Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis , worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent. N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ∼35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma . We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)—[Su-(var)3–9; E(z); Trithorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)—are required for H3K27me3, whereas the fourth component, Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7 , resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an ;350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines.

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