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Lab Report #1

Forward Genetics:
An approach to the mapping and identification
of a mutation in trichome morphogenesis of
Arabidopsis thaliana

Michael Wade Jackson


BICD 101: Eukaryotic Genetics
4/24/2003

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Introduction:
The desired outcome of this investigation was to apply a forward genetics
approach to identify and map a possible genetic mutation in the trichome morphogenesis
of Arabidopsis thaliana. The principle of a forward genetics approach is that one takes a
phenotypic mutation present in the Arabidopsis and then through the use of both a
complementation test and genetic mapping the gene or genes of interest in the sample
population can be located. Forward genetics is: to start with a mutation and then identify
the responsible gene.
The mutation of interest to this inquiry is the process of trichome morphogenesis
which is first isolated in a process of mutant screening. The mutant of specific interest is
the two prong forming mutation which would play a role in trichome development and be
inhibited in some fashion by some type of genetic mutation which would not be found in
the phenotypically normal plant. The trichome is an outgrowth of the epidermis such as a
hair and in the Arabidopsis thaliana the trichome is formed by one large cell. The two
prong mutation once mapped to a specific site on one of the five chromosomes of
Arabidopsis will provide insight into the developmental process behind trichome
morphogenesis.
The benefits of understanding a developmental process such as this is both to aide
in a greater understanding of the Arabidopsis plant but as well the Arabidopsis is itself a
model organism of genetic research and the mapping of its genome will allow
comparisons to be made amongst newly discovered research subjects and pre-existing
model organisms. Arabidopsis is used as a model organism due to its robust nature and
ease of growth in laboratory conditions as well as its cost effective nature. The
knowledge gained through the process of mutation identification and localization will
provide a framework by which the developmental process of the trichome can be better
understood and mutation that may arise may be targeted to prevent fatal mutations to the
Arabidopsis population.
The process of mapping the mutation in Arabidopsis involved the use of both a
coarse mapping and a fine mapping utilizing different genetic markers (polymorphisms).
The coarse map was created by using eleven SSLPs (simple sequence length

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polymorphisms) which utilize the pre-existing presence of tandem repeats of one- two- or
three-nucleotide motifs. These are commonly called microsatellites and identification
can be made by the variance in the number of repeats between two PCR primers. The
number and size of repeats is polymorphic between ecotypes which will allow the marker
to be used as a co-dominant genetic marker. The benefit of SSLPs in the coarse genetic
mapping is that they do not utilize restriction enzyme digests which add to the possible
locations in which the experiment can go wrong.
The fine mapping utilized CAPS (cleaved amplified polymorphic sequences)
markers of which there were three. The CAPS markers utilize the presence of restriction
sites in sequences of DNA which is comparable to that of RFLPs (restriction fragment
length polymorphisms). The benefit of using CAPS to RFLPs is that the CAPS can be
assayed on agarose gels by electrophoresis and that the CAPS utilize PCR for
hybridization instead of blot techniques. The fine map is developed once the
chromosomal location of the mutation is known and linkage is shown to a SSLP marker
and then CAPS surrounding the marker will be utilized based on sequence specific
knowledge.
The main diagnostic techniques used for both marker types are PCR and agarose
gel electrophoresis which allow for amplification of a specific region of the genome and
then visualization of the size differences by the movement of DNA through agarose gel.
The variance between the two markers is the need for restriction endonucleases in the
creation of diagnostic bands for CAPS markers.
The trichome morphogenesis mutation of interest once mapped will allow a
window into the development of trichomes in Arabidopsis thaliana which will provide
insight into future organisms of interest and elucidate the possible pitfalls and
developmental network of trichome development.

Materials and Methods:


The first step in mapping the mutation in trichome morphogenesis is the selection
of mutant phenotypes from a population of plants in a process known as a mutant screen.
The mutant screen is created from irradiated seeds which produce mutations in single
cells within the developing plant creating a mosaic Arabidopsis. The mosaic plant is then

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outcrossed to a heterozygous plant (wild-type) creating the parental generation. The F1
generation of that cross is then all heterozygous and this population is then selfed to
produce a doubly recessive mutation (homozygous) plant. The homozygous mutant (F2)
is then taken to the mapping population to begin the process of isolating genes of interest.
The ecotype of the homozygous mutant is Columbia (Col) and this mutant is then crossed
with a Lanceberg erecta (Ler) to produce the F3 generation. The progeny of the F3
generation are then selfed to produce the F4 generation plants which are utilized in the
mapping process involving the SSLPs and CAPS markers.
The F4 generation plants are then isolated from there pots and the DNA is then
extracted from the plants. Refer to “Preparation of plant genomic DNA for PCR 4/3/03”
for exact procedural protocols for the process. The outcome is the production of ten
samples of the mutant phenotype from a plant population and then two controls one Ler
and one Col ecotype plants.
The next step is the PCR amplification of the genomic DNA for the use of SSLPs
in the process of coarse mapping analysis. Refer to “PCR with primers for SSLP
markers” protocol for specific procedural techniques. This process involved the
production of ten samples which consisted of two controls and eight mutants of interest.
Once the amplification was completed the PCR products were run on agarose gel through
the process of electrophoresis. The data from the SSLP analysis then targeted a specific
chromosome and the fine mapping with CAPS markers was then initiated.
The CAPS marker analysis involved the preparation of thirteen mutant samples as
well as two controls by PCR amplification and included the extra step of a restriction
digest which had to be completed before agarose gel electrophoresis could occur. Refer
to “Protocol for PCR with CAPS marker primers” for procedural specifics involved in
preparation of samples. The CAPS marker PCR products were then ran on a gel and
analyzed, allowing for the production of a higher definition fine map which allowed for a
more accurate localization of mutation 1 on chromosome five.
The complementation test which will be completed at the end of this laboratory
course will involve the crossing of apf3 mutant females to apf3 heterozygous males to
identify if the five of the six mutations of interest are linked. The crosses and how to
cross the Arabidopsis plant are referenced in “Doing crosses in Arabidopsis thaliana.”
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Results:
Mutant Screen:

Table 1: Mutant Phenotypes

Group # Phenotype Picture Wild-Type function


1,9 2 prongs Develop a three
prong trichome

2,8 2 prongs Develop a three


prong trichome

3,10 2 prongs Develop a three


prong trichome

4,7 2 prongs Develop a three


prong trichome

5,11 2 prongs Develop a three


prong trichome

6 4 prongs Inhibition and


regulation of
trichome
development

There are six possible mutations producing six phenotypes that were the focus of the
mutant screen. Of the mutations presented in the screen the phenotype of interest is the
two prong trichome which appears to have a mutation effecting the proper formation of
three prongs of a wild-type trichome. A complementation test underway, the results of
which will show whether or not the mutation is linked to the same gene or independent
gene mutations. The two prong phenotype has been selected for the mutation mapping
by SSLP and CAP markers.

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Coarse Mapping: SSLP marker results
Photo 1: Agarose Gel SSLP marker NGA280

Diagram #1 : SSLP marker NGA280 Gel Diagram

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Ladder Ler Col 6A 6B 6C 6D 6E 6F 6G 6H Ladder

PCR product TAIR website values: NGA280


Ler 85 bp Col 105 bp

The gel electrophoresis of the SSLP marker PCR products did not produce a viable
picture for analysis. However, diagram #1 is a representation of the gel and it should be
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noted that the Col band for control was extrapolated from the two bands in lane 6 from
sample 6B. The Col control did not show up under UV illumination of the gel with
ethidium bromide. The gel had little success with regards to sample resolution which
may have been related to a loading issue concerning sample size of 6 microliters versus
filling the sample wells to capacity. The gel did however yield appropriately sized bands
according to the ladder, a Promega 50bp DNA step ladder. The fragment sizes of 105 bp
and 85 bp were accordingly staggered on the gel and were within the given region of the
gel according to the ladder. The Col band was at 105 bp and the Ler band was at 85 bp.

Table #2: Group 6: Marker NGA 280


Marker Type A Type B Type C X^2 p value Linkage
2 parental 1P&1R 2 Recombinant
NGA280 3.84 0.5>p>0.1 No
Susan 0 3 4
Michael 1 1 1
2001-02 1 5 3

The results of the SSLP linkage analysis within the NGA280 samples produced 1 A type,
4 B type, and 5 C type which when compiled with the previous years data displayed no
sign of linkage to mutation 1. The NGA280 SSLP marker is not located on the
appropriate chromosome for the mutation as therefore the mutation does not travel with
the marker in a linked fashion. The compilation of data with the class and the previous
year displayed linkage to the marker MTH12 which resides on chromosome 5.

Table #3: Class Combined SSLP Marker Data

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Marker Type Type B Type C X^2 p value Linkage
A
CIW3 4 8 20 24 p<0.005 No
Chromosome 2
CIW11 4 3 3 1.80 0.5>p>0.1 No
Chromosome 3
CIW7 3 9 0 4.50 p~ 0.1 No
Chromosome 4
MTH12 13 4 0 24.64 p<0.005 Yes
Chromosome 5
NGA248 1 3 2 0.33 0.9>p>0.5 No
Chromosome 1
NGA280 2 9 8 3.84 0.5>p>0.1 No
Chromosome 1
NGA168 14 7 3 14.25 p<0.005 No
Chromosome 2
ATHFUS6 5 8 14 10.47 0.01>p>0.005 No
Chromosome 3
ATHPHYC 12 10 5 5.44 0.1>p>0.05 Yes
Chromosome 5
NGA12 3 13 3 2.57 0.5>p>0.1 No
Chromosome 4
NGA151 4 12 4 0.80 0.9>p>0.5 No
Chromosome 5

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Table #4: Linkage Analysis SSLP Markers:
Marker Parental Recombinant Total % / cM value
CIW3 16 48 64 75% / 50 cM
CIW11 11 9 20 45% / 45 cM
CIW7 15 9 24 37.5% / 38 cM
MTH12 30 4 34 11.8% / 12 cM
NGA248 5 7 12 58.3% / 50 cM
NGA280 13 25 38 65.8% / 50 cM
NGA168 35 13 48 27.1% / 27 cM
ATHFUS6 18 36 54 66.7% / 50 cM
ATHPHYC 34 20 54 37% / 37 cM
NGA12 19 19 38 50% / 50 cM
NGA151 20 20 40 50% / 50 cM

The linkage analysis results provided by the class as well as the pooling of data from
previous year (2001-2002) produced the appearance of linkage to the SSLP marker
MTH12 on chromosome 5. The linkage was also established with SSLP marker
AthPHYC which is also resident on chromosome 5. From these results and the
information gathered at the Tair database (http://www.arabidopsis.org) the process of
coarse mapping can occur.

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Diagram #2: Map of 5 Chromosomes of Arabidopsis thaliana

Chromosome 1: 135 cM/ 31083 Kbp Conversion 230.24 Kbp/ cM

NGA248 (42.17 cM/ 9709 Kbp) NGA280 (83.83 cM / 20461 Kbp)

Chromosome 2: 97 cM / 20298 Kbp Conversion 209.26 Kbp/ cM

CIW3 (30.35 cM/ 6351 Kbp) NGA168 (73.77 cM/ 16240 Kbp

Chromosome 3: 101 cM / 24248 Kbp Conversion 240.08 Kbp/ cM

CIW11 (40.72 cM / 9775 Kbp) ATHFUS6 (81.64 cM / 19600 Kbp)

Chromosome 4: 125 cM / 18134 Kbp Conversion 145.07 Kbp/ cM

NGA12 (22.92 cM / 3325 Kbp) CIW7 (72.3 cM / 10488 Kbp)

Chromosome 5: 139 cM / 27579 Kbp Conversion 198.41 Kbp/ cM

NGA151(29.62cM/4669Kbp) ATHPHYC(69.15cM/13721Kbp) MTH12(119.94cM/23797Kbp)

The production of the chromosome maps of the five chromosomes of Arabidopsis


thaliana was made possible by the data provided by the Tair database at
http://www.arabidopsis.org . The red circles signify the centromere of the chromosome
and the vertical colored lines correspond to the appropriate SSLP marker of interest. The
chromosome of interest in this analysis proved to be chromosome 5.

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Diagram #3: SSLP Rough Map Chromosome 5:

Chromosome 5 139 cM 27579 Kbp Conversion Factor 198.41 Kbp/cM

NGA 151 (29.62 cM) AthPHYC (69.15 cM) Mutation(107 cM)/(132cM) MTH12 (119.94
cM)
12 cM
MTH12 & Mutation
36-38 cM separation
AthPHYC & Mutation

AthPHYC (69.15 cM) Mutation (106-108 cM) / (132 cM) MTH12 (119.94 cM)

The process of coarse mapping focused on the recombination frequency of the SSLP
markers and produced a map which focused on chromosome 5 as the site of mutation 1.
The separation from MTH12 was shown to be approximately 12 cM and from this data
the location of the mutation can be placed at 12 cM from the location of MTH12 which is
accordingly ~107 cM or ~132 cM. Chromosome 5 is 139 cM in length and could be
resident to approximately 5500 genes. The process of coarse mapping does not have
sufficient resolution to approximate the location of the mutation, therefore fine mapping
by CAPS marker analysis is required to produce a more precise location of mutation 1.

Fine Mapping: CAPS Markers and Chromosome 5

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Photo #2: CAP Marker Gel K14B20

PCR
Products

K14B20

Ler 700bp
~100bp lost

One Cut
Alw26I

Col 800bp

No Cut
Alw26I

Source
Dr. Gus

Table #5: Caps marker Analysis

CAPS Type A Type B Type C Parental Recombinant % Recombination


Marker
K14B20
Michael 7 0 0 1/152=0.66 %
Class 68 1 0
Total 75 1 0 151 1 0.66 cM
EG7F2
Susan 5 0 1 8/70=11.4%
Class 23 6 0
Total 28 6 1 62 8 11.4 cM
PDC2 18/98=18.4%
Class 31 18 0
Total 31 18 0 80 18 18.4 cM

The fine mapping by CAPS marker analysis of the PCR products involved the utilization
of three CAPS markers to define the recombination frequency involved to develop cM
distances between the markers to produce a higher resolution map of the mutation. The

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results from the analysis of marker K14B20 which is a BAC itself provided 7 A type or
double parental allele individuals which displayed it high degree of linkage to mutation
one when the data was collated with the class which produced 75 A type and 1 B type
individual which produced a percent recombination of 0.66% or a separation distance of
0.66 cM. This data then allowed for the mapping of the mutation on chromosome 5 in
much higher resolution.
Diagram #4: CAPS marker mapping of chromosome 5

Chromosome 5: Fine mapping by CAPS markers

Bac MBG8 Bac MTH12 Bac MAF19 Bac K14B20

PDC2(111cM/22024 Kbp) MTH12(119cM/23797Kbp) EG7F2(122 cM/24340Kbp) K14B20(130cM/25951 Kbp)


MUTATION 1: (129.34 cM (25662.3 Kbp) ~ 130.66 cM (25924.3 Kbp ))

Through the linkage analysis the conclusion can be made that the mutation surrounds
marker K14B20 by 1.32 cM which in relation to the number of genes per cM in
Arabidopsis thaliana would yield the possibility of 55 genes in this region. However,
approximately 15 genes have been mapped to the BAC K14B20 and this could reduce the
number to 15 if the gene of interest has already been discovered. This however may not
be the case and further mapping with CAPS closer to mutation 1 are in order. The fine
mapping ability is limited to a region of 1.32cM or 261Kbp which is not of a resolution
adequate to gain significant insight into the location of the mutation of interest to pinpoint
a specific gene.

Discussion:
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The goal of discovering the a mutation involved in trichome morphogenesis was
accomplished through the use of forward genetic technique which utilized the selection of
mutant phenotypes and then the analysis of the subsequent F4 progeny by both SSLP and
CAPS marker analysis of the PCR products to produce a fine map localization of
mutation 1 on chromosome 5. The purpose of this process is to locate and analyze the
mutation in the Arabidopsis and then compare that sequence once gained to the wild-type
individuals and discover if and what type of loss of function mutation has occurred.
The precision involved in the map produced by the CAPS markers leaves a range
of 1.32 cM of distance between marker K14B20 and mutation 1 which is not a high level
of precision considering that in 1 cM the Arabidopsis typically has 42 genes. The
K14B20 BAC however does contain approximately 15 genes that have been isolated
form the region of interest and could possibly be the gene that has the mutation involved
in trichome morphogenesis. This, though not a highly likely possibility, would allow for
the reduction of the possible number of comparisons of mutant to wild-type from 55
genes to that of 15. The analysis of 15 genes though is not a effortless process and is of a
time consuming nature that makes it impractical for the laboratory environment found in
this class.
The outcome of the forward genetics approach to mapping and analysis of
mutation 1 yielded a semi-high resolution map which is significantly better than the one
produced by the SSLP PCR products and gives insight into the location of the gene of
interest. The compressed nature of this course allowed for an expedited process of
mutant screening and then subsequent genetic analysis utilizing PCR, gel electrophoresis,
and compiled genetic maps of Arabidopsis researchers around the world from the Tair
database. These are all skills utilized frequently in a genetics lab and are invaluable in
the development of proper laboratory technique with a high degree of accuracy and
expertise.

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