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Karyotype analyses and studies on the nuclear DNA content in 30 genotypes of potato (Solanum tuberosum) L.
Cell Biology International 28 (2004) 625e633 www.elsevier.com/locate/cellbi
Karyotype analyses and studies on the nuclear DNA content in 30 genotypes of potato (Solanum tuberosum) L. I.C. Mohantya, D. Mahapatraa,*, S. Mohantyb, A.B. Dasb a
Department of Agricultural Biotechnology, University Main Building, Orissa University of Agriculture and Technology, Bhubaneswar 751003, India b Regional Plant Resource Centre, Nayapally, Bhubaneswar 751015, India Received 27 July 2003; revised 19 April 2004; accepted 24 May 2004
Abstract The cytophotometric estimation of 4C DNA content, and karyotypic and somatic chromosome number analyses were carried out in 30 genotypes comprising seven cultivars and 23 advanced breeding lines of Solanum tuberosum. Detailed karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosomes of the genome, with a rare phenomenon of aneusomatic (2n = 4x C 2 = 50) condition in cv.K. Chandramukhi. The origin of this variation could be attributed to mitotic non-disjunction in the shoots giving rise to aneusomatic roots. Highly significant variations in the genome length, volume and total form percentage were noted at the cultivar level. The total chromosome length varied from 84.56 mm in cv.K. Pukhraj to 127.62 mm in MS/89-60, with an average value of 100.94 mm G 1.82. Total chromosome volume varied from 57.22 mm3 in MS/ 92-1090 to 132.64 mm3 in JW-160. Significant variations in the 4C DNA content (7.28e15.83 pg) were recorded at the cultivar level, with an exceptionally high DNA content (22.24 pg) in cv.K. Chandramukhi. This could be due to the aneusomatic condition of this genotype. Correlation studies revealed interdependence between the chromosomal and nuclear parameters of the genotypes. Structural alterations in the chromosomes, as well as loss or addition of highly repetitive sequences in the genome, caused variations in DNA content at the cultivar level. Variations in genomic structure and nuclear DNA content of the 48-chromosome genotypes suggest a genetic drift during microevolution, leading to the development of new cultivars. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Potato; Genome length; Genome volume; Cytophotometry; Karyotype; Nuclear DNA content
1. Introduction Potato, a widely adopted crop throughout the world, is a high yielding and short duration crop. Potato has a great potential to meet the increasing world food demand, as it produces the highest dry matter and protein amount per unit area and unit time among the major food crops. The nutrient value of potato is high, providing carbohydrates, proteins, minerals, vitamin C, a number of B-group vitamins and high quality dietary * Corresponding author. E-mail address: dmahapatra2@rediffmail.com (D. Mahapatra).
fibres (Gopalan et al., 1972). In terms of quantity, potato is fourth in the list of crop species that are important for human nutrition worldwide after rice, wheat and corn. Potatoes are produced for three main purposes: for the fresh food market, which provides more edible food annually than the combined world output of fish and meat; for the food processing industry, in the form of frozen French fries and potato chips; and for non-food industrial uses in processing and manufacturing of starch and alcohol. Industrial or nonfood uses of potato have potential in the future in the context of renewable resources and new special products, such as modified starches.
1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.05.004
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Potato production is expanding at an unprecedented rate and a large number of cultivars have been released. Cultivar identification and techniques to assess cultivar homogeneity are essential to commercial seed production, crop certification and registration. In addition, the new potato cultivars created from the restricted gene pool are genetically quite similar and hence difficult to characterize. Therefore, genetic identities of cultivars and inter-cultivar relationships are useful to maintain germplasms and to breed new cultivars. Karyotype analysis provides valuable information related to the mechanisms of genome evolution. There are few cytological studies reported to date in either cultivated or wild potatoes, because of the small and relatively numerous chromosomes (Wilkinson, 1994). The somatic chromosome number of Solanum tuberosum is 2n = 4x = 48 (Fish and Karp, 1986; Sree Ramulu et al., 1983). Comparison of nuclear DNA provides useful data in many cytotaxonomic and evolutionary studies (Price, 1976). It has been suggested that nDNA could play an important role in tolerance/ resistance to low temperatures and in response to ozone depletion or global warming (Bennet and Leitch, 1995). Keeping this in mind, the current investigation was undertaken to generate some information on molecular cytology, to help characterize various potato genotypes and establish the cytotaxonomic and evolutionary relationship.
2. Materials and methods Plants of 30 different cultivars and advanced breeding lines of Solanum tuberosum ssp.tuberosum and Solanum tuberosum ssp.andigena used in this study were obtained from the Central Potato Research Institute, Shimla, through the All India Coordinated Potato Improvement Project, OUAT, Bhubaneswar (Table 1), and were grown at the experimental site of the AICPIP, OUAT, for three cropping seasons (1999e2002). For chromosome preparation, root tips from the sprouted tubers were pre-treated in a saturated solution of pDB with aesculine for 3 h at 18 C, followed by overnight fixation in 1:3 aceto-alcohol. Chromosomes were stained in 2% aceto-orcine after cold hydrolysis in 5 N HCl for 5 min. The root tips were then squashed in 45% propionic acid. Ten well-scattered metaphase plates from each genotype were selected for karyotype analysis. The genomic chromosome length and karyotype volume were determined using the method of Das and Mallick (1993). The form percentage of individual chromosomes was calculated using the method of Levan et al. (1964), and the total form percentage (TF%) was taken as the average of the sum total of F% of a karyotype.
To score interphase nuclear volume (INV), 20 root tips of about 2.5 mm long from each genotype were fixed in acetic acid:ethanol (1:3) for 24 h at 25 C and hydrolysed in 1 N HCl at 4 C for 15 min. After thorough washing, the root tips were placed in Schiff’s reagent for 1 h at 20 C and kept in the dark for staining. Squash preparations were carried out in 45% acetic acid. Ten randomly selected nuclei were scored from each root tip. Under an oil immersion objective, the mean of the two diameters of nuclei from somatic cells, obtained by measuring at right angles to each other, was used to calculate the volume (Das and Mallick, 1993). Correlation coefficients of the different chromosomal parameters were calculated. For Feulgen cytophotometric estimation of 4C DNA, fixed root tips from each genotype were hydrolysed in 1 N HCl, stained in Schiff’s reagent for 2 h at 14 C and squashed in 45% acetic acid. The 4C DNA was estimated from metaphase chromosomes using a Nikon Optiphot microdensitometer, following the method of Sharma and Sharma (1980) with monochromatic light at 550 nm. In situ DNA values were obtained on the basis of optical density converted to picograms (pg) using Van’t Hof’s (1963) 4C nuclear DNA values for Allium cepa cv. Deshi (67.1 pg) as standard. To find out the significant differences in 4C DNA content between different genotypes, analysis of variance (ANOVA) (Sokal and Rohlf, 1973) was performed. Correlation coefficient analyses between different chromosomal parameters were carried out to find out the relationship between different genomic characteristics. Genetic relatedness among the different genotypes was calculated by grouping the genotypes via the numerical taxonomic procedure of cluster analysis. Cytological parameters, such as total chromosome length (TCL), total chromosome volume (TCV), total form percentage (TF%), interphase nuclear volume (INV), 4C nuclear DNA content, number of secondary constricted chromosomes and somatic chromosome number, were considered. Gower’s similarity index (Gower, 1985) was used to find out the similarity indices. Cluster analysis was carried out following the SHAN technique, and the dendrogram was developed by the unweighted pair group method with arithmetic mean algorithm (UPGMA).
3. Results 3.1. Chromosome characteristics Detailed analysis of somatic chromosomes of 30 genotypes of S. tuberosum of the family Solanaceae showed somatic chromosome number 2n = 4x = 48 in all the lines except cv.K. Chandramukhi, where an aneusomatic set of chromosomes (2n = 4x = 48 C 2)
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I.C. Mohanty et al. / Cell Biology International 28 (2004) 625e633 Table 1 Experimental material used in this study Sl. no.
Name of the germplasm
Parentage
Year of release/selection
Remarks
1. 2.
K. Jyoti cv.K. Pukhraj
1968 1999
Released as a cultivar Released as a cultivar
3. 4. 5. 6. 7. 8. 9. 10. 11.
K. Sutlej K. Badshah JW-160 K. Jawahar K. Ashoka JX-90 JX-576 85P-718 EX/A-680-16
30690/(4) ! 2814a (1) Craig’s defiance ! JEX/B-687 (EM/C-1021 ! CP-1468) K. Bahar ! K. Alankar K. Jyoti ! K. Alankar OB/A-9-120 ! CP-1462 K. Neelamani ! K. Jyoti EM/C-1021 ! CP-1468 CP-1346 ! MS/78-62 JE-812 ! K. Jyoti K. Bahar ! K. Jyoti An andigena clone
1996 1979 1995 1996 1996 1993 1993 1992 1972
12.
MF-1
K. Jyoti ! K. Alankar
1984
13.
TPS-13
Selection from EX/A-680-16
1984
14. 15. 16. 17.
cv.K. Chandramukhi JX-161 85P-670 JTH/C-107a
1968 1993 1992 1992
18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
HPS-I/13 92PT-27 MTP-I DTP-1 DTP-II MTP-II MS/92-1090 MS/89-60 MS/92-2105 JX-371 MS/92-3146 MS/92-3128 MS/92-209
Sd 4485 ! K. Kuber JE/812 ! K. Jyoti K. Jyoti ! PS4904 Meiotic tetraploid from K. Jyoti MF-1 ! TPS-13 83P-47 ! TPS/D-150 TPS-7 ! MST-1 JE-118 ! EX/A-680-16 K. Sutlej ! EX/A-680-16 JTH/C-107 ! MST-1 K. Jyoti ! PH/F-7545 ON.1645 ! MS/78-79 K. Lalima ! MS/82-797 JE/812 ! K. Jyoti MS/82-638 ! MS/80-758 MS/82-638 ! MS/80-758 CP-2400 ! PH/F-4518
Released as a cultivar Released as a cultivar Advance breeding line Released as a cultivar Released as a cultivar Adv. breeding line Adv. breeding line Adv. breeding line Parental line for TPSb Parental line for TPS Parental line for TPS Released as a cultivar Advance breeding line Advance breeding line Parental line for TPS Released as a cultivar Advance TPS population Advance TPS population Advance TPS population Advance TPS population Advance TPS population Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line
1985 1994 1999 1999 1999 1999 1998 1995 1999 1993 1999 1998 1999
a It is a meiotic tetraploid of K. Jyoti which evolved from a cross between K. Jyoti (4x) with IVP-35 a clone of S.Phureja (2x) via 2n (unreduced gamete) formation. b TPS, true potato seed.
was found (Fig. 1 (2e8)). On the basis of the size of the chromosomes and the position of the constrictions, four common chromosome types were found. A general description of the representative types of chromosomes is shown in Fig. 1 (1). Type A chromosomes are large to medium sized with two constrictions in nearly median to median and nearly sub-median to sub-median positions. Type B chromosomes are large to medium sized chromosomes with two constrictions in nearly sub-median to sub-median positions. Type C chromosomes are medium to small with nearly median to median primary constrictions. Type D chromosomes are medium to small chromosomes, with nearly sub-median to sub-median primary constrictions. Although all four types of chromosomes were present in all the studied genotypes, numerical differences did occur. The karyotype formulae of all the genotypes revealed definite differences in their chromosome structure (Table 2).
The type A secondary constricted chromosomes were present in all the genotypes in equal number. Type B secondary constricted chromosomes were present in 10 genotypes only e K. Ashoka, JX-90, JX-576, JX-161, HPS I/13, 92PT-27, MTP-I, DTP-III, MS/89-60 and JX-371. Furthermore, dose differences in the median and sub-median constricted C and D type chromosomes were found in all the cultivars. Type C chromosomes were the most numerous in all the genotypes, ranging from 24 (EX/A-680-16) to 40 (K. Jyoti, JW-160, and K. Jawahar). The number of sub-median D type chromosomes varied from four to 20. The highest number of D type chromosomes was found in the genotype EX/ A-680-16. Detailed analysis of the somatic complements and different genomic characteristics indicated genotype-specific variations in the genome (Fig. 2). Total chromosome length varied from 84.56 mm in K. Pukhraj to 127.62 mm in MS/89-60, whereas total chromosome volume varied from 57.22 mm3 in
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Fig. 1. (1) Standard types of chromosomes (AeD) present in different potato genotypes; (2e8) Somatic metaphase chromosomes (2n = 48, 2n = 48 + 2 ).
MS/92-1090 to 132.64 mm3 in JW-160. The average chromosome length varied from 1.762 mm in cv.K. Pukhraj to 2.659 mm in MS/89-60. The popular cultivar cv.K. Chandramukhi had comparatively larger chromosomes (2.487 mm) than the others. The centromeric index of chromosomes of all genotypes varied from 34.48% in MF-1 to 48.62% in K. Jyoti. Significant differences in chromosome length (F = 304.3, P ! 0.01), volume (F = 223.1, P ! 0.01) and TF % (F = 303.9, P ! 0.01) were seen from the analysis of variance. 3.2. INV and nuclear DNA content Interphase nuclear volume (INV) varied significantly (F = 3.07, P ! 0.01) from 738.58 mm3 in K. Sutlej to
1487.57 mm3 in JTH/C-107, and 4C DNA content varied (F = 21.19, P ! 0.01) from 7.28 pg in MS/92-1090 to 22.24 pg in cv.K. Chandramukhi (Table 2). In the genotypes MS/92-1090 and cv.K. Chandramukhi, genomic chromosome length was 85.10 mm and 119.38 mm, and genomic chromosome volume was 57.22 mm3 and 110.82 mm3, respectively. The greatest DNA content was found in the aneuploid cv.K. Chandramukhi, which has an extra pair of chromosomes in addition to the normal 48. The nuclear DNA content was significantly correlated to the genomic chromosome length, but not to total chromosome volume or INV (Table 3). Significant correlations were also found between TCL and TCV; TCL and INV; TCV and INV (Table 3).
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I.C. Mohanty et al. / Cell Biology International 28 (2004) 625e633 Table 2 Comparative chromosomal parameters and 4C nDNA content in 30 genotypes of Solanum tuberosum Sl. no Genotypes
Somatic chromo- Karyotype formula some no.
NSC TCL (mm)
TCV (mm3)
TF%
4C DNA (pg)
INV (mm3)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
48 48 48 48 48 48 48 48 48 48 48 48 48 48 C 2 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48
4 4 4 4 4 4 8 8 8 4 4 4 4 4 6 4 4 8 6 6 4 4 8 4 8 4 8 4 4 4
75.55 G 0.57 58.64 G 0.39 76.24 G 0.32 80.15 G 0.29 132.64 G 1.21 60.69 G 1.34 81.66 G 1.18 98.34 G 1.31 90.69 G 1.15 83.53 G 1.08 91.47 G 1.17 79.12 G 1.06 88.35 G 1.41 110.82 G 0.97 84.10 G 1.73 96.84 G 1.40 99.07 G 1.24 60.47 G 1.13 79.37 G 1.26 74.41 G 1.14 80.99 G 1.13 69.19 G 1.51 79.88 G 1.28 57.22 G 1.01 116.44 G 1.06 68.65 G 1.37 95.42 G 1.39 104.71 G 1.08 108.07 G 1.88 92.23 G 1.41
48.62 G 0.03 42.20 G 0.02 42.61 G 0.03 38.57 G 0.05 45.79 G 0.04 44.97 G 0.04 43.32 G 0.09 45.33 G 0.07 42.37 G 0.08 36.96 G 0.17 34.48 G 0.06 44.48 G 0.07 45.40 G 0.10 45.32 G 0.06 44.02 G 0.07 44.73 G 0.07 42.52 G 0.04 45.33 G 0.09 47.64 G 0.05 46.26 G 0.09 42.22 G 0.03 43.58 G 0.05 43.98 G 0.08 44.92 G 0.08 42.42 G 0.05 43.51 G 0.05 41.46 G 0.09 42.74 G 0.06 44.76 G 0.08 42.39 G 0.10
13.18 G 0.42 9.56 G 0.22 15.83 G 0.51 11.92 G 0.52 10.32 G 0.41 10.31 G 0.40 11.19 G 0.68 10.55 G 0.51 8.74 G 0.22 14.38 G 0.84 9.23 G 0.60 8.72 G 0.21 12.28 G 0.84 22.24 G 0.57 12.72 G 0.59 15.29 G 0.93 14.98 G 1.03 9.42 G 0.54 12.53 G 0.69 10.84 G 0.64 10.27 G 0.61 9.80 G 0.71 11.12 G 0.64 7.28 G 0.87 13.76 G 0.85 8.79 G 0.57 11.21 G 0.72 10.37 G 0.66 10.46 G 0.64 9.71 G 0.68
1442.60 G 155.10 1026.02 G 086.68 738.58 G 060.30 1061.76 G 089.12 1283.43 G 112.39 856.05 G 041.47 939.18 G 067.81 913.81 G 092.58 916.63 G 066.27 949.14 G 085.88 1055.85 G 112.38 1406.37 G 132.30 1096.67 G 130.91 1408.22 G 090.16 1120.42 G 103.14 1087.43 G 088.83 1487.57 G 142.90 1164.89 G 097.25 1259.72 G 118.01 1019.38 G 097.97 1156.85 G 105.88 1114.85 G 098.49 1174.86 G 096.01 1172.14 G 101.50 1014.50 G 104.10 1044.61 G 087.20 1055.71 G 098.96 1106.82 G 106.80 924.71 G 047.71 1138.44 G 093.20
K. Jyoti cv.K. Pukhraj K. Sutlej K. Badshah JW-160 K. Jawahar K. Ashoka JX-90 JX-576 85P-718 EX/A-680-16 MF-1 TPS-13 K.C.M. JX-161 85P-670 JTH/C-107 HPS I/13 92PT-27 MTP-I DTP-I DTP-II MTP-II MS/92-1090 MS/89-60 MS/92-2105 JX-371 MS/92-3146 MS/92-3128 MS/92-209
4A C 40C C 4D 4A C 36C C 8D 4A C 36C C 8D 4A C 32C C 12D 4A C 40C C 4D 4A C 40C C 4D 4A C 4B C 36C C 4D 4A C 4B C 32C C 8D 4A C 4B C 36C C 4D 4A C 32C C 12D 4A C 24C C 20D 4A C 28C C 16D 4A C 32C C 12D 4A C 42C C 4D 4A C 4B C 36C C 4D 4A C 28C C 16D 4A C 32C C 12D 4A C 4B C 36C C 4D 4A C 4B C 36C C 4D 4A C 4B C 32C C 8D 4A C 36C C 8D 4A C 36C C 8D 4A C 4B C 36C C 4D 4A C 36C C 8D 4A C 4B C 36C C 4D 4A C 36C C 8D 4A C 4B C 32C C 8D 4A C 36C C 8D 4A C 36C C 8D 4A C 36C C 8D
102.59 G 0.21 84.56 G 0.23 98.40 G 0.52 106.10 G 0.34 106.10 G 0.78 93.74 G 0.53 112.16 G 0.38 115.56 G 0.41 109.60 G 0.48 100.32 G 0.31 97.86 G 0.29 95.11 G 0.37 98.16 G 0.43 119.38 G 0.82 100.96 G 0.64 106.92 G 0.48 97.32 G 0.51 89.16 G 0.97 100.48 G 1.02 103.24 G 0.41 99.11 G 0.74 86.96 G 0.48 91.24 G 0.38 85.10 G 0.51 127.62 G 0.35 89.13 G 0.63 104.73 G 0.80 99.75 G 0.68 97.78 G 0.71 109.13 G 0.73
NSC: number of secondary constricted chromosomes; TCL: total chromosome length; TCV: total chromosome volume; TF%: total form percentage; INV: interphase nuclear volume.
3.3. Phylogenetic analysis The similarity matrices from all the cytological parameters calculated by Gower’s ranging method showed the highest coefficient of 0.96 between DTP-II and MS/92-2105, followed by 0.93 between TPS-13 and EX/A-680-16 and 92PT-27 and DTP-I (Table 4). The lowest coefficient of 0.25 occurred between cv.K. Pukhraj and c.v.K. Chandramukhi. The seven cytological parameters divided all potato genotypes into two clear phenetic clusters at 60 phenons (Fig. 3).
4. Discussion 4.1. Karyotype, genome length, and nuclear DNA content Detailed karyotype analysis in 30 genotypes of S. tuberosum revealed some interesting facts at the cultivar level. The chromosome number 2n = 4x = 48 was constant in all the studied genotypes, except cv.K. Chandramukhi (2n = 4x = 48 C 2), where an aneuploid set of chromosomes was found in the root tip cells. But
the type of chromosomes and the number of secondary constricted chromosomes varied with genotype. A, C and D type chromosomes were common in all genotypes, with high variability in terms of chromosomes in each category. With respect to karyotype formula, there were no differences between cv.K. Pukhraj and K. Sutlej; DTP-I and DTP-II; JW-160 and K. Jawahar; 92PT-27 and MTP-I. In contrast, genotype chromosome length and volume varied significantly. Detailed karyotype analysis revealed the preponderance of C type chromosomes in all the genotypes studied. Total F% analysis showed symmetrical karyotypes having median to nearly median chromosomes with a moderate fluctuation in F % values (from 34.48% in MF-I to 48.62% in K. Jyoti.). The gradual alterations to and shifting of TF % values may be due to chromosomal anomalies. The structural alterations in chromosome morphology, as well as the variations of secondary constricted chromosomes in the genotypes, may be due to duplication of chromosomes or translocations between chromosomes with or without secondary constrictions at a very early stage of evaluation (Das, 1991; Das et al., 1998; Mohanty et al., 1997).
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Fig. 2. Line diagram showing the pattern of variation in different cytological parameters.
Total chromosome length and volume differed markedly among the genotypes. Minute observations indicated a significant correlation between total chromosome length and volume, suggesting a high interdependence between them at the cultivar level. These facts indicate the predetermined genetic control of chromosome coiling. Evidently, differences in chromosome length or volume were due to differential condensation and spiralization of the chromosome arms. In addition, the genotype-specific compaction of DNA threads along with nucleosomes or additional gene sequences with altered non-histone proteins in the chromosome may have played an important role in the chromosomal architecture of the genotypes (Chattopadhyay and Sharma, 1990; Das et al., 1997).
Table 3 Correlation coefficient among the different cytological parameters of potato cultivars
TCL TCV INV
TCV
INV
4C DNA
0.694*
0.836* 0.609*
0.521* 0.374 ns 0.208 ns
TCL: total chromosome length; TCV: total chromosome volume; INV: interphase nuclear volume.
4.2. Diversification of DNA content Nuclear DNA content varied significantly from 7.28 pg in euploid MS/92-1090 to 15.83 pg in K. Sutlej. The aneusomatic cv.K. Chandramukhi had a much higher value of 22.24 pg, which may be due to the extra pair of chromosomes revealed by the karyotype analysis. The variability in the 4C DNA content in different genotypes might be attributed to the loss or addition of many repeats in the genome through alterations in the micro- and macroenvironment during the selection of new cultivars (Price et al., 1980). The significant correlation coefficient between genomic length and DNA content is in agreement with findings in other plant genera (Das et al., 1995; Das and Das, 1994; Das et al., 1999). The analysis of DNA content at the cultivar level in repeated experiments revealed stable 4C DNA in each genotype. By contrast, the DNA amount differed significantly among the genotypes. Flavell et al. (1997) reported that differences in DNA content depend on the repetitive DNA content. We agree that variability of DNA content can be attributed to loss or addition of highly repetitive DNA sequences rather than the AT- or GC-rich sequences in a genome (Martel et al., 1997), which reached a certain level and became
Table 4 Similarity matrix (SG) for seven cytological parameters of 30 potato genotypes Similarity matrix for cytological parameters 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0.65 0.77 0.87 0.62 0.69 0.79 0.63 0.71 0.80 0.74 0.85 0.78 0.59 0.87 0.76 0.81 0.70 0.89 0.88 0.82 0.78 0.82 0.65 0.54 0.76 0.92 0.74 0.68 0.76
0.71 0.69 0.38 0.78 0.65 0.49 0.58 0.64 0.67 0.68 0.63 0.25 0.68 0.54 0.61 0.81 0.73 0.76 0.79 0.87 0.76 0.83 0.44 0.87 0.65 0.68 0.60 0.68
0.80 0.45 0.74 0.78 0.61 0.69 0.82 0.72 0.70 0.74 0.43 0.80 0.71 0.75 0.63 0.84 0.83 0.83 0.73 0.76 0.58 0.57 0.75 0.74 0.76 0.70 0.73
0.66 0.73 0.91 0.76 0.83 0.86 0.82 0.79 0.83 0.56 0.91 0.85 0.72 0.70 0.88 0.93 0.86 0.80 0.86 0.65 0.65 0.81 0.92 0.81 0.75 0.85
0.53 0.59 0.72 0.69 0.63 0.70 0.73 0.71 0.67 0.66 0.73 0.60 0.57 0.61 0.61 0.59 0.51 0.60 0.49 0.58 0.50 0.71 0.70 0.74 0.66
0.71 0.68 0.75 0.80 0.81 0.78 0.78 0.41 0.75 0.67 0.57 0.89 0.69 0.78 0.72 0.81 0.80 0.84 0.40 0.83 0.69 0.66 0.77 0.62
0.82 0.86 0.83 0.76 0.72 0.76 0.57 0.84 0.78 0.66 0.63 0.81 0.89 0.82 0.73 0.78 0.58 0.69 0.75 0.87 0.77 0.74 0.85
0.89 0.78 0.80 0.65 0.79 0.73 0.74 0.84 0.61 0.62 0.64 0.72 0.65 0.58 0.66 0.54 0.69 0.60 0.81 0.73 0.82 0.76
0.85 0.88 0.76 0.82 0.61 0.81 0.86 0.63 0.67 0.71 0.80 0.74 0.68 0.74 0.64 0.62 0.71 0.85 0.75 0.84 0.85
0.88 0.80 0.89 0.58 0.92 0.87 0.73 0.73 0.82 0.84 0.80 0.74 0.82 0.68 0.58 0.75 0.83 0.77 0.85 0.74
0.84 0.93 0.58 0.87 0.85 0.73 0.79 0.77 0.81 0.81 0.77 0.83 0.74 0.56 0.79 0.87 0.83 0.89 0.80
0.81 0.56 0.83 0.72 0.76 0.80 0.83 0.79 0.81 0.81 0.89 0.77 0.44 0.81 0.75 0.73 0.75 0.72
0.62 0.90 0.85 0.74 0.79 0.81 0.80 0.81 0.75 0.84 0.71 0.55 0.75 0.84 0.80 0.85 0.76
0.58 0.71 0.61 0.44 0.53 0.49 0.47 0.38 0.49 0.37 0.65 0.37 0.60 0.57 0.62 0.57
0.85 0.77 0.74 0.90 0.88 0.88 0.81 0.89 0.70 0.61 0.79 0.89 0.84 0.80 0.82
0.76 0.65 0.75 0.78 0.73 0.66 0.74 0.60 0.67 0.66 0.89 0.79 0.82 0.81
0.57 0.83 0.71 0.80 0.66 0.73 0.53 0.63 0.65 0.78 0.84 0.72 0.77
0.70 0.73 0.72 0.87 0.84 0.92 0.35 0.87 0.66 0.64 0.68 0.63
0.88 0.93 0.78 0.85 0.66 0.61 0.77 0.82 0.85 0.71 0.84
0.88 0.83 0.84 0.69 0.62 0.85 0.89 0.82 0.76 0.83
0.80 0.86 0.68 0.61 0.80 0.83 0.88 0.74 0.88
0.89 0.85 0.45 0.96 0.76 0.74 0.69 0.71
0.80 0.50 0.88 0.81 0.78 0.76 0.75
0.30 0.53 0.61 0.59 0.64 0.58
0.47 0.69 0.71 0.63 0.72
0.77 0.74 0.88 0.71 0.83 0.71 0.88
28
29
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0.86 0.86 0.72
631
632
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Fig. 3. Dendrogram based on Gower’s similarity index and UPGMA generated by seven cytological parameters for 30 genotypes.
stabilized selection.
during
micro-evolution
and
gradual
4.3. Phylogenetic analysis The phylogenetic tree constructed from the cytological parameters through the SHAN technique of cultivar analysis by the UPGMA method showed two distinct phenetic clusters, which may very well be subdivided into four groups e IA, IB, IIA, and IIB. Group IA includes six cultivars e cv.K. Pukhraj, K. Jawahar, HPS I/13, DTP-II, MS/92-1090 and MS/92-2105. The second group (IB), the major group, comprises 13 numbers of genotypes, involving a total of 30 genotypes. This group includes the cultivar K. Jyoti, four of its derivatives and others. The third group (IIA) includes eight genotypes, and the fourth, the smallest, includes three genotypes. A good clustering of K. Jyoti and its derivatives, K. Badshsh, MF-1, JTH/C-107 and JX-371, was to be expected. Similarly, genotypes JX-576 and JX-161, two different clones derived from a single cross, have shown close similarity and are included in the same group (IIA). What stands out as very curious is the large distance between K. Jyoti and 85P-718 in Group IIA, since the latter is partly derived from the former. Another curious feature is the close similarity (0.84) of a red-skinned mutant, MS/92-2105, with DTP-II, a white skinned genotype. In fact, close similarities between certain genotypes agree well with present taxonomic judgements. It is gratifying to mention that, by and large, the results from this experimental study correlate remarkably well with those based on morphology. However, further work on DNA polymorphism is needed to resolve close similarities displayed between different genotypes and to establish genetic unrelatedness among cultivars/breeding lines with a common genotype in their parentage. This analysis could help breeders to
choose diverse parents for heterosis breeding programmes aimed at varietal improvement, keeping in zmind the tetrasomic nature of inheritance in this crop. Acknowledgements We acknowledge the help of the Director, Central Potato Research Institute, Shimla, India, for providing the germplasm used in this investigation. We also thank Dr. P. Das for facilitating the laboratory work for this investigation at RPRC, Bhubaneswar, India. References Bennett M, Leitch I. Nuclear DNA amounts in angiosperms. Ann Bot 1995;76:113e76. Chattopadhyay D, Sharma AK. Chromosome studies and microspectro-photometric estimation of nuclear DNA in different strains of Coriandum sativum L. Cytobios 1990;64:43e51. Das AB. Chromosomal variability in relation with 4C DNA content in the sub tribe carinae. Cytologia 1991;56:627e32. Das AB, Das P. Estimation of 4C DNA content and karyotype analysis in edible varieties of banana (Musa acuminata). Cytobios 1994;78:213e20. Das AB, Mallick R. Nuclear DNA chromosome changes within the tribe Ammineae. Cytobios 1993;74:197e207. Das AB, Basak UC, Das P. Karyotype diversity and genomic variability in some Indian tree mangroves. Caryologia 1995;48: 319e28. Das AB, Mohanty S, Das P. Meiotic behaviour and nuclear DNA variation in some species of Mammilaria (Cactaceae). Cytologia 1997;62:253e7. Das AB, Mohanty S, Marrs RH, Das P. Somatic chromosome number and karyotype diversity in fifteen species of Mammilaria of the family Cactaceae. Cytobios 1999;97:141e51. Das AB, Rai S, Das P. Karyotype analysis and 4C DNA content in some cultivars of ginger (Zingiber officinale Ross). Cytobios 1998; 93:175e84. Fish N, Karp A. Improvements in regeneration from protoplasts of potato and studies on chromosome stability. Theor Appl Genetics 1986;72:405e12.
I.C. Mohanty et al. / Cell Biology International 28 (2004) 625e633 Flavell RB, Rinpau J, Smith DB. Repeated sequence DNA relationships in four cereal genomes. Chromosome (Berl) 1997; 63:205e22. Gopalan C, Ramashashtri BV, Balsubramanian SC. Nutritive value of Indian foods. Hyderabad, India: National Institute of Nutrition (Indian Council of Medical Research); 1972. Gower JC. Measures of similarity, dissimilarity and distance. In: Kotz S, Johnson NL, editors. Encyclopedia of statistical sciences, vol. 5. New York, USA: Wiley; 1985. p. 397e405 [Original ref]. Levan A, Fredyak Sandberg A . Nomenclature for centromeric position on chromosome. Hereditas 1964;52:201e20. Martel E, Denay D, Siljakyakovtev S, Brown S, Sarr A. Genome size variation and basic chromosome number in pearl millet and fourteen related Pennisetum species. J Heredity 1997;88:139e43. Mohanty S, Das AB, Das P. Studies on nuclear DNA and meiotic chromosome in 8 species of Mammilaria. Cytologia 1997;62: 331e6.
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Price HJ. Evaluation of DNA content in plants. Bot Rev 1976;42: 27e52. Price HJ, Bachman K, Cihambers KL, Riggs J. Detection of intraspecific variation in nuclear DNA content of Microseris douglasii. Bot Gaz 1980;141:195e8. Sharma AK, Sharma A. Chromosome techniques: theory and practice. 3rd ed. London: Butterworths; 1980. Sokal PR, Rohlf FJ. Introduction to biostatistics. San Francisco: Freeman; 1973. Sree Ramulu KP, Dijkhuis P, Roest S. Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato. Theor Appl Genetics 1983;65:329e38. Van’t Hof J, Sparrow AH. A relationship between DNA content, nuclear volume, and minimum mitotic cycle time. Proc Natl Acad Sci U S A 1963;49:897e902. Wilkinson MJ. Genome evolution in potatoes. In: Bradshaw , Mackay , editors. Potato genetics. University Press; 1994. p. 43e70.
Karyotype analyses and studies on the nuclear DNA content in 30 genotypes of potato (Solanum tuberosum) L. I.C. Mohantya, D. Mahapatraa,*, S. Mohantyb, A.B. Dasb a
Department of Agricultural Biotechnology, University Main Building, Orissa University of Agriculture and Technology, Bhubaneswar 751003, India b Regional Plant Resource Centre, Nayapally, Bhubaneswar 751015, India Received 27 July 2003; revised 19 April 2004; accepted 24 May 2004
Abstract The cytophotometric estimation of 4C DNA content, and karyotypic and somatic chromosome number analyses were carried out in 30 genotypes comprising seven cultivars and 23 advanced breeding lines of Solanum tuberosum. Detailed karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosomes of the genome, with a rare phenomenon of aneusomatic (2n = 4x C 2 = 50) condition in cv.K. Chandramukhi. The origin of this variation could be attributed to mitotic non-disjunction in the shoots giving rise to aneusomatic roots. Highly significant variations in the genome length, volume and total form percentage were noted at the cultivar level. The total chromosome length varied from 84.56 mm in cv.K. Pukhraj to 127.62 mm in MS/89-60, with an average value of 100.94 mm G 1.82. Total chromosome volume varied from 57.22 mm3 in MS/ 92-1090 to 132.64 mm3 in JW-160. Significant variations in the 4C DNA content (7.28e15.83 pg) were recorded at the cultivar level, with an exceptionally high DNA content (22.24 pg) in cv.K. Chandramukhi. This could be due to the aneusomatic condition of this genotype. Correlation studies revealed interdependence between the chromosomal and nuclear parameters of the genotypes. Structural alterations in the chromosomes, as well as loss or addition of highly repetitive sequences in the genome, caused variations in DNA content at the cultivar level. Variations in genomic structure and nuclear DNA content of the 48-chromosome genotypes suggest a genetic drift during microevolution, leading to the development of new cultivars. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Potato; Genome length; Genome volume; Cytophotometry; Karyotype; Nuclear DNA content
1. Introduction Potato, a widely adopted crop throughout the world, is a high yielding and short duration crop. Potato has a great potential to meet the increasing world food demand, as it produces the highest dry matter and protein amount per unit area and unit time among the major food crops. The nutrient value of potato is high, providing carbohydrates, proteins, minerals, vitamin C, a number of B-group vitamins and high quality dietary * Corresponding author. E-mail address: dmahapatra2@rediffmail.com (D. Mahapatra).
fibres (Gopalan et al., 1972). In terms of quantity, potato is fourth in the list of crop species that are important for human nutrition worldwide after rice, wheat and corn. Potatoes are produced for three main purposes: for the fresh food market, which provides more edible food annually than the combined world output of fish and meat; for the food processing industry, in the form of frozen French fries and potato chips; and for non-food industrial uses in processing and manufacturing of starch and alcohol. Industrial or nonfood uses of potato have potential in the future in the context of renewable resources and new special products, such as modified starches.
1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.05.004
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Potato production is expanding at an unprecedented rate and a large number of cultivars have been released. Cultivar identification and techniques to assess cultivar homogeneity are essential to commercial seed production, crop certification and registration. In addition, the new potato cultivars created from the restricted gene pool are genetically quite similar and hence difficult to characterize. Therefore, genetic identities of cultivars and inter-cultivar relationships are useful to maintain germplasms and to breed new cultivars. Karyotype analysis provides valuable information related to the mechanisms of genome evolution. There are few cytological studies reported to date in either cultivated or wild potatoes, because of the small and relatively numerous chromosomes (Wilkinson, 1994). The somatic chromosome number of Solanum tuberosum is 2n = 4x = 48 (Fish and Karp, 1986; Sree Ramulu et al., 1983). Comparison of nuclear DNA provides useful data in many cytotaxonomic and evolutionary studies (Price, 1976). It has been suggested that nDNA could play an important role in tolerance/ resistance to low temperatures and in response to ozone depletion or global warming (Bennet and Leitch, 1995). Keeping this in mind, the current investigation was undertaken to generate some information on molecular cytology, to help characterize various potato genotypes and establish the cytotaxonomic and evolutionary relationship.
2. Materials and methods Plants of 30 different cultivars and advanced breeding lines of Solanum tuberosum ssp.tuberosum and Solanum tuberosum ssp.andigena used in this study were obtained from the Central Potato Research Institute, Shimla, through the All India Coordinated Potato Improvement Project, OUAT, Bhubaneswar (Table 1), and were grown at the experimental site of the AICPIP, OUAT, for three cropping seasons (1999e2002). For chromosome preparation, root tips from the sprouted tubers were pre-treated in a saturated solution of pDB with aesculine for 3 h at 18 C, followed by overnight fixation in 1:3 aceto-alcohol. Chromosomes were stained in 2% aceto-orcine after cold hydrolysis in 5 N HCl for 5 min. The root tips were then squashed in 45% propionic acid. Ten well-scattered metaphase plates from each genotype were selected for karyotype analysis. The genomic chromosome length and karyotype volume were determined using the method of Das and Mallick (1993). The form percentage of individual chromosomes was calculated using the method of Levan et al. (1964), and the total form percentage (TF%) was taken as the average of the sum total of F% of a karyotype.
To score interphase nuclear volume (INV), 20 root tips of about 2.5 mm long from each genotype were fixed in acetic acid:ethanol (1:3) for 24 h at 25 C and hydrolysed in 1 N HCl at 4 C for 15 min. After thorough washing, the root tips were placed in Schiff’s reagent for 1 h at 20 C and kept in the dark for staining. Squash preparations were carried out in 45% acetic acid. Ten randomly selected nuclei were scored from each root tip. Under an oil immersion objective, the mean of the two diameters of nuclei from somatic cells, obtained by measuring at right angles to each other, was used to calculate the volume (Das and Mallick, 1993). Correlation coefficients of the different chromosomal parameters were calculated. For Feulgen cytophotometric estimation of 4C DNA, fixed root tips from each genotype were hydrolysed in 1 N HCl, stained in Schiff’s reagent for 2 h at 14 C and squashed in 45% acetic acid. The 4C DNA was estimated from metaphase chromosomes using a Nikon Optiphot microdensitometer, following the method of Sharma and Sharma (1980) with monochromatic light at 550 nm. In situ DNA values were obtained on the basis of optical density converted to picograms (pg) using Van’t Hof’s (1963) 4C nuclear DNA values for Allium cepa cv. Deshi (67.1 pg) as standard. To find out the significant differences in 4C DNA content between different genotypes, analysis of variance (ANOVA) (Sokal and Rohlf, 1973) was performed. Correlation coefficient analyses between different chromosomal parameters were carried out to find out the relationship between different genomic characteristics. Genetic relatedness among the different genotypes was calculated by grouping the genotypes via the numerical taxonomic procedure of cluster analysis. Cytological parameters, such as total chromosome length (TCL), total chromosome volume (TCV), total form percentage (TF%), interphase nuclear volume (INV), 4C nuclear DNA content, number of secondary constricted chromosomes and somatic chromosome number, were considered. Gower’s similarity index (Gower, 1985) was used to find out the similarity indices. Cluster analysis was carried out following the SHAN technique, and the dendrogram was developed by the unweighted pair group method with arithmetic mean algorithm (UPGMA).
3. Results 3.1. Chromosome characteristics Detailed analysis of somatic chromosomes of 30 genotypes of S. tuberosum of the family Solanaceae showed somatic chromosome number 2n = 4x = 48 in all the lines except cv.K. Chandramukhi, where an aneusomatic set of chromosomes (2n = 4x = 48 C 2)
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I.C. Mohanty et al. / Cell Biology International 28 (2004) 625e633 Table 1 Experimental material used in this study Sl. no.
Name of the germplasm
Parentage
Year of release/selection
Remarks
1. 2.
K. Jyoti cv.K. Pukhraj
1968 1999
Released as a cultivar Released as a cultivar
3. 4. 5. 6. 7. 8. 9. 10. 11.
K. Sutlej K. Badshah JW-160 K. Jawahar K. Ashoka JX-90 JX-576 85P-718 EX/A-680-16
30690/(4) ! 2814a (1) Craig’s defiance ! JEX/B-687 (EM/C-1021 ! CP-1468) K. Bahar ! K. Alankar K. Jyoti ! K. Alankar OB/A-9-120 ! CP-1462 K. Neelamani ! K. Jyoti EM/C-1021 ! CP-1468 CP-1346 ! MS/78-62 JE-812 ! K. Jyoti K. Bahar ! K. Jyoti An andigena clone
1996 1979 1995 1996 1996 1993 1993 1992 1972
12.
MF-1
K. Jyoti ! K. Alankar
1984
13.
TPS-13
Selection from EX/A-680-16
1984
14. 15. 16. 17.
cv.K. Chandramukhi JX-161 85P-670 JTH/C-107a
1968 1993 1992 1992
18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
HPS-I/13 92PT-27 MTP-I DTP-1 DTP-II MTP-II MS/92-1090 MS/89-60 MS/92-2105 JX-371 MS/92-3146 MS/92-3128 MS/92-209
Sd 4485 ! K. Kuber JE/812 ! K. Jyoti K. Jyoti ! PS4904 Meiotic tetraploid from K. Jyoti MF-1 ! TPS-13 83P-47 ! TPS/D-150 TPS-7 ! MST-1 JE-118 ! EX/A-680-16 K. Sutlej ! EX/A-680-16 JTH/C-107 ! MST-1 K. Jyoti ! PH/F-7545 ON.1645 ! MS/78-79 K. Lalima ! MS/82-797 JE/812 ! K. Jyoti MS/82-638 ! MS/80-758 MS/82-638 ! MS/80-758 CP-2400 ! PH/F-4518
Released as a cultivar Released as a cultivar Advance breeding line Released as a cultivar Released as a cultivar Adv. breeding line Adv. breeding line Adv. breeding line Parental line for TPSb Parental line for TPS Parental line for TPS Released as a cultivar Advance breeding line Advance breeding line Parental line for TPS Released as a cultivar Advance TPS population Advance TPS population Advance TPS population Advance TPS population Advance TPS population Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line Advance breeding line
1985 1994 1999 1999 1999 1999 1998 1995 1999 1993 1999 1998 1999
a It is a meiotic tetraploid of K. Jyoti which evolved from a cross between K. Jyoti (4x) with IVP-35 a clone of S.Phureja (2x) via 2n (unreduced gamete) formation. b TPS, true potato seed.
was found (Fig. 1 (2e8)). On the basis of the size of the chromosomes and the position of the constrictions, four common chromosome types were found. A general description of the representative types of chromosomes is shown in Fig. 1 (1). Type A chromosomes are large to medium sized with two constrictions in nearly median to median and nearly sub-median to sub-median positions. Type B chromosomes are large to medium sized chromosomes with two constrictions in nearly sub-median to sub-median positions. Type C chromosomes are medium to small with nearly median to median primary constrictions. Type D chromosomes are medium to small chromosomes, with nearly sub-median to sub-median primary constrictions. Although all four types of chromosomes were present in all the studied genotypes, numerical differences did occur. The karyotype formulae of all the genotypes revealed definite differences in their chromosome structure (Table 2).
The type A secondary constricted chromosomes were present in all the genotypes in equal number. Type B secondary constricted chromosomes were present in 10 genotypes only e K. Ashoka, JX-90, JX-576, JX-161, HPS I/13, 92PT-27, MTP-I, DTP-III, MS/89-60 and JX-371. Furthermore, dose differences in the median and sub-median constricted C and D type chromosomes were found in all the cultivars. Type C chromosomes were the most numerous in all the genotypes, ranging from 24 (EX/A-680-16) to 40 (K. Jyoti, JW-160, and K. Jawahar). The number of sub-median D type chromosomes varied from four to 20. The highest number of D type chromosomes was found in the genotype EX/ A-680-16. Detailed analysis of the somatic complements and different genomic characteristics indicated genotype-specific variations in the genome (Fig. 2). Total chromosome length varied from 84.56 mm in K. Pukhraj to 127.62 mm in MS/89-60, whereas total chromosome volume varied from 57.22 mm3 in
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Fig. 1. (1) Standard types of chromosomes (AeD) present in different potato genotypes; (2e8) Somatic metaphase chromosomes (2n = 48, 2n = 48 + 2 ).
MS/92-1090 to 132.64 mm3 in JW-160. The average chromosome length varied from 1.762 mm in cv.K. Pukhraj to 2.659 mm in MS/89-60. The popular cultivar cv.K. Chandramukhi had comparatively larger chromosomes (2.487 mm) than the others. The centromeric index of chromosomes of all genotypes varied from 34.48% in MF-1 to 48.62% in K. Jyoti. Significant differences in chromosome length (F = 304.3, P ! 0.01), volume (F = 223.1, P ! 0.01) and TF % (F = 303.9, P ! 0.01) were seen from the analysis of variance. 3.2. INV and nuclear DNA content Interphase nuclear volume (INV) varied significantly (F = 3.07, P ! 0.01) from 738.58 mm3 in K. Sutlej to
1487.57 mm3 in JTH/C-107, and 4C DNA content varied (F = 21.19, P ! 0.01) from 7.28 pg in MS/92-1090 to 22.24 pg in cv.K. Chandramukhi (Table 2). In the genotypes MS/92-1090 and cv.K. Chandramukhi, genomic chromosome length was 85.10 mm and 119.38 mm, and genomic chromosome volume was 57.22 mm3 and 110.82 mm3, respectively. The greatest DNA content was found in the aneuploid cv.K. Chandramukhi, which has an extra pair of chromosomes in addition to the normal 48. The nuclear DNA content was significantly correlated to the genomic chromosome length, but not to total chromosome volume or INV (Table 3). Significant correlations were also found between TCL and TCV; TCL and INV; TCV and INV (Table 3).
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I.C. Mohanty et al. / Cell Biology International 28 (2004) 625e633 Table 2 Comparative chromosomal parameters and 4C nDNA content in 30 genotypes of Solanum tuberosum Sl. no Genotypes
Somatic chromo- Karyotype formula some no.
NSC TCL (mm)
TCV (mm3)
TF%
4C DNA (pg)
INV (mm3)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
48 48 48 48 48 48 48 48 48 48 48 48 48 48 C 2 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48
4 4 4 4 4 4 8 8 8 4 4 4 4 4 6 4 4 8 6 6 4 4 8 4 8 4 8 4 4 4
75.55 G 0.57 58.64 G 0.39 76.24 G 0.32 80.15 G 0.29 132.64 G 1.21 60.69 G 1.34 81.66 G 1.18 98.34 G 1.31 90.69 G 1.15 83.53 G 1.08 91.47 G 1.17 79.12 G 1.06 88.35 G 1.41 110.82 G 0.97 84.10 G 1.73 96.84 G 1.40 99.07 G 1.24 60.47 G 1.13 79.37 G 1.26 74.41 G 1.14 80.99 G 1.13 69.19 G 1.51 79.88 G 1.28 57.22 G 1.01 116.44 G 1.06 68.65 G 1.37 95.42 G 1.39 104.71 G 1.08 108.07 G 1.88 92.23 G 1.41
48.62 G 0.03 42.20 G 0.02 42.61 G 0.03 38.57 G 0.05 45.79 G 0.04 44.97 G 0.04 43.32 G 0.09 45.33 G 0.07 42.37 G 0.08 36.96 G 0.17 34.48 G 0.06 44.48 G 0.07 45.40 G 0.10 45.32 G 0.06 44.02 G 0.07 44.73 G 0.07 42.52 G 0.04 45.33 G 0.09 47.64 G 0.05 46.26 G 0.09 42.22 G 0.03 43.58 G 0.05 43.98 G 0.08 44.92 G 0.08 42.42 G 0.05 43.51 G 0.05 41.46 G 0.09 42.74 G 0.06 44.76 G 0.08 42.39 G 0.10
13.18 G 0.42 9.56 G 0.22 15.83 G 0.51 11.92 G 0.52 10.32 G 0.41 10.31 G 0.40 11.19 G 0.68 10.55 G 0.51 8.74 G 0.22 14.38 G 0.84 9.23 G 0.60 8.72 G 0.21 12.28 G 0.84 22.24 G 0.57 12.72 G 0.59 15.29 G 0.93 14.98 G 1.03 9.42 G 0.54 12.53 G 0.69 10.84 G 0.64 10.27 G 0.61 9.80 G 0.71 11.12 G 0.64 7.28 G 0.87 13.76 G 0.85 8.79 G 0.57 11.21 G 0.72 10.37 G 0.66 10.46 G 0.64 9.71 G 0.68
1442.60 G 155.10 1026.02 G 086.68 738.58 G 060.30 1061.76 G 089.12 1283.43 G 112.39 856.05 G 041.47 939.18 G 067.81 913.81 G 092.58 916.63 G 066.27 949.14 G 085.88 1055.85 G 112.38 1406.37 G 132.30 1096.67 G 130.91 1408.22 G 090.16 1120.42 G 103.14 1087.43 G 088.83 1487.57 G 142.90 1164.89 G 097.25 1259.72 G 118.01 1019.38 G 097.97 1156.85 G 105.88 1114.85 G 098.49 1174.86 G 096.01 1172.14 G 101.50 1014.50 G 104.10 1044.61 G 087.20 1055.71 G 098.96 1106.82 G 106.80 924.71 G 047.71 1138.44 G 093.20
K. Jyoti cv.K. Pukhraj K. Sutlej K. Badshah JW-160 K. Jawahar K. Ashoka JX-90 JX-576 85P-718 EX/A-680-16 MF-1 TPS-13 K.C.M. JX-161 85P-670 JTH/C-107 HPS I/13 92PT-27 MTP-I DTP-I DTP-II MTP-II MS/92-1090 MS/89-60 MS/92-2105 JX-371 MS/92-3146 MS/92-3128 MS/92-209
4A C 40C C 4D 4A C 36C C 8D 4A C 36C C 8D 4A C 32C C 12D 4A C 40C C 4D 4A C 40C C 4D 4A C 4B C 36C C 4D 4A C 4B C 32C C 8D 4A C 4B C 36C C 4D 4A C 32C C 12D 4A C 24C C 20D 4A C 28C C 16D 4A C 32C C 12D 4A C 42C C 4D 4A C 4B C 36C C 4D 4A C 28C C 16D 4A C 32C C 12D 4A C 4B C 36C C 4D 4A C 4B C 36C C 4D 4A C 4B C 32C C 8D 4A C 36C C 8D 4A C 36C C 8D 4A C 4B C 36C C 4D 4A C 36C C 8D 4A C 4B C 36C C 4D 4A C 36C C 8D 4A C 4B C 32C C 8D 4A C 36C C 8D 4A C 36C C 8D 4A C 36C C 8D
102.59 G 0.21 84.56 G 0.23 98.40 G 0.52 106.10 G 0.34 106.10 G 0.78 93.74 G 0.53 112.16 G 0.38 115.56 G 0.41 109.60 G 0.48 100.32 G 0.31 97.86 G 0.29 95.11 G 0.37 98.16 G 0.43 119.38 G 0.82 100.96 G 0.64 106.92 G 0.48 97.32 G 0.51 89.16 G 0.97 100.48 G 1.02 103.24 G 0.41 99.11 G 0.74 86.96 G 0.48 91.24 G 0.38 85.10 G 0.51 127.62 G 0.35 89.13 G 0.63 104.73 G 0.80 99.75 G 0.68 97.78 G 0.71 109.13 G 0.73
NSC: number of secondary constricted chromosomes; TCL: total chromosome length; TCV: total chromosome volume; TF%: total form percentage; INV: interphase nuclear volume.
3.3. Phylogenetic analysis The similarity matrices from all the cytological parameters calculated by Gower’s ranging method showed the highest coefficient of 0.96 between DTP-II and MS/92-2105, followed by 0.93 between TPS-13 and EX/A-680-16 and 92PT-27 and DTP-I (Table 4). The lowest coefficient of 0.25 occurred between cv.K. Pukhraj and c.v.K. Chandramukhi. The seven cytological parameters divided all potato genotypes into two clear phenetic clusters at 60 phenons (Fig. 3).
4. Discussion 4.1. Karyotype, genome length, and nuclear DNA content Detailed karyotype analysis in 30 genotypes of S. tuberosum revealed some interesting facts at the cultivar level. The chromosome number 2n = 4x = 48 was constant in all the studied genotypes, except cv.K. Chandramukhi (2n = 4x = 48 C 2), where an aneuploid set of chromosomes was found in the root tip cells. But
the type of chromosomes and the number of secondary constricted chromosomes varied with genotype. A, C and D type chromosomes were common in all genotypes, with high variability in terms of chromosomes in each category. With respect to karyotype formula, there were no differences between cv.K. Pukhraj and K. Sutlej; DTP-I and DTP-II; JW-160 and K. Jawahar; 92PT-27 and MTP-I. In contrast, genotype chromosome length and volume varied significantly. Detailed karyotype analysis revealed the preponderance of C type chromosomes in all the genotypes studied. Total F% analysis showed symmetrical karyotypes having median to nearly median chromosomes with a moderate fluctuation in F % values (from 34.48% in MF-I to 48.62% in K. Jyoti.). The gradual alterations to and shifting of TF % values may be due to chromosomal anomalies. The structural alterations in chromosome morphology, as well as the variations of secondary constricted chromosomes in the genotypes, may be due to duplication of chromosomes or translocations between chromosomes with or without secondary constrictions at a very early stage of evaluation (Das, 1991; Das et al., 1998; Mohanty et al., 1997).
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Fig. 2. Line diagram showing the pattern of variation in different cytological parameters.
Total chromosome length and volume differed markedly among the genotypes. Minute observations indicated a significant correlation between total chromosome length and volume, suggesting a high interdependence between them at the cultivar level. These facts indicate the predetermined genetic control of chromosome coiling. Evidently, differences in chromosome length or volume were due to differential condensation and spiralization of the chromosome arms. In addition, the genotype-specific compaction of DNA threads along with nucleosomes or additional gene sequences with altered non-histone proteins in the chromosome may have played an important role in the chromosomal architecture of the genotypes (Chattopadhyay and Sharma, 1990; Das et al., 1997).
Table 3 Correlation coefficient among the different cytological parameters of potato cultivars
TCL TCV INV
TCV
INV
4C DNA
0.694*
0.836* 0.609*
0.521* 0.374 ns 0.208 ns
TCL: total chromosome length; TCV: total chromosome volume; INV: interphase nuclear volume.
4.2. Diversification of DNA content Nuclear DNA content varied significantly from 7.28 pg in euploid MS/92-1090 to 15.83 pg in K. Sutlej. The aneusomatic cv.K. Chandramukhi had a much higher value of 22.24 pg, which may be due to the extra pair of chromosomes revealed by the karyotype analysis. The variability in the 4C DNA content in different genotypes might be attributed to the loss or addition of many repeats in the genome through alterations in the micro- and macroenvironment during the selection of new cultivars (Price et al., 1980). The significant correlation coefficient between genomic length and DNA content is in agreement with findings in other plant genera (Das et al., 1995; Das and Das, 1994; Das et al., 1999). The analysis of DNA content at the cultivar level in repeated experiments revealed stable 4C DNA in each genotype. By contrast, the DNA amount differed significantly among the genotypes. Flavell et al. (1997) reported that differences in DNA content depend on the repetitive DNA content. We agree that variability of DNA content can be attributed to loss or addition of highly repetitive DNA sequences rather than the AT- or GC-rich sequences in a genome (Martel et al., 1997), which reached a certain level and became
Table 4 Similarity matrix (SG) for seven cytological parameters of 30 potato genotypes Similarity matrix for cytological parameters 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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0.65 0.77 0.87 0.62 0.69 0.79 0.63 0.71 0.80 0.74 0.85 0.78 0.59 0.87 0.76 0.81 0.70 0.89 0.88 0.82 0.78 0.82 0.65 0.54 0.76 0.92 0.74 0.68 0.76
0.71 0.69 0.38 0.78 0.65 0.49 0.58 0.64 0.67 0.68 0.63 0.25 0.68 0.54 0.61 0.81 0.73 0.76 0.79 0.87 0.76 0.83 0.44 0.87 0.65 0.68 0.60 0.68
0.80 0.45 0.74 0.78 0.61 0.69 0.82 0.72 0.70 0.74 0.43 0.80 0.71 0.75 0.63 0.84 0.83 0.83 0.73 0.76 0.58 0.57 0.75 0.74 0.76 0.70 0.73
0.66 0.73 0.91 0.76 0.83 0.86 0.82 0.79 0.83 0.56 0.91 0.85 0.72 0.70 0.88 0.93 0.86 0.80 0.86 0.65 0.65 0.81 0.92 0.81 0.75 0.85
0.53 0.59 0.72 0.69 0.63 0.70 0.73 0.71 0.67 0.66 0.73 0.60 0.57 0.61 0.61 0.59 0.51 0.60 0.49 0.58 0.50 0.71 0.70 0.74 0.66
0.71 0.68 0.75 0.80 0.81 0.78 0.78 0.41 0.75 0.67 0.57 0.89 0.69 0.78 0.72 0.81 0.80 0.84 0.40 0.83 0.69 0.66 0.77 0.62
0.82 0.86 0.83 0.76 0.72 0.76 0.57 0.84 0.78 0.66 0.63 0.81 0.89 0.82 0.73 0.78 0.58 0.69 0.75 0.87 0.77 0.74 0.85
0.89 0.78 0.80 0.65 0.79 0.73 0.74 0.84 0.61 0.62 0.64 0.72 0.65 0.58 0.66 0.54 0.69 0.60 0.81 0.73 0.82 0.76
0.85 0.88 0.76 0.82 0.61 0.81 0.86 0.63 0.67 0.71 0.80 0.74 0.68 0.74 0.64 0.62 0.71 0.85 0.75 0.84 0.85
0.88 0.80 0.89 0.58 0.92 0.87 0.73 0.73 0.82 0.84 0.80 0.74 0.82 0.68 0.58 0.75 0.83 0.77 0.85 0.74
0.84 0.93 0.58 0.87 0.85 0.73 0.79 0.77 0.81 0.81 0.77 0.83 0.74 0.56 0.79 0.87 0.83 0.89 0.80
0.81 0.56 0.83 0.72 0.76 0.80 0.83 0.79 0.81 0.81 0.89 0.77 0.44 0.81 0.75 0.73 0.75 0.72
0.62 0.90 0.85 0.74 0.79 0.81 0.80 0.81 0.75 0.84 0.71 0.55 0.75 0.84 0.80 0.85 0.76
0.58 0.71 0.61 0.44 0.53 0.49 0.47 0.38 0.49 0.37 0.65 0.37 0.60 0.57 0.62 0.57
0.85 0.77 0.74 0.90 0.88 0.88 0.81 0.89 0.70 0.61 0.79 0.89 0.84 0.80 0.82
0.76 0.65 0.75 0.78 0.73 0.66 0.74 0.60 0.67 0.66 0.89 0.79 0.82 0.81
0.57 0.83 0.71 0.80 0.66 0.73 0.53 0.63 0.65 0.78 0.84 0.72 0.77
0.70 0.73 0.72 0.87 0.84 0.92 0.35 0.87 0.66 0.64 0.68 0.63
0.88 0.93 0.78 0.85 0.66 0.61 0.77 0.82 0.85 0.71 0.84
0.88 0.83 0.84 0.69 0.62 0.85 0.89 0.82 0.76 0.83
0.80 0.86 0.68 0.61 0.80 0.83 0.88 0.74 0.88
0.89 0.85 0.45 0.96 0.76 0.74 0.69 0.71
0.80 0.50 0.88 0.81 0.78 0.76 0.75
0.30 0.53 0.61 0.59 0.64 0.58
0.47 0.69 0.71 0.63 0.72
0.77 0.74 0.88 0.71 0.83 0.71 0.88
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0.86 0.86 0.72
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Fig. 3. Dendrogram based on Gower’s similarity index and UPGMA generated by seven cytological parameters for 30 genotypes.
stabilized selection.
during
micro-evolution
and
gradual
4.3. Phylogenetic analysis The phylogenetic tree constructed from the cytological parameters through the SHAN technique of cultivar analysis by the UPGMA method showed two distinct phenetic clusters, which may very well be subdivided into four groups e IA, IB, IIA, and IIB. Group IA includes six cultivars e cv.K. Pukhraj, K. Jawahar, HPS I/13, DTP-II, MS/92-1090 and MS/92-2105. The second group (IB), the major group, comprises 13 numbers of genotypes, involving a total of 30 genotypes. This group includes the cultivar K. Jyoti, four of its derivatives and others. The third group (IIA) includes eight genotypes, and the fourth, the smallest, includes three genotypes. A good clustering of K. Jyoti and its derivatives, K. Badshsh, MF-1, JTH/C-107 and JX-371, was to be expected. Similarly, genotypes JX-576 and JX-161, two different clones derived from a single cross, have shown close similarity and are included in the same group (IIA). What stands out as very curious is the large distance between K. Jyoti and 85P-718 in Group IIA, since the latter is partly derived from the former. Another curious feature is the close similarity (0.84) of a red-skinned mutant, MS/92-2105, with DTP-II, a white skinned genotype. In fact, close similarities between certain genotypes agree well with present taxonomic judgements. It is gratifying to mention that, by and large, the results from this experimental study correlate remarkably well with those based on morphology. However, further work on DNA polymorphism is needed to resolve close similarities displayed between different genotypes and to establish genetic unrelatedness among cultivars/breeding lines with a common genotype in their parentage. This analysis could help breeders to
choose diverse parents for heterosis breeding programmes aimed at varietal improvement, keeping in zmind the tetrasomic nature of inheritance in this crop. Acknowledgements We acknowledge the help of the Director, Central Potato Research Institute, Shimla, India, for providing the germplasm used in this investigation. We also thank Dr. P. Das for facilitating the laboratory work for this investigation at RPRC, Bhubaneswar, India. References Bennett M, Leitch I. Nuclear DNA amounts in angiosperms. Ann Bot 1995;76:113e76. Chattopadhyay D, Sharma AK. Chromosome studies and microspectro-photometric estimation of nuclear DNA in different strains of Coriandum sativum L. Cytobios 1990;64:43e51. Das AB. Chromosomal variability in relation with 4C DNA content in the sub tribe carinae. Cytologia 1991;56:627e32. Das AB, Das P. Estimation of 4C DNA content and karyotype analysis in edible varieties of banana (Musa acuminata). Cytobios 1994;78:213e20. Das AB, Mallick R. Nuclear DNA chromosome changes within the tribe Ammineae. Cytobios 1993;74:197e207. Das AB, Basak UC, Das P. Karyotype diversity and genomic variability in some Indian tree mangroves. Caryologia 1995;48: 319e28. Das AB, Mohanty S, Das P. Meiotic behaviour and nuclear DNA variation in some species of Mammilaria (Cactaceae). Cytologia 1997;62:253e7. Das AB, Mohanty S, Marrs RH, Das P. Somatic chromosome number and karyotype diversity in fifteen species of Mammilaria of the family Cactaceae. Cytobios 1999;97:141e51. Das AB, Rai S, Das P. Karyotype analysis and 4C DNA content in some cultivars of ginger (Zingiber officinale Ross). Cytobios 1998; 93:175e84. Fish N, Karp A. Improvements in regeneration from protoplasts of potato and studies on chromosome stability. Theor Appl Genetics 1986;72:405e12.
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Price HJ. Evaluation of DNA content in plants. Bot Rev 1976;42: 27e52. Price HJ, Bachman K, Cihambers KL, Riggs J. Detection of intraspecific variation in nuclear DNA content of Microseris douglasii. Bot Gaz 1980;141:195e8. Sharma AK, Sharma A. Chromosome techniques: theory and practice. 3rd ed. London: Butterworths; 1980. Sokal PR, Rohlf FJ. Introduction to biostatistics. San Francisco: Freeman; 1973. Sree Ramulu KP, Dijkhuis P, Roest S. Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato. Theor Appl Genetics 1983;65:329e38. Van’t Hof J, Sparrow AH. A relationship between DNA content, nuclear volume, and minimum mitotic cycle time. Proc Natl Acad Sci U S A 1963;49:897e902. Wilkinson MJ. Genome evolution in potatoes. In: Bradshaw , Mackay , editors. Potato genetics. University Press; 1994. p. 43e70.