Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2015
Project End Date
Aug 31, 2018
Grant Year
Project Director
Guo, X.
Recipient Organization
Performing Department
Marine and Coastal Sciences
Non Technical Summary
Oyster farming is one of the most important aquaculture industries in the US. One of the most significant advances in oyster aquaculture is the development of triploid and tetraploid oysters. Triploids, because of their extra set of chromosomes, are sterile and possess several characteristics that are ideal for aquaculture. Triploid oysters grow fast and maintain high meat quality in summer. Sterility prevents interbreeding between cultured and wild populations making aquaculture more environmental friendly. Triploids are produced by crossing diploids and tetraploids, and the development of tetraploid oysters has led to rapid commercial production of triploids. Now triploid oysters produced from tetraploids have become an important part of oyster aquaculture in the US and around the world, accounting for 30 - 50% of aquaculture production. However, tetraploid genomes are unstable and performances of triploids are inconsistent. Some tetraploids can quickly degenerate into aneuploids, triploids and mosaics mostly in somatic tissue but may also in gonads producing aneuploid gametes. Aneuploidy, which negatively affects growth and survival, may be responsible for the inconsistent performance of triploid oysters. This study aims to improve tetraploid and triploid eastern oysters by investigating: 1) whether genome instability in tetraploids produces aneuploid gametes; 2) whether aneuploid gametes from tetraploids affect the performance of triploid progeny; and 3) whether genome stability of tetraploids is heritable and can be genetically improved. This project may identify superior tetraploids that produce best-performing triploids and contribute significantly to the sustainable development of oyster aquaculture in the US and beyond.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3723 - Oysters;

Field Of Science
1080 - Genetics; 1081 - Breeding;
Goals / Objectives
Triploid oysters have become an important part of the oyster aquaculture, although their field performance is variable probably due to genome instability of their tetraploid parent. The goal of this project is to improve genome stability of tetraploid oysters and the field performance of triploid oysters. We will do so by studying the reproductive genetics of tetraploid oysters and possibly identifying the best tetraploids that show stable chromosome inheritance and produce superior triploids. The specific objectives are to investigate:1) Whether somatic genome instability of tetraploids as measured by DNA content is correlated with the production of aneuploid gametes;2) Whether the performance of triploid progeny is affected by chromosome loss in tetraploid fathers or aneuploidy; and3) Whether genome stability of tetraploids is heritable and can be improved.
Project Methods
The goal of this project is to improve the stability of tetraploids and performance of triploids in support of oyster aquaculture. Our hypothesis is that genome instability in tetraploids observed in somatic tissue also exists to some extent in germline cells and results in chromosome loss in gametes and triploid progeny, which negatively affects the performance of triploids. We further hypothesize that genome stability of tetraploids is variable, inheritable and can be improved through selection. We will test these hypotheses by producing a set of triploid and tetraploid families using tetraploids with different levels of somatic reversion. We will determine somatic chromosome loss in tetraploids by flow cytometry and germline chromosome loss by direct chromosome counting of resultant embryos. We will study the relationship among somatic chromosome loss, germline chromosome loss, chromosome integrity of triploids, and the performance of triploid and tetraploid progeny. We will identify the most stable tetraploid families that produce the best performing triploids for the oyster industry.

Progress 09/01/15 to 08/31/18

Target Audience:Shellfish farmers, shellfish breeders, oyster researchers, biologists, geneticists, educators, aquaculturists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunity was provided to three graduate students, two postdoctoral researchers and six undergraduate students. How have the results been disseminated to communities of interest?Results of this project has been presented at national and local meetings. Improved tetraploid oysters have been used by hatcheries for commercialproduction of triploids for oyster farmers in NJ and Northeastern States. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Oyster farming is an important industry in the US and around the globe. It has been expanding along much of the Atlantic coast partly due to the use of triploid oysters. Triploid oysters grow significantly faster than normal diploids. Fast growth means that triploids reach market size earlier than diploids, which reduces production cost and losses to disease. Triploid oysters now account for about 40-50% of the aquaculture production in the US. Triploid oysters are produced by crossing diploids with tetraploids that contain four sets chromosomes. The performance of triploids may be influenced by the quality of tetraploids whose genome is not always stable. The goal of this study is to determine whether the performance of triploid oysters is affected by genome instability of tetraploids and can be improved by improving tetraploids. Results of this project show that tetraploid oysters have a profound effect on the performance of triploids. Triploid oysters produced from large tetraploids grow significantly faster than triploids produced from small or average-sized tetraploids. Larger tetraploids also produced larger tetraploids. Genome instability of tetraploids were limited to somatic cells. Tetraploids with chromosome loss in somatic cells did not show similar chromosome loss in gametes. Genotype of diploid parents also significantly affected the performance of triploids. These results indicate that genetics background of diploid and tetraploid parents is more important than somatic mosaicism or aneuploidy in determining the performance of triploids. This project produced our new lines of tetraploids using the best-performing triploids and tetraploids. These new lines have been transferred to the industry for commercial production of improved triploids. A new strategy for genetic improvement of tetraploids, systematic interploidy breeding, was also developed which may have wide applications in the genetic improvement of tetraploid and triploid shellfish. Objective 1. Somatic genome instability and aneuploid gametes Somatic genome of tetraploid oysters is unstable. Somatic cells of tetraploid oysters may lose chromosomes over time and become mosaics or revert to triploids or diploids. To determine whether somatic chromosome loss is related to germline aneuploidy, we identified three classes of tetraploids based on flow cytometry of somatic cells:"good" (3.8-4.0n), "low" (3.5-3.7n) and "mosaic" (3n/4n) tetraploids. Males of the three classes of tetraploids were crossed with the same diploid females. Chromosome number in 2-4 cell embryos was used to determine the level of aneuploidy in different classes of tetraploid males. No significant difference in the level of aneuploid sperm was observed among the three classes of tetraploids. This result suggests that the chromosome loss in tetraploids is mostly limited to somatic cells, and the germline cells are not significantly affected by chromosome loss. This finding means that genome instability in somatic cells is not a serious concern for the breeding of tetraploids because it is not directly correlated with chromosome loss in gametes. Objective 2. Effects of tetraploids on the performance of triploids To determine if different tetraploids affect the performance of triploids, triploids were produced from large, small and average-sized tetraploids and tetraploids showing somatic mosaicism. Five groups of triploids were produced using diploid females from two different stocks: the disease-resistant stock NEH and a stock from Maine. The five groups of triploids and two diploid controls were deployed in triplicate at Cape Shore, New Jersey, for field evaluation. At most sampling date, the size of triploid progeny is correlated with the size of their tetraploid father, i.e., the largest tetraploids produced the largest triploids, and verse visa. At two years of age, triploids produced with the largest tetraploids (top 10%) produced 12.3 g of meat, while triploids produced with the smallest tetraploids (bottom 10%) produced 9.9 g of meat, compared with 6.7 g of meat for diploid controls. Thus, the size of tetraploids has a significant influence on the size of triploids, suggesting the fast-growing tetraploids produce fast-growing triploids. The triploid group fathered by triploid-tetraploid mosaics contained higher levels of diploid-triploid mosaics. The finding that mosaic tetraploids tend to produce more mosaics in triploids is interesting, although somatic mosaicism does not appear to affect growth or germline cells. Further, all triploid groups produced with diploid females from the NEH showed significantly higher survival (63%) than triploids produced with the diploid females from the Maine stock (18%). For the two diploid controls, the NEH stock had higher survival (70%) than the Maine stock (21%). NEH is a disease-resistant stock and well adapted to the Delaware Bay environment, while the Maine stock is susceptible to diseases (MSX and Dermo) and stressors at the Delaware Bay site. Thus, it is not surprising that the Maine stock had lower survival than NEH. It is interesting that the difference in survival between the two diploid lines is completely transfer to their triploid progeny, suggesting that genotype rather than chromosome loss is more important for the performance of triploids. Objective 3.Genome stability and improvement of tetraploids To determine if tetraploids can be genetically improved, we produced tetraploids by using the largest tetraploids as parents. Results show that tetraploids produced with large tetraploids are significantly larger than that produced with small tetraploids. This finding suggests that the growth of tetraploids can be improved by selective breeding. At 6 months of age, tetraploids produced with largest tetraploids (top 10%) weighed 11.2 g, while tetraploids produced from smallest tetraploids (bottom 10-20%) weighed 5.3 - 8.4 g. We also produced tetraploids with the largest triploids. Although we did not produce tetraploids with the smallest triploids, the use of largest triploids and tetraploids appeared to have led to improvement of tetraploids over time. Tetraploids produced in 2016 were significantly larger than diploids and triploids at 6 months of age, while tetraploids produced in 2005 were smaller than diploids and triploids. Thus, a new strategy was developed for genetic improvement of tetraploids through systematic interploidy breeding (SIB). In SIB, tetraploids are produced from tetraploid x tetraploid mating with strong selection on tetraploids, as well as de novo from triploids and diploids with strong selections. The mated and de novo tetraploids were mated every three generations to combine selection on tetraploids, triploids and diploid. The SIB strategy brings not only selection from the triploid and diploid phases into tetraploids, but also new genetic material to maintain genetic diversity. A patent has been filed for the new bring method. This project generated four new lines of tetraploids through SIB. These new lines have been distributed to three hatcheries (one in New Jersey and two in Maine) for commercial production of tetraploids. These tetraploids lines are produced from Rutgers disease-resistant strains. They will provide valuable materials for further improvement of tetraploid eastern oysters. The new method may contribute to genetic improvement of tetraploids in other shellfish species.


  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Guo, X., C. Li and H. Wang. 2018. Diversity and evolution of living oysters. J. Shellfish Res., 37(4):755-771.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu, M. and X. Guo. 2017. A novel and stress adaptive alternative oxidase derived from alternative splicing of duplicated exon in oyster Crassostrea virginica. Scientific Reports, 7:10785.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Guo, X. 2018. Observations on tetraploid eastern oysters, Crassostrea virginica and implications in genetic improvement of triploids. Presented at 110th Annual Meeting of National Shellfisheries Association, March 18 - 22, 2018, Seattle, USA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Guo, X. 2019. Progress in genetic improvement of eastern oysters. Accepted for presentation at the Northeast Aquaculture Conference & Exposition and the Milford Aquaculture Seminar in Boston, Massachusetts, January 9-11, 2019.
  • Type: Other Status: Under Review Year Published: 2019 Citation: Yang, H., X. Guo and J. Scarpa. 2019. Oyster Tetraploid Induction and Establishment of Breeding Stocks for All-Triploid Seed Production. Gainesville: University of Florida Institute of Food and Agricultural Sciences, EDIS.

Progress 09/01/16 to 08/31/17

Target Audience:Shellfish farmers, hatchery managers, shellfish biologists and breeders, aquaculturists, geneticists, educators and students Changes/Problems:We requested a one-year non-cost extension to complete field evaluation to market size and to produce another generation of tetraploids in 2018. What opportunities for training and professional development has the project provided?The project provided training opportunities to twoundergraduate students, twograduate students, one postdoctoral researcher and 3 visiting scientists. How have the results been disseminated to communities of interest?Results of this project has been disseminated through presentation at conferences, talking to hatchery managers and farmers. Triploids produced from this project were given to four farmers for their evaluation. Tetraploids were provided to hatcheries for commercial production of triploids. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete field evaluation of triploids and tetraploids. We will produce another generation of tetraploids in summer of 2018. Initial data suggest the growth performance of the triploids are significantly improved. We plan to file a patent application for the improved oysters.

What was accomplished under these goals? In the past year, we have been evaluating the growth and survival of triploid oysters produced in 2016. The growth of triploids was significantly faster than that of diploids. Survival of triploids were variable and mostly the same as that ofdiploids. No difference in genome stability was observed among triploid groups, although the evaluation is still at early stages. We will conduct a full assessment of genome stability of triploids and tetraploids in summer of 2018.In 2017, new tetraploids and triploids are produced and deployed for field evaluations. Partly support by this project, we tested triploid induction in the hard clam and developed a protocol that can produce high percentages of triploid hard clams by inhibiting polar body II. The protocol may lead to commercial production of triploid hard clams for aquaculture.


  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Yang, H. and X. Guo. 2017. Triploid hard clams Mercenaria mercenaria produced by inhibiting polar body I or polar body II. Aquaculture Research, 49(1):449-461.

Progress 09/01/15 to 08/31/16

Target Audience:Shellfish farmers, shellfish biologists and breeders, geneticists, educators and students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development opportunities were provided to 2 graduate students, 1 postdoctoral research and 4 visiting scientists. How have the results been disseminated to communities of interest?The results have been communicated to farmers through the Oyster Grower Forum and will be presented to the community at the Annual Meeting of the National Fisheries Association, March 26-30, 2017, Knoxville, TN, USA. What do you plan to do during the next reporting period to accomplish the goals?We will collect cytogenetics and performance data on triploids and tetraploids produced in 2016 and produce the next generation of tetraploids in summer of 2017.

What was accomplished under these goals? In June 2016, we produced families and linesof tetraploids and triploids using large, small and mosaic tetraploids. Triploids and tetraploids are deployed for field evaluation. At 6 months post-fertilization, triploids and tetraploids produced from large tetraploid grow significantly faster than these produced from small and mosaic tetraploids.These results show that the genotype oftetraploids have a significant effect on the performance of their tetraploid and triploid progeny, and superior triploids and tetraploidscan be produced by selecting and using the best-performing tetraploids. We are collecting cytogenetics data to determine if different tetraploids produce different levels of aneuploids and if genome stability can be improved.


  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Guo, X., M. Liu, B. Xu, Y. Chen, Y. Dong, L. Lv, M. Whiteside and G. DeBrosse. Superior triploid eastern oysters produced by selecting tetraploids. Accepted by 109th Annual Meeting of National Shellfisheries Association, March 26-30, 2017, Knoxville, TN, USA.