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SOIL ANALYSIS
SOIL ANALYSIS
Our three main objectives for analyzing our soil samples from St. Croix in the lab are to:
I. Discover what is possible with the archived soil samples and test their utility for DNA extraction, PCR, and DNA sequencing.
II. Relate the role carbon-nitrogen plays in soil biodiversity and detect the amount of carbon-nitrogen in fresh soil samples to study the specific impact of plantation usage.
III. Compare the soil eDNA and carbon-nitrogen content between old-growth and secondary-growth forests from the archived and fresh soil samples.
We would achieve these goals by:
Conducting eDNA analysis on the archived soil samples.
Conducting elemental analysis on the fresh soil samples.
The soil samples analyzed for eDNA analysis were collected by CEL on June 30, 2022, and July 1, 2022 from the Estate Little Princess (ELP) site in St. Croix. They were received by the Eagle Lab at UCLA on February 12, 2024. All these samples were taken from old-growth (OG) and secondary-growth (SG) areas. These areas were determined based on observable characteristics OG areas comprised older, wider, and larger trees with greater species diversity. The characteristics of SG areas included bushy areas that looked younger and had greater density, especially with snake plants and were generally more difficult to pass through.
Methods
Methods
eDNA Extraction & Quantification
We extracted eDNA from soil samples 1-5 (OG1), 21-23 (OG2), and 36-38 (SG2) using the Qiagen PowerSoil Pro kit. We used fresh soil samples collected in different parts of campus as our controls (C1, C2, & C3) to compare the efficiency of eDNA extraction between archived and fresh soil samples. Then, we quantified the concentration of extracted eDNA.
PCR & Gel Electrophoresis
PCR (polymerase chain reaction) is a method that makes millions to billions of copies of a specific DNA sample. The results of a PCR reaction are usually visualized and made visible using gel electrophoresis, which is a technique in which fragments of DNA are pulled through a gel matrix by an electric current and separated according to size. We performed PCR on the archived soil samples using a 16S microbe primer targeting the bacterial 16S ribosomal RNA gene, specifically 806R, and 515F, indicating the presence of bacteria in the soil eDNA.
Preparation for Sequencing - Indexing and Pooling
To prepare the archived soil samples for sequencing, we indexed the samples (#1-5, 21-24, and 36-38), the controls (C1 and C2), and the blanks (B1, B2, and B3) and pooled them all together.
Results
Results
eDNA Extraction & Quantification
When we quantified the extracted eDNA, we found that the average DNA concentration found in the archived soil samples was greater than in the fresh control soil samples, a significant difference (p = 0.017 < 0.05). The average DNA concentration in archived soil samples from OG2 was greater than the soil samples from OG1 and SG2 (see Figure 4).
Here are some figures illustrating the results of our soil analysis regarding DNA concentration. The rest can be found under Figures on the Data page and our final report.
Average DNA Concentration between all Archived & Fresh (Control) Soil Samples
Note. Archived soil samples are in blue while fresh (control) soil samples are in orange. Archived average was taken from archived soil samples #1-5 (OG1), #21-23 (OG2), & #36-38 (SG2). Fresh average was taken from 3 soil samples collected at UCLA (C1, C2, C3).
Average DNA Concentration in Archived Soil Samples from OG1, OG2, and SG2
Note. The average DNA concentration in archived soil samples from OG2 (x̄ = 13.2 ng/μL) is greater than the samples from OG1 (x̄ = 5.16 ng/μL) and SG2 (x̄ = 4.5 ng/μL).
Average DNA Concentration between OG (#1-5, 21-23) and SG (#36-38) Archived Soil Samples
Note. The average DNA concentration in OG (x̄ = 8.17 ng/μL) vs. in SG (x̄ = 4.5 ng/μL). p = 0.14 > 0.05 shows that the difference in average DNA concentration between OG and SG is not significant.
DNA Concentration in ELP Archived Soil Samples
Note. Highlighted in blue are the archived soil samples from ELP and highlighted in orange are the fresh (control) soil samples from UCLA.
PCR & Gel Electrophoresis
After several attempts at PCR, we successfully amplified the 806R and 515F gene in all of our samples as confirmed by the gels via gel electrophoresis (see Figure 11). PCR is
Here are some figures illustrating the results of our soil analysis regarding PCR and Gel electrophoresis, Indexing, and sequencing. The rest can be found under Figures on the Data page and our final report.
Results from gel electrophoresis after PCR with 16S microbe primer
Results from gel electrophoresis after PCR with 16S microbe primer
on samples #1-5, #36-38, C1, C2, B1, and B2.
Note. The DNA from the samples after PCR is shown to be 300bp.
Preparation for Sequencing - Indexing and Pooling
We successfully indexed the samples (#1-5, 21-24, and 36-38). Explain indexing.... The controls- the samples not collected on St. Croix, but rather on UCLA campus (C1 and C2), and the blanks- samples with no DNA present (B1, B2, and B3) (see Figure 12). Then, we pooled them all together based on their DNA concentration, meaning that we (see Figure 14).
Results after PCR indexing on samples #1-5, #21-24, #36-38, C1, C2, B1, B2, & B3.
Results after PCR indexing on samples #1-5, #21-24, #36-38, C1, C2, B1, B2, & B3.
Note. All samples except #1, 3, 5, 24, & B3 amplified. The DNA from the samples after PCR indexing is shown to be 400-500bp.
Results after PCR indexing on samples #1, 3, 5, 24, B3.
Note. The DNA from samples #1, 3, 5, 24, & B3 after PCR indexing is shown to be 350-400bp.
DNA Concentration in PCR Indexed Archived Soil Samples from ELP and Fresh (Control) Soil Samples at UCLA
DNA Concentration in PCR Indexed Archived Soil Samples from ELP and Fresh (Control) Soil Samples at UCLA
Note. We introduced blanks (B1, B2, and B3), which are shaded in gray. This means they have PCR indexes but not soil eDNA.
Elemental Analysis
Isotope Analysis of Archived Soil Samples
We utilized the elemental analysis data of the archived soil samples from Estate Little Princess to examine the soil's carbon:nitrogen (C:N) ratio.
C:N Ratio of Archived Soil Samples taken from Estate Little Princess, with Old Growth 1 (OG1) soil samples #1-5 having the highest C:N ratio, seen between a C:N ratio range of 15-25. While Old Growth 2 (OG2) soil samples #21-23 come second with a C:N ratio range of 10-15, and Secondary Growth 2 (SG2) soil samples #36-28 come last with the lowest soil samples running from 7-13.
Average C:N Ratio for each Archived Soil Sample plot taken from Estate Little Princess: OG1 indicates soil samples collected from Plot 1 with Old Growth forests presently growing, OG2 indicating soil samples collected from Plot 2 with Old Growth Forest presently growing, and SG2 indicates soil samples collected from Plot 2 with Secondary Growth Forest presently growing.
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