The Use of Microsatellite Markers to Characterize Oxalis Cultivars




Chelsea L. Butcher

Introduction

Although it is suggested that urbanization decreases biodiversity and increases the number of invasive species colonizing and establishing the area, several studies have shown that increasing the amount of green space mitigates these effects (Melles et al. 2003; Matteson et al. 2008). One way to accomplish this increase in urban habitat is by installing green roofs on buildings. Green roofs benefit the surrounding flora and fauna by serving as a unique habitat (Oberndorfer et al. 2007). Native birds and invertebrates use green roofs as habitats (Baumann 2006; Kadas 2006). Plant species, such as the native yellow wood sorrel (Oxalis stricta) also use green roofs as habitats (Butcher et al. 2012). However, little is known about the genetic diversity of species in urban environments. Of the few studies that have been conducted on urban species, only animals have been examined (Munshi-South and Kharchenko 2010). Furthermore, the genetic diversity has not yet been examined on any green roof species.

Therefore, my long-term research goal is to expand on the topic of green roofs as plant habitat and the effects of urbanization on the genetic diversity to investigate gene flow and population structure of O. stricta on green roofs in the five boroughs of New York City. In order to accomplish this goal, I needed to first identify microsatellite primer pairs that would successfully amplify regions of microsatellite DNA in plants of the genus Oxalis. In this study, I used four microsatellite primer pairs originally created for Oxalis alpina for use on eight other Oxalis cultivars of varying morphologies. Specifically, my objective for this project was to use these four primer pairs to characterize eight Oxalis cultivars with agarose gels and one fluorescently labeled primer pair that allowed me to determine each cultivars' allele size at one locus.

Materials and Methods

A total of eight Oxalis cultivars were used in this analysis. Seven cultivars of various morphologies were purchased from florists and greenhouses in New York, Delaware, and Rhode Island in March 2013. One O. stricta individual was collected from a suburban landscape in Rhode Island in April 2013.

DNA was extracted from live plant tissue and primers from Tsyusko et al. (2007) were used to amplify four microsatellite loci (Oxa22, Oxa46, Oxa62, and Oxa84). A separate PCR was also conducted using primer pairs that were fluorescently labeled with FAM. This product was purified with a QIAQuick PCR Purification Kit (Qiagen, Hilden, Germany) and was sent to Genewiz, Inc (South Plainfield, NJ) for Oxa62 allele size determination. The microsatellite output was analyzed using Peak Scanner 1.0 (Applied Biosystems, Foster City, CA).

Results

Successful PCR amplification occurred for four samples (samples 2, 4, 7, and 8) using 5ng of DNA. PCR product (5 µL) was loaded and run on a 1% agarose gel (Figure 1). Likely, the reason for this inconsistent amplification was the presence of PCR inhibitors. In plants, PCR inhibitors such as complex polysaccharides, present in high concentrations in the cell wall, can be extracted and co-purified with DNA (De Boer et al. 1995). To increase resolution of the samples, I loaded and ran 1 µL of the four samples that showed amplification with 5ng of DNA on a 4% agarose gel (Figure 2). Differentiation ability did not improve as differences between samples 2, 4, and 7 were still unclear.

Reducing the amount of DNA added to each PCR reaction has been shown to decrease the effects of inhibitors (De Boer et al. 1995). Therefore, I repeated the PCR with fluorescently labeled primers for locus Oxa62 using 1ng of DNA. Decreasing the amount of DNA resulted in successful amplification of all eight samples (Figure 3). Furthermore, in order to differentiate between all eight samples, I used a the PCR purified product from this PCR reaction to determined allele sizes for each of the eight samples. Three alleles (i.e. 163, 164, and 172 base pair alleles) and three allele combinations (i.e. genotypes) were found at the Oxa62 locus (Figure 4).

Discussion

In this study, four primer pairs originally created for O. alpina were optimized for use on other Oxalis cultivars. To account for the small differences in microsatellite repeats, fluorescently labeled primers were used to identify the exact size of each plant Oxa62 allele. This allowed me to differentiate between all samples and determine their allele size(s) at the Oxa62 microsatellite locus.

A total of three alleles were found across all eight samples. Samples 1, 2, 3, 5, 6, and 7 were all homozygous for the 164 base pair allele. Sample 4 showed two allele sizes indicating that this sample is heterozygous at this locus. However, the 163 base pair allele is surprising since length changes in microsatellite loci are typically attributed to replication slippage that results in the addition or deletion of repeat units (Ellegren 2004). The repeat motif of Oxa62 is that of “CTCTCT….,” therefore, the changes in allele sizes should be in multiples of two (Tsyusko et al. 2007). Although repeat mutations are most common in microsatellite DNA, single-base pair mutations also occur in this portion of the genome (Du et al. 2012). This indicates that this unique genotype was a result of a single-base pair mutation rather than replication slippage. Sample 8 also showed a unique genotype. While the major allele size was found to be the 172 base pair allele, four other allele sizes were detected (160, 168, 180, and 184). Further work will need to be conducted to determine the reason for multiple peaks in this sample.

References

Baumann, N. 2006. Ground-nesting birds on green roofs in Switzerland: preliminary observations. Urban Habitats 4:37-50.

Butcher, C.L., J.M. Dannenhoffer, B.J. Swanson. 2012. Factors influencing colonization of native and invasive plant species on green roofs in Michigan, USA. Unpublished Data.

De Boer, S.H., L.J. Ward, X. Li, and S. Chittaranjan. 1995. Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO. Nucleic Acids Research 23:2567-2568.

Du, Q., C. Gong, W. Pan, and D. Zhang. 2012. Development and application of microsatellites in candidate genes related to wood properties in the Chinese white poplar (Populus tomentosa Carr.). DNA Research 20:31-44.

Kadas, G. 2006. Rare invertebrates colonizing green roofs in London. Urban Habitats 4:66-86

Matteson, K.C., J.S. Ascher, and G.A. Langellotto. 2008. Bee richness and abundance in New York City urban gardens. Annals of the Entomological Society of America 101:140-150.

Melles, S., S. Glenn, and K. Martin. 2003. Urban bird diversity and landscape complexity: species-environment associations along a multiscale habitat gradient. Conservation Ecology 7:5-26.

Munshi-South, J. and K. Kharchenko. 2010. Rapid, persuasive genetic differentiation of urban white-footed mice (Peromyscus leuopus) populations in New York City. Molecular Ecology 19:4242-4254.

Oberndorfer, E., J. Lundholm, B. Bass, et al. 2007. Green roofs as urban ecosystems: ecological structures, functions, and services. Bioscience 57:823-833.

Tsyusko, O.V., T.D. Tuberville, M.B. Peters, et al. 2007. Microsatellite markers isolated from polyploidy wood-sorrel Oxalis alpina (Oxalidaceae). Molecular Ecology Notes 7:1284-1286.

Figures


Figure 1-PCR product visualized on a 1% agarose gel from microsatellite markers Oxa22, Oxa46, Oxa62, and Oxa84. PCR was performed using 5ng of DNA for all samples.


Figure 2-PCR product visualized using a 4% agarose gel from microsatellite markers Oxa22, Oxa46, Oxa62, and Oxa84. Only samples showing amplification were loaded on this gel.


Figure 3-PCR product visualized on a 1% agarose gel from microsatellite marker Oxa62 fluorescently labeled with FAM. PCR was performed using 1ng of DNA for all samples.


Figure 4-Allele sizes for each of the eight samples.


Abstract

It is suggested that urbanization decreases biodiversity and increases the number of invasive species colonizing and establishing the area. However, several studies have shown that increasing the amount of green space, such as the installation of green roofs on buildings, can mitigate these effects. Plant species, such as the native yellow wood sorrel (Oxalis stricta), colonize and use green roofs as habitats; however, no studies have been conducted on the genetic diversity of this species. Therefore, my long-term research goal is to investigate the genetic diversity and population structure of O. stricta on green roofs in the five boroughs of New York City. In order to accomplish this goal, I needed to first identify microsatellite primer pairs that would successfully amplify regions of microsatellite DNA in plants of the Oxalis genus. In this study, I used four microsatellite primer pairs originally created for Oxalis alpina for use on eight other Oxalis cultivars of varying morphologies. Four microsatellite primer pairs were identified that successfully amplified regions of microsatellite DNA in Oxalis cultivars. Differentiation between individuals was accomplished using agarose gel electrophoresis and a fluorescently labeled primer pair that allowed me to determine each individual’s allele size at one locus. Three different alleles at one locus were found across the eight samples. Furthermore, seven of the samples were homozygous and one of the samples was heterozygous at this locus.

Full Paper

Acknowledgments

I would like to thank Kate Reid and Catharina Grubaugh for their patience and guidance throughout this project. I would also like to thank Dr. Sylvia Anderson for her help in collecting Oxalis cultivars from various florists and greenhouses as well as assistance with genotyping. Additionally, I would like to thank my advisor, Dr. Jim Lewis, for his support and encouragement while taking this course and completing this research project. Finally, I would like to thank Dr. Berish Rubin for the support and knowledge provided throughout this course and research project.


This document was last modified 05/05/2013.
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