Development of Disease-Resistance-Associated Microsatellite DNA Markers for Selective Breeding of Tilapia (Oreochromis spp.) Farmed in Taiwan

There are numerous means to improve the tilapia aquaculture industry, and one is to develop disease resistance through selective breeding using molecular markers. In this study, 11 disease-resistance-associated microsatellite markers including 3 markers linked to hamp2, 4 linked to hamp1, 1 linked to pgrn2, 2 linked to pgrn1, and 1 linked to piscidin 4 (TP4) genes were established for tilapia strains farmed in Taiwan after challenge with Streptococcus inae. The correlation analysis of genotypes and survival revealed a total of 55 genotypes related to survival by the chi-square and Z-test. Although fewer markers were found in B and N2 strains compared with A strain, they performed well in terms of disease resistance. It suggested that this may be due to the low potency of some genotypes and the combinatorial arrangement between them. Therefore, a predictive model was built by the genotypes of the parental generation and the mortality rate of different combinations was calculated. The results show the same trend of predicted mortality in the offspring of three new disease-resistant strains as in the challenge experiment. The present findings is a nonkilling method without requiring the selection by challenge with bacteria or viruses and might increase the possibility of utilization of selective breeding using SSR markers in farms.

Chromosome morphology and cytomolecular characteristics of the perennial rye cultivar ‘Kriszta’

The perennial Secale cereanum cultivar ‘Kriszta’ is an artificial hybrid of S. cereale and S. strictum ssp. anatolicum. From the cross between the wheat line Mv9kr1 and ‘Kriszta’, which aimed the transfer of beneficial traits from rye to wheat, numerous translocation lines have been produced. For the identification of the translocated chromosomes, the unambiguous differentiation between chromosome arms of ‘Kriszta’ is essential. The identification of its short chromosome arms using conventional FISH probes is easy, but because of their similar hybridization patterns, its long arms cannot be distinguished. The present study aimed to create the detailed karyotype of ‘Kriszta’, especially that of long arms, by both chromosome measurements and FISH using highly repetitive, as well as subtelomeric tandem repeat, and synthetic microsatellite DNA sequences. Our results indicate that the chromosome complement of ‘Kriszta’ is not a simple combination of the chromosomes of the parental rye species but is composed of rearranged chromosomes. It is also showed that an adequate pair-wise combination of the DNA sequences pSc119.2, pSc200, pSc250, and (AAC)5 makes it possible to identify any of the long arms of S. cereanum cv. Kriszta chromosomes. The future usability of the identified wheat- ‘Kriszta’ translocated chromosomes is also discussed.

Characterisation of the Mitochondrial Genome and the Population Genetics of Polyprion Oxygeneios (Hapuku) from Around New Zealand

<p><b>Polyprion oxygeneios (hapuku) is an important commercial and recreational fishery species within New Zealand. Moreover, P. oxygeneios are currently being developed as a high-value New Zealand aquaculture species. There have been no previous studies on New Zealand’s P. oxygeneios that have been able to detect genetic differences among samples, which may be of use to either broodstock or fisheries managers. An understanding of the genetic structure of commercially harvested species maximises the potential for sustainable harvesting through effective management schemes. The primary goal of this thesis was to investigate the population genetic structure of P. oxygeneios using molecular markers to analyse samples collected from sites within New Zealand’s Exclusive Economic Zone (EEZ).</b></p> <p>The DNA sequence of the whole mitochondrial genome of P. oxygeneios was determined and it showed a similar structure and gene organisation to that of other species across a wide range of taxa. A set of species-specific control region primers was developed for P. oxygeneios and Polyprion americanus, and additional primers were designed for the 16S and ND6 genes of P. oxygeneios. A ~488 bp portion of the mitochondrial DNA (mtDNA) control region sequence from 274 individuals, and genotypes from 259 individuals using nine polymorphic microsatellite loci, were used to investigate the phylogeography and population genetic structure of P. oxygeneios. The mitochondrial DNA data failed to detect any significant differentiation between sample sites. However, the microsatellite DNA analyses showed that individuals sampled from the west coast of the South Island (Hokitika) were genetically distinct from individuals sampled at all other New Zealand sites. These two groups might be representative of two discrete populations of P. oxygeneios within New Zealand’s EEZ. These results suggest that the west coast South Island P. oxygeneios fishery should continue to be managed as a separate stock, with some possible revision of the Cook Strait fishery required. Analyses of the mtDNA and microsatellite DNA data of P. oxygeneios broodstock held at NIWA’s Bream Bay Aquaculture Park showed that they were not significantly differentiated from the wild populations (excluding Hokitika). Simulations also described the appropriate sampling efforts required to capture an appropriate level of genetic diversity when either establishing a new broodstock or supplementing an existing broodstock with new individuals. Continued management of the broodstock will be required to maintain the high levels of genetic diversity that have been captured in the founding broodstock in future generations.</p>

A population genetic analysis of the New Zealand spotty (Notolabrus celidotus) using mitochondrial DNA and microsatellite DNA markers

<p>Notolabrus celidotus (the New Zealand spotty) is a common rocky reef species that is endemic to New Zealand. This species is the most abundant demersal reef fish in New Zealand, and is distributed throughout the North and South Islands, and Stewart Island. Notolabrus celidotus consumes a wide variety of small invertebrates, and juveniles are reliant on coastal kelp forests as nursery habitats. Because N. celidotus is such a common species on New Zealand rocky reefs it is a good model species for population genetic studies. The primary goal of this research was to investigate new genetic markers and add new sample locations to bolster previous genetic population data from N. celidotus. The thesis research utilised DNA sequences obtained from a 454 massively parallel DNA sequencer and reports six new microsatellite loci for N. celidotus. These loci are the first microsatellite DNA markers to be developed for this species. Additional mitochondrial DNA (mtDNA) control region sequences were obtained from new samples of N. celidotus and combined with previously reported mtDNA sequences. Increasing the sample size improved the genetic coverage of N. celidotus populations around coastal New Zealand. The mtDNA sequences were analysed to examine the population connectivity and demographic history of N. celidotus. The microsatellite DNA loci reported in this study were also used to examine the levels of genetic diversity and population structure in N. celidotus. Results of the combined genetic analyses revealed extremely high levels of genetic diversity among the population sample of the mtDNA control region. Both the mitochondrial DNA and microsatellite DNA analyses showed a distinct lack of population genetic structuring, which suggests there is constant mixing of N. celidotus among sites. The results of this study have the potential to inform the expectations about the genetic structure of closely related wrasse species, such as Notolabrus fucicola, as well as other coastal species that have a similar life history, dispersal power, and New Zealand-wide distribution.</p>

Population genetic structure of New Zealand blue cod (Parapercis colias) based on mitochondrial and microsatellite DNA markers

<p>Parapercis colias (blue cod) is an endemic temperate reef fish that supports an important commercial and recreational fishery in New Zealand. However, concerns have been raised about localized stock depletion, and multiple lines of evidence have suggested P. colias may form several biologically distinct populations within the New Zealand Exclusive Economic Zone. Mark and recapture studies along with otolith and stable isotope studies have indicated that individuals are sedentary with very limited movement beyond the scale of 10-20km. The primary goal of this research was to advance the current knowledge of P. colias population genetic structure. This information can be incorporated into stock assessment models with the aim of improving the management of the P. colias fishery. This study made use of 454 pyrosequencing technology to isolate and develop the first set of microsatellite DNA markers for P. colias. These seven microsatellite loci, along with mitochondrial control region sequences, were used to determine the levels of genetic variation and differentiation between sites around the New Zealand coastline, including the Chatham Islands. Significant differentiation was observed between the Chatham Islands and mainland New Zealand sample sites, indicating that these two regions form distinct populations. Interpretation of the results for the mainland sites was more complex. Mitochondrial sequence data detected no significant pairwise differentiation between mainland sites, although a pattern of isolation-by-distance was observed. However, evidence for genetic differentiation among mainland sites was weak based on the microsatellite DNA analysis. Although pairwise Gѕт levels were significant in some sites, this was not reflected in principal component analysis or Bayesian structure analysis. It is likely that through long range dispersal, migration is at or above the threshold for genetic connectivity, but below a level necessary for demographic connectivity. This is indicated by both the genetic structure reported here, along with previous studies showing limited dispersal of P. colias.</p>

A population genetic analysis of the New Zealand spotty (Notolabrus celidotus) using mitochondrial DNA and microsatellite DNA markers

<p>Notolabrus celidotus (the New Zealand spotty) is a common rocky reef species that is endemic to New Zealand. This species is the most abundant demersal reef fish in New Zealand, and is distributed throughout the North and South Islands, and Stewart Island. Notolabrus celidotus consumes a wide variety of small invertebrates, and juveniles are reliant on coastal kelp forests as nursery habitats. Because N. celidotus is such a common species on New Zealand rocky reefs it is a good model species for population genetic studies. The primary goal of this research was to investigate new genetic markers and add new sample locations to bolster previous genetic population data from N. celidotus. The thesis research utilised DNA sequences obtained from a 454 massively parallel DNA sequencer and reports six new microsatellite loci for N. celidotus. These loci are the first microsatellite DNA markers to be developed for this species. Additional mitochondrial DNA (mtDNA) control region sequences were obtained from new samples of N. celidotus and combined with previously reported mtDNA sequences. Increasing the sample size improved the genetic coverage of N. celidotus populations around coastal New Zealand. The mtDNA sequences were analysed to examine the population connectivity and demographic history of N. celidotus. The microsatellite DNA loci reported in this study were also used to examine the levels of genetic diversity and population structure in N. celidotus. Results of the combined genetic analyses revealed extremely high levels of genetic diversity among the population sample of the mtDNA control region. Both the mitochondrial DNA and microsatellite DNA analyses showed a distinct lack of population genetic structuring, which suggests there is constant mixing of N. celidotus among sites. The results of this study have the potential to inform the expectations about the genetic structure of closely related wrasse species, such as Notolabrus fucicola, as well as other coastal species that have a similar life history, dispersal power, and New Zealand-wide distribution.</p>

Characterisation of the Mitochondrial Genome and the Population Genetics of Polyprion Oxygeneios (Hapuku) from Around New Zealand

<p><b>Polyprion oxygeneios (hapuku) is an important commercial and recreational fishery species within New Zealand. Moreover, P. oxygeneios are currently being developed as a high-value New Zealand aquaculture species. There have been no previous studies on New Zealand’s P. oxygeneios that have been able to detect genetic differences among samples, which may be of use to either broodstock or fisheries managers. An understanding of the genetic structure of commercially harvested species maximises the potential for sustainable harvesting through effective management schemes. The primary goal of this thesis was to investigate the population genetic structure of P. oxygeneios using molecular markers to analyse samples collected from sites within New Zealand’s Exclusive Economic Zone (EEZ).</b></p> <p>The DNA sequence of the whole mitochondrial genome of P. oxygeneios was determined and it showed a similar structure and gene organisation to that of other species across a wide range of taxa. A set of species-specific control region primers was developed for P. oxygeneios and Polyprion americanus, and additional primers were designed for the 16S and ND6 genes of P. oxygeneios. A ~488 bp portion of the mitochondrial DNA (mtDNA) control region sequence from 274 individuals, and genotypes from 259 individuals using nine polymorphic microsatellite loci, were used to investigate the phylogeography and population genetic structure of P. oxygeneios. The mitochondrial DNA data failed to detect any significant differentiation between sample sites. However, the microsatellite DNA analyses showed that individuals sampled from the west coast of the South Island (Hokitika) were genetically distinct from individuals sampled at all other New Zealand sites. These two groups might be representative of two discrete populations of P. oxygeneios within New Zealand’s EEZ. These results suggest that the west coast South Island P. oxygeneios fishery should continue to be managed as a separate stock, with some possible revision of the Cook Strait fishery required. Analyses of the mtDNA and microsatellite DNA data of P. oxygeneios broodstock held at NIWA’s Bream Bay Aquaculture Park showed that they were not significantly differentiated from the wild populations (excluding Hokitika). Simulations also described the appropriate sampling efforts required to capture an appropriate level of genetic diversity when either establishing a new broodstock or supplementing an existing broodstock with new individuals. Continued management of the broodstock will be required to maintain the high levels of genetic diversity that have been captured in the founding broodstock in future generations.</p>

Population genetic structure of New Zealand blue cod (Parapercis colias) based on mitochondrial and microsatellite DNA markers

<p>Parapercis colias (blue cod) is an endemic temperate reef fish that supports an important commercial and recreational fishery in New Zealand. However, concerns have been raised about localized stock depletion, and multiple lines of evidence have suggested P. colias may form several biologically distinct populations within the New Zealand Exclusive Economic Zone. Mark and recapture studies along with otolith and stable isotope studies have indicated that individuals are sedentary with very limited movement beyond the scale of 10-20km. The primary goal of this research was to advance the current knowledge of P. colias population genetic structure. This information can be incorporated into stock assessment models with the aim of improving the management of the P. colias fishery. This study made use of 454 pyrosequencing technology to isolate and develop the first set of microsatellite DNA markers for P. colias. These seven microsatellite loci, along with mitochondrial control region sequences, were used to determine the levels of genetic variation and differentiation between sites around the New Zealand coastline, including the Chatham Islands. Significant differentiation was observed between the Chatham Islands and mainland New Zealand sample sites, indicating that these two regions form distinct populations. Interpretation of the results for the mainland sites was more complex. Mitochondrial sequence data detected no significant pairwise differentiation between mainland sites, although a pattern of isolation-by-distance was observed. However, evidence for genetic differentiation among mainland sites was weak based on the microsatellite DNA analysis. Although pairwise Gѕт levels were significant in some sites, this was not reflected in principal component analysis or Bayesian structure analysis. It is likely that through long range dispersal, migration is at or above the threshold for genetic connectivity, but below a level necessary for demographic connectivity. This is indicated by both the genetic structure reported here, along with previous studies showing limited dispersal of P. colias.</p>

Population genetics of the school shark (Galeorhinus galeus) in New Zealand, Australian and Chilean waters

<p>The school shark (Galeorhinus galeus) is a coastal bentho-pelagic species that is highly migratory and has a widespread distribution in temperate waters. This species matures late, has a relatively low fecundity and is slow growing, which makes it vulnerable to overfishing. They are commercially fished throughout their distribution, and some global stocks have been under pressure because of poor management. In Australia, longline and gillnet fisheries targeted pregnant females and juveniles around Victorian and Tasmanian nursery grounds, resulting in loss of historical inshore nursery habitat. School shark tagging programmes have reported migration between Australian and New Zealand stocks, but preliminary genetic studies have suggested that there are slight genetic differences between the stocks. Currently, the Australian and New Zealand school shark fisheries are assessed and managed as separate stocks. However, the question of whether this species is comprised of a single population or multiple sub-populations in the South Pacific remains unresolved. Given the commercial importance of the school shark fisheries and the concern about stock levels on the regional and trans-Tasman scales, knowledge of stock structure is essential for effective management. The aim of this thesis research was to determine the levels of genetic diversity and population structure of G. galeus in New Zealand and Australia, and compare these to a population in Chile, using mitochondrial DNA (mtDNA) sequencing and microsatellite DNA markers. The DNA sequence of an 893 base pair region of the mtDNA control region (CR) was determined using 475 school shark samples and nine microsatellite DNA loci were genotyped in 239 individuals. Analyses of the data revealed strong evidence of genetic differentiation between G. galeus populations in Australasia and Chile, suggesting restricted gene flow among populations in the western and eastern areas of the Pacific Ocean. The FST values ranged from 0.188 to 0.300 for CR mtDNA, and 0.195 to 0.247 for microsatellite DNA in G. galeus. However, there was no evidence of stock differentiation among New Zealand/Australian sample sites for either mtDNA or microsatellite DNA data. These results support the model of a single panmictic stock across the Tasman Sea. The similarity of the results obtained from the maternally inherited mtDNA and biparental inherited microsatellite loci did not support the suggestion of sex-biased dispersal of G. galeus in the New Zealand/Australia region and it was concluded that females and males had similar patterns of dispersal. Sharks can be either monogamous or polygamous, which is important when considering stock assessments and harvesting models. Multiple paternity has been reported in several shark species, however, the number of sires per litter varies considerably among species. An investigation of multiple paternity (MP) was conducted in G. galeus by assessing the levels of relatedness within progeny arrays using six polymorphic microsatellite DNA markers. Five “families” (mother and litters) were sampled from the North Island of New Zealand and a parentage analysis was conducted. The minimum number of males contributing to each progeny array was estimated by identifying the putative paternal alleles by allele counting and reconstructing multilocus genotypes method. The analysis showed the occurrence of genetic polyandry in G. galeus; two of five litters showing multiple sires involved in the progeny arrays (40%). The minimum number of sires per litter ranged from one to four. Although MP was only detected in two litters, this finding is consistent with the known reproductive characteristics of G. galeus. It can potentially store sperm for long periods of time and has a specific mating season when males and females typically mix on the edge of the continental shelf. Detecting MP within a litter has highlighted the importance of the post-copulatory selective processes in the G. galeus mating system, and this has implications for the management and conservation of genetic diversity.</p>