Genetic Diversity of Armenian Grapevine (Vitis vinifera L.) Germplasm: Molecular Characterization and Parentage Analysis

Armenia is an important country of origin of cultivated Vitis vinifera subsp. vinifera and wild Vitis vinifera subsp. sylvestris and has played a key role in the long history of grape cultivation in the Southern Caucasus. The existence of immense grapevine biodiversity in a small territory is strongly linked with unique relief and diverse climate conditions assembled with millennium-lasting cultural and historical context. In the present in-depth study using 25 nSSR markers, 492 samples collected in old vineyards, home gardens, and private collections were genotyped. For verification of cultivar identity, the symbiotic approach combining genotypic and phenotypic characterization for each genotype was carried out. The study provided 221 unique varieties, including 5 mutants, from which 66 were widely grown, neglected or minor autochthonous grapevine varieties, 49 turned out to be new bred cultivars created within the national breeding programs mainly during Soviet Era and 34 were non-Armenian varieties with different countries of origin. No references and corresponding genetic profiles existed for 67 genotypes. Parentage analysis was performed inferring 62 trios with 53 out of them having not been previously reported and 185 half-kinships. Instability of grapevine cultivars was detected, showing allelic variants, with three and in rare cases four alleles at one loci. Obtained results have great importance and revealed that Armenia conserved an extensive grape genetic diversity despite geographical isolation and low material exchange. This gene pool richness represents a huge reservoir of under-explored genetic diversity.

Parentage Assignment Reveals Multiple Paternity in the Critically-Endangered Guatemalan Beaded Lizard (Heloderma Charlesbogerti)

Within captive management programs for species of conservation concern, understanding the genetic mating system is of fundamental importance, given its role in generating and maintaining genetic diversity and promoting opportunities for sperm competition. If a goal of a conservation program is reintroduction, knowledge of the mating system may also inform prediction models aimed at understanding how genetic diversity may be spatially organized, thus informing decisions regarding where and which individuals should be released in order to maximize genetic diversity in the wild population. Within captive populations, such information may also influence how animals are maintained in order to promote natural behaviors. Here we investigate the genetic mating system of the Guatemalan beaded lizard, Heloderma charlesbogerti, a member of a genus lacking such information. A group of adult male and female H. charlesbogerti were co-habited for five years during the species perceived breeding season. Through genomic parentage analysis, 50% of clutches comprising multiple offspring were found to result from polyandry, with up to three males siring offspring within single clutches. Furthermore, males were found to be polygamous both within and across seasons, and females would exhibit promiscuity across seasons. As such, within this captive environment, where opportunities existed for mating with multiple sexual partners, the genetic mating system was found to be highly promiscuous, with multiple paternity common within clutches. These findings are novel for the family Helodermatidae, and the results have broader implications about how reproductive opportunities should be managed within captive conservation programs.

Landscape barriers to pollen and seed flow in the dioecious tropical tree Astronium fraxinifolium in Brazilian savannah

Gene flow studies provide information on gene exchange between populations, which is essential for developing genetic conservation strategies. Such analyses enable a better understanding of the life history and seed and pollen dispersal mechanisms of plant species. In this study, we investigate pollen and seed flow in a regenerant population of the pioneer species Astronium fraxinifolium in an area degraded during the construction of a hydroelectric dam. We mapped, sampled, sexed, and genotyped 386 individuals in the regenerant population (RP), as well as 128 adult trees located along two highways adjacent to the degraded area; one in Mato Grosso do Sul State (MS) and other in São Paulo State (SP). Parentage analyses was carried out for 370 individuals of the RP population, using as putative parents 348 individuals from RP and all 128 individuals sampled in MS and SP. Based on parentage analysis and eight microsatellite loci, our analyses revealed that for individuals of the RP with an identified father (pollen donor), 1.1% of the pollen was dispersed up to 532 m, while for those with an identified mother (seed donor), 0.5% of seeds were dispersed up to 4,782 m. However, a large proportion of pollen (76.5%) and seeds (57%) immigrated from trees outside the sampled populations. Pollen and seeds were dispersed through a pattern of isolation by distance. Genetic diversity was significantly similar between adults of both highway populations and individuals from RP, with significant levels of inbreeding detected only in RP. Our results demonstrate that the nearest trees contributed pollen and seeds for the recovery of the degraded area, indicating reproductive spatial isolation among the sampled populations due to the damming of the river. Such results help to understand the process of regeneration for A. fraxinifolium in regenerant populations to inform strategies for conservation and environmental recovery with this species.

Comparative analysis of single nucleotide polymorphisms and microsatellite markers for parentage verification and discovery within the equine Thoroughbred breed

Short tandem repeat (STR), also known as microsatellite markers are currently used for genetic parentage verification within equine. Transitioning from STR to single nucleotide polymorphism (SNP) markers to perform equine parentage verification is now a potentially feasible prospect and a key area requiring evaluation is parentage testing accuracies when using SNP based methods, in comparison to STRs. To investigate, we utilised a targeted equine genotyping by sequencing (GBS) panel of 562 SNPs to SNP genotype 309 Thoroughbred horses - inclusive of 55 previously parentage verified offspring. Availability of STR profiles for all 309 horses, enabled comparison of parentage accuracies between SNP and STR panels. An average sample call rate of 97.2% was initially observed, and subsequent removal of underperforming SNPs realised a pruned final panel of 516 SNPs. Simulated trio and partial parentage scenarios were tested across 12-STR, 16-STR, 147-SNP and 516-SNP panels. False-positives (i.e. expected to fail parentage, but pass) ranged from 0% for 147-SNP and 516-SNP panels to 0.003% when using 12-STRs within trio parentage scenarios, and 0% for 516-SNPs to 1.6% for 12-STRs within partial parentage scenarios. Our study leverages targeted GBS methods to generate low-density equine SNP profiles and demonstrates the value of SNP based equine parentage analysis in comparison to STRs - particularly when performing partial parentage discovery.

Parentage Analysis in Giant Grouper (Epinephelus lanceolatus) Using Microsatellite and SNP Markers from Genotyping-by-Sequencing Data

Pedigree information is necessary for the maintenance of diversity for wild and captive populations. Accurate pedigree is determined by molecular marker-based parentage analysis, which may be influenced by the polymorphism and number of markers, integrity of samples, relatedness of parents, or different analysis programs. Here, we described the first development of 208 single nucleotide polymorphisms (SNPs) and 11 microsatellites for giant grouper (Epinephelus lanceolatus) taking advantage of Genotyping-by-sequencing (GBS), and compared the power of SNPs and microsatellites for parentage and relatedness analysis, based on a mixed family composed of 4 candidate females, 4 candidate males and 289 offspring. CERVUS, PAPA and COLONY were used for mutually verification. We found that SNPs had a better potential for relatedness estimation, exclusion of non-parentage and individual identification than microsatellites, and > 98% accuracy of parentage assignment could be achieved by 100 polymorphic SNPs (MAF cut-off < 0.4) or 10 polymorphic microsatellites (mean Ho = 0.821, mean PIC = 0.651). This study provides a reference for the development of molecular markers for parentage analysis taking advantage of next-generation sequencing, and contributes to the molecular breeding, fishery management and population conservation.

The smell of success: Reproductive success related to rub behavior in brown bears

Several species of bears are known to rub deliberately against trees and other objects, but little is known about why bears rub. Patterns in rubbing behavior of male and female brown bears (Ursus arctos) suggest that scent marking via rubbing functions to communicate among potential mates or competitors. Using DNA from bear hairs collected from rub objects in southwestern Alberta from 2011–2014 and existing DNA datasets from Montana and southeastern British Columbia, we determined sex and individual identity of each bear detected. Using these data, we completed a parentage analysis. From the parentage analysis and detection data, we determined the number of offspring, mates, unique rub objects where an individual was detected, and sampling occasions during which an individual was detected for each brown bear identified through our sampling methods. Using a Poisson regression, we found a positive relationship between bear rubbing behavior and reproductive success; both male and female bears with a greater number of mates and a greater number of offspring were detected at more rub objects and during more occasions. Our results suggest a fitness component to bear rubbing, indicate that rubbing is adaptive, and provide insight into a poorly understood behaviour.

Distance-based exclusion method for parentage analysis using microsatellites (SSR) markers

Genotyping mistakes represent a challenge in parental assignment where even small errors can lead to significant amounts of unassigned siblings. Different parental assignment algorithms have been designed to approach this problem. The Exclusion method is the most applied for its reliability and biological meaning. However, the resolving power of this method is the lowest for data containing genotyping errors. We introduce a new distance-based approach which we coin as Distance-Based Exclusion (DBE). The DBE method calculates the distance between the offspring haplotype and haplotype of each of the potential fathers. The father with the lowest distance is then assigned as candidate father according to a distance ratio (α). We have tested the Exclusion and DBE methods using a real dataset of 1230 offsprings subdivided into families of 25 individuals. Each family had six potential fathers and one known mother. Compared with the Exclusion method, the DBE method is able to solve 4.7% more individuals (64.4% Exclusion vs 69.1% DBE) using the most restrictive α tested without errors. DBE method can also be used together with the Exclusion method for error calculation and to further solve unassigned individuals. Using a two-step approach, we were able to assign 98.1% of the offsprings with a total predicted error of 4.7%. Considering the results obtained, we propose the use of the DBE method in combination with the Exclusion method for parental assignment.

Performing parentage analysis for polysomic inheritances based on allelic phenotypes

Polyploidy poses several problems for parentage analysis. We present a new polysomic inheritance model for parentage analysis based on genotypes or allelic phenotypes to solve these problems. The effects of five factors are simultaneously accommodated in this model: (1) double-reduction, (2) null alleles, (3) negative amplification, (4) genotyping errors and (5) self-fertilization. To solve genotyping ambiguity (unknown allele dosage), we developed a new method to establish the likelihood formulas for allelic phenotype data and to simultaneously include the effects of our five chosen factors. We then evaluated and compared the performance of our new method with three established methods by using both simulated data and empirical data from the cultivated blueberry (Vaccinium corymbosum). We also developed and compared the performance of two additional estimators to estimate the genotyping error rate and the sample rate. We make our new methods freely available in the software package polygene, at http://github.com/huangkang1987/polygene.