Identifying St. Augustinegrass Off-Types in Sod Production Fields J.L. Buhlman 1 K.E. Kenworthy 1 , P.F. Harmon 2 , S.R. Milla-Lewis 3 , and E.F. Rios 1 1 Agronomy Department, University of Florida, Gainesville, FL, USA 2 Plant Pathology Department, University of Florida, Gainesville, FL, USA 3 Deparment of Crop Science, North Carolina State University, Raleigh, NC, USA Materials & Methods Background/ Rationale • ‘Bitterblue’ and ‘Floratam’ are the two oldest commercial cultivars of St. Augustinegrass produced in Florida. Floratam: aneuploid, 2n = 32 Bitterblue: triploid, 2n = 3x = 27 • 2007, 81% of St. Augustinegrass sod sold in Florida was Floratam • There are no original stock fields for either cultivar still in existence, and both cultivars have been on the market for many years. It is possible that sod entering the market is variable with respect to both cultivars. • Objective 1: Determine the extent of genetic diversity between Floratam samples, between Bitterblue samples, and determine the source of off-types, if possible. • Sugarcane mosaic virus (SCMV) has been shown to be part of Lethal Viral Necrosis, a new disease of St. Augustinegrass • Complete kill occurs on infected tissue of Floratam • Other cultivars susceptible to infection by SCMV but infection does not lead to death • Bermudagrass Latent Virus (BGLV) also suspected to be part of disease • Objective 2: Screen all samples submitted for SCMV infection. Overview • Samples were submitted by sod farms from Floratam and Bitterblue production fields (including suspected areas of contamination, when possible) • Mature stolons within a sample displaying phenotypic variation (Fig.1A) were separated and recoded as subsamples (Fig.1B) • Sample were identify using morphological measurements, Flow Cytometry (FC), Simple Sequence Repeat (SSR) markers • Samples were tested for the presence of SCMV SCMV Double Antibody Sandwich (DAS) Enzyme-Linked Immunosorbent Assay (ELISA)(Fig.2) • SCMV DAS ELISA kit from Agdia used to test for the presence of SCMV, and plates analyzed using a SpectraMax Plus Microplate Reader Morphological Study • Single pot entries, 3 replications, Randomized Complete Block Design (RCBD)(Fig.3) • 68 samples and 17 cultivars • Measurements taken after 1 month: • # of stolons per pot (TS), stolon length (SL), 4 th internode length (IL), stolon diameter (SD), leaf length (LL) and leaf width (LW) • Data was analyzed using Analysis of Variance (ANOVA) and Tukey’s HSD used to separate means, in R 3.5.1 • Principle Component Analysis (PCA) performed in R 3.5.1 • SSR Marker groups applied as group labels Flow Cytometry • Extracted nuclei from single meristem per sample and stained with Propidium Iodide (PI), using Sysmex FC Kit • Sorghum and Maize co-chopped as internal standards with each sample • Analyzed on Accuri C6 Flow Cytometer (Fig.4) • Total picograms were calculated based on internal standards and ploidy determined using diploid 1n value SSR Markers • Samples were ran with 9 of 22 selected primer pairs • Polymerase Chain Reactions (PCRs) performed using a thermocycler and PCR products analyzed using capillary electrophoresis on a Advance FS96/AB3730 system • Bands were scored in PeakAnalyzer (Fig.5) and transformed into a binary matric in Excel • A dissimilarity matrix was calculated and UPGMA used to construct a dendogram in NTSYSpc 2.2 Summary & Future Research Results Objective 1: Sample identification • Morphological measurements were able to somewhat distinguish between diploids and polyploid cultivars. • Flow cytometry does not provide an accurate estimation of DNA content in polyploid lines • SSR Markers ran thus far were able to differentiate between most cultivars, except for close relatives. • Floratam appears to have robust genetic integrity within and among production fields in Florida • The two most common off-types in Floratam fields are likely Palmetto and Seville • No trend was seen for Bitterblue samples using any of the 3 identification methods, suggesting significant differences between production fields Objective 2: Screening for SCMV • Future Research - A protocol for amplifying Bermudagrass Latent Virus (BGLV) has been developed and all samples should be screen for the presence of BGLV Special Thanks To: Camisha Alexis, Florian Buhlman, Katie Cox, and Ken Quesenberry for help completing the work related to this project Figure 1b. Single stolon pots of two phenotypes from submitted Floratam Sample F39: F39A and F39B Figure 1a. Submitted Floratam Sample F14, containing two phenotypes of St. Augustinegrass Figure 2. Diagram showing the DAS-ELISA procedure Figure 3. Morphological Trial. 3 reps, _ entries per rep. F39A F39B Captiva, 2X control Figure 4. Flow Cytometry results for subsamples F39A and F39B from submitted Floratam sample F39 and Captiva as a diploid control. Figure 5. Four banding patterns observed for Primer 1646 Marker Results (Fig.6) Group 1 • Predominately diploid cultivars with smaller cultivar groups • Palmetto groups includes: • 1 Bitterblue and 6 Floratam samples • Seville group includes: • 3 Bitterblue and 7 Floratam samples Group 2 • Predominately polyploid cultivars • Tight cluster of 38 Floratam samples • Includes Floralawn and 5 Bitterblue samples • Floratine grouped near 1 Bitterblue sample and 2 Floratam samples Outliers • FX10 arbitrarily fused to Group 1 • Tamstar and Sapphire somewhat fused to Group 2 • B05D, B06A and F48A grouped together but failed to group closely to any cultivar standards Flow Cytometry • Picogram estimation of diploids cultivars was consistent • Picogram estimation of suspected and known polyploid samples varied greatly, with ploidy estimates ranging from diploid to tetraploid Morphological Study Results • ANOVA (data not shown) - significant difference between sample means for • stolon length, internode length, stolon diameter, leaf length, leaf width • PCA (Fig. 7) • Genetic groups identified also show morphological clustering • Loose clustering of Floratam • No clustering of Bitterblue samples As previously reported, all samples were negative for SCMV Group 2 FC Results • Pg range: 0.99-2.32 • Estimated ploidy: Diploid to Tetraploid Group 1 FC Results • Pg range: 0.83-1.14 • Ploidy: Diploid Palmetto Seville Likely Floratam Figure 6. Dendogram using UPGMA of SSR marker data for submitted samples and cultivar checks Figure 7. PCA of morphological study, with groups from maker data applied.
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Identifying St. Augustinegrass Off-Types in Sod Production Fields
J.L. Buhlman1 K.E. Kenworthy1, P.F. Harmon2, S.R. Milla-Lewis3,
and E.F. Rios1
1Agronomy Department, University of Florida, Gainesville, FL, USA 2Plant Pathology Department, University of Florida, Gainesville, FL, USA
3Deparment of Crop Science, North Carolina State University, Raleigh, NC, USA
Materials & Methods
Background/ Rationale
• ‘Bitterblue’ and ‘Floratam’ are the two oldest commercial cultivars of St.
Augustinegrass produced in Florida.
Floratam: aneuploid, 2n = 32
Bitterblue: triploid, 2n = 3x = 27
• 2007, 81% of St. Augustinegrass sod sold in Florida was Floratam
• There are no original stock fields for either cultivar still in existence, and both
cultivars have been on the market for many years. It is possible that sod
entering the market is variable with respect to both cultivars.
• Objective 1: Determine the extent of genetic diversity between Floratam
samples, between Bitterblue samples, and determine the source of off-types, if
possible.
• Sugarcane mosaic virus (SCMV) has been shown to be part of Lethal Viral
Necrosis, a new disease of St. Augustinegrass
• Complete kill occurs on infected tissue of Floratam
• Other cultivars susceptible to infection by SCMV but infection does not
lead to death
• Bermudagrass Latent Virus (BGLV) also suspected to be part of disease
• Objective 2: Screen all samples submitted for SCMV infection.
Overview
• Samples were submitted by sod farms from Floratam and
Bitterblue production fields (including suspected areas of
contamination, when possible)
• Mature stolons within a sample displaying phenotypic
variation (Fig.1A) were separated and recoded as
subsamples (Fig.1B)
• Sample were identify using morphological measurements,
Flow Cytometry (FC), Simple Sequence Repeat (SSR)
markers
• Samples were tested for the presence of SCMV
SCMV Double Antibody Sandwich (DAS) Enzyme-Linked
Immunosorbent Assay (ELISA)(Fig.2)
• SCMV DAS ELISA kit from Agdia used to test for the
presence of SCMV, and plates analyzed using a SpectraMax
Plus Microplate Reader
Morphological Study
• Single pot entries, 3 replications, Randomized Complete
Block Design (RCBD)(Fig.3)
• 68 samples and 17 cultivars
• Measurements taken after 1 month:
• # of stolons per pot (TS), stolon length (SL), 4th
internode length (IL), stolon diameter (SD), leaf length
(LL) and leaf width (LW)
• Data was analyzed using Analysis of Variance (ANOVA) and
Tukey’s HSD used to separate means, in R 3.5.1
• Principle Component Analysis (PCA) performed in R 3.5.1
• SSR Marker groups applied as group labels
Flow Cytometry
• Extracted nuclei from single meristem per sample and
stained with Propidium Iodide (PI), using Sysmex FC Kit
• Sorghum and Maize co-chopped as internal standards with
each sample
• Analyzed on Accuri C6 Flow Cytometer (Fig.4)
• Total picograms were calculated based on internal standards
and ploidy determined using diploid 1n value
SSR Markers
• Samples were ran with 9 of 22 selected primer pairs
• Polymerase Chain Reactions (PCRs) performed using a
thermocycler and PCR products analyzed using capillary
electrophoresis on a Advance FS96/AB3730 system
• Bands were scored in PeakAnalyzer (Fig.5) and transformed
into a binary matric in Excel
• A dissimilarity matrix was calculated and UPGMA used to
construct a dendogram in NTSYSpc 2.2
Summary & Future Research
Results
Objective 1: Sample identification
• Morphological measurements were able to somewhat distinguish
between diploids and polyploid cultivars.
• Flow cytometry does not provide an accurate estimation of DNA content
in polyploid lines
• SSR Markers ran thus far were able to differentiate between most
cultivars, except for close relatives.
• Floratam appears to have robust genetic integrity within and among
production fields in Florida
• The two most common off-types in Floratam fields are likely Palmetto and
Seville
• No trend was seen for Bitterblue samples using any of the 3 identification
methods, suggesting significant differences between production fields
Objective 2: Screening for SCMV
• Future Research - A protocol for amplifying Bermudagrass Latent Virus
(BGLV) has been developed and all samples should be screen for the
presence of BGLV
Special Thanks To: Camisha Alexis, Florian Buhlman, Katie Cox, and Ken
Quesenberry for help completing the work related to this project
Figure 1b. Single stolon pots of two
phenotypes from submitted
Floratam Sample F39: F39A and
F39B
Figure 1a. Submitted Floratam Sample F14, containing two phenotypes of St. Augustinegrass
Figure 2. Diagram showing the DAS-ELISA procedure
Figure 3. Morphological Trial. 3 reps, _ entries per
rep.
F39A F39B Captiva, 2X control
Figure 4. Flow Cytometry results for
subsamples F39A and F39B from submitted
Floratam sample F39 and Captiva as a diploid
control.
Figure 5. Four banding patterns observed for
Primer 1646
Marker Results (Fig.6)
Group 1
• Predominately diploid cultivars with
smaller cultivar groups• Palmetto groups includes:
• 1 Bitterblue and 6 Floratam samples
• Seville group includes:
• 3 Bitterblue and 7 Floratam samples
Group 2
• Predominately polyploid cultivars• Tight cluster of 38 Floratam samples
• Includes Floralawn and 5 Bitterblue
samples
• Floratine grouped near 1 Bitterblue sample
and 2 Floratam samples
Outliers
• FX10 arbitrarily fused to Group 1
• Tamstar and Sapphire somewhat
fused to Group 2
• B05D, B06A and F48A grouped
together but failed to group closely to
any cultivar standards
Flow Cytometry
• Picogram estimation of diploids
cultivars was consistent
• Picogram estimation of suspected and
known polyploid samples varied
greatly, with ploidy estimates ranging
from diploid to tetraploid
Morphological Study Results
• ANOVA (data not shown) - significant
difference between sample means for• stolon length, internode length, stolon
diameter, leaf length, leaf width
• PCA (Fig. 7)
• Genetic groups identified also show
morphological clustering
• Loose clustering of Floratam
• No clustering of Bitterblue samples
As previously reported, all samples were negative for SCMV
Group 2FC Results
• Pg range: 0.99-2.32
• Estimated ploidy:
Diploid to Tetraploid
Group 1FC Results
• Pg range: 0.83-1.14
• Ploidy: Diploid
Palmetto
Seville
Likely Floratam
Figure 6. Dendogram using UPGMA of SSR marker data for submitted samples
and cultivar checks
Figure 7. PCA of morphological study, with groups from maker data
applied.
Related Documents
