Identification And Application Of Disease-resistance Markers In The Astern Oyster (Crassostrea Virginica)

The eastern oyster (Crassostrea virginica Gmelin), naturally occurring along the coasts ofAtlantic Ocean and the Gulf of Mexico, supports important fishery and aquaculture industries inthe United States. The oyster industry has been seriously affected by two major diseases: MSXand Dermo since1950s. The two diseases, along with over-fishing and habitat destruction, areamong the leading causes for the collapse of the oyster fisheries in the mid-Atlantic region. Inthis study, disease-resistance markers with candidate genes and RAD sequencing technique areidentified. Further, with the putative disease-resistance and neutral markers, how diseasesmodify the population structure of spat population in Delaware Bay is analyzed. Finally, theeffective population sizes (Ne) of eastern oyster populations in Delaware Bay are estimated.1. Association between the cvSI-1gene and disease resistance in the eastern oysterSNP198of cvSI-1gene was genotyped in disease-resistant and susceptible strains, wildpopulations with different disease exposure histories as well as the populations within the sameestuary but with different diseases pressures. At SNP198, the C allele consistently increases infrequency after Dermo-caused mortalities. As SNP198is synonymous, we study whether itslinkage to polymorphism at the promoter region can explain the resistance. A631bp fragment ofthe5’ flanking region is cloned by genome-walking and re-sequenced, revealing22SNPs andthree insertion/deletions (indels). A25bp indel at-404is genotyped along with SNP198forassociation analysis using before and after mortality samples. After mortalities that wereprimarily caused by P. marinus, the frequency of deletion allele at-404indel increases by14.4%(p=0.0595), while that of SNP198C increases by only3.4%(p=0.5756). The resistance alleles at the two loci are linked in80.3%of the oysters. Oysters with the deletion allele at-404indelshows significant (p=0.017) up-regulation of cvSI-1under P. marinus challenge. Our resultssuggest that mutation at the promoter region causes increased transcription of cvSI-1, which inturn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenicproteases from the parasite.2. Association between the cvSI-2gene and disease resistance in the eastern oysterA new serine protease inhibitor from the eastern oyster, cvSI-2, has been shown to inhibit themajor extracellular protease of the Dermo pathogen P. marinus. In this study, we focus on theassociation between cvSI-1genetic variations and disease resistance in the eastern oyster.BLAST search on the National Center for Biotechnology Information (NCBI) databases returns33expressed sequence tag (EST) using cDNA sequence of cvSI-2as query. After discardingsequences with poor quality and too short length,27sequences are aligned for SNP discovery.Five SNPs, including four synonymous and one non-synonymous are identified in the258bpcoding region. The non-synonymous SNP, SNP226is genotyped in families before and afterdisease-caused mortalities, disease-resistant and susceptible strains developed from the samepopulation, as well as wild populations within the same estuary but with different diseasepressures. The G allele frequency of SNP226consistently increases after Dermo-causedmortalities in families and are enriched in the disease-resistant strain. In addition, within thesame estuary, populations with high prevalence of Dermo disease have more G allele than that ofthe populations with no Dermo. These results indicate cvSI-2gene is under natural selectionupon Dermo disease and the G allele of SNP226is associated with Dermo resistance in theeastern oyster. SNP226is a non-synonymous mutation, causing a valine-to-ileline (Val/Ilerespectively) substitution. The G allele (Val genotype) may result in increased expression ofcvSI-2or higher affinity of cvSI-2for perkinsin.3. Constructing high density linkage map and mapping disease-resistance QTLs in theeastern oysterThe high density linkage map of eastern oyster is constructed with RAD markers. The putativedisease-resistance QTLs are identified by post-mortality genotype frequency shifts analysis. Thefemale map containing1,350markers in12linkage groups, is1,081.24cM in total length withaverage interval distance of0.8cM and98.14%in genome coverage. The male map containing 1,236markers in10linkage groups, is723.61cM in total length with average interval distanceof0.59cM and98.28%in genome coverage. Significant post-mortality shifts in genotypefrequency are detected at88and80markers in female and male maps, respectively. Linkageanalysis reveal that most markers (~70%) showing frequency shifts are closely linked to eachother on the genetic map. This finding suggests that post-mortality shifts in genotype frequencyare not random, but link to disease-resistance QTLs.11putative disease-resistance QTLs areidentified in female map, distributing in five linkage groups with the8thlinkage group containingthe most of four. Five putative disease-resistance QTLs are identified in male map, distributingin four linkage groups with the7thlinkage group containing two.4. Analysis of how diseases affecting the eastern oyster spat populations structure inDelaware BaySpat oysters are collected from Hope Creek (HC)、Round Island (RI)、Beadons (BD) andCape Shore (CS) in Delaware Bay and genotyped in eight putative disease-resistance and nineputative neutral SSR markers. Genetic differentiation between populations (Fst) for all pairs ofpopulations are analyzed. Pair-wise comparisons between the four sample locations revealtypically low, not significant Fst estimates (-0.0016-0.0015) in all of the six pair-wisecomparisons with17markers. Not only the neutral markers but also the putative disease-resistance marker don’t detect population differentiation among the four populations. Thissuggests that the spat populations in Delaware Bay are a large and homogenous population.5. Ne estimation of eastern oyster populations in Delaware BayThe eastern oyster (Crassostrea virginica) may be prone to sweepstake reproductive success(SRS) due to its high fecundity and type III survivorship. To test if SRS occurs in the easternoyster, we study temporal and spatial genetic variation of oyster populations in Delaware Bay.Adults and spats are collected from five locations in different years and genotyped with sevenmicrosatellite markers. Slight genetic differences are revealed by Fst statistics between the adultpopulations and spat recruits, while the adult populations are spatially homogeneous andtemporally stable. No changes in allele richness are evident among adult and spat collections,suggesting no strong SRS. Ne estimates obtained with five methods are variable and oftenwithout upper confidence limits. When confidence limits are available, Ne estimates for spatcollections (140–440) are consistently smaller than that for adult populations (589–2,779). Analysis of pooled adult samples across all sites suggests that Nefor the whole Bay may be verylarge, which is also indicated by the lack of upper confidence limits in many estimates. Ourresults suggest that Nemay be small for a given spat fall due to moderate SRS, but the entireadult population may have large Neand temporally stable as they are accumulations of many spatfalls per year and over many years.