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.

Five steps for mapping 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

What is the loci polymorphic or monomorphic between the pools? Parents P1 P2 F2 Bulks F1 Locus rr RR _ _ _ _ _ A B C D polymorphic monomorphic F2 Individuals rr rr Rr Rr Rr Rr Rr RR RR _ _ A B C D _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

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 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:

Step2: F1 Heterozygote Test Step1: Parents ♀ Ler X Col ♂ Ler Col Selenate (50uM) Step2: F1 Heterozygote Test WS Col Ler x 150 120 bp 102

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/Expected Dominant:(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 Values Degrees of Freedom 5% Critical Value Ler (7mm) 1 2 3 3.841 5.991 7.815 Number of plants

Chi-Square Test: Table1: F2 segragation on selenate X2 = (Observed-Expected)2/Expected If Se sensitive is dominant: X2 = (208-207)2/207+(68-69)2/69 = 0.019 < 3.84 Se sensitive is dominant and Se tolerance is recessive.

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)

Step5: 22 SSLP markers for bulked segregant analysis : Representation of 22 SSLP marker positions used in the genetic map experiment

Table1: 22 SSLP markers for bulked segregant analysis

The molecular markers ciw1 and nga280 linked 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

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 50-100 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.

A example of finding the flanking markers Se tolerance homozygous F2 plants number ………..80 Col Ler Marker A Marker B Ler Col 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% 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)

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)

Constructing Transgenic Plants 12 02 2004