Collection, Characterizaton and Conservation of Genetic Resources of Yam Cultivars From Ekiti State, Nigeria

This study was initiated to restore the genetic resources of yam which had been decimated in a core yam-producing community in Ekiti State, Southwestern Nigeria. Twenty cultivars, consisting of Dioscorea rotundata, D. cayenensis, D. alata , and D. dumetorum were identified, collected, characterized and multiplied using an On-farm Participatory Method. The yam cultivars were characterized for quality and yield of pounded yam ( iyan ) as well as starch properties. They were later conserved in the Teaching and Research Farm of the Obafemi Awolowo University Ile-Ife. Morphological characterization separated Dioscorea alata ( Ewura ) by its winged vines while Dioscorea dumetorum ( Esuru ) was separated by its pubescent spines, trifoliate leaf with acute apex and base and the clustered, irregularly-shaped tuber. All the tubers of the yam cultivars had high storability. Pounded yam quality rated the Ikumo and Ajimokun cultivars as best while Odo was rated average and this was attributed to the swelling properties and amylose content of these cultivars. Yam cultivars with high granules had low swelling capacities. The Brittle Fraction Index of the starch from all the cultivars was lower than 1.0 explaining why the iyan they produced had no crust on the surface and kept for long hours after preparation. From this study, it was concluded that the local yam cultivars collected represent the core of yam genetic resources for utilization in the region. These cultivars are therefore recommended for prioritization in further studies on propagation, conservation and improvement so that a narrow genetic base of cultivars is not encouraged, for example, by promoting cultivars whose vines perform well in tuberization.


INTRODUCTION 1
The family Dioscoreaceae is the yam family comprising of five genera and 750 species (Murti 2001). Caddick 2 et al. (2000a, b) reported that the family includes four genera: Dioscorea, Trichopus, Taca and Stenomeris. The 3 genus Dioscorea is the largest of the genera with about 90% of the species in the family. These species are 4 represented in all the geographical regions in which representatives of the family occur (Smith 1937;Onwueme 5 1978).

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The genus Dioscorea are principally tuber-bearing plants and they have economic value as food in the tropics.

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About 600 species are distributed in the subtropical and tropical areas of Africa, America, Asia and Polynesia.
8 About 90 of these species are edible; 10 of them are cultivated for food in West and Central Africa (Coursey 9 1967;Ayensu 1972;Mignouna et al. 2003;Adegbite et al. 2006;Quain et al. 2011). Nigeria has been reported 10 to be the largest producer of yam in the world while Ghana and Cote d'Ivoire are competing for the second and 11 third positions (Nweke et al. 1991;FAO 2010FAO , 2013.

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The most important Dioscorea species cultivated for consumption in the West African belt are D. cayenensis, D.
13 rotundata and D. alata (IITA, 2009). The tuber of the yam crop is particularly important as a source of 14 carbohydrates, proteins, minerals and vitamins. Their low glycemic index gives better protection against obesity 15 and diabetes (Bell 1983;Eni 2008). Yam is also important for its cultural uses, pharmaceutical products and as 16 a major source of income generation for the people in yam-growing areas.

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The physico-chemical properties of the cultivars of yam have been investigated as a means of understanding the 18 food quality of the major cultivars used for food for generations. Some properties like steaming time and size of 19 starch particles have been reported to affect the functional qualities of yam flour (FAO 1991;Iwuoha and 20 Nwakanma 1998;Iwuoha 1999). Malomo and Jayeola (2010) however suggested that the chemical properties 21 of yam starch depend less on granule dimensions but more on molecular properties and associative forces 22 intrinsic in the cultivars. Iwuoha and Nnanemere (2003) canvassed the need for a comprehensive evaluation of 23 post-extraction and handling of yam flour in the classification of tubers of cultivars of yam.

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Yam is a major staple food in Nigeria. The germplasm of yam is a tested combination of cultivars which 25 farmers have used over generations during for which they have built a massive array of protocols in yam care, 26 cultural practices, propagation techniques and preservation through indigenous knowledge.

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Yam research in Nigeria has focused on propagation, the state of the seed yam system, conservation of yam 28 germplasm and improvement through selection and breeding. The work on yam propagation has addressed mini 29 setts (Balogun 2004(Balogun , 2007(Balogun , 2009, micropropagation through plantlets raised in regular cultures and aeroponics, 30 microtuber production (Balogun 2014;Aighewi et al. 2015), generation of plantlets through regular callus 31 production and somatic embryogenesis, production of encapsulated embryos and propagation from regular 32 botanic seeds. Yam production in Nigeria is restricted to the South-Eastern, South-Western, Middle-Belt and

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East of River Niger zones where the soil fertility, humidity and rainfall permit its production.

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This paper reports a phenomenon of genetic erosion of yam in Ekiti State, Nigeria which is known as a major 35 centre for yam marketing and consumption as staple food when pounded into a paste known as iyan. This 36 phenomenon began about 15 years ago when migrants from the Middle Belt region of Nigeria migrated to Ekiti State to farm (Oluwasusi and Tijani 2013;Agoyi 2013). The indigenous farmers in the State adopted many of 38 their cultivars which were inferior in pounded yam quality and agro-botanical attributes. By the time they 39 pulled back, their yam germplasm had suffered substantial genetic erosion.

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This project was located in Omu-Ekiti in the Oye Local Government Area of Ekiti State which is an epicentre of 41 this loss of genetic resources.

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The specific objectives of this study were to:

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These cultivars were moved to the Teaching and Research Farm at the Obafemi Awolowo University, Ile-Ife for 56 the validation of the morphological characters of the cultivars, multiplication and conservation for future use.

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Morphological description of the yam cultivars was done according to the IPGRI Yam Descriptors (1997) with 58 slight modifications. The yam cultivars were described at the young and mature vegetative stages based on their 59 vine characters (habit, colour, number; presence or absence of wings, spines, pigmentation). The leaves were 60 characterized by shape, length, breadth, presence or absence of hairs, colour and multiple characters of the 61 petiole (spine, hairiness, length, breadth and colour). The tubers were characterized based on their length, 62 circumference, shape, number of tubers per hill, hair density and distribution pattern, number of cusps.

Quality and Yield of Pounded Yam (iyan) from the Cultivars Collected.
Yam tubers stored for 6 months and freshly-harvested yam tubers (less than 2 weeks) were used in the 65 preparation of yam paste (iyan) using the traditional pestle and mortar system (see Plate 1). Professional 66 pounded yam sellers operating within the Obafemi Awolowo University campus were engaged for this part of 67 the study. Tubers of the yam cultivars collected were peeled and sliced and then made into pounded yam. The 68 parameters recorded are weight of the tubers before and after peeling, yam cooking time, volume of water used 69 in pounding and final weight of the pounded yam paste (iyan) for each cultivar. The iyan from the yam cultivars 70 were rated good, average and poor by the pounders based on the volume of water required by the cooked tuber 71 during pounding, swelling of the pastes and the paste elasticity and viscosity.

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Starch was extracted from peeled, weighed and diced yam tubers soaked in distilled water for 48 hours to soften 78 according to the modified method of Farhat et al. (1999). The soaked diced pieces were blended and the slurry 79 was poured into a container filled with distilled water for 24 hours followed by sieving to obtain the extracted   where, b = minimum Feret diameter, l = maximum Feret diameter, A = projected area of the particle and p = 98 perimeter of the particle.

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The starch particle density was determined using the liquid pycnometer method according to Alebiowu and 100 Itiola (2002). In this method, acetone was used as the displacement fluid. The bulk density of each starch 101 powder at zero pressure (loose density) was determined by pouring the powder at an angle of 45º through a 102 funnel into a glass measuring cylinder with a diameter of 31 mm and a volume of 10 mL (Paronen, 1983;Itiola, 103 1991). Determination was done in triplicate. The relative density, D0, of each starch powder was obtained from 104 the ratio of its loose density to its particle density. The Hausner's ratio (Herman, 1989), determined as the ratio 105 of the initial bulk volume to the tapped volume, was obtained by applying 100 taps to 30 g of each starch sample 106 in a graduated cylinder at a standardized rate of 38 taps per minute according to the British Standard Institution 107 (1979). The packing properties were obtained using a modification of Kawakita equation and the degree of 108 volume reduction due to tapping was calculated from Equation (8).
where, N = the number of taps and C = volume reduction due to tapping, Vo and VN are the powder bed volumes 111 at initial and nth tapped states, respectively.

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Swelling Capacity and Water Retention Capacity

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The method described by Bowen and Vadino (1984) was used. Five grams of each starch was poured into a 100 115 mL measuring cylinder and the bulk volume measured (V1). Deionized water (90 mL) was added and the 116 suspensison was well shaken for 5 min. Water was added to make up to 100 mL. The suspension was left for 24 117 h before the sedimentation volume was read (V2). The swelling capacity was calculated as V2/V1.

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Determinations were done in triplicate, using the method of Ring (1985). To 5 g of each starch in a 100 mL 119 measuring cylinder was added 90 mL of deionized water and the suspension was well shaken for 5 mins. Water 120 was then added to make 100mL. Fifteen milliliters of the suspension was centrifuged (Optima Centrifuge type, BHG 500, Germany) for 25 mins at 5000 rpm. The supernatant was discarded and the residue weighed (W1).

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The residue was then dried at 70 o C to constant weight (W2) in a hot air oven. The water retention capacity was 123 computed as W1/W2. Determination was done in triplicate.

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Amylose content was determined in triplicate for each cultivar starch powder using the method of Juliano

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The twenty cultivars of four Dioscorea species studied were dileanated based on the morphology of the vine, 154 leaf and tuber of the cultivars (Plates 2, 3). Table 3 shows the character states of the vines (smooth, spined, 155 pubescent; branched/unbranched), leaf forms (shape, colour, base type) and tuber forms (shape, hairiness and its 156 distribution patterns, occurrence of cusps, colour of flesh). These characters dilienated all the cultivars studied.

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Dioscorea alata (Ewura) was separated by its winged vines while Dioscorea dumetorum (Esuru) was separated 158 by its pubescent spines, trifoliate leaf with acute apex and base and the clustered, irregularly-shaped tuber.

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The mortar and pestle are the major tools for the preparation of iyan; both are carved from wood: Vitellaria 162 paradoxa C.F. Gaertn. for the mortar and Blighia sapida K.D. Koenig used to be prime wood materials for the 163 pestle. The protocol used in this study was decoded from the wealth of experience of the professionals used in 164 this aspect of the study. The entire process of iyan production from peeling of yam through boiling to the 165 production of the final paste is open and completely under the control of the professionals.

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The first step in the production process of iyan is peeling of yam which involves a total removal of the bark and 167 all rotten parts and dead spots on the tuber. This is followed by the boiling process which is strictly monitored 168 by inspecting the tuber pieces.

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The major steps involved in achieving a good quality pounded yam is the crushing of the boiled yam (Tite and 170 Wiwo) which ensures the mashing of the boiled yam before proceeding to pounding it for homogeneity. This is 171 followed by a Check Point which involves the removal of lumps. Further pounding is done to achieve a smooth 172 paste and then water (hot or cold) is added as required followed by gentle pestle work, pounding and kneading 173 to ensure a hot final iyan paste. The mortar in Plate 1 shows two ladies doing the pounding; this ensures that the 174 process can be fast, efficient and monitorable to achieve the desired paste quality for the paste.

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All the yam cultivars stored well. Yam tubers were traditionally stored in the open, under shade in the olden 176 days and they stored well round-the-year. Tubers will also store well in well-ventilated store rooms. As the 177 tubers started to germinate, the vines were removed to prevent weight loss. Ikumo had the least percentage loss

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The shape of the starch granules was mainly oval-oblong; a few are oblong granules (   (Table 6) .

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Ten of the cultivars are greater than 40 µm in starch particle length which corresponds to the Equivalent Circle 193 diameter. The values for circularity and irregularity (IR) confirm the oval-oblong shape of the starch particles.

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The mean bulk density of the starch particles are around 0.5 while the Porosity is between 0.6 and 0.

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At the highest tapping, Anika and Gaungaun had the lowest flowability ratings. At low tapping, the starch 209 particles with smaller particle sizes were still better packed.

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The highest pasting temperatures were observed in Boki and Ilesu which means that the cultivars required high 211 gelatinization temperatures which translates into longer cooking time. Peak Viscosity values rated Anika, Obabi,

Gambari
Multiple vines, spinous dark green, cordate cylindrical, many hairs with thorns throughout the tuber, no cusps.

Riro
Firmness and elasticity of dough Paste is like puree; tends to regain shape after deformation. This term defines the five textural attributes of pounded yam (springiness, cohesiveness, hardness, smoothness and stickiness) which the traditional professionals have mastered and used for quality assurance.

Crushing
The first stage of pounding to reduce the tuber into smaller fragments.

Tite
Pounding Initial pounding to convert the smaller fragments of yam into coarse paste.

Gigun
Pounding More vigorous pounding to achieve a smooth paste.

Rin
Kneading Dexterous sideways working of pestle to convert the paste into a smooth, uniform mass.

Iyan
Pounded Yam Fine paste, the final product.

DISCUSSION
The yam cultivars were clearly designated on the bases of the morphological characters of the vines (smooth, branched/multiple; spined or smooth), leaf shape; tuber shape, presence or absence of cusps, presence or absence of hairiness and its distribution patterns as detailed out in Table 3. Farmers had no difficulties whatsoever in identifying their yam cultivars but these identities were not that clear with marketers of yam who rely essentially on hairiness of tubers, particularly its occurrence, density and distribution patterns, for the identification of the cultivars.
The vines of yam have very useful agrobotanical characters as the aerial part of the yam crop. The ability to climb stakes and trees enable the vines to display the leaves of the above-ground biomass for the interception of sunlight. All the cultivars studied are climbers but Boki, Gaungaun and Areyingbakumo can make do with spreading their branches on heaps or stubs of trees or low-hanging branches of neighbouring dead trees.
Multiple vines are a major advantage for the yam crop; first because it can result in the production of multiple tubers and second, because it can ensure the survival of at least one vine when drought causes vines to die back.
Multiple vines occur when the first bud produced by the corm of the yam seed does not exert apical dominance on successive ones.
Yam is propagated through yam sets which are obtained by cutting matured tubers into sizeable units. The second crop is obtained after harvesting the first tuber without damaging the corm and existing roots. This enables the corm to produce another tuber which is often used as yam sets. All the yam cultivars studied have this character but the main tuber of Gambari does not produce good yam sets; only the secondary tuber does.
This is the main reason, apart from the fact that it does not store well, that the mass adoption of this cultivar created problems for farmers in Ekiti State. Cultivar Gambari is however rated highly among consumers because of its suitability for the production of iyan. The poor storability is ameliorated by careful digging during harvesting to ensure that the tubers are not injured; they are not even washed for the same reason!

Standardization of Pounded yam (Iyan) Quality of the Cultivars Studied
Pounded yam is an important food in Africa and a prominent traditional food consumed throughout Nigeria with D. rotundata being the major species widely used (Otagbayo et al. 2007;Adeola et al. 2012). In Ekiti State where this study was domiciled, pounded yam is almost a mandatory meal for all supers. The expertise to make good iyan is an acquired habit in women from their formative years. Professional caterers do not lack expertise to hire because there are many experts that the iyan tradition has engendered in the population.
The highest cooking time was recorded for Ikumo and Okunmodo for fresh and stored yam tubers, respectively, which contradicts the result of the Rapid Viscometer Analysis (RVA) peak time and temperature. This could be as a result of the lower temperature intensity used in cooking these yam cultivars when there is less urgency for a meal. The RVA result revealed that most of the cultivars that required high cooking time also had significantly-high peak temperatures for their starch pasting. The respondents identified optimal cooking as a major factor for quality pounded yam while over-cooking reduced paste quality. The result obtained for pasting time of the starch from the Rapid Viscometer Analysis (RVA) is in conformity with the report of Otegbayo et al. (2006).
The quality of a good pounded yam is the function of the textural quality of the yam used which adept caterers have mastered. The specific elements of this quality are attributes like springiness, cohesiveness, hardness, smoothness and stickiness (Bogunjoko 1992;Mensah 1995;Otegbayo et al. 2006;Nindjin et al. 2007). These pasting attributes are ensured by standard cooking practices.
The yam cultivars used in this study demonstrated good textural qualities as evidenced by their high peak viscosity, breakdown viscosity, final viscosity and setback viscosity. Pasting temperatures were also low for fresh yam as reported by Otegbayo et al. (2006). Starch gelatinization resulted in the syrupy nature of the water left after cooking which is always used in the pounding process thereby recycling the gelatinised starch particles.
The pounding process was supervised by physically assessing the paste for the diagnostic attributes with the sense of touch. The term riro has been decoded in Table 3 to define the states of textural attribute of pounded yam which the traditional professionals have mastered and used at various check points in the pounding process for quality assurance. The final iyan is rolled into a ball of smooth paste that does not stick to the mortar.
Of the twenty cultivars identified and characterised in this study, three (Dioscorea dumetorum, Dioscorea alata and Dioscorea cayenensis) were not used for the preparation of iyan because they are not so-used in most of Southwestern Nigeria. As a matter of fact, it is taboo to use D. dumetorum for iyan in most of this region. It is also important to place the fact that iyan is the most hygienic food prepared from yam, as a result of the precision required in its processing from peeling through cooking to pounding, on record. The other preparation from yam is the yam flour, traditionally known as elubo, and it is used to prepare amala which is another

Starch Analysis of the Yam Cultivars
High irregularity (IR) has been attributed to the presence of a large amount of amylose which can inhibit the swelling of pounded yam (Gallant and Bouchet 1986;Zeleznak and Hesney 1997;Otegbayo et al. 2011). None of the yam powders had good flowability but Ilesu, followed by Lolo Ayin recorded fair values of this parameter. Flowability is complex because it depends on many properties (Adeoye and Alebiowu, 2014) among which are size and shape of starch particles. In this regard, the particle size, flowability, swelling and amylose contents of Gambari and Gaungaun starch powders were high. This contradicts the findings of Alebiowu and Itiola (2002), Otegbayo et al. (2011) and Zhu (2015) which reported a direct relationship between the parameters. The highest and lowest water retention was recorded for Ilesu and Anika powders. Generally, D.
rotundata cultivars have closely-associated starch polymers which compare with polymers of other native starches (Falade and Ayetigbo, 2014). Rasper and Coursey (1967) showed that the influence of amylopectin can affect the pasting and gel properties of starches with similar amylose contents. Amylopectin is mainly responsible for water uptake; peak viscosity and associated parameters are generally negatively correlated with amylose content. Final Viscosity can be an important parameter in predicting the textural quality of pounded yam. In this regard, Gambari is outstanding.
The major objectives of this study have been achieved. Twenty cultivars of Dioscorea rotundata including some from D. cayenensis, D. alata and D. dumetorum have been collected, characterised and will be conserved through regular propagation for further studies and utilization. The morphological and physical properties of the starch granules of the yam cultivars studied and their chemical compositions are in agreement with the studies of Otegbayo et al. (2006). Higher Final Viscosity, Breakdown Viscosity and Setback Viscosity were recorded for all the cultivars. This trend explains the stability of their pastes (Oduro et al. 2000). The pasting temperatures are in the range of 76.28ºC and 81.53ºC which, with the other physical properties, explains the high quality of the paste of the yam cultivars in pounded yam.
The next phase of research on this base collection will be focused on the areas of propagation, conservation and improvement (raising of plantlets from nodal cuttings which will be nurtured through tuberization) as proposed by Balogun et al. (2004;, the use of biotechnology (regular tissue culture techniques such as callus production and differentiation to produce plantlets) and also through somatic embryogenesis, selection of cultivars that produce botanic seeds and generation of hybrids through regular breeding processes.
Previous reports on propagation highlighted the effects of explant, species and genotype on tuberization (Balogun, 2004;). This has limited the adoption of these techniques to cultivars/genotypes that are amenable. The immediate danger in promoting plants that are amenable to specific propagation methods for adoption is the narrowing of the genetic base of materials available for cultivation. The adoption of the core cultivars used by local farmers should not be controlled by the ease of tuberization for a few cultivars/ genotypes. This can be ensured by encouraging peasant farmers to keep their prime cultivars rather than inducing them to adopt the new products of research exclusively. The way forward for yam conservation is to prioritize all the core cultivars for further studies using all the methods available to advance each cultivar for easier propagation, conservation and improvement.