Computer simulations to determine the efficacy of different genome resource banking strategies for maintaining genetic diversity

Genome resource banks (GRBs) and assisted reproductive techniques are increasingly recognized as useful tools for the management and conservation of biodiversity, including endangered species. Cryotechnology permits long-term storage of valuable genetic material. Although, the actual application to endangered species management requires technical knowledge about sperm freezing and thawing, a systematic understanding of the quantitative impacts of various germ plasm storage and use scenarios is also mandatory. In this study, various GRB strategies were analyzed using the historical data from three managed populations of endangered species with varied pedigrees (Eld's deer, Przewalski's horse, and Sumatran tiger). The following types of sperm banks were assessed: (1) a "Wild Bank" consisting of sperm (i.e., genes) from 5 to 10 males unrelated to the managed population and to each other; and (2) a "Best Male" bank containing sperm from only the most genetically valuable males alive in the ex situ population at the time the bank was established. These different bank types were then used to evaluate the effectiveness of different bank usage frequencies. The efficiency of each scenario was assessed by examining the level of inbreeding and gene diversity in the population. Overall, a sperm usage frequency of five times per year was determined to be the most efficient and "wild banks" were highly successful at enhancing genetic diversity. The value of a GRB established from the ex situ population depends on how closely related the banked males are to future generations. A GRB will have significantly less benefit when banked males also produce many successful offspring, or when donors are already genetically over-represented in the population at the time of establishing the GRB.     

Strategies for maintaining genetic diversity in captive populations through reproductive technology

The maintenance of genetic diversity in captive populations is a primary goal of captive breeding plans, and it is becoming increasingly apparent that reproductive technology has much to offer captive breeding programs in attaining this goal. Reproductive technology can best assist captive breeding programs in this task by developing strategies that effectively increase the genetic contribution of new wild founders to a population as well as increase the reproductive life span of existing founders and their close descendents. This will act to reduce genetic drift and inbreeding effects in the population and thereby minimize the loss of genetic diversity. Considering only one aspect of reproductive technology, semen collection, this paper examines some of the genetic considerations that might be used for choosing which males in a population to collect semen from, assuming the goal of the captive breeding program is the preservation of genetic diversity. It is shown that semen collection and preservation, with future intent of artificial insemination, can make significant contributions to the maintenance of genetic diversity if careful consideration is given to the selection of donor males. Finally, the pedigree of the captive population of Asian lions (Panthera leo persica) is used to illustrate some of these genetic concepts that might be important in selecting males as semen donors.     

Population structure and genetic diversity of wild <Emphasis Type="Italic">Helianthus</Emphasis> species from Mozambique

The production of sunflower suffered a major decline in Mozambique after its independence in 1975. Civil war, human activities and environmental damage subjected the species to an ecological stress contributing to reduce the number and size of wild populations. As this reduction is often related to a loss of genetic variation we estimated the genetic diversity within and among populations of wild Helianthus from five districts of Mozambique using RAPD markers. The 44 accessions studied grouped into four major clusters exhibiting structured variability with regard to geographic origin. A high level of genetic diversity (He = 0.350 and I = 0.527) was retained at the population level. The genetic variation among populations was high (59.7%), which is consistent with low gene flow (Nm = 0.338). The proportion of total genetic diversity residing among these populations should be kept in mind to devise different conservation strategies in order to preserve these populations. Currently wild Helianthus genetic resources present in Maputo and Sofala are on the edge of extinction mainly due to excessive urbanization. Therefore, conservation of what remains of this plant genetic diversity is essential for sustainable utilization and can be useful for breeding programs.     

Genetic assessment of a conservation breeding program of the houbara bustard (Chlamydotis undulata undulata) in Morocco,based on pedigree and molecular analyses

Protection and restoration of species in the wild may require conservation breeding programs under genetic management to minimize deleterious effects of genetic changes that occur in captivity, while preserving populations' genetic diversity and evolutionary resilience. Here, through interannual pedigree analyses, we first assessed the efficiency of a 21-year genetic management, including minimization of mean kinship, inbreeding avoidance, and regular addition of founders, of a conservation breeding program targeting on Houbara bustard (Chlamydotis undulata undulata) in Morocco. Secondly, we compared pedigree analyses, the classical way of assessing and managing genetic diversity in captivity, to molecular analyses based on seven microsatellites. Pedigree-based results indicated an efficient maintenance of the genetic diversity (99% of the initial genetic diversity retained) while molecular-based results indicated an increase in allelic richness and an increase in unbiased expected heterozygosity across time. The pedigree-based average inbreeding coefficient F remained low (between 0.0004 and 0.003 in 2017) while the proportion of highly inbred individuals (F > .1) decreased over time and reached 0.2% in 2017. Furthermore, pedigree-based F and molecular-based individual multilocus heterozygosity were weakly negatively correlated, (Pearson's r = −.061 when considering all genotyped individuals), suggesting that they cannot be considered as alternatives, but rather as complementary sources of information. These findings suggest that a strict genetic monitoring and management, based on both pedigree and molecular tools can help mitigate genetic changes and allow to preserve genetic diversity and evolutionary resilience in conservation breeding programs.     

The cryopreservation of ovarian tissue: uses and indications in veterinary medicine

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.     

Sperm cryopreservation in wild animals

Extinction of a species represents the loss of a resource evolved through eons of mutations and natural selection. Reproductive technologies, including artificial insemination, embryo transfer, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, and genome resource banking (GRB) have all been developed with the aim of solving existing problems and preserving genetic material for conservation purposes. Although protocols from domestic or non-threatened related species have been extrapolated to nondomestic and endangered species, usually these reproductive technologies are species-specific and inefficient in many nondomestic species because of insufficient knowledge on their basic reproduction biology and the need for species-specific customization. Since spermatozoa are usually more accessible and come in large numbers compared to oocytes and embryos, they are considered the primary cell type preserved in most emerging GRBs. For this purpose, semen from endangered species is currently cryopreserved to avail long-term storage. Due to the intractability of most exotic species, semen collection without chemical restraint is limited to only a handful of species and individuals. Viable epididymal spermatozoa can be obtained from dead or castrated animals, but this resource is limited. Electroejaculation, artificial vagina, abdominal massage, and/or transrectal, ultrasound-guided, massage of the accessory sex glands of living animals are viable alternative methods of semen collection. The ultimate goal is to adapt and optimize collection and cryopreservation protocols for each species, making it feasible, among other things, to collect gametes in the wild and introduce them into captive or isolated populations to increase genetic diversity. Recent advances in these fields have allowed the establishment of GRBs for many threatened species.     

Developing core collections to optimize the management and the exploitation of diversity of the coffee Coffea canephora

The management of diversity for conservation and breeding is of great importance for all plant species and is particularly true in perennial species, such as the coffee Coffea canephora. This species exhibits a large genetic and phenotypic diversity with six different diversity groups. Large field collections are available in the Ivory Coast, Uganda and other Asian, American and African countries but are very expensive and time consuming to establish and maintain in large areas. We propose to improve coffee germplasm management through the construction of genetic core collections derived from a set of 565 accessions that are characterized with 13 microsatellite markers. Core collections of 12, 24 and 48 accessions were defined using two methods aimed to maximize the allelic diversity (Maximization strategy) or genetic distance (Maximum-Length Sub-Tree method). A composite core collection of 77 accessions is proposed for both objectives of an optimal management of diversity and breeding. This core collection presents a gene diversity value of 0.8 and exhibits the totality of the major alleles (i.e., 184) that are present in the initial set. The seven proposed core collections constitute a valuable tool for diversity management and a foundation for breeding programs. The use of these collections for collection management in research centers and breeding perspectives for coffee improvement are discussed.     

AFLP assessment of genetic diversity among Indian Mucuna accessions

Amplified fragment length polymorphism (AFLP) marker was used to assess diversity in germplasm collection of Mucuna species which has gained tremendous attention in the recent past due to its promising nutritional, agronomic and medicinal attributes. Twenty five accessions comprising five species, collected from seven states of India were evaluated with twelve AFLP primer combinations that generated a total of 1,612 fragments with an average of 134 fragments per primer combination. The values of polymorphic information content (PIC), marker index (MI) and the resolving power (Rp) demonstrated the utility of the primer combinations used in the present study for discriminating the Mucuna accessions. UPGMA and Principal coordinate analysis (PCoA) of the genotypic data revealed clustering of accessions as per phenetic and genetic relationships. The Jaccard’s similarity coefficient values suggested good variability among the M. pruriens accessions indicating their utility in breeding programs. Molecular diversity presented in this study combined with the datasets on other morphological/agronomic traits will be highly useful for selecting appropriate accessions for plant improvement through conventional as well as molecular breeding approaches and for evolving suitable conservation strategies.     

Genetic structuring in wild populations of two important medicinal plant species as inferred from AFLP markers

Systematic analysis of germplasm diversity and genetic relationship among cultivars is critical for development of appropriate conservation and breeding strategies. This approach has been applied to gain an insight about the molecular variance that exists in wild population of two important medicinal plant species of India that have a long history of therapeutic usage in herbal medicine. Adhatoda vasica and Andrographis paniculata, members of the family Acanthaceae, have wide geographical and climatic distribution across India suggesting a large amount of genetic diversity available for resource management and breeding programs. In this study we have assessed the genetic diversity of both these species distributed in five varied geo-environmental regions, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting with selected primer combinations and statistical analysis. Cluster analysis and analysis of molecular variance also suggested a very high genetic variation. Detailed analyses of the results predict that the genetic variation found in A. vasica was more discrete that reflected strongly in the populations studied, whereas the genetic variation in A. paniculata was relatively uniform. Considering significantly large sample size and distinctive characteristics of the selected populations, this work contributes valuable insights that can be used to engineer conservation and utilization strategies for these species.     

The USDA Barley Core Collection: Genetic Diversity,Population Structure,and Potential for Genome-Wide Association Studies

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define ‘mini-core’ sets of accessions capturing the majority of the allelic diversity present in the core collection. These ‘mini-core’ sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of ‘hull cover’, ‘spike row number’, and ‘heading date’ demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.     

The origin and genetic diversity of Pinus radiata in Australia

Summary Despite the fact that forest trees are in early stages of domestication there has been little direct evaluation of either the origin of, or genetic diversity within the breeding material in tree improvement programs. Allozyme variation was used to compare the total genetic diversity in the breeding programs of P. radiata within Australia and the five wild populations in North America. The current breeding populations were very similar genetically and were essentially homogenous with only 1.8% of the variation among programs. The total genetic diversity in the species was 0.12, which is a low estimate compared to most conifers. Overall in the Australian material the genetic diversity was somewhat less. The comparison of allelic frequencies in the five native populations with the Australian material indicates that the Monterey and Año Nuevo populations were probably the major source of the original introductions and that a substantial portion of the genetic diversity in the two populations has been captured in current breeding programs. The three southern populations do not appear to be currently represented in the breeding programs. The implications for future breeding strategies are discussed.     

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae)

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees.     

Conservation of genetic diversity in old‐growth forest communities of the southeastern United States

Question: How do studies of the distribution of genetic diversity of species with different life forms contribute to the development of conservation strategies? Location: Old‐growth forests of the southeastern United States. Methods: Reviews of the plant allozyme literature are used to identify differences in genetic diversity and structure among species with different life forms, distributions and breeding systems. The general results are illustrated by case studies of four plant species characteristic of two widespread old‐growth forest communities of the southeastern United States: the Pinus palustris – Aristida stricta (Longleaf pine – wiregrass) savanna of the Coastal Plain and the Quercus – Carya – Pinus (Oak‐hickory‐pine) forest of the Piedmont. Genetic variation patterns of single‐gene and quantitative traits are also reviewed. Results: Dominant forest trees, represented by Pinus palustris(longleaf pine) and Quercus rubra (Northern red oak), maintain most of their genetic diversity within their populations whereas a higher proportion of the genetic diversity of herbaceous understorey species such as Sarracenia leucophylla and Trillium reliquum is distributed among their populations. The herbaceous species also tend to have more population‐to‐population variation in genetic diversity. Higher genetic differentiation among populations is seen for quantitative traits than for allozyme traits, indicating that interpopulation variation in quantitative traits is influenced by natural selection. Conclusion: Developing effective conservation strategies for one or a few species may not prove adequate for species with other combinations of traits. Given suitable empirical studies, it should be possible to design efficient conservation programs that maintain natural levels of genetic diversity within species of conservation interest.     

Stocking activities for the Arctic charr in Lake Geneva: Genetic effects in space and time

Artificial stocking practices are widely used by resource managers worldwide, in order to sustain fish populations exploited by both recreational and commercial activities, but their benefits are controversial. Former practices involved exotic strains, although current programs rather consider artificial breeding of local fishes (supportive breeding). Understanding the complex genetic effects of these management strategies is an important challenge with economic and conservation implications, especially in the context of population declines. In this study, we focus on the declining Arctic charr (Salvelinus alpinus) population from Lake Geneva (Switzerland and France), which has initially been restocked with allochtonous fishes in the early eighties, followed by supportive breeding. In this context, we conducted a genetic survey to document the evolution of the genetic diversity and structure throughout the last 50 years, before and after the initiation of hatchery supplementation, using contemporary and historical samples. We show that the introduction of exotic fishes was associated with a genetic bottleneck in the 1980–1990s, a break of Hardy–Weinberg Equilibrium (HWE), a reduction in genetic diversity, an increase in genetic structure among spawning sites, and a change in their genetic composition. Together with better environmental conditions, three decades of subsequent supportive breeding using local fishes allowed to re‐establish HWE and the initial levels of genetic variation. However, current spawning sites have not fully recovered their original genetic composition and were extensively homogenized across the lake. Our study demonstrates the drastic genetic consequences of different restocking tactics in a comprehensive spatiotemporal framework and suggests that genetic alteration by nonlocal stocking may be partly reversible through supportive breeding. We recommend that conservation‐based programs consider local diversity and implement adequate protocols to limit the genetic homogenization of this Arctic charr population.     

Genetic analysis of 50-year old Brazilian pine (Araucaria angustifolia) plantations: implications for conservation planning

In order to determine the implications of including planted populations in conservation planning, we investigate the structure and genetic diversity, mating system, and effective population size within families in three planted and one natural Araucaria angustifolia populations. The study area is a Brazilian National Forest in southern Brazil, established in the 1950s. Genetic analysis was investigated using nine allozyme loci. Significant and positive fixation index was detected for the adults of all studied populations. Although we observed lower levels of genetic diversity and biparental inbreeding in some populations, their pattern of mating, similar to that expected in panmixia, indicates that all populations can be used in conservation planning. Furthermore, the presence of unique alleles and the significant genetic differentiation among all adult populations (F _ST = 0.404) reinforces that all populations are important conservation resources. The average paternity correlation was high in almost all populations, showing that open-pollinated families are composed of mixtures of half- and full-sibs. With regards to conservation strategies, our results suggest that seed collection from at least 133 seed trees is necessary to retain a reference effective population size of 500. Our results confirm that the planted Araucaria forests in southern Brazil can be useful as a genetic resource for conservation, breeding and reforestation strategies for this threatened species.     

The breeding of great apes at the Jersey Wildlife Preservation Trust and a look into the future

Details of breeding orangutans and gorillas at the Jersey Wildlife Preservation Trust are reviewed, with special reference to the diversity of managerial requirements and reproductive parameters for the two species of great apes represented. The importance of recording and publishing as much data as possible to provide important guidelines for others involved with the same or related species in captivity, as well as such information contributing directly to conservation in the wild, is underlined. The Jersey Trust's involvement with cooperative and coordinated breeding programs for the two species on both a national and international basis is reported, and prospects for future long-term management strategies are outlined.     

Analysis of conservation priorities of Iberoamerican cattle based on autosomal microsatellite markers

Background

Determining the value of livestock breeds is essential to define conservation priorities, manage genetic diversity and allocate funds. Within- and between-breed genetic diversity need to be assessed to preserve the highest intra-specific variability. Information on genetic diversity and risk status is still lacking for many Creole cattle breeds from the Americas, despite their distinct evolutionary trajectories and adaptation to extreme environmental conditions.

Methods

A comprehensive genetic analysis of 67 Iberoamerican cattle breeds was carried out with 19 FAO-recommended microsatellites to assess conservation priorities. Contributions to global diversity were investigated using alternative methods, with different weights given to the within- and between-breed components of genetic diversity. Information on Iberoamerican plus 15 worldwide cattle breeds was used to investigate the contribution of geographical breed groups to global genetic diversity.

Results

Overall, Creole cattle breeds showed a high level of genetic diversity with the highest level found in breeds admixed with zebu cattle, which were clearly differentiated from all other breeds. Within-breed kinships revealed seven highly inbred Creole breeds for which measures are needed to avoid further genetic erosion. However, if contribution to heterozygosity was the only criterion considered, some of these breeds had the lowest priority for conservation decisions. The Weitzman approach prioritized highly differentiated breeds, such as Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú, while kinship-based methods prioritized mainly zebu-related breeds. With the combined approaches, breed ranking depended on the weights given to the within- and between-breed components of diversity. Overall, the Creole groups of breeds were generally assigned a higher priority for conservation than the European groups of breeds.

Conclusions

Conservation priorities differed significantly according to the weight given to within- and between-breed genetic diversity. Thus, when establishing conservation programs, it is necessary to also take into account other features. Creole cattle and local isolated breeds retain a high level of genetic diversity. The development of sustainable breeding and crossbreeding programs for Creole breeds, and the added value resulting from their products should be taken into consideration to ensure their long-term survival.     

From microsatellites to single nucleotide polymorphisms for the genetic monitoring of a critically endangered sturgeon

The use of genetic information is crucial in conservation programs for the establishment of breeding plans and for the evaluation of restocking success. Short tandem repeats (STRs) have been the most widely used molecular markers in such programs, but next‐generation sequencing approaches have prompted the transition to genome‐wide markers such as single nucleotide polymorphisms (SNPs). Until now, most sturgeon species have been monitored using STRs. The low diversity found in the critically endangered European sturgeon (Acipenser sturio), however, makes its future genetic monitoring challenging, and the current resolution needs to be increased. Here, we describe the discovery of a highly informative set of 79 SNPs using double‐digest restriction‐associated DNA (ddRAD) sequencing and its validation by genotyping using the MassARRAY system. Comparing with STRs, the SNP panel proved to be highly efficient and reproducible, allowing for more accurate parentage and kinship assignments' on 192 juveniles of known pedigree and 40 wild‐born adults. We explore the effectiveness of both markers to estimated relatedness and inbreeding, using simulated and empirical datasets. Interestingly, we found significant correlations between STRs and SNPs at individual heterozygosity and inbreeding that give support to a reasonable representation of whole genome diversity for both markers. These results are useful for the conservation program of A. sturio in building a comprehensive studbook, which will optimize conservation strategies. This approach also proves suitable for other case studies in which highly discriminatory genetic markers are needed to assess parentage and kinship.     

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species ( Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations ( Oryza glumaepatula ) recently collected in the Amazon forest and western Brazil rivers. F -statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.     

Unravelling genetic diversity and cultivar parentage in the Danish apple gene bank collection

Characterization of apple germplasm is important for conservation management and breeding strategies. A set of 448 Malus domestica accessions, primarily of local Danish origin, were genotyped using 15 microsatellite markers. Ploidy levels were determined by flow cytometry. Special emphasis was given to pedigree reconstruction, cultivar fingerprinting and genetic clustering. A reference set of cultivars, mostly from other European countries, together with a private nursery collection and a small set of Malus sieversii, Malus sylvestris and small-fruited, ornamental Malus cultivars, was also included. The microsatellite markers amplified 17–30 alleles per loci with an average degree of heterozygosity at 0.78. We identified 104 (23%) duplicate genotypes including colour sports. We could infer first-degree relationships for many cultivars with previously unknown parentages. STRUCTURE analysis provided no evidence for a genetic structure but allowed us to present a putative genetic assembly that was consistent with both PCA analysis and parental affiliation. The Danish cultivar collection contains 10% duplicate genotypes including colour sports and 22% triploids. Many unique accessions and considerable genetic diversity make the collection a valuable resource within the European apple germplasm. The findings presented shed new light on the origin of Danish apple cultivars. The fingerprints can be used for cultivar identification and future management of apple genetic resources. In addition, future genome-wide association studies and breeding programmes may benefit from the findings concerning genetic clustering and diversity of cultivars.