EFFECT OF GAMMA RADIATION ON MORPHOLOGICAL AND MOLECULAR CHARACTER OF Sphenostylis stenocarpa (Hoechst. ex. A. Rich.) Harms

Introduction Globally, several species of plants, that are cultivated for food production and generation of revenue are underutilized and or neglected, although they perform an important role in the livelihood of poor people in rural settlements (Magbagbeola et al., 2010). These species of crops which includes Sphenostylis stenocarpa (Hochst.ex A.Rich.) Harms are rich in nutrient. Consequently, their extinction will have adverse effect on food security hence the need to improve them through crop improvement programs to increase their nutritional values and to promote food security, especially among the poorer communities (Dansi et al., 2012). Sphenostylis stenocarpa (Hochst.ex A.Rich.) Harms also referred to as African yam bean (AYB), is grown mostly in Africa for their seeds and tubers especially in West Africa. The protein content found in the seed and tubers ranges from 15 to 29% and its amino acid values are more than those found in other legumes like; cowpea, bambara groundnut and pigeon pea (Adewale & Dumet, 2010). Although, it is well known in the eastern part of Nigeria, for its nutritious beans and revenue ABSTRACT Genetic variation in two varieties of Sphenostylis stenocarpa (Hochst.ex A.Rich.) Harms was created using different doses of gamma-rays with the aim of increasing variability among the landraces for better morpho-agronomic characteristics. The two varieties responded differently to the doses of the treatment. The mutagen stimulated the germination percentage in both varieties. The highest germination percentage was observed at 200Gy for Tss86 while for Tss10 it was observed at 25Gy. It showed variation on the germination and growth parameters evaluated. The highest plant height (74.17±4.42) for Tss10 was observed at 25Gy while for Tss86, 100Gy gave the highest mean of 79.6±8.02. Molecular evaluation revealed variation in the patterns of RAPD profile between irradiated plants and control. The RAPD primers showed 82.9% polymorphism. Also, as observed from the similarity coefficient values, the trend of the genetic variability was not proportional to the doses of the gamma ray. The similarity indices ranged from 0.09 to 0.60 with 3 distinct cluster groups identified at 0.64. The variant of Tss86 at 250Gy was found to be the most diverse. Thus, this integrated approach can be used for carrying out the mutation-assisted breeding and subsequent selection of desired mutants using molecular markers in AYB.

generation, it is still highly underutilized in most Nigerian homes due to long pre-cooking soaking to manually remove the seed coat followed by long duration (4-6 hours) of cooking of the beans (Thomas et al., 2005). The use of mutation breeding to create variation in the genome of crop plants for desired traits has been well reported in literatures (Melchinger, 2015;Asare et al., 2017). However, the use of mutation induction can be associated with creation of undesired traits alongside the desired (Roychowdhury & Tah, 2011;Oladosu et al., 2016). Mutation breeding can be achieved using three types of mutagenesis: mutagenesis using physical mutagens (irradiation); mutagenesis using chemical mutagens and biological mutagens (transposable elements). Among the physical mutagens, gamma (γ -rays) have so far been used extensively for mutation breeding aimed at improving agronomic traits of different crop species (Akhtar, 2014;Laskar et al., 2018). The application of molecular markers for analysis of genetic diversity and variety identification is considered best because it is not affected by stage of development of the crop, crop's environmental or management practices. In resent times, the molecular makers commonly used are Polymerase chain reaction (PCR)based markers which includes Random Amplified Polymorphic DNA (RAPD) (Selvi et al., 2007;Abebe et al., 2020).
According to Selvi et al. (2007) genetic variability and diversity analysis in African Yam Bean (AYB) has been achieved using identifiable phenotypic traits. Also, Popoola et al. (2017) has reported the use of RAPD in the evaluation of intraspecific variability among some African Yam Bean accessions. However, no report has been made on the use of molecular markers to estimate genetic variability and diversity studies created as a result of mutation induction using gamma rays. The success in the use of RAPD in determination of genetic diversity in crops has been reported hence, its use in this study for diversity studies. RAPD has been employed in diversity study of crops like rice, maize, wheat and barley (Salem et al. 2007). This study aimed to create genetic variation in African yam bean (Sphenostylis stenocarpa) using gamma rays for crop improvement and germplasm conservation and to evaluate the variation as a result of the irradiation using RAPD.

Planting materials
Two hundred and fifty healthy certified seeds each of accessions TSs10 and TSs86 of African yam bean used for this research were collected from the Gene bank of the International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria. The description and geographic origin of the varieties selected according to the International Institute of Tropical Agriculture are presented in (Table  1).

Gamma source and irradiation of seeds
About two hundred and twenty-four (224) seeds of each accession of AYP were irradiated. Thirty-two seeds (32)  Germination tests of irradiated seeds was carried out in the laboratory on Whatman No.1 filter paper in eight (8) replicates (per dose per accession) of 5 cm Petri dishes previously moistened with water. Each replicate contains a total of four (4) seeds making it Thirty-two (32) seeds per dose per accession.
For the field experiment, six seedlings in three replicates per dose per accession were sown 1m apart on a single role plot of length 10m. Weeding of the experimental plots was done at an interval of three weeks, pest control practices were also observed two weeks after planting following the method of Adewale et al. (2012). Staking was done three weeks after germination using strong stake each measuring about 3m high. Each seedling was independently staked to prevent mixing the accessional yields.

Experimental design
The laboratory experiment was carried out in a Completely Randomized Design (CRD) with eight replicates, while Randomized Completely Block Design (RCBD) with three replicates, was adopted for the field experiment.

Evaluation of agronomic and morphological traits
After 11 days of sowing, the germination percentage was evaluated, the length of hypocotyl and radicle were measured using a metre rule and the number of lateral roots were also counted. A total of six (6) morphological characters were evaluated using IITA descriptors of AYB. These characters are: plant height, number of branches, number of leaves, terminal leaf length, terminal leaf width and petiole length.

Determination of germination percentage and rate
Estimation of the germination percentage (GP) and rate of germination (MGT) were calculated following the method adopted by Khaje-Hosseini et al. (2003). GP = Number of germinated seeds after 11 days/ Total number of seeds planted X 100 MGT= Where; MGT= mean germination time; F= the number of newly germinated seeds on each day; X= the day of counting.

DNA extraction and amplification
The method used for the DNA extraction was as described by Dellaporta et al. (1983). Young leaves (0.2 g) of each accession were lyophilized and grounded into fine powder. The protocol incorporates a plant extraction buffer (PEB). The PEB is composed of 637.5ml of double distilled water, 100ml of 1 M Tris-HCl (pH 8.0), 100 ml of 0.5M Methylenediaminetetraacetic acid (EDTA) (pH 8.0), 100 ml of 5 M Nacl 2 and 62.5 ml of 20% sodiumdodecylsulphate (SDS). Seven hundred (700) ul of hot (65 o c) of PEB was poured into Eppendorf tube. The PEB is pre-warmed and just before use, one percent b-mercaptoethanol was added. The tube was then capped and gently inverted six to seven times to mix the buffer with the sample. To homogenize the samples, the solution was incubated at 65°c in water bath for 20 mins with occasional mixing. The samples were then removed from the water bath, the caps were opened and left for 2 minutes at room temperature to cool. Five hundred (500 ul) of 5M of potassium acetate was added to the content and covered, followed by gentle mix by inverting the tubes six to seven times. The samples were incubation on ice for 20 minutes after which the tubes were spin at 12,000 rpm for 10 minutes using a centrifuge (Kecheng HI-16k, China) at 4°C. The supernatant was transferred to a new clean 1.5ml Eppendorf tubes using a 1000 µl micropipette, care being taken to avoid the debris. Seven hundred (700 µl) chloroform: isoamyl alcohol was added to the supernatant and then spin at 10,000 rpm for 10 minutes using a centrifuge. The supernatant was collected and transferred into new marked tubes, followed by the addition of 700-µl ice-cold isopropanol and mixed gently by inverting the tubes 6-10 times. The tubes were then stored at -20°C for at least 1 hour to precipitate the DNA. In order not to dislodge the pellets from the bottom of the tubes; the supernatant was discharge with care and then drained on a clean paper towel inverted for 1 hour or more. Pellets of the DNA were double purified in 100 µl, cold 70% ethanol for 20 minutes and thoroughly air dried, after which 60 µl of 1×TE [10mM Tris-HCL (pH 8.0), 1mM EDTA (Ph-8.0)] was poured onto the pellets, followed by 2µl of 10ng/ml Rnase to extract the RNA. The solution was incubated for 40 minutes at 37°C with gentle mix at 10 minutes intervals.
PCR amplification was done using a thermal Cycler (Eppendorf Mastercycler pro S (US) with first denaturation step for 5 min at 94°C, followed by 35 cycles each of 1 minute at 94°C (denaturation), 1 min at 59.3°C (annealing), 2 min at 72°C (extension) followed by one final extension of 7 minutes at 72°C.The products of the amplification were electrophoresed in 1.8% agarose gels in 0.5× TBE (10× stock containing 1 M Tris, 0.8 M boric acid, 0.5 M EDTA) and stained with ethidium bromide (0.5 μg/ml). The gels placed were placed under a Gel Documentation System (Perkin Elmer Geliance 2000, state country of origin). The bands were scored for presence (score of 1) or (score of 0) for analysis.

Molecular analysis
Seven (7) RAPD primers (Table 6) showing polymorphism were employed in scoring and estimating the level of polymorphism. The gel bands were scored as present (1) or absent (0) and were assembled in a binary matrix. The data generated were analysed using Numerical Taxonomic System of Statistics (NTSYS version 2.21) and Power Marker Software (version 3.25).
Jaccard coefficient of similarity was used to estimate genetic distance and to construct dendogram using Unweighted Pair Group Method of Arithmetic Means (UPGMA) to reveal phonetic representation of genetic relationship between mutants and control.

Statistical analysis
Data analysis was done using Statistical Analysis Software (SAS version 9.1). Means of ANOVA analysis showing significant differences were separated by Duncan Multiple Range Test (DMRT) at 95% (p˂ 0.05). Table 1 shows the description and geographic origin of the varieties selected according to the International Institute of Tropical Agriculture are presented. The percentage germination at the different doses of treatment is all higher than the control except Tss 86 at 25Gy which has the minimum percentage germination and at 150Gy the two accessions had a germination percentage lower than the control (Fig. 1). Thus, Gamma irradiation showed a stimulatory effect on African Yam Bean Seeds.

Results and discussion
A differential response of the mean germination time of the two varieties of AYB studied was reported in Table 2. For Tss10 in Table 2, 200 Gy had the lowest mean germination time which differed significantly from the other dose treatments while the same dose in Tss86 had the highest but only differed significantly from 250 Gy. Dhumal & Bolbhat (2010) reported a reduction in germination of gamma irradiated horsegram plant with increase in dose treatment which was observed in Tss10 accession. Varied mutagenic sensitivity to different doses of the mutagenic treatment, in different genotypes has also been reported in production of viable mutants in Nigella sativa L. and in Vignamungo L. following gamma ray irradiation (Goyal & Khan, 2010). For Tss86, 150 Gy had the lowest MGT however, it did not differ significantly from other dose treatments applied (25, 50, 100, 200 and 250Gy). The irradiation dose had significant effect (p≤0.001) on the hypocotyls length, the number of lateral roots and the root length (Table 3). The varietal effect on the hypocotyl's length was significant (p≤0.001) while the interaction between variety and dose had significant effect (p≤0.05) on hypocotyls length but not significant effect on the number of lateral roots and the root length, this can be compared to the work of Borzouei et al.        Values in the same column with different letters as superscripts are significantly different. PHplant height, NL-number of leaves, PL-petiole length, LL-leave length, LW-leave width, NBnumber of branches. Table 6 shows the primers used and their sequences, as well as the total number of bands produced by each primer. The DNA polymorphism obtained in the two varieties of AYB studied with the seven (7) primers revealed variation as a result of the irradiation. The polymorphic information content (PIC) as shown in (Table 7), ranged from 0.6953 (OPB-01and OPT-06) to 0.9124 . The highest PIC (0.9124) was showned by primer OPT-20 followed by OPT-07 (0.9008). Thus, these primers provided useful information which can be employed for further studies. Figs. 2 to 8 present the banding profile of irradiated plant materials and control with different RAPD primers. There was variation in banding pattern between the irradiated and non-irradiated beans which could be attributed to the mutagenic effect of the treatment which is similar to the work of Eze et al. (2015) who reported genetic variability on sodium azide induced maize plant. These effects of gamma rays may be due to structural re-arrangements in DNA strand caused as result damages (Selvi et al., 2007;Gill et al., 2015). The present findings are in agreement with reports of Erdem & Oldcay (2004) and Hussain et al. (2008).    The Jaccard's coefficient of similarity (Table 8) reflected the genetic relationship between the samples. The highest level of similarity was found between two irradiated plantlets; 25Gy of Tss10 and 50Gy of Tss10, with a similarity coefficient of 0.600, while a minimum similarity coefficient (0.095) was observed between 250Gy of Tss86 and 150Gy of Tss86 (the highest genetic distance). The dendrogram based on Pearson's coefficients of Gamma ray-induced plants showed that three variants (V2 of 250Gy, V 1 of 100Gy and V 2 of 150Gy) were more distinct from the control, whereas others were quite similar to each other and with the control (Fig.  9). It can be deduced from these results that RAPD successfully detected variation between the non-irradiated and irradiated plants, which were morphologically indiscernible. This similar to the findings of Dhaksanamoorthy et al., (2011) who reported polymorphism detection using RAPD on irradiated Jatropha curcas.
V1-Tss10, V2-Tss86 Fig. 9. UPGMA dendrogram based on Pearson's coefficient showing the genetic similarities and distance among Variety x Treatment of the two varieties of AYB studied.

Conclusion
Gamma ray was able to induce genetic variability in the accessions of African yam bean and could be used as basis for mutation induction. selection in plant breeding considering the range of 25Gy dose through to 200Gy dose can be considered. There were differential responses of the two varieties to the mutagenic treatment. The gammarays exposure influenced the patterns of RAPD profile compared to the control, thus, proving the exploitation of RAPD analysis as a molecular marker for the identification of polymorphism in breeding of AYB useful. Progressive molecular characterization of irradiated plantlets may result in the establishment of an efficient mutation induction protocol, for example, identifying proper doses of mutagen by assaying DNA damage. The irradiated varieties with differences in their morphological characters, also, presented DNA polymorphism in PCR profile amplified by RAPD marker. It can be recommended that further analysis like proximate analysis be done on the seeds produced by irradiated plants to determine if there is a decrease or increase in protein content compared to the control.