Cheung Poster

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1. Castilleja hispida: 4 photo taken by Tom Kaye a. Wolf Haven (WH) b. Joint Base Lewis-McChord (JBLM) c. Scatter Creek South (SCS) d. Bald Hill (BH) 2. Assume hybrids [Glacial Heritage]: a. Maternal Plants (F1) b. Presumed F2 3. Castilleja levisecta: 4 photo taken by Tom Kaye a. Ebey’s Landing b. Fort Lewis c. Rocky Point d. Naas Molecular Markers Distinguish Hybridization Patterns in Castilleja Lisa Cheung 1 ; Jeremie Fant 2 ; Andrea Kramer 2 ; Adrienne Basey 2 1 Carleton College, Northfield, MN 55057; 2 Chicago Botanic Garden, Glencoe, IL 60022 Introductions Materials Materials/Methods Results Discussion References Castilleja levisecta (golden paintbrush) is an endangered flowering plant restricted in the Pacific Northwest United States (Figure 1). This species is undergoing restoration in prairies native to another Castilleja species, C. hispida, which is being used to restore habitat to an endangered butterfly 5 . However, it has been observed that hybridization of the two Castilleja species can occur 2 , which could cause the genetic swamping of C. levisecta. Hybridization between the two species can occur on the top and bottom inflorescence. Although C. levisecta flowers first, C. hispida’s flowering time overlaps with C. levisecta’s. Due to flowering phenology, outcrossing between the two can result in hybridization differences between the top and bottom [earlier and later, respectively] inflorescence 3 . It is hypothesized that there will be a distinguishable variance in the microsatellite regions between the two species. Additionally, these variances will allow us to understand the likelihood of hybridization related to the position of the inflorescence. To determine whether C. levisecta and C. hispida had detectable differences in their microsatellite regions, I ran a Structure analysis on 21 populations. Based on the restults, the 21 populations were assigned into two distinct clusters, distinguished as levisecta (yellow) and hispida (red), respectively. From Figure 2, there are notable differences between the two Castilleja species, displaying a confidence over 90% as either C. levisecta or C. hispida. Comparing these differences to the Structure analysis from population 7 showed that the assumed F1 hybrids from Glacial Heritage were a levisecta-hispida mix; however, the analysis also indicated that some of the hybrids were not F1. Assuming the hybrids were all F1, the Structure analysis would display an approximately linear pattern at 50% confidence across the population.Yet, some of the hybrids displayed a 75% confidence towards levisecta and others a 25%, suggesting that the hybrids are backcrossing to their pure species (Figure 4). Additionally, these results suggest that not all hybrids are distinguishable. To determine whether hybrids displayed a noticeable hybridization pattern, I compared the Maternal F1 to the F2.Yet, comparison could only be analyzed for M1, M2, M15, M22 (see Figure 3), and their respective offspring due to missing Maternal F1 samples or polyploidy issues within the F2. However, the Maternal F1 and F2 lines that were analyzed for both the top and bottom inflorescence indicated that a directional hybridization pattern was not observed (Figure 5). For example, M2 and M22 were backcrossing towards levisecta. Although the bottom inflorescence for their respective offspring backcrossed towards hispida, M2’s offspring’s top inflorescence backcrossed towards levisecta; whereas, M22’s offspring’s backcrossed towards hispida. This implies that there is no clear hybridization pattern. Nevertheless, pollination is random and less specific, therefore, there is no pattern for which inflorescence becomes pollinated by time or position 2 . 1 Basey, A. 2015. Genetic changes associated with native plant propagation: case study in Castilleja levisecta. Master’s thesis, Northwestern University, Evanston, IL, and Chicago Botanic Garden, Glencoe, IL. 2 Clark, L. A. 2015. Bee-crossed lovers and a forbidden Castilleja romance: cross-breeding between C. hispida and endangered C. levisecta in prairie restoration sites. Master’s thesis, University of Washington, Seattle, WA. 3 Fisher, L. L., Bakker, J. D., and Dunwiddie, P. W. 2015. An assessment of seed production and viability of putative Castilleja levisecta x C. hispida hybrids. Report prepared fr the Center for Natural Lands Management, University of Washington, Seattle, WA. 4 Kaye, T. N., and Blakeley-Smith, M. 2008. An Evaluation of the Potential for Hybridization Between Castilleja levisecta and C. hispida. Unpublished report. Institute of Applied Ecology, Corvallis, OR. 5 Lawrence, B. A., and T. N. Kaye. 2009. Reintroduction of Castilleja levisecta: Effects of ecological similarity, source population genetics, and habitat quality. Restoration Ecology. doi:10.1111/j.1526-100X.2009.00549.x. DNA Extraction: Extracted DNA from leaf tissues and seed capsules using a CTAB protocol Assessed extracted DNA concentration using a Nanodrop 2000. PCR Amplification: Amplified 7 microsatellite regions of the extracted DNA using a Castilleja polymerase chain reaction (PCR) program 1 . Genetic Scoring: Analyzed and scored microsatellite products with Beckman Coulter CEQ 8000 Genetic Analysis System to determine allele size in two species from six populations total. Analysis: Ran data gathered from the Beckman on a Structure program to assign individuals into species and populations. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% M1 1BA1 1BB1 1BB24 1BB30 1BB32 1BB4 1BB43 1TA1 1TA14 1TA27 1TA42 1TA44 1TB3 % Confidence Sample Levisecta Hispida 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% M2 2BA23 2BA39 2BA5 2BA58 2BA70 2T13 2T16 2T18 2T5 2T6 2T7 2T8 2T9 % Confidence Sample Levisecta Hispida 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% M15 15B11 15B12 15B13 15B3 15B4 15B5 15B6 15B8 15TA1 15TA2 15TA4 15TA6 15TA7 15TA8 15TB1 15TB2 * Confidence Sample Levisecta Hispida 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% M22 22B1 22B10 22B2 22B3 22B5 22B6 22B7 22TB1 22TB1022TB1122TB15 22TB3 22TB4 22TB6 22TB8 % Confidence Sample Levisecta Hispida Popula’on Species Hispida Levisecta Individuals Sample ID 1 Levisecta 2% 98% 30 2 Levisecta 3 Levisecta 2% 99% 35 4 Levisecta 1% 99% 30 5 Hispida 97% 3% 26 6 Hispida 98% 2% 29 7 F1 Hybrids 54% 46% 16 Maternal F1 (M) 8 F2 Hybrids 62% 39% 7 1 BoDom (1B) 9 F2 Hybrids 26% 74% 6 1 Top (1T) 10 F2 Hybrids 13% 87% 5 2 BoDom (2B) 11 F2 Hybrids 2% 99% 8 2 Top (2T) 12 F2 Hybrids 29% 71% 2 3 BoDom (3B) 13 F2 Hybrids 8% 92% 3 3 Top (3T) 14 F2 Hybrids 23% 77% 8 14 BoDom (14B) 15 F2 Hybrids 54% 46% 8 15 BoDom (15B) 16 F2 Hybrids 19% 81% 8 15 Top (15T) 17 F2 Hybrids 75% 25% 7 22 BoDom (22B) 18 F2 Hybrids 89% 11% 8 22 Top (22T) 19 F2 Hybrids 26% 74% 8 24 BoDom (24B) 20 F2 Hybrids 6% 94% 5 25 BoDom (25B) 21 F2 Hybrids 14% 86% 8 25 Top (25T) Acknowledgments I’d like to thank Jeremie Fant and Andrea Kramer for their assistance and mentorship throughout the research project, as well as Adrienne Basey for providing the C. levisecta data. I would also like to thank Rebecca Nelson and Jennifer Fischer for their help with DNA extraction and NSG-REU grant DBI-1461007 for support funding this project. Figure 3. Summary of % confidence, number of individuals, and population for wild and reintroduced C. hispida, C. levisecta, and F1 and F2 hybrids. Figure 1. Generalized map of wild and reintroduced populations of C. hispida, C. levisecta, and F1 and F2 hybrids. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 % Confidence Population Levisecta Hispida 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% M1 M2 M4 M7 M11 M12 M13 M14 M15 M17 M18 M19 M20 M21 M22 M23 % Confidence Sample Levisecta Hispida Figure 5. Structure analysis of M1, M2, M15, M22, and their respective offspring. Each graph represents genetic composition of maternal plants followed by the genetic composition of 5-8 seeds collected from the top and bottom capsules. Figure 4. Structure analysis for putative hybrid maternal lines assigned into clusters of K=2. A straight black line at 50% represents F1 and dotted line at 75% and 25% represents backcross to C. hispida and C. levisecta, respectively 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 9 9 9 9 9 9 1010101010 1111111111111111 1212 131313 1414141414141414 1515151515151515 1616161616161616 17171717171717 1818181818181818 1919191919191919 2020202020 2121212121212121 % Confidence Population Levisecta Hispida Figure 2. Top: structure analysis for C. hispida and C. levisecta. Assigned into clusters of K=2, yellow corresponds to % confidence of C. levisecta and red corresponds to % confidence of C. hispida. Bottom: structure analysis for F1 and F2 hybrids. Assigned into clusters of K=2, the mix of yellow and red determine the % confidence towards C. hispida or C. levisecta.

Transcript of Cheung Poster

Page 1: Cheung Poster

1. Castilleja hispida: 4photo taken by Tom Kaye

a. Wolf Haven (WH)

b. Joint Base Lewis-McChord (JBLM)

c. Scatter Creek South (SCS)

d. Bald Hill (BH)

2. Assume hybrids [Glacial Heritage]:

a. Maternal Plants (F1)

b. Presumed F2

3. Castilleja levisecta: 4photo taken by Tom Kaye

a. Ebey’s Landing

b. Fort Lewis

c. Rocky Point

d. Naas

Molecular Markers Distinguish Hybridization Patterns in Castilleja Lisa Cheung1; Jeremie Fant2; Andrea Kramer2; Adrienne Basey2

1Carleton College, Northfield, MN 55057; 2Chicago Botanic Garden, Glencoe, IL 60022

Introductions

Materials

Materials/Methods

Results

Discussion

References

Castilleja levisecta (golden paintbrush) is an endangered flowering plant restricted in the Pacific Northwest United States (Figure 1). This species is undergoing restoration in prairies native to another Castilleja species, C. hispida, which is being used to restore habitat to an endangered butterfly5. However, it has been observed that hybridization of the two Castilleja species can occur2, which could cause the genetic swamping of C. levisecta. Hybridization between the two species can occur on the top and bottom inflorescence. Although C. levisecta flowers first, C. hispida’s flowering time overlaps with C. levisecta’s. Due to flowering phenology, outcrossing between the two can result in hybridization differences between the top and bottom [earlier and later, respectively] inflorescence3. It is hypothesized that there will be a distinguishable variance in the microsatellite regions between the two species. Additionally, these variances will allow us to understand the likelihood of hybridization related to the position of the inflorescence.

To determine whether C. levisecta and C. hispida had detectable differences in their microsatellite regions, I ran a Structure analysis on 21 populations. Based on the restults, the 21 populations were assigned into two distinct clusters, distinguished as levisecta (yellow) and hispida (red), respectively. From Figure 2, there are notable differences between the two Castilleja species, displaying a confidence over 90% as either C. levisecta or C. hispida. Comparing these differences to the Structure analysis from population 7 showed that the assumed F1 hybrids from Glacial Heritage were a levisecta-hispida mix; however, the analysis also indicated that some of the hybrids were not F1. Assuming the hybrids were all F1, the Structure analysis would display an approximately linear pattern at 50% confidence across the population. Yet, some of the hybrids displayed a 75% confidence towards levisecta and others a 25%, suggesting that the hybrids are backcrossing to their pure species (Figure 4). Additionally, these results suggest that not all hybrids are distinguishable.   To determine whether hybrids displayed a noticeable hybridization pattern, I compared the Maternal F1 to the F2. Yet, comparison could only be analyzed for M1, M2, M15, M22 (see Figure 3), and their respective offspring due to missing Maternal F1 samples or polyploidy issues within the F2. However, the Maternal F1 and F2 lines that were analyzed for both the top and bottom inflorescence indicated that a directional hybridization pattern was not observed (Figure 5). For example, M2 and M22 were backcrossing towards levisecta. Although the bottom inflorescence for their respective offspring backcrossed towards hispida, M2’s offspring’s top inflorescence backcrossed towards levisecta; whereas, M22’s offspring’s backcrossed towards hispida. This implies that there is no clear hybridization pattern. Nevertheless, pollination is random and less specific, therefore, there is no pattern for which inflorescence becomes pollinated by time or position2.

1Basey, A. 2015. Genetic changes associated with native plant propagation: case study in Castilleja levisecta. Master’s thesis, Northwestern University, Evanston, IL, and Chicago Botanic Garden, Glencoe, IL.

2Clark, L. A. 2015. Bee-crossed lovers and a forbidden Castilleja romance: cross-breeding between C. hispida and endangered C. levisecta in prairie restoration sites. Master’s thesis, University of Washington, Seattle, WA.

3Fisher, L. L., Bakker, J. D., and Dunwiddie, P. W. 2015. An assessment of seed production and viability of putative Castilleja levisecta x C. hispida hybrids. Report prepared fr the Center for Natural Lands Management, University of Washington, Seattle, WA.

4Kaye, T. N., and Blakeley-Smith, M. 2008. An Evaluation of the Potential for Hybridization Between Castilleja levisecta and C. hispida. Unpublished report. Institute of Applied Ecology, Corvallis, OR.

5Lawrence, B. A., and T. N. Kaye. 2009. Reintroduction of Castilleja levisecta: Effects of ecological similarity, source population genetics, and habitat quality. Restoration Ecology. doi:10.1111/j.1526-100X.2009.00549.x.

DNA Extraction:

Extracted DNA from leaf tissues and seed

capsules using a CTAB protocol

Assessed extracted DNA concentration using a Nanodrop

2000.

PCR Amplification:

Amplified 7 microsatellite regions of the extracted DNA

using a Castilleja polymerase chain reaction (PCR)

program1.

Genetic Scoring:

Analyzed and scored microsatellite products with Beckman Coulter

CEQ 8000 Genetic Analysis System to

determine allele size in two species from six

populations total.

Analysis:

Ran data gathered from the Beckman on a Structure program to assign individuals into

species and populations.

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Popula'on   Species   Hispida   Levisecta   Individuals   Sample  ID  1   Levisecta   2%   98%   30   -­‐  2   Levisecta   -­‐   -­‐   -­‐   -­‐  3   Levisecta   2%   99%   35   -­‐  4   Levisecta   1%   99%   30   -­‐  5   Hispida   97%   3%   26   -­‐  6   Hispida   98%   2%   29   -­‐  7   F1  Hybrids   54%   46%   16   Maternal  F1  (M)  8   F2  Hybrids   62%   39%   7   1  BoDom  (1B)  9   F2  Hybrids   26%   74%   6   1  Top  (1T)  10   F2  Hybrids   13%   87%   5   2  BoDom  (2B)  11   F2  Hybrids   2%   99%   8   2  Top  (2T)  12   F2  Hybrids   29%   71%   2   3  BoDom  (3B)  13   F2  Hybrids   8%   92%   3   3  Top  (3T)  14   F2  Hybrids   23%   77%   8   14  BoDom  (14B)  15   F2  Hybrids   54%   46%   8   15  BoDom  (15B)  16   F2  Hybrids   19%   81%   8   15  Top  (15T)  17   F2  Hybrids   75%   25%   7   22  BoDom  (22B)  18   F2  Hybrids   89%   11%   8   22  Top  (22T)  19   F2  Hybrids   26%   74%   8   24  BoDom  (24B)  20   F2  Hybrids   6%   94%   5   25  BoDom  (25B)  21   F2  Hybrids   14%   86%   8   25  Top  (25T)  

Acknowledgments I’d like to thank Jeremie Fant and Andrea Kramer for their assistance and mentorship throughout the research project, as well as Adrienne Basey for providing the C. levisecta data. I would also like to thank Rebecca Nelson and Jennifer Fischer for their help with DNA extraction and NSG-REU grant DBI-1461007 for support funding this project.

Figure 3. Summary of % confidence, number of individuals, and population for wild and reintroduced C. hispida, C. levisecta, and F1 and F2 hybrids.

Figure 1. Generalized map of wild and reintroduced populations of C. hispida, C. levisecta, and F1 and F2 hybrids.

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Figure 5. Structure analysis of M1, M2, M15, M22, and their respective offspring. Each graph represents genetic composition of maternal plants followed by the genetic composition of 5-8 seeds collected from the top and bottom capsules.

Figure 4. Structure analysis for putative hybrid maternal lines assigned into clusters of K=2. A straight black line at 50% represents F1 and dotted line at 75% and 25% represents backcross to C. hispida and C. levisecta, respectively

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Figure 2. Top: structure analysis for C. hispida and C. levisecta. Assigned into clusters of K=2, yellow corresponds to % confidence of C. levisecta and red corresponds to % confidence of C. hispida. Bottom: structure analysis for F1 and F2 hybrids. Assigned into clusters of K=2, the mix of yellow and red determine the % confidence towards C. hispida or C. levisecta.