural daylight. After the seeds had germinated, the seedlings limitless tertiary genepool for soybean improvement. usually entered a stationary phase lasting up to 6 weeks. Once the shoots had resumed growth, plantlets were repotted into Given this, the relatively small size of the genus, and the 15 cm diameter plastic pots and supported as required with establishment of seed collections for these species, the either canes or plastic netting (Fig. 1A). Plants were maintained present study was established to characterize the genus in a glasshouse on 55 x 55 x 5-5 cm plastic gravel trays, each further by initiating a preliminary investigation into the containing 8-0mm diameter pea gravel to a depth of 40cm. DNA content of Glycine species. This paper presents flow Plants, which were sensitive to waterlogging, were watered sparingly by keeping the gravel moist. During the months of cytometric estimations of the DNA amounts of 48 geno- October to June, natural daylight was supplemented, for 12 h types from the genus and evidence of intraspecific vari- per day, with eight Thorn 85 W, 3500 white fluorescent tubes, ation in the DNA amounts of G. canescens. 95 cm above the level of the gravel. On occasions when the top growth of G. latrobeana died back to the tap root, the plants MATERIALS AND METHODS were watered very sparingly until new shoots appeared. Seeds were collected from the single axillary pods (Fig. ID) which Seed sources developed from cleistogamous flowers. Seeds were stored in Seeds of Glycine accessions with a 'G' prefix were obtained paper envelopes at room temperature (22 °C) and relative from J. P. Grace, CSIRO Division of Plant Industry, P.O. Box humidity.

FIG. 1. (A, B) Plant of the prolific twining species, G. tomenteila 9941 (A) and the scrambling species, G.falcata P1246591 (B) (XO-09; XO17). (C, D) Floral arrangements in perennial Glycine species showing chasmogamous flowers of G. argyrea G1626 arranged along an extended rachis (c), and pod development from a single, axillary, cleistogamous flower of G. argyrea G1626 (D) (x 0-88; x 0-32). Hammatt et al.—DNA Amounts in Glycine Species 661 Production of seedlings for DNA analysis 1-2-3, a commercial spreadsheet program, for further analysis. Seeds of all perennial species were surface sterilized in a The position of the first bead peak was used as an estimate of 10% v/v 'Domestos' bleach solution (Lever Bros., London) for zero-offset drift (Vindelov, Christiansen, and Nissen, 1983) and 20 min, followed by four rinses, each of 10 min duration, in an offset correction factor from channel 148 calculated. As log sterile tap water. Prior to soaking in sterile water (48 h with a histograms had been acquired, this factor was added to the change after 24 h), a piece of the testa was removed from each means of the other three peaks. Log to linear conversion was seed using a scalpel. Soybeans and seeds of G. soja were sterilized performed using the method of Schmid, Schmid, and Giorgj for 10 min in 10% v/v 'Domestos', and rapidly rinsed in sterile (1988) in which ^=1-027*, where y is the linear equivalent of water. A piece of the testa was removed from seeds of G. soja, log channel x. The quantity of fluorescence for the 2C DNA but the seeds were not soaked in water prior to sowing. Seeds amount was calculated as the difference between the linear 3 of all species were sown in 175 cm capacity, screw-capped, means of the GQ/GJ and G2/M peaks. powder round jars (Beatson Clarke, Rotherham, UK), each containing 30 cm3 of agar-solidified (80 g dm"3) half-concentra- tion medium of Gamborg, Miller, and Ojima (1968), with the Estimation of DNA content omission of growth regulators. The seeds were germinated by Before calculating the DNA contents of Glycine species from Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 placing jars at 27 °C on glass shelves, 30 cm below Thorn their relative fluorescence,i t was determined that the technique Pluslux 3500 cool white 36 W fluorescent tubes, giving an used resulted in a proportional, straight-line relationship irradiance of 1-6 W m"2 at seed level. between the relative fluorescence obtained for the standard Seeds of A Ilium cepa, Hordeum vulgare, Pisum sativum, Secale species and the DNA content as assessed previously by micro- cereale, and Triticum aestivum were sown on 7-0 cm diameter, densitometry (Bennett and Smith, 1976). In determinations of Whatman filter paper circles in 9-0 cm diameter plastic Petri the DNA amounts in Glycine, one accession for each of the 3 dishes, each containing 2-0 cm of tap water. Seeds were germin- AA, A2A2, BB, B^j and B2B2 genomes was selected (marked ated at 27 °C in the dark. Seedlings of Senecio vulgar is were with an asterisk in Table 1), and their DNA contents were selected from a wild population growing on the University of estimated from at least three independent comparisons of relat- Nottingham campus. ive fluorescence with that produced by 4C Allium nuclei, which are known to contain 67 pg DNA (Bennett and Smith, 1976). Preparation of nuclei for flow cytometry The DNA amounts of the remaining accessions with A and B A suspension of nuclei was obtained by chopping cotyledons genomes, with the exception of G. argyrea (AiAt) were calcu- from 7-d-old Glycine seedlings or leaves from 5-d-old seedlings lated by comparing mean relative fluorescence determined from of Allium, Hordeum, Pisum, Secale, Senecio, and Triticum, on at least three separate experiments, with that derived from nuclei ice, into 1-0 cm3 of ice-cold buffer. The buffer solution consisted of the standard accession chosen for each species. Subsequently, the DNA amounts for AjAt and the remaining Glycine genomes of 45 mM MgCl2, 30 mM sodium citrate, 20 mM morpholino- propanesulphonate, 10 g dm"3 Triton X-100 (Galbraith et al., were measured by direct comparison of relative fluorescence to 1983) and 100/igcm"3 of ethidium bromide. All reagents were the Allium standard. When the DNA content for a given from Sigma Chemical Co. The resulting nuclear suspension was accession was calculated by direct comparison to Allium, in passed through a 15 /xm nylon mesh, and kept on ice. Approxim- order to estimate variation attributable to different runs on the ately 50 mnrof a 1:1 mixture of 50% bright and DNA check machine, coefficients of variation were calculated from at least fluorescent beads (Coulter) were added to each sample of nuclei three separate experiments. prior to analysis, no later than 5 min after extraction. RESULTS Flow cytometry Nuclei isolated from all of the species examined emitted Samples were analysed with a Coulter EPICS 541 flow cytometer (Coulter Electronics Ltd., Luton, UK) equipped with fluorescence after staining with ethidium bromide, which a 5 W argon ion laser tuned to 488 nm, and a power output of was quantitated electronically by the flow cytometer. 100 mW. Sheath fluid consisted of 0-9% saline and was passed When relative mean integral fluorescence from nuclei of through a 150 pm flow cell tip at a pressure of 7 psi. Fluorescence the DNA standards, was plotted against the DNA content emissions were collected through a 473 nm band stop filter and a 508 nm long pass filter. For the species used as DNA stand- calculated by microdensitometry (Bennett and Smith, ards, histograms of linear integral fluorescence were collected. 1976), a significant (P=0-001) straight-line relationship Log integral fluorescencehistogram s were acquired from 10 000 (r = 0-996) was observed (Fig. 2). A typical histogram of particles for each Glycine sample and Allium, when used as a relative fluorescence is presented in Fig. 3, in this case for standard with Glycine species. G. argyrea G2004. The coefficients of variation (cvs) for peaks A and B, which comprise GQ/GJ and G /M nuclei Data analysis 2 are 2-72% and 1-66%, respectively. These values varied Histograms of log integral fluorescence for Glycine species from between 0-89% and 3-2% in most of the histograms and Allium, or linear integral fluorescence for the standards, were transferred to the EASY 88 computer system (IBM XT obtained from Glycine species. compatible) and analysed with Coulter Easy 2 software. Using Variation in mean relative fluorescence was detected the Intgra program, four ranges were defined to correspond to between experiments in all of the genotypes examined. the two standard fluorescent bead peaks, when present, and the The run-to-run coefficient of variation for nuclei from GQ/G! nuclei and the G2/M nuclei (Fig. 3). The mean, standard deviation and number of particles were calculated for each the Allium standard was calculated as 3-27% from five range and stored as part of the data files. For all samples, this separate experiments. For Glycine genotypes in which the analytical information was extracted and transferred to Lotus DNA content was estimated by direct comparison to 662 Hammatt et al.—DNA Amounts in Glycine Species Allium, the between experiment cvs ranged from 1-93% in G. latrobeana G1387 to 7-2% in G. tomentella G1300. TrtUcum The estimated 4C DNA amounts for the Glycine geno- types studied are presented in Table 1. In all four experi- ments, the relative fluorescence of G. clandestine! (A2A2) (mean 4-88 ± 0-08 pg) was always more than for G. canescens (AA) (mean 4-20 + 0-33 pg). In two experi- ments in which G. argyrea (A^) was compared to the AA and A2A2 genomes, the three accessions studied (G1626, G2004, and G2010) emitted greater fluorescence than any accession with an AA or A2A2 genome. In the 20 30 40 50 60 70 80 four experiments with G. canescens, accession Gl 171 con- Known DNA amount (po per 4C nudeus) sistently emitted 28-7 + 4-0% more fluorescence than Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 FIG. 2. Relationship between mean relative integral fluorescence of Gl 114, while G1249 produced an additional 18 9+ 1-84%. nuclei from six plant species and their known DNA content as assessed by microdensitometry (Bennett and Smith, 1976). No consistent trends in the DNA contents of B genome species were detected from the individual experiments. Upon further analysis (Fig. 4), among the diploid spe-

/w - cies, two groups appeared, based on their DNA amounts. The first were those with the A, B, DD, EE, and GG 600 - CD genomes, whose range covered 3-80 to 5-29 pg. The second 500- group of diploids (Fig. 4) consisted of G. curvata, •3 G. cyrtoloba (CC), and G.falcata (FF) with higher DNA 1 400- amounts spanning 5-27 to 6-59 pg. The FF genome, with 3 z 300 - r /u Fluorescent 6-24 to 6-59 pg was the most unusual. Each of the A ^2/M beads genome species appeared to contain a specific and exclus- 200 - I i\ 1 ive range of DNA amounts. Likewise, with the exception 100 - Lj of G. tabacina G1317, the B genome species appeared to u 1 •• occur in exclusive areas within the range of DNA amounts 0 - 40-81 0 120 160 200 240 in the genus. Relative fluorescence DISCUSSION FIG. 3. Histogram showing the distribution of log integral fluorescence It can be concluded, from the data presented here, that for G0/Gi and G2/M nuclei isolated from cotyledons of G. argyrea G2004, with 50% and DNA check fluorescent beads, used as non- the procedure for isolating, staining and analysing fluor- biological, internal standards. escent nuclei from Glycine species, resulted in quantitative

5 0 6.0 7.0 8 0 90 Estimated mean DNA content (pg pet 4C nucleus) FIG. 4. Figure to illustrate the variation in DNA amounts found in each genome of the genus Glycine. ARE—G. arenaria, CUR—G. curvata, LAT—G. latrobeana, •—indicates the position of a species represented in these studies by only one genotype. Hammatt et al.—DNA Amounts in Glycine Species 663 TABLE 1. Flow cytometric estimations of the DNA content of Glycine species

Species Genome Accession Origin Number of DNA content CV independent (pg per 4C (%) - —»-- measurements nucleus)

G. canescens AA Gil 14 NSW 4 •3-80 2-0 F. J. Herm G117I NSW 3 4-83 G1240 NSW 4 4-06 G1249 NSW 4 4-52 G130I NT 3 3-99 G1340 NT 4 418 G1699 NSW 4 4-00 Mean 4-20 ±0-33 7-9 G. argyrea A,A, G1626 Q 3 516 5-2 Tind. G2004 Q 2 5-05 G2010 Q 3 5-06 4-6 Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 G. clandestine! A2A2 G1003 NSW 4 4-91 Wendl. G1019 ACT 4 4-98 G1145 NSW 3 4-76 " G1201 NSW 4 •4-83 6-3 G1231 NSW 4 4-94 Mean 4-88 ±0-08 1-6 G. microphylla BB 307 NSW 3 3 91 (Benth.) Tind. 312 NSW 4 •3-99 6-5 Mean 3-95 G. latifolia B,B, G1137 Q 3 4-31 (Benth.) Newell G1233 NSW 4 4-27 and Hymowitz G1343 Q 3 4-24 G1426 NSW 3 *4-28 7-2 G1456 Q 3 4 31 Mean 4-28 ±0-026 0-6 G. tabacina B2B2 PI373986 NSW 3 •4-02 4-0 2/7 = 40 G1138 NSW 4 418 (Labill.) Benth. G1195 NSW 3 417 G1206 NSW 3 415 G1317 NSW 4 4-46 Mean 4-20±0-14 3-3§ G. cyrtoloba CC 317 NSW 2 5-27 Tind. 480 Q 2 6-25 G. tomentella DD G1300 Q 4 412 7-8 2/7 = 40 G1381 PNG 4 4-51 6-4 Hayata G. lomenlella EE PI373987 NSW 3 4-33 4-7 2/7 = 38 Hayata G.falcata FF G1246 Q 3 6-24 51 Benth. G2082 Q 2 6-35 G2086 Q 2 6-59 G. max (L.) Merr. GG ESSEX USA 2 4-70 G. soja Sieb. GG 3080 T 2 4-56 and Zucc. G. tabacina AABJBJ/ G1080 NSW 1 811 2/7 = 80 BBB2B2 G1255 NSW 1 8-96 (Labill.) Benth. G1258 NC 1 810 G. lomentella DDEE PI373988 NSW 3 9-03 2/7 = 78 Hayata G. tomentella AADD G1134 Q 1 8-54 2/7 = 80 G1146 Q 3 8-38 Hayata G. arenaria G1305 WA 3 4-81 7-5 Tind. G1931 WA 3 4-64 6-0 G. curvata — G1396 Q 2 5-98 Tind. G. latrobeana — G125I V 2 5-40 (Meissn.) Benth. G1387 V 3 517 1-0

ACT, Australian Capital Territory; CV, coefficient of variation; NC, New Caledonia; NSW, New South Wales; NT, Northern Territory; PNG, Papua New Guinea; Q, Queensland; T, Taiwan; V, Victoria; WA, Western Australia; * indicates the accession from each species used as a DNA standard for comparison with Alliwn cepa (see Materials and Methods); §, calculated without accession G1317 which was different from other G. tabacina. The genomes of G. latrobeana, G. arenaria, and G. curvata have yet to be determined, although studies of chromosome pairing in interspecific hybrids have revealed some affinity of G. latrobeana to the AA genome (Grant et al., 1984). The newly described species G. albicans, G. hirticaulis, and G. lactovirens (Tindale and Craven, 1988) were not examined. 664 Hammatt et al.—DNA Amounts in Glycine Species staining and detection of nuclear fluorescence in plant one another by a paracentric inversion and that this has DNA standard species whose 4C DNA amounts ranged formed the basis for speciation. This was also thought to from 5-88 to 69-27 pg. distinguish the BB, BjB,, and B2B2 genomes. The present Variation in DNA content was detected between runs study suggests that the isolation of these species has been on the flow cytometer for onion and Glycine genotypes, paralleled or even assisted by changes in DNA amounts but since other workers have not quantified run-to-run in the different genomes. variation, it is difficult to compare the present findings. As expected, the polyploid Glycine genomes contained While nuclear DNA estimation by Feulgen densitometry approximately the sum of the DNA amounts found in is more labour intensive than flow cytometry, Bennett the respective diploid species from which they are thought and Smith (1976) obtained coefficients of variation in to have derived. DNA content consistently less than 2%, albeit from a Following concern that flow cytometry may not be larger number of replicates than those used in these sufficiently sensitive to detect intraspecific variation in the experiments with flow cytometry. Given the degree of DNA content of plants, Rayburn et al. (1989) used this Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 variation obtained in experiments with the flow cytometer, technique to demonstrate variation in DNA amounts confidence in calculating absolute DNA content would between varieties of corn (Zea mays). The variation have demanded a larger sample size. To survey the whole observed in the intraspecific DNA amounts for Glycine genus in this manner would be logistically impractical, species could have derived principally from the experi- but for the purposes of this study, estimation of relative mental protocol rather than biological factors. However, DNA content, rather than the absolute DNA amount the coefficient of variation observed, particularly for was an equally useful parameter. Flow analysis revealed accessions of G. canescens and constant differences that the DNA amounts in the genus Glycine are all found between the DNA amounts between accessions in the at the lower end of the range so far measured in plants. taxon for most of the experiments, suggest that a bio- The present estimation of nuclear DNA content of the logically significant degree of variation in DNA content GG genome of G. max and G. soja was greater than the existed in the AA genome of Glycine. The results 4C value of 3-7 pg calculated for soybean by Doerschug, with G. canescens support the view of Rayburn et al. Miksche, and Palmer (1978). (1989), that flow cytometry is sufficiently sensitive to The FF genome of G.falcata shows little homology detect intraspecific variation in the DNA contents of with other Glycine genomes (Singh et al., 1988), and the plants. exceptional nature of this species, compared to other This study has shown that the differences between the diploid Glycine taxa, can also be seen in a range of DNA contents of Glycine accessions within a species and morphological and biochemical characteristics. The those between species are variable. The significance of unusual DNA amounts found in G.falcata, substantiate these results could be further elucidated by studies with the view that this genome is distantly related to the a greater number of genotypes. It is hoped that this, genomes of other species. An increase in DNA content coupled with advances in the understanding of Glycine may have contributed to the low levels of pairing observed systematics, will indicate the significance of changes in between FF and other Glycine genomes (Singh et al., DNA content in the process of speciation in Glycine and 1988) which, in turn, may have resulted in the isolation other genera. and definition of this species. This may also be true for the CC genome of G. cyrtoloba. ACKNOWLEDGEMENTS As the A and B genomes contain six perennial species Figure 1 was prepared with the photographic assistance which predominate the genus, they were studied in greater of B. V. Case. This analysis was performed on the flow detail using a large number of accessions than those cytometer purchased by the British Technology Group genomes with only one species. The difference between and the University of Nottingham. the amount of DNA in the lowest AA and the highest A!A! accession was greater than the equivalent value for B species over the limited geographical range examined. LITERATURE CITED These data, albeit from a small sample of genotypes, BENNETT, M. D., and SMITH, J. B., 1976. Nuclear DNA amounts suggest that the B genome DNA content may be more in Angiosperms. Philosophical Transactions of the Royal Soci- ety of London, 11 A, 227-74. conserved than that of the A genome. DE LAAT, A. M. M., G6HDE, W., and VOGELZANG, M. J. D. C, Based on the evidence from the germplasm examined 1987. Determination of ploidy of single plants and popula- and excepting the unusual data collected from G. tabacina tions by flow cytometry. Plant Breeding, 99, 303-7. G1317, each of the A and B genome species contains an DOERSCHUG, E. B., MIKSCHE, J. P., and PALMER, R. G., 1978. 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E., 1984. Interspecific hybridization in Glycine Willd. subgenus complexes of Glycine tabacina (Labill.) Benth. and Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021 Glycine (Leguminosae). Australian Journal of Botany, 32, G. tomentella Hayata revealed by cytogenetic analysis. Gen- 655-63. ome, 29, 490-7. HAMMATT, N., JONES, B., and DAVEY, M. R., 1989. Plant regen- 1988. Further data on the genomic relationships eration from seedling explants and cotyledon protoplasts of among wild perennial species (2/i = 40) of the genus Glycine Glycine argyrea Tind. In Vitro Cellular and Developmental Willd. Ibid. 30, 166-76. Biology, 25, 669-72. TINDALE, M. D., and CRAVEN, L. A., 1988. Three new species HYMOWITZ, T., and SINGH, R. J., 1988. Biosystematics of the of Glycine (: Phaseolae) from North-Western Aus- genus Glycine. Soybean Genetics Newsletter, 15, 97-8. tralia, with notes on amphicarpy in the genus. Australian MiKSCHE, J. P., 1971. Intraspecific variation of DNA per cell Systematic Botany, 1, 399-410. between Picea sitchensis (Bonq.) Can. Provenances. Chromo- ULRICH, I., FRITZ, B., and ULRICH, W., 1988. Application of soma, 32, 343-52. DNA fluorochromes for flow cytometric DNA analysis of NEWELL, C. A., and HYMOWITZ, T., 1983. Hybridization in the plant protoplasts. Plant Science, 55, 151-8. genus Glycine subgenus Glycine Willd. (Leguminosae, Papi- VINDELOV, L. L., CHRISTIANSEN, I. J., and NISSEN, N. I., 1983. lionoideae). American Journal of Botany, 70, 334—48. Standardization of high resolution flow cytometric DNA PRICE, H. J., CHAMBERS, K. L., and RIGGS, J., 1980. Detection analysis by the simultaneous use of chicken and trout red of intraspecific variation in nuclear DNA content in Micro- blood cells as internal reference standards. Cytometry, 3, seris douglasii. Botanical Gazette, 141, 195-8. 328-31. RAYBURN, A. L., AUGER, J. A., BENZINGER, E. A., and HEPBURN, WATSON, E. M., 1987. Nuclear DNA content in the Australian A. G., 1989. Detection of intraspecific DNA content variation Bulbine (Liliaceae). Genome, 29, 225-34. Downloaded from https://academic.oup.com/jxb/article/42/5/659/471287 by guest on 25 September 2021