fast plants final paper

Spring 2014 Biol 2202: Genetics Lab 5pm

Dante Moroni

Proposed Molecular Markers for the Anthocyaninless Gene in Brassica Rapa Fast Plants

Introduction

Observing the relationship between an organism’s expressed traits and the underlying

gene mechanisms has been a focus in many genetic studies. Identification of different alleles and

their locations on chromosomes can give further insight into their function. In order to determine

the location of a gene locus on a chromosome we can use molecular markers to distinguish the

coding sequence from others. These markers are able to select for specific segments of DNA that

are of interest to researchers. They may prove applicable in future studies that address gene

mapping and heredity patterns.

In this study, we used the Brassica rapa Fast Plant. The short generation time of 14 to 18

days and ease of pollination suited B. rapa as an ideal subject for our lab setting (Williams and

Hill, 1986). Focus was on a gene that influenced stem color in the plants. This anthocyaninless

(anl) gene controls stem color by producing anthocyanins, flavonoid pigments which give the

stem a purple coloration. Recessive mutations for the anl gene have been shown to result in a

stem without the purple pigment (Burdzinski and Wendell, 2007). The anthocyaninless gene has

been determined to have a locus on B. rapa’s chromosome 9 (Fast Plants Molecular Markers I,

UMD 2014) However; the exact location has been elusive and few B. rapa anthocyaninless loci

have been characterized at the molecular level (Burdzinski and Wendell, 2007).

The goal of our study was to identify a good molecular marker for the anl gene in order

to determine its chromosomal location. We worked with two proposed molecular markers,

D9BrapaS4 and Park9, both of which are less than 50 cM away from the anl gene (Fast Plants

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Molecular Markers I, UMD 2014). Our hypothesis for each, D9BrapaS4 and Park9, is that they

will be useful in producing sections of DNA that distinguish between the alleles of the anl gene.

DNA extracted from Fast Plants was amplified using the Polymerase Chain Reaction

procedure, and then analyzed for differences in gene sequence length via gel electrophoresis. The

length of isolated DNA sequences allowed us to make inferences about relationships between

both dominant and recessive alleles, in order to further understand their function. The UV gel

image results were not identical for each marker. D9Brapas4 supported the hypothesis while

Park9 did not.

DNA Extraction

50mg of leaf tissue was harvested from a F1 generation (purple stem) juvenile B. rapa

plant. This F1 generation plant was the offspring resulting from mating of true breeding parental

lines, DWRCBr76 (purple stem) and DWRCBr53 (green stem). The harvested leaf was

physically homogenized and put into solution. The solution was then processed using the

DNeasy mini plant procedure (QIAGEN, 2012). Final flow-through was collected, which

contained the purified plant DNA.

DNA Amplification

The Polymerase Chain Reaction technique was used to amplify the purified DNA. A

mixture of 2.0 μL processed plant DNA, 5.5 μL sterile water, 2.5 μL of forward and reverse

D9BrapaS4 primers, and 12.5 μL Syzygy Taq 2X Master Mix was prepared in a 200μL tube.

This mixture targeted the DNA and propagated copies of our sequence of interest, a proposed

molecular marker. Once prepared, the mixture was amplified using a thermal cycler. Changes in

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temperature necessary to denature, anneal, and synthesize along with using Taq polymerase gave

us a product which may show separate alleles through our next steps.

Gel Electrophoresis

Amplified DNA was loaded into the third lane of a 1.2% agarose gel situated in TAE

buffer. Along with the F1 generation, amplified DNA segments obtained using the same

procedures as above were also loaded: parent 76 (purple stem) into lane four and parent 53

(green stem) into lane two. Along with the plant DNA, a ladder was added to the first lane for

referencing DNA fragment lengths. The gel ran at 80 volts for 30 minutes and the final results

were analyzed in a gel imager.

Results

Getting a more accurate location of recessive and dominant anthocyaninless genes on

chromosome 9 was the goal of this experiment. Resolution between the two alleles may be

revealed by a good molecular marker. Fast Plant molecular marker D9brapaS4 and Park9 DNA

fragments were amplified and imaged using an Ultraviolet Transilluminator. The results revealed

two different fragments, 1 and 2; with lengths of 462 and 318 base pairs respectively (Fig. 2).

The 462 bp fragment was analogous to the green stemmed parental line, and the 318 bp fragment

matched up with the purple stemmed parental line. The F1 generation (purple stem) had both

DNA fragments present which may indicate a heterozygous nature. Using the D9brapaS4 as a

molecular marker gave us defined resolution between alleles. DNA fragments produced from the

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Park 9 molecular marker all had an equivalent length of 1022 base pairs. We were unable to

distinguish between alleles (Fig. 2).

Discussion

Finding a good molecular marker for the anthocyaninless gene in Brassica rapa Fast

Plants may help us to better find an accurate anl gene location on the chromosome. We tested

two markers, Park9 and D9BrapaS4, to evaluate their effectiveness in distinguishing between the

dominant and recessive alleles at the anthocyaninless locus. The D9BrapaS4 molecular marker

proved useful in providing resolution between the alleles. Having a reliable molecular marker

such as D9BrapaS4 allows us to visualize dominant and recessive alleles which supports and

subsequently helps to solidify our understanding genotypes and phenotypes.

The UV image produced via D9BrapaS4 after gel electrophoresis revealed DNA

fragments with different length indicating two separate alleles (Fig. 1). The Park9 marker was

not effective as its UV image resulted in gene fragments of equivalent size which did not show

the different alleles (Fig. 2). The UV images produced were of high resolution and relayed the

information with clarity. Further experiments that intent to find accurate molecular markers can

provide more information on the right steps to take other than guessing, in hypothesizing a

predictable molecular marker. The areas reliable molecular markers can be applied are diverse

and can include gene mapping, population genetics, phylogenic reconstruction, and several

others (Schlotterer, 2004).

The hypothesis was supported for D9BrapaS4 and rejected for Park9. If we were to

conduct this experiment again we may try to find an even more accurate molecular marker than

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D9BrapaS4. Both proposed markers were about 50 centiMorgans away from the ANL gene; this

is still a significant distance and their relation to the alleles cannot be 100% credible (Fast Plants

Molecular Markers I, UMD 2014). If possible, finding a marker with a distance even closer to

the gene could provide us with even more precision and confidence in our results.

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References

Biology 2202 Lab Manual. 2014. Fast plants Molecular Markers I – DNA Extraction Lab.

University of Minnesota Duluth, MN.

Biology 2202 Lab Manual. 2014. Fast plants Molecular Markers II – PCR Lab. University of

Minnesota Duluth, MN.

Biology 2202 Lab Manual. 2014 Fast plants Molecular Markers III – Gel Lab. University of

Minnesota Duluth, MN.

Burdzinski C., Wendell D. L. 2007. Mapping the anthocyaninless (anl) locus in rapid-

cycling Brassica rapa (RBr) to linkage group R9. BMC Genet. 8, 64.10.1186/1471-2156-

8-64.

QIAGEN. 2012.DNeasy Plant Handbook (Mini Procedure).

Schlotterer, C. 2004. The evolution of molecular markers — just a matter of fashion? J. Nature.

Vol 5: 69

Williams, P.H., Hill, C.B. 1986. Rapid-cycling populations of Brassica. J Science 1986,

232:1385-1289.

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Figure 1. Gel electrophoresis image of Fast Plant D9BrapaS4 DNA fragments. Lane 1, 100 bp

ladder; Lane 2, (462/462 bp) true breeding green stemmed parent line DWRCBr53; lane 3,

(318/462 bp) purple stemmed F1 generation DWRCBr53 X DWRCBr76; and lane 4, (318/318

bp) true breeding purple stemmed parent line DWRCBr76. The F1 generation has two different

alleles (heterozygous) while the parent lines have only one allele (homozygous).

Figure 2. Fast Plant Park9 molecular marker DNA fragments. . Lane 1, 100 bp ladder; Lane 2,

(1022/1022 bp) true breeding green stemmed parent line DWRCBr53; lane 3, (1022/1022 bp)

purple stemmed F1 generation DWRCBr53 X DWRCBr76; and lane 4, (1022/1022 bp) true

breeding purple stemmed parent line DWRCBr76. All fragments were of the same length and

multiple alleles are indistinguishable.

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fast plants final paper

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