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 Lab #1 A02-92-6779 -2-

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 Lab #1 A02-92-6779 -3-

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.” Lab #1 A02-92-6779 -4-

Results:
Mutant Screen: Table 1: Mutant Phenotypes Group # 1,9 2,8 3,10 4,7 5,11 6 Phenotype 2 prongs 2 prongs 2 prongs 2 prongs 2 prongs 4 prongs Picture Wild-Type function Develop a three prong trichome Develop a three prong trichome Develop a three prong trichome Develop a three prong trichome Develop a three prong trichome 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 Lab #1 A02-92-6779 -6-

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

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 Lab #1 A02-92-6779 -7-

Marker CIW3 Chromosome 2 CIW11 Chromosome 3 CIW7 Chromosome 4 MTH12 Chromosome 5 NGA248 Chromosome 1 NGA280 Chromosome 1 NGA168 Chromosome 2 ATHFUS6 Chromosome 3 ATHPHYC Chromosome 5 NGA12 Chromosome 4 NGA151 Chromosome 5

Type A 4 4 3 13 1 2 14 5 12 3 4

Type B 8 3 9 4 3 9 7 8 10 13 12

Type C X^2 20 3 0 0 2 8 3 14 5 3 4 24 1.80 4.50 24.64 0.33 3.84 14.25 10.47 5.44 2.57 0.80

p value p<0.005 0.5>p>0.1 p~ 0.1 p<0.005 0.9>p>0.5 0.5>p>0.1 p<0.005

Linkage No No No Yes No No No

0.01>p>0.005 No 0.1>p>0.05 0.5>p>0.1 0.9>p>0.5 Yes No No

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Table #4: Linkage Analysis SSLP Markers: Marker CIW3 CIW11 CIW7 Parental 16 11 15 Recombinant 48 9 9 Total 64 20 24 % / cM value 75% / 50 cM 45% / 45 cM 37.5% / 38 cM

MTH12
NGA248 NGA280 NGA168 ATHFUS6

30
5 13 35 18

4
7 25 13 36

34
12 38 48 54

11.8% / 12 cM
58.3% / 50 cM 65.8% / 50 cM 27.1% / 27 cM 66.7% / 50 cM

ATHPHYC
NGA12 NGA151

34
19 20

20
19 20

54
38 40

37% / 37 cM
50% / 50 cM 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) Chromosome 2: 97 cM / 20298 Kbp

NGA280 (83.83 cM / 20461 Kbp) Conversion 209.26 Kbp/ cM

CIW3 (30.35 cM/ 6351 Kbp) Chromosome 3: 101 cM / 24248 Kbp

NGA168 (73.77 cM/ 16240 Kbp Conversion 240.08 Kbp/ cM

CIW11 (40.72 cM / 9775 Kbp) Chromosome 4: 125 cM / 18134 Kbp

ATHFUS6 (81.64 cM / 19600 Kbp) Conversion 145.07 Kbp/ cM

NGA12 (22.92 cM / 3325 Kbp) Chromosome 5: 139 cM / 27579 Kbp

CIW7 (72.3 cM / 10488 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) cM)

AthPHYC (69.15 cM) Mutation(107 cM)/(132cM) MTH12 (119.94 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 Lab #1 A02-92-6779 - 11 -

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 Marker K14B20 Michael Class Total EG7F2 Susan Class Total PDC2 Class Total Type A 7 68 75 5 23 28 31 31 Type B 0 1 1 0 6 6 18 18 Type C 0 0 0 1 0 1 0 0 Parental Recombinant % Recombination 1/152=0.66 % 151 1 0.66 cM 8/70=11.4% 62 80 8 18 11.4 cM 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 Lab #1 A02-92-6779 - 12 -

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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