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Project Work Plan

Department of Interior USGS GE PES
Fiscal Year 2014 Study Work Plan

TITLE: Genomic sequencing of African Jewelfish
INVESTIGATOR: Maggie Hunter Co-Investigator: Pam Schofield

STATEMENT OF PROBLEM: Eradicating already-established and dispersed nonindigenous species is a significant challenge. A new generation of biologically-based eradication strategies focuses on the introduction of genetically-modified individuals into a wild population to reduce reproductive capacity. These strategies have the potential to effectively control and eradicate widespread nonindigenous species with minimal effort in the field while also posing little to no risk to native species or habitats. One example is termed The Trojan Y Chromosome strategy, and it is being developed at the SESC as a means to eradicate African jewelfish (Hemichromis letourneuxi) from Everglades National Park. Efforts are currently underway to generate Trojan African jewelfish broodstock to first test this strategy in mesocosm studies before it is attempted in the Park.

Broodstock construction involves the use of hormone-induced sex reversal of fish followed by selective breeding in order to generate fish with two Y chromosomes. Because breeding YY fish is a time- and labor- intensive activity, it is essential to define sex-specific chromosome markers from African jewelfish to effectively track the Y chromosome through genetic crosses. With a sex-specific marker for the Y chromosome, the chromosomal genotype of a fish can be defined solely based upon its DNA (and not by progeny ratios resulting from test crosses). Finding a sex-specific marker for the African jewelfish is thus essential for development of YY broodstock fish and the ultimate testing of the eradication strategy.

Current efforts to define a sex-specific marker for African jewelfish involve RAPD PCR screening of pooled male-specific and female-specific DNA using short random oligonucleotide primers. Since the primers tested are random DNA sequences, it is a matter of chance that one will be identified that is sex-specific. Only rarely is it expected that a random primer will amplify a DNA sequence close to a sex-determination gene and produce a PCR product that is sex-specific. This strategy was used successfully to isolate a sex-specific marker for the Y chromosome of common carp Cyprinus carpio. For common carp, it was necessary to screen 240 primers before a male-specific DNA marker was defined. It was necessary to screen a large number of primers because most random primers tested amplify regions of the genome that are not closely linked to a sex-determination gene. Currently, approximately 200 primers have been tested in an effort to define a sex-specific DNA marker for African jewelfish and no sex-specific markers have been identified to date. The high level of uncertainty regarding the number of additional primers that will have to be tested prior to identifying the sex-specific marker is now causing difficulties in project planning, budget allocations, timing and execution.

If a large proportion of the genomic DNA sequence of male and female African Jewelfish was available, it could allow a more efficient screen for sex-linked DNA markers, reducing the number of primers to be screened and shortening the time required to identify a sex-specific marker. By comparing the male genomic sequence to the female genomic sequence using DNA analysis software, all the sequences that are common to both sexes could be eliminated as a source of DNA sequences for the design of new PCR primers. Instead of generating random primer sequences, primers could be designed from DNA sequences that are found only in the genomic DNA sequences of males. It would thus be advantageous to obtain sex-specific genomic DNA sequences for African jewelfish in order to enhance the screening efforts for sex-specific DNA markers and increase the likelihood of success.

OBJECTIVES: We plan to develop Restriction site Associated DNA (RAD) markers using Next generation sequencing to conduct high-throughput and efficient screening of sex-linked DNA markers.

METHODOLOGY: The technology of genomic DNA sequencing has greatly advanced. Large number of genomic loci can be sequenced rapidly at a relatively low cost. Next generation sequencing using the Illumina MiSeq will rapidly produce male and female sex-specific genomic DNA sequences for African jewelfish. This produces thousands of sequences at a coverage level sufficient to provide the resolution of sex-specific sequences, if present, and distinguishing those differences from individual DNA sequence variation. If possible, sequencing will be conducted in collaboration with a USGS genetics laboratory.

Develop back crosses of genetically similar brood stocks. February 2014-August 2014
Develop DNA libraries of genetically similar brood stocks. July-August 2014
Next generation sequencing. August 2014
Sequence data analysis. August 2014-December 2014
Report and publication (if applicable) preparation. January 2015-June 2015

KEYWORDS: Invasive species, molecular tools, Next generation sequencing, invasive species, control and irradiation, Everglades National Park

Written report. June 2015
Scientific publication (if applicable). June 2015
Genomic data/metadata. December 2015

RELEVANCE AND BENEFITS: Dozens of non-native fish species have established throughout south Florida (including Everglades National Park, Big Cypress National Preserve, Biscayne National Park and various state and private lands). Thus far, research on these species has focused on documenting their distributions (e.g., Fuller et al. 1999; Adams & Wolfe 2007; Shafland et al. 2008; USGS 2009), natural history (Nico & Muench 2004; Bergmann & Motta 2005) and physiological tolerances (Schofield et al. 2007; Langston et al. 2009; Schofield & Nico 2009; Schofield et al. 2009 a&b). Research is beginning to emerge on interactions of native species with non-natives (e.g., Loftus et al. 2006; Brooks & Jordan 2009) although it is only in the early stages. Research on control of non-native fishes in South Florida is also lacking, although it is potentially the most important and useful to Federal and State natural resource managers.

At present, the only management techniques available to control non-native fishes are physical removal, dewatering or ichthyocides (e.g., Schofield & Nico 2007). Unfortunately, all of these methods negatively impact native fauna as well as the targeted non-native fishes and require a great deal of effort (and therefore, funding). Herein, we propose a research program focused on applying a genetic technique common in aquaculture to control of non-native fishes.

COMMUNICATION PLAN, TECHNOLOGY AND INFORMATION TRANSFER: The information and results of this project will be openly available in the form of one or more electronic internet publications.

COOPERATORS AND PARTNERS: The USGS's Greater Everglades Priority Ecosystems Science program is funding this project.

Dr. Hunter's qualifications are listed on her USGS Professional Page:

FACILITIES, EQUIPMENT, AND STUDY AREAS: Office and laboratory facilities and equipment, and any needed field gear or supplies, will be provided by the Southeast Ecological Science Center.

JOB HAZARD ANALYSES (JHA): Office work, Working in the Laboratory, Formeldehyde Use

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Last updated: 17 April, 2015 @ 09:54 AM (KP)