Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 3-9, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-840 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Matthew Jo, Faluyi Jo (2021) Chromosomal analysis of eight culti- vars in three species of cultivated Yam (Dioscorea L.) species in Nigeria. Car- yologia 74(2): 3-9. doi: 10.36253/caryolo- gia-840 Received: January 02, 2020 Accepted: April 26, 2021 Published: October 08, 2021 Copyright: © 2021 Matthew Jo, Faluyi Jo. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID MJ: 0000-0003-3401-9327 Chromosomal analysis of eight cultivars in three species of cultivated Yam (Dioscorea L.) species in Nigeria Joshua Matthew1,2,*, Julius Oloaye Faluyi1 1 Botany Department, Obafemi Awolowo University, Ile-Ife, Nigeria 2 National Horticultural Research Institute, Ibadan, Nigeria *Corresponding author, jolumatthew@gmail.com Abstract. The genus Dioscorea comprises of economically-important plant species known for their starch throughout the world; it is also a major source of food and income in Africa. The most important Dioscorea species cultivated and consumed in the West Africa belt include D. cayenensis, D. rotundata and D. alata. The plant materi- als used in this study were collected from Omu-Ekiti, Oye Local Government of Ekiti State, Nigeria using the On Farm Participatory Method (OFPM). Mitotic chromo- some studies were carried out on three species viz, Dioscorea alata (‘ewura’), D. cay- enensis (‘igangan’) and D. rotundata (‘areyingbakumo’, ‘gaungaun’, ‘ikumo’, ‘ogunmole’ and ‘sandpaper’). Mitotic chromosome studies were carried out on each of the cultivars using the root tip squash method made in modified Orcein stain (FLP-Orcein). Dor- mant tubers were cut to mini-setts and placed in carbonised rice husk for rooting. This study reports the basic chromosome number of x = 8, i.e. 2n = 4x =32 (D. alata), 2n = 4x =38 (D. rotundata) and 2n = 8x = 68 (D. cayenensis) for Dioscorea suggesting that both D. rotundata and D. cayenensis are aneuploids. Keywords: Dioscorea, Chromosomes, aneuploids, polyploids, mixoploidy. INTRODUCTION The genus Dioscorea L. belongs to the family Dioscoreaceae which includes about 90% of the species in the family (Murti 2001). The genus Dioscorea is principally tuber-bearing and has great economic value in the tropics as food, pharmaceutical, starch, socio-cultural uses and source of income to farmers in West and Central Africa (Asiedu et al. 1998; Séka et al. 2009). The most important Dioscorea species cultivated and consumed in the West Africa belt include D. cayenensis Lam., D. rotundata Poir. and D. alata L. (IITA 2009). West African countries produce over 90% of world Dioscorea, of which, Nigeria is the largest producer of Dioscorea in the world, producing over 60% of the world Dioscorea (FAO 2016; 2017). Dioscorea has presented a challenge to systematists for many years due to its great morphological diversity, its reproductive biology: dioecy, small flowers, low or no seed set and tuber propagation (Wilkin et al. 2005). Nor- 4 Joshua Matthew, Julius Oloaye Faluyi man et al. (2012) has reported the difficulty of chromo- some studies in yams (Dioscorea spp.) due to the small dot-like chromosomes and few dividing cells in the root tips. Also, mixoploidy has been reported to be charac- teristic of many highly productive commercial cultivars with small chromosome sizes (Kunakh 2005; Kunakh et al. 2008). Mixoploidy was reported by Baquar (1980) in some Dioscorea species studied which he tagged “odd chromosome numbers” Dioscorea alata was reported to have the highest diversity in polyploidy ranging between 2n = 30 and 2n = 80 (Sharma and Deepesh 1956; Franklin and Oritz 1963; Egesi et al. 2012). The ploidy levels of D. cayenen- sis have been reported to range between a tetraploid (2n = 4x = 40) and an octoploid (2n = 8x = 80) on a basic chromosome number of 10 (Baquer 1980; Gamiette et al. 1999; Dansi et al. 2001). A basic chromosome number of 20 has been suggested using D. cayenensis (Dora et al. 2005) and D. alata (Arnau et al. 2009). Flow cytometry has offered some advantages in ploidy level analysis (Babil et al. 2010). Two ploidy lev- els (4x and 8x) were detected by flow cytometry in two populations of D. cayenensis and D. rotundata culti- vars (Dansi et al. 2000; Babil et al., 2010). However, this technique has failed to detect aneuploidy in this popu- lation. Therefore, Babil et al. (2010) recommended the use of classical chromosome studies to determine ploidy levels and solve the complication of mixoploidy in the genus Dioscorea. Babil et al. (2010) then recommended that determination of the basic chromosome number of Dioscorea spp. requires further investigations. Norman et al. (2012) advocated the need for chromosome stud- ies which is necessary to clarify the structure, function, organisation and evolution of yam genomes. The aim of this study was to investigate the chro- mosome number of the cultivars in the three major Dioscorea species that are present in Nigeria using the squash technique. The results presented will be useful both in the identification and understanding of the phy- logenetic relationship among the major cultivars in the three major species of Dioscorea. METHODOLOGY The plant materials used in this study were collected from Omu-Ekiti (N 07.90497’ E 005.39092’) in the Oye Local Government of Ekiti State, Nigeria. This commu- nity typifies an epicentre of loss of genetic resources as a result of mass adoption of introduced Dioscorea cultivars by migrant farmers from the Middle Belt of Nigeria in the last twelve years. Mitotic chromosome studies were carried out on seven cultivars in three species: Dioscorea alata, D. cayenensis and D. rotundata. Dormant tubers were cut into mini-setts placed in carbonised rice husk for rooting. The root tips were harvested between 10.30– 11.30 am, rinsed in water and transferred into 1:3 Ace- tic Ethanol fixative. This was left on the working bench for 24 h at room temperature before keeping in the refrigerator for future usage. The root tips for examina- tion were hydrolysed in 18% HCl for 10 min, squashed and stained with FLP-Orcein. Photomicrographs of the good mitotic chromosome spreads were documented under oil immersion (x1000) objective magnification using BK Series Phase Contrast Microscope (PW-BK 5000T) equipped with a DCM510 5 Megapixel camera. The chromosome numbers were based on five consistent counts. RESULTS Phenotypic variation in leaf characters Table 1 and Figure 1 show the forms and shapes of the leaves of some of the Dioscorea species stud- ied. All the cultivars studied had simple, glabrous, cor- date leaves. The petiole of D. alata (‘ewura’) had purple wing which was absent in other species. The leaves of D. rotundata (‘areyingbakumo’) and D. cayenensis (‘igan- gan’) cultivars had orbiculate (broad cordate) leaves. Table 1. Leaf characteristics of the Yam cultivars studied. Species Local name Foliar description Dioscorea alata Ewura Green colour, ovate shape, acuminate apex, cordate base and winged petiole. Dioscorea cayenensis Igangan Light green colour, orbicular shape, acuminate apex and cordate base. Dioscorea rotundata Areyingbakumo Green colour, orbicular shape, acuminate apex and cordate base. Gaungaun Green colour, ovate shape, acuminate apex and sagittate base. Ikumo Green colour, orbicular shape, acuminate apex and cordate base. Ogunmole Green colour, ovate shape, acuminate apex and cordate base. Sandpaper Dark green colour, long ovate shape, acuminate apex and sagittate base. 5Chromosomal analysis of eight cultivars in three species of cultivated Yam (Dioscorea L.) species in Nigeria However, the leaf colour of D. cayenensis was light green while that of D. rotundata were dark green. Only, D. rotundata (‘sandpaper’) had long cordate leaves. The cul- tivars had acuminate leaf apices. The leaf bases observed in D. rotundata, (‘gaungaun’ and ‘sandpaper’ cultivars) were sagittate while other D. rotundata cultivars studied had cordate leaf bases. Chromosome number and morphology The mitotic chromosome counts observed in the Dioscorea species studied were shown in Figures 2 and 3. A mitotic chromosome count of 2n = 32 was observed in D. alata cultivar while a mitotic chromosome count of 2n = 68 was observed in the D. cayenensis cultivar (Figure 2). The mitotic chromosome count observed in all the D. rotundata cultivars was 2n = 38 (Figures 2 and 3). Table 2 shows the mitotic chromosome counts in this study and the previous chromosome counts. The mor- phology of the chromosomes could not be ascertained in this study because of their small sizes. DISCUSSION The findings of this study revealed morphological variations between and within the leaves of the cultivars of the Dioscorea species studied. As established in Table 1 and Figure 1, the distinct by its winged petiole of D. alata distinguishes it from delimit the D. rotundata and D. cayenensis cultivars while can be. Also, leaf colour Figure 1. Leaf form and shape in some the cultivars of Dioscorea studied. (a) D. cayenensis (b) D. rotundata (c) D. rotundata (d) D. rotun- data 6 Joshua Matthew, Julius Oloaye Faluyi serves as a delimiting character, D. cayenensis has light- green leaves while D. rotundata has dark-green leaves and D. alata leaves are green. The orbicular leaf shape of D. cayenensis distinguish it from some D. rotundata and D. alata cultivars. However, in the morphological char- acterisation of Dioscorea, there is possibility of overlap of characters, therefore, the use of multiple delimiting fea- tures is important for their characterisation. This study established a chromosome number of 2n = 38 for the four cultivars of Dioscorea rotundata, 2n = 32 for D. alata and 2n = 68 for D. cayenensis. None of the mitotic counts observed in this study is in agree- ment with the previous mitotic chromosome num- bers reported for yam (Table 1). (Bousalem et al. 2006) reported that the dot-like and varying chromosome sizes that occurred in the mitotic cells of Dioscorea made the definite determination of chromosome numbers dif- ficult. The mitotic chromosome counts reported in this study were smaller compared to the mitotic chromo- some numbers earlier reported. Asiedu et al. (1998) had reported the occurrence of smaller chromosome num- bers and polyploidy levels in the species of Dioscorea from Asia and Africa. This study reports the basic chromosome number of x = 8 in D. alata (2n = 4x =32), D. rotundata, (2n = 4x =38) and D. cayenensis (2n = 8x = 68). The x = 8 basic chromosome number agrees with the findings of (Dansi et al. 2001). There was no mixoploidy observed. Baquar (1980) reported odd chromosome numbers that were not direct multiples of their basic chromosome numbers which he tagged “odd chromosome numbers”. The study cytogenetics of Dioscorea using roots from tubers could give a better result in terms chromosome morphology and stainability compared to vines generated roots. The findings indicate that D. rotundata (2n = 38) and D. cayenensis (2n = 68) are distinct species. This Figure 2. Mitotic metaphase spreads in the Dioscorea species studied. A. D. alata (Ewura), 2n = 32 (Arrows show mitotic chromosome overlaps); B. D. alata (Ewura), 2n = 32; C. D. cayenensis (Igangan), 2n = 68 (Arrow shows mitotic chromosome overlap); D. D. cayenensis (Igangan), 2n = 68; E. D. rotundata (Ikumo), 2n = 38 (Arrows show mitotic chromosome overlap); F. D. rotundata (Ikumo), 2n = 38. 7Chromosomal analysis of eight cultivars in three species of cultivated Yam (Dioscorea L.) species in Nigeria study affirms that D. cayenensis is a distinct species from D. rotundata thus corroborating the results of Bressan et al. (2014) who classified the two species separately through isozymatic analysis. D. cayenensis might be a speciated polyploid of D. rotundata based on the leaf morphology and the chromosome count reported in this study. Dioscorea is principally propagated vegetatively, hence, D. cayenensis could have arose through the pro- cess somatic cell divisions and polyploidy. The prob- ability that this occurrence could have been as a result of abnormality in the somatic cell divisions of the plant- ing materials (Sharma and Deepesh 1956; Stebbins 1971; Baquar 1980) is considered remote. Polyploidy has been reported in domesticated plants which include Dioscorea species (Lewis 1980; Leitch and Leitch 2008; Jeredi et al. 2012). Based on a basic number of x = 8 (Dansi et al., 2001), D. alata (2n = 4x =32) is a tetraploid and D. rotundata (2n = 38) can only be an aneuploid trisomic for six linkage groups while D. cay- enensis (2n = 68) would be an octaploid with four tri- somic sets. CONCLUSION The chromosome numbers reported in this work are based on five consistent counts for all the cultivars. It is difficult to agree that mixoploidy is an issue in the chro- mosome numbers of the cultivars studied because the analysable cells did not show wide variations in chro- mosome number. On the other hand, for a crop that is maintained by clonal propagation, the occurrence of multiple chromosome numbers is not impossible, espe- cially since a cultivar is not a taxonomic hierarchy. Rath- er, it is characterized by a cluster of valuable food and agronomic attributes that have distinguished it for selec- tion and conservation through generations of cultivation by peasant farmers. ACKNOWLEDGEMENTS The authors are grateful to Dr S.O. Azeez (Botany Dept. OAU) and Mr O.F. Oyelami (IITA, Ibadan) for the information and suggestions provided in the course of this study. Figure 3. Mitotic metaphase spread in Dioscorea species studied. A. D. rotundata (Gaungaun), 2n = 38 (Arrows show chromosome over- laps); B. D. rotundata (Gaungaun), 2n = 38; C. D. rotundata (Sandpaper), 2n = 38 (Arrows show chromosome overlaps); D. D. rotundata (Ogunmole), 2n = 38 (Arrows show chromosome overlaps); E. D. rotundata (Areyingbakumo), 2n = 38; D. rotundata (Areyingbakumo), 2n = 38 (Arrow shows chromosome overlap) 8 Joshua Matthew, Julius Oloaye Faluyi REFERENCES Abraham, K., Nair, P. G., 1991. Polyploidy and sterility in relation to sex in Dioscorea alata L. (Dioscoreaceae). 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Taxonomic and phylogenetic significance of leaf venation characteristics in Dioscorea plants. Archives of Biological Sciences 70(2): 397-407. Wilkin, P., Scholes, P., Mark, W.C., Kongkanda, C., Car- ol, A.F., Suzy, H., Franck, R., Erik, S., Chirdsak, T., 2005. A plastid gene phylogeny of the yam genus, Dioscorea: roots, fruits and Madagascar. Syst. Bot. 30(4): 736-749. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 1 - 2021 Firenze University Press