美洲牡蛎（Crassostrea virginica）抗病相关基因标记的筛选及应用
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摘要
美洲牡蛎(Crassostrea virginica)天然分布于大西洋西岸和墨西哥湾，是美国重要的经济贝类。上个世纪中期，美洲牡蛎曾遭受两种原生动物病害(MSX和Dermo)的重创，无论养殖群体还是野生群体，均出现高感染率、高死亡率的现象，这使美国东海岸的养殖业几乎崩溃。本研究通过候选基因法和RAD测序的方法筛选美洲牡蛎抗病相关基因标记，并利用已知抗病相关的分子标记和中性的分子标记分析寄生虫病对特拉华湾美洲牡蛎群体遗传结构的影响。
1.丝氨酸蛋白酶抑制因子1基因(cvSI-1)和美洲牡蛎抗病性状的相关性
通过分析美洲牡蛎cvSI-1基因的SNP198位点在抗病品系、易感品系及其野生基群体，具有不同病害感染史的野生群体和处于同一流域，但具有不同病害选择压力的野生群体中和抗病性状的相关性，本研究发现SNP198位点C等位基因和美洲牡蛎的抗Dermo性状紧密相关。但SNP198是个同义突变，不改变编码氨基酸的序列，所以本研究还分析了SNP198位点和cvSI-1基因5’调控区多态性位点的连锁性来解释其抗病相关性。通过染色体步移和重测序的方法，获得长631bp的5’调控区，包含22个SNPs和3个插入/缺失多态性位点。利用野生群体死亡前和死亡后的材料，分析5’调控区-404位，长25bp的插入/缺失位点和SNP198位点的连锁性。在Dermo造成大部分死亡的死亡后群体中，-404插入/缺失的缺失等位基因上升了14.4%(p=0.0595)，而SNP198的C等位基因仅上升了3.4%(p=0.5756)。缺失等位基因和C等位基因在80.3%的牡蛎中具有连锁性。具有缺失等位基因的牡蛎经Dermo病原体Perkinsus marinus感染后，其cvSI-1的表达量显著提高(p=0.017)。本研究结果提示我们，5’调控区的变异会提高cvSI-1的转录活性，因此通过抑制寄生虫的蛋白酶活性而对其产生抗性。2.丝氨酸蛋白酶抑制因子2基因(cvSI-2)和美洲牡蛎抗病性状的相关性一种新的丝氨酸蛋白酶抑制因子，cvSI-2，已经从美洲牡蛎的细胞质中分离出来，其能够体外抑制P. marinus的活性。本研究分析了cvSI-2基因的多态性和美洲牡蛎抗病性状的相关性。利用cvSI-2的cDNA序列搜索NCBI数据库，共得到33条美洲牡蛎的EST序列。将低质量的序列去除后，27条高质量的EST序列用于比对和SNP位点的筛查，获得5个高质量SNP位点，包括4个同义突变和1个非同义突变。随后，对非同义突变SNP226(A/G)和美洲牡蛎的抗病相关性进行分析。SNP226的GG基因型在两个家系死亡后的材料中分别上升17.0%(p=0.0005)和16.9%(p=0.0634)。另外，SNP226的G等位基因在选育的抗病品系中的频率最高，为93.5%，显著高于其野生基群体CTW (69.6%)和两个选育的易感品系FMF (63.0%)和UMFS (51.3%)。在Martha’s Vineyard、特拉华湾和切斯皮克湾三个不同水域，病害选择压力大的野生群体G等位基因频率都要高于病害选择压力小的群体。SNP226的G等位基因编码缬氨酸，A等位基因编码异亮氨酸。此位的缬氨酸可能能够增强cvSI-2蛋白和Dermo病原体丝氨酸蛋白酶结合的亲和性，从而增强了抑制其活性的能力，最终在美洲牡蛎的抗Dermo病害方面发挥重要作用。3.美洲牡蛎高密度遗传连锁图谱的构建及抗病性状的QTL定位利用RAD测序技术，构建了美洲牡蛎高密度遗传连锁图谱。通过分析标记死亡前后基因型频率的变化，定位抗病性状相关的QTL。雌性连锁图谱含12个连锁群，共1,350个标记，覆盖1,081.24cM，最大图距12.24cM，平均图距0.80cM，覆盖率为98.14%。雄性连锁图谱含10个连锁群，共1,236个标记，覆盖723.61cM，最大图距10.05cM，平均图距0.59cM，覆盖率为98.28%。雌性图谱中，88个标记在死亡后群体中基因型频率发生显著变化(p <3.74×10~(-5)，经Bonferroni校正后的P值)，其中有62个(76.3%)标记是成对或者成簇分布。雌性图谱检测到11个高精度QTL，分布在5个连锁群，其中第8号连锁群的QTL数目最多，为4个。雄性图谱中，80个标记在死亡后群体中基因型频率发生显著变化(p <4.04×10~(-5)，经Bonferroni校正后的P值)，其中有57个(71.3%)标记是成对或者成簇分布。雄性图谱检测到5个高精度的QTL，分布在4个连锁群，在第7号连锁群检测到2个。4.病害对特拉华湾美洲牡蛎幼体群体遗传结构的影响
本研究利用8个抗病相关的SSR标记和9个中性的SSR标记对特拉华湾由北向南Hope Creek (HC)、Round Island (RI)、Beadons (BD)及Cape Shore (CS)4个采样点的美洲牡蛎幼体群体进行了群体遗传学分析。如果用17个微卫星位点分析两两群体间的遗传分化系数(Fst)，Fst的分布范围为-0.0016-0.0015。最南部群体CS和最北部群体HC间的Fst值最大，为0.0015，但是和0没有显著性差异(p>0.0083，经Bonferroni校正后的P值)。如果用8个抗病相关的微卫星位点进行分析， Fst的分布范围为-0.0052-0.0013。CS群体和HC群体间的Fst值最大，为0.0013。但是其Fst值仍和0没有显著性差异(p=0.8417)。同样地，中性的微卫星位点也没有检测到这4个群体间的遗传分化。这说明特拉华湾幼体群体是个均一的大群体，没有出现遗传分化。5.特拉华湾美洲牡蛎群体有效群体大小(Ne)的估算
具有高繁殖力和III型存活类型的美洲牡蛎中极有可能存在“少数个体维持大群体”的现象。为了验证特拉华湾美洲牡蛎群体中是否存在这种现象，本研究估算了其有效群体的大小(Ne)，并比较特拉华湾的Ne在不同病害选择压力群体间和时间上的变化。对特拉华湾5个采样点，2006年和2009年成体牡蛎和幼体牡蛎群体样品在7个中性的SSR位点进行分型，利用三个双样品法和两个单样品法估测特拉华湾美洲牡蛎群体的Ne。结果显示，成体牡蛎和幼体牡蛎间的基因丰富度没有显著性差异，从而不支持“少数个体维持大群体”的理论。5种方法估算得到的Ne值差异较大，总体而言，所得Ne值较小，并且通常没有获得有效的上限估算值。幼体群体的Ne值分布范围是140-440，比成体群体的Ne值要小(589–2,779)。通过混合所有成体的样品所估算的Ne值提示着整个海湾的有效群体大小可能较大。本研究结果表明，特拉华湾某一批幼体牡蛎可能是由少数的成体牡蛎繁殖而来的，并存在一定的遗传漂变。但是这种小范围的遗传漂变并不影响整个湾成体群体的遗传变化，因为成体群体是每年很多批幼体群体积累多年的结果。
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 oyster
SNP198of 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 oyster
A 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 oyster
The 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 Bay
Spat 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 Bay
The 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.
1.丝氨酸蛋白酶抑制因子1基因(cvSI-1)和美洲牡蛎抗病性状的相关性
通过分析美洲牡蛎cvSI-1基因的SNP198位点在抗病品系、易感品系及其野生基群体，具有不同病害感染史的野生群体和处于同一流域，但具有不同病害选择压力的野生群体中和抗病性状的相关性，本研究发现SNP198位点C等位基因和美洲牡蛎的抗Dermo性状紧密相关。但SNP198是个同义突变，不改变编码氨基酸的序列，所以本研究还分析了SNP198位点和cvSI-1基因5’调控区多态性位点的连锁性来解释其抗病相关性。通过染色体步移和重测序的方法，获得长631bp的5’调控区，包含22个SNPs和3个插入/缺失多态性位点。利用野生群体死亡前和死亡后的材料，分析5’调控区-404位，长25bp的插入/缺失位点和SNP198位点的连锁性。在Dermo造成大部分死亡的死亡后群体中，-404插入/缺失的缺失等位基因上升了14.4%(p=0.0595)，而SNP198的C等位基因仅上升了3.4%(p=0.5756)。缺失等位基因和C等位基因在80.3%的牡蛎中具有连锁性。具有缺失等位基因的牡蛎经Dermo病原体Perkinsus marinus感染后，其cvSI-1的表达量显著提高(p=0.017)。本研究结果提示我们，5’调控区的变异会提高cvSI-1的转录活性，因此通过抑制寄生虫的蛋白酶活性而对其产生抗性。2.丝氨酸蛋白酶抑制因子2基因(cvSI-2)和美洲牡蛎抗病性状的相关性一种新的丝氨酸蛋白酶抑制因子，cvSI-2，已经从美洲牡蛎的细胞质中分离出来，其能够体外抑制P. marinus的活性。本研究分析了cvSI-2基因的多态性和美洲牡蛎抗病性状的相关性。利用cvSI-2的cDNA序列搜索NCBI数据库，共得到33条美洲牡蛎的EST序列。将低质量的序列去除后，27条高质量的EST序列用于比对和SNP位点的筛查，获得5个高质量SNP位点，包括4个同义突变和1个非同义突变。随后，对非同义突变SNP226(A/G)和美洲牡蛎的抗病相关性进行分析。SNP226的GG基因型在两个家系死亡后的材料中分别上升17.0%(p=0.0005)和16.9%(p=0.0634)。另外，SNP226的G等位基因在选育的抗病品系中的频率最高，为93.5%，显著高于其野生基群体CTW (69.6%)和两个选育的易感品系FMF (63.0%)和UMFS (51.3%)。在Martha’s Vineyard、特拉华湾和切斯皮克湾三个不同水域，病害选择压力大的野生群体G等位基因频率都要高于病害选择压力小的群体。SNP226的G等位基因编码缬氨酸，A等位基因编码异亮氨酸。此位的缬氨酸可能能够增强cvSI-2蛋白和Dermo病原体丝氨酸蛋白酶结合的亲和性，从而增强了抑制其活性的能力，最终在美洲牡蛎的抗Dermo病害方面发挥重要作用。3.美洲牡蛎高密度遗传连锁图谱的构建及抗病性状的QTL定位利用RAD测序技术，构建了美洲牡蛎高密度遗传连锁图谱。通过分析标记死亡前后基因型频率的变化，定位抗病性状相关的QTL。雌性连锁图谱含12个连锁群，共1,350个标记，覆盖1,081.24cM，最大图距12.24cM，平均图距0.80cM，覆盖率为98.14%。雄性连锁图谱含10个连锁群，共1,236个标记，覆盖723.61cM，最大图距10.05cM，平均图距0.59cM，覆盖率为98.28%。雌性图谱中，88个标记在死亡后群体中基因型频率发生显著变化(p <3.74×10~(-5)，经Bonferroni校正后的P值)，其中有62个(76.3%)标记是成对或者成簇分布。雌性图谱检测到11个高精度QTL，分布在5个连锁群，其中第8号连锁群的QTL数目最多，为4个。雄性图谱中，80个标记在死亡后群体中基因型频率发生显著变化(p <4.04×10~(-5)，经Bonferroni校正后的P值)，其中有57个(71.3%)标记是成对或者成簇分布。雄性图谱检测到5个高精度的QTL，分布在4个连锁群，在第7号连锁群检测到2个。4.病害对特拉华湾美洲牡蛎幼体群体遗传结构的影响
本研究利用8个抗病相关的SSR标记和9个中性的SSR标记对特拉华湾由北向南Hope Creek (HC)、Round Island (RI)、Beadons (BD)及Cape Shore (CS)4个采样点的美洲牡蛎幼体群体进行了群体遗传学分析。如果用17个微卫星位点分析两两群体间的遗传分化系数(Fst)，Fst的分布范围为-0.0016-0.0015。最南部群体CS和最北部群体HC间的Fst值最大，为0.0015，但是和0没有显著性差异(p>0.0083，经Bonferroni校正后的P值)。如果用8个抗病相关的微卫星位点进行分析， Fst的分布范围为-0.0052-0.0013。CS群体和HC群体间的Fst值最大，为0.0013。但是其Fst值仍和0没有显著性差异(p=0.8417)。同样地，中性的微卫星位点也没有检测到这4个群体间的遗传分化。这说明特拉华湾幼体群体是个均一的大群体，没有出现遗传分化。5.特拉华湾美洲牡蛎群体有效群体大小(Ne)的估算
具有高繁殖力和III型存活类型的美洲牡蛎中极有可能存在“少数个体维持大群体”的现象。为了验证特拉华湾美洲牡蛎群体中是否存在这种现象，本研究估算了其有效群体的大小(Ne)，并比较特拉华湾的Ne在不同病害选择压力群体间和时间上的变化。对特拉华湾5个采样点，2006年和2009年成体牡蛎和幼体牡蛎群体样品在7个中性的SSR位点进行分型，利用三个双样品法和两个单样品法估测特拉华湾美洲牡蛎群体的Ne。结果显示，成体牡蛎和幼体牡蛎间的基因丰富度没有显著性差异，从而不支持“少数个体维持大群体”的理论。5种方法估算得到的Ne值差异较大，总体而言，所得Ne值较小，并且通常没有获得有效的上限估算值。幼体群体的Ne值分布范围是140-440，比成体群体的Ne值要小(589–2,779)。通过混合所有成体的样品所估算的Ne值提示着整个海湾的有效群体大小可能较大。本研究结果表明，特拉华湾某一批幼体牡蛎可能是由少数的成体牡蛎繁殖而来的，并存在一定的遗传漂变。但是这种小范围的遗传漂变并不影响整个湾成体群体的遗传变化，因为成体群体是每年很多批幼体群体积累多年的结果。
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 oyster
SNP198of 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 oyster
A 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 oyster
The 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 Bay
Spat 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 Bay
The 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.
引文
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