Preliminary assessment of the genetic diversity in Lamyropsis microcephala (Asteraceae)

Endangered species with small and isolated populations has been a key topic of conservation biology studies in the last decade. Lamyropsis microcephala is among the most significant narrow endemic plants in the Mediterranean region, lying on the Gennargentu massif of the Sardinia island (Italy). Due to heavy threat factors, this species has rapidly become threatened with extinction. The inter-simple sequence repeat technique was used to assess the genetic variation and structure of the individuals growing in the four remnant localities known to date, with the aim to implement further conservation strategies. Results indicated a degree of differentiation among the four subpopulations, in particular for the Fonni one. The estimates of Nei's genetic diversity (H) ranged from 0.0563 (Fonni) and 0.1104 (Bau ‘e Laccos). Analysis of molecular variance values showed that 53% of the total variation may be attributed to the individuals within subpopulations, while 47% is due to differences among subpopulations (P < 0.001). Results also highlighted a scarce gene flow (N_m = 0.503).     

CHIIMP: An automated high‐throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees

Short tandem repeats (STRs), also known as microsatellites, are commonly used to noninvasively genotype wild‐living endangered species, including African apes. Until recently, capillary electrophoresis has been the method of choice to determine the length of polymorphic STR loci. However, this technique is labor intensive, difficult to compare across platforms, and notoriously imprecise. Here we developed a MiSeq‐based approach and tested its performance using previously genotyped fecal samples from long‐term studied chimpanzees in Gombe National Park, Tanzania. Using data from eight microsatellite loci as a reference, we designed a bioinformatics platform that converts raw MiSeq reads into locus‐specific files and automatically calls alleles after filtering stutter sequences and other PCR artifacts. Applying this method to the entire Gombe population, we confirmed previously reported genotypes, but also identified 31 new alleles that had been missed due to sequence differences and size homoplasy. The new genotypes, which increased the allelic diversity and heterozygosity in Gombe by 61% and 8%, respectively, were validated by replicate amplification and pedigree analyses. This demonstrated inheritance and resolved one case of an ambiguous paternity. Using both singleplex and multiplex locus amplification, we also genotyped fecal samples from chimpanzees in the Greater Mahale Ecosystem in Tanzania, demonstrating the utility of the MiSeq‐based approach for genotyping nonhabituated populations and performing comparative analyses across field sites. The new automated high‐throughput analysis platform (available at https://github.com/ShawHahnLab/chiimp ) will allow biologists to more accurately and effectively determine wildlife population size and structure, and thus obtain information critical for conservation efforts.     

Pluripotent Stem Cells for Uncovering the Role of Mitochondria in Human Brain Function and Dysfunction

Mitochondrial dysfunctions are a known pathogenetic mechanism of a number of neurological and psychiatric disorders. At the same time, mutations in genes encoding for components of the mitochondrial respiratory chain cause mitochondrial diseases, which commonly exhibit neurological symptoms. Mitochondria are therefore critical for the functionality of the human nervous system. The importance of mitochondria stems from their key roles in cellular metabolism, calcium handling, redox and protein homeostasis, and overall cellular homeostasis through their dynamic network. Here, we describe how the use of pluripotent stem cells (PSCs) may help in addressing the physiological and pathological relevance of mitochondria for the human nervous system. PSCs allow the generation of patient-derived neurons and glia and the identification of gene-specific and mutation-specific cellular phenotypes via genome engineering approaches. We discuss the recent advances in PSC-based modeling of brain diseases and the current challenges of the field. We anticipate that the careful use of PSCs will improve our understanding of the impact of mitochondria in neurological and psychiatric disorders and the search for effective therapeutic avenues.     

Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats

The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16–20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.     

A microsatellite marker in Helianthus annuus L.

The importance of genetic polymorphism detected by microsatellites is now well established in mammalian genomes. Sequences with a CA repeat, specific to sunflower, Helianthus annuus L., were found by screening a genomic library in M13. After amplification, some polymorphism was detected on these sequences within a sample of cultivars and populations.     

The age of Alu subfamilies

Using Kimura's distance measure we have calculated the average age of all major Alu subfamilies based on the most recent available data. We conclude that AluJ sequences are some 26 Myr older than previously thought. Furthermore, the origin of the FLA (Free Left Arm) Alu family can be traced back to the very beginning of the mammalian radiation.One new minor subfamily is reported and discussed in the context of sequence diversity in major Alu subfamilies. Correspondence to: J. Jurka     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.     

FTO demethylase activity is essential for normal bone growth and bone mineralization in mice

The Fto gene locus has been linked to increased body weight and obesity in human population studies, but the role of the actual FTO protein in adiposity has remained controversial. Complete loss of FTO protein in mouse and of FTO function in human patients has multiple and variable effects. To determine which effects are due to the ability of FTO to demethylate mRNA, we genetically engineered a mouse with a catalytically inactive form of FTO. Our results demonstrate that FTO catalytic activity is not required for normal body composition although it is required for normal body size and viability. Strikingly, it is also essential for normal bone growth and mineralization, a previously unreported FTO function.