Differential expression profiles of microRNAs in musk gland of unmated and mated forest musk deer (Moschus berezovskii)

Background The formation of musk is a complex biophysical and biochemical process that change with the rut of male forest musk deer. We have reported that the mating status of male forest musk deer might result to the variations of chemical composition and microbiota of musk and its yields. Critical roles for microRNAs (miRNAs) of multi-tissues were profiled in our previous study; however, the role for miRNAs of the musk gland remains unclear in this species. Methods In this study, we used Illumina deep sequencing technology to sequence the small RNA transcriptome of unmated male (UM) and mated male (UM) of Chinese forest musk deer. Results We identified 1,652 known miRNAs and 45 novel miRNAs, of which there were 174 differentially expressed miRNAs between UM and MM. chi-miR-21-5p, ipu-miR-99b and bta-miR-26a were up-regulated in UM among the 10 most differentially expressed miRNAs. Functional enrichment of the target genes showed that monosaccharide biosynthetic process, protein targeting, cellular protein catabolic process enriched higher in MM. Meanwhile, structural molecule activity, secretion by cell, regulated exocytosis and circulatory system process enriched more in UM, hinting that the formation of musk in UM was mediated by target genes related to exocytosis. The miRNA-mRNA pairs such as miR-21: CHD7, miR143: HSD17B7, miR-141/200a: Noc2 might involve in musk gland development and musk secretion, which need to be verified in future study.

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2′,3′-cGAMP-degrading enzyme family.

Transcriptomic Analysis of the Brown Planthopper, Nilaparvata lugens, at Different Stages after Metarhizium anisopliae Challenge

Nilaparvata lugens is one of the major pests of rice and results in substantial yield loss every year. Our previous study found that the entomopathogenic fungus Metarhizium anisopliae showed effective potential for controlling this pest. However, the mechanisms underlying M. anisopliae infection of N. lugens are not well known. In the present study, we further examined the transcriptome of N. lugens at 4 h, 8 h, 16 h, and 24 h after M. anisopliae infection by Illumina deep sequencing. In total, 174.17 Gb of data was collected after sequencing, from which 23,398 unigenes were annotated by various databases, including 3694 newly annotated genes. The results showed that there were 246 vs 75, 275 vs 586, 378 vs 1055, and 638 vs 182 up- and downregulated differentially expressed genes (DEGs) at 4 h, 8 h, 16 h, and 24 h after M. anisopliae infection, respectively. The biological functions and associated metabolic processes of these genes were determined with the Clusters of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The DEGs data were verified using RT-qPCR. These results indicated that the DEGs during the initial fungal infection appropriately reflected the time course of the response to the fungal infection. Taken together, the results of this study provide new insights into the molecular mechanisms underlying the insect host response to fungal infection, especially during the initial stage of infection, and may improve the potential control strategies for N. lugens.

De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae)

Background: Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigate the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results: Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs and DEUs) indicated that they were mostly involved in metabolism and signal transduction, as well as DNA repair and plant-pathogen interactions, which may be of adaptive importance. In addition, comparison analysis between non-flowering and flowering tissues revealed that expressions of FmFT and FmHd3a, two putative F. macclureana orthologs, were differently regulated in NF- vs F- leaves, and carbohydrate metabolism and signal transduction were two major KEGG pathways that DEUs were enriched in. Finally, we detected 9,296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions: F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes.

De novo sequencing of the transcriptome of Fargesia macclureana (Poaceae) reveals regulators of the floral transition and ecological adaptations to high altitude

Background: Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigate the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results: Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs and DEUs) indicated that they were mostly involved in metabolism and signal transduction, as well as DNA repair and plant-pathogen interactions, which may be of adaptive importance. In addition, comparison analysis between non-flowering and flowering tissues revealed that expressions of FmFT and FmHd3a, two putative F. macclureana orthologs, were differently regulated in NF- vs F- leaves, and carbohydrate metabolism and signal transduction were two major KEGG pathways that DEUs were enriched in. Finally, we detected 9,296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions: F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes.

De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae)

Background Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2000 ~ 3800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigate the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs and DEUs) indicated that they were mostly involved in metabolism and signal transduction, as well as DNA repair and plant-pathogen interactions, which may be of adaptive importance. In addition, comparison analysis between non-flowering and flowering tissues revealed that expressions of FmFT and FmHd3a, two putative F. macclureana orthologs, were differently regulated in NF- vs F- leaves, and carbohydrate metabolism and signal transduction were two major KEGG pathways that DEUs were enriched in. Finally, we detected 9296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes.

De novo sequencing of the transcriptome of Fargesia macclureana (Poaceae) reveals regulators of the floral transition and ecological adaptations to high altitude

Background Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigated the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs, and DEUs) and a weighted gene co-expression network analysis (WGCNA) revealed that changes in expressions of unigenes related to the circadian cycle may account for the differences in the floral transition of F. macclureana after being transplanted from the QTP to a laboratory outside. In addition, there were differences in active carbohydrate metabolism and signal transduction between the flowering and non-flowering plants. Moreover, we detected the expression of unigenes related to DNA repair and plant-pathogen interactions, which may be of adaptive importance. Finally, we detected 9,296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes.