(AFLP) Amplified Fragment Length Polymorphism

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Information about (AFLP) Amplified Fragment Length Polymorphism

Published on October 1, 2014

Author: mahanteshbiradar45405

Source: authorstream.com

slide 1: 1 Almost forgotten or Latest practice AFLP applications analyses and advances in crop improvement MAHANTESH BIRADAR Phd Scholar UAS GKVK Bangalore slide 2: 2 Contents AFLP principle Steps Applications Advances Case studies Conclusion slide 3: 3 Amplified fragment length polymorphism AFLP are generated by complete restriction endonuclease digestion of genomic DNA followed by selective reamplification and electrophoresis of a subset of fragments and resulting in unique reproducible fingerprint for each individual. What is AFLP slide 4: 4 Restriction fragment Genomic DNA Rare cutter EcoRI Frequent cutter Mse I Step I. Restriction Digestion confirmation slide 5: 5 Restriction Fragment Adapter ECORI Adapter Mse i Restriction fragments with adapters are called as Tagged Restriction Fragments TRF Step II. Ligation of Adapters slide 6: 6 Restriction Fragment Adapter Adapter Restriction Fragment Adapter Adapter Restriction Fragment Adapter Adapter Types of TRFs and preamplification confirmation slide 7: 7 Restriction Fragment Adapter Adapter A Selective Amplification T C G A T C G confirmation slide 8: 8 slide 9: 9 AFLP gel with silver staining An AFLP gel run with fluorescent dyes slide 10: 10 Comparison of different markers types slide 11: 11 No prior sequence information High genomic heterogenity Genetic variability low Rapid generation of data High quality DNA No established marker available Access to appropriate facilities eg. PAGE AFLP technique widely used slide 12: 12 AFLP applications Linkage mapping Parentage analysis Measuring genetic diversity Population genetics Development of single locus markers from AFLP for MAS slide 13: 13 Development of simple single locus markers SCAR Sequence characterized amplified region CAPS Cleaved amplified polymorphic sites SNPs by AFLP SBA – A rapid SNP isolation strategy cDNA - AFLP Transcript profiling Advances in AFLP slide 14: 14 SNPs by AFLP SBA : a rapid isolation strategy for non model organisms SNPs – limited to humans and genetical model organisms Reason : lack of available sequence data in non model organisms SNP – most abundant resource of genetic variation among individuals of a species slide 15: 15 SNPs mostly discovered by homologous fragments of genomic DNA Strategies for developing SNPs LSA locus specific amplification and comparative sequencing Expensive ESTs sequencing Whole genome short gun sequencing AFLP based SNP isolation strategy – No cloning required and less cumbersome slide 16: 16 SNP by AFLP SBA Steps Isolation of bands of interest Gel pieces incubated 2 hr at 68 0 c and then stored at 4 0 c Excised bands from dried gel placed in 50ul of 1X PCR buffer AFLP analysis slide 17: 17 Band re- amplification Candidate SNP identification by sequencing SNP first step validation SNP second step validation Primer for candidate SNP and then sequenced slide 18: 18 Conversion of AFLP markers into simple single locus markers Need to convert AFLP into SLA CAPS or SCARs AFLP markers less suitable AFLP mediated mini sequencing Reamplification of AFLP fragment in a less complex fingerprint and excision of the AFLP fragment Direct sequencing of the excised and reamplifed AFLP fragment Design for internal locus specific primers Screening of additional internal polymorphic sites Steps slide 19: 19 Overview of steps of the protocol to convert any AFLP markers into simple locus PCR based marker assay slide 20: 20 slide 21: 21 slide 22: 22 AFLP based transcript profiling cDNA AFLP for genomic wide expression Definition : cDNA AFLP is a gel based transcript profiling method to generate quantitative gene expression for any organism. slide 23: 23 Microarray based techniques cDNA AFLP Closed system prior sequence information required Open system No prior sequence information Restricted to species with genome sequence information Used in any organism High start up cost Low start up cost Rare transcript cannot be detected Rare transcript to be measured at great accuracy EST database by sequencing TDF Comparison of cDNA- AFLP with microarrays slide 24: 24 cDNA-AFLP procedure slide 25: 25 Major Applications of cDNA-AFLP Expression QTL mapping eQTL mapping Gene discovery slide 26: 26 Development of AFLP and derived CAPS markers for root-knot nematode resistance in cotton Wang et al.2006 Objective Identify markers linked to nematode resistance gene to facilitate Marker assisted selection slide 27: 27 Phenotype of parents to Root-knot nematode in cotton slide 28: 28 slide 29: 29 susceptible Resistant slide 30: 30 NemX x SJ2 slide 31: 31 slide 32: 32 slide 33: 33 slide 34: 34 slide 35: 35 Development of high throughput markers Dominant marker E-AAG/MCCG327cloned and sequenced to produce 300 bp sequence Comparison with NCBI database with blast search revealed that about 80bp sequence was conserved with GTP binding protein in Arabidopsis thaliana Primer amplified 300 bp fragment in both parents slide 36: 36 slide 37: 37 Conclusion PCR analysis of DNA from 60 F 2:7 NemX x SJ-2 revealed that the AFLP marker and CAPS GHAAC1 cosegregated with resistance for use in MAS. Picture showing segregation of marker for RKN disease slide 38: 38 Case study 1 Relationship between hybrid performance and AFLP based genetic distance in highland maize inbred lines Legesse et al.2007 slide 39: 39 slide 40: 40 slide 41: 41 slide 42: 42 slide 43: 43 Dendrogram derived from UPGMA analysis based on 32 maize inbred lines slide 44: 44 Conclusion Significant positive correlation manifested between GDs and hybrid performance for most of the traits in inbred line x tester combinations slide 45: 45 Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L. Julio et al. 2006 Lack of molecular diversity using RAPDs led to difficulty in developing markers for three major diseases Blue mold disease Black root rot disease Potato necrotic disease slide 46: 46 K 326 sus. to PVY BR x ITB 32 Res PVY BR 103 106 RILs BB16 res - BM x TN86 sus- BM 17 DHLs Plant populations slide 47: 47 slide 48: 48 AFLP peak generated by two varieties slide 49: 49 marker Disease RILs segregation Genetic distance Chall - 2 BR 109 71S:35R 1.0cM MiI 275 BM 17 11HR:6S - PVYME1 PV 103 54R:44S 5.1cM Table showing markers linked to three disease and their genetic distance slide 50: 50 Technical advances: Genome wide cDNA- AFLP analysis in Arabidopsis genome Volkmuth et al. 2004 Arabidopis thaliana subjected to different environmental conditions Cold treatment Nitrate starved conditions Salicylic acid treatment Light treatment mRNA 27 different tissues Reverse transcriptase cDNA slide 51: 51 Expression profiling done in cDNA using different TaqI/MseI AFLP enzyme combinations in 27 different tissues and then isolation of fragments and sequencing cDNA-AFLP database A.thaliana Arabidopsis genome intiative2000 25498 Ceres genome annotation of Arabidiopsis 2001 30880 65527 bands isolated 44367 matched Microarray database of A.thaliana slide 52: 52 Expression of mRNAs of high sequence similarity can be distinguished by cDNA-AFLP Differential genes acetyl-coenzyme A carboxylase 1 ACC 1 acetyl-coenzyme A carboxylase 2 ACC 2 90 sequence similarity across the whole length of their ORFs of 6765 and 7128 nts Sequence similarity between ACC1 and ACC2 slide 53: 53 A. cDNA-AFLP profiling of pooled samples B. Size distribution of cDNA-AFLP fragments isolated from the profiling of pooled samples C. Distribution in the number of detected AFLP fragments per cDNA. slide 54: 54 cDNA-AFLP analysis correlate with those generated by the hybridization of microarrays Scatter plot showing correlation between cDNA- AFLP and microarrays slide 55: 55 Conclusion Far from being ‘ almos t fo r g o tten ’ AFLP is a highly useful technique and if fostered by parallel development of new analysis methods will continue to be at the forefront in answering important scientific questions.

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