Parentage assignment in Haliotis midae L.: a precursor to future genetic enhancement programmes for South African abalone

The establishment and evaluation of family lines using pedigree information provides an advanced understanding of the variability that exists for complex, economically valuable traits and is a necessary step in the execution of an effective breeding programme. The aim of this study was to assign parentage to mass‐spawned Haliotis midae juveniles using species‐specific microsatellite markers. Screening of wild abalone individuals revealed that the nine loci selected complied with the minimum requirements for parentage analyses: a null allele frequency <5% as well as a high number and frequency of alleles per locus. A total of 598 individuals were genotyped (198 breeding individuals and 400 F1 progeny) from two farms, with parentage results yielding 91% and 90% successful assignment for Farms A and B respectively. This study, therefore, provided the necessary pedigree information required for controlled breeding of individual adult abalone and indicated the usefulness of the panel of microsatellite markers selected for parentage assignment.     

中华绒螯蟹微卫星多重PCR体系的建立及其亲子鉴定应用

利用自主开发的微卫星标记对中华绒螯蟹(Eriocheir sinensis)进行微卫星多重PCR体系构建,最终成功建立4组多重体系,每组体系包含4个微卫星位点,并成功应用于3个家系的亲子鉴定中。结果显示:(1)本研究筛选的16个微卫星标记,平均观测杂合度为0.8206,平均期望杂合度为0.8164,平均多态信息含量为0.7927,具有丰富的多态性;(2)运用Cervus 3.0软件对已知系谱信息的3个中华绒螯蟹家系共95个子代个体进行亲子鉴定分析,结果显示,选用任意两组微卫星多重PCR体系时,累积实际正确鉴定率均超过94.74%;使用任意三组微卫星多重PCR体系时,累积实际正确鉴定率均大于98.95%;使用四组微卫星多重PCR体系时,累积实际正确鉴定率达到100%。并且当选用组合1、2和组合3,或者选用组合1、3和组合4时,对3个家系的累积正确鉴定率达到了100%,因此选用这些组合不但可以获得准确的系谱信息,还能减少工作量,降低成本。本研究构建的微卫星多重PCR体系能为中华绒螯蟹的种群选育和家系管理提供便捷、高效的途径。     

大黄鱼微卫星多重PCR体系的建立及其应用

利用本实验室开发的微卫星标记,通过优化退火温度、引物浓度、循环次数等条件,建立了3组大黄鱼微卫星多重PCR体系,每组包含3个微卫星位点,用3组微卫星多重PCR分析了一个大黄鱼选育群体JD-01的遗传多样性。结果显示,该群体的平均等位基因为12.111,平均有效等位基因为7.408,平均多态信息含量为0.846,平均观测杂合度和期望杂合度分别为0.825、0.868,香农多样性指数为2.165。运用Cervus 3.0软件,对已知系谱关系的9个大黄鱼家系及对应亲本进行了亲子鉴定分析,以验证3组微卫星多重PCR在亲子鉴定中的准确性。结果显示,使用该3组微卫星多重PCR体系进行亲子鉴定准确率为100%。大黄鱼微卫星多重PCR体系的建立为分析群体的遗传多样性、亲子鉴定和辅助家系管理提供了一种高效的技术手段。     

Development and evaluation of a high‐throughput single nucleotide polymorphism multiplex assay for assigning pedigrees in common carp

Accurate pedigree information is critical when managing animal breeding programmes and ensure the highest rate of genetic gain. The abundance of available genomic data and the development of high‐throughput genotyping platforms have facilitated the use of single nucleotide polymorphisms (SNPs) as the best DNA markers for genomic selection studies. Furthermore, the superior qualities of SNPs compared with those of microsatellite markers allow standardization between laboratories, which is crucial for developing an international set of markers for use in traceability studies. The objective of this study was to develop a high‐throughput SNP array to assign common carp pedigrees accurately. A 48‐SNP array was developed based on the Fluidigm genotyping platform, and a phylogenetic analysis was performed to distinguish different pedigrees. A likelihood‐based approach was used to infer parental pairs, and the pair with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non‐parentage) was assigned. The SNP genotypes of the offspring and candidate parents tested were collected, and 94% of the offspring were assigned to the most‐likely parent pair, which was consistent with the actual pedigree records. Using this SNP assay will allow implementation of offspring testing at large commercial farms where improved accuracy of pedigree assignments and genetic evaluations will increase genetic gains in the common carp aquaculture industry.     

Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

Parentage analysis of tropical spiny lobster (Panulirus homarus) by microsatellite markers

Parentage analysis is of vital importance for hatchery production and breeding programmes. Two multiplex PCR protocols including seven tropical spiny lobster (Panulirus homarus) microsatellites (Pho‐G06, Pho‐G25, Pho‐G53, Pho‐G62, Pho‐G74, Pho‐G89 and Pho‐G100) were introduced for parentage assignment. All loci were polymorphic with allele sizes from 113 to 337 base pairs (bp), observed alleles (k) from two to seven and polymorphic information content (PIC) of 0.25 to 0.73. Twenty‐four stage‐3 phyllosoma larvae (39 days posthatching) were collected for paternity exclusion study using selected microsatellites. Exclusion‐based parentage analysis unambiguously assigned 83% of fry (20 of 24) to a single female parent. Of ten putative female parents, five have contributed to the 20 allocated offspring, with one being true parent of 11. Four others were assigned to two or more potential female parents, probably due to genotyping error or the presence of null allele. The exclusion power (EP) for the seven loci varied between 13% and 54% with known genotypes of one parent (P1) and 19% and 71% for given the genotypes of both parents (P2). The theoretical combined parentage exclusion (cEP) power for all seven microsatellites was P1 = 95% and P2 = 99%. The power of discrimination for each locus varied between 0.18 and 0.86. This report presents the first study to utilize microsatellite markers for successful parentage assignment of P. homarus. The selected microsatellites provide a practical tool for parentage analysis in hatchery production of juveniles as well as in future commercial breeding programme of tropical spiny lobsters.     

凡纳滨对虾SNP标记开发与家系亲缘关系验证分析

本研究基于转录组序列开发了37个SNP标记,并对22个凡纳滨对虾(Litopenaeus vannamei)家系进行了遗传多样性、家系聚类和亲子鉴定分析,探讨SNP标记在对虾家系选择育种中的应用途径。结果显示,37个SNP位点在凡纳滨对虾22个家系中的平均期望杂合度(He)和平均观测杂合度(Ho)分别为0.38和0.34;平均多态信息含量(PIC)为0.30,属于中度多态性(0.25相似文献   

Evaluating spawning performance among captive Florida pompano Trachinotus carolinus broodstock using microsatellite‐based parentage assignment

Florida pompano has been identified as a promising candidate for commercial‐scale aquaculture production, but to date, little information is available regarding captive broodstock spawning characteristics. Genetic markers were tested for their power in monitoring mating outcomes and potential in analysing heritability of rapid growth trait in Trachinotus carolinus. A total of 20 unrelated adults (10 females and 10 males) were chosen for a hormone‐induced mass spawning event. The 515 fastest growing and 485 slowest growing fish of the total 4852 offspring were considered a selected progeny stock, and fish were collected at 45 days post hatch based on their growth traits. Parentage analyses based on the 20 breeders and 1,000 selected progeny were performed using a total of nine microsatellite markers, a 100% assignment rate was achieved, and a four‐marker set was the minimum number for the parentage assignment. The effective breeding number for the selected progeny was 11 (six females and five males), among which three females and two males were predominant contributors with the total contribution of 95.8% and 94.7% respectively. The proportion of fast‐growing offspring from broodfish and each mating cross (sire/dam) was used for detecting whether variation in growth of the offspring was related to parental stocks. Results showed that three adults and their mated combination exhibited the greatest fast‐growing offspring proportion (69.73% and 55.95%). This research provided new information regarding spawning performance and parental contribution during mass spawning events; both important first steps towards developing improved management strategies for captive Florida pompano broodstock.     

Development and evaluation of microsatellite markers for identification of individual Greenshell™ mussels (Perna canaliculus) in a selective breeding programme

A set of 49 microsatellite loci isolated from the endemic New Zealand Greenshell™ mussel, Perna canaliculus, were evaluated for inclusion in a parentage assignment marker suite by assessing their ease of PCR amplification, allele scoring and conformity to Mendelian inheritance in hatchery-produced families. Ten polymorphic loci (mean H_e = 0.78 and polymorphic information content (PIC) = 0.72) were identified as being suitable for parentage assignment. These 10 microsatellite loci gave a combined non-exclusion probability of < 0.001 (probability that an unrelated parent pair will not be excluded from parentage of an arbitrary offspring), based on allele frequencies from 16 broodstock mussels. Simulations predicted an assignment success rate of 99.9% with all 10 loci and > 95% with the best 5 or more loci (mean PIC = 0.84). In actual parentage assignments, 124 offspring from 8 full-sib families were assigned to the correct parent pair with 4 or more loci. We found evidence for null alleles and extensive size homoplasy in many loci, highlighting the importance of thoroughly characterizing and evaluating microsatellite markers prior to parentage assignment and other applications.     

Assignment of parentage in triploid species using microsatellite markers with null alleles,an example from Pacific oysters (Crassostrea gigas)

Triploid production in aquaculture is increasing because of their more profitable growth and reproduction traits. Triploids are mostly produced through mass spawning techniques, meaning that exact pedigree is unknown. The ability to trace the pedigree of high performing triploids would allow selection of broodstock to perpetuate triploids of greater economic value. This study aimed to develop a method of determining parental assignment in triploids and test its accuracy on triploid oysters. Using a likelihood approach and accounting for null allele frequencies, a method was developed which proved to be efficient at determining the pedigree of triploid oysters. This method was able to provide accurate pedigree on simulated data and two commercial cohorts of triploid oysters. The analysis of the triploid cohorts showed that mass spawning to produce triploid oysters, like that for diploid and tetraploids, results in a strong bias in parental contributions, with the effective population size being 34‐49% lower than the census population. This highlights the need for pedigree control in breeding programs and indicates that the ability to determine parentage of triploids will be a valuable tool for breeding programs.     

A Microsatellite DNA Marker Developed for Identifying Disease‐resistant Population of Giant Black Tiger Shrimp,Penaeus monodon

White spot disease caused by white spot syndrome virus (WSSV) poses major problems that result in huge economic losses each year in shrimp aquaculture throughout the world. In the present study, microsatellite‐based DNA fingerprints have been compared between naturally occurring WSSV disease‐resistant and susceptible populations of giant black tiger shrimp, Penaeus monodon, to find DNA markers. For the first time, we report here a microsatellite locus, which, after amplification by polymerase chain reaction, provides a highly statistically significant DNA fingerprint of 71 bp, only in disease susceptible populations but not in disease‐resistant shrimp populations, whereas a 317 bp band is common in both. The absence of the former DNA marker will be very useful to identify disease‐resistant broodstock of P. monodon for marker‐assisted selection in breeding programs to generate disease‐free shrimps (P. monodon) in the aquaculture industry.     

荧光标记微卫星技术用于凡纳滨对虾不同家系亲权关系鉴定

应用荧光标记微卫星技术对凡纳滨对虾(Litopenaeus vannamei)家系进行亲权鉴定。挑选14个多态信息含量较高的微卫星位点,以人工选育建立的凡纳滨对虾5个全同胞家系为试验材料,采用Cervus 3.0进行亲权分析,并根据家系内个体间的遗传距离进行UPGMA聚类分析。结果表明,14个微卫星位点的平均等位基因数为6,平均多态信息含量为0.6896,平均期望杂合度为0.7309,平均观测杂合度为0.7661,第一亲本、第二亲本和双亲的累计排除率分别为0.99721733、0.99996559和0.99999997;进一步模拟分析表明,要达到亲权鉴定的要求,在双亲性别已知时至少需要5个微卫星位点,双亲性别未知时至少需要6个微卫星位点;模拟分析及试验验证所选用的14个微卫星位点最多可以鉴定1 954个已知性别的亲本或1 203个未知性别的亲本。所选用的14个微卫星标记可在生产及科研试验中用于获取凡纳滨对虾系谱信息。     

Effects of dietary supplementation of a commercial prebiotic Previda® on survival,growth, immune responses and gut microbiota of Pacific white shrimp,Litopenaeus vannamei

A 35‐day feeding trial was conducted to evaluate growth, bacterial populations of the gastrointestinal tract (GIT) and immune responses of Litopenaeus vannamei fed diets containing the commercial prebiotic Previda^®. Diets were formulated to contain Previda^® at 0, 0.2, 0.5, 1.0 or 1.6 g kg^?1 by weight. At the end of the study, differences in weight gain and survival among treatments were not significant (P > 0.05), but denaturing gradient gel electrophoresis analysis revealed that the microbial communities in the GIT changed significantly with the inclusion of dietary Previda^® at different levels. Previda^® was therefore able to selectively modify the microbial communities in the shrimp's GIT. Although individual bacterial species were not identified, enteric populations in shrimp fed the prebiotic at similar levels of inclusion were genetically similar. In addition, shrimp fed Previda^® at 1.6 g kg^?1 responded significantly (P < 0.05) better immunologically with respect to hemocyte phagocytic capacity, haemolymph protein, hyaline cell counts and haemolymph glucose compared with shrimp fed the basal diet. Although shrimp were not exposed to virulent pathogens in this study, the observed upregulation of some of imm‐une responses upon prebiotic supplementation indicates that an improved outcome of such challenges may be anticipated in Previda^®‐fed shrimp under commercial conditions.     

Parentage determination of the mud crab Scylla paramamosain using microsatellite markers

This study evaluated the properties of nine Scylla paramamosain microsatellite loci screened by us previously for inclusion in a parentage assignment marker suite. These nine highly polymorphic markers (mean He=0.847 and PIC=0.830) were determined as being suitable for parentage assignment. Simulations based on allele frequency data from 15 known maternal families (165 individuals) demonstrated that at least four loci were required to assign >95% of offspring to maternal parents with 95% confidence. In actual parentage assignments, all progenies were assigned to the maternal parents with six or more loci, which was similar to the simulation predictions. Our results suggest that this set of microsatellites provide a powerful and efficient tool for identifying pedigree information for selective breeding programmes of S. paramamosain.     

黄河鲤全同胞家系的微卫星标记亲子鉴定

本研究利用11个高度多态的微卫星标记对26个黄河鲤(Cyprinus carpio haematoperus)全同胞家系中的417尾体重较大的正常体色个体(黄河鲤较大个体)和52尾红色个体(红鲤表型个体)进行了亲子鉴定。11个微卫星座位的平均等位基因数、平均观测杂合度(H_o)、平均期望杂合度(H_e)和平均多态信息含量(PIC)分别为8.2、0.792、0.792和0.76。当双亲未知且置信度为95%时,11个座位的累积排除概率达99.79%。除29尾黄河鲤较大个体以外,388尾黄河鲤较大个体和52尾红鲤表型个体准确地找到了其父母本,实际鉴定率为93.82%。通过对子代数目超过20尾的7个黄河鲤家系生长性状的比较分析,本研究成功鉴定出子代生长性状优良的黄河鲤亲本组合及其家系,同时也鉴定出包含隐性红色基因的黄河鲤杂合亲本,为进一步选育表型和体色纯正且生长快速的黄河鲤新品种提供了理论依据和技术手段。     

Characterization of single‐nucleotide polymorphism markers in the Mediterranean mussel,Mytilus galloprovincialis

The Mediterranean mussel, Mytilus galloprovincialis, is one of the most important aquaculture species in Europe. Appropriate molecular markers are required to evaluate genetic resources and to trace genealogies in breeding programmes for improving mussel culture. Microsatellites have been commonly applied to this purpose in other species. However, Mediterranean mussel microsatellites have demonstrated high frequencies of null alleles that hamper accurate estimates of population parameters and confident parentage inferences. As alternative markers, we have characterized in silico 25 potential single‐nucleotide polymorphism (SNP) markers in the Mediterranean mussel from expressed sequence tag (EST) public databases. The genotyping of SNPs was performed using a single‐base extension approach. Their polymorphism was evaluated in 47 individuals from an Atlantic population. Out of the 25 potential SNPs tested, 12 were technically feasible (producing a single amplicon) and polymorphic. All were biallelic and had an unbiased heterozygosity ranging from 0.160 to 0.504. One SNP was from a mitochondrial gene. The combined potential of nuclear SNPs for parentage assignment gave an exclusion probability of a false couple of parents of 0.9471. These markers will be useful for evaluating resources and tracing genealogies in genetic breeding programmes implemented to solve the main problems of mussel culture.     

A highly accurate, single PCR reaction for parentage assignment in Senegal sole based on eight informative microsatellite loci

From a battery of microsatellite markers (100 loci), recently identified by our group, we have selected eight for parentage assignment in Senegal sole ( Solea senegalensis ). This tool is based on microsatellite loci obtained from four genomic DNA libraries and one cDNA library. Within the eight loci (six from anonymous genomic DNA sequences and two located in expressed sequence tags of known genes), we have found, in an analysis of a reproductive broodstock, between nine and 16 alleles. The expected heterozygosity was between 0.616 and 0.860. In addition, we have optimized the polymerase chain reaction (PCR) conditions to amplify all loci simultaneously in a single multiplex PCR reaction, and we have tested three lots of male and female (five to six individuals) and three offspring (50–60 larvae each). The use of the eight microsatellite loci, the possibility of amplifying them in a single PCR reaction and the high value of the exclusion probability (0.9992) make this multiplex PCR method a unique tool for parentage assignment.
Finally, analysing one meiotic gynogenetic progeny, we have determined the relative distance of six of these loci to the centromere, and we have also found that all of them are unlinked. All these characteristics confer this tool with a high accuracy for parentage studies and genetic population analyses of Senegal sole.     

Practical Considerations of Molecular Parentage Analysis in Fish

Pedigree information is important in selective breeding programs and hatchery management of aquaculture. However, in aquaculture species, tagging of young fish with physical tags is very difficult. Molecular parentage analyses have been used in reconstructing pedigrees in aquaculture species since the 1990s. Some studies have been carried out to optimize every step to reduce the cost and improve the efficiency of molecular parentage analysis. However, molecular parentage analysis of a large number of individuals is still too expensive because some steps of molecular parentage analysis are very cost‐intensive. In this review, we have summarized the techniques of major steps in parentage analysis, and provided a guide for aquaculture scientists on how to cost‐effectively and rapidly extract DNA from a large number of samples, select the right types of DNA markers, optimize multiplex polymerase chain reaction, and select appropriate methods and software to analyze the genotyping data for reconstructing pedigrees. In addition, we discuss the challenges and future directions of molecular parentage analysis in aquaculture species.     

Development of a Multiplex Microsatellite PCR Assay Based on Microsatellite Markers for the Mud Carp,Cirrhinus molitorella

In this study, we developed a multiplex microsatellite polymerase chain reaction (PCR) assay that consisted of 13 pairs of primers selected from previously developed microsatellite markers in mud carp data obtained in our laboratory. We adjusted the number of cycles, the time of extension, and the primer ratios to make the multiplex PCR system as valuable as possible. Using this system, we performed a genetic analysis and parentage assignment for 2 male and 5 female mud carps and 146 of their progeny. The results showed a range of alleles of 2 < K < 5, with an average of 3.54. The polymorphism information content ranged from 0.271 to 0.697, with an average of 0.4954. The observed heterozygosity ranged from 0.183 to 0.810, with an average of 0.6008. The expected heterozygosity ranged from 0.324 to 0.741, with an average of 0.5572. The null allele frequency was 1.29–27.68%. The computer‐simulated identification was 99%, while the combined exclusion power using all loci was 100% for actual offspring. Thus, this multiplex PCR assay is a new technological tool for selective breeding and a genetic resource for studying the mud carp, Cirrhinus molitorella.