Louisiana Marsh Dieback Genetic Marker Data for Amplified Fragment Length Polymorphism Data (Brown Marsh Fingerprint Data)

Louisiana State University, Department of Agronomy and Environmental Management
Publication_Date: 2005
Louisiana Marsh Dieback Genetic Marker Data for Amplified Fragment Length Polymorphism Data (Brown Marsh Fingerprint Data)
Geospatial_Data_Presentation_Form: Digital tabular data
Barataria-Terrebonne National Estuary Program Scientific-Technical Committee
Publication_Date: 20010207
Requests for submission of Scope of Services for Marsh Dieback and Nutria Control Emergency Response
Geospatial_Data_Presentation_Form: document
Publication_Place: Thibodaux, LA
Barataria-Terrebonne National Estuary Program Scientific-Technical Committee
Online_Linkage: <http://www.brownmarsh.net/reports/brown_marsh_rfp.pdf>
This data set consists of the data for 693 AFLP markers generated by a technique knows as amplified fragment length polymorphism on 44 brown marsh accessions. The data were used to measure genetic diversity among the brown marsh accessions.
The bands in different brown marsh accession were scored and then these were analyzed using software known as NTSYS-PC to measure the genetic diversity among them. Those accessions with the presence of a particular band or marker can be judged similar in their genetic make up.
Beginning_Date: 2001
Ending_Date: 2003
Currentness_Reference: ground condition
Progress: Complete
Maintenance_and_Update_Frequency: None planned
Description_of_Geographic_Extent: Coastal Louisiana
West_Bounding_Coordinate: -93.910681
East_Bounding_Coordinate: -88.777417
North_Bounding_Coordinate: 30.509791
South_Bounding_Coordinate: 28.892107
Theme_Keyword_Thesaurus: None
Theme_Keyword: emergent vegetation
Theme_Keyword: brown marsh
Theme_Keyword: dead zone
Theme_Keyword: transition zone
Theme_Keyword: dieback
Theme_Keyword: genetic diversity
Theme_Keyword: accession
Theme_Keyword: saltmarsh
Theme_Keyword: amplified fragment length polymorphism
Theme_Keyword: AFLP
Place_Keyword_Thesaurus: None
Place_Keyword: Louisiana
Place_Keyword: Coastal Louisiana
Place_Keyword: LA
Place_Keyword: Southern Louisiana
Place_Keyword: Terrebonne Basin
Place_Keyword: Barataria Basin
Taxonomic_Keyword_Thesaurus: None
Taxonomic_Keywords: Spartina alterniflora
Taxonomic_Keywords: species
Taxon_Rank_Name: Kingdom
Taxon_Rank_Value: Plantae
Taxon_Rank_Name: Division
Taxon_Rank_Value: Magnoliophyta
Taxon_Rank_Name: Class
Taxon_Rank_Value: Liliopsida
Taxon_Rank_Name: Subclass
Taxon_Rank_Value: Commelinidae
Taxon_Rank_Name: Order
Taxon_Rank_Value: Cyperales
Taxon_Rank_Name: Family
Taxon_Rank_Value: Poaceae
Taxon_Rank_Name: Genus
Taxon_Rank_Value: Spartina
Taxon_Rank_Name: Species
Taxon_Rank_Value: alterniflora
Applicable_Common_Name: smooth cordgrass
Data generated by the Brown Marsh principal investigators will be available only to the project’s other principal investigators, following the established QA/QC review, for a period of two years. After two years from the finalization of the data, it shall be made available to the public. Please contact the principal investigator for exact date of availability. It is strongly recommended that these data be directly acquired from the Brown Marsh Database Information Management System server and not indirectly through other sources which may have changed the data in some way.
Acknowledgement of the principal investigator as a data source would be appreciated in products developed from these data, and such acknowledgement as is standard for citation and legal practices for data source is expected by users of these data. Users should be aware that comparison with other data sets for the same area from other time periods may be inaccurate due to inconsistencies resulting from changes in mapping conventions, methodologies, data collections and computer processes over time. The principal investigator and the distributor shall not be liable for improper or incorrect use of these data, based on the description of appropriate/inappropriate uses described in this metadata document. The principal investigator and distributor make no claims as to the data’s suitability for other purposes. These data are not legal documents and are not to be used as such.
Louisiana State University, Department of Agronomy and Environmental Management
Contact_Person: Prasanta K. Subudhi
Contact_Position: Assistant Professor
Address_Type: Physical
Address: 215 Sturgis Hall
City: Baton Rouge
State_or_Province: LA
Postal_Code: 70803
Country: USA
Contact_Voice_Telephone: 225 578-1303
Contact_Facsimile_Telephone: 225 578-1403
Contact_Electronic_Mail_Address: psubudhi@agcrt.lsu.edu
Native_Data_Set_Environment: Microsoft Excel 2000
Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau
Publication_Date: 199511
Title: AFLP: a new technique for DNA fingerprinting
Geospatial_Data_Presentation_Form: journal article
Publication_Place: Oxford, UK
Publisher: Oxford University Press
Series_Name: Nucleic Acids Research
Issue_Identification: Vol. 23, Issue 21 4407-4414
Analytical_Tool_Description: PTC-100 Thermocycler - thermal cycler
Online_Linkage: <http://www.mjr.com>
Tool_Access_Instructions: See website for information
Contact_Organization: Bio-Rad Laboratories, INC
Address_Type: Mailing
Address: 590 Lincoln Street
City: Waltham
State_or_Province: MA
Postal_Code: 02451
Country: USA
Contact_Voice_Telephone: 888 735-8437
Contact_Facsimile_Telephone: 617 923-8080

The accuracy of the attributes is determined by the accuracy of the DNA processing and analysis. Only good quality genomic DNA was used for analyses; quality of the DNA was determined by running a 1% agarose gel and an adjustment of the final concentration to 50ng/µl. The AFLP protocol (Vos, et al. 1995) was followed.
Sites were chosen according to the dieback zones, and distances of two sampling sites varied from 10 to 15 meters. All samples were labeled according to the zones where collected occur. Six additional sampling sites (outside of the dieback region in coastal Louisiana) were chosen for normal healthy marsh environment. All equipment were calibrated according to the manual or standard operating procedure.
Completeness_Report: There are no known omissions in the data.
Methodology_Type: Field/Lab
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: sites
Methodology_Keyword: dieback zones
Methodology_Keyword: samples
Methodology_Keyword: greenhouse
Methodology_Keyword: container
Methodology_Keyword: reestablished
Methodology_Keyword: ramets
Methodology_Keyword: S. alterniflora
Methodology_Keyword: young green tissue
Methodology_Keyword: labeled
Sample collection - From nineteen dieback sites across the Terrebonne and Barataria basins, surviving ramets of S. alterniflora were collected from the interior dead zone (where dieback impact was severe) and from transition zones on the periphery of the dead zones (where dieback impact was less severe). Samples were also collected from six normal and healthy marsh sites in coastal Louisiana (four in the south-west part of the state and two in close promixity of the dieback area.) Individual samples were labeled and brought back to the Golden Meadows Plant Materials Center in Galliano. The samples were reestablished in containers. From each of these 40 samples, young green leaf tissue (approximately 5-10gm) were collected and stored at -80 degrees C until DNA extraction.
Methodology_Type: Lab
Methodology_Keyword_Thesaurus: None
Methodology_Keyword: liquid nitrogen
Methodology_Keyword: microcentrifuge tube
Methodology_Keyword: agarose gel
Methodology_Keyword: concentration
Methodology_Keyword: genomic DNA
Methodology_Keyword: incubation
Methodology_Keyword: AFLP protocol
Methodology_Keyword: AFLP bands
Methodology_Keyword: thermocycler
Methodology_Keyword: intervals
DNA isolation and AFLP protocol - Leaf tissues from each individual were ground into a fine powder in liquid nitrogen using a mortar and pestle, and approximately 100mg of the powder were transferred to a microcentrifuge tube. Good quality genomic DNA was extracted using the GenElute Plant Genomic DNA kit. AFLP protocol was followed as described by Vos et al. (1995). Five hundred nanograms of genomic DNA of each S. alterniflora individual was digested with 5U of EcoRI (Invitrogen, Carlsbad, CA) and 5U of MseI (New England Biolabs, Inc., Beverly, MA) in a reaction volume of 30 µl at 370 C for 2 hours in a restriction ligation buffer (10mM Tris-HAc, pH 7.5, 10 mM MgAc2, 50 mM KAc, 5mM DTT, and 0.005% BSA), followed by incubation at 700 C for 15 minutes to inactivate the restriction enzymes. Twenty microliters of the digested DNA was then mixed with 10 µl of ligation mix containing 1 µl of EcoRI adapter (5 pMol), 1 µl MseI adapter (50 pMol), 1 U T4 DNA ligase, 6 µl of 5X ligation buffer with ATP, and incubated again at 200 C in a PTC-100 Thermocycler (MJ Research, Inc., Waltham, Mass.) for 3 hours. Ligated DNA products were diluted 10 times, and diluted ligated DNA was preamplified using EcoRI-Pre and MseI-Pre primers following 30 cycles (940 C 30 Sec, 560 C 60 Sec, 720 C 60 Sec) of amplification. The adapter and pre-amplification primers were synthesized according to Vos et al. (1995). The preamplified PCR products were run on a 1.5% agarose gel in which a low-molecular weight smear was visible, indicating the presence of abundant PCR products. Then the preamplified PCR products were diluted 20 times with sterile water for selective amplification. Selective amplification PCR was performed in 12-µl reaction volumes containing 25 ng each of EcoRI+3 and MseI+3 primers, 3 µl of template DNA, 0.3 mM of all four dNTP, 2.5 mM MgCl2, 0.25 U Taq polymerase (Promega Corporation, Madison, WI) and 1x PCR buffer supplied with the Taq polymerase. Twelve EcoRI+MseI primer combinations (Table 2) were used for selective amplification using the PTC-100 thermal cycler. The thermocycler was programmed as follows: cycle 1 for 30 seconds at 940 C, 1 minute at 650 C, 1 minute at 720 C; cycles 2-13: annealing temperature was reduced by 0.70 C in every successive cycle; cycles 14-36: annealing at 560 C; final extension for 5 minutes at 720 C and hold at 40 C. AFLP reaction products (12 µl) were added to an equal volume of loading dye (0.025% each of Xylene Cyanol and Bromophenol blue, deionised formamide, 10 mM EDTA). Denaturing of the samples was done for 4 minutes at 950 C immediately before loading and then placed on ice while loading into the gel. Three microliters of the AFLP reaction products were analyzed on 6% denaturing polyacrylamide gels and electrophoresed in 0.5x TBE at 500 C, 110 W for 2 hours. A Bio-Rad Sequi-Gen® (38 x 50 cm) unit was used to run the gels. Silver staining of the gels was done manually following the procedure of Fritz et al. (1999). After the gels were developed, a sheet of 3 mm Whatman white paper was placed under the developed gel to visualize the AFLP bands and then scanned directly using a scanner.
Process_Description: See methodology
Process_Date: 2000-2005

The tabular data for the brown marsh fingerprinting is organized according to the number of genotypes analyzed (44) and the number of markers generated (693). The corresponding markers and its genotype were assigned either "1", "2", or "999" to indicate the presence (1) or absence (2) of the AFLP band (999 indicates no data). Markers were assigned ascending numbers for identification purposed, while the genotypes were assigned an alpha-numerical identification tag. The alpha codes designate if the sample was collected from "D" (dead zone), "T" (transition zone), "AD" (adjacent to a dead zone), or "AT" (adjacent to a transition zone.) Other genotype identification tags were assigned for those samples outside the brown marsh area, those of normal healthy coastal Louisiana marshes. Tags 44, 49, 75, and 90 were from sites in south-west Louisiana and tags 10 and Vermiliion were taken from sites closer to the brown marsh area.
Entity_and_Attribute_Detail_Citation: Prasanta K. Subudhi, written comm. 5-27-05

Contact_Organization: U.S. Geological Survey, National Wetlands Research Center
Contact_Person: Scott Wilson
Contact_Position: Electrical Engineer
Address_Type: Mailing and Physical
Address: 700 Cajundome Blvd
City: Lafayette
State_or_Province: LA
Postal_Code: 70506
Country: USA
Contact_Voice_Telephone: 337 266-8664
Contact_Facsimile_Telephone: 337 266-8513
Contact_Electronic_Mail_Address: scott_wilson@usgs.gov
Although these data have been processed successfully on a computer system at the designated principal investigator’s facility, no warranty expressed or implied is made regarding the accuracy or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. This disclaimer applies both to individual use of the data and aggregate use with other data. It is strongly recommended that these data are directly acquired from the official Brown Marsh Database Information Management System server or from the principal investigator, and not indirectly through other sources which may have changed the data in some way. It is also strongly recommended that careful attention be given to the contents of the metadata file associated with these data. The Brown Marsh committee shall not be held liable for improper or incorrect use of the data described and/or contained herein. So, these data are provided as is and without any express or implied warranties. Also, use of trade names or commercial products in the metadata file is solely for the purpose of providing specific information, and does not imply recommendation or endorsement. Any downloading and use of these data signifies a user’s agreement to comprehension and compliance of the disclaimer. Insure all portions of metadata are read and clearly understood before using these data in order to protect both user and principal investigators interests.
Format_Name: Microsoft Excel
Format_Version_Number: 2000+
File_Decompression_Technique: none
Network_Resource_Name: <http://www.brownmarsh.net>
Fees: None, if available online
Custom_Order_Process: None

Metadata_Date: 20050531
Contact_Organization: U.S. Geological Survey/NWRC
Contact_Position: Metadata specialist
Address_Type: Mailing and Physical
Address: 700 Cajundome Blvd
City: Lafayette
State_or_Province: LA
Postal_Code: 70506
Country: USA
Contact_Voice_Telephone: 337 266-8500
Contact_Facsimile_Telephone: 337 266-8513
Metadata_Standard_Name: Content Standard for Digital Geospatial Metadata
Metadata_Standard_Version: FGDC-STD-001-1998
Biological Data Profile of Content Standard for Digital Geospatial Metadata
Online_Linkage: <http://www.fgdc.gov/standards/status/sub5_2.html>
Metadata_Access_Constraints: None
Acknowledgement of the National Wetlands Research Center as the metadata source would be appreciated. Please cite the original metadata when using portions to create another record for slightly altered data, such as reprojection.

Generated by mp version 2.7.33 on Fri Jun 03 08:54:26 2005