Molecular Parentage Analysis Research Articles

Odor cues rather than personality affect tadpole deposition in a neotropical poison frog.

Animals constantly need to evaluate available external and internal information to make appropriate decisions. Identifying, assessing, and acting on relevant cues in contexts such as mate choice, intra-sexual competition, and parental care is particularly important for optimizing individual reproductive success. Several factors can influence decision-making, such as external environmental cues and the animal's own internal state, yet, we have limited knowledge on how animals integrate available information. Here, we used an entire island population (57 males, 53 females, and 1,109 tadpoles) of the neotropical brilliant-thighed poison frog Allobates femoralis to investigate how 2 factors (olfactory cues and personality traits) influence the ability of males to find and use new resources for tadpole deposition. We experimentally manipulated the location of tadpole deposition sites and their associated olfactory cues, and repeatedly measured exploration and boldness in adult males. We further reconstructed tadpole deposition choices via inferred parent-offspring relationships of adult frogs and tadpoles deposited in our experimental pools using molecular parentage analysis. We found that the discovery and use of new rearing sites were heavily influenced by olfactory cues; however, we did not find an effect of the measured behavioral traits on resource discovery and use. We conclude that in highly dynamic environments such as tropical rainforests, reliable external cues likely take priority over personality traits, helping individuals to discover and make use of reproductive resources.

Molecular Parentage Analysis in Aquaculture: Principles, Applications, and Challenges: A Review

Molecular parentage analysis is a powerful tool for reconstructing pedigrees and estimating genetic parameters in aquaculture species. It is based on the comparison of DNA marker genotypes between offspring and potential parents. This review provides a concise overview of molecular parentage analysis in aquaculture, covering its principles, methods, applications, challenges, and limitations. It describes common DNA markers, including microsatellites and single nucleotide polymorphisms (SNPs), used in parentage analysis and the criteria for their selection. The software and statistical methods for assigning parentage and evaluating the accuracy and power of the assignment are also discussed. This review demonstrates applications for estimating genetic parameters, investigating inbreeding, evaluating reproductive success, and improving selective breeding programs. In conclusion, while molecular parentage analysis is a valuable tool for improving genetic management in aquaculture, careful planning, implementation, and interpretation are essential.

Odor cues rather than personality affect tadpole deposition in a neotropical poison frog.

Animals constantly need to evaluate available external and internal information to make appropriate decisions. Identifying, assessing, and acting on relevant cues in contexts such as mate choice, intra-sexual competition, and parental care is particularly important for optimizing individual reproductive success. Several factors can influence decision-making, such as external environmental cues and the animal's own internal state, yet, we have limited knowledge on how animals integrate available information. Here, we used an entire island population (57 males, 53 females, and 1,109 tadpoles) of the neotropical brilliant-thighed poison frog Allobates femoralis to investigate how 2 factors (olfactory cues and personality traits) influence the ability of males to find and use new resources for tadpole deposition. We experimentally manipulated the location of tadpole deposition sites and their associated olfactory cues, and repeatedly measured exploration and boldness in adult males. We further reconstructed tadpole deposition choices via inferred parent-offspring relationships of adult frogs and tadpoles deposited in our experimental pools using molecular parentage analysis. We found that the discovery and use of new rearing sites were heavily influenced by olfactory cues; however, we did not find an effect of the measured behavioral traits on resource discovery and use. We conclude that in highly dynamic environments such as tropical rainforests, reliable external cues likely take priority over personality traits, helping individuals to discover and make use of reproductive resources.

Development of an informative SNP panel for molecular parentage analysis in large yellow croaker (Larimichthys crocea)

Large yellow croaker is a marine fish with high industrial value. Its traditional breeding in China is established through mass selection and family-based selective breeding. Pedigree records are essential in aquaculture for estimating genetic merits when family-based breeding is applied. Pedigree records can be traced through individual tagging, which is costly and not practical for small fish. Alternatively, the parentage of an individual can be tracked by using DNA markers. Single nucleotide polymorphisms (SNPs) have been widely used in aquaculture for parentage assignments due to their even distribution across the genome and are highly adaptive for robust genotyping of a large number of animals. This study aimed to develop an informative SNP panel for paternity assignment in large yellow croaker and evaluate its accuracy using individuals (n = 635) with known pedigrees. A total of 635 samples randomly selected from 11 families and a mixed group were genotyped using the “NingXin-III 55 K” SNP array. High-quality SNPs with high polymorphism were targeted. The informativeness of the final SNP panel was assessed based on genetic diversity (observed (Hobs) and expected heterozygosity (Hexp) and marker parameters such as discrimination power (DP), probability of exclusion (PE), and polymorphism information content (PIC)). The final panel consisted of 141 highly informative SNPs (DP: 0.610–0.625, PE: 0.111–0.125, PIC: 0.361–0.375). The cumulative probability of exclusion (CPE) for the selected panel (CPE > 0.999999989) met the standard criteria for animal DNA testing (CPE > 0.999). A successful parentage assignment for 635 individuals was performed using the selected SNPs with a probability >99.99%. In addition, the results were validated with principal component analysis and phylogenetic tree. The findings prove that the developed SNP panel enables accurate parentage assignment in the breeding population of large yellow croaker.

Breeding Asian seabass to increase survival against big belly disease and growth

Asian seabass (Lates calcarifer) is an important foodfish species. It has been cultured in Singapore since the 1980s. A selective breeding program to improve growth, meat quality and disease resistance has been ongoing in Singapore since 2004. In 2012, outbreaks of the big belly disease (BBD) occurred in two batches of offspring generated by a mass-cross among F1 brooders in a hatchery. After the outbreaks, only 437 survived in 6000 F2 juveniles. Molecular parentage analysis with 10 microsatellites revealed that four parents (F1) produced high percentage of offspring that survived the BBD outbreaks. Eighty-two BBD survivor offspring (F2) were selected based on their growth performance at 90 days post hatch (dph), family origin and allelic diversity. They were used as brooders to generate F3. In a period of five years, these F2 brooders produced six batches of offspring, where no outbreak of BBD occurred. In the same period, outbreaks of BBD occurred in 4 out of 18 batches produced by F2 brooders selected for growth, with mortality rates ranging from 85.1% to 95.4%. In F3, the average body weight (57.1 ± 1.09 g, n = 3068) of the line for BBD survival was slightly lower than that (60.6 ± 2.29 g, n = 4280) of the line for growth at 90 dph. In both lines, the body weight of F3 was much higher than that (46.90 ± 0.19 g, n = 12117) of F2. The genetic diversity in the line for BBD survival (F3) was much lower than that in F3 line for growth due to small founder population. These data indicate that the selection of BBD survival during BBD outbreaks using molecular parentage analysis is effective to reduce BBD outbreaks. Avoiding further reduction of genetic diversity is essential in the line selected for increased survival against BBD.

Occurrence of dorsal fin and opercular deformities and their effects on body weight in Asian seabass

Asian seabass is an important aquaculture species. Dorsal fin and opercular deformities have been observed in hatcheries of this fish. However, it is unclear what causes these anomalies, and whether they affect growth performance. To quantify the occurrence of these anomalies, analyse their effect on growth, and trace their family origins, 8535 offspring generated from 13 independent mass-crosses were genotyped with 10 microsatellites. Dorsal fin and opercular deformities were recorded. Body weight at 90 dph were measured for each offspring. The occurrence frequencies of dorsal fin and opercular deformities were 2.30% and 2.16% respectively, with the former ranging from 1.38% to 5.16%, and the latter ranging from 1.38% to 4.84% for different batches of fish. These deformities significantly reduced the body weight, indicating that these deformities not only affected the appearance, but also decreased growth performance. Molecular parentage analysis revealed that fish with either dorsal fin or opercular deformities came from most of the families in each mass-cross, rather than from a few families or a few brooders, which suggests that heritabilities of these deformities might be low.

Where are the beachmasters? Unexpectedly weak polygyny among southern elephant seals on a South Shetland Island

AbstractIntraspecific variation in animal mating systems can have important implications for ecological, evolutionary and demographic processes in wild populations. For example, patterns of mating can impact social structure, dispersal, effective population size and inbreeding. However, few species have been studied in sufficient detail to elucidate mating system plasticity and its dependence on ecological and demographic factors. Southern elephant seals (Mirounga leonina) have long been regarded as a textbook example of a polygynous mating system, with dominant ‘beachmaster’ males controlling harems of up to several hundred females. However, behavioural and genetic studies have uncovered appreciable geographic variation in the strength of polygyny among elephant seal populations. We, therefore, used molecular parentage analysis to investigate patterns of parentage in a small satellite colony of elephant seals at the South Shetland Islands. We hypothesised that dominant males would be able to successfully monopolise the relatively small numbers of females present in the colony, leading to relatively high levels of polygyny. A total of 424 individuals (comprising 33 adult males, 101 adult females and 290 pups) sampled over 8 years were genotyped at 20 microsatellites and reproductive success was analysed by genetically assigning parents. Paternity could only be assigned to 31 pups (10.7%), despite our panel of genetic markers being highly informative and the genotyping error rate being very low. The strength of inferred polygyny was weak in comparison to previous genetic studies of the same species, with the most successful male fathering only seven pups over the entire course of our study. Our results show that, even in a species long regarded as a model for extreme polygyny, male reproductive skew can vary substantially among populations.

Patterns of reproductive success among reintroduced Atlantic salmon in two Lake Champlain tributaries

Reintroduction programs are increasingly implemented to regenerate self-sustaining salmon populations. The extent to which returning adults successfully produce surviving offspring is useful for informing reintroduction efforts but is often unknown, as is the genetic makeup of those offspring. We investigated the patterns of reproductive success (RS) among returning Atlantic salmon reintroduced as juveniles in two tributaries of Lake Champlain, by combining extensive river surveys and a molecular parentage analysis. Tissue samples collected from returning adults and their young-of-the-year (YOY) offspring produced in the wild were genotyped using microsatellite loci. Our results suggest that a low proportion of returning adults produced the small numbers of YOY detected in either tributary (range 9.9–13.7%, or 17 of 124 and 8 of 81 returning adults, respectively) and that 81.6% and 1.2% of YOY assigned to a pair of sampled returning adults. These results demonstrate that adults (of different life history strategies) can successfully reproduce in both of the studied tributaries (Winooski and Boquet Rivers) and that unsampled individuals could have recolonized a given tributary. Nevertheless, a larger number of breeders would need to produce surviving offspring in subsequent years to demographically augment the reintroduced population whilst avoiding short-term issues associated with low genetic diversity and inbreeding. Our results also point to underlying ecological problems that may have reduced RS and potential genetic issues within the hatchery-reared population being used for supplementation. Further adaptive management will be needed to ensure self-sustaining populations in this system.

Genetic monogamy despite frequent extrapair copulations in "strictly monogamous" wild jackdaws.

“Monogamy” refers to different components of pair exclusiveness: the social pair, sexual partners, and the genetic outcome of sexual encounters. Avian monogamy is usually defined socially or genetically, whereas quantifications of sexual behavior remain scarce. Jackdaws (Corvus monedula) are considered a rare example of strict monogamy in songbirds, with lifelong pair bonds and little genetic evidence for extrapair (EP) offspring. Yet jackdaw copulations, although accompanied by loud copulation calls, are rarely observed because they occur visually concealed inside nest cavities. Using full-day nest-box video surveillance and on-bird acoustic bio-logging, we directly observed jackdaw sexual behavior and compared it to the corresponding genetic outcome obtained via molecular parentage analysis. In the video-observed nests, we found genetic monogamy but frequently detected forced EP sexual behavior, accompanied by characteristic male copulation calls. We, thus, challenge the long-held notion of strict jackdaw monogamy at the sexual level. Our data suggest that male mate guarding and frequent intrapair copulations during the female fertile phase, as well as the forced nature of the copulations, could explain the absence of EP offspring. Because EP copulation behavior appeared to be costly for both sexes, we suggest that immediate fitness benefits are an unlikely explanation for its prevalence. Instead, sexual conflict and dominance effects could interact to shape the spatiotemporal pattern of EP sexual behavior in this species. Our results call for larger-scale investigations of jackdaw sexual behavior and parentage and highlight the importance of combining social, sexual, and genetic data sets for a more complete understanding of mating systems.

Molecular Genetics Unveiled Unknown Family Relationships and Hybrids in an Ex-Situ Colony of African Penguins (Spheniscus demersus).

Genealogical relationships among colony members, inbreeding status, and presence of hybrids are crucial data that can assist zoo curators in captive colony management and decision-making on relocation for reproduction. This study employed molecular markers to study a large colony (n = 56) of African Penguin hosted in an Italian biopark. A panel of 15 STRs (single tandem repeats) was selected, and genotype data were analyzed using COLONY software to determine parentage relationships and compare the existing studbook information to a pedigree built from genetic analyses. The existence of extra-pair mating and the presence of hybrids were investigated: discrepancies in kinship relationships emerged following molecular parentage analysis and 10 unknown genetic relationships were revealed. Infidelity of one member of the pair was observed in 6 cases and extra-pair copulation was assessed by genetic analysis in 2 episodes. One member of the colony was found to be a hybrid (S. demersus × S. humboldti); his progeny, derived by extra-pair copulation, was traced. Three other hidden hybrids were discovered and assessed using the identified candidate private alleles. Overall, our results demonstrate that molecular methods to confirm parentage and analyze relatedness among colony members are a valuable tool to complement studbook-based genetic management of African penguin captive populations. Because a variety of behavioral dynamics (e.g., extra-pair mating) can make observations ineffective in some species and because molecular markers outperform studbook in identifying the presence of hybrids, reliance on studbook information alone is not recommended.

Who eats whom, when and why? Juvenile cannibalism in fish Asian seabass

While juvenile cannibalism plays an important role in the evolution of organisms in natural populations, it is a serious problem in aquaculture. A number of genetic and environmental factors result in different rates of cannibalism. Whether there is kin recognition in juvenile cannibalism in fish is poorly understood. We studied cannibalism and kinship recognition in juveniles of Asian seabass using molecular parentage analysis with polymorphic microsatellites. In the three mass crosses, under an ordinary feeding scheme without size grading, the rate of juvenile loss due to cannibalism was 1.08% per day. In the group without feeding for 24 h, 2.30% ± 0.43% of offspring per day were lost within 24 h due to cannibalism. We detected that juveniles avoided cannibalizing their siblings when they were not hungry, whereas cannibalism among siblings increased when they were hungry. These data suggest that there is kin discrimination in fish cannibalism. Raising genetically closely related offspring in the same tanks and appropriate levels of feeding may reduce the rate of cannibalism. We hypothesized that the chemical cues for kin discrimination might be secreted by fish skins. To test this hypothesis, we analyzed gene expression profiles in the skins of juveniles under slightly and very hungry conditions using RNA sequencing and bioinformatics analysis. Genes differently expressed under slightly and very hungry conditions were identified. Among them, genes from the trypsin family were significantly down-regulated under starved conditions, suggesting that they may play a role in kin discrimination.

Genetic parameter estimates and genotype by environment interaction analyses for early growth traits in grass carp (Ctenopharyngodon idella)

Despite the importance of grass carp (Ctenopharyngodon idella) in aquaculture worldwide, selective breeding programs for growth traits remain limited. This study aimed to assess the genetic effects and extent of genotype by environment interactions (G × E) for early growth traits in grass carp. Fifty-eight full-sib grass carp families (n = 1704; 20 dams and 15 sires), cultured in two ponds, were reconstructed using molecular parentage analysis. Based on the animal model and restricted maximum likelihood (REML) algorithm, the heritability of standard length (SL), body weight (BW), and condition factor (K) of grass carp at 40 dph (days post-hatch) were calculated as 0.304, 0.307, and 0.150, respectively (P 0.05). A significant interaction effect was found between family and environment (P < 0.01), while Pearson’s correlations for SL and BW of grass carp in different ponds were 0.773 and 0.630, respectively. Differential stabilities among families were responded to environments. These results indicate that the early growth traits of grass carp could be improved by exploiting additive genetic effects through selective breeding. Environmental effects and family stability should be taken into account in practical breeding programs.

Testing and validating a modified CTAB DNA extraction method to enable molecular parentage analysis of fertilized eggs and larvae of an emerging South African aquaculture species, the dusky kob Argyrosomus japonicus.

This study describes the successful implementation of a modified cetyltrimethyl ammonium bromide (CTAB) protocol to isolate genomic DNA and amplify 14 microsatellite markers from fertilized eggs and larvae of an emerging South African farmed marine fish species, the dusky kob Argyrosomus japonicus. To test and validate the efficiency of this method, genetic data were utilized to resolve parentage and kinship of first-generation (F1) offspring produced in mass-spawning events of wild broodstock fish in a commercial hatchery.

Practical Considerations of Molecular Parentage Analysis in Fish

AbstractPedigree 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 extractDNAfrom a large number of samples, select the right types ofDNAmarkers, 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.

Genetic monogamy despite variable ecological conditions and social environment in the cooperatively breeding apostlebird

Mating strategies may be context-dependent and may vary across ecological and social contexts, demonstrating the role of these factors in driving the variation in genetic polyandry within and among species. Here, we took a longitudinal approach across 5 years (2006–2010), to study the apostlebird (Struthidea cinerea), an Australian cooperatively breeding bird, whose reproduction is affected by ecological “boom and bust” cycles. Climatic variation drives variation in the social (i.e., group sizes, proportion of males and females) and ecological (i.e., plant and insect abundance) context in which mating occurs. By quantifying variation in both social and ecological factors and characterizing the genetic mating system across multiple years using a molecular parentage analysis, we found that the genetic mating strategy did not vary among years despite significant variation in rainfall, driving primary production, and insect abundance, and corresponding variation in social parameters such as breeding group size. Group sizes in 2010, an ecologically good year, were significantly smaller (mean = 5.8 ± 0.9, n = 16) than in the drought affected years, between 2006 and 2008, (mean = 9.1 ± 0.5, n = 63). Overall, apostlebirds were consistently monogamous with few cases of multiple maternity or paternity (8 of 78 nests) across all years.

Molecular Parentage Analysis Is Essential in Breeding Asian Seabass

In aquaculture species, maintaining pedigree information and genetic variation in each generation is essential, but very difficult. In this study, we used nine microsatellites to genotype 2,520 offspring from four independent full-factorial crosses (10 males ×10 females) of Asian seabass to reconstruct pedigree and monitor the change of genetic variations. In all four crosses, over 96.8% of the offspring could be assigned to their parents, indicating the high power of the nine microsatellites for parentage assignment. This study revealed several interesting results: (1). In all four crosses, the contribution of parents to offspring was significantly uneven, and some dominant breeding fishes (i.e. brooders) were found; (2). In two mass crosses where the brooders were carefully checked for reproductive status, a majority (≥90%) of brooders contributed to offspring, whereas in another two crosses, where the brooders were randomly picked without checking reproductive status, only a few brooders (40.0–45.0%) produced offspring; (3). Females had more problems in successful spawning compared to males; and (4). In the two crosses where a few brooders produced offspring, there was a substantial loss in allelic (24.1–34.3%) and gene (20.5–25.7%) diversities in offspring, while in the other two crosses, the majority of allelic (96.8–97.0%) and gene diversities (94.8–97.1%) were maintained. These observations suggest that a routine molecular parentage analysis is required to maintain both allelic and gene diversity in breeding Asian seabass.

Reduced fitness of Atlantic salmon released in the wild after one generation of captive breeding

Salmonids rank among the most socioeconomically valuable fishes and the most targeted species by stocking with hatchery-reared individuals. Here, we used molecular parentage analysis to assess the reproductive success of wild- and hatchery-born Atlantic salmon over three consecutive years in a small river in Québec. Yearly restocking in this river follows a single generation of captive breeding. Among the adults returning to the river to spawn, between 11% and 41% each year were born in hatchery. Their relative reproductive success (RRS) was nearly half that of wild-born fish (0.55). RRS varied with life stage, being 0.71 for fish released at the fry stage and 0.42 for fish released as smolt. The lower reproductive success of salmon released as smolt was partly mediated by the modification of the proportion of single-sea-winter/multi-sea-winter fish. Overall, our results suggest that modifications in survival and growth rates alter the life-history strategies of these fish at the cost of their reproductive success. Our results underline the potential fitness decrease, warn on long-term evolutionary consequences for the population of repeated stocking and support the adoption of more natural rearing conditions for captive juveniles and their release at a younger stage, such as unfed fry.

Precision of host sanctions in the fig tree–fig wasp mutualism: consequences for uncooperative symbionts

Host sanctions that reduce the relative fitness of uncooperative symbionts provide a mechanism that can limit cheating and thus stabilise mutualisms over evolutionary timescales. Sanctions have been demonstrated empirically in several mutualisms. However, if multiple individual symbionts interact with each host, the precision with which individual cheating symbionts are targeted by host sanctions is critical to their short- and long-term effectiveness. No previous empirical study has directly addressed this issue. Here, we report the precision of host sanctions in the mutualism between fig trees and their pollinating wasps. Using field experiments and molecular parentage analyses, we show that sanctions in Ficus nymphaeifolia act at the level of entire figs (syconia), not at the level of the individual flowers within. Such fig-level sanctions allow uncooperative wasps, which do not bring pollen, to avoid sanctions in figs to which other wasps bring pollen. We discuss the relevance of sanction precision to other mutualisms.

Clones, hermaphrodites and pregnancies: nature's oddities offer evolutionary lessons on reproduction

AbstractI love the term ‘natural history’ because it encapsulates the sentiment that nature's operations have evolutionary etiologies. Charles Darwin was a natural historian par excellence and his elucidation of natural selection, artificial selection, and sexual selection fundamentally changed how scientists interpret the origins of biological features previously ascribed to sentient craftsmanship by supernatural agents. Darwin's insights on evolutionary forces grew from his exceptional knowledge of natural history, yet two key topics steeped in natural history – sex and reproductive genetics – remained poorly understood (and probably even shunned) in Darwin's Victorian era. That situation changed dramatically in the latter half of the 20th century with societal awakenings about sexuality that also happened to coincide with the introduction of molecular parentage analyses that unveiled a plethora of formerly hidden ‘sexcapades’ throughout the biological world. Here I summarize some of the evolutionary revelations that have emerged from selection theory as applied to genetic and phylogenetic information on clonality, hermaphroditism, and pregnancy, three procreative phenomena that are relatively rare in vertebrate animals and thus offer alternative evolutionary perspectives on standard reproductive modes. Collectively, these three peculiarities of nature illustrate how the abnormal in biology can enlighten evolutionary thought about the norm.

Multiple mating and clutch size in invertebrate brooders versus pregnant vertebrates

We summarize the genetic literature on polygamy rates and sire numbers per clutch in invertebrate animals that brood their offspring and then compare findings with analogous data previously compiled for vertebrate species displaying viviparity or other pregnancy-like syndromes. As deduced from molecular parentage analyses of several thousand broods from more than 100 "pregnant" species, invertebrate brooders had significantly higher mean incidences of multiple mating than pregnant vertebrates, a finding generally consistent with the postulate that clutch size constrains successful mate numbers in species with extended parental care. However, we uncovered no significant correlation in invertebrates between brood size and genetically deduced rates of multiple mating by the incubating sex. Instead, in embryo-gestating animals otherwise as different as mammals and mollusks, polygamy rates and histograms of successful mates per brooder proved to be strikingly similar. Most previous studies have sought to understand why gestating parents have so many mates and such high incidences of successful multiple mating; an alternative perspective based on logistical constraints turns the issue on its head by asking why mate numbers and polygamy rates are much lower than they theoretically could be, given the parentage-resolving power of molecular markers and the huge sizes of many invertebrate broods.