Analysis of intraspecies diversity reveals a subset of highly variable plant immune receptors and predicts their binding sites

The evolution of recognition specificities by the immune system depends on the generation of receptor diversity and on connecting the binding of new antigens with the initiation of downstream signaling. In plant immunity, the innate Nucleotide-Binding Leucine Rich Repeat (NLR) receptor family enables antigen binding and immune signaling. In this study, we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies that show high allelic diversity. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine Rich Repeat domains, but the locations of the clusters vary among NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for the emergence of direct and indirect pathogen-sensing receptors and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and can inform NLR engineering efforts.

A Microsatellite Multiplex Assay for Profiling Pig DNA in Mosquito Bloodmeals

Genetic profiling has been used to link mosquito bloodmeals to the individual humans, but this analysis has not been done for other mammalian bloodmeals. In this study, we describe a microsatellite-based method for identifying individual pigs in mosquito bloodmeals based on their unique multilocus genotypes. Eleven tetranucleotide microsatellites and a sex-specific marker were selected based on Smith-Waterman DNA sequence alignment scores from the reference genome and primers were designed with features that reduce primer dimers, promote complete adenylation, and enable fluorescent labeling of amplicons. A multiplex polymerase chain reaction (PCR) assay was optimized and validated by analyzing DNA of individual pigs from several nuclear families and breeds before it was used to analyze genomic DNA of pig-derived mosquito bloodmeals from villages of Papua New Guinea. Population analysis of the nuclear families showed high expected and observed heterozygosity. The probability of observing two unrelated or sibling individuals sharing the same genotype at a single microsatellite locus or a combination of loci was vanishingly low. Samples had unique genotypes and gender was accurately predicted. Analysis of 129 pig bloodmeals identified 19 unique genotypes, which varied greatly in frequency in the mosquito bloodmeal samples. The high allelic diversity of the microsatellite loci and low probability of false attribution of identity show that this genotyping method reliably distinguishes distantly and closely related pigs and can be used to identify individual pigs from genotyped mosquito bloodmeals.

AmpUMI: Design and analysis of unique molecular identifiers for deep amplicon sequencing

MotivationUnique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments.ResultsBased on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user-friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis. AmpUMI is open-source and freely available at http://github.com/pinellolab/[email protected]

Criteria for evaluating molecular markers: Comprehensive quality metrics to improve marker-assisted selection

Despite strong interest over many years, the usage of quantitative trait loci in plant breeding has often failed to live up to expectations. A key weak point in the utilisation of QTLs is the “quality” of markers used during marker-assisted selection (MAS): unreliable markers result in variable outcomes, leading to a perception that MAS products fail to achieve reliable improvement. Most reports of markers used for MAS focus on markers derived from the mapping population. There are very few studies that examine the reliability of these markers in other genetic backgrounds, and critically, no metrics exist to describe and quantify this reliability. To improve the MAS process, this work proposes five core metrics that fully describe the reliability of a marker. These metrics give a comprehensive and quantitative measure of the ability of a marker to correctly classify germplasm as QTL[+]/[-], particularly against a background of high allelic diversity. Markers that score well on these metrics will have far higher reliability in breeding, and deficiencies in specific metrics give information on circumstances under which a marker may not be reliable. The metrics are applicable across different marker types and platforms, allowing an objective comparison of the performance of different markers irrespective of the platform. Evaluating markers using these metrics demonstrates that trait-specific markers consistently out-perform markers designed for other purposes. These metrics also provide a superb set of criteria for designing superior marker systems for a target QTL, enabling the selection of an optimal marker set before committing to design.

Formation of novel PRDM9 allele by indel events as possible trigger for tarsier-anthropoid split

PRDM9 is currently the sole speciation gene found in vertebrates causing hybrid sterility probably due to incompatible alleles. Its role in defining the double strand break loci during the meiotic prophase I is crucial for proper chromosome segregation. Therefore, the rapid turnover of the loci determining zinc finger array seems to be causative for incompatibilities. We here investigated the zinc finger domain-containing exon of PRDM9 in 23 tarsiers. Tarsiers, the most basal extant haplorhine primates, exhibit two frameshifting indels at the 5’-end of the array. The first mutation event interrupts the reading frame and function while the second compensates both. The fixation of this peculiar allele variant in tarsiers led to hypothesize that de‐ and reactivation of the zinc finger domain drove the speciation in early haplorhine primates. Moreover, the high allelic diversity within Tarsius point to multiple effects of genetic drift reflecting their phylogeographic history since the Miocene.