B599 - Genetic Sampling for Tree Species, Rocky Mountain Research Station, Forest Inventory Analysis

Genetic testing of tree needle samples to determine tree species and provide information regarding their level of certainty in the accuracy for each sample according to the below testing protocol and in accordance with the statement of work included in the attached combined solicitation/synopsis. Testing protocol description:

1. Receive the samples, organize them, prepare for DNA testing and data entry.

2. Extract total genomic DNA from a 1–2 mm segment of a single pine needle from each fascicle using either: i. the Sigma Aldrich Extract?N?Amp™ Plant PCR Kit (St. Louis, Missouri) or secondarily ii. the Qiagen DNeasy Plant Mini Kit (Valencia, California).

3. Quantify DNA concentration to confirm extraction success. Repeat extraction in case of insufficient DNA.

4. Use the polymerase chain reaction (PCR) to amplify two regions of the pine chloroplast DNA (cp DNA) with diagnostic restriction enzyme cut site differences (i.e., chloroplast matK BsmAl and psbA-trnH Psil loci).

5. Add restriction enzyme to a 10 ?l aliquot of the PCR reaction (restriction digestion).

6. Use agarose gel electrophoresis to visualize the presence/absence of restriction cut sites. a. Presence of a cut site in cpDNA region 1 is diagnostic of whitebark pine. Presence of a cut site in cpDNA region 2 is diagnostic of limber pine.

7. Record cut site results and species ID to whitebark or limber pine in a CSV?formatted file.

8. In case of ambiguities (both or neither region cuts), repeat steps 1?6. If still ambiguous, record as unknown.

9. Record notes relevant to any anomalies found while assessing the species. Examples include, but are not limited to, - “PsiI bands faint ? redo with more DNA in PCR rxn. 3rd BsmAI run again yielded a blank.” - “blank on BsmAI 14 July 2021” - “1 band 6 July 2021” - “needles not fascicled; 3 bands faint 6 July 2021; blank on BsmAI 14 July 2021” - “needles all separate and not in fascicles” 10. Return results to RMRS?FIA in the form of a spreadsheet and/or CSV file via email

05/21/2024

(UPDATED) - Amendment 0001 to solicitation #1240BG24Q0027. The purpose of this amendment is to notify the public of the following:

1) This solicitation period is extended until 05/29/2024 at 1:00PM EDT.

2) Questions must be received no later than 05/27/2024 at 1:00PM EDT.

3) Interested vendors are asked to review the testing methods specified in this solicitation at the link included in this notice, reference: (Alongi et.al. 2019)