Composition and Expression of Conserved MicroRNA Genes in Diploid Cotton (Gossypium) Species

MicroRNAs are ubiquitous in plant genomes but vary greatly in their abundance within and conservation among plant lineages. To gain insight into the evolutionary birth/death dynamics of microRNA families, we sequenced small RNA and 50-end PARE libraries generated from two closely related species of Gossypium. Here, we demonstrate that 33 microRNA families, with similar copy numbers and average evolutionary rates, are conserved in the two congeneric cottons. Analysis of the presence/absence of these microRNA families in other land plants sheds light on their depth of phylogenetic origin and lineage-specific loss/gain. Conserved microRNA families in Gossypium exhibit a striking interspecific asymmetry in expression, potentially connected to relative proximity to neighboring transposable elements. A complex correlated expression pattern of microRNA target genes with their controlling microRNAs indicates that possible functional divergence of conserved microRNA families can also exist even within a single plant genus

The asparagus genome sheds light on the origin and evolution of a young Y chromosome

Several models have been proposed to explain the emergence of sex chromosomes. Here, through comparative genomics and mutant analysis, Harkess et al. show that linked but separate genes on the Y chromosome are responsible for sex determination in Asparagus, supporting a two-gene model for sex chromosome evolution

Integrated, scalable tools for small RNA genomics: novel algorithms and their application to characterize germline-associated sRNA pathways in diverse species

Meyers, Blake C.Cells associated with the male germline, specifically in rice and maize (grasses), produce diverse and numerous “phased” 21-nt and 24-nt siRNAs. These phased siRNAs (phasiRNAs) show striking similarity to mammalian Piwi-interacting RNAs (piRNAs) in terms of their abundance, biogenesis and timing of accumulation. Both the plant phasiRNA and mammalian piRNA pathways are emerging as factors crucial for reproductive success. However, since the first report of germline-associated plant phasiRNAs, no systematic study of their evolutionary origins has yet been reported; in this context, the meiotic (24-nt) phasiRNAs are particularly interesting, as they have only been described in grasses, a group of monocots that speciated ~71 million years ago (MYA). Grasses include the most important staple crops: rice, maize and wheat. Given the importance of reproductive success to crop yield, a deeper understanding of phasiRNA pathway is crucial. ☐ This dissertation traces the prevalence and origins of phasiRNA pathways in monocot evolution, while simultaneously it addresses a broad range of key computational gaps and algorithmic limitations in leveraging small RNA data for the study of small RNA in plants. First, I present a new set of tools for identifying and validating miRNA targets, and a new suite for computational characterization of phasiRNAs, which together comprise important methods for studies of plant sRNA field. These next generation tools efficiently scale to the increasing volume of high-throughput data, and are fast, sensitive and feature-rich compared to the existing options. Next in my work, I deployed these tools to investigate phasiRNAs in a recently sequenced genome, that of Asparagus officinalis. The common ancestor of asparagus and the grasses diverged approximately 109 MYA. My work then further expanded to study two other non-grass monocots, Lilium (Lilium maculatum) and daylily (Hemerocallis lilioasphodelus), which diverged from Asparagus ~111 MYA. In this dissertation, I demonstrate that both pre-meiotic and meiotic phasiRNAs are prevalent across the monocots that I studied, establishing their origins well before grasses. In addition to male germline, I find evidence for their accumulation in female and somatic tissues, perhaps suggesting that the narrow accumulation of reproductive phasiRNAs in anthers is either not a general characteristic or it is the product of evolutionary refinement in the grasses. I show that the miRNA trigger for pre-meiotic (21-nt) phasiRNAs likely shifted in evolutionary time from targeting pathogen-defense genes to long, non-coding RNAs (observed in grasses) via specialization and sub-functionalization versus neo-functionalization. I also demonstrate that exceptions to the canonical mechanism of biogenesis of phasiRNAs exist in monocot evolution, whereby phasiRNAs are produced apparently without a miRNA trigger. I conclude that plants show substantial variation in their composition and biogenesis of reproductive phasiRNAs, which have broad roles in plant germline development.Ph.D.University of Delaware, Center for Bioinformatics and Computational Biolog

Identification, Validation and Utilization of Novel Nematode-Responsive Root-Specific Promoters in Arabidopsis for Inducing Host-Delivered RNAi Mediated Root-Knot Nematode Resistance

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants

Composition and Expression of Conserved MicroRNA Genes in Diploid Cotton (Gossypium) Species

MicroRNAs are ubiquitous in plant genomes but vary greatly in their abundance within and conservation among plant lineages. To gain insight into the evolutionary birth/death dynamics of microRNA families, we sequenced small RNA and 50-end PARE libraries generated from two closely related species of Gossypium. Here, we demonstrate that 33 microRNA families, with similar copy numbers and average evolutionary rates, are conserved in the two congeneric cottons. Analysis of the presence/absence of these microRNA families in other land plants sheds light on their depth of phylogenetic origin and lineage-specific loss/gain. Conserved microRNA families in Gossypium exhibit a striking interspecific asymmetry in expression, potentially connected to relative proximity to neighboring transposable elements. A complex correlated expression pattern of microRNA target genes with their controlling microRNAs indicates that possible functional divergence of conserved microRNA families can also exist even within a single plant genus.This article is from Genome Biology and Evolution 5 (2013): 2449, doi:10.1093/gbe/evt196. Posted with permission.</p

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These phasiRNAs are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots

Celf1 CLIP assay on mouse lens (Fig 3G Fig 4K Fig 5C)

Data for cross-linked immunoprecipitation (CLIP) using Celf1 antibody on wild-type mouse lens followed by RT-qPCR for specific mRNAs: Dnase2b, p27Kip1, Sptb (Fig 3G, Fig 4K, Fig 5C in Siddam et al. 2018 PLOS Genetics

Quantification of lamin A/C immunofluorescence in Celf1 cKO mouse lens (S11 Fig)

Data for quantification of immunofluorescence signal of lamin A/C in control and Celf1 cKO mouse lens (Supplementary Fig. S11 in Siddam et al. 2018 PLOS Genetics

RT-qPCR of Dnase2b in mouse lens (Fig 3E)

RT-qPCR assay data for Dnase2b mRNA in WT and Celf1 cKO mouse lens (Fig 3E in Siddam et al. 2018 PLOS Genetics