Lab Report #1 BICD 101

Lab #1 A02-92-6779

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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


3,10 2 prongs


4,7 2 prongs


5,11 2 prongs


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

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

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

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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

2 parental

Type B

1 P & 1 R

Type C

2 Recombinant

X^2 p value Linkage

NGA280

Susan

Michael

2001-02

0

1

1

3

1

5

4

1

3

3.84 0.5>p>0.1 No

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

A

Type B Type C X^2 p value Linkage

CIW3

Chromosome 2

4 8 20 24 p<0.005 No

CIW11

Chromosome 3

4 3 3 1.80 0.5>p>0.1 No

CIW7

Chromosome 4

3 9 0 4.50 p~ 0.1 No

MTH12

Chromosome 5

13 4 0 24.64 p<0.005 Yes

NGA248

Chromosome 1

1 3 2 0.33 0.9>p>0.5 No

NGA280

Chromosome 1

2 9 8 3.84 0.5>p>0.1 No

NGA168

Chromosome 2

14 7 3 14.25 p<0.005 No

ATHFUS6

Chromosome 3

5 8 14 10.47 0.01>p>0.005 No

ATHPHYC

Chromosome 5

12 10 5 5.44 0.1>p>0.05 Yes

NGA12

Chromosome 4

3 13 3 2.57 0.5>p>0.1 No

NGA151

Chromosome 5

4 12 4 0.80 0.9>p>0.5 No

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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

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.

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

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

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

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


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


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


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

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Diagram #3: SSLP Rough Map Chromosome 5:

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

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)

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

36-38 cM separation AthPHYC & Mutation

12 cM MTH12 & Mutation

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Photo #2: CAP Marker Gel K14B20

Table #5: Caps marker Analysis CAPS Marker

Type A Type B Type C Parental Recombinant % Recombination

K14B20 Michael Class Total

7 68 75

0 1 1

0 0 0

151

1

1/152=0.66 %

0.66 cM EG7F2 Susan Class Total

5 23 28

0 6 6

1 0 1

62

8

8/70=11.4%

11.4 cM PDC2 Class Total

31 31

18 18

0 0

80

18

18/98=18.4%

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

PCR Products

K14B20

Ler 700bp ~100bp lost

One Cut Alw26I

Col 800bp

No Cut Alw26I

Source Dr. Gus

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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

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:

Chromosome 5: Fine mapping by CAPS markers

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 ))

Bac K14B20 Bac MAF19 Bac MTH12 Bac MBG8

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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.

Documents

Lab Report #1 BICD 101