1. 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.

2. 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

3. What is the loci polymorphic or monomorphic between the pools?
Parents
P 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
_ _ _ _
_ _ _ _ _
_ _ _ _
_ _ _ _
_ _
_ _ _ _

4. 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

5. 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

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

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/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

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

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
………..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.

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 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.

18. 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)

19. Constructing Transgenic Plants