Progress 01/01/14 to 12/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:This problem/change was reported in the prior progress report. In Years 1 and 2, the Thorgaard lab had difficulty producing doubled haploid trout for this project and generated only 80 individuals, which is not a sufficient sample size for the project. Doubled haploid trout are produced by irradiating eggs from a non-homozygous domesticated female to destroy the maternal DNA, fertilizing the eggs with sperm from a WRxAR male, and stopping the first cell division to restore diploidy. This process has high mortality but general produces sufficient numbers of doubled haploid offspring. Because of this, we have proceeded more cautiously with the genome-wide genotyping, which is a major portion of the materials and supplies budget. Near the end Year 2, we switched to an alternative strategy to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. A family of these fish was successfully produced and arrived at UMass Dartmouth in January 2016, and the corresponding growth study was completed in August 2016. We have extracted DNA from these samples, and they have been sequenced for RAD genotyping. Data analysis is currently underway. What opportunities for training and professional development has the project provided?The grant provided support in the form of a research assistantship for Dr. Soniya Balli (former PhD. student) who worked on the project as part of her PhD research. As a result, Dr. Balli received training in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. She performed the experimental rearing procedures on the 2014 experimental trout population and refined techniques for the genetic analysis. Dr. Balli graduated in September 2016. Mr. Ryan Higgins (Biology Masters candidate) has taken over where Dr. Balli left off. Over the course of the project, Mr. Higgins has been trained in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. He performed the experimental rearing procedures on the 2016 experimental trout population, extracted DNA, and processed all samples for RAD sequencing. Mr. Joseph McCabe (Biology undergraduate) helped care for the trout during the growth experiment and is assisting with genotyping using microsatellites. Mr. McCabe has gained experience with fish care, molecular genetic laboratory techniques, experimental troubleshooting, and data analysis. He is also being mentored regarding careers in genetics and aquaculture. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Nontechnical project summary Fish experience a variety of stressors in aquaculture, including overcrowding, poor water quality, and handling by humans. These stressors are often long-term, can negatively impact growth, health, and reproduction, and therefore reduce aquaculture production. Fish that are genetically resistant to stress are expected to grow more rapidly in captivity, but there has been little direct evidence to support this prediction. Our project will test the hypothesis that there is a genetic link between resistance to stress and growth rate in rainbow trout, an important aquaculture species. We predict that this link will be easiest to detect when fish are reared under more stressful conditions. Our objectives are to (1) measure growth rates of captive rainbow trout that experience increased stress in the form of daily handling, and (2) identify locations in the genome associated with increased growth under stress. We predict that genes in these regions will have greater influence on growth when fish are raised with increased stress and may not be detectable under normal rearing conditions. The ultimate goals of this research are to identify genes associated with improved growth under aquaculture conditions, and to use this information to improve aquaculture production. Nontechnical Impact Statement: Fish experience a variety of stressors in aquaculture, including overcrowding, poor water quality, and handling by humans. These stressors are often long-term, can negatively impact growth, health, and reproduction, and therefore reduce aquaculture production. Fish that are genetically resistant to stress are expected to grow more rapidly in captivity, but there has been little direct evidence to support this prediction. Our project will test the hypothesis that there is a genetic link between resistance to stress and growth rate in rainbow trout, an important aquaculture species. We predict that this link will be easiest to detect when fish are reared under more stressful conditions. 1) Major activities completed / experiments conducted In Year 1 (2014), we completed a preliminary growth study with 80 doubled haploid (completely homozygous) rainbow trout from the laboratory of Dr. Gary Thorgaard (subcontractor, Washington State University). For Year 2 (2015), Dr. Thorgaard's laboratory worked to produce additional doubled haploids so that we will have a sufficient sample size and power for the QTL analysis. Unfortunately, there was poor survival and no success in obtaining additional doubled haploid trout for the experiment. Instead, we are proceeding with our alternative plan to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. Eggs were fertilized using this approach in November 2015, and the resulting juveniles arrived at UMass Dartmouth in January 2016. In Year 3 (2016), we conducted the growth trial on the outbred x WRxAR family using an experimental design similar to our Year 1 study with the doubled haploids. 320 fish were divided among eight tanks in a recirculating system (40 fish per tank). Half of the tanks were subjected to the daily stressor while the other half were no-stress controls. The growth trial was terminated in August 2016, and fish were euthanized for genetic analysis. Over the past months, DNA was extracted from all 380 individuals (plus parents) from both the Year 1 and Year 3 growth experiments. In Year 4 (2017), we prepared sequencing libraries for genotyping SNPs in 322 individuals and the two parental strains. The libraries were sequenced at TUCF Genomics Core Facility (Tufts University, Boston, MA) on an Illumina HiSeq 2500 high throughput sequencer. 2) Data collected In Year 1, we collected standard length, body mass, and genetic samples for ~80 doubled haploid individuals. In Year 3, we collected standard length, body mass, and genetic samples for ~320 offspring of the outbred x WRxAR cross. In Year 4, we acquired between 2.4 billion sequence reads for 322 individuals and the two parental lines. These data are currently being processed for SNP identification and genome-wide genotyping. 3) Summary statistics and discussion of results Data analysis is underway but will be completed in 2018. QTL results will not be available until we have completed processing of the genome-wide SNP data and performed the QTL analyses. 4) Key outcomes or other accomplishments realized. Data analysis is underway but will be completed in 2018.
Publications
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:This problem/change was reported in the Year 2 progress report. In Years 1 and 2, the Thorgaard lab had difficulty producing doubled haploid trout for this project and generated only 80 individuals, which is not a sufficient sample size for the project. Doubled haploid trout are produced by irradiating eggs from a non-homozygous domesticated female to destroy the maternal DNA, fertilizing the eggs with sperm from a WRxAR male, and stopping the first cell division to restore diploidy. This process has high mortality but general produces sufficient numbers of doubled haploid offspring. Because of this, we have proceeded more cautiously with the genome-wide genotyping, which is a major portion of the materials and supplies budget. Near the end Year 2, we switched to an alternative strategy to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. A family of these fish was successfully produced and arrived at UMass Dartmouth in January 2016, and the corresponding growth study was completed in August 2016. We have extracted DNA from these samples and processed them for RAD sequencing. What opportunities for training and professional development has the project provided?The grant provided support in the form of a research assistantship for Dr. Soniya Balli (former PhD. student) who worked on the project as part of her PhD research. As a result, Dr. Balli received training in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. She performed the experimental rearing procedures on the 2014 experimental trout population and refined techniques for the genetic analysis. Dr. Balli graduated in September 2016. Mr. Ryan Higgins (Biology Masters candidate) has taken over where Dr. Balli left off. Over the course of the project, Mr. Higgins has been trained in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. He performed the experimental rearing procedures on the 2016 experimental trout population, extracted DNA, and processed all samples for RAD sequencing. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Near the end Year 2, we switched to an alternative strategy to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. A family of these fish was successfully produced and arrived at UMass Dartmouth in January 2016 and the corresponding growth study was completed in August 2016 on 320 individuals. We have extracted DNA from these samples, and processed them for RAD sequencing using high-throughput Illumina sequencing. Preparation of these samples took longer than expected so we have requested a second no-cost extension to complete the RAD sequencing and QTL analysis. Our planned timeline is as follows: December 2016-January 2017: RAD sequence data will be generated using high-throughput Illumina sequencing at the Tufts University Genomics Core facility. February-March 2017: The RAD sequence data will be processed, genotyping each individual at hundreds of SNPs. The locations of these SNPs will be determined using linkage mapping and comparisons to the draft rainbow trout genome. April-June 2017: Final quantitative trait locus (QTL) analysis will be performed to identify regions of the genome associated with variation in growth rate under stressful conditions. Final results will be prepared for publication.
Impacts
What was accomplished under these goals?
Nontechnical Impact Statement: Fish experience a variety of stressors in aquaculture, including overcrowding, poor water quality, and handling by humans. These stressors are often long-term, can negatively impact growth, health, and reproduction, and therefore reduce aquaculture production. Fish that are genetically resistant to stress are expected to grow more rapidly in captivity, but there has been little direct evidence to support this prediction. Our project will test the hypothesis that there is a genetic link between resistance to stress and growth rate in rainbow trout, an important aquaculture species. We predict that this link will be easiest to detect when fish are reared under more stressful conditions. 1) Major activities completed / experiments conducted In Year 1 (2014), we completed a preliminary growth study with 80 doubled haploid (completely homozygous) rainbow trout from the laboratory of Dr. Gary Thorgaard (subcontractor, Washington State University). For Year 2 (2015), Dr. Thorgaard's laboratory worked to produce additional doubled haploids so that we will have a sufficient sample size and power for the QTL analysis. Unfortunately, there was poor survival and no success in obtaining additional doubled haploid trout for the experiment. Instead, we are proceeding with our alternative plan to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. Eggs were fertilized using this approach in November 2015, and the resulting juveniles arrived at UMass Dartmouth in January 2016. We requested and received a no-cost extension for an additional year to complete the growth study and genome-wide genotyping of these fish. In Year 3 (2016), we conducted the growth trial on the outbred x WRxAR family using an experimental design similar to our Year 1 study with the doubled haploids. 320 fish were divided among eight tanks in a recirculating system (40 fish per tank). Half of the tanks were subjected to the daily stressor while the other half were no-stress controls. The growth trial was terminated in August 2016, and fish were euthanized for genetic analysis. Over the past months, DNA was extracted from all 380 individuals (plus parents) from both the Year 1 and Year 3 growth experiments, and we have completed processing the samples for RAD sequencing using high-throughput Illumina sequencing. 2) Data collected In Year 1, we collected standard length, body mass, and genetic samples for ~80 doubled haploid individuals. In Year 3, we collected standard length, body mass, and genetic samples for ~320 offspring of the outbred x WRxAR cross. 3) Summary statistics and discussion of results The project is still underway. QTL results will not be available until we have completed the genome-wide SNP genotyping and performed the QTL analyses. 4) Key outcomes or other accomplishments realized. The project is still underway.
Publications
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Progress 01/01/15 to 12/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:In Years 1 and 2, the Thorgaard lab had difficulty producing doubled haploid trout for this project and generated only 80 individuals, which is not a sufficient sample size for the project. Doubled haploid trout are produced by irradiating eggs from a non-homozygous domesticated female to destroy the maternal DNA, fertilizing the eggs with sperm from a WRxAR male, and stopping the first cell division to restore diploidy. This process has high mortality but general produces sufficient numbers of doubled haploid offspring. Because of this, we have proceeded more cautiously with the genome-wide genotyping, which is a major portion of the materials and supplies budget. Near the end Year 2, we switched to an alternative strategy to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. A family of these fish was successfully produced and arrived at UMass Dartmouth in January 2016. The growth study is currently underway. What opportunities for training and professional development has the project provided?The grant provided support in the form of a research assistantship for PhD candidate Soniya Balli who is working on the project as part of her PhD research. As a result, Ms. Balli has received training in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. She performed the experimental rearing procedures on the 2014 experimental trout population and is currently refining techniques for the genetic analysis. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Near the end Year 2, we switched to an alternative strategy to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. A family of these fish was successfully produced by Dr. Thorgaard's laboratoryand arrived at UMass Dartmouth in January 2016. The growth study is currently underway. Our revised timeline is as follows: March 2016: Commence the 6-month growth study. Forty fish will be randomly assigned to each of eight tanks. Half of the tanks will receive a daily stressor in the form of chasing with a net for 5 mins, while the remaining four tanks will be controls. Growth will be monitored monthly by measuring standard length and wet mass. A genetic sample (small fin clip) will also be taken. These samples will be genotyped using microsatellites so that samples can be matched up across time points and growth rates can be determined. PhD student Soniya Balli is graduating in May 2016 so master's student Ryan Higgins has taken over the experiment. June-August 2016: Mid-experiment fin clip samples will be analyzed using RAD sequencing. Mid-experiment growth rate data will be analyzed using QTL analysis. September-December 2016: The growth study will be completed. Finally growth measurements will be taken, and fish will be euthanized for final genetic analysis. The final QTL analysis will be performed on the complete growth rate data set. Final results will be written up for publication
Impacts
What was accomplished under these goals?
Nontechnical project summary Fish experience a variety of stressors in aquaculture, including overcrowding, poor water quality, and handling by humans. These stressors are often long-term, can negatively impact growth, health, and reproduction, and therefore reduce aquaculture production. Fish that are genetically resistant to stress are expected to grow more rapidly in captivity, but there has been little direct evidence to support this prediction. Our project will test the hypothesis that there is a genetic link between resistance to stress and growth rate in rainbow trout, an important aquaculture species. We predict that this link will be easiest to detect when fish are reared under more stressful conditions. Our objectives are to (1) measure growth rates of captive rainbow trout that experience increased stress in the form of daily handling, and (2) identify locations in the genome associated with increased growth under stress. We predict that genes in these regions will have greater influence on growth when fish are raised with increased stress and may not be detectable under normal rearing conditions. The ultimate goals of this research are to identify genes associated with improved growth under aquaculture conditions, and to use this information to improve aquaculture production. Nontechnical Impact Statement: Fish experience a variety of stressors in aquaculture, including overcrowding, poor water quality, and handling by humans. These stressors are often long-term, can negatively impact growth, health, and reproduction, and therefore reduce aquaculture production. Fish that are genetically resistant to stress are expected to grow more rapidly in captivity, but there has been little direct evidence to support this prediction. Our project will test the hypothesis that there is a genetic link between resistance to stress and growth rate in rainbow trout, an important aquaculture species. We predict that this link will be easiest to detect when fish are reared under more stressful conditions. 1) Major activities completed / experiments conducted In Year 1 (2014), we completed a preliminary growth study with 80 doubled haploid (completely homozygous) rainbow trout from the laboratory of Dr. Gary Thorgaard (subcontractor, Washington State University). For Year 2, Dr. Thorgaard's laboratory worked to produce additional doubled haploids so that we will have a sufficient sample size and power for the QTL analysis. Unfortunately, there was poor survival and no success in obtaining additional doubled haploid trout for the experiment. Instead, we are proceeding with our alternative plan to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. A mapping family was produced using this approach in November 2015 and arrived at UMass Dartmouth in January 2016. We requested and received a no-cost extension for an additional year to complete the growth study and genome-wide genotyping of these fish. 2) Data collected In Year 1 we collected standard length, body mass, and genetic samples for ~80 doubled haploid individuals. A growth study is currently underway for an additional 320 fish using the alternative strategy. In Year 2, PhD student Soniya Balli optimized sample preparation methods for RAD sequencing and genotyped the 80 fish from Year 1 using microsatellites. 3) Summary statistics and discussion of results The project is still underway. QTL results will not be available until we have completed the genome-wide SNP genotyping and performed the QTL analyses. In addition, a larger sample size is needed; this is being addressed by repeating the experiment in Year 3 (see below). 4) Key outcomes or other accomplishments realized. The project is still underway.
Publications
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Progress 01/01/14 to 12/31/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:In Year 1, the Thorgaard lab had difficulty producing doubled haploid trout for this project and generated only 80individuals, which is not a sufficient sample size for the project. Doubled haploid trout are produced by irradiating eggs from a non-homozygous domesticated female to destroy the maternal DNA, fertilizing the eggs with sperm from a WRxAR male, and stopping the first cell division to restore diploidy. This process has high mortality but general produces sufficient numbers of doubled haploid offspring. Paul Wheeler, manager of the Thorgaard aquaculture facility, believes that the poor success in Year 1 was mainly caused by poor egg quality. Because of this, we have proceeded more cautiously with the genome-wide genotyping, which is a major portion of the materials and supplies budget. For Year 2, Dr. Thorgaard's group is currently working to produce additional doubled haploids so that we will have a sufficient sample size and power for the QTL analysis. The Thorgaard lab believes that they have addressed the issues from Year 1 and will have improved double haploid production in Year 2. If these issues continue, our alternative plan is to fertilize untreated eggs from a domesticated female to produce offspring that include genetic contributions from both the WRxAR male and the domesticated female. This will complicate genotyping somewhat, as we will need to track the inheritance of additional alleles, but this is commonly done in QTL mapping experiments when it is not possible to produce doubled haploids. Likewise QTL analysis will be complicated by these multiple alleles and by dominance effects, but again this is common in other species. Because of this delay, we plan to request a no-cost extension to complete genome-wide genotyping of fish from Year 2. What opportunities for training and professional development has the project provided?The grant provided support in the form of a research assistantship for PhD candidate Soniya Balli who is working on the project as part of her PhD research. As a result, Ms. Balli has received training in trout aquaculture, experimental design and analysis, and advanced genetic analysis techniques. She performed the experimental rearing procedures on the 2014 experimental trout population and is currently refining techniques for the genetic analysis. Ms. Balli also attended the 2015 International Plant and Animal Genome Meeting, where we discussed project goals and strategies with colleagues in aquaculture genomics from the USDA and NOAA. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In summer 2015, the Thorgaard lab at Washington State University is producing additional experimental trout for this experiment. Once these fish have been produced, we will continue with the QTL analysis as planned.
Impacts
What was accomplished under these goals?
1) Major activities completed / experiments conducted In Summer 2014, we obtained 80 doubled haploid rainbow trout from the laboratory of Dr. Gary Thorgaard (subcontractor, Washington State University). These doubled haploid trout were descended from a cross between a wild, stress-sensitive clonal line (Whale Rock female) and domesticated, stress-tolerant line (Arlee, male). The fish were shipped to UMass Dartmouth and divided evenly between two 40 gal tanks in a custom recirculating system. One tank was raised under normal aquaculture conditions while the other received a stress treatment consisting of being chased around the tank for 2 minutes at a random time during each day. Food consumption and appetite was monitored daily for each tank. At 4-week intervals, individual fish were anesthetized and measured for standard length and body mass, and a fin clip was taken. Data points from these sample points will be matched up using microsatellite genotypes. After monitoring growth for 5 months, the fish were measured for the last time and euthanized (December 2014). DNA has been extracted from these fish and is currently being analyzed using whole genome SNP genotyping approaches. 2) Data collected We currently have standard length and body mass data for all ~80 individuals, and are currently working to match up data from sampling time points using microsatellite genotypes so that we can analyze growth rate data. Genetic samples were collected from five time points, and whole frozen fish are available from the last time point. These samples are currently be analyzed using microsatellites and whole genome SNP genotyping methods. 3) Summary statistics and discussion of results The project is still underway. QTL results will not be available until we have completed the genome-wide SNP genotyping and performed the QTL analyses. In addition, a larger sample size is needed; this is being addressed by repeating the experiment in Year 2 (see below). 4) Key outcomes or other accomplishments realized. The project is still underway.
Publications
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