in 4x, 6x and 8x ploidy levels. No diploid clone was found. Chromosomes counts and flow cytometry data led to the same results. The flow cytometry histograms ...
Genetic Resources and Crop Evolution 46: 19–27, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
19
Ploidy determination of some yam species (Dioscorea spp.) by flow cytometry and conventional chromosomes counting F. Gamiette1, F. Bakry2 & G. Ano1 1 INRA
– URPV, B.P.: 515, 97165 Pointe a` Pitre Cedex; 2 CIRAD – FLHOR, B.P.: 5035, 34032 Montpellier Cedex
01. Received 1 July 1998; accepted in revised form 8 July 1998
Key words: chromosomes count, Dioscorea, flow cytometry, polyploidy, yam.
Abstract Chromosomes counting and flow cytometry were used for assessing the ploidy level of various yam accessions of the INRA collection located in Guadeloupe. About 85 different clones were evaluated in the D. alata and D. cayenensis-rotundata and four other wild species related to D. cayenensis-rotundata. All the studied clones fitted in 4x, 6x and 8x ploidy levels. No diploid clone was found. Chromosomes counts and flow cytometry data led to the same results. The flow cytometry histograms for D. cayenensis-rotundata were not separated from those of its related wild species. Polyploidisation by fusion of 2n+n gamets was found to be unlikely for the two species D. cayenensis-rotundata and D. alata. Moreover, these results lead to the conclusion that the D. cayenensis-rotundata cultigens and the wild species analysed in this study may belong to the same gene pool.
Introduction Yams, Dioscorea spp., monocotyledons, order Dioscoreales, family Dioscoreacae (Cronquist) are tuberous vines producing edible tubers. Yams provide 12% of the energetic food for tropical populations, after cassava (20%), and before taro (4%) and sweet potato (2%), (Coursey & Martin, 1970). Its world-wide production is increasing, and in 1994, was estimated at 30 millions metric tons. Africa, with 29 million tons and especially Nigeria with 22 million tons, is the first world producer (F.A.O., 1994). Dioscoreas having economic importance belong to the section Enantiophyllum with the two main cultigens, D. alata L. and the complex D. cayenensis-rotundata (D. cayenensis Lam. and D. rotundata Poir.). They are native from Africa (D. cayenensis-rotundata) and South-East Asia (D. alata), and are now widely distributed all over the tropics. In recent decades, cropping techniques have evolved from a traditional to an industrial way of cultivation. Therefore, yams selected nowadays must meet economic and alimentary needs of producers and consumers.
Yam is a dioecious, vegetatively propagated plant. In the past, yam improvement, mainly involved clonal selection but limited progress was achieved, since no variety combined all the desirable characteristics. Therefore, significant yam improvement will necessarily go through sexual reproduction which is the usual way for combining desirable characteristics. In order to start such a program, basic knowledge about genetic resources is essential; especially for yams, the ploidy level of the clones must be known. A literature survey shows that, in yams, the basic chromosome number is related to the origin of the clone. In the genus Dioscorea, all the Asian clones, 52% of the African clones, and 13% of the American clones have a basic number x = 10. Most of the remaining American clones display a basic number x = 9 (Essad, 1984). As many vegetatively propagated crops, many polyploid clones of yam have been found (2n = 4x, 6x, 8x). Diploid clones have never been found for D. alata and D. cayenensis- rotundata (Essad, 1984; Martin & Ortiz, 1963; Ramachandran, 1968; Abraham & Nair, 1991; Zoundjiekpon et al., 1990). Moreover, Sharma, (1956, 1957) found levels of ploidy to be 3x, 5x and 7x by chromosome count.
20 Table 1. Accessions of yam investigated for ploidy level Species
Number of studied accessions
D. alata Linn´e D. cayenensis -rotundata D. mangenotiana Mi`ege D. abyssinica Hoch D. praehensilis Benth. D. burkilliana Mi`ege
73 12 1 1 1 1
Recently, in D. alata, by flow cytometry, a continuous range of ploidy (3x, 3.5x, 4.5x, 6x, 7x, 8.5x), has been reported (Hamon et al., 1992). The aim of this paper is to establish the ploidy level of the accessions of the collection at the INRA station in Guadeloupe, using chromosomes counting and flow cytometry for D. alata, D. cayenensis-rotundata and its related wild species.
Material The studied species are presented in Table 1. The great morphological variability of the D. alata species as already described by several authors (Prain & Burkill, 1939; Velayudhan et al., 1989) is obvious among our 73 accessions of D. alata collected from the West Indies, Nigeria, Ivory Coast, India, and New Caledonia. On the other hand Hamon et al. (1986) have described 20 clusters of D. cayenensis-rotundata, but only two clusters of D. cayenensis-rotundata (‘Kangba’ and ‘Yaobadou’) are represented in our collection. For some clones there is some doubt as to their belonging to any cluster. The assumed related wild species of D. cayenensis-rotundata studied in this paper are mentioned in Table 1.
Methods Chromosomes counting In order to study the mitotic chromosomes, root tips were excised from tuber cuttings planted in sawdust kept wet in a greenhouse. The root tips were taken off from plants soon after sunrise, before 7 a.m., placed in a hydroxyquinoline solution (2mM) open to the air at 20–25 ◦ C for 5 h and fixed in acetic acid-alcohol
(1/3) for 48 h. They were stained in Feulgen reagent in darkness for at least 2 h after 45 min hydrolysis in 5N HCl stopped by a 10 min immersion in N HCl. After squashing in 45% acetic acid, slides were stored at -20 ◦ C. After removing the cover-slip, drying the slides in absolute ethanol (30 – 60 min), and ventilating (15 min), the slides were over-stained in a Giemsa solution (1% Giemsa solution, in a phosphate buffer, pH = 6.7), rinsed in distilled water (2 min) and dried again before mounting. Several root tips were excised from one plant per clone. From one slide or more, seven metaphases were inspected. Chromosome counts were performed at a 1000 × magnification. An average of the seven counts was calculated for each clone. Flow cytometry Flow cytometry is a rapid and robust technique that allows accurate determination of DNA content in a large number of nuclei. This technique quantifies the intensity of light emitted by isolated nuclei stained with DNA specific fluorochromes after excitation. The fluorescent signal of each individual nucleus is proportional to the amount of fluorochrome stoichiometrically bound to the DNA. To isolate and stain nuclei, 30 – 50 mg of young leaves from vitro plants were chopped with a razor blade in a plastic petri dish containing 2 ml LB01 fresh lysis buffer (Dolezel et al., 1994). The suspension of released nuclei was passed through a 15 µm nylon mesh and supplemented with 15 µl of a DAPI solution (1mg/ml) which is an AT base pair binding fluorescent dye. Preparations were kept on ice in the dark for 5 min before analysis. Measures were performed on a Bryte HS flow cytometer using UV provided by a 75 W Xenon-Mercury lamp. DAPI is exited by UV waves’ length. Histograms of intensity were recorded over 256 channels on a linear scale and studied on a 486DX33 Dell PC using WinBryte software Ver.1.01. As it is useful to work with an internal reference (Petit et al., 1986; Brown et al., 1991a and b), a reference plant was chopped with the sample (1/3 of weight of chopped leaves comes from the internal reference, the remainder from the sample). For D. alata the clone ‘Tahiti M’ was used as internal reference. But when its peaks were not separated from those of the sample, it was replaced by the clone ‘Ti Joseph 2’. For D. cayenensis-rotundata and the other species the clone ‘22 OR’ was used as internal reference.
21
Figure 1. Cell at metaphasic stage, (D. alata) hexaploid clone ‘Tahiti M’.
Let F.I. (Fluorescent Index) be the mean position of the G1 peak. The ploidy level (P.L.) for each clone is given by the formula: (F.I. / F.I.R.) × (ploidy of internal reference), where F.I.R.(Fluorescent Index of internal Reference) is the index value of the internal reference.
Results Chromosome count results: Figure 1 presents a cell at metaphasic stage of the clone ‘Tahiti M’(D. alata). Results are given in Table 2. According to Essad (1984), because of their very small size, their tendency to stick together, and their satellites sometimes as large as the chromosomes themselves, yam chromosomes count is difficult. This may explain the great variation in our counts. Indeed, the standard deviation varies from 0.76 to 5.19. Nevertheless, without any doubt, 3 clusters of D. alata have been pointed out. Their average, 38.89, 58.89 and 77.93, fit respectively with tetraploid, hexaploid and octoploid level. One clone of D. cayenensis-rotundata is tetraploid, the other is octoploid. The clones used as internal reference, ‘Ti Joseph 2’ and ‘22OR’ are tetraploid; ‘Tahiti M’ is hexaploid.
Flow cytometry results: In a young leaf a majority of cells is not in division. These cells are in the G0 stage. Their nuclear DNA content reflects the ploidy level of the plant. Cells in division pass from the G0/G1 stage, to the G2 stage where they have a double DNA content (De Laat et al., 1987). Figure 2a shows the DNA histograms obtained from the clone ‘Tahiti M’. Distribution of nuclei over G0/G1 in channel 92 and G2 in channel 181 can be clearly recognised. When the preparation, involves both the sample and the internal reference, the peaks of the internal reference can be recognised by their smaller area, (Figure 2c), three different cases can be observed: - two peaks are present: the internal reference and the sample have the same DNA content - three peaks are present: G2 of the one is overlapped by G1 of the other with a double DNA content (Figure 2b) - four peaks are present: two peaks for the stages G1 and two for the stage G2 of each clone. Results are given in Tables 3 and 4. The use of an internal reference revealed to be essential for an accurate comparison of DNA content of the samples. The use of this method allowed to classify, without any doubt, our D. alata clones in three clusters: a cluster comprising the clones which have the same DNA con-
22
Figure 2. Views of flow cytometric data. Plant materials that are chopped together are indicated. Numbers at the top of the peak are the mean values of their channel of fluorescence.
tent as the internal reference, a cluster having 1.5 times the content and another the double. The coefficients of variation with this method ranged from 0.4% to 6.4%. Concerning D. cayenensis-rotundata, out of the 12 tested clones, 8 clones have the same DNA content as the internal reference, 2 have 1.5 times the content and 2 the double. Concerning the wild species presumably related to D. cayenensis-rotundata, the peaks of D. praehensilis, D. mangenotiana and D. abyssinica overlapped with those of the D. cayenensis-rotundata reference. It was shown that the peaks of D. burkilliana and those of the clone ‘Igname jaune’ belonging to the ‘Yaobadou’ cluster overlap (Table 5 and Figure 3). As shown in Figure 4, four peaks were observed for the sample involving the D. alata and D. cayenensis-rotundata mixed.
Figure 3. DNA histogram of the two species overlap on the channel 95 for the G1 stage, and on the channel 185 for the G2 stage.
23 Table 2. Results and interpretation of chromosomes counts of 18 clones of D. alata and 2 clones of D. cayenensis-rotundata C.V.: coefficient of variation.
D. alata Brazzo fuerte Fenakue Igname rouge Kokoeta Pyramide SEA 144 SEA 191 Ste Catherine Ti Joseph 1 Ti Joseph 2 Vino purple form Vino white form 65 Caillade 2 De Agua Tahiti M AIA 443 Noumea D. cayenensis-rotundata 22 OR Igname jaune (varietal cluster: Yaobadou)
Number of counted chromosomes in individual metaphases 1 2 3 4 5 6
Results
Interpretation
7
Average
σ
C.V.
Ploidy level
40 40 40 41 45 37 40 44 43 39 39 40 56 59 62 63 74 73
44 41 36 42 40 38 40 43 38 40 40 41 53 61 56 59 76 82
36 41 37 42 40 38 40 39 40 40 39 42 56 62 53 55 83 85
36 41 41 39 38 41 43 39 40 41 43 40 60 61 58 62 78 72
43 42 38 37 40 42 41 42 36 39 40 43 57 58 62 61 76 76
38 40 40 42 36 39 39 40 37 40 41 37 58 60 59 60 82 74
39 40 40 36 42 39 39 41 41 39 37 41 59 59 61 59 78 82
39.43 40.43 38.86 39.86 40.14 39.14 40.29 41.14 39.29 39.71 39.86 40.57 57.00 60.00 58.71 59.86 78.14 77.71
3.15 1.13 1.86 2.54 2.85 1.77 1.38 1.95 2.43 0.76 1.86 1.90 2.31 1.41 3.35 2.61 3.29 5.19
7.9 2.8 4.8 6.4 7.0 4.5 3.4 4.7 6.1 1.9 4.6 4.6 4.0 2.0 5.7 4.3 4.2 6.6
4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 6x 6x 6x 6x 8x 8x
40 78
39 76
35 82
39 83
42 81
37 77
36 82
38.29 79.86
2.43 2.79
6.3 3.4
4x 8x
Discussion
Figure 4. Data showing a different proportion of A-T base pairs in the genome of D. cayenensis-rotundata and D. alata.
According to the chromosome count results, the internal reference ‘Ti joseph 2’ is tetraploid and ‘Tahiti M’ hexaploid. DAPI is an AT base pairs binding dye; however as the internal reference belongs to the studied species, it is possible to compare their DNA content (Dolezel, 1992). The use of flow cytometry allows to classify, our D. alata clones in three clusters corresponding to 4x, 6x and 8x ploidy levels, (1x, 1.5x, 2x, the DNA content of the internal reference). Concerning D. cayenensis-rotundata, as its internal reference, ‘22OR’ is tetraploid, it is possible to assess that, out of the 12 tested clones, 8 are tetraploid, 2 hexaploid and 2 octoploid. For assessing the level of ploidy estimated by both methods, there is a close relationship between results achieved by conventional counts and flow cytometry (Tables 2 and 3, Tables 2 and 4). Whether one method or the other is used the coefficients of variation are similar. Since chromosomes counting in
24 Table 3. Asssessment of the level of ploidy of 73 clones of D. alata using the fluorescence index of the internal reference (F.I.R.) and the fluorescent index of the sample (F.I.) – C.V.: coefficient of variation of F.I. – P.I.R.: Ploidy of the Internal Reference Studied clones Clone
Provided by
72 76 AIA 445 Asmhore Bacala 2 Bete Bete Brazzo fuerte Bresil 1 Buet Campêche 2 Caplaou Cinq Cuba 1 Cuella Largo DA 28 DA 32 Divin 2 Fenakue Florido Gordito Grand Etang 1 Hawaï branched Igname couleuvre Igname rouge Kabusa - SEA 190 Kinabayo Kokoeta Lupias Morado Muriapo white Oriental Pacala Guyane Pacala station Purple Lisbon Pyramide Saint Sauveur 1 Saint Vincent Blanc 1 Saint Vincent Violet Sainte Catherine Smooth statia Ti Joseph 1 Ti Joseph 2 Toki - SEA 119 Venezuela Vino purple form Vino white form Wahamana bis
Guadeloupe/IRAT Guadeloupe/IRAT Guadeloupe/IRAT Guadeloupe/IRAT Nigeria/IITA Trinidad Haïti Ivory Coast Puerto Rico Brasil New Caledonia Guadeloupe Puerto Rico Guadeloupe Cuba Puerto Rico French Guiana French Guiana Guadeloupe New Caledonia Puerto Rico Puerto Rico Guadeloupe Puerto Rico Guadeloupe Guadeloupe Philippines Philippines / Porto Rico New Caledonia Trinidad Barbados French Guiana Guadeloupe Puerto Rico India / Puerto Rico Guadeloupe Martinique Martinique Guadeloupe Porto Rico Haïti Haïti New Caledonia Venezuela Puerto Rico Puerto Rico New Caledonia
Value of the internal reference F.I. C.V. 72 72 72 73 67 68 76 71 67 71 72 79 75 75 73 76 75 69 76 71 69 76 71 66 66 74 68 73 66 63 74 75 78 73 66 71 73 68 68 77 75 77 71 66 66 78 69
4.1 4.2 4.0 2.7 0.4 2.9 3.0 3.2 5.0 4.1 3.4 2.8 3.1 2.9 2.3 3.4 3.4 3.9 2.9 2.9 3.4 3.4 6.4 4.7 4.4 3.7 3.2 4.3 5.1 5.2 3.6 3.1 3.4 3.1 4.7 2.9 3.7 5.0 3.9 2.6 3.1 2.6 3.8 4.7 4.6 3.1 4.7
Interpretation F.I.R.
P.I.R.
Ploidy level
108 108 107 106 96 102 112 104 102 105 105 118 110 106 105 114 110 100 109 108 101 110 106 98 100 111 100 108 100 95 111 110 115 109 98 106 104 100 99 114 110 115 105 96 97 116 103
6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x
4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x
25 Table 3. contd. Wasa 65 66 67 Belep Caillade 2 DA 26 DA 27 De Agua Feo Goana Pakutrany Puneji bis Renta Yam Saint Martin Sassa 1 Sassa 2 Tahiti cultiv´e Tahiti French Tahiti Messaien Telemaque Wenefela bis AIA 443 Noumea Tana Wabe
New Caledonia Guadeloupe / IRAT Guadeloupe / IRAT Guadeloupe / IRAT New Caledonia Haïti French Guiana French Guiana Puerto rico Puerto rico New Caledonia New Caledonia New Caledonia Jamaica Martinique Martinique Martinique Guadeloupe Guadeloupe Guadeloupe Martinique New Caledonia Nigeria / IITA New Caledonia New Caledonia New Caledonia
72 99 97 97 94 107 105 99 104 108 102 108 104 105 104 101 103 101 104 106 105 108 137 75 135 137
4.2 5.6 4.3 3.0 2.5 3.0 1.3 2.6 2.0 2.0 1.7 2.4 5.7 2.7 5.6 2.9 3.8 2.5 2.4 2.9 3.1 3.4 2.3 3.2 2.8 3.3
108 67 97 97 62 71 70 68 70 75 70 108 104 70 71 68 103 70 71 69 71 72 103 37 71 103
6x 4x 6x 6x 4x 4x 4x 4x 4x 4x 4x 6x 6x 4x 4x 4x 6x 4x 4x 4x 4x 4x 6x 4x 4x 6x
4x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 6x 8x 8x 8x 8x
Table 4. Assessment of the level of ploidy of 12 clones of D. cayenensis rotundata by using the fluorescence index of the internal reference and the fluorescent index of the sample (F.I.). – C.V.: coefficient of variation of F.I. Studied clones Species
Clones
D. cayenensis-rotundata 85 OR AFU 1 Grosse Caille 3 mois Grosse Caille St Dom. Mozella Yam Piquant Pognon Portugaise e´ pineuse Igname poule blanc Salagnac 1 Igname jaune Igname poule jaune
Origin
Nigeria /IITA Trinidad Guadeloupe Guadeloupe Jamaica French Guiana French guiana Martinique Martinique Haïti Guadeloupe Martinique
Internal reference Varietal cluster
Kangba Kangba kangba
Kangba
Yaobadou Yaobadou
Interpretation Ploidy level
F.I.
C.V.
F.I.R.
P.I.R.
50 55 52 49 53 50 53 51 77 71 95 96
2.5 3.7 3.9 5.9 2.9 4.8 3.3 3.2 1.6 3.9 3.4 2.2
50 55 52 49 53 50 53 51 54 52 50 52
4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x
4x 4x 4x 4x 4x 4x 4x 4x 6x 6x 8x 8x
26 Table 5. F.I. value of 4 wild yam species, and of the internal reference (F.I.R.) Studied clone Species D. praehensilis
Results clone
Provided by: F.I. C.V. F.I.R.
Ref: 331 PH-15 ORSTOM
49 3.7
49
Ref: 185MG-01 ORSTOM
48 4.8
48
Ref: 296 AB-02 ORSTOM
50 4.5
50
Ref: 333-BU-08 ORSTOM
99 2.7
54
D. mangenotiana D. abyssinica D. burkilliana
yams is difficult and flow cytometry is a quick and robust technique, it is easier to assess ploidy levels for a large number of accessions or seedlings with the latter method. The average coefficient of variation of the measure of flow cytometry is 3.6% in linear scale. The resolution achieved by the method described in this paper was better than described before by other authors working on yam, Hamon (1992), Zoundjiekhpon (1992). As we included an internal reference in each sample, it is possible to conclude that, 4x, 6x, and 8x are the only ploidy levels of the tested clones. Therefore, for D. alata, we could not confirm Sharma’s (1956, 1957), and Hamon’s (1992) results, since in our study, levels 3x, 3.5x, 4.5x, 7x, 8.5x were not observed. The difference may be due to differences between accessions, or the lack of an internal reference in the analysis of previous authors. The levels 3x, 5x, and 7x are at least rare in D. alata and unknown for D. cayenensis-rotundata. Thus, this results suggest that, polyploidisation by fusion of 2n+n gamets is rare in these two species. As previously described for other clones, we did not find any diploid within the tested clones. Maybe they do not exist and these species are allopolyploid. This, is supported by the observation of twenty bivalents during meiosis of a tetraploid D. alata (Ramachandran, 1968). Reports about the level of ploidy of the wild species are rare and often contradictory. Thus, using flow cytometry Hamon (1992) found that D. abyssinica Hochst., D. mangenotiana Miège, D. praehensilis Benth., have the same DNA content. Unlike the latter, by chromosome counting, Essad (1984)
found D. abyssinica, tetraploid, D. praehensilis and D. mangenotiana, octoploid. D. mangenotiana was considered to be hexaploid and D. burkilliana Miège, tetraploid by Zoundjiekpon (1993). In our study, peaks of D. cayenensis-rotundata were not separated from those of wild species. Let’s assume that the proportions of AT base pairs of D. cayenensis – rotundata DNA and its related wild species are the same. In that case, the D. burkilliana DNA content is the double of that of the tetraploid clone‘22OR’; the other wild species have the same DNA content as the tetraploid clone ‘22OR’. These contradictory results concerning the wild species obtained by different authors might be due to the fact that only few clones, not representative of the entire variability of each species, were studied. According to the phylogenetic scheme suggested by Terauchi (1992), the gene pool D. cayenensisrotundata, may result from crosses between wild species followed by domestication. These results suggest that D. cayenensis-rotundata and the studied wild species belong to a gene pool. Four peaks were observed for the sample involving the D. alata and D. cayenensis-rotundata mixed. As DAPI is an AT base pairs binding fluorescent dye, the difference can be related to different DNA content or to different proportions of AT base pairs between the two species. This, would be specified by using intercalators binding to DNA. For phylogenetic study or breeding programs, an accurate knowledge of the ploidy level of the clones is required. The use of an appropriate method of flow cytometry giving results in agreement with chromosome counts offers the most suitable tool for this purpose. As a conclusion, the method of flow cytometry described in this paper offers a reliable tool for determination of ploidy of our Dioscorea collection. It provides necessary data for yam breeding, based on sexual reproduction.
Acknowledgement Thanks to Dr B. Malaurie (ORSTOM, in vitro genetic resources) for the supplying of the wild species and to Mrs L. Rousseau for her precious help.
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