Duabanga Moluccana
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Development and Polymorphism of Simple Sequence Repeats (SSRs) markers using ISSR-PCR for Duabanga moluccana Supervisors: Dr. Ho Wei Seng (UNIMAS) Mr. Julaihi Abdullah (SFC) Prepared by: Liew Kit Siong (08021302) Forest Genomics & Informatics Laboratory (FGiL) Department of Molecular Biology Faculty of Resource Science & Technology Universiti Malaysia Sarawak Content 1 Introduction 2 Materials and Methods 3 Results and Discussion 4 Conclusions Introduction Planted Forest Sarawak government targets 1.0 million hectares for planted forest to be established by year 2020 (within a period of 15 years) 30 million of planting materials are required annually Planted forest Forest genetics & tree improvement (R&D) Pusaka, 2009 high-quality timber in maximum amounts in the shortest period of time at a reasonable cost. Simple sequence repeats (SSRs) Repeated arrays of short motifs (1-6 base pairs in length) generally found near a gene of interest (ATT)4 Forward Primer (ATT)7 (ATT)3 Reverse Primer Adapted from Pioneer, 2001 Simple sequence repeats (SSRs) Such variations in DNA sequence are tracked as SSR markers and potentially be used to identify desired genes Criteria Conventional tree Molecular tree breeding breeding Selection Phenotypic traits DNA Speed Mature Tree 2-3 months (> 15 years) (seedling stage) Pioneer, 2001 Environmental effects Yes No Advantages SSR markers SSRs presented a higher level of polymorphism and a greater information content than RAPDs and AFLPs SSR RAPD AFLP Advantages SSR markers - Four microsatellite loci were able provides sufficient diagnostic power to discriminate virtually all individuals in a population. - The probability of 2 individuals in the population having the same profile was approximately one in 500,000 (2.06 x 10-6). Advantages SSR markers Other advantages: (a) Co-dominant (b) PCR-based markers (c) Widespread distribution (d) Locus-specificity - Four microsatellite loci were able provides sufficient diagnostic power to discriminate virtually all individuals in a population. (e) Cross-species transferability - The probability of 2 individuals in the population having the same profile was approximately one in 500,000 (2.06 x 10-6). Applications of SSR markers 1 DNA fingerprinting and Parentage Analysis 2 Seed dispersal 3 Consensus Linkage Map 4 Germplasm characterization 5 Seed orchard management Development of SSRs ISSR-PCR suppression method Advantages (1) does not require skillful (2) without enrichment technique / relatively easy and screening to perform procedures (Lian et al., 2001) Development of SSRs 1 2 3 Selection of Species Studied Duabanga moluccana Family- Sonneratiaceae Sawih Fast-growing speciesSelection of Species Studied Commercial Values - plywood - blockboard - veneer - interior joinery Objectives To establish an efficient protocol for isolating pure and high-molecular weight genomic DNA from Sawih To develop a set of SSR markers specific for genotyping Sawih trees To determine the polymorphism of each newly developed SSR marker Content 1 Introduction 2 Materials and Methods 3 Results and Discussion 4 Conclusions Materials and Methods SSR markers development 1st method – Lian et al. (2001) Materials and Methods 2nd method – Lian et al. (2006) 1 x primer design 1 x cloningCloning, & sequencing sequencing of ISSR fragments & primer design SSR markers validation IP1 SSR SSR (AC)6(AG)5 SSR SSR (TC)6(AC)5 IP2 st sequencing 1 SSR IP1 nd SSR SSR 2 IP 2 SSR Content 1 Introduction 2 Materials and Methods 3 Results and Discussion 4 Conclusions Results and Discussion A. Original DNA isolation protocol (Doyle & Doyle, 1990) DNA band was not clearly observed – minute amount of DNA was obtained the isolated DNA was very viscous and could not be resolved on agarose gel. This is caused by anomalous reassociation kinetics between polysaccharides and DNA sample during alcohol precipitation (Do and Adams, 1991). Figure genomic DNA of Sawih on 0.8% agarose gel. Results and Discussion Re-optimized DNA isolation protocol Modifications 1. 1% β-mercaptoethanol in the extraction buffer 2. DNA precipitation in the presence of 1/3 vol of 5M NaCl together with isopropanol; 3. adding a CIA extraction step. Results and Discussion Re-optimized DNA isolation protocol Modifications 1. 1% β-mercaptoethanol in the Polymerase Chain Reactionextraction (PCR) buffer Restriction2. DNA precipitation enzyme (RE) analysisin the presence of 1/3 vol of 5M NaCl together with isopropanol; 3. adding a CIA extraction step. Results and Discussion Re-optimization CTAB method SSR markers Good yield and (Doyle and Doyle, quality DNA development 1990) Sawih DNA Marker: the way forward….. Results and Discussion SSR markers development Twenty SSR markers (48%) - Lian et al. (2001) and Twenty-four markers (52%) - Lian et al. (2006) Total identified SSR markers: 44 52% 48% Lian et al., 2006 Lian et al., 2001 Results and Discussion (i) SSR regions identification No. of identified SSR loci vs ISSR primers 16 (TC)6(AC)5 14 Major 12 (AG)10 (AC) (AG) 10 6 5 Minor (AC)10 8 6 Frequency 4 (GTG)6 2 0 ISSR primer Figure Frequency of SSR loci obtained from five different ISSR primers Results and Discussion No. identified SSR loci vs repeat motifs 72.8% 25 compound 20 9% Interrupted 15 18.2% 10 simple Frequency 5 0 Repeat motifs Figure Frequency of different types of SSR repeats Results and Discussion Most abundant dinucleotide: - (AG)19 Other plant studies: Shorea curtisii (Ujino et al., 1998), Caryocar brasiliense (Collevatti et al., 1999), Hevea brasiliensis (Roy et al., 2004) and Pinus resinosa (Boys et al., 2005). Results and Discussion (ii) SSR markers validation The polymorphism of each newly developed SSR markers was validated with 50 individual Sawih samples Twenty-seven SSR markers (61.4%) amplified the expected fragments size. The remaining primers (38.6%) showed either: no amplification, unexpected PCR products or multiple bands. Results and Discussion e.g. DMAG05 – (AG)19/(CT)19 One to three bands (165 - 210bp). M2 M1 225bp 150bp M1= 100bp ladder M2 25bp ladder Results and Discussion Additions of Additions of two CT point 6bp repeat units (SSR mutations nucleotides region) Results and Discussion Tree 1 Tree 2 Core repeat : (CT)2CC(CT)14 Core repeat : (AG)19 Product size : 164bp Product size : 174bp Additions of 10bp Results and Discussion No PCR bands detected - amplification conditions or mutations in the primer-binding sites (or also called as null alleles). ~ re-amplification on such samples were carried out for confirmation in order to exclude the possibility of failure in PCR. Multiple unspecific products was also observed, as a consequence of multiple priming sites along the genome (Rallo et al., 2000). Results and Discussion The polymorphism of each newly developed marker was determined by PowerMarker (V3.25) - No. of alleles PIC – polymorphic information content Hardy Weinberg Equilibrium (HWE test) Results and Discussion (iv) Scoring of PCR bands Results and Discussion Table 1 Characteristics of microsatellite markers DMAG05 shows highly variable and detect more No. of T (°C) PIC alleles than those with a short repeatSSR type length. a Marker Core repeat Alleles longer repeats are more mutable due to the increased (GA) CACC(GA) Interrupted probabilityDMAC01 of slippage3 (Ellergen7 , 2004). 5 55 0.625 Slip-strand mispairing model DMAC02 (GT)5GC(GT)2 Interrupted 5 52 0.562 Compound DMAC04 (AT) (GT) 4 0.563 3 8 perfect 53 DMAG04 (GAT)3GTT(GAT)3 Compound 5 59 0.713 imperfect DMAG05 (CT)19 Simple perfect 8 54 0.782 Mean 5.4 0.6489 Ta = optimal annealing temperature (°C); PIC – Polymorphism Information Content Results and Discussion It is found that all loci shows significant deviation from HWE test (P < 0.05) because of the excess of homozygotes (Lian et al., 2006). The deviation from HWE may be also affected by: a. the presence of null alleles, b. large allele dropout in the amplification, c. small sample size or d. size homoplasy (Li et al., 2010). Content 1 Introduction 2 Materials and Methods 3 Results and Discussion 4 Conclusions Conclusions An array of SSR markers were successfully developed for Sawih Data Analysis will be performed for other SSR markers Generation of baseline genetic information for effective selection of plus trees and establishment of seed production areas (SPAs) of Sawih in the selected forest reserves for planted forest development and tree improvement activities Seed orchard management References Craft, K.J., Owens, J.D. & Ashley. 2007. Application of plant DNA markers in forensic botany: Genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites. Forensic Science International 165: 64-70. Kim, J., Jo, B.H., Lee, K.L., Yoon, E.S., R.,G. H & Chung, K.W. 2007. 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