Genetic diversity of functional food species Spinacia ...

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Genetic diversity of functional food species Spinacia oleracea L. by protein markers a

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M. Rashid , Z. Yousaf , M.S. Haider , S. Khalid , H.A. Rehman , A. a

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Younas & A. Arif a

Department of Botany, Lahore College for Women University, Jail Road, Lahore, Pakistan b

Institute of Agricultural Sciences, University of Punjab, Lahore, Pakistan Published online: 05 Feb 2014.

To cite this article: M. Rashid, Z. Yousaf, M.S. Haider, S. Khalid, H.A. Rehman, A. Younas & A. Arif , Natural Product Research (2014): Genetic diversity of functional food species Spinacia oleracea L. by protein markers, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.881359 To link to this article: http://dx.doi.org/10.1080/14786419.2014.881359

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.881359

Genetic diversity of functional food species Spinacia oleracea L. by protein markers M. Rashida, Z. Yousafa*, M.S. Haiderb, S. Khalida, H.A. Rehmana, A. Younasa and A. Arifa a

Department of Botany, Lahore College for Women University, Jail Road, Lahore, Pakistan; Institute of Agricultural Sciences, University of Punjab, Lahore, Pakistan


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(Received 7 November 2013; final version received 27 December 2013) Exploration of genetic diversity contributes primarily towards crop improvement. Spinacia oleracea L. is a functional food species but unfortunately the genetic diversity of this vegetable is still unexplored. Therefore, this research was planned to explore the genetic diversity of S. oleracea by using morphological and protein markers. Protein profile of 25 accessions was generated on sodium dodecyl sulphate polyacrylamide gel. Total allelic variation of 27 bands was found. Out of these, 20 were polymorphic and the rest of the bands were monomorphic. Molecular weights of the bands ranged from 12.6 to 91.2 kDa. Major genetic differences were observed in accession 20541 (Peshawar) followed by 20180 (Lahore) and 19902 (AVRDC). Significant differences exist in the protein banding pattern. This variation can further be studied by advanced molecular techniques, including two-dimensional electrophoresis and DNA markers. Keywords: electrophoresis; genetic diversity; monomorphic; polymorphic; Spinacia oleracea

1. Introduction Spinacia oleracea L. (spinach) belongs to the family Chenopodiaceae. It is native to Central and south-western Asia. Spinach is an annual herb and is grown all over the world over almost 800,000 hectares (Hu et al. 2007). It can survive winters in temperate regions. Spinach is highly nutritious and rich in minerals. It is a good source of vitamins, magnesium, manganese, iron, calcium, potassium, copper, protein, phosphorus, zinc, selenium, antioxidants and fatty acids (Ball 2006). Vitamin B9 was first isolated from spinach in 1941 (Ismail et al. 2013). It is a good source of carotenoids, flavonoids, apocyanin and p-coumaric acid (Burton 1976; Zennie et al. 1977). Besides this, spinach is also a plant with medicinal importance. The whole plant is used for remedy of urinary calculi while its leaves are used especially for bowel, lung inflammation, febrile affliction and cooling (Jain & De-Fillipps 1991). Many authors have reported a number of powerful water-soluble natural antioxidants from the leaf extract of spinach possessing biological activities (Lomnitski et al. 2000). Agronomic, morphological and physiological traits were mostly used to characterise the different varieties; however, this type of data is unable to provide accurate genetic diversity estimation due to multiple alleles. Besides, the technique of field testing and evaluation is somehow difficult and time consuming, whereas biochemical markers are considered more authentic and less time consuming for the exploration of genetic diversity (Rahman et al. 2004). Among these biochemical techniques, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is more simple, valid, practically reliable and extensively used

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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for genetic characterisation of germ plasm (Ghafoor et al. 2002). Electrophoretic protein profiles could be used as genotype markers, due to their high stability and independence of ecological conditions. In spite of a considerable progress in genetic improvement of S. oleracea L., its genetic variability at molecular level has not been investigated. Therefore, the main objective of this research was the assessment of genetic diversity of spinach accessions (collected from different areas of Pakistan) by using SDS-PAGE technology and molecular markers. 2. Results and discussion Seed proteins are usually more stable and unaffected by environmental fluctuations, due to which their study has been extensively used to characterise the taxonomic and evolutionary features of different crops (Mennella et al. 2001; Ghafoor et al. 2002; Yousaf et al. 2006). Seed proteins of 25 accessions of S. oleracea L. were analysed by using SDS-PAGE technique. This is comparatively a simple, practically reliable and widely used biochemical method to analyse the genetic variability of different accessions (Ghafoor et al. 2002). Germination period, germination rate, seed shape, colour and weight, plant height, stem colour and length, number of leaves per plant, petiole length, leaf shape and size were selected as important indicating markers (Supplementary Tables S2 and S3). All quantitative characters have revealed considerable variation, while qualitative characters revealed slight variation among 25 accessions. Other systematic scientists are also of the opinion that the geographical condition has more influence on quantitative characters than qualitative (Sneath & Sokal 1973). Banding pattern for seed proteins of spinach on 12% polyacrylamide gel represented variation in number of bands/accession and intensities of protein bands (Supplementary Figure S1). The whole banding pattern was divided into three distinct zones (A, B and C) on the basis of intensity of bands, that is dark stained, medium and lightly stained. Total allelic variation obtained, ranged from 12.6 to 91.2 kDa, which were spread into A, B and C zones. Total number of bands was 27 of which 20 were polymorphic and 4 were monomorphic. Zone A contained nine lightly stained bands. These ranged from 53.7 to 91.2 kDa among which five bands were polymorphic and one was monomorphic. Zone B contained seven bands with both dark and lightly stained bands. The bands of this zone ranged from 29.5 to 50.1 kDa with four polymorphic and three monomorphic bands. Zone C consisted of 11 bands including both medium and lightly stained bands. These bands ranged from 12.6 to 28.2 kDa, all of which were polymorphic. Protein band classification based on the number parallel to staining intensity (representing concentration) was also used for systematic observations by different researchers for different plant species and accessions (Tabassum & Ashraf 2006; Ghafoor & Arshad 2008; Miskoska-Milevska et al. 2008; Turi et al. 2010; Win et al. 2011; Barpete et al. 2012). Win et al. (2011) found 14 polymorphic bands out of 18 protein bands in 68 Myanmar cowpea accessions. Ghafoor and Arshad (2008) observed 62% variation in the protein profiles of 67 pea genotypes. It was observed that accessions varied significantly in their intensities as well as the number of protein bands. Most of the accessions contained 12 protein bands while number of bands varied in other accessions with a range of 10 – 18. Protein bands with molecular weights 79.4, 66.1 and 63.1 kDa were the only bands present in all accessions. These bands may be considered as species specific and used for the identification of spinach. Besides this, bands of 53.7, 35.5, 33.1 and 29.5 kDa molecular weights were monomorphic. These were detected only in accession 20541. This accession was collected from Peshawar. Hence, this band may be an important ecological marker for this vegetable. The other 20 bands were polymorphic. It is also believed that the polymorphism in storage proteins of seeds is greatly linked with the geographical regions of that particular species or accession (Ghafoor et al. 2003; Nisar et al. 2007). One accession (19902) from AVRDC and two local accessions (20180 and 20541) from Lahore and


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Figure 1. Dendrogram of morphological characters of 25 accessions of S. oleracea L.

Peshawar, respectively, demonstrated major genetic differences in protein banding pattern (Figure 1). However, protein profile of some accessions from Haripur, Hafizabad, Attock, Gujranwala, Mansehra, Khanewal and Bahawalpur indicated great similarity among them. Therefore, it may be concluded that the genetic diversity of spinach accessions is affected by climatic conditions of various regions. Genetic linkage among 25 accessions of S. oleracea L. was carried out by cluster analysis and a dendrogram was constructed based on protein profiles (Figure 2). Different kinds of

Figure 2. Dendrogram of protein profiles of 25 accessions of S. oleracea L.


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proteins in diverse plant genotypes may become responsible to distinguish them at seed level and to make record of genetic resources (Rahman et al. 2004). According to the clusters formed, all accessions were divided into two major groups 1 and 2. Group 1 contained accession 20541, while group 2 was re-divided into subgroups 2A and 2B. Subgroup 2A consisted of accessions 19902 and 20180, while 2B was again divided into subgroups 2Bi and 2Bii. Subgroup 2Bi included six accessions of which three accessions (20095, 20209 and 20308) had 100% similarity, while the other three (20118, 20068 and 20334) were also 100% same in their protein profiles. In addition, subgroup 2Bii was divided into two subgroups: 2Biia and 2Biib. Subgroup 2Biia contained seven accessions from which accessions 20322, 20540 and 20188 and 20402, 20110 and 20240 had 100% similarity with respect to one another, while accession 20125 was different from them. Subgroup 2Biib consisted of nine accessions in which two accessions (20074 and 20066) were same and the other seven (20476, 20256, 20502, 20219, 20127, 20314 and 20100) were identical to one another. Furthermore, on the basis of morphological markers and protein markers, a relationship was built among 25 accessions of S. oleracea L. (Table 1). Protein profiles of all accessions divided them into different subclusters (Figure 1). Accession 20541 was different from accessions 19902 and 20180 in their protein profiles. Accession 20541 contained 53.7, 33.1 and 29.5 kDa protein bands which were absent in other two accessions. In addition, accession 19902 contained 69.2, 25.7 and 14.1 kDa and accession 20180 had 28.2 kDa distinguishing protein bands. Similarly, their morphological characters also differentiated them from one another in their seed shapes. All these had prickly seeds with different spine numbers, that is 2 –6. In their protein profiles, Table 1. Cluster-based relationship among 25 accessions of S. oleracea L.

Cluster 1 2

Sub Clusters 2A 2B

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-

2Ba

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2Bb 2Bbi

2Bbii

Accessions in SubClusters 20541 19902 20180 20095 20209 20308 20118 20068 20334 20322 20540 20188 20402 20110 20240 20125 20476 20256 20502 20219 20314 20127 20100 20066 20074

Morphological Variation

Protein Profile

Prickly seeds with up to 6 spines Prickly seeds with up to 3 spines Prickly seeds with up to 2 spines Same

53.7, 33.1, 29.5 69.2, 25.7,14.1 28.2 Same

15.5 Brown

21.4

Yellowish Brown

15.5

Irregular seeds with 5 ends

Same 13.2

Irregular seeds with 4 ends Same Irregular seeds with 5 ends

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one band was found to be at similar location (29.5, 25.7 and 28.2 kDa) which can be considered to be responsible in creating difference among spine numbers. Morphology of all other 22 accessions was found to be similar with slight differences among their protein banding patterns. These minor differences included presence of 15.5, 21.4 and 13.2 kDa protein bands in some accessions while absent in others. These findings indicated the relationship between morphological and protein markers among different accessions. Variations in seed proteins can also become responsible to create differences in morphological appearance of accessions. This outcome was also supported by researches of different authors (Yousaf et al. 2006; Jatoi et al. 2011). In contrast to these reports, Ghafoor et al. (2003) found no genetic basis to differentiate small and large seeds of lentils. 3. Methodology Different accessions of spinach (S. oleracea L.) were analysed genetically and morphologically to explore the existing genetic diversity among them. Seeds of 25 spinach accessions were obtained from gene bank of Institute of Agri-biotechnology and Genetic Resources (IABGR), National Agriculture Research Centre (NARC), Islamabad, Pakistan (Supplementary Table S1). Morphological markers such as seed shape, colour, weight, germination rate, plant height, stem colour, length, number of leaves, petiole length, leaf tip, base, length and width were considered. Proteins of 25 accessions of S. oleracea L. were analysed by using SDS-PAGE. Electrophoresis was performed according to the modified Laemmli method (1970) by vertical slab gel in discontinuous buffer system. Protein profile was studied after staining and de-staining of the gel (detail in Supplementary material). Dissociated polypeptide sample weight can be determined by plotted standard curve to calculate the Rf value for standard protein markers. Standard curve was used to determine the molecular weight of unknown protein band. Presence (1) or absence (0) of band was scored and entered in a simple statistics, for which a dendrogram was built with the help of computerised statistical software (Minitab). 4. Conclusion From the obtained results, it is concluded that the genetic variation found in spinach accessions is significant. These genetic differences were directly linked with that of morphological differences. Therefore, it is recommended that the DNA-based markers and two-dimensional electrophoresis must be explored for more comprehensive assessment of genetic diversity. Supplementary material Supplementary material relating to this article is available online, alongside Tables S1 –S3 and Figure S1. References Ball GFM. 2006. Vitamins in foods: analysis, bioavailability, and stability. Food science and technology. Boca Raton (FL): CRC Press; p. 236. Barpete S, Dhingra M, Parmar D, Sairkar P, Sharma NC. 2012. Intraspecific genetic variation in eleven accessions of Grass Pea using seed protein profile. Sci Secure J Biotech. 1:20– 27. Burton TB. 1976. Human nutrition. In: Lapedes DN, editor. Encyclopaedia of food agriculture and nutrition. 4th ed. New York, NY: H.J. Heinz [by] McGraw-Hill; p. 1634. Ghafoor A, Arshad M. 2008. Seed protein profiling of Pisum sativum L., germplasm using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for investigation of biodiversity. Pak J Bot. 40:2315–2321.


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Ghafoor A, Ahmad Z, Qureshi AS, Bashir M. 2002. Genetic relationship in Vigna mungo (L.) Hepper and V. radiata (L.) R. Wilczek based on morphological traits and SDS-PAGE. Euphytica. 123:367–378. Ghafoor A, Gulbaaz FN, Afzal M, Ashraf M, Arshad M. 2003. Inter-relationship between SDS-PAGE markers and agronomic traits in chickpea (Cicer arietinum L.). Pak J Bot. 35:613–624. Hu J, Mou B, Vick BA. 2007. Genetic diversity of 38 spinach (Spinacia oleracea L.) germplasm accessions and 10 commercial hybrids assessed by TRAP markers. Genet Resour Crop Evol. 54:1667–1674. Ismail F, Farah NT, Memon AN. 2013. Determination of water soluble vitamin in fruits and vegetables marketed in Sindh Pakistan. Pak J Nutr. 12(2):197–199. Jain SK, De-Fillipps RA. 1991. Medicinal plants of India. Algonac, MI: Reference Publication; p. 227–230. Jatoi SA, Javaid A, Iqbal M, Sayal OU, Masood MS, Siddiqui SU. 2011. Genetic diversity in radish germplasm for morphological traits and seed storage proteins. Pak J Bot. 43:2507– 2512. Laemmli UK. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature. 227:680–685. Lomnitski L, Carbonatto M, Ben-Shaul V, Peano S, Conz A, Corradin L. 2000. The prophylactic effects of natural water soluble antioxidant from spinach and apocynin in a rabbit model of lipopolysaccharide induced endotoxemia. Toxicol Pathol. 28:588–600. Mennella G, Onofaro SV, D’Alessandro A, Coppola R, Poma I. 2001. Tomato ecotype characterization by anionic exchange high performance liquid chromatography analysis of endosperm seed proteins. Acta Horticulturae. 546:453–457. Miskoska-Milevska E, Dimitrievska B, Poru K, Popovski ZT. 2008. Differences in tomato seed protein profiles obtained by SDS-PAGE analysis. J Agric Sci. 53:13–23. Nisar M, Ghafoor A, Khan MR, Ahmad H, Qureshi AS, Ali H. 2007. Genetic diversity and geographic relationship among local and exotic chickpea germplasm. Pak J Bot. 39:1575–1581. Rahman MM, Hirata Y, Alam SE. 2004. Genetic variation within Brassica rapa cultivars using SDS-PAGE for seed protein and isozyme analysis. J Biol Sci. 4:239–242. Sneath PHA, Sokal RR. 1973. Numerical taxonomy: the principles and practice of numerical classification. San Francisco: W.H. Freeman & Co.; p. 573. Tabassum S, Ashraf M. 2006. Qualitative analysis of seed storage protein of shisham (Dalbergia sissoo). J Chem Soc Pak. 28(3):266–270. Turi NA, Farhatullah, Rabbani MA, Khan NU, Akmal M, Pervaiz ZH, Aslam MU. 2010. Study of total seed storage protein in indigenous Brassica species based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afr J Biotechnol. 9:7595–7602. Win KT, Oo AZ, New KL, Thein MS, Yutaka H. 2011. Diversity of Myanmar cowpea accessions through seed storage polypeptides and its cross compatibility with the subgenus Ceratotropis. J Plant Breed Crop Sci. 3:87–95. Yousaf Z, Masood S, Shinwari ZK, Khan MA, Rabbani A. 2006. Evaluation of taxonomic status of medicinally important species of the genus Solanum and Capsicum based on acrylamide gel electrophoresis. Pak J Bot. 38(1): 99–106. Zennie M, Thomas D, Ogzewalk C. 1977. Ascorbic acid and vitamin A content of edible wild plants of Ohio and Kentucky. J Econ Bot. 31:76–79.