Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2021 18(1): 795-804. OPEN ACCESS

Diversity in Australis through morphological and biochemical markers

Izhar Ullah1, Murad Ali1*, Ali Asghar2, Waqar Khan3, Nausheen Nazir1, Mohammad Sohail4 and Mohammad Nisar1*

1Department of Botany, University of Malakand, Pakistan 2Department of Biological Sciences, Sunway University, Malaysia 3Nanjing agriculture University, China 4 Departments of Botany, Abdul Wali Khan University, Mardan, Pakistan

*Correspondence: [email protected], [email protected] Received 17-10-2020, Revised: 07-03-2021, Accepted: 10-03-2021 e-Published: 15-03-2021 Celtis is grown for various uses like fodder, fuel, timber and other uses in or around agricultural fields in rainfed agriculture. It play a vital role in socioeconomic structure of hill people by supplying highly palatable, nutritious and tannin-free green fodder particularly during the period of scarcity of green fodder to livestock. This study was carried out to explore the genetic diversity through morphological and biochemical markers using SDS-PAGE. A total of 11 morphological characters were recorded. The genetic diversity was observed. In quantitative traits maximum of genetic diversity was found in 10 seed weight (89.85%). Total of 14 protein bands was detected. Total of 18% genetic diversity were recorded. Significant level of Genetic diversity observed in B1 (0.68%), B2 (0.63%) while in B3 (0.49%). Keywords: Celtis australis L., Genetic diversity, morphology, PCA, SDS-PAGE

INTRODUCTION 2009; Sommavilla et al. 2012). The Celtis is Celtis australis L. belongs to family Ulmaceae, mainly grown for fodder. It is lopped during lean is indigenous to Western Himalaya.Family periods (October to mid-January) and provides Ulmaceae containing 15 genera and 200 species. ample supply of highly palatable, nutritious, and Celtis is the largest genus includes about 60 tannin-free fodder during peak periods (Bisht and species distributed in the temperate and tropical Yadav, 2015). A yellow dye is obtained from the zones (Luna, 1996). It is widely distributed and bark (Polunin, 1969). The fruits are used as grows in 500-2500 altitudinal range in North remedies for amenorrhea, colic, heavy menstrual Eastern Himalayan region (Gaur, 1999). In these and intermenstrual bleeding (Duke and Ayensu, species Celtis australis L. and 1985; Chopra et al. 1956). L. were cultivated for shade purposes in Egyptian For different crop germplasm and genotypes gardens. It grows well along stream banks, on genetic diversity is much important step for the sloping hillsides and on clay loam soil with competent and successful maintenance and sufficient moisture (Luna, 1996). Celtis is used for utilization (Ghafoor, 1999). For all breeders fodder, timber and various other uses (Subba et the evaluation and characterization by agro- al. 1996). morphological parameters are therefore very The leaves of Celtisa ustralis contain rare important (Martins et al. 2006). In excess of the flavonoid c-glycosides, such as acacetin 7-O- most recent few decades, the genetic diversity in glucoside, isovitexin, and cytisoside (Spitaler et al. several depends on their morphological Izharullah et al. Assessing diversity in celtis austrailus characters and through it were evaluated (Nisar et Seed storage proteins were evaluate on slab al. 2016). In propagation increases the type SDS-PAGE smaller than normal gel (mini probabilities for success in developing highly gel) apparatus AE-6530, Atto Japan according to productive new cultivars with moral quality per Laemmli (1970) using 12.25% polyacrylamide properties over a long period of time due the use gel. of highly diverse germplasm (Horsley et al. 1992). Presence and absence of polypeptide bands Genetic diversity of interrelated uninhabited was observed for all the samples run on gels. The species or crop ancestors can also be central to bands were scored as 1 for presence and 0 for use in breeding to resolve difficulties related to absence and binary data (1, 0) were recorded in crop disappointment (Geleta, 2007). Heritable Binary data matrix (M.S Excel 2013 sheet) for all variety inside and among crop populaces is genotypes. Similarity coefficients were generated resolute using procedures such as morphological using presence absence pattern based pair-wise depiction, electrophoresis, and DNA (or comparison of cultivars, both for unique or shared molecular) marker analysis (Nisar et al. 2008; polypeptide bands. Ghafoor et al. 2008; Ali et al. 2017). Aim of the research work of celtis austrailus was evaluated to identify the genetic diversity RESULTS among genotype of celtis austrailus with the help of morphological traits both (qualitative and Agro-morphological evaluation quantitative), and also enlist and conserve the During the study total 80 landraces of Celtis celtis austrailus plant. australis were studied. We chose total of 11 agro- morphological characters (Four qualitative and MATERIALS AND METHODS Seven quantitative) to be study. The frequency distribution for agro-morphological traits Exploration and collection containing kernel color, seed shape, shell texture, During October 2017 different exploratory seed color, leaf length, leaf width, petiole length, trips were arrange to 80 different agro ecological internode length, seed length, seed width, 10 zones (50 genotypes from Malakand and 30 seeds weight. genotypes from Swat). A total of 80 genotypes of Celtis australis were collected and processed for Qualitative characters morphological and biochemical characterization. In qualitative characters kernel colors normally yellow for some genotypes while green Morphological characterization in some landraces. The seed shape is variable in Morphological characterization was carried the collected 80 genotypes such as round, oval out for estimation of inter and intra species while the shell texture is hard, semi hard, soft and genetic diversity, a total of 11 morphological seed color is white, black, yellow. characters were scored. Out of 11 characters 4 were qualitative and 7 quantitative. Qualitative Quantitative characters trait includes Kernal colour (KC), Seed shape (SS), Shell texture (ST), Seed colour (SC), Leaf length Quantitative traits includes Leaf length (LL), Leaf Leaf length was divided into three categories width (LW), Petiole length (PL), Internode length to range from 4.0cm to 15.0cm with a mean value (IL), Seed length (SL), Seed width (SW), Weight of 8.46 and sample variance 5.05. Frequency of 10 seeds. distribution of leaf length is given in figure.

Evaluation of Seed Storage Protein Leaf width A total of 80 samples of Celtis australis were The range of leaf width of plant is 1.0cm to collected from different ecological regions. For the 7.0cm, the mean value was recorded 3.6 and extraction of protein, with the help of pestle and sample variance 1.39. Minimum value was 1cm mortar the seed of single genotype was ground to and maximum value was 7 cm were found. The the fine powder. From each sample 0.01g powder frequency distribution for leaf width plant is given were taken in 1.5ml in eppendorf tube and 400μl in figure. protein extraction buffer were add and centrifuge. As a supernatant the crude proteins were Petiole length extracted and kept in 4ºC till electrophoresis. In this case the petiole length divided into

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Izharullah et al. Assessing diversity in celtis austrailus three groups, range from 0.5cm to 4.5cm the 21 Correlation coefficient values were observed in mean value 1.36 sample variance 0.28 the which 17 were found positive and 4 were found maximum value 3 and minimum1. negative. Correlation of plant leaf length (0.454**), leaf Internode length width, petiole length also positive correlated with The internodes length was studied in the leaf length (.282*), leaf length were positive population. The internodes length ranges from correlated with 100 seed weigh .289**t, seed 0.5cm with a mean value 2.21 sample variance width was strong positive correlated with seed 0.78 .The maximum values of 6 while the length .677**, and also positive correlate with 100 minimum value was 0.5cm. The frequency seed weight .246*, and seed width also positive distribution of this genotype divided the total correlate with 100 seed width .236*, petiole length studied genotype into three groups. negative correlate with leaf length -0.015, inter node length were negative correlate with seed Seed length width -0.135. The seed length was studied in the population showing the following variation. The fruit length Cluster analysis for agro-morphological ranges from 4 to 11mm with a mean value 7.49, characters sample variance 3.01 .The maximum value of Cluster analysis of 80 genotypes for agro- was 11cm while the minimum value of 4.The morphological characters was conducted using frequency distribution of these genotype divided Ward’s Method as shown in Figure (). The the total studied genotype into three groups. dendogram divided into two linkages, the linkage further divided into 11 clusters. Linkage 1 consists Seed width of 8 cluster and linkage 2 in 3 clusters. In the present study three types of seed width were listed. Range from 4 to 11 with mean value Biochemical analysis 7.48 sample variance 2.91. 80 genotypes of celtis australis were tested for protein profiling through SDS-PAGE. 14 bands 1000 seed weight were observed in all selected genotypes (Fig 5 The 1000 seed weight was studied in the and 7). A total of 68% genetic diversity was found population. The 1000 seed weight from 10 to 50g the outmost level of variation was found in B1 with a mean 2.47 value sample variance 0.73. (0.68%), B2 (0.63%), B3 (0.49%), B5 (0.48%), B9 The maximum value of 50g while the minimum (0.43%), B6 and B8 (0.42%) were polymorphic value of 8g .The frequency distribution of these respectively. Similarly B12 (0.39%) revealed low divided the total studied genotype into three level genetic diversity B4 and B7 (0.38%), B13 groups. (0.35%), B11 (0.28%), B10 (0.27%) and B14 (0.20%). Protein binary data matrix based genetic Correlation tree grouped the 80 genotypes into different Correlation analysis was conducted for seven clusters. Three linkages L-1, L-2 and L-3 were agro-morphological characters of 80 Celtis noted in dendogram as shown in (figure 5). australis landraces by MS Excel (2013). A total of

Table 1: Frequency distribution for qualitative traits

Traits Type F %age Kernel covered Green 10 12.5 Yellow 70 87.5

Seed shape Oval 8 10 Round 72 90

Shell texture Soft 4 5 Semi hard 74 92.5

Hard 2 2.5

Seed color Black 30 37.5 White 46 57.5

Yellow 4 5

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Table 2: Descriptive statistic of seven quantitative traits.

Std. Std. Sample Traits Mean Minimum Maximum CV% Error Deviation Variance LL 8.46 0.25 2.24 5.04 4 15 78.24 LW 3.51 0.13 1.18 1.39 1 7 84.75 PL 1.36 0.06 0.53 0.28 1 3 87.43 IL 2.21 0.10 0.88 0.78 1 5 13.44 SL 7.49 0.19 1.74 3.01 4 11 30.46 SW 7.48 0.19 1.71 2.91 4 11 34.45 10S/w 2.47 0.10 0.86 0.73 0.77 4.8 89.85

Table 3: Correlation of 7 quantitative traits.

Traits LL LW PL IL SL SW 10S/w LL 1

LW .454** 1

PL -0.015 0.144 1

IL .282* 0.077 0.103 1

SL 0.097 -0.08 0.025 0.039 1

SW 0.157 -0.009 0.017 -0.135 .677** 1

100S/w .289** 0.095 -0.004 0.083 .246* .236* 1 **. Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

Table 4: Cluster analysis for agro-morphological characters

CLUSTER GENOTYPE C-1 CA01, CA11, CA49, CA20, CA21, CA33, CA30 C-2 CA12,CA54, CA26, CA27, CA29, CA65, CA25, CA74, CA76, CA79 CA10, CA60, CA15, CA46, CA64, CA75, CA72, CA34, C-3 CA69, CA29, CA62, CA63, CA80 C-4 CA35, CA38, CA41 C-5 CA42, CA53, CA52, CA45, CA44, CA55, CA47 C-6 CA02, CA66, CA78, CA07, CA73 C-7 CA04, CA24, CA14, CA19, CA66, CA16, CA77, CA17, CA32, CA28 C-8 CA03, CA22, CA13, CA68, CA05, CA57,CA58, CA61, CA59, CA08, CA09, CA70, CA31, CA18 C-9 CA37 C-10 CA35, CA71, CA56, CA67, CA40, CA43, CA39, CA48 C-11 CA56, CA51

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Figure 1: Frequency distribution of qualitative traits

Figure:2: Graphic shown distribution of frequency for seven quantitative traits

Figure 3: Dendogram tree for quantitative characters

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Figure 4: PCA for quantitative traits

Figure 5: Two way cluster Dendogram for seed storage protein

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Figure 6: PCA for seed storage protein

Figure 7: Gel picture of some genotypes of Celtis australis L. yield and biomass indicated the scope of DISCUSSION improvement through simple selection that has Celtis australis L. and many other 42 been suggested in various legumes (Coulibaly et multipurpose and shrubs species are al. 2002). cultivated in agroforestry systems for fodder, fuel, In the present research work a total of 80 fibre, timber and various uses (Bhatt, 2002). Its genotypes of celtis austrailus were evaluated to timber is used for making tool and whip-handles, identify the genetic diversity among genotype of cups, spoons, churners, sports goods, oars, snake bean with the help of morphological traits. canoes, agricultural implements, and for carving. A total of 11 agro-morphological characters (Four The material also used for paper, pulp and its qualitative and Seven quantitative) were studied. wood is used fuelwood. The sweet drupes are The frequency distribution for agro-morphological eaten by birds, squirrels, monkeys and rodents traits containing kernel color, seed shape, shell and thus help in seed dispersal. Celtis australis ia texture, seed color, leaf length, leaf width, petiole an ornamental tree, for urban areas because of length, internode length, seed length, seed width, resistance to drought and parasites. 10 seeds weight and qualitative traits as round, The primary objective of the study was to oval while the shell texture is hard, semi hard, soft investigate the amount of genetic diversity in and seed color is white, black, yellow. ). For these snake bean germplasm. High variability for grain character descriptive statistics, correlation and

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Izharullah et al. Assessing diversity in celtis austrailus cluster analysis was done the quantitative traits SDS PAGE provided valid evidence for detecting was observed with significant variation for all the intra specific variation and assessing inter specific traits. Even though quantitative characters are relationships (Hameed et al. 2009; Emre et al. influenced by the environmental conditions, they 2010).according to (Zeeshan et al. 2012) says should not be excluded or neglected by that he worked on 200 accession of barley, but conservationists in different processes of crop the bands were not clear, SDS PAGE data was diversity studies (Yao, 2007).the result was not suitable for analysis. But celtis australus as similarly investigate in cereal crops such as wild plant and also investigate through protein (Ebrahim et al. 2015) who reported the same profiling which consist of the protein profile of 80 result for the agro morphological traits he divided genotypes of celtis australis tested through SDS- into 4 cluster. Ghafoor et al. (2005) reported PAGE revealed fourteen reproducible bands. The Quantitative traits change is reflected by the electrophoregram is consists of 80 genotypes of diversity within and between regions, the celtis australis protein with 14-bands, A total of geographical source of genetic variability is 18% genetic diversity was found the outmost level determined. of variation was found in B1 (0.68%) followed by The correlation, mean and standard variation B2 (0.63%) and B3 (0.49%), B5 (0.48%), B9 was calculated by using the software (0.43%), B6 and B8 (0.42%) polymorphism STATISTICA and SPSS, and observe magnificent respectively. Similarly B12 (0.39%) revealed low result, among the eight quantitative traits was level of i.e.B4 and B7 (0.38%) and B13 (0.35%), show strong correlation Correlations between B11 (0.28%), B10 (0.27%) and B14 (0.20%). morphological variables of Celtis australis: The Significant level of Genetic diversity observed correlation matrix of Pearson (n) indicates that within banding pattern is an advantage for plant there are a negative correlations and other breeder’s in future breeding activities. Similarly positive between variables taken two by two (Ali et al. 2017) reported the same result for (Table 2). Some correlations were negatively high genetic diversity of Zea mays L. For present study between morphological characteristics of trees of PCA (scatter plot) were also draw which show different provenances. The Total height of the tree variation among the genotype which divided into shows strong negative correlations between different group on the basis of their similarities crown height (r = -0.747) and the diameter of the and differences. crown (-0.508) (Hameed et al. 2009). In our only The present genotypes for the research work for the seven quantitative traits were analyze for were collected from different regions of Malakand correlation which are Correlation of plant leaf and Swat district, Khyber Pakhtunkhwa, Pakistan. length (0.454**), leaf width, petiole length also positive correlated with leaf length (.282*), leaf CONCLUSION length were positive correlated with 100 seed It was concluded that for investigation of weigh .289**t, seed width was strong positive genetic divergence, four qualitative traits found for correlated with seed length .677**, and also kernel color, seed shape, shell texture, seed color. positive correlate with 100 seed weight .246*, and In case of quantitative characters, maximum seed width also positive correlate with 100 seed variation was leaf length, leaf width, petiole length, width .236*, petiole length negative correlate with internodes length, seed length, seed width, 10 leaf length -0.015, inter node length were negative seeds weight. A considerable variation was correlate with seed width -0.135 . observe in SDS-PAGE analyses of seed storage The biochemical analysis using SDS-PAGE protein were also done which give significant has great contribution for the estimation of genetic result, in total of 14 bands were observed found diversity (Zahoor et al. 2015). Through SDS variation among the cluster. It was suggested that PAGE various grain legumes crop plants have representative genotype from different clusters been elaborated previously through could be chosen for the future hybridization electrophoresis of seed protein, for example program. Chickpea (Nisar et al. 2007; Hameed et al. 2009). For the protein profiling SDSPAGE is a CONFLICT OF INTEREST biochemical technique which is widely used for The authors declared that present study was identification genetic structure of different crops performed in absence of any conflict of interest. because it is simple, reliable and free from fluctuation (Ghafoor et al. 2005).the researchers ACKNOWLEGEMENT approved that biochemical analysis of protein by The authors acknowledged the Higher

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Education Commission of Pakistan for financial areas using agromorphological traits and support. hordein. African Journal of Biotechnology. 14(22): 1886-1896. AUTHOR CONTRIBUTIONS Emre, İ., Turgut-Balik, D., Genç, H., and Sahin, A. IU and MN designed the experiments. MA, AA (2010) Total seed storage protein patterns of and WK perform the experiments and wrote the some Lathyrus species growing in Turkey manuscript. NN and MS reviewed the manuscript using SDS-PAGE. Pak. J. Bot. 42(5): 3157- and data analysis. All authors read and approved 3163. the final version. Gaur, R. D. (1999) Flora of the District Garhwal Copyrights: © 2021@ author (s). Northwest Himalaya with Ethnobotanical This is an open access article distributed under the Notes. Transmedia Publication Centre, terms of the Creative Commons Attribution License Srinagar Garhwal, Uttaranchal, India, pp. (CC BY 4.0), which permits unrestricted use, 811. distribution, and reproduction in any medium, Ghafoor, A. (1999) Genetic diversity and gene- action in Vigna mungo based on provided the original author(s) and source are morphological and biochemical credited and that the original publication in this markers (Doctoral dissertation, Quaid-i-Azam journal is cited, in accordance with accepted University Islamabad, Pakistan). academic practice. No use, distribution or Ghafoor, A., Ahmad, Z., & Afzal, M. (2005) Use of reproduction is permitted which does not comply SDS-PAGE markers for determining with these terms. quantitative traits loci in blackgram [Vigna mungo (L.) Hepper] germplasm. Pakistan REFERENCES Journal of Botany. 37(2): 263. Ali, M., Alam, M., Wadood , S. F., Khan, W., Ghafoor, A., and Arshad, M. (2008) Seed protein Uddin, N., Zaman, S. U., Nazir, N., Karim, N., profiling of Pisum sativum L., germplasm Ali, Z., and Nisar, M. (2017) Biochemical using sodium dodecyl sulphate characterization of Pakistani Zea mays polyacrylamide gel electrophoresis (SDS- landraces growing in the remote areas of PAGE) for investigation of biodiversity. Pak. Khyber Pakhtunkhwa. International Journal J. Bot. 40(6): 2315-2321. of Biosciences. 11(3): 51-58. Hameed, A., Shah, T. M., Atta, B. M., Iqbal, N., Bhatt, B. P. (2002) some multipurpose tree Haq, M. A., and Ali, H. (2009) Comparative species for agroforestry systems. ICAR seed storage protein profiling of Kabuli Research Complex for NEH Region, Umiam, chickpea genotypes. Pak. J. Bot. 41(2): 703- Meghalaya. 710. Bisht, J. K., and Yadav, R. P. (2015) Horsley, R. D., Schwarz, P. B., Faue, A. C., and Ethanobotanical utilization of trees in Manthey, F. A. (1992) Survey of beta-glucan kumaon region, North West Himalaya, India. content of barley cultivars adapted to North Not Available. Dakota. North Dakota farm research-North Chopra, R. N., and Nayar, S. L. (1956) Glossary Dakota, Agricultural Experiment Station of Indian medicinal plants. Council of (USA). Scientific And Industrial Research; New Luna, R. K. (1996) Plantation trees. International Delhi. Book Distributors. Coulibaly, S., Pasquet, R. S., Papa, R., and Martins, S. R., Vences, F. J., de Miera, L. S., Gepts, P. (2002) AFLP analysis of the Barroso, M. R., and Carnide, V. (2006) phenetic organization and genetic diversity of RAPD analysis of genetic diversity among Vigna unguiculata L. Walp. reveals extensive and within Portuguese landraces of common gene flow between wild and domesticated white bean (Phaseolus vulgaris L.). Scientia types. Theoretical and Applied Horticulturae. 108(2): 133-142. Genetics. 104(2-3): 358-366. Nisar, M., Ghafoor, A., Khan, M. R., Ahmad, H., Duke, J. A., and Ayensu, E. S. (1985) Medicinal Qureshi, A. S., and Ali, H. (2007) Genetic plants of China(Vol. 2). Reference diversity and geographic relationship among Publications. local and exotic chickpea germplasm. Pak. J. Ebrahim, S., Shiferaw, E., and Hailu, F. (2015) Bot. 39(5): 1575-1581. Evaluation of genetic diversity in barley Nisar, M., Ghafoor, A., Ahmad, H., Khan, M. R., (Hordeum vulgare L.) from Wollo high land Qureshi, A. S., Ali, H., and Islam, M. (2008)

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