Microsatellite fingerprinting, enzymes activity and chlorophyll profiling of local lines of air potato yam ( Dioscorea bulbifera L . ) for salt tolerance

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Introduction
Air potato yam (Dioscorea bulbifera) is an indigenous edible crop propagated for its cheap source of the nutrient.It is fast-growing and can adapt to different soil types, weather and habitat conditions, therefore having the potential of tolerating salt stress thus providing food security in an era of famine occasioned by climate change (Clare and Jean, 1984).Air potato yam is differentiated from all other species of Dioscorea by possessing specialized aerial bulbils at the base of petioles (Martin, 1974).The plant is a herb, growing vigorous with twining vine and nonspiny stems that grow up to 20m in length and branch freely above.The stems are round or slightly angled in cross-section.D. bulbifera is a perennial vine with broad leaves.The plant is dioecious, inflorescences are axillary and simple fasciculate.Flowers are white or greenish tinged, diminutive and sessile (Han and Hozio, 1993;Miller, 2003).Tuberization of this plant is solely aerial at the top (Miege and Demissew, 1997), with some varieties possessing edible underground bulbils.Most of the varieties of D. bulbifera are cultivated for human consumption in tropical and subtropical countries of Asia, Africa and America, where their edible tubers are important human food sources for man and livestock (Malapa et al., 2005).Air potato yam is an economically important food crop (Hammer, 1998).Some cultivars of D. bulbifera have been reported to contain diosgenin, a steroid that is the major bioactive component employed in the synthesis of most steroidal hormones used in the production of birth control pills (Oboh et al., 2001;Dansi et al., 2005).Air potato yam has also been used in herbal medicine systems in Africa Asia and South America and has been reportedly utilized in the treatment of dysentery, diarrhea, conjunctivitis, fatigue and depression among other ailing health conditions (Dansi et al., 1991).Notwithstanding its nutritional, medicinal and economic potentials of D. bulbifera, the crop is yet to receive the much-desired research attention it deserved.Commercial production and development of this aerial yam have not made any significant impact, probably because it is only utilized by rural resource-poor farmers.The development of Air potato yam has been reportedly hindered by poor genetic make-up in terms of resistance, which subjects most local cultivars to adverse biotic and abiotic stress (Aggarwal et al., 2008).Identification of resistance genes in local cultivars using molecular markers will be of immense benefit for the crop development since genetic markers are not influenced by environment and plant morphology and remain a basis for genetic improvement and discriminating among cultivars with similar morphological attributes.Molecular markers are also indispensable in determining the evolutionary relationship among local cultivars for plant identification (Aggarwal et al., 2008).Some of the genetic markers adopted in Dioscorea research included Amplified Fragment Length Polymorphsm (AFLP) (Mignouna et al., 2003;Malapa et al., 2005), Random Amplified Polymorphism DNA (RAPD) (Asemota et al., 1996), and Simple Sequence Repeats (SSR) (Scarcelli et al., 2006).These markers have been widely used to detect and develop resistant genes in crops that enhance growth and productivity in the face of abiotic stress and challenges.Recent studies by Kalu et al. (2021), revealed that abiotic stress contributes to the low productivity of crops (Shanker and Venkateswarly, 2011) and has been estimated to account for about 70% losses in total yields of crop plants (Rengasmy, 2010).According to Gónmez-Bellot et al. (2013), salinity, as a major abiotic stress in agriculture has affected an estimated 45 million hectares of arable irrigated land and a projected continued increase as a result of climate change (Kumar et al., 2013).It has been estimated that >50% of the world Arable lands would be unproductive by the year 2040 if salinity is not checked (Wang et al., 2013).Soil salinity is an environmental problem affecting agriculture (Jouyban, 2012) and has greatly contributed to low productivity of plants worldwide (Kumar et al., 2013).Saline soil causes physiological and metabolic imbalance resulting in poor development, growth and yield of plants (Jouyban, 2012).Salinity has been reported severally to negatively influence plant growth and reproduction.It also causes a reduction in chlorophyll concentrations, catalyzed or reduced enzymatic reactions and other physiological reactions occasioned by increased salt concentrations in the plant cell sap (Kalu et al., 2021).This study, therefore, seeks to evaluate the tolerance of air potato yam to sodium chloride through molecular, biochemical and physiological studies.

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Asian J Agric & Biol.2022(3).Planting and treatment of the plant Bulbils were planted in polythene bags and placed in a greenhouse in a randomized complete block design with three replicates.Three bulbils were planted per bag and watered with 250 ml of distilled water daily until sprouting was established after which the plants were thinned to one per bag.After four weeks of growth sprouting, the plants were treated with three different concentrations of sodium chloride solution (0, 100, 200 and 300mM), twice a week for six weeks during which agro-morphological data of vine length, number of leaves per plant, leaf area, petiole length and leaf length ascertained.Physiological parameters such as chlorophyll a and b concentrations, peroxidase and glucose-6 phosphate dehydrogenase enzymatic activities were measured using the atomic absorption spectrophotometer from leaf extracts of the plant samples.

Experimental site
The fieldwork was carried out in the screen house, University of Calabar.Enzymes activity profile, Chlorophyll a and b concentrations and molecular screening of the local lines were all carried out at the molecular biology laboratory in the Department of Biological Sciences, University of Calabar.Sodium chloride and other reagents were purchased from a commercial chemical store.All reagents were of analytical grade.The University of Calabar is on Longitude 004 0 E and Latitude 008 0 N.

Physiological characterization studies Preparation of extracts
Leaf samples were weighed, frozen and ground using mortar and pestle.Grounded samples were hydrated with 20 ml of 80% acetone and phosphate buffer for Chlorophyll contents and enzyme's activities determination respectively.Each of the mixtures was incubated and centrifuged at 15000 rpm for four minutes.Supernatants were collected and stored on ice till further studies.

Chlorophyll a and b concentrations assay Chlorophyll determination
The methods for the determination of chlorophyll contents from the D. bulbifera were adopted from Shanker and Venkateswarly, 2011. Five (5ml) suspension of algae was obtained at three (3) days interval from reaction prepared.Each algal suspension was centrifuged at 3500 rpm for 15minutes.The supernatant was easily discarded and the residual pellet was treated with 95% Dimethyl sulphate (DMSO).Five (5) ml of DMSO was then added to 5ml and homogenized.These were kept in a water bath at 70 o C for 5minutes, removed and left to cool to room temperature.The extract was centrifuged at 3500 rpm for 5mins and the pigment read at a 660nm wavelength at photo spectrometer using distilled water as blank and readings recorded.Each of the sample extracts was transferred into a cuvette and absorbance was read at a wavelength of 645nm and 650nm.Chlorophyll concentrations were calculated from the formula as described by Kumar et al., 2013 andJayeola andOyebola, 2013.Chlorophyll a contents of the leaves of yam varieties sown at the different concentrations were measured at the standard wavelengths of 645 and 650nM using the Atomic Absorption Spectrophotometer (AAS), Perkin Elmer, 7000-series, Machinio, USA.Chlorophyll b contents of the leaves of rice varieties sown at the different concentrations were measured at the standard wavelengths of 645 and 650nM using the AAS.

Peroxidase activity assay
The extract (0.6 ml), hydrogen peroxide (0.1 ml), guaiacol (0.1 ml) and buffer (2 ml) were added together in a cuvette for a reaction and the absorbance value was read at a wavelength of 436nm after one minute, two, three, four and five minutes.The values were used to determine the peroxidase activity.

Glucose-6 phosphate dehydrogenase activity (Enzyme) assay
The reaction mixture for glucose-6 phosphate dehydrogenase activity included 0.6ml extract, 2.0 ml buffer, 0.1 ml glucose -6 phosphate and 0.1 ml beta Nicotinamide adenine dinucleotide phosphate (NADP).The absorbance values were read at a wavelength of 340nm at one minute, two, three, four and five minutes.The values were used to determine the glucose-6 phosphate dehydrogenase activity.

Linolytic enzyme assay
This enzyme was assayed using Manganese peroxidase (MnP), Laccase (Lac) and Manganeseindependent peroxidase (MIP).Each of these was determined through the measurement of the respective absorbance change at 470 nm relative to the rate of oxidation of 2,6-dimethoxyphenol in malonate buffer (pH 4.5).However, Lignin peroxidase (LiP) activity was measured by the oxidation of veratryl alcohol in succinate buffer (pH 3.0) at 310 nm.

Xylanase and Cellulase Enzymes assay
The modified procedures of Bisswanger, 2014 spiced with Oat spent xylem (1%) as substrate at 50 °C and a 5.3 pH was adopted for Xylanase and cellulose enzymes assay.Cellulase activity was measured using reducing sugars obtained from carboxymethly cellulose (2% w/v, low viscosity).At the end of laboratory procedures, Xylose and glucose standard curves were used to calculate the xylanase and cellulase activities from the samples (Terauchi et al., 1992).

Data Analysis
The Analysis of variance (ANOVA) option of the Genstat V 12of ICRAF of 2018 was used to analyze the data and mean with significant differences were separated using the Fishers least significant difference (LSD) at 5% level of probability.

Molecular Screening of Dioscorea bulbifera local lines using Simple Sequence Repeats (SSR) analysis Collection of Plant materials
Five (5) g of apical fresh leaves of forty local lines of Dioscorea bulbifera representing entries from the different localities in Nigeria (Table 1) were used for the molecular study.The leaves were collected from Plants after two weeks of growth cultivated in 30cm size pots filled with sterilized top soil and maintained in a screen house at the University of Calabar research farm, Nigeria for genomic DNA extraction.Ta = annealing temperature

DNA extraction and quantification
Genomic DNA was extracted from fresh leaf apex of young leaves using modified cetyltrimethylammonium bromide (CTAB) procedure as described by Mignouna et al. (2003) in the molecular laboratory of the Department of Genetics and Biotechnology, University of Calabar.The quality and concentration of DNA was assessed by gel electrophoresis using 1% agarose with known concentrations of undigested lambda DNA (Sigma St. Louis, MO, USA).Quantification of DNA was done using a Nanodrop spectrophotometer (Beckman Coulter DU 530) at 260nm.Extracts were diluted in water to obtain a DNA concentration of 25ng/L.

Polymerase chain reaction and fragment analysis
A total of ten SSR primer pairs were used in the study (Table 2).PCR reaction was conducted in a 20l volume in a 96-well microtitre plate using an automated thermal cycler (Peltier Thermal Cycler 200).The reaction volume contained 25ng of template DNA, 100m each of dNTP, 2.5mM MgCL 2 , 0.5M each of fluorescently labeled forward primer and unlabeled reverse primer, 1X reaction buffer and 2 units of Taq DNA polymerase (Invitrogen).The forward primer was 5 1 -labeled with one of the four fluorochromes PET, 6-FAM, NED and VIC.The PCR programme consisted of denaturation at 94 O C for 5 mins, followed by 35 cycles of 94 O C for 30s, 55 or 45 O C for 20s and 72 O C for 30 s, with a final extension step at 72 O C for 7 min.Capillary electrophoresis with a semi-automated system ABI/PRISM 3100 genetic analyzer was used to separate amplified PCR products.Samples for amplified product separation were prepared by adding 1l of diluted PCR products to 9.4l formamide and 0.1l Gen size-500 LIZ.This was dispensed in ABI 96-well plates and denatured at 94 O C for 5 min and allowed to cool down on the ice.
Primers used for the study Ten (10) primer sets (forward and reverse) previously used by researchers working on Dioscorea bulbifera (Table 1) in the International Institute for tropical agriculture (IITA) Ibadan were sourced from Operon, USA and used for the study.

SSR fragment analysis
The fragment sizes in base pairs for each genotype across SSR markers were converted to binary data where alleles were transformed into presence (1) or absence (0) of an SSR band.Genetic diversity indices such as the number of alleles for locus; gene diversity and Polymorphic Information Content (PIC) were estimated using Power Marker version 3.25 software Genetica, The Netherlands.The cluster pattern of the genetic diversity was constructed using DARWIN 5.0 SIB, Switzerland.

Genetic diversity indices analysis
Table 3 presents the summary of genetic diversity parameters generated by 10 SSR markers.DBSSR 2 recorded the highest number of alleles detected (12 alleles).This was followed by DB5 (10 alleles) DB3 and DBSSR4, respectively (Table 3).The discriminatory power of each SSR primer was assessed by calculating Polymorphic Information Content (PIC) values.The mean PIC value for all markers used was 0.74 and ranged between 0.37 and 0.85 in loci DB7 and DBSSR4, respectively (Table 3).Markers with more alleles but lower allele frequency had larger PIC as found in DBSSR4 (9 alleles and the highest PIC of 0.85, followed by DB5 with 10 alleles and PIC of 0.84, respectively (Table 3).Again, it can be established from the results that marker DB7 had the highest allele frequency of 0.60 while markers DB3, DB5 and DBSSR4 had the lowest frequency of the major allele (0.24).The average frequency of the major allele was 0.33 (Table 3).Gene diversity was high, ranging from0.48 in DB7 to 0.86 in DBSSR4 with a mean of 0.77.Generally, the allele frequency of all the primers was below 0.95, indicating that they were all polymorphic in character (Table 3).Plates 1& 2 show SSR marker (DBSSR2 & DB5) profiles generated for the D. bulbifera local accessions used in the study.Generally, the pattern of movement of the DNA bands in the gel distinguishes one accession from the other.Results observed from allelic frequency analysis proved that all the 10 primers were polymorphic (Table 3).No rare alleles (alleles with allelic frequencies of less than 0.l005) were obtained.This was possibly due to the genetic closeness of the local lines studied.Major allele frequency ranged between 0.2400 in DB3 to 0.3600 in DBSSR 5 with a mean value of 0.3320.The number of alleles per locus varied from as low as 2 obtained with DB7 to 12.00 obtained with DBSSR 2 markers with a mean number of 7.400.Nei's gene diversity was 0.4800 in DB7 and 0.8608 in DBSSR 4 with average gene diversity of 0.7725.The AMOVA presented in

Figure-4: Scattergram showing distribution of 40 Air potato yam (Dioscorea bulbifera) accessions along the major principal coordinate axes
Table 6 presents the results of Five ( 5) principal components axes used to delineate 40 accessions of local D. bulbifera lines based on their contributions to the observed variations in their response to salt and physiological stress.The total variations observed among the local accessions to salt tolerance using this approach were 97.48% somewhat close to 100% obtained with the analysis of molecular variance.
Principal component 1 showed an eigenvalue of 0.06581 with the highest percentage contribution of 48.63% of the total 97.48% variations in salt tolerance observed among all the accessions.The Among Pops 0% Within Pops 100%  The network analysis of the 40 local lines of Dioscorea bulbifera clearly delineates the lines into ten groups with specific genetic distances and relatedness within and among all lines (Fig. 6).Values are mean of chlorophyll a and b determinations.

Percentages of Molecular Variance
Means with the same superscripts on the same row are not significantly different (p<0.05).
Twenty (20) lines that showed tolerance for common salt at 400, 300 and 200 bps were selected and evaluated for Chlorophyll a and b profiles or contents.The results as presented in Table 8 and figure 7 revealed that all the evaluated lines had more Chlorophyll b contents compared to Chlorophyll a.
The results also showed that the concentrations and profiles of both Chlorophyll a and b decreases with increasing concentration of common salt in soil solution.The local line Dbor 1 obtained from Oron, in Akwa Ibom state had the highest Chlorophyll a and b profiles at all the given concentrations and control while Dboh 6 showed the least profiles of Chlorophyll a and b contents in all the given concentrations and control.
Results of enzymatic activities for ten local lines with high salt tolerance potentials are presented in Table 8.
The result shows that the activities of all evaluated enzymes in the local lines increased with increase in the concentrations of salts in the soil solution.The concentration of cellulose was highest in all the local lines evaluated while peroxidase was the least active enzyme in terms of concentrations.The results revealed that increase in the concentrations of enzymes was proportional to increasing stress initiated by increasing salt concentrations.Enzymes activity showed increasing activity with increasing concentration of the salt in solution.All the enzymes G6DP, peroxidase, Xylanase, cellulose and lignolytic enzymes all showed increased activities with increasing salt concentrations.

Discussion
Sodium chloride was observed to cause an increase in cellulose, xylanase, lignolytic, glucose-6 phosphate dehydrogenase and peroxidase activities in D. bulbifera.An increase in these enzyme activities suggests the formation of excess hydrogen peroxide by the salt and consequent oxidative stress.Therefore, as a defense response to oxidative stress, the peroxidase and glucose-6 phosphate dehydrogenase activities were increased.This agrees with Terauchi et al., 1992, who reported an increase in anti-oxidative enzymes under salt stress..This suggests that enzymatic activities in Dioscorea bulbifera increase with increased salt stress or concentrations.The cellulase enzyme activity was found to be the most affected due to its involvement in the plant photosynthetic activity especially during the day.Despite the reduction in growth and yield of crop plants by salinity, some species of plants such as rice and maize are reported to have the capacity of tolerating salinity of various concentrations.These abilities to tolerate salinity is made possible through an increase in the activities of enzymes associated with salt stress (Terauchi et al., 1992).The increased enzyme activity acts as a defensive mechanism against cell damage and contributes to the tolerance response of plants (Mignouna et al., 2003).The profile of Chlorophyll a and b was found to decrease with an increase in salt concentrations in soil solutions.Not many variations in chlorophyll contents were obtained relative to the control experiment with zero concentration of salt.The present study also revealed higher concentrations of Chlorophyll b compared to Chlorophyll a.It also revealed a decreasing total chlorophyll content with increasing concentrations of common salt in solution.This finding is in line with the reports of Kalu et al., 2021 who posited that chlorophyll contents increase with decreasing abiotic stress (Ashraf et al., 2014;Asiedu et al., 1998).Sodium chloride reduced the phenomic parameters in Dioscorea bulbifera as enlisted in Table 2. Specifically, it decreases the vine length, number of leaves and number of bulbils of the D. bulbifera.The decrease increases with increasing concentration of sodium chloride and was more significant at 300mM concentration of the sodium chloride.At 100mM, the height and number of leaves of D. bulbifera of some of the lines was at higher value, although not significantly different from that of the controls.
From this, it can be deduced that a higher concentration of sodium chloride (>100mM) caused a reduction in the growth of D. bulbifera and lower concentration of sodium chloride (≤100mM) tend to stimulate the growth of D. bulbifera.The reduction in the growth of Dioscorea bulbifera by sodium chloride agrees with authors who reported a reduction in growth and yield of plants exposed to salt stress (Ben and Yene, 2013;Karp et al., 1997. Malapa et al. (2005) reported a reduction in the growth of Euryae marginata by salt stress as evidenced by reduction in fresh weight, leaf water content and Chlorophyll a and b content.Gónmez-Bellot et al. ( 2013) reported a decrease in total root length of E. japonica treated with salt.Jouyban (2012) also reported the same decrease in Echium amoenum.Sodium chloride was observed to cause a loss in fresh root weight of maize, an effect that increases with a higher level of salinity (Jouyban, 2012).Jampeetong and Brix (2009) reported decrease in leaf area and root length with salinity in Salvinia natans.Salinity also causes a reduction in chlorophyll concentrations of plants.The leaf yellowing was more conspicuous at a 300mM concentration of sodium chloride.This was an indication of a deficiency of some essential nutrients (sulphur, iron, magnesium and nitrogen) in the plants.Saline soils of higher concentration (above 200mM) could lead to interference in the absorption of nutrients by the root hairs of the plants.Bulbils with spindle or elongated shapes could be said to be less tolerant to the salt treatment compared to the round-shaped lines which were more tolerant to salt treatments.The results of principal component analysis of molecular attributes studied revealed five principal components, which explained 97.48% while analysis of molecular variance attributes explained 100 % of germplasm using DNA based markers like SSR is important for efficient conservation and utilization.The results demonstrated genetic polymorphism in D. bulbifera germplasm from all local sources in the study area.Variations observed in the cluster groups suggest that the mutation rate is high and many of the mutations are not neutral showing and having an effect on phenotype.This is expressed in morphological variation among the cultivars presenting different colours and shapes for both aerial and underground organs (Obidiegwu et al., 2009).

Conclusion
Thirty (30) local lines of Disocorea bulbifera showed fingerprinted at 100, 200, 300 and 400 bps respectively.Ten of the local lines did not show any affinity to the salt-tolerant marker of gene and this was reflected in their low Chlorophyll a and b profiles and reduced enzyme activities.The forty (40) lines of D. bulbifera were also assessed for genetic diversity using ten microsatellite markers or loci.A total of 74 alleles were detected with an average allele number of 7.4 per locus.Polymorphic Information Content (PIC) mean value of 0.74 showed the existence of variability among the accessions.Cluster and principal component analysis showed 2 major and 10 sub-cluster groups.There was 97.48 % relationship between the relatedness of the lines and their local area of collection.Ultimately, microsatellite fingerprinting markers proved to be effective in identifying salt-tolerant lines of the studied D. bulbifera germplasm.The study could help in improving the available local lines of D. bulbifera for salt tolerance, breeding programs and cultivar development.The study had revealed the size of the possible genes which can be harnessed and developed as a salt-tolerant marker for Dioscorea species in this locality and beyond.The study revealed that D. bulbifera lines secrete more enzymes with increasing concentration of salts.Cellulose enzymes being the major enzyme involved in photosynthesis was at the highest concentration and activity.Chlorophyll b showed the highest-profile compared to Chlorophyll b across all the salt-tolerant lines.A combination of biochemical, physiological and molecular approaches in this study could prove a viable tool in the determination of genetic diversity and relationships among D. bulbifera accessions which is critical to the genetic improvement of the crop.

Figure- 3 :
Figure-3: Percentage molecular variation of Air potato yam (Discorea bulbifera) population major contributing local line to this principle component is Dbak -3 from Akpabuyo, Cross River State with a loading value of 0.4479.Principal component 2 showed an eigenvalue of 0.03448 with a percentage contribution of 25.00% out of the total 97.48% variations observed in salt tolerance ability among the accessions.The major contributing local line to principal component 2 is Dbak -4 from Akpabuyo, Cross River State with a loading value of 0.4832.Principal component 3 showed an eigenvalue of 0.02603 with a percentage contribution of 11.32% out of the total 97.48% variations observed in salt tolerance attributes of the local accessions.The major contributing local line to principal component 3 isDbok -3 from Okobo, Akwa Ibom State with a significant loading value of 0.3470.The results for principal component 4 showed an eigenvalue of 0.01583 with a percentage contribution of 6.89% out of the total 97.48% variations observed in salt tolerance attributes of the local accessions.The major contributing local accession to principal component 4 is Dbok -8 from Okobo, Akwa Ibom State with a significant loading value of 0.3092.The results for the last Principal component 5 revealed an eigen value of 0.01296 with percentage contribution of 5.64% out of the total 97.48% variations observed in salt tolerance attributes of the local accessions.The major contributing local accession to principal component 5 is Dbor -8 from Oron, Akwa Ibom State with a significant loading value of 0.2445.The results of analysis of molecular data using DARwin 5.0 software delineated the 40 local lines of Dioscorea bulbifera into two (2) major clusters and ten (10) subclusters.Major cluster one has 20 local lines partitioned into 6 subclusters.Subclusters 1 has Dboh 4 and Dboh 2 local lines with the same genetic distance of 41.Sub-cluster 2 has 4 local lines of Dbak 10, Dboh 3, Dbor 3 and Dboh 4 respectively having the same genetic distance of 43.Sub-cluster 3 has 2 lines namely Dbok 1 and Dbor 5 with genetic distance of 47. Sub-cluster 4 has DBok 6 and Dbak 2 with the same genetic distance of 46.Sub-cluster five has five lines of Dbok 1, Dbor 5, Dboh 8, Dbok 8 and Dbor 8 all with the same genetic distance of 47.The last sub-cluster 6 in major cluster 1 has five lines including Dboh 9, Dbor 9, Dbor 6, Dbor 2 and Dbak 5 with genetic distance of 48 (fig.5).Major cluster 2 has four sub-clusters of 7, 8, 9 and 10.Sub-cluster 7 has two lines Dbok 7 and Dbok 5 with a genetic distance of 45.Sub-cluster 8 has two lines also, Dbor 4 and Dboh 1 with a genetic distance of 46.Sub-cluster 9 has the highest number of 15 local lines with a genetic distance of 44.Local lines in this sub-cluster includes Dboh 10, Dbok 10, Dbok 9, Dbak 8, Dbor 8, Dbak 6, Dboh 7, Dboh 6, Dbor 1, Dbak 2, Dbak 1, Dboh 5, Dboh 3 and Dboh 2 respectively.The sub-cluster with the least number of local lines is sub-cluster 10 with Dbak 4 line and has a genetic distance of 60 similar with the clade.

Figure- 5 :
Figure-5: Dendrogram showing genetic distance of 2 major clusters and 10 sub-clusters from 40 accessions of Air potato yam (Dioscorea bulbifera) evaluated for salt tolerance using microsatellite markers

Figure- 7 :
Figure-7: Radar plots showing Chlorophyll a and b in five lines of Air potato yam with high salt tolerance potentials Line 1= Dboh 2; Line 2 = Dbok 3; Line 3= Dboh 2; Line 4 = Dbak 2 and Line 5 = Dbor 1 variations in the population to molecular attributes with most of the loading score coming from Dbor , Dbok and Dbak.Cluster patterns based on squared Euclidean distance, 0.05, using Ward's method revealed two major clusters each with 6 and 4 sub clusters respectively with varying genetic distances indicating a close genetic relationship and relatedness among the local lines.The results also showed that all the local lines developed from a common ancestor.The results are in consonance with the positions of Brisibe et al., 2021 who reported similar results with Musa species AAB genome.Assessing the genetic diversity of D. bulbifera Success Eni Kalu et al 13/14 Asian J Agric & Biol.2022(3).
Bulbils of D. bulbifera with two distinguished shapes were obtained from local farmers in Abia State, Akwa Ibom State and Cross River State and established in the Biological Sciences Research Farm, University of Calabar, Calabar for the study.Forty (40) accessions of Air potato yam (Dioscorea bulbifera) were sourced and obtained.Ten (10) each of the following accessions were obtained from Ohafia in Abia State, Oron in Akwa Ibom State, Okobo in Akwa Ibom State and Akpabuyo in Cross River State, Nigeria.The accessions used and their source of germplasm are designated as Dboh for Ohafia, Dbak for Akpabuyo, Dbor for Oron and Dbok for Okobo lines respectively and their shapes as presented in Table 1;

Table - 5: AMOVA table for SSR analysis of 40 accessions of Dioscorea bulbifera accessions evaluated for salt tolerance Percentage molecular variation of the Dioscorea bulbifera local populations
Key: phqt = estimate of the population genetic differentiation based on Permutation.

Table - 6: Eigen value and percentage variations of five principal coordinates obtain from 40 Accessions of Air potato yam (Dioscorea bulbifera) evaluated for salt tolerance
Success Eni Kalu et al 10/14 Asian J Agric & Biol.2022(3).