Dr. Pat Gaffney - Abstracts
Elfstrom, C. M., P. M. Gaffney, C. T. Smith, and J. E. Seeb. 2005.
Abstract: We describe 27 single nucleotide polymorphisms (SNPs) in a commercially important bivalve, the weathervane scallop ( Patinopecten caurinus ), identified using a targeted-gene approach. We further characterize 12 of these using 5'-nuclease and allele-specific PCR assays. Polymorphisms were identified in both mitochondrial and nuclear genes. These are the first SNPs developed for delineating population structure in the weathervane scallop and will provide a useful complement to currently available genetic markers.
Milbury, C. A., and P. M. Gaffney. 2005.
Abstract: The complete mitochondrial genome of the eastern oyster Crassostrea virginica (GenBank accession number AY905542) is 17,243 bp in length and contains two ribosomal genes, 12 protein-coding genes, and 23 tRNAs. The arrangement of protein-coding genes is identical to that of the congeneric Pacific oyster, C. gigas , but tRNA genes show several duplications and extensive rearrangements between the species. Unique features in C. virginica include an additional trnM gene, the absence of an ATPase subunit 8 ( atp8 ) gene and an inferred translational frameshift within the cytochrome b ( cob ) gene. In both species, the large subunit ribosomal RNA gene is encoded by two separate regions of the mitochondrial genome, the first reported case of a split ribosomal RNA gene in a metazoan. Translation of protein-coding genes in both species is initiated with methionine, with the exception of cob , which uses leucine. In C. virginica , translation of all protein-coding genes (except possibly cob ) terminates with TAA, with polyadenylation completing the primary transcript in cytochrome oxidase subunit 3 ( cox3 ) and NADH dehydrogenase subunit 4L ( nad4L ), whereas C. gigas employs stop codons TAA and TAG equally. Interspecific divergence of mitochondrially encoded proteins is considerable, with amino acid identities ranging from 47-92%. A single major noncoding region representing the putative control region is found in both species.
Gaffney, P. M., J. C. Pierce, A. G. Mackinlay, D. A. Titchen, and W. K. Glenn. 2003.
Abstract: While genome sequencing projects have discovered numerous types of transposable elements in diverse eukaryotes, there are many taxa of ecological and evolutionary significance that have received little attention, such as the molluscan class Bivalvia. Examination of a 0.7-MB genomic sequence database from the cupped oyster Crassostrea virginica revealed the presence of a common interspersed element, CvA. CvA possesses subterminal inverted repeats, a tandemly repeated core element, a tetranucleotide microsatellite region, and the ability to form stable secondary structures. Three other less abundant repetitive elements with a similar structure but little sequence similarity were also found in C. virginica. Ana-1, a repetitive element with similar features, was discovered in the blood ark Anadara trapezia by probing a genomic library with a dimeric repeat element contained in intron 2 of a minor globin gene in that species. All of these elements are flanked by the dinucleotide AA, a putative target-site duplication. They exhibit structural similarity to the sea urchin Tsp family and Drosophila SGM insertion sequences; in addition, they possess regions of sequence similarity to satellite DNA from several bivalve species. We suggest that the Crassostrea repetitive elements and Ana-1 are members of a new MITE-like family of nonautonomous transposable elements, named pearl. Pearl is the first putative nonautonomous DNA transposon to be identified in the phylum Mollusca.
Geneomic approaches to marker development and mapping in the eastern oyster, Crassostrea virginica. Pp. 84-91 in N. Shimizu, T. Aoki, I. Hirono and F. Takashima, eds. Aquatic Geneomics Steps Towards a Great Future. Springer-Verlag, Tokyo.
Gaffney, P.M. 2002.
Abstract: Crassostrea virginica (Gmelin) is the primary oyster species in eastern North America, with economically significant wild harvests and culture operations from Canada to Mexico. A century of decline resulting from overfishing, habitat degradation and disease has sparked interest in genetically improved lines for aquaculture and stock enhancement. In view of the limited success of traditional breeding methods, we hope to apply molecular breeding methods to accelerate the pace of selective breeding via identification of quantitative trait loci (QTL), marker-assisted selection and marker-assisted introgression. A first step in this process is the construction of a genetic linkage map for mapping QTL, which would enable focused selective breeding programs and identification of candidate genes for important traits. The map would also be relevant to parallel efforts in the Pacific oyster (Crassostrea gigas), one of the most important aquaculture species worldwide. To further these goals, identification of both type 1 and type 11 markers is desirable. From a survey of 0.7 MB of C. virginica genomic DNA, we discuss the distribution of simple sequence repeats and common repetitive elements. One repetitive element, CvA, is particularly abundant and contains a tetranucleotide microsatellite (ACRG)n.
Smith, P.J., P.M. Gaffney and M. Purves. 2001.
Abstract: Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrail DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.
Chromosomal location of the major ribosomal RNA genes in Crassostera virginica and Crassostrea gigas. Veliger 44:79-83.
Xu, Z., X. Guo, P.M. Gaffney and J.C. Pierce. 2001.
Abstract: The chromosomal location of the major ribosomal RNA genes (rDNA) was studied in the eastern oyster, Crassostera virginica (Gmelin, 1791), and the Pacific oyster, C. gigas (Thunberg, 1793) by fluorescence in situ hybridization (FISH). Probes were made by PCR amplification of the intergenic transcribed spacers between the 18S and 5.8S (ITS1), and between 5.8S and 28S RNA genes (ITS2), and labeled by PCR incorporation of DIG-11-dUTP. FISH with either probe on interphase nuclei showed two strong signals in both species. In C. virginica, FISH signals were located on the short arms of Chromosome 2, the second longest, with a centromere index of 0.39. In C. gigas, the rDNA probe hydridized to the long arms of Chromosome 10, the shortest chromosome. In both species, the FISH signals were confined to the telomere region of the chromosomes. All Crassostrea species have a haploid number of 10, and C. virginica and C. gigas share an almost indistinguishable karyotype. The size of the rDNA-bearing chromosome is the first major difference reported between the two karyotypes. This result suggests that considerable chromosomal evolution is possible in oysters despite the highly conserved haploid number.
Abstract: During the last decade, methods
for detecting DNA polymorphisms have proliferated at a bewildering pace.
Today the investigator must choose among various types of genetic markers
as well as among a variety of methods for discovering and screening polymorphisms.
Polymorphisms useful for the analysis of population structure are found
in both mitochondrial and nuclear genomes. Marker development may proceed
along two routes: 1) discovery of species-specific markers, and 2) application
of universal methods. Species-specific markers are based on sequence data
from the target species or close relatives, whereas universal markers
are based on the use of polymerase chain reaction (PCR) primers targeted
to regions highly conserved across diverse taxa. Markers commonly employed
include mitochondrial DNA polymorphisms, microsatellites, anonymous nuclear
loci and known genes (both coding and noncoding regions). Methods for
detecting polymorphisms range from technically simple (RFLP analysis)
to more sophisticated mutation-scanning methods.
We review the application of these approaches to several key Antarctic species (the Patagonian toothfish Dissostichus eleginoides, the mackerel ice fish Champsocephalus gunnari and the squid Martialia hyadesi) and present preliminary data on genetic polymophisms in toothfish and icefish.
Protease inhibitory activity in selectively bred families of eastern oysters. Journal of Aquatic Animal Health 12:136-145.
Oliver, J.L., P.M. Gaffney, J.S.K. Allen, M. Faisal and S.L. Kaattari. 2000.
Abstract: Selected stocks of eastern oysters, Crassostrea virginica, were interbred to produce ten families with high inter- and intra-family genetic variability. In an effort to identify potential biochemical markers for resistance to Dermo, a disease afflicting eastern oysters, subsamples of two year old oysters from each of these families were assessed for protease inhibitory activity (PI) against proteases of Perkinsus marinus, the causative agent of Demo disease. Prevalence and intensity of the pathogen were assessed via Ray's Fluid Thioglycollate Medium (RFTM) test. Family survival under field challenge, disease intensity, and protease inhibitory activity were compared to evaluate family performance. In the laboratory, three families had the highest survival, lowest average number of parasite cells, and highest average PI. In field challenges, the same three families exhibited the lowest cumulative mortality. Among all families, disease intensity was negatively correlated with protease inhibitory activity. Mortality patterns also indicated an inverse relationship between disease intensity and protease inhibitory activity.
Orbacz, E.A. and P.M. Gaffney. 2000.
Abstract: The tautog (Tautoga onitis) is one of two temperate labrid species commonly inhabiting the coastal marine and estuarine waters of the mid-Atlantic coast of the United States. To delineate population structure throughout its primary range, we examined samples collected from three sites (Rhode Island, Delaware, Virginia). Five regions of the mitochondrial genome (COI, ATPase 6, cyt b, ND2 and control region) and one nuclear intron were amplified by PCR and screened for sequence variation with a battery of restriction enzymes (RFLP analysis), or by denaturing gradient gel electrophoresis (DGGE). With RFLP analysis an average of 129 restriction sites per individual were revealed and 532 bases per individual were surveyed. Polymorphisms were observed in the ND2 and control region fragments, but not in the COI, ATPase 6, or cyt b fragments. Mean within- sample haplotype diversity was 0.6905 (plus or minus 0.00184), within the range of values reported for other marine species. However, mean nucleotide diversity was 0.000782, one of the lowest values reported for a marine teleost. Corrected nucleotide divergence between samples was essentially zero, suggesting the absence of population structuring along the mid-Atlantic Coast. DGGE analyses of COI, cyt b, and a lactate dehydrogenase (LDH) intron revealed little additional variation; each product possessed a single common haplotype and occasional rare variants. The low level of genetic diversity observed in the tautog may reflect a small effective population size resulting from historical population bottlenecks or large variance in reproductive success. The apparent absence of geographic differentiation suggests that tautog from Rhode Island to Virginia form a single genetic stock; data from additional genetic polymorphisms are needed to confirm or disprove this conclusion.
Pierce, J.P., G. Tan and P.M. Gaffney. 2000.
Abstract: The Delaware Bay contains the world's largest population of horseshoe crabs, which constitute an ecologically significant component of this estuarine ecosystem. The North Atlantic species Limulus polyphemus has an extensive geographical distribution, ranging from New England to the Gulf of Mexico. Recent assessments of the Delaware Bay population based on beach spawning and trawling data have suggested a considerable decrease in the number of adult animals since 1990. Considerable debate has centered on the accuracy of these estimates and their impact on marine fisheries management planning. Compounding this problem is the lack of information concerning the genetic structure of Atlantic horseshoe crab populations. This study assessed patterns of genetic variation within and between the horseshoe crab populations of Delaware Bay and Chesapeake Bay, using both Random Amplification of Polymorphic DNA (RAPD) and DNA sequence analysis of the mitochondrial cytochrome oxidase I gene (COI). We examined 41 animals from Delaware Bay and 14 animals from the eastern shore of Chesapeake Bay. To provide high quality, uncontaminated genomic DNA for RAPD analysis, DNA was isolated from hemocytes by direct cardiac puncture, purified by spin column chromatography, and quantified by agarose gel electrophoresis. RAPD fingerprints revealed a relative paucity of polymorphic fragments, with generally homogeneous banding patterns both within and between populations. DNA sequence analysis of 515 bases of the 5' portion of the mitochondrial COI gene showed haplotype diversity in the Chesapeake Bay sample to be significantly higher than in the Delaware Bay sample, despite the larger size of the latter. Haplotype analysis indicates minimal contemporary gene flow between Delaware Bay and Chesapeake Bay crab populations, and further suggests that the Delaware Bay population is recovering from a recent population decline.
Lankford, T.E., Jr., T.E. Targett and P.M. Gaffney. 1999.
Abstract: Genetic population structure in Atlantic croaker (Micropogonias undulatus Linnaeus) was examined by using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). Juvenile croaker from three U.S. Atlantic localities (Delaware, North Carolina, and Florida) and one Gulf of Mexico locality (Louisiana) were screened to document the magnitude and spatial distribution of mtDNA variation in M. undulatus; to evaluate the integrity of Cape Hatteras, North Carolina, as a genetic stock boundary; and to estimate levels of gene flow among Atlantic localities to provide an improved basis for future decisions regarding coastwide management of this fishery resource. RFLP analysis of the ATPase 6 and D-loop mtDNA regions revealed a total of 15 composite haplotyes in 93 individuals. Monte Carlo simulations revealed no geographic heterogeneity in mtDNA haplotype frequencies among Atlantic localities and no evidence that juveniles collected north and south of Cape Hatteras originated from separate gene pools (net sequence divergence=-0.002%). There was significant heterogeneity between Atlantic and Gulf of Mexico samples, suggesting restricted gene flow between these two regions. Analysis of molecular variance also indicated regional (Atlantic versus Gulf) population structure, but provided no evidence that Cape Hatteras represents a genetic stock boundary. ANOVA indicated relatively high gene flow (N sub(e)m sub(female), = 12-23 effective female migrants per generation) among Atlantic localities. These findings are consistent with 1) a single genetic stock of M. undulatus on the Atlantic coast and 2) separate, weakly differentiated stocks in the Atlantic and Gulf of Mexico.
Ó Foighil, D., P.M. Gaffney, A.E. Wilbur and T.J. Hilbish. 1998.
Abstract: The Portuguese oyster Crassostrea angulata was long assumed to be native to the northeastern Atlantic, however, a number of lines of evidence now indicate that it is a close relative, or identical, to the Asian Pacific oyster C. gigas. Three hypotheses have been proposed to explain how this strikingly disjunct geographic distribution may have come about: ancient vicariance events, recent anthropogenic introduction to Asia and recent anthropogenic introduction to Europe. We have performed a molecular phylogenetic analysis of C. angulata based on mitochondrial DNA sequence data for 579-nucleotide fragment of cytochrome oxidase I. Our results show that Portuguese oyster haplotypes cluster robustly within a clade of Asian congeners and are closely related, but not identical, to C. gigas from Japan. The mitochondrial data are the first to show that Portuguese oysters are genetically distinct from geographically representative samples of Japanese Pacific oysters. Our phylogenetic analyses are consistent with a recent introduction of C. angulata to Europe either from a non-Japanese Asian source population or from a subsequently displaced Japanese source population. Genetic characterization of Pacific oysters throughout their Asian range is necessary to fully reveal the phylogenetic relationships among Portuguese and Pacific oysters.
Wilbur, AE, E.A. Orbacz, J.R. Wakefield and P.M. Gaffney. 1997.
Abstract: Restriction fragment-length polymorphism analysis was used to evaluate genetic variation in a Siberian population of the Japanese scallop (Patinopecten yessoensis), and the results were compared with those of a similar study of populations in Japan and British Columbia. The polymerase chain reaction was used to amplify three coding regions of the mitochondrial genome (1.5 kilobases [kb] of the ATP synthetase subunit 6 and cytochrome c oxidase, subunit 3, a 1.1-kb region including the tRNA gene for threonine, and 1.4 kb of the cytochrome b apoenzyme) in 20 scallops collected in Peter the Great Bay (Primorye region of Siberia, Russia). Digestion of each region with 11 restriction enzymes revealed 22 polymorphic sites. Haplotype diversity and within-population nucleotide diversity were high in the Siberian sample (0.98 and 0.015, respectively). Both estimates are much greater than those reported by others for a Canadian hatchery population (haplotype diversity = 0.53, nucleotide diversity = 0.0012) and two Japanese populations (mean haplotype diversity=0.72, mean nucleotide diversity = 0.0017). Genetic divergence between regions (Japan and Siberia) was calculated on the basis of a subset of restriction sites common to both studies. Estimates of divergence were low (0.004-0.018), and the variation between regions was not significant (analysis of molecular variance, 1.7%; p=0.14). Haplotype frequency distributions, however, were significantly different among regions (p=0.028, log-likelihood exact test).
Wilbur, AE and P.M. Gaffney. 1997.
Abstract: The bay scallop, Argopecten irradians, exhibits extensive variation in morphology among geographically separated populations, resulting in the recognition of three major subspecies (A. i. irradians, A. i. concentricus, A. i. amplicostatus). The extent to which morphological variation results from differing environmental conditions is unknown. In the present study, bay scallops from Massachusetts, North Carolina, Florida, and Texas were collected, spawned and the offspring reared in a common garden experiment to determine if scallops cultured under similar environmental conditions exhibited the morphology expected given the geographic origin of their parents. Significant differences among populations were indicated by ANOVA in both the wild-caught (13/14 morphological characters) and cultured (11/14 characters) scallops. Principal components analysis clustered wild-caught scallops according to geographic origin and cultured scallops according to geographic ancestry. The morphological characters most influential in resolving groups were plical width, plical spacing, number of plicae and valve convexity. Geographic variation in morphology apparently has a strong genetic basis, and reflects significant differentiation among disjunct populations of bay scallops.
Gaffney, P.M., V.P. Rubin, D. Hedgecock, D.A. Powers, G. Morris and L. Hereford. 1996.
Abstract: Numerous attempts have been made to restore the declining California abalone fisheries by outplanting of hatchery-produced seed. Poor survival of planted seed has been generally attributed to predation and emigration, lessening enthusiasm for reseeding efforts. We present genetic evidence that one large-scale outplanting of red abalone (Haliotis rufescens) in southern California was effective, and document changes in the genetic composition of red abalone populations accompanying hatchery propagation. Allozymes appeared to behave as neutral and therefore effective markers for tracking population bottlenecks in hatchery production and reseeded populations. Careful monitoring of hatchery breeding practices is essential for both commercial production and artificial reseeding programs.
Gaffney, P.M. and D. Bushek. 1996.
Abstract: Like other phenotypic traits, resistance to disease is generally subject to underlying genotypic variability. This may come about indirectly, as a result of variation in overall physiology or in life history characteristics, or may be more directly attributable to variation in cellular or biochemical mechanisms. We summarize here our current understanding of genetic influences in physiological and life history variation in Crassostrea and review the evidence available to date on intra- and interspecific genetic variation in disease resistance, with emphasis on Perkinsus marinus and MSX. We also describe our current view of population structure in Crassostrea virginica, and how it may affect the evolution of disease resistance. Finally, we explore approaches to the development of disease-resistant oysters that capitalize on the genetic variability inherent in C. virginica and within the genus Crassostrea.
Differences in mitochondrial 16S ribosomal gene sequences allow discrimination among American [Crassostrea virginica Gmelin] and Asian [C. gigas (Thunberg) C. ariakensis Wakiya] oyster species. Journal of Experimental Marine Biology and Ecology 192:211-220.
Ó Foighil, D.O., P.M. Gaffney and T.J. Hilbish. 1995.
Abstract: Limited field trials for potentially disease resistant Asian cupped oysters were recently initiated in the central Atlantic coastal region of North America. We have developed a simple and sensitive genetic assay that unambiguously distinguishes the native Crassostrea virginica (Gmelin) from two of its exotic Asian congeners, C. gigas (Thuberg) and C. ariakensis (Wakiya). homologous fragments (443 nt) of the mitochondrial rRNA large subunit were amplified from, and sequenced for, these three commercially important species of oysters. C. virginica exhibits 85.5 and 84% sequence similarity to C. gigas and C. ariakensis (respectively) and contains seven additional nucleotides in the amplified 16S gene fragment. The two Asian species do not differ in gene length and display 95.1% sequence similarity. Numerous endonuclease restriction site differences are apparent among the three oysters species and we present a polymerase chain reaction/restriction fragment length polymorphism strategy that can distinguish these species. This methodology will prove valuable in monitoring the ecological consequences, at all life history stages, of trial introductions of Asian Crassostrea species into this region.