Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Identification of novel miRNAs and their target genes in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Despite Eucalyptus grandis being the most widely planted hardwood tree globally, along with the availability of a sequenced genome and easily accessible functional genetic tools, the quantities and roles of miRNA in its developmental processes remains largely unknown. In this study, we constructed small RNA libraries by high-throughput sequencing from Eucalyptus grandis samples, and 386 novel miRNAs were identified by miRDeep2. We found 179 novel miRNAs, 41 miRNA families, and 456 target genes in leaf samples, and 257 novel miRNAs, 61 miRNA families, and 483 target genes in stem samples. The function of the MIR396 family of miRNAs in Eucalyptus grandis was found to be mainly associated with the process of cell growth. By annotation analysis of miRNA targets, we found that some target genes, such as GRF, expansin-A15, and RPS2, had a close correlation in stem. Finally, the three randomly selected members of the MIR396 family were confirmed to express in Eucalyptus grandis by qRT-PCR, indicating that our reported miRNAs were existed. The identification of miRNAs and their target genes will lead to a greater understanding of the role of miRNAs in the physiology, growth, and development of Eucalyptus grandis trees.     

Five new species of calea (Compositae: Heliantheae) from planaltine Brazil

Described from the Planalto of Brazil are:Calea quadrifolia, C. heteropappa, andC. abbreviata of sectionMeyeria,C. intermedia of sectionLemmatium, andC. lutea of sectionCalea. The relationships of each are discussed and a key to the nearest relatives of each is provided. Additionally, all butC. intermedia are illustrated.     

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, ??miRNA primer designer?? to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php, respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.     

Overexpression of a novel salt stress-induced glycine-rich protein gene from alfalfa causes salt and ABA sensitivity in Arabidopsis

Key message

We cloned a novel salt stress-induced glycine-rich protein gene ( MsGRP ) from alfalfa. Its overexpression retards seed germination and seedling growth of transgenic Arabidopsis after salt and ABA treatments.

Abstract

Since soil salinity is one of the most significant abiotic stresses, salt tolerance is required to overcome salinity-induced reductions in crop productivity. Many glycine-rich proteins (GRPs) have been implicated in plant responses to environmental stresses, but the function and importance of some GRPs in stress responses remain largely unknown. Here, we report on a novel salt stress-induced GRP gene (MsGRP) that we isolated from alfalfa. Compared with some glycine-rich RNA-binding proteins, MsGRP contains no RNA recognition motifs and localizes in the cell membrane or cell wall according to the subcellular localization result. MsGRP mRNA is induced by salt, abscisic acid (ABA), and drought stresses in alfalfa seedlings, and its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants confers salinity and ABA sensitivity compared with WT plants. MsGRP retards seed germination and seedling growth of transgenic Arabidopsis plants after salt and ABA treatments, which implies that MsGRP may affect germination and growth through an ABA-dependent regulation pathway. These results provide indirect evidence that MsGRP plays important roles in seed germination and seedling growth of alfalfa under some abiotic stress conditions.     

Metadenopsis Matesova, 1966 and similar mealybug genera (Homoptera,Coccinea: Pseudococcidae) from Russia and neighboring countries

Seven mealybug genera, Kalaginella gen. n., Archanginella gen. n., Metadenopsis Matesova, Metadenopus ?ulc, Glycycnyza Danzig, Inopicoccus Danzig, and Volvicoccus Goux, all with a simplified anal apparatus and lacking cerarii, are revised and illustrated. Kalaginella intermedia gen. et sp. n. and Archanginella kyzylkumica gen. et sp. n. are described from the Kara Kum and Kyzyl Kum deserts, correspondingly. Mirococcopsis stipae Borchsenius, 1949 is considered a junior synonym of Volvicoccus volvifer Goux, 1945.     

Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo

Key message

Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.

     

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga,a halophytic species

Key message

QTL mapping in F ₂ population [ V. luteola × V. marina subsp. oblonga ] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL.

Abstract

The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F₂ population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F_2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50 % of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.