Presentation on theme: "Identification of markers linked to Selenium tolerance genes by bulked segregant analysis in Arabidopsis thaliana Bulked segregant analysis is a rapid."— Presentation transcript:
Identification of markers linked to Selenium tolerance genes by bulked segregant analysis in Arabidopsis thaliana Bulked 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.
1: Create F2 mapping population 2: Establish linkage using bulked segregant analysis 3: Identify flanking PCR markers 4: Screen recombinants by PCR analysis of a large mapping population with flanking markers 5: Fine mapping and mutation genes identification Five steps for mapping
What is the loci polymorphic or monomorphic between the pools? Parents P 1 P 2 Locus ABCDABCD ____ ____ F1 ________ F2 Bulks rr RR ______ ________ polymorphic monomorphic F 2 Individuals ABCDABCD rr _ Rr Rr Rr Rr Rr RR RR _ _ _ _ _ _ _ _ _ _
Types of molecular markers used in the mapping SSLPs: Simple sequence length polymorphisms RFLPs: Restriction Fragment Length Polymorphism CAPS: Cleaved Amplified Polymorphic Sequence RAPDs: Random Amplified Polymorphic DNA SNPs: Single nucleotide polymorphisms AFLPs: Amplified fragment length polymorphisms
Advantage Disadvantage SSLP No digestion required Sequence information required RFLP Versatile robust Southern blotting required CAPS Easy to detect Sequence information required RAPD Easy to find Dominant/recessive Poor reproducibility SNP Frequent in the genome Detection less reliable AFLP Easy to find Detection labor intensive
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 heterozygoteF 1. The F 2 segregation was produced on MS medium including 50uM selenate. The process of identification of genetic markers linked to Se tolerance was performed as following:
Step1: Parents ♀ Ler X Col ♂ LerCol Selenate (50uM) Step2: F1Heterozygote Test WSCol Ler WS x Col WS x Col Ler x Col bp 102
Step3 : F 2 Segregation on Selenate (50uM) and Chi-Square Test to determine if the observed results fit or deviate from the expected ratio. Ler (7mm) Col (13mm) Root length (mm) Number of plants F2 distribution Fig. Chi-Square Test: X 2 = (Observed-Expected) 2 /Expected Dominant:(S:R=1:3) Recessive:(S:R=3:1) Incomplete dominant: (S:I:R=1:2:1) Degrees of Freedom5% Critical Value Table of Chi-Square(x 2 ) 5% Critical Values
Table1: F2 segragation on selenate Chi-Square Test: X 2 = (Observed-Expected) 2 /Expected X 2 = ( ) 2 /207+(68-69) 2 /69 = < 3.84 If Se sensitive is dominant: Se sensitive is dominant and Se tolerance is recessive.
Step4: Pooled DNA preparation: Sample A: Heterozygous F 1 plant (used to generate the F 2 mapping population) Sample B: 100 Homozygous resistant F 2 plants Sample C: 100 Homozygous susceptible F 2 plants (Aliquots 2.5ug of each individual DNA)
Representation of 22 SSLP marker positions used in the genetic map experiment Step5: 22 SSLP markers for bulked segregant analysis :
Table1: 22 SSLP markers for bulked segregant analysis
Ler specific band linked with tolerant phenotype The molecular markers ciw1 and nga280 linked with the interesting gene. This indicates that the mutation maps to the lower arm of chromosome 1
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 F 2 plants with markers from the region. A small mapping population of about individual homozygous of F 2 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. Fine mapping:
A example of finding the flanking markers Se tolerance homozygous F 2 plants number Col Ler Col Marker A Marker B ……….. 80 Calculating the Recombination frequency to find two markers on opposite sides of Se tolerance gene which the “r” is < 5%. These two markers are called flanking marker.
Step6: Converting genetic distance to physical distance Recombination frequercy (r, measured in ?%) =Recombination gametes/Non-recombination gametes X100% Genetic distance (D, measured in centiMorgan: cM) Physical distance (Measured in base pairs of DNA: bp, kb, MB) In Arabidopsis, when r 10%, use a mapping function:D=25ln[(100+2r)/(100-2r)] to convert the r to D. In Arabidopsis, average length of 1cM=200kb (10X10 7 basepair/500cM=200kb/cM)
Step7: Screen for recombinants: 1000 plants will be analysised by PCR with flanking markers. The recombinants will be used for further mapping. The genetic interval containing the mutation is narrowed down as much as possible by creating and analyzing new markers in the region. Ideally, markers that are only one recombinant apart from the mutation are identified.
Step8: Identify the interesting genes Methods for identifying the gene: Transformation: The most direct evidence that a particular clone corresponds to the target gene is by complementation of the mutant phenotype by transformation with the gene. The interesting gene is expected to be contained in one or more of the clones contig.(A contig is a set of contiguous clones) High-resolution mapping to demonstrate co-segregation of the candidate gene with the phenotype.(This methods is used when the plant species is not easy to transform)