GENETIC DIVERSITY IN PRUNUS PERSICA L (BATSCH) REPORTED FROM MALAKAND DIVISION, KHYBER PAKHTUNKHWA, PAKISTAN Mohammad Nisar and Ihsan Ullah Department of Botany, University of Malakand, Chakdara (Dir Lower), Khyber Pakhtunkhwa, Pakistan Email: mnshaalpk@yahoo.com Abstract A total of 20 genotypes were collected from unexplored area of Malakand division, NWFP, Pakistan and evaluated on the basis of morphological traits and SDS-PAGE. In morphological traits significant level of genetic diversity was observed in leaf size (29.46% CV), fruit weight (11.36% CV), flower color and flower shape. The data both on morphological traits and SDS-PAGE were analyzed through cluster analysis. In morphological traits (both qualitative and quantitative traits) it was evident that there was cluster analysis not sorted the collected germplasm into no consistence in the grouping of the cultivars and were inter-spread within clusters. Similarly, SDS- PAGE divide the collected cultivars into two main groups i.e. L 1 and L 2; however at linkage distance 2.4 tree was sub-divided into 2 clusters C 1 and C 2. C 1 sorted three cultivars: Ammy, Dogar, 7-No and 8-No and C 2 encircled cultivar 5-No only. On the other-hand L 2 is divided into 2 clusters (C 3 and C 4 ) at linkage distance 2.0; while C 3 sorted cultivar 5-No-sample and C 4 5-No-China. Keywords: Peaches Pakistani Cultivars, Biochemical Analysis, Cluster Analysis INTRODUCTION Peach (Prunus persica) is known as a species of Prunus native to China that bears an edible juicy fruit. The tree is deciduous in nature belonging to the subfamily Prunoideae of the family Rosaceae. It is classified with the almond in the subgenus Amygdales within the genus Prunus, distinguished from the other subgenera by the corrugated seed shell. The leaves are lanceolate, 7 15 cm long (3-6 in), 2 3 cm broad, pinnately veined. The flowers are produced in early spring before the leaves; they are solitary or paired, 2.5 3 cm diameter, pink, with five petals. The fruit has yellow or whitish flesh, a delicate aroma, and a skin that is either velvety (peaches) or smooth (nectarines) in different cultivars. The flesh is very delicate and easily bruised in some cultivars, but is fairly firm in some commercial varieties, especially when green. The single, large seed is red-brown, oval shaped, approximately 1.3-2 cm long, and a wood-like husk. The tree is small, and up to 15 ft tall (Huxley, 1992). Peach (Prunus persica) is the most preferred species among the stone fruits and is temperate in nature. This species is a traditional crop of Northern area of Pakistan and occupies an area of 4543 hectares with the production of 48284 tones. Quettea, Kalat, Peshawar, Swat valley and certain parts of Kohistan hills are the main major growing areas of peach. It is considered to be very delicious and attractive in flavour and aroma (Annual report of ARI Tarnab, 2008). Peach plants grow very well in a fairly limited range, since they have a chilling requirement that tropical areas cannot satisfy, and they are not very cold-hardy. The trees themselves can usually tolerate temperatures ranging from 26 C to 30 C, although the following season's flower buds are usually killed at these temperatures, leading to no crop that summer. Flower bud starts rupturing between 15 C and 25 C depending on the cultivar (some are more cold-tolerant than others) and the timing of the cold, with the buds becoming less cold tolerant in late winter. Certain cultivars are more tender and others can tolerate a few degrees colder. In addition, a lot of summer heat is Co-Published By: Human Resource Management Academic Research Society 150
required to mature the crop, with mean temperatures of the hottest month between 20 C and 30 C. The trees tend to flower fairly early in spring while the blooms often damaged or killed by freezes; typically, if temperatures drop below freezing point ( 4 C), However, if the flowers are not fully open, they can tolerate a couple of degrees colder. (Szalay, et al., 2000). Genetic diversity as the germplasm of plants, animals, or other organisms containing useful characters of actual or potential values, especially when these characters provide the variation in genes and genotypes between and within species or populations (Cromwell et al., 1999). According to Nisar (2008) a number of methods were applied for the estimation of genetic diversity in desirable traits for germplasms evaluation that includes morphological characterization, biochemical marker at protein (SDS PAGE) and DNA level. Among these different techniques Acrylamide gel electrophoresis in presence of sodium dodecyl sulfate has become one of the most widely used techniques, which separate and characterize proteins. Genetic diversity is our heritage its conservation and effective evaluation is the key point to deal with the scarcity of food and to develop elite genotypes/cultivars for future generation. Therefore an attempt was been made in the present study to evaluate the level of genetic diversity based morphological and biochemical marker (SDS- PAGE) in Peach germplasm from different agro-ecological zones of Pakistan. The aim of the study was to develop catalogue information for bio-technological treatment using advance level of molecular markers (RAPD, SCAR, SSR markers). The present report well is helpful for the plant breeder to develop different combination for selection of desirable traits. Materials and Methods Plant Materials A total of 20 genotypes of Prunus persica L (Batsch) were collected during 2008-2009, from different zones of Malakand division, NWFP, Pakistan. The collected germplasm was evaluated on the basis of morphological traits and SDS-PAGE analysis. Morphological Traits Morphological traits were further divided into qualitative and quantitative treats. Qualitative traits were scored through general visualization. A total of nine qualitative traits: flower color, flower type, selected plant size, leaf color, leaf type, leaf venation, fruit color, fruit type, fruit shape and disease status of the plant were studied at the relevant stage of development in the available germplasm. Likewise, quantitative traits were recorded for leaf size (cm), flower size (cm), weight of fruit -1 and fruits -10. SDS-PAGE Analysis The collected germplasm was also evaluated using SDS-PAGE, to explore the genetic diversity based on protein profile. For SDS-PAGE analysis, seeds of each genotype were crushed into a fine powder with mortar and pestle. Protein extraction buffer (400 l) was added to 0.01g of seed flour and mixed well with Automatic Lab-Mixer DH-10. The extraction buffer contained the following final concentrations: 0.5M Tris HCl (ph 6.8), 2.5% SDS, 10% glycerol and 5% 2- mercaptoethanol. Bromophenol blue (BPB) was added to the protein extraction buffer as tracking dye to watch the movement of protein in the gel. The molecular weight of the dissociation polypeptides was determined by using molecular weight protein marker MW-SDS-70 Kit (Sigma). The SDS-PAGE of total seed protein was carried out in the discontinuous buffer system following to the method outline of Laemmli, (1970). Acrylamid gel concentration 12.5% and 6 l of sample were used for analyzing germplasm. After staining and de-staining, the gel was dried using (Atto, Rapidery-Mini Japan) gel drier. In order to check the reproducibility of the method two separate gels were run under similar electrophoretic conditions. DATA ANALYSIS Co-Published By: Human Resource Management Academic Research Society 151
Morphological data were analyzed for descriptive statistics, coefficient of variation and correlation was calculated to depict genetic diversity of indigenous germplasm for these traits. Each band in case of SDS-PAGE was treated as a unit character and were scored as preset (1) or absent (0) for each of the band-genotype combination in a binary matrix. Estimates of genetic similarity were calculated between all pairs of genotypes according to Sneath and Sokal (1973). The similarity matrix thus generated was used to assess genetic relationship among the genotypes with a cluster analysis using unweighted pair-group method with arithmetic average (UPGMA). All computation was carried using the computer program STAISTICA vir-2.0. RESULTS Morphological Traits Analysis Varying degree of genetic polymorphism was observed in qualitative traits. A total of six traits were recorded in collected germplasm. Out of which, seven alleles were responsible for fruit color and four for fruit shape. Similarly, three alleles each were scored for flower color, leaf color and fruit test, while two alleles scored for flower type (Fig 1). In the collected cultivars Simple-#-6 were large fruit size; Suhani and Golden completely round shape fruit; Ever-Fruit palm shaped fruit; while all other cultivars were normal in fruit size. Out of cultivar 2 nd - Arhy, Disi-(Wild), Jetty and Baber all the remaining were tasteful (Fig 1) ). Significant level of genetic variation was calculated in leaf size (29.46%) and fruit weight (11.36%), while flower size and fruit weight /10 showed low level of CV% (Table 1). Correlation is the statistical relationship between two or more random variables or observed data. In the present study 4 quantitative traits were computed for traits correlation. It was found that fruit weight /1 highly significantly positive (p-value < 0..01) correlated with flower size only and fruit weight/ 10 with flower color, leaf size and fruit weight (Table 2) Fig 1 Qualitative traits alleles distribution among 20 different genotypes of peaches cultivars collected from Malakand Agency, NWFP, Pakistan Table 1: Descriptive Statistics of Four Quantitative Traits in Peaches Reported from Pakistan Mean± St EE St. Dev Range CV% Co-Published By: Human Resource Management Academic Research Society 152
Traits Minimum Maximum Flower Size 3.26±0.05 0.24 2.80 3.73 7.38 Leaf Size 11.59±0.76 3.41 6.25 21.00 29.46** Fruit weight 97.50±2.48 11.08 54.33 108.50 11.36** Fruit weight/10 967.90±17.76 79.44 672.67 1034.00 8.21 Standard Error - St EE: Standard Deviation - St. Dev: CV Coefficient of Variation Table 2: Correlation Coefficient in Four Quantitative Traits in 20 Different Genotypes of Peaches Collected from Different parts of Pakistan S.No Quantitative traits Flower Size Leaf Size Fruit weight Fruit weight/10 1 Flower Size 1 2 Leaf Size 0.13 1 3 Fruit weight 0.43** 0.36* 1 4 Fruit weight/10 0.57** 0.50** 0.85** 1 ** - highly significantly correlated with p-value< 0.01; * - significantly correlated with p-value < 0.05. Cluster Analysis Based on Morphological Traits Genetic similarity based on qualitative traits in local Peach germplasm was estimated through cluster analysis. The analysis sorted the total germplasm into two main Lineages i.e. Lineage- I and Lineage-II at linkage distance 50. Likewise, at linkage distance 20, the total population was splits into three clusters. C-1 grouped six cultivars i.e. No.6-(Golden), No.7, No.6.69, No.5-(Suhani) and Baber. While C-2 grouped five cultivars i.e. No.8, Early-Green, No.5, No.4 and No.3-(Ary). Similarly, C-3 sorted 9 cultivars and subdivided into two clusters C-3a and C-3b. Sub cluster C-3a clustered cultivar No.4 and No.2-(Ary) and C-3b were grouped No.5-(Golden), No.5-(Basary), No.5- (China), Jetty, Everfruit and Disi-(Wild) (Fig 2). Data scored on quantitative traits were also subjected to cluster analysis and divided the collected germplasm into Lineage-I and Lineage-II at linkage distance 0.12. While at linkage distance 0.03, the total population was split into four clusters. C-1 grouped seven cultivars: No.3, No.6- (Golden), No.5-(Basary), No.5-(China), No.6.69, No.5-(Suhani) and No.8. While C-2 sorted seven cultivars: No.6, Early-Green, No.5, No.4, No.4.5, No.5-(Dogar) and No.3-(Ary). Similarly, C-3 clustered 5 cultivars which were further divided into two sub clusters C-3a and C-3b. C-3a grouped Jety, Baber and No.2-(Ary) and C-3b sorted Jety, Ever-Fruit and Disi-(Wild) (Fig 3). Co-Published By: Human Resource Management Academic Research Society 153
50 40 L -1 L -2 30 20 C -1 C -2 C -3 10 C -3 a C -3 b C -3 c Linkage Distance 0 No. 7 No. 6 No. 6.69 No. 6 (Golden) No. 8 Baber No. 5 No. 4.5 Early Grain No. 5 (Suhani) No. 4No. 3 No. 3 (Ary) Jety Ever Fruit No. 2 (Ary) Disi (Wild) No. 5 No. (Golden) 5 No. (Basary) 5 (China) Fig 2 Genetic similarity in Peaches using Ward s Method based on qualitative traits reported from Pakistan L-1 L-2 C-1 C-2 C-3 C-4 Fig 3 Genetic similarity in Peaches using Ward s Method based on quantitative traits reported from Pakistan Molecular Characterization Genotypes evaluated for morphological traits were also tested through molecular characterization using SDS-PAGE, in order to estimate the picture of genetic diversity in seed storage protein profile. The SDS-PAGE was carried out in various combinations and was optimized that 15% acrylamide gel concentration and 6 µl samples gave the best resolution. To check the reproducibility, all the genotypes were run in two replicates. A total of 23 bands were scored for each genotype, the protein banding profile is given in (Fig. 4). The binary data matrix of 7 genotypes was computed for the construction of phylogenetic tree (dendrogram) using UPGMA. Dendrogram was divided into two lineages i.e. Lineage-1 and Lineage-2 at linkage distance 2.4 the trees was subdivided into two clusters C-1 and C- 2. C-1 sorted four germplasms Ammy, Dogar, 7-No and 8-No; while C-2 sorted only L1 and L2 for 5-No Simple. Similarly, cluster 3 and C4 grouped a single genotype each i.e. 5-No-simpe and 5-No- China respectively (Fig 5 and Table 3). Co-Published By: Human Resource Management Academic Research Society 154
2.5 2.0 Linkage Distance 1.5 1.0 0.5 0.0 Ammy Dogar 7-No 8-No 6-No Simple 5-No Simple 5-No China Fig 5 Cluster distributions of 7 genotypes of Peach collected from Malakand division Table 3: Cluster Distribution of 7 Genotypes of Peach Collected from Malakand Division. S/No Cluster Genotypes 1 C-1 Ammy, Dogar, 7-No, 8-No 2 C-2 6-No Simple 3 C-3 5-No Simple 4 C-4 5-No China C- Cluster 5-No China 5-No Simple 6-No Simple 8-No 7-No Dogar Ammy Bands 4 1 7 14 18 23 Fig: 4 Protein bands of 7 germplasm of Peach collected from Malakand division Co-Published By: Human Resource Management Academic Research Society 155
The table 4 regarding to the allelic distribution reflected that cultivars 5-No-Simple and 5-No-China showed 83% alleles homology while No. 6-Simple, No. 8, No.7, Dogar and Ammy were 96% similarity. Table 4: Allelic Distribution among 7 Germplasms Collected from Malakand Division Bands # No.50-China No.5- (Simple) No.6- (Simple) No.8 No.7 Dogar Ammy B1 0 1 1 1 1 1 1 B2 0 1 1 1 1 1 1 B3 1 1 1 1 1 1 1 B4 0 0 1 1 1 1 1 B5 0 0 1 1 1 1 1 B6 1 1 1 1 1 1 1 B7 1 1 1 1 1 1 1 B8 1 1 1 1 1 1 1 B9 1 0 0 1 1 1 1 B10 1 1 1 1 1 1 1 B11 1 0 1 1 1 1 1 B12 1 1 1 1 1 1 1 B13 1 1 1 1 1 1 1 B14 1 1 1 0 0 0 0 B15 1 1 1 1 1 1 1 B16 1 1 1 1 1 1 1 B17 1 1 1 1 1 1 1 B18 1 1 1 1 1 1 1 B19 1 1 1 1 1 1 1 B20 1 1 1 1 1 1 1 B21 1 1 1 1 1 1 1 B22 1 1 1 1 1 1 1 B23 1 1 1 1 1 1 1 present allele 19 19 22 22 22 22 22 Absent allele 4 4 1 1 1 1 1 % similarities 83 83 96 96 96 96 96 Total variation 17 17 4 4 4 4 4 B- Band, 1- Presence, 0- Absence Co-Published By: Human Resource Management Academic Research Society 156
DISCUSSION Investigation on the extant of genetic diversity is vital to maintain, evaluate and utilize germplasm effectively. Germplasm is the main source used by the plant breeder to develop new cultivars, while characterization of germplasm determines the purity of cultivar (Barenger, 2004; Nisar et al., 2008). Present study was planned to investigate the un-seen genetic potential of Pakistani peaches cultivars. It was investigated that flower color is found under the control of three different alleles as previously reported by Edward et al., (1994), while the fruits were downy drupe and succulent with deeply furrowed. High level of diversity were found in fruit shape, among these the Cultivar-6 had large fruit like mango shape, Suhani and Golden completely round shape fruit like orange; Ever-fruit small drupe like Prunus domestica L., while the remaining are all large drupe. In general observation and comparative normal human taste the cultivar No-6, No-7, China-8, Arhay-3 are more tasteful; while Arhy-2, Disi (Wild), Jetty and Baber are with sour taste. The studied developed catalogue information based on morph-metric which is the first step for further study. It is suggested that physical, chemical and sensorial parameter is employed to explore the exact nature of the important germplasm (Infante et al., 2008). Highly diversity in the flowering season and shedding (Days to flowering) in different cultivars with reference to different locality was found. The duration between flowerings to shedding were found fix in all cultivars. In higher elevation zones ( Meerabad-Shangla, Sakhra-Swat and Manja-Swat) the flowering season start one week later than then lower elevation zones of the selected area (higher temperature zones) like Abuha-Swat, Thana- Malakand, Chakdara and Ouch- Dir (Lower). Cluster analysis based on qualitative traits sort the whole germplasm, with respect to allele s homology; with the exception of No.6, which grouped separately while cultivars Disi (wild), Ever-Fruit and Jetty on same clusters group. Peaches cultivars No.5 - (China), No.5-(Golden) and No.5-(Basary) were grouped in same cluster. Cultivars No. 2-(Ary), No.3 and No.4 are in same cluster, Cultivars No.3-(Ary), No.4.5, No.5-(Simple), Early-Green and No.8 were grouped in same cluster. Baber, No.5-(Suhani), No.6, No.6.69 and No.7 occur in same cluster. The clustered cultivars were similar because they have similar qualitative characteristics like flowering season, flower type, fruit size, fruit taste and fruit ripening season. Cluster analysis based on quantitative traits sort the whole germplasm, with respect to alleles homology, with the exception of No.6.69 which grouped in separately. Cultivars No. 2 (Ary) and No. 3 were grouped in same cluster, cultivars No. 3(Ary), No. 4, No.4.5 and No.5 (Dogar), grouped in same cluster. Cultivars No.5, No.6 and No.7 occur in same cluster. Early-Green was found in separate cluster because of their unique characters that it is an early flowering and fruiting plant but have similar fruit to that of No.8.Peaches cultivars No.3, No.5 (China), No.5 (Golden), No.5 (Basary) and No.8 are found in same cluster while No.6.69 found in separate cluster. These have same quantitative characteristics like flowering season, flower size, leaf size and fruit weight. In correlation study, all the yield contributed traits were high significantly positively correlated which reflect the strength of the important germplasm reported from Pakistan. Disease which observed in Peach plant were shot hol, root rot, flat heeded peach borer, powdery mildew, fruit fly disease and scale disease. It was found that early flowering plant was less susceptible to diseases as compare to the late flowering because of low temperature and less raining in the selected areas. Genotypes evaluated for morphological traits were also tested through SDS-PAGE. Allelic distribution exhibit that cultivar 5-No-Simple and 5-No China were 83% alleles similar in Co-Published By: Human Resource Management Academic Research Society 157
protein profile. While cultivar No.6-Simple, No. 8, No.7, Dogar and Ammy were 96% similar in allelic distribution. Low level of genetic diversity was observed in the local peaches cultivar REFERENCES Annual Report (2008) Evaluation of different Peach (Prunus persica) varies under the soil and climatic condition of Peshawar. Horticulture Section. Agriculture Research Istitute Tarnab Peshawar: 6-13. Barenger, A.G., Aubert, Arnau. AI. Iaine G. Deniot, J. Poteir (2004) Genetic diversity in Pisum stivum using protein and PCR-based markers. Theoretical and apploed genitics 108:1309-1321 Cromwell. E.D.,Cooper. P., Mulvany (1999) Agricultural biodiversity and livelihood: issue and entry point paper for DFID linking policy and practice on biodiversity project. Fishman. S. and M. Génard (1998) A biophysical model of fruit growth. Simulation of seasonal and diurnal dynamics of mass. Plant, cell and environment 21:739 752. Huxley, A., (1992). New RHS dictionary of gardening. Macmillan ISBN 0-333-474945. Mcfadyen. L.M., Hutton. R. J. and Barlow E.W.R.(1996). Effects of crop load in fruit water relations and fruit growth in Peach. Journal of horticultural science 71:469 480 Mantell. A., Goldschmidt. E., Monselise S. (1980). Turnover of titivated water in calamondin plants. Journal of the American society for horticultural science 105:741 744. Nisar M (2008) Genetic diversity among Pea (Pisum sativum L.) Ph.D theses. Quaid-i-Azam University Islamabad. Szalay, L., Papp, J., and Szaóbo, Z. (2000). Evaluation of frost tolerance of Peach varieties in artificial freezing tests. in: geibel, m., fischer, m., & fischer, c. (Eds.). Eucarpia symposium on fruit breeding and genetics. Acta horticulturae 538. abstract. Edward F. G. and D. G. Watson (1994). Fact Sheet St-513, A series of the environmental horticulture department, Florida cooperative extension service, institute of food and agricultural sciences, University of Florida Co-Published By: Human Resource Management Academic Research Society 158
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