(AFLP) Amplified Fragment Length Polymorphism

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AFLP, Applications, Advantages, Disadvantages, Conclusion

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1 Almost forgotten or Latest practice AFLP applications analyses and advances in crop improvement MAHANTESH BIRADAR Phd Scholar UAS GKVK Bangalore

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2 Contents AFLP principle Steps Applications Advances Case studies Conclusion

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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

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4 Restriction fragment Genomic DNA Rare cutter EcoRI Frequent cutter Mse I Step I. Restriction Digestion confirmation

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5 Restriction Fragment Adapter ECORI Adapter Mse i Restriction fragments with adapters are called as Tagged Restriction Fragments TRF Step II. Ligation of Adapters

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6 Restriction Fragment Adapter Adapter Restriction Fragment Adapter Adapter Restriction Fragment Adapter Adapter Types of TRFs and preamplification confirmation

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7 Restriction Fragment Adapter Adapter A Selective Amplification T C G A T C G confirmation

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9 AFLP gel with silver staining An AFLP gel run with fluorescent dyes

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10 Comparison of different markers types

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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

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12 AFLP applications Linkage mapping Parentage analysis Measuring genetic diversity Population genetics Development of single locus markers from AFLP for MAS

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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

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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

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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

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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

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17 Band re- amplification Candidate SNP identification by sequencing SNP first step validation SNP second step validation Primer for candidate SNP and then sequenced

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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

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19 Overview of steps of the protocol to convert any AFLP markers into simple locus PCR based marker assay

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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.

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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

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24 cDNA-AFLP procedure

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25 Major Applications of cDNA-AFLP Expression QTL mapping eQTL mapping Gene discovery

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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

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27 Phenotype of parents to Root-knot nematode in cotton

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29 susceptible Resistant

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30 NemX x SJ2

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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

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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

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38 Case study 1 Relationship between hybrid performance and AFLP based genetic distance in highland maize inbred lines Legesse et al.2007

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43 Dendrogram derived from UPGMA analysis based on 32 maize inbred lines

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44 Conclusion Significant positive correlation manifested between GDs and hybrid performance for most of the traits in inbred line x tester combinations

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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

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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

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48 AFLP peak generated by two varieties

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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

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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

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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

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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

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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.

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54 cDNA-AFLP analysis correlate with those generated by the hybridization of microarrays Scatter plot showing correlation between cDNA- AFLP and microarrays

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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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