Presentation on theme: "Identification of markers linked to Selenium tolerance genes"— Presentation transcript:
1 Identification of markers linked to Selenium tolerance genes by bulked segregant analysis in Arabidopsis thalianaBulked segregant analysis is a rapid procedure for identifying interesting genes in specific regions of the genome.The method involves comparing two pooled DNA samples of individuals from a segregating population originating from a single cross.Within each pool, or bulk, the individuals are identical for the trait or gene of interest but are arbitrary for all other genes. Two pools contrasting for a trait (e.g., resistant and sensitive to a particular disease.) are analyzed to identify markers that distinguish them.Markers that are polymorphic between the pools will be genetically linked to the loci determining the trait used to construct the pools.
2 Five steps for mapping 1: Create F2 mapping population 2: Establish linkage using bulked segregant analysis3: Identify flanking PCR markers4: Screen recombinants by PCR analysis of a largemapping population with flanking markers5: Fine mapping and mutation genes identification
3 What is the loci polymorphic or monomorphic between the pools? ParentsP P2F2 BulksF1LocusrrRR_____ABCDpolymorphicmonomorphicF2 Individualsrr rrRr Rr Rr Rr Rr RR RR_ _A B CD_ _ _ __ _ _ _ __ _ _ __ _ _ __ __ ___
4 Types of molecular markers used in the mapping SSLPs: Simple sequence length polymorphismsRFLPs: Restriction Fragment Length PolymorphismCAPS: Cleaved Amplified Polymorphic SequenceRAPDs: Random Amplified Polymorphic DNASNPs: Single nucleotide polymorphismsAFLPs: Amplified fragment length polymorphisms
5 Advantage Disadvantage SSLP No digestion required Sequence information requiredRFLP Versatile robust Southern blotting requiredCAPS Easy to detect Sequence information requiredRAPD Easy to find Dominant/recessivePoor reproducibilitySNP Frequent in the genome Detection less reliableAFLP Easy to find Detection labor intensive
6 SSLP markers linked to Selenium tolerance genes by bulked segregant analysis in Arabidopsis thaliana In Arabidopsis thaliana, cross between Selenium sensitive ecotype Landsberg ( Ler) and Selenium tolerance ecotype Columbia (Col) was made in greenhouse.Genetic SSLP marker nga151 was used to identify the heterozygoteF1.The F2 segregation was produced on MS medium including 50uM selenate. The process of identification of genetic markers linked to Se tolerance was performed as following:
7 Step2: F1 Heterozygote Test Step1: Parents ♀ Ler X Col ♂LerColSelenate (50uM)Step2: F1 Heterozygote TestWSColLerx150120bp102
8 Step3: F2 Segregation on Selenate (50uM) and Chi-Square Test to determine if the observed results fit or deviate from the expected ratio.F2 distribution Fig.Chi-Square Test:X2 = (Observed-Expected)2/ExpectedDominant:(S:R=1:3)Recessive:(S:R=3:1)Incomplete dominant:(S:I:R=1:2:1)Col (13mm)Root length (mm)Table of Chi-Square(x2) 5% Critical ValuesDegrees of Freedom5% Critical ValueLer (7mm)1233.8415.9917.815Number of plants
9 Chi-Square Test: Table1: F2 segragation on selenate X2 = (Observed-Expected)2/ExpectedIf Se sensitive is dominant:X2 = ( )2/207+(68-69)2/69= < 3.84Se sensitive is dominant and Se tolerance is recessive.
10 Step4: Pooled DNA preparation: Sample A: Heterozygous F1 plant (used to generate the F2 mapping population)Sample B: 100 Homozygous resistant F2 plants(Aliquots 2.5ug of each individual DNA)Sample C: 100 Homozygous susceptible F2 plants(Aliquots 2.5ug of each individual DNA)
11 Step5: 22 SSLP markers for bulked segregant analysis : Representation of 22 SSLP marker positions used in the genetic map experiment
12 Table1: 22 SSLP markers for bulked segregant analysis
13 The molecular markers ciw1 and nga280 linked Ler specific band linked with tolerant phenotypeThe molecular markers ciw1 and nga280 linkedwith the interesting gene. This indicates that themutation maps to the lower arm of chromosome 1
14 Fine mapping:Although a bulked segregant analysis is a very effective way to detect linkage, it usually does not allow determination of the order of closely linked loci on the chromosome. It is necessary to examine individual F2 plants with markers from the region.A small mapping population of about individual homozygous of F2 plants will be tested with SSLPs (Simple sequence length polymorphisms), RFLPs (Restriction Fragment Length Polymorphisms) or CAPS (Cleaved Amplified Polymorphic Sequence) as genetic markers, which located in that region.
15 A example of finding the flanking markers Se tolerance homozygous F2 plants number………..80ColLerMarker AMarker BLerColCalculating the Recombination frequency to find two markerson opposite sides of Se tolerance gene which the “r” is < 5%. Thesetwo markers are called flanking marker.
16 Step6: Converting genetic distance to physical distance Recombination frequercy (r, measured in ?%)=Recombination gametes/Non-recombination gametes X100%In Arabidopsis, when r<10%, r=D.When r>10%,use a mapping function:D=25ln[(100+2r)/(100-2r)]to convert the r to D.Genetic distance (D, measured in centiMorgan: cM)In Arabidopsis, average length of 1cM=200kb(10X107 basepair/500cM=200kb/cM)Physical distance (Measured in base pairs of DNA: bp, kb, MB)
17 Step7: Screen for recombinants: 1000 plants will be analysised by PCR with flanking markers. The recombinantswill be used for further mapping.The genetic interval containing the mutation is narrowed down as much as possibleby creating and analyzing new markers in the region.Ideally, markers that are only one recombinant apart from the mutation are identified.
18 Step8: Identify the interesting genes Methods for identifying the gene:Transformation: The most direct evidence that a particularclone corresponds to the target gene is by complementationof the mutant phenotype by transformation with the gene.The interesting gene is expected to be contained in one ormore of the clones contig.(A contig is a set of contiguousclones)High-resolution mapping to demonstrate co-segregation ofthe candidate gene with the phenotype.(This methods isused when the plant species is not easy to transform)