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J. Duhok Univ., Vol.14, No.1 (Pure and Eng. Scienes), Pp 17-24, 2011 GENETIC DIVERSITY ASSESSMENT AND VARIETY IDENTIFICATION OF PEACH (Prunus persica) FROM KURDISTAN REGION-IRAQ USING AFLP MARKERS SHAYMAA H. ALI Scientific Research Center, University of Duhok. Kurdistan Region-Iraq (Received: January 25, 2010; Accepted for publication: February 27, 2011) ABSTRACT The peach (Prunus persica) is an important member of the Rosaceae family, which contains many fruit, nut, and ornamental species. It has a basic chromosome number of 8. Amplified fragment length polymorphisms (AFLP) markers were used to determine the level of genetic diversity, genetic relationships, and fingerprinting of peach varieties cultivated in Kurdistan- Iraq. A total of 21 samples have been collected from different districts of Kurdistan including Duhok, Erbil and Sulaymani. The samples were analyzed by using AFLP markers. Two primer combinations generated a total of 124 bands and among them 109 (87.9%) were polymorphic. Using UPGMA clustering analysis method based on the similarity coefficient, varieties were separated into three major genetic clusters. The first genetic cluster mostly includes (Korneet asfer, Floredasin, Motaem bkornet mobaker (ahmer), Sprink time, Nectar 4, Nectar 6, Ahmer myse, Badree, Mskee, Tenee, Esmailly, Migrant, Abo-zalma, Silverking). The second genetic cluster includes (Read heaven, Sharly shapor). While the third genetic cluster contains (Zard, Elberta, Zaefaran, Dixired, j. h. hale). Genetic distance among 21 peach varieties were ranged from 0.0073 to 0.8572. The lowest genetic distance (0.0073) was found between varieties (Tenee) and (Esmailly) which were collected from Erbil, whereas the highest genetic distance (0.8572) was found between varieties num (Badree) and (Elberta) collected from Duhok and Sulaymani respectively. The results obtained in this study may assist peach cultivation and peach breeding programs in the region. KEYWORDS: - Peach (Prunus persica), Genetic Diversity, AFLP-Markers. T INTRODUCTION he peach (Prunus persica) is one of the species of genus, Prunus, native to China that bears an edible juicy fruit. It is a deciduous tree growing to 5-10m tall, and it is an important member of the Rosaceae family, which contains many fruit, nut, and ornamental species having a basic chromosome number of 8 (Wang et al. 2002). The scientific name persica, along with the word "peach" itself and its cognates in many European languages, derives from an early European belief that peaches were native to Persia. The modern botanical consensus is that they originate in China, and were introduced to Persia and the Mediterranean region along the Silk Road before Christian times (Huxley 1992). Polymerase chain reaction (PCR)-based methods for genetic diversity analyses have been developed, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and inter simple sequence repeat (ISSR/SSR). Each technique is not only differed in principal, but also in the type and amount of polymorphism detected. AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three basic steps: (1) restriction of the DNA and ligation of oligonucleotide adapters, (2) selective amplification of sets of restriction fragments, and (3) gel analysis of the amplified fragments (Vos et al. 1995). Recently, the use of Amplified fragment length polymorphisms (AFLP) in genetic marker technologies has become the main tool due to its capability to disclose a high number of polymorphic markers by single reaction, high throughput, and cost effective (Jones et al. 1997). AFLP have been widely utilized markers for constructing genetic linkage maps and genetic diversity analysis. It is a useful technique for breeders to accelerate plant improvement for a variety of criteria, by using molecular genetic maps to undertake markerassisted selection and positional cloning for special characters. Molecular markers are more reliable for genetic studies than morphological characteristics because the environment does not affect them (Vos et al. 1995). AFLP markers have successfully been used for analyzing genetic diversity in some other plant species. It has been proven the most efficient technique estimating diversity in barley (Russel et al. 1997), provides detailed estimates of the genetic variation of papaya (Kim et al. 2002), and have been used to analyze the genetic 17
J. Duhok Univ., Vol.14, No.1 (Pure and Eng. Scienes), Pp 17-24, 2011 diversity of various plants such as tea (Lai et al. 2001), eggplant (Mace et al. 1999), peach (Manubens et al. 1999), apple (Guolao et al. 2001), rapeseed (Lombard et al. 1999), wild radish (Man and Ohnishi 2002), Musa sp. (Wong et al. 2001; Ude et al. 2002), peanut (Herselman, 2003), soybean (Ude et al. 2003), and maize (Lübberstedt et al. 2000). A little work has been done on peach using AFLP molecular techniques for evaluating genetic diversity in relatedness with geographical origin. The objectives of this study are to use AFLP markers for varietal identification and to estimate genetic relationships among the peach varieties from Kurdistan region of Iraq. MATERIALS AND METHODS Sample Collection Leaf samples of the local peach varieties were collected from different districts in Kurdistan region and analyzed for AFLP. These samples were obtained from Duhok, Erbil and Sulaymani. The varieties of peach selected for this study were: Korneet asfer, Floredasin, Motaem bkornet mobaker (Ahmer), Sprink time, Nectar 4, Nectar 6, Ahmer myse, Badree, Mskee, Tenee, Esmailly, Migrant, Abo-zalma, Silverking, Read heaven, Sharly shapor, Zard, Elberta, Zaefaran, Dixired, and J. H. Hale. DNA Extraction From each variety, approximately 3g of young leaf tissue was collected and grounded to a fine powder using liquid nitrogen. DNA was extracted as reported by Weigand et al., (1993). This method was based on the use of 10ml of pre-heated (60 o c) 2x CTAB extraction buffer (2x CTAB, 5M NaCl, 1M Tris-HCl, 0.5 M EDTA), mixed well, and incubated at 60 ° c in shaking water bath. After 30 min of incubation, the mixture was extracted with an equal volume of choloroform/isoamyl alcohol (24:1, v/v). The mixture was then centrifuged (at 4000 rpm for 30min). The aqueous phase was transferred into another tube and precipitated with 0.66 volume of isopropanol, and then TE- buffer was added to dissolve the nucleic acids. The samples of DNA obtained were loaded on to a 0.8% agarose gel, and DNA concentration was estimated by comparing the florescence with �DNA standard. PCR Amplification of AFLP- primers The AFLP analysis was performed according to Vos et al. (1995) method with minor modifications. Initially, genomic DNA (500ng of each sample) were digested with 5U each of two restriction enzymes, Tru91 (recognition site 5’T↓TAA3’) and PstI (recognition site 5’CTGCA↓G3’) in 30µl a final volume of 18 reaction mix containing, 1x one phor-all buffer (pharmacia Bioteh, Uppsala, Sweden) incubating three hours at 37 o C. The DNA fragments were then ligated with PstI and Tru91 adapter. This was achieved by adding 50 pmol of Tru91adapter, 5 pmol of PstI-adapter,in a reaction containing1U of T4-DNA ligase, 1mM rATP in 1x one phore-all buffer and incubating for 3 hours at 37 o C. After ligation, the reaction mixture was diluted to 1:5 using sterile distilled water. Pre-selective PCR amplification was performed in a reaction volume of 20µl containing 50ng of each of the oligonucleotid primers (P00, M43) corresponding to the Tru91 and PstI adapters (P00 primer corresponding for Pst1 adapter and M43 primer corresponding for Mse1(Tru91) adapter), 2µl of template- DNA, 1U Taq DNA polymerase, 1x PCR buffer and 5mM dNTPs, in a final volume 20µl. The PCR reaction was performed in a thermal cycler using following temperature: 30 cycles of 30sec at 94 ºC, 6 at 60ºC, 1min at 72ºC. After that, the pre-amplification product was diluted to 1:5 and 2µl used as template for selective amplification. Selective amplification was conducted using Tru91 and Pst1 primers combinations. The Pre-amplification and selective amplification primer combinations that used in this study are (P101+M181, P101+M184). Amplification was performed in thermo cycler programmed for 36 cycles with the following cycle profile: a 30sec DNA denaturation step at 94ºC, 30 sec annealing step (see below) and a 1min extension step at 72ºC. The annealing temperature was varied in the first few cycle it was 65ºC; in each subsequent cycle for the next 12 cycle it was reduced by 0.7ºC (touchdown PCR), and for the remaining 23 cycles, it was 56ºC. The selective amplification products were loaded onto 6% denaturating polyacrylamid gels, and DNA fragments were visualized by silver staining kit (Promega, Madison, Wis) as described by the supplier, silver-stained gels were scaned to capture digital images of the gels after air dryin. Data analysis Total bands were scored visually and polymorphic bands were recorded for presence (1) or absence (0). The polymorphic adapt was used to estimate Jaccard coefficient of dissimilarity (Rohlf, 1993). The similarity coefficient was used for construction of dendrogram base on Unweighted Pair-Group Method Arithmetic (UPGMA). The dissimilarity coefficient estimation and dedrogram construction were performed using NTSYS-pc ver. 1.8 software (Rohlf, 1993).
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