FY24 Genetic ID_Statement of Work -Draft_Redacted.pdf

DRAFT O

NLY

US Department of Agriculture (USDA)

Statement of Work

Contents Project

General Information

1.0 Scope of Work

2.0 Background

Contractor Requirements

3.0 Technical Requirements / Tasks

4.0 Government Furnished

5.0 Deliverables / Schedule

6.0 Travel

7.0 Contractor’s Key Personnel

8.0 Security Requirements

9.0 Data Rights

10.0 Section 508 – Electronic and Information Technology Standards

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Project Genetic Sampling for tree species that are difficult to reliably identify in the field.

General Information

1.0 Scope of Work

Goal: To improve the accuracy of tree species data in FIA for whitebark (Pinus albicaulis) and limber (Pinus flexilis) pine tree species, which havefew (if any) field identifiable characteristics, particularly during the early life stages.

Objectives: This will be accomplished by sending genetic tree tissue samples to labs which they will use to:

1. perform genetic testing to determine the species of the tree tissue samples.

2. assess lab data quality, by testing a small (5% or less) portion of samples at two labs.

2.0 Background

FIA’s nationally standardized Measurement Quality Objective (MQO) for tree species states that FIA crews will correctly identify tree species 95% of the time. This is not possible for some species where the key characteristics needed to identify the species are ephemeral. This is particularly true for whitebark and limber pine. For these two similar species, genetic sampling can help us to meet our species accuracy targets in areas where the two species overlap.

The similarity of whitebark (listed as threatened in December 2022 under the Endangered Species Act) and limber pine’

(also in decline, but not yet listed) needles, bark, and overall morphology make it very difficult to distinguish the two species when cones are not present in the field. Cones are often not present, because they are a highly sought-after food source for animals, and whitebarks do not produce cones during the first 80 years of their lives.

RMRS- conducted a pilot study to perform genetic analysis of 371 whitebark and limber pine tree needle samples to determine their species. Results indicated that 83% of the field identifications were correct in areas of range overlap for whitebark and limber pine (89% for trees and 78% for seedlings). Field-identified samples were correct 60% of the time for limber pine. All these rates of correct field identification of tree species fall well below our nationally standardized MQO for species identification of correct tree species identification 95% of the time.

The process is as follows: RMRS-FIA sends tree tissue samples collected on FIA plots/trees to lab(s) capable of doing plant genetic testing. The lab(s) perform the tests and send the results back to RMRS- . A portion of the samples are sent to two labs, to retain a measure of repeatability and precision in lab methods.

Contractor Requirements

3.0 Technical Requirements / Tasks

Overall description: The lab(s) will perform genetic testing of tree needle samples and provide information regarding their level of certainty in the accuracy for each sample.

Methods to be used to identify whitebark pine and limber pine from provided needle samples (from Alongi et.al. 2019):

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 μl 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 to

The results expected back from the lab(s) are:

• The lab identification of species found through genetic testing for the samples in each envelope, delivered in an excel file via email. That excel file will have the following fields:

o CN: This will be the field provided to the contractor by RMRS- / This control number (or CN) identifies the sample envelope with a number that can be traced back to a particular tree in the FIA dataset.

o psbA-trnH_PsiI_pattern: This field will be used by the lab to identify the number of bands used at the psbA-trnH Psil loci to determine the species.

o Date 1: will be used to indicate when results were recorded for the psbA-trnH_PsiI_pattern.

o matK_BsmAI_pattern: This field will be used by the lab to identify the number of bands used at the matK BsmAl loci to determine the species.

o Date 2: will be used to indicate when results were recorded for the matK_BsmAI_pattern.

o DNA: this column will be used to indicate which DNA extraction kit was used (Qiagen or Sigma Aldrich) o Genetic_Identification: This field will have the species that the lab confirmed. It will be populated with

“albicaulis”, flexilis” or “unknown” o Certainty: this field indicates if there were irregularities in the test or other reasons to question the results. It will be populated with “low” or “high”. Genetic identifications with “low” certainty require a note in the “Lab_comment” field.

o Internal_QC: This field will be used to record if a sample is processed twice by the same lab as part of an internal quality assurance protocol o Lab_comment: The lab may use this field to record any helpful notes or anomalies.

4.0 Government Furnished

The Government (i.e., RMRS- ) will supply:

Samples:

• RMRS- will provide suspected whitebark and limber pine needle samples. Samples will contain at least one needle per tree, but the preferred sample will be one fascicle per tree. Although a single needle is generally sufficient for DNA extraction, the extra needles provide backup tissue.

• RMRS- will place each needle sample in a small sample envelope, at least 2”x 3”, folded and unsealed.

• Up to 12 coin envelopes will then be placed in a plastic or paper bag ¼ to ½ cup of silica gel may be used to ensure the samples dry, particularly if using a plastic bag. This can be left to the vendor’s preference.

• Each coin envelope will be marked with a unique set of numbers and letters, called a CN or control number, which will act as a unique identifier for that needle sample.

• RMRS- will repeat the process of bundling and bagging 12 needle samples until at least 92 samples are collected per batch. Batches will be sent in with at least 92 samples per batch. Batches larger than 92 samples will be sent in multiples of 8 or 12, to help keep lab efficiency up and costs down.

• The prepared sample bags will then be mailed to the lab/vendor at the government’s expense.

Tracking spreadsheet:

• RMRS will send a tracking spreadsheet via email with each batch of tree tissue samples in envelopes labeled with an identifying control number (aka, “CN”, or unique index number associated with each individual needle sample)

• This spreadsheet will also include any information regarding the sample that may help the lab (e.g., “only one fascicle in envelope”)

• The FIA field crew tree species identification will not be shared with the labs prior to genetic testing to preventbias.

5.0 Deliverables / Schedule

Tree tissue genetic sampling and Associated Deliverables:

Deliverables:

1. The final product from the lab will be an excel spreadsheet table, delivered via email to and/or any email box specified by the government, with the following columns:

CN psbA-trnH

_PsiI_pattern Date1 matK_BsmAI

_pattern Date2 DNA Genetic

_Identification Internal_QC Certainty Lab_comment

1 1 band 6-Jul-

2 bands 14- Jun-21

Qiagen albicaulis N high 1 band faint 5 July 2021;

blank on BsmAI 14 July

2 3 bands 11- Jun-21

1 band 7-Jun-

Qiagen flexilis Y high needles not fascicled

2. Progress report: A simple emailed progress report will be required in the following two situations:

a. if lab results have taken more than three months to produce genetic testing results, or

b. if it is not possible to return all lab genetic testing results by January 15st of any year. (see timeline, below)

The progress report shall cover all work already completed along with the work yet to be accomplished. This report shall also identify any problems that arose and provide a statement explaining how the problem can be/was resolved. This report shall also identify any problems that have arisen but have not been completely resolved and provide an explanation.

3. Invoice: Upon delivery of the genetic sample information for each sample, the lab(s) will provide an invoice for the number of samples processed according to the per-unit rate as specified in the agreement.

• Each invoice must include, at a minimum:

• The lab’s name, address, and telephone number

• U.S. Forest Service contract number

• Address for RMRS-

• Invoice date

• Performance dates of the work completed (start & end)

• Total invoice amount for the billing period

• Cumulative amount of U.S. Forest Service payments to date for the USFS Fiscal Year (October 1st through September 30th).

• Statement that the invoice is a request for payment.

• Invoice Number, if applicable

Timeline:

• The standard amount of time expected for labs to conduct needle sample genetic testing upon receiving the genetic tissue samples is two months.

• There is flexibility in this two-month expectation, provided that the labs understand FIA’s need to get results back by January 31st of every calendar year.

• RMRS- will not expect a turnaround time of less than two months from the date the samples arrive at the lab. For example: RMRS- may not expect that needle samples sent in on December 15th be guaranteed to be delivered by January 15th.

6.0 Travel

No travel is expected for this project.

7.0 Contractor’s Key Personnel

Lab manager: Must be capable of performing genetic testing of plant materials. At a minimum, they need to be able to accurately perform and troubleshoot where necessary, the genetic testing methods described above. Additional lab technicians may be helpful, but their presence is dependent upon a proficient lab manager.

8.0 Security Requirements

No security requirements or clearance needed. The lab will neither need, nor have, any direct access to FIA plot data or coordinates. They will only receive the simple spreadsheet with the “CN” listed in one column for each tree tissue sample, which they will return to FIA with added columns about the tree species identification and process.

9.0 Data Rights

RMRS- retains exclusive rights to the data provided by the lab. The lab has no rights to publish results based on this genetic sampling of data from FIA plots/trees without express permission from RMRS- , in writing. This is a fee-for-service endeavor, where the lab is paid to provide a service and retains no rights to the data (aka, the service provided).

10.0 Section 508 – Electronic and Information Technology Standards

Basic minimum 508 compliance for an excel spreadsheet is required. This should be no additional work.

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