Scoring microsatellite loci

Microsatellites have been utilized for decades for genotyping individuals in various types of research. Automated scoring of microsatellite loci has allowed for rapid interpretation of large datasets. Although the use of software produces an automated process to score or genotype samples, several sources of error have to be taken into account to produce accurate genotypes. A variety of problems (from extracting DNA to entering a genotype into a database) which can arise throughout this process might result in erroneous genotype assignment to one or more samples, potentially confounding the conclusions of your study. Correctly assigning a genotype to a sample requires knowledge of the chemistry you use to generate the data as well as the software you use to analyze these results. In this chapter we describe the critical and more common points that researchers experience when scoring microsatellite loci. More importantly we provide insight from an experienced perspective for these challenges

A new approach to improve the scoring of mononucleotide microsatellite loci

Premise of the study: Mononucleotide microsatellites markers are useful for detecting genetic variation among individuals; however, scoring can be error-prone. We developed a new approach to improve the accuracy of allele scoring. Methods and Results: A set of 14 mononucleotide microsatellite primers of the chloroplast were developed based on published Pinus spp. chloroplast genomes. Due to substantial scoring error for mononucleotide repeats ≥ 10 bp, we included part of the microsatellite in the reverse primer to reduce slippage and improve the scoring of these polymorphic markers. For markers 10-20 bp, the error rate in scoring with this method has a binomial 95% confidence interval of 0.7-2.1%. Conclusions: These new primers provide variable chloroplast markers in species of subsection Cembroides and other Pinus spp. with more accurate assignment of the alleles. This approach can be used to improve the allele scoring of mononucleotide or dinucleotide repeats from nuclear and chloroplast genomes

Microsatellite primers in the foundation tree species Pinus edulis and P. monophylla (Pinaceae)

Premise of the study: Microsatellite primers were developed in the foundational tree species Pinus edulis to investigate population differentiation of P. edulis and hybridization among closely related species. Methods and Results: Using a hybridization protocol, primer sets for 11 microsatellite loci were developed using megagametophyte tissue from P. edulis and scored for polymorphism in three populations of P. edulis and a single P. monophylla population. The primers amplified simple and compound di-, tri-, and pentanucleotide repeats with two to 18 alleles per locus. Conclusions: These results demonstrate the utility of the described primers for studies of population differentiation within and among P. edulis populations as well as across putative hybrid zones where P. edulis may coexist with sister species

Unpacking boxes: Integration of molecular, morphological and ecological approaches reveals extensive patterns of reticulate evolution in box eucalypts

Reticulate evolution by hybridization is considered a common process shaping the evolution of many plant species, however, reticulation could also be due to incomplete lineage sorting in biodiverse systems. For our study we selected a group of closely related plant taxa with contrasting yet partially overlapping geographic distributions and different population sizes, to distinguish between reticulated patterns due to hybridization and incomplete lineage sorting. We predicted that sympatric or proximal populations of different species are more likely to have gene flow than geographically distant populations of the same widespread species. Furthermore, for species with restricted distributions, and therefore, small effective population sizes, we predicted complete lineage sorting. Eastern grey box eucalypt species (Eucalyptus supraspecies Moluccanae) provide an ideal system to explore patterns of reticulate evolution. They form a diverse, recently evolved and phylogenetically undefined group within Eucalyptus, with overlapping morphological features and hybridization in nature. We used a multi-faceted approach, combining analyses of chloroplast and nuclear DNA, as well as seedling morphology, flowering time and ecological spatial differentiation in order to test for species delimitation and reticulate evolution in this group. The multiple layers of results were consistent and suggested a lack of monophyly at different hierarchical levels due to multidirectional gene flow among several species, challenging species delimitation. Chloroplast and nuclear haplotypes were shared among different species in geographic proximity, consistent with hybridization zones. Furthermore, species with restricted distributions appeared better resolved due to lineage sorting in the absence of hybridization. We conclude that a combination of molecular, morphological and ecological approaches is required to disentangle patterns of reticulate evolution in the box eucalypts

Tree genotype influences ectomycorrhizal fungal community structure : ecological and evolutionary implications

Although the eco-evolutionary dynamics of multicellular organisms are intertwined with the microorganisms that colonize them, there is only a rudimentary understanding of how a host's genotype influences its microbiome. We utilize Populus angustifolia to test whether communities of essential symbionts, ectomycorrhizal fungi (EMF), vary among host genotypes. Further, we test whether EMF communities covary among tree genotypes with the chemistry of senescent leaves and aboveground biomass, traits important to tree fitness, and carbon and nutrient cycling. We found: 1) EMF composition, colonization and the Basidiomycota to Ascomycota ratio varied among tree genotypes (broad-sense heritability = 0.10–0.25). 2) EMF composition did not covary among genotypes with aboveground biomass but it did covary with senescent leaf chemistry (rho = 0.29), primarily due to a single genotype. These findings demonstrate a link between tree genotype and EMF communities, which has implications for fungal diversity, host-symbiont interactions and aboveground-belowground linkages in ecological and evolutionary contexts

Sexual stability in the nearly dioecious Pinus johannis (Pinaceae)

Premise of the study: Even though dioecy is a dominant sexual system among gymnosperms, little is known about its evolutionary history. Pinus johannis may represent a model system because unisexual and monoecious individuals compose its populations. The presence of unisexual individuals in other Pinus species is a consequence of sexual lability. Here we determined whether P. johannis represents the first example of a dioecious or nearly dioecious reproductive system in conifers by evaluating its sexual stability. Methods: To assess the stability of sexual expression, we quantified the proportion of male vs. female reproductive structures produced by trees over multiple years and tested for the presence of sexual dimorphism. Sexual lability hypotheses were also examined by looking at the relationship between environmental factors and sexual expression and by comparing the reproductive behavior of P. johannis with its closest labile relative, P. edulis. Key results: Pinus johannis is nearly dioecious: ∼99% of individuals are unisexual or express a low proportion of the opposite gender with few changes in sexual expression through time. We found sexual dimorphism consistent with sexual stability. Sexual expression did not vary with tree size/age, abiotic environment, or herbivore removal, providing evidence against sexual lability. Individuals of P. johannis tended to produce only male or female strobili, whereas those of P. edulis were mainly monoecious with a gradient in the female to male strobili ratio. Conclusions: This study represents the first report of a nearly stable dioecious Pinus species. The variety of sexual morphs coexisting in the same population makes P. johannis a model for studying the evolution of dioecy in gymnosperms

Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae)

Background and AimsThe sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described.MethodsThe spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD.Key resultsPCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption.ConclusionsTemporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species