Genetic analysis of endangered orchids suggests genetic diversity and healthy populations.

Hi! My name is Jane Cowie and I currently live in Hanover, NH. I'm a sophomore at Hopkins School in New Haven, CT. I started out with the NHAS in 2017 with the after-school program studying C. elegans. Since then, I have also researched water quality, bacteria colonies, and the propagation of Cypripedium arietinum. I started genetic research of Cypripedium reginae in 2020, and learned how to do PCR, Gel Electrophoresis, DNA Sequencing and more from Zoom lectures held by the NHAS. The researched outlined in this paper was completed in August of 2021. I have always been interested in biology, but the NHAS made it possible for me to further my understanding of scientific concepts, as well as laboratory methods and procedures.

Cypripedium reginae or the Showy Lady’s Slippers are native to North America and are classified as endangered in New Hampshire. [1] Showy Lady’s Slippers are endangered due to habitat loss and a lack of genetic diversity within populations. Showy populations can be found in partially shaded forests or bogs with moist soil. [2] On each plant there are usually two flowers. Each flower has a pouch, sepals, and petals. On the stem of each plant there are three to six leaves, and the plants can reach three feet tall at full maturity.

These orchids can reproduce sexually through pollination or vegetatively through underground rhizomes. Showy Lady’s Slippers rely on bees and other insects to pollinate them. Cypripedium reginae reproduce asexually by the branching of underground roots and rhizomes that produce genetically identical plants or clones. Plants that are closer together are likely to be genetic clones, whereas plants farther apart are likely diverse because of seed dispersal. [3] If an entire fen consisted solely of clones, a threat such as a pest or change in environment that threatens to kill one plant could wipe out the entire orchid population in the fen. [4]

Genetic diversity confers upon a population a higher tolerance of environmental changes, creating a more sustainable population. We can test for genetic diversity by amplifying regions of the genome identified by microsatellite primers using polymerase chain reaction (PCR) and sequencing the DNA. Microsatellites are sequences of up to a few hundred nucleotides with repeats that mutate more quickly than coding regions of the genome, allowing for detection of changes among individual plants. The genome of Cyp. reginae has not yet been sequenced, so primers previously established for looking at microsatellites in a similar species, Cyp. tibeticum, were used as a starting point. [5] Twenty primer sets from that study were previously tested for their abilities to amplify regions in Cyp. reginae, with 8 sets found to work (private communication with L. Schaner and V. Rastogi, NHAS). These microsatellite primers: 172, 209, 164, 880, 289, 182, 233, and 130 were used to amplify specific areas of the DNA in the plant samples tested here.

Methods

Leaf samples were collected from three Showy Lady’s Slipper populations in New Hampshire and Vermont. Thirteen leaf samples were collected on July 30, 2021 from the Strafford fen located in Strafford, VT. The samples came from individual plants clumped into five clusters. Seven samples from three clumps were harvested from the Eshqua bog in Woodstock, VT in June of 2020. These seven samples were analyzed for genetic diversity in 2020 using the 172 and 209 microsatellite primers. [6] Finally, three samples were collected from the artificial Crossroads Academy fen in Lyme, NH on August 3, 2021. Leaf samples were collected from individual plants clumped together, and distances between clumps in the Eshqua and Strafford fens varied between 1-5 meters. Individuals in the Crossroads fen are 1-3 feet apart. The Strafford and Eshqua populations are naturally-occurring. The Crossroads Lady Slipper fen was man-made with transplanted plants from the Eshqua bog. DNA was extracted from all samples and the twenty-three DNA samples were each amplified with eight microsatellite primers.

The Monarch Genomic DNA Purification Kit from New England Biolabs (NEB) was used based on the manufacturer's instructions to extract DNA from the leaf samples. [7] DNA concentrations ranged between 27.5-170.2 ng/μL. A Vortex-Genie 2, an Eppendorf 5415 Centrifuge, and Gilson micropipettes were used in this step, as well as others.

To prepare the samples for PCR, ultimate mixes, which are uniform among multiple PCR tubes in a group, were made, one for each DNA sample. These had 25μL NEB OneTaq 2x Master Mix, 1μL of template DNA extracted from leaf samples, and 22 μL sterile water for each single reaction.  Then, 48μL of the Ultimate mix was distributed into each 0.2mL PCR tube and 2μL of forward/reverse primer solution was added separately to each tube, so that each DNA sample was tested with all eight primers. The tubes were then run in the Mini 8 Thermal Cycler (miniPCR) [8] or the MyCycler Thermal Cycler System (Bio-Rad Laboratories). [9] The initial denaturation was run at 95°C for 120 seconds. The repeated denaturation step was 95°C for 30 seconds. The annealing temperature was 49°C for the main portion of the testing, but was also run at 47°C to troubleshoot the PCR. It always ran for 30 seconds. The extension step was 68°C for 30 seconds. This three-step cycle was run 35 times. Then, the final extension was run at 68°C for 60 seconds.

PCR reactions were then run on 2% agarose gels made with Gel Green dye (MiniPCR). Into each gel, 12μL of each sample as well as 12μL of a 100 bp ladder were loaded. The gels and samples were run in the Bluegel electrophoresis system (miniPCR) according to the manufacturer's instructions. Photos of the gels were taken 15 minutes into the process and every five minutes after, until 35 minutes had passed. If single bands in the 200-300 base pair range appeared on the gel, the PCR sample from that lane was selected for cleanup and sequencing. In lanes with double bands, gel extraction was performed to cut out each band and purify the DNA with the Monarch DNA Gel Extraction Kit according to the manufacturer’s instructions. [10]

To clean up the PCR product, New England Biolabs Monarch PCR and DNA Cleanup Kit was used according to the manufacturer's instructions. [11] The eluted 15μL of purified DNA was then sent to Eton Bioscience for sequencing. The chromatograms sent back were viewed using FinchTV. [12] If the chromatogram had relatively low baseline noise and evenly spaced peaks, a section was copied into a text editor. Multiple sequences were copied and submitted to Clustal Omega [13] for alignment, along with the Cyp. tibeticum sequence from NCBI catalog. [14] Spots in the alignment where nucleotides differ from the rest indicate a possible mutation and can be confirmed by referencing the chromatogram. The chromatogram can also show heterozygous positions in the sequence which are indicated by double peaks, showing two bases can be found at that position. Results from Clustal Omega were viewed in Jalview. [15]

Discussion

Figure 1 summarizes the end results of PCRs performed in this study and suggests that the microsatellite primers used varied in effectiveness for analyzing regions of the Showy Lady’s Slipper genome. The 209 primer produced the highest yield of 61% efficacy across all samples. The 164, 233, and 880 primers showed the lowest yield in this experiment. Primers that do not successfully amplify Showy Lady’s Slipper DNA may indicate that those microsatellite regions have diverged between Cyp. tibeticum and Cyp. reginae, at least in the regions where the primers should bind. However, it is also possible that unsuccessful reactions were due to human or system error.

Chromatograms were analyzed for double peaks, indicating heterozygous positions which provide context for bases that may have been called differently among sequences and provide more detail on the genetic makeup of each individual. These double peaks can be compared to other samples for the same position to indicate how identical different individuals are to each other. The experiment will need to be repeated to confirm any double peak is real and not noise in the chromatogram. Baseline noise and double peaks in the chromatogram are suppressed in the sequence read-out used for alignments, so direct percent identity comparisons between chromatogram data could provide further evidence (Figure 2).

The data in Figure 3 indicates potential genetic diversity present within the Strafford fen (P3C1 compared to P2C4) and even within a single cluster (P2C1 compared to P1C1 and P3C1).

With the data provided in this experiment, phylogenetic tree analysis (Figure 4) suggests that there is genetic diversity between the Strafford and Eshqua bog populations based on the results of primer 172. Additional samples from the Crossroads fen tested with the 172 microsatellite primer could begin to provide enough data to support bootstrap analysis. Combining data from multiple microsatellite regions will also bolster confidence in genetic diversity between individuals and populations.

Still, the data in Figure 5 visually suggests that the individuals within the Crossroads fen are genetically diverse and there is “distance” between the Strafford and Crossroads populations (9 nucleotide differences out of a total aligned sequence length of approximately 200 base pairs). Future research could compare the genetic diversity of the artificial Crossroads fen and the original Eshqua fen.

Further analysis of more areas of the genome will also lend more information to support a conclusion of whether the populations are largely identical or diverse. The results from Eshqua bog will also be compared with results from the artificial Crossroads fen.

The data presented here suggests that the Cypripedium reginae samples from the Eshqua bog, Crossroads fen, and Strafford fen have mutations in their microsatellite regions compared to the Cypripedium tibeticum samples from the catalog. Preliminary evidence of mutational differences between populations as well as within the Strafford fen, Eshqua bog, and Crossroads fen suggests that all populations have genetic diversity. Genetic diversity is an indicator for a healthy population, suggesting that the Cyp. reginae species can adapt to a changing environment. Sequencing reactions will need to be repeated, and process yield will need to improve to increase confidence in conclusions and finally quantify the genetic diversity of these endangered orchids.