ORIGINAL ARTICLE

Karyotype analysis of ten draught resistant cultivars of Indiantaro - Colocasia esculenta cv. antiquorom Schott

Arundhati Das & Anath Bandhu Das

Received: 6 February 2014 /# Archana Sharma Foundation of Calcutta 2014

Abstract Somatic chromosome number, detailed karyotypeand genome size analysis were made to assess genetic diver-sity in ten drought resistant cultivars of Indian Taro (Colocasiaesculenta var antiquorom Schott.).Karyotype analysis revealedgenotype specific chromosomal characteristics and structuralalterations in chromosomes of the genome, with variations ofploidy from 2n=2×=28 (cv. Mothan, cv. Muktakeshi, cv. SreeKiran, cv. Sree Pallavi, cv. Sunajhili) to 3n=3×=42 (cv. Banky,cv. DP-25, cv. Duradin, cv. H-3, cv. Telia). Highly significantvariations in the genomic length, volume and total form (TF)%were noted at variety level. Total genomic chromosome lengthvaried from 46.96 μm in cv. Sree Kiran to 100.49 μm in cv.Duradin. Total genomic chromosome volume varied from18.22 μm3 in cv. Sunajhili to 38.22 μm3 in cv. Duradin. Totalform percentage was varied from 24.94 % in cv. Sree Kiran to39.04 % in cv. H-3 confirming mostly of near metacentric tometacentric chromosomes in the karyotype. Significant varia-tions in the 4C DNA content noted among the cultivars thatranged from 7.24 pg in cv. Sree Kiran to 18.24 pg in cv.Duradin; accordingly, genome size varied from ~7,095 to17,875 Mbp. High genome size in all the triplod varieties with3 x=42 chromosomes could be due to the presence of extra setof chromosomes in the genome or high amount of repetitiveDNA. The variation in the genome size at the cultivar levelmay be attributed to loss or addition of highly repetitivesequences in the genome. Detailed chromosomal analysis often drought resistant cultivars could help breeders to choosethe diverse parents for breeding programme along with high

yielding drought susceptible varieties for future crop improve-ment programme.

Keywords 4CDNA content . Chromosomal variation .

Karyotype . Polyploidy

Introduction

Taro (Colocasia esculenta var antiquorom Schott.), a memberof the family Araceae is a traditional root crop of the tropicsgrown for its edible corms and leaves and is believed to be oneof the earliest cultivated root crops in the world [19]. World-wide production is on the increase, with Food and AgricultureOrganization (FAO) records indicating that taro productionhas doubled over the past decade [12] and is now the fifthmost-consumed root vegetable worldwide. Cultivated typesare mostly diploid (2n=2x=28) with some triploids having2n=3x=42 chromosomes. Two major taxonomical varietiesare found i.e. dasheen type (Colocassia esculenta cv.esculentus) which has large central corm with sucker andstolons and the second is the eddoe type (Colocasia esculentacv. antiquorom) which has small central corm and large num-ber of small cornels [29]. Taro is the major food crop forMelanesian and Polynesian people, and is grown vegetatively,rarely from seed, for both domestic consumption and export.There are growing concerns over the narrow genetic base oftaro cultivars in the Pacific islands, particularly with the out-break of taro leaf blight (Phytophthera colocasiae) in Samoaand American Samoa in 1993–1994. This has led to theinitiation of several breeding programs with the aim of broad-ening the genetic base of breeding populations, in addition toselection for resistance to taro leaf blight. Since, taro cultiva-tion need good irrigation for its crop yield, some of thedrought susceptible high yielding varieties required geneticimprovement through breeding. It necessitates chromosome

A. Das :A. B. Das (*)Department of Agricultural Biotechnology, College of Agriculture,Orissa University of Agriculture & Technology,Bhubaneswar 751003, Odisha, Indiae-mail: a_b_das@hotmail.com

A. Dase-mail: arundahtidas@gmail.com

NucleusDOI 10.1007/s13237-014-0113-0

number and karyotype determination of some of the populardrought resistant cultivars of Indian taro. Some of the studiesusing molecular techniques, specifically isozyme [20] andRAPD [15] were reported which have indicated that theOceanian cultivars, particularly the Polynesian cultivars, showvery little diversity and have stressed the importance of broad-ening the base of existing breeding programs.

The development of molecular markers for taro germplasmcharacterization is required for efficient breeding programmeto speed the integration of new genetic material into elitegermplasm. In addition, taro germplasm characterizationusing molecular marker will provide information on thegenetic relationships between accessions of the wild and cul-tivated genepool, and hence facilitate the breeding of tarocultivars to satisfy market needs and to respond to diversebiotic (e.g., taro leaf blight) and abiotic (e.g., drought) chal-lenges. Microsatellites have proven to be particularly usefulfor inbreeding crops with low level of intraspecific diversity[33] and are increasingly useful for root crops that are fre-quently vegetatively propagated such as cassava [4,32], sweetpotato [2] and yam [43].

Cultivar identification and techniques to assess cultivarhomogeneity are important for seed production, germplasmmaintenance, crop certification and registration. The newcultivars obtained from the restricted gene pool are likely tobe genetically quite similar and hence difficult to differentiatemorphologically. Therefore, genetic identification of cultivarsand varieties are useful in maintaining germplasm and plan-ning of breeding programme for new cultivar production tocater the demand of this crop to grow in different agroclimaticenvironment. Karyotype analysis provides valuable informa-tion related to the mechanisms of genome evolution. There arefew cytological studies in taro because of large and relativelynumerous chromosomes [46]. Abundant cytological data wererepor ted for Colocasia in many ear l ie r reports[11,14,16–18,23–26,31,34,38,39,45,47,48] but cytologicaldata are mainly associated with different populations ofC. esculenta for its variation of chromosome numbers. Re-cently, Yang et al. [47] also reported three diploid (2n=28)species of Colocasia like C. gongii, C. gaoligongensis andC. gigantea from Yunnan, China. However, no detailed infor-mation on karyotypes of Indian taro cultivars are available forbreeding purpose.

Genome size is an important character of fundamentalsignificance that provides useful data in many cytotaxonomicand evolution studies [27]. It plays an important role intolerance/resistance to low temperature and in response toozone depletion or to the effect of global warming [1]. DNAcontent report is also very meager in taro. In the presentinvestigation, an attempt has been made to utilize abovementioned techniques for assessment of genetic variation inten drought resistant cultivars of taro (Colocasia esculenta varantiquorom) for detailed analysis of chromosome structure

and ploidy determination that complement breeders to choosebreeding partner in the crop improvement programme of thisimportant minor tuber crop.

Materials and methods

Ten cultivars of drought resistant advanced breeding lines ofColocasia esculenta var antiquorom Schott. were obtainedfrom the Central Tuber Crop Research Institute, Bhubanes-war, India through the curtsy of Dr. S. K. Naskar, Director,CTCRI, Bhubaneswar (Table 1). They were grown in theexperimental green house of Orissa University of Agricultureand Technology, Bhubaneswar.

Karyotype analysis

For chromosome preparation, root tips from the sproutedtubers were pre-treated in saturated solution of pDB (para-dichlorobenzene) with aesculine for 3 h at 18 °C followed byovernight fixation in 1:3 acetic acid : ethanol. Chromosomeswere stained in 2 % aceto-orcine after cold hydrolysis in5 N HCl for 5 min. The root tips were then squashed in45% propionic acid. Ten well scattered metaphase plates fromeach genotype were selected for karyotype analysis. Thegenomic chromosome length and volume of a karyotype weredetermined following the method of Das and Mallick [10].The total genomic chromosome length was ascertained byadding the length of haploid set of chromosomes in thekaryotype and the total genomic chromosome volume ofkaryotype was calculated by applying the formula πr2h, where‘r’ and ‘h’ represents the radius and length of the chromo-some, respectively. The form % (F%) of individual chromo-somes was calculated following the method of Levan et al.[21], and the total form percentage (TF %) was the average ofthe sum total of F% of a karyotype. The mean values of totalgenomic chromosome length and total genomic chromosomevolume with standard error were calculated.

4C nuclear DNA content and genome size

For Feulgen cytophotometric estimation of 4C DNA content,ten fixed root-tips from each genotypes were fixed in 1:3acetic acid:ethanol for overnight in room temperature, hydro-lysed in 1 N HCl for 12 min at 60 °C. Hydrolysed root tipswere washed in distilled water and stained in Schiff’s reagentfor 2 h at 14 °C. Each root-tip squash was prepared in 45 %acetic acid. In situ nuclear DNA content was estimated frommetaphase chromosomes using a Nikon Optiphot microscopefitted with a microspectrophotometer using monochromaticlight at 550 nm following the method of Sharma and Sharma[35], with ten scorings made from each slide. In situ DNAcontent were obtained on the basis of optical density, which

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was converted to picograms (pg) using the 4C nuclear DNAvalues (67.1 pg) for Allium cepa cv. Deshi as a standard [44].The genome size of different genotypes was calculated fromtheir 4C DNA values and according to their ploidy level.Genome size = (4C DNAvalue/ploidy level) pg×980 Mbp=value in pg×980 Mbp. To find out the significant differencesin chromosome length, volume and genome size amongdifferent cultivars, if any, analysis of variation (ANOVA)was performed [36].

Results

Chromosome characteristics and 4C DNA content

Detailed analysis of somatic chromosomes of ten varieties ofC. esculenta cv. antiquorom of the family Araceae showedsomatic chromosome number 2n=2x=28 chromosome incv. Mothan (Figs. 9 & 9a) cv. Muktakeshi (Figs. 8 & 8a),cv. Sree Kiran (Figs. 4 & 4a), cv. Sree Pallavi (Figs. 5 & 5a),cv. Sunajhili (Figs. 10 & 10a) and 2n=3x=42 chromosomein cv. Banky (Fig. 1 & 1a), cv. DP-25 (Figs. 6 & 6a), cv.Duradin (Figs. 7 & 7a), cv. H-3 (Figs. 3 & 3a), cv. Telia(Figs. 2 & 2a). On the basis of the size of the chromosomeand the position of the constrictions, a number of chromo-some types were found common with the genotypes studiedthough they differed from each other in the minute structuraldetails of the karyotype.

A general description of the representative types of chro-mosomes is given below.

Type A Chromosomes are large to medium sized with twoconstrictions in nearly median to median and near-ly sub median to sub median in positionrespectively.

Type B Large to medium sized chromosomes with oneconstriction comprised with median to nearly me-dian primary constriction.

Type C Chromosomes are medium to small with sub me-dian to nearly median primary constrictions.

Type D Chromosomes are comparatively smaller to Type‘C’ chromosome with nearly sub median to submedian primary constrictions.

All the four types of chromosomes were present only incv. Banky, but the Types A, B and C chromosomes werepresent in rest of the cultivars (Table 1). The karyotypeformula of all the genotypes revealed definite differences inthe chromosome structure. Type A chromosomes were pres-ent in all the genotypes where as Type Dwere only present incv. Banky. Furthermore, dose differences in Type A, B and Cchromosomes in all the genotypes were found. Type CT

able1

Somaticchromosom

enumber,karyotypicparametersand4C

DNAcontento

ftengenotypesof

C.esculenta

cv.antiquorom

Cultiv

ars

Som

aticchromosom

eNo.(2n=2x,3x)

Karyotype

form

ula

Totalg

enom

icchromosom

elength

inμm

±S.D.

Totalg

enom

icchromosom

evolumein

μm

3±S.D.

F%

4CDNA

content±

S.D.

DNAcontentp

erchromosom

eGenom

elength

Mbp

Banky

424A

+12B+16C+10D

77.00±1.23

29.59±0.35

41.02±0.54

15.22±0.08

0.362

14915.6

Telia

422A

+18B+22C

95.19±1.55

36.07±1.30

37.94±0.65

16.98±0.09

0.404

16640.4

H-3

424A

+18B+20C

90.64±0.98

31.06±0.98

39.04±0.92

16.10±0.06

0.383

15778.0

Sree

Kiran

282A

+12B+14C

46.96±2.34

22.24±1.22

24.94±0.56

07.24±0.05

0.258

7095.2

Sree

Pallavi

282A

+12B+14C

53.02±1.25

24.56±1.24

33.29±0.82

08.22±0.08

0.293

8055.6

DP-25

426A

+10B+26C

86.35±2.55

29.03±0.96

34.10±0.45

17.64±0.51

0.420

17287.2

Duradin

424A

+18B+20C

100.49

±3.24

38.22±1.50

34.25±0.55

18.24±0.25

0.434

17875.2

Muktakesi

284A

+12B+12C

71.20±1.26

28.65±0.66

38.47±0.97

09.25±0.06

0.330

9065.0

Mothan

282A

+10B+16C

55.04±0.95

20.24±0.75

38.43±0.39

08.11±0.08

0.289

7947.8

Sunajhili

282A

+10B+16C

51.50±2.51

18.22±0.67

35.25±0.76

08.55±0.12

0.305

8379.0

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chromosomes were the most numerous in all the genotypesthat varied from 14 in cv. Sree Kiran and cv. Sree Pallavi to 26in cv. DP-25. The number of Type B chromosomes were notvaried that much as compared to Type C; cv. Muktakesi, cv.

Mothan and cv. Sunajhili possessed 10 numbers were as cv.Banky, cv. Sree Kiran and cv. Sree Pallavi showed 12 numbersand cv. Telia, cv. H-3 and cv. Duradin had 18 numbers. Thehighest number of Type C chromosomes was found in the

Figs. 1–10 Somatic metaphasechromosomes of 10 cultivars oftaro (C. esculenta var.antiquorom) (×1942). 1=cv.Banky (2n=42), 2=cv. Telia (2n=42) 3=cv. H-3 (2n=42), 4=cv.Sree Kiran (2n=28) 5=cv. SreePallavi (2n=28), 6=cv. DP-25(2n=42), 7=cv. Duradin (2n=42), 8=cv. Muktakeshi (2n=28),9=cv. Mothan (2n=28) and 10=cv. Sunajhili (2n=28)

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genotype cv. DP-25 a triploid. Detailed analysis of the somaticcompliments and the different genomic characteristics showedgenotype specific variations in chromosome structure(Table 1). The total genomic chromosome length varied from

46.96 μm in cv. Sree Kiran to 100.49 μm in cv. Duradin andthe total genomic chromosome volume varied from18.22 μm3 in cv. Sunajhili to 38.223 in cv. Duradin. Thecentromeric index in the chromosomes of all the genotypesvaried from 24.94 % in cv. Sree Kiran to 38.472 % in cv.Muktakeshi. Significant variations in chromosome length,volume and TF% were observed among the studied tarocultivars from the analysis of variance.

4C DNA content varied from 7.24 p.g. in cv. Sree Kiran to18.24 p.g. in cv. Duradin. In diploid varieties (2n=28), theminimum nuclear DNA obtained 7.24 p.g. in cv. Sree Kiranand maximum 9.25 p.g. in cv. Muktakeshi whereas, amongtriploid varieties (3n=42) DNA content was varied from15.22 p.g. in cv. Banky to 18.24 p.g. in cv. Duradin. However,DNA content per chromosome ranged from 0.258 in cv. SreeKiran to 0.434 in cv. Duradin. Accordingly, the calculatedgenome length also varied from 7095.2 to 17875.2 Mbp(Tables 1 and 2).

Discussion

Karyotype, chromosome length, and genome size

Detailed karyotype analysis in 10 cultivars of C. esculenta cv.antiquorom revealed some interesting facts at inter-cultivarlevel. The chromosome number (2n=2x=28) was constant in

Figs. 1a-10a Karyotypes of tencultivers of taro. 1=cv. Banky(2n=42), 2=cv. Telia (2n=42) 3=cv. H-3 (2n=42), 4=cv. SreeKiran (2n=28) 5=cv. Sree Pallavi(2n=28), 6=cv. DP-25 (2n=42),7=cv. Duradin (2n=42), 8=cv.Muktakeshi (2n=28), 9=cv.Mothan (2n=28) and 10=cv.Sunajhili (2n=28)

Table 2 ANOVA table of 4C DNA content, chromosome length, chro-mosome volume and F% in different cultivars of taro

Source DF SS MS F

4C DNA content

Between cultivars 9 55.24 6.14 31.20**

Within cultivars 90 18.20 0.20 –

Total 99

Chromosome length

Between cultivars 9 45.26 5.02 8.96**

Within cultivars 40 22.45 0.56 –

Total 49

Chromosome volume

Between cultivars 9

Within cultivars 40 53.25 5.91 18.46**

Total 49 13.14 0.32 –

Total Form Percentage (TF%)

Between cultivars 9 62.55 6.95 11.03**

Within cultivars 40 25.23 0.63 –

Total 49

**Significant at p≥0.001, DF, degree of freedom; SS, sum squares; MS,mean squares; F, variance ratio

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cv. Muktakeshi, cv. Mothan, cv. Sree Kiran, cv. Sree Pallaviand cv. Sunajhili and (2n=3x=42) was constant in rest of thestudied varieties (Table 1) having triploid set of chromosomesin the root tip cells. Type of chromosomes and the number ofsecondary constricted chromosomes varied significantly withthe genotypes. Types A, B and C chromosomes were commonin all the genotypes, with high variability in terms of chromo-somes in each category. However, Type D chromosomes, veryshort sub-median type, was only present in a triploid cv.Banky (Table 1, Figs. 1 & 1a). Furthermore, in respect ofkaryotype formula, there were no differences between cv. SreeKiran and cv. Sree Pallavi having 2n=28 chromosomes. Incontrast, the genotype chromosome length and volume variedsignificantly. Detailed karyotype analysis revealed the numbersecondary constricted chromosomes i.e. Type A chromo-somes, were varied from 2 to 6. Median chromosomes (TypeB) were comparatively less in number as compared to sub-median chromosomes (Type C) in diploids whereas of medianchromosomes in triploids. Type B obtained in triploid varie-ties was 18 except cv. DP-25 (10 numbers) and cv. Banky (12numbers) whereas in diploid cultivars it was 10 in numberexcept cv. Sree Kiran and cv. Sree Pallavi having 12 numbers.The variation of Type B and Type C chromosomes were notpronounced among diploid genotypes cv. Muktakeshi, cv.Mothan and cv. Sunajhili as well as cv. Sree Kiran and cv.Sree Pallavi but a significant variation was observed amongtriploid genotypes (Table 1). Total F% analysis showed sym-metric karyotype having median to nearly median chromo-somes with a moderate fluctuation of F% value from 33.29 to41.02 % in cv. Sree Pallavi to cv. Banky respectively exceptcv. Sree Pallavi with 24.56 %. The sifting of median chromo-some of genome to highly sub-median chromosomes in cv.Sree Pallavi as compared to the other studied cultivars mightbe due to the break and reunion of the more chromosomes inevolution for stabilization of these vegetatively propagatedplant. The gradual alterations and shifting of TF% valuesmight be due to the chromosomal alteration in the genome.The structural alterations in the chromosome morphology aswell as variations of secondary constricted chromosomes inthe genotypes might be due to duplication of chromosomes ortranslocations between the chromosomes with or withoutsecondary constrictions at a very early stage of evaluation [7].

Total chromosome length and volume differed markedlyamong the genotypes. Minute observations showed a propor-tional increase in chromosome length with an increase inchromosome volume. A correlation coefficient of 0.69 wasfound between the total chromosome length and total chro-mosome volume suggesting a high interdependence betweenthem at the cultivar level. These facts indicate thepredetermined genetic control of chromosome coiling. Evi-dently, differences in chromosome length or chromosomevolume were due to differential condensation and spiralizationof the chromosome arms. In addition, the genotype specific

compaction of DNA threads along with nucleosomes or theadditional gene sequences with altered non-histone proteins inthe chromosome played an important role in the chromosomalarchitecture of the genotypes [3]. Earlier cytological studieson Colocasia indicated some confusion concerning the basicchromosome number of the genus, and some different chro-mosome numbers were estimated, such as 2n=28, 36 and 42,and x=7, 12 and 14 were suggested as the basic chromosomenumber of Colocasia by some previous researchers[5,6,24,30,31]. But, cultivated genotypes of C. esculenta var.tiquorom possess the same chromosome number, 2n=2x=28and 2n=3x=42 as reported earlier. According to chromosomebehaviour in meiosis [24,45], the basic chromosome numberof Colocasia could be taken as x=14. The populations ofC. esculenta with 42 chromosomes are triploid, 3x=42 whichis in accordance with Yang et al. [47] for 2n=28. The fact thatplants with 42 chromosomes are sterile is one of evidences;x=12 were suggested as a basic chromosome number ofColocasia based on the observations of Rao [31]. However,none of the more recent studies on Colocasia have confirmedx=12 as a base number. It therefore seems that the plantsobserved with a base number of 12 were either misidentifiedas Colocasia species, or that the chromosome counts wereinaccurate. Three species studied by Yang et al. [47] i.e.C. gongii, C. gigantean and C. esculenta were diploid with2n=2x=28. C. esculenta is the only species inColocasiawithvarious chromosome numbers and various basic chromosomenumbers. The varieties with chromosome number of 2n=42were triploid with a basic chromosome number of x=14 butwere not hexaploid with a basic chromosome number of x=7.The chromosome number of varieties cultivated ofC. esculenta may vary due to long history and the variousconditions of cultivation. Differences in the numbers of me-dian and sub-median chromosomes as well as satellite-chromosomes among species were reported by Yang et al.[47] in inter-specific level as well as varietal level ofC. esculenta, which showed diversity not only in the chromo-some number but also in karyotypes as reported earlier [40].So, karyotypes cannot be compared between C. gongii, C.gigantean and C. esculenta. It is very necessary to study otherspecies in Colocasia for revealing phylogenetic relationshipsof whole genus. Chromosome number reported from root tipcells revealed that diploids (2n=28) and triploids (2n=42)occur in Indian taros in almost equal proportion. The frequen-cy of the ploidy types showed clear difference in ploidy-wisedistribution in the different zones of the country. Althoughboth the types occur in all the regions, the diploids predomi-nate in South India over the triploids while the triploidsconvincingly out-numbered the diploids in the north [41].Several factors are known to influence the frequency of poly-ploids in different eco-geographical regions. However, poly-ploids in general have larger dimensions and greater adapt-ability which apparently enable them to thrive better in a wide

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range of higher latitudinal and altitudinal zones. As in the caseof Indian taros, Zhang and Zhang [49] also observed a greaterpercentage of triploid forms in higher altitude regions of China.Initial screening of the germless accessions for tuber yieldrevealed the superiority of triploids compared to diploid acces-sions in several characters such as plant height, tillering habit,number and size of leaves, corm and cormel yield. The cormand cormel yield showed very promising and impressive in-crease in the higher ploidy types. This implies that for selectinghigh yielding types in taro, it is desirable to consider thetriploids rather than diploids [42]. The same was found to betrue in another tuber crop viz. cassava which showed signifi-cant increase in tuber yield and starch content in the artificiallyproduced triploids which might be due to triploidy per se [37].

Diversification in genome size

The genome size varied significantly from ~7,095 Mbp in cv.Sree Kiran to ~17,875 cv. Duridan. The triploid cultivars had amuch larger genome size as compared to diploids which mightbe due to the extra set of chromosomes as well as largechromosome size as revealed in the karyotype. The variabilityin the genome size in different genotypes might be attributed tothe loss or addition of many repeats in the genome throughalterations in the micro- and macro-environment during evolu-tion in the selection of new cultivars [28]. The correlationcoefficient between total chromosome length and genome sizeshowed significant correlation (r=0.521). This clearly suggeststhat the genome size is positively correlated with the totalchromosome length. Such variations are in agreement withthe findings of other workers [7–9,11]. The analysis of genomesize at the cultivar level in repeated experiments revealed thestable genome size in each genotype. On the other hand thegenome size differed significantly among the genotypes.Flavell et al. [13] reported that differences in genome sizedepend on the repetitive DNA amount. We agree that variabil-ity of genome size can be attributed to loss or addition of highlyrepetitive DNA sequences rather than the AT- or GC-richsequences in a genome [22] which reached a certain level andgot stabilized during micro-evolution and gradual selection.

Acknowledgments The Central Tuber Crop Research Institute, Bhu-baneswar, India is highly acknowledged for supplying different varietiesof Colocasia esculenta (var antiquorom Schott) germplasms, the neces-sary research material for starting this work. The Department of Agricul-tural Biotechnology, College of Agriculture, OUAT, Bhubaneswar hasalso been acknowledged for providing laboratory facilities for this work.

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