Author: Shannon Robert Loughnan
Loughnan, Shannon Robert, 2014 Capturing and Maintaining Genetic Diversity for the Establishment of a Long-Term Breeding Program for Barramundi (Lates Calcarifer) Aquaculture, Flinders University, School of Biological Sciences
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Mass spawning hatchery practices using small broodstock populations, in addition to the cannibalistic nature of some fish species, contribute to a reduction of genetic diversity from parent to offspring and throughout the juvenile grow-out stages. This is of concern when establishing a selective breeding program for such species because the genetic diversity that is captured in the start-up and initial generations of the program is the basic ingredient for future genetic improvement. The aim of this thesis was to examine methods for capturing and conserving genetic diversity in mass spawning barramundi (Lates calcarifer), when constructing a base population for a long-term selective breeding program for the species. Involving 21 males and 12 females, the transfer of genetic diversity from broodstock to offspring in a large commercial mass spawn was investigated in chapter 2. Previous studies had indicated that substantial amounts of genetic diversity were lost using mass spawning techniques, which are normal practice for the commercial barramundi industry. A high participation rate of parents was detected among the large spawning group used in this study (n = 31). Broodstock contributions were skewed and the contribution by individual dams and sires was as high as 48% and 16% respectively at one day post hatch (dph). Barramundi progeny were monitored throughout the juvenile stages to investigate the conservation of genetic diversity, during the periods of larval metamorphosis and size grading (to inhibit cannibalism). A reduction in allelic richness (Ar) was identified from broodstock to offspring at 1 dph, (Ar was 3.94 among broodstock and 3.52 among offspring sampled). However, no further loss of Ar or genetic diversity was detected in the offspring from 1 to 90 dph, which included the period of metamorphosis, multiple size grading events and losses through size culling, mortalities and the sale of juveniles. The effective population size (Ne) in the broodstock group ranged from 10.1 - 16.7, well below the broodstock census size of 33, whereas the rate of inbreeding was less than 5%. The results from the mass spawn provided reproductive and demographic parameters that could be used to inform the design of a base population for a barramundi selective breeding program. In chapter 3, 407 mature captive broodstock under current use in eight commercial barramundi hatcheries were pedigree tested using 17 microsatellite markers, to determine their suitability for inclusion into a base population. Levels of genetic diversity within each hatchery and the degree of relatedness between individuals were estimated and compared. Genetic diversity was moderate within each broodstock group (Ar ranged from 2.67 - 3.42) and heterozygosity ranged from 0.453 - 0.537. Relatedness estimates within hatcheries were generally low and ranged from -0.003 to 0.273. Structure analysis revealed that captive Australian broodstock were broadly divided into two genetic stocks and suggested that hatchery individuals were either sourced from the two stocks or represented an admixture between them. From the results, an assessment was made of the genetic suitability of existing domesticated broodstock as contributors to the base population. Chapter 4 sampled 1205 barramundi individuals from 48 wild sites covering a broad distribution range. Levels of wild genetic diversity were estimated and compared to captive groups from chapter 3. The wild collections were found to cover two broad ranging genetic stocks, an eastern and western stock and a central stock of genetic admixture (FST = 0.076). The majority of captive individuals were assigned to the eastern stock (59%), followed by the western stock (23%) and central region of admixture (13%). Levels of genetic diversity, as determined by allelic richness (Ar), were slightly lower in the captive groups (average Ar = 3.15) when compared to the wild populations (average Ar = 3.40). Some genetic variation was unrepresented in the captive groups and it was concluded that the inclusion of wild individuals would enhance overall levels of genetic diversity in a base population for selective breeding. Finally, a computer simulation model was developed in chapter 5 and used to compare different options for sourcing genetic variation for inclusion into the base population. It was assumed that the primary goal when establishing the base population would be to maximise genetic diversity. Candidates for inclusion into the synthetic base populations were selected according to levels of genetic diversity and relatedness. A range of options were tested, which included the use of candidates from both wild and captive populations. There was a significant reduction in the level of Ar between broodstock and offspring for many of the options. The best options for retaining genetic diversity were from the base populations constructed from an even representation of wild samples from genetic stocks (WSAr, broodstock and offspring Ar was 5.21 and 4.75 respectively) and to select captive broodstock according to the lowest mean kinship levels (Cmkr, broodstock and offspring Ar was 5.05 and 4.69 respectively). Five alternate base population sizes (Nc) were tested to estimate the effective population size (Ne) based on the variance of parental contribution and unequal sex ratio. Ne was 76, 85, 98, 105 and 115 from an Nc of 150, 180, 200, 230 and 250 respectively, and the rate of inbreeding (∆F) ranged from 0.4 - 0.7%. Under the model presented in this study, an Nc of more than 213 broodstock individuals is required to achieve an Ne greater than 100 and ∆F less than 0.5%. The results suggested that a mixture of both wild and captive barramundi should be included in the base population at the commencement of a selective breeding program for barramundi. This thesis investigated the effects of hatchery practices, such as mass spawning and size grading on the conservation of genetic diversity. In addition, options for selecting candidates to compose a founding population were explored, and recommendations made to promote the longevity and impact of a selective breeding program for barramundi. The Australian industry has on hand a large number of mature captive broodstock that would be suitable for inclusion into a base population for barramundi selective breeding. However, it would be beneficial to include a selection of wild individuals from regions of high genetic diversity to strengthen the fitness of a base population at the commencement of a selective breeding program.
Keywords: Lates calcarifer,barramundi,size grading,mass spawning,parentage,selective breeding,genetic improvement,relatedness,assignment
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
Completed: 2014
School: School of Biological Sciences
Supervisor: Prof. Luciano Beheregaray