Morphological and Molecular Variability in Some Iranian Almond
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1 Morphological and molecular variability in some Iranian almond 2 genotypes and related Prunus species and their potentials for rootstock 3 breeding 4 5 Kianoush Nikoumanesh a,*, Ali Ebadi a, Mehrshad Zeinalabedini b, Yolanda Gogorcena c 6 7 a Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran, P.C. 8 31587-77871, Karaj, Iran 9 b Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), Seed 10 and Plant Improvement Institutes, Mahdasht Road, Karaj, Iran 11 c Departamento de Pomología, Estación Experimental de Aula Dei (CSIC), Apartado 13034 , E- 12 50080 Zaragoza, Spain 13 14 15 1 16 A B S T R A C T 17 In this study, in order to know the variability for a rootstock breeding program genetic diversity 18 and relationships among 55 Iranian almond genotypes and seven related Prunus species were 19 investigated. Morphological and molecular analyses were used. Principal component analysis 20 showed that three components explained 67.6% of the total morphological variation for the first 21 year and 68.06% for the second year of the study. Leaf traits were predominant in the first 22 component and contributed most of the total variation. Leaf length and width, as well as, leaf 23 area were highly correlated with each other and correlated to vigor. Also a negative correlation 24 was found among leaf length/width ratio and vigor. Ward’s method was used to construct cluster 25 from morphological data which allocated individuals into their respective species. Out of 100 26 pre-screened RAPD primers, 16 with reproducible bands and maximum polymorphism were 27 selected. Two-hundred and sixty bands were scored which 250 of them were polymorphic. 28 Average value of polymorphism per primer was 95.81% and maximum value for polymorphism 29 (100%) was obtained from TIBMBA-14, TIBMBA-17, TIBMBB-05, TIBMBB-08, TIBMBD- 30 09, TIBMBD-10. On the other hand, the minimum value was obtained from TIBMBB-16 (86%). 31 Primer TIBMBB-5 gave the maximum number of bands (25 fragments) and the minimum 32 obtained from TIBMBE-18 (11 fragments). Genetic similarity based on Jaccard’s coefficient 33 ranged from 0.28 to 0.79 with an average of 0.53. Molecular analysis revealed a high degree of 34 separation among samples regarding their geographical origin. Correlation between two 35 approaches was low (R= -0.38). High molecular and morphological variability indicated that this 36 collection includes rich and valuable plant materials for almond rootstock breeding. 37 Keywords: Almond - Rootstock - RAPD - Vegetative traits - Related Prunus spp. 38 2 39 *Corresponding author. Email: [email protected], [email protected] Tel: +98 261 40 2248721; Cell phone: +989125370535; Fax: +98 261 2248721 41 3 42 1. Introduction 43 44 Almonds are deciduous trees and/or shrubs adapted to arid and semi-arid environments. 45 They also encompass a wide range of values from nutritional to ecological applications 46 (Martínez-Gómez et al., 2007; Zeinalabedini et al., 2008). The cultivated almond, which is one 47 of the oldest nut crops [Prunus dulcis (Mill.) D.A.Webb; syn. P. amygdalus Batsch], is thought 48 to have originated in the arid mountainous regions of Central Asia (Grasselly, 1976b) and is 49 grown commercially worldwide. Iran, due to a diverse variability in geographical regions such as 50 mountain ranges and deserts spreading throughout the country and hence diverse kinds of 51 climates, is one of the origin of almond (Grasselly, 1976a,b; Kester and Gradziel, 1996; 52 Ladizinsky, 1999; Martínez-Gómez et al., 2007). Based on the latest statistics (FAO STAT Data 53 sources, 2008) world production of almond was approximately 2,420,000 Tons, of which Iran 54 produced 110,000 Tons and stands in the fifth place. Almond has been cultivated in Iran for 55 millennia and its production is mostly based on orchards with traditional managements. These 56 clones, local cultivars and seedlings as well as related wild species constitute a valuable source 57 of genetic diversity and an excellent potential for improvement. Owing to the responsibility of 58 rootstock for a wide range of fruit tree properties (Cummins and Aldwinckle, 1983; Layne, 1987; 59 Zarrouk et al., 2005; Jiménez et al., 2007), they have an important role in modern horticulture 60 and commercial orchards. Therefore, a suitable rootstock should have a range of characters from 61 compatibility with cultivars to adaptation to biotic and abiotic stresses. Evaluation of genetic 62 diversity and relationships among cultivated almond and its related wild species is of great 63 importance in determining gene pools and developing better strategies in conservation and 64 identification of genetic resources (Gradziel et al., 2001; Hend et al., 2009; Tahan et al., 2009) 4 65 and owing to the absence of crossing barriers, the possibility of interspecific hybridization 66 supplies almond with a rich germplasm and 67 a valuable source for improvement programs (Browicz and Zohary, 1996; Gradziel et al., 2001; 68 Martínez Gómez et al., 2003b). 69 Although traditional approaches for germplasm characterization are based on 70 morphological observations, they are time consuming, affected by environmental conditions, 71 particularly due to the long generation time and large tree size. Also there is low level of 72 variability in morphological traits (Casas et al., 1999; Sorkheh et al., 2007; Zeinalabedini et al., 73 2008; Bouhadida et al., 2009; Sorkheh et al., 2009b). In this concept, in order to supplement the 74 morphology-based results, several molecular techniques including isoenzyme (Vezvaei, 2003), 75 and DNA-based markers such as, ISSRs (Martins et al., 2003), SSRs (Cipriani et al., 1999; 76 Martínez-Gómez et al., 2003a,b; Mnejja et al., 2004; Xu et al., 2004; Xie et al., 2006; 77 Zeinalabedini et al., 2008; Bouhadida et al., 2009) and AFLPs (Sorkheh et al., 2007), which are 78 not affected by environmental changes have been used for describing diversity and genetic 79 characterization of Prunus germplasm throughout the world. Random amplified polymorphic 80 DNA (RAPD) technique has been used to study the genetic diversity of Prunus, including 81 almonds, in several studies (Gogorcena et al., 1993; Casas et al., 1999; Mir Ali and Nabulsi, 82 2003; Gouta et al., 2008; Shiran et al., 2007) and is considered as a quick, inexpensive and less 83 laborious approach for studying genetic diversity. Previous studies showed that results of RAPD 84 analysis are reliable and comparable to markers like SSR (Baránek et al., 2006; Shiran et al., 85 2007; Bouhadida et al., 2009; Gouta et al., 2010) and AFLP (Sorkheh et al., 2009a). 86 A few studies have conducted on the assessment of genetic diversity and relationships 87 among Iranian cultivated and wild almond genotypes with regard to molecular and 5 88 morphological analyses (Zeinalabedini et al., 2008; Sorkheh et al., 2007; 2009a). In other words, 89 ongoing researches on Iranian Almond germplasm are increasing our knowledge about the 90 morphological/molecular duality in these valuable and closely related plant materials. 91 Hence, the aim of present study was to investigate the genetic diversity and relationships among 92 Iranian almond genotypes and relative wild Prunus species, from the breeding program at the 93 University of Tehran, for their conservation, management, utilization and future selection of 94 rootstocks, by comparing morphological and RAPD analyses. 95 2. Materials and methods 96 97 2.1. Plant Material 98 99 Samples in this study were classified into two groups: first, Prunus dulcis genotypes 100 (cultivated almond) and second, wild species being relatives of almond (Fig. 1). In main 101 production regions of the country an individual open pollinated almond tree was selected due to 102 characters such as drought tolerance and vigor and considered as mother plant. Then 103 approximately 30 seeds were collected from them, stratified and sown in the experimental 104 orchard of the University of Tehran ( Longitude: 50.56 and latitude: 35.47). All seedlings in each 105 row were considered as a family and evaluated based on length and diameter of main trunk (two 106 important vegetative traits for rootstock). At that point, the most vigorous seedlings (ranging 107 from five to ten in each family) were selected for further morphological study. The samples were 108 named with initials of cities at collection sites and/or local names. This material is growing in the 109 experimental field of the Department of Horticulture, College of Agriculture, University of 110 Tehran in Karaj. Second group, consisted of wild almonds species (P. scoparia, P. arabica, P. 111 eburnea, P. erioclada, P. lycioides, P. orientalis and P. communis) characterized by high 6 112 tolerance to harsh environmental conditions (Kester et al., 1991; Gradziel et al., 2001) collected 113 from natural habitats. Both groups together, included 62 genotypes (Table 1, Fig. 2). 114 115 2.2. Morphological Evaluation 116 117 During two growing seasons (2008 and 2009), fifteen morphological traits (eight 118 quantitative and seven qualitative), were evaluated based on almond descriptors developed by 119 the International Plant Genetic Resources Institute (IPGRI) (Gulcan, 1985), in the experimental 120 orchard as well as in natural ecosystems. Leaf and branch traits were measured on samples from 121 open-grown canopy, around the tree and in case of leaves, from middle part of normal current 122 season’s shoots. Diameter of main trunk was measured in the height of 15 cm above the ground 123 which is the common height for grafting in nurseries. 124 125 126 127 128 129 130 2.3. Molecular Evaluation 131 132 2.3.1. DNA Isolation 133 7 134 Young leaves were freeze-dried immediately after sampling by liquid nitrogen. For total 135 DNA extraction approximately 50 mg of young leaves were ground in a 1.5 ml Eppendorf tube 136 with 750 ml of CTAB extraction buffer (100 mM Tris–HCl, 1.4 M NaCl, 20 mM EDTA, 2% 137 CTAB, 1% PVP, 0.2% mercaptoethanol, 0.1% NaHSO3) (Doyle and Doyle, 1987).