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Salsola drummondii is a perennial habitat-indifferent halophyte growing in saline and non-saline habitats of the Arabian hyperarid deserts. It offers an invaluable opportunity to examine the molecular mechanisms of salt tolerance. The present study was conducted to elucidate these mechanisms through transcriptome profiling of seedlings grown from seeds collected in a saline habitat. The Illumina Hiseq 2500 platform was employed to sequence cDNA libraries prepared from shoots and roots of non-saline-treated plants (controls) and plants treated with 1200 mM NaCl. Transcriptomic comparison between salt-treated and control samples resulted in 17,363 differentially expressed genes (DEGs), including 12,000 upregulated genes (7870 in roots, 4130 in shoots) and 5363 downregulated genes (4258 in roots and 1105 in shoots), and 272,643 unigenes were functionally annotated. The majority of identified DEGs are known to be involved in transcription regulation (79), signal transduction (82), defense metabolism (101), transportation (410), cell wall metabolism (27), regulatory processes (392), respiration (85), chaperoning (9), and ubiquitination (98) during salt tolerance. This study identified potential genes associated with the salt tolerance of S. drummondii and demonstrated that this tolerance may depend on the induction of certain genes in shoot and root tissues. These gene expressions were validated using reverse-transcription quantitative PCR, the results of which were consistent with transcriptomics results. To the best of our knowledge, this is the first study providing genetic information on salt tolerance mechanisms in S. drummondii. This article is protected by copyright. All rights reserved.

With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants. In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq. This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.

Background Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium. Conclusion A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.

Cymbidium geringii has high ornamental and economic importance. Its traits, including flower shape, size, and color, are highly sought by orchid breeders. Gaining insights into the molecular basis of C. geringi flower development would accelerate genetic improvement of other orchids. Methods and Results: Here, C. goeringii RNA was purified from normal and peloric mutant flowers, and cDNA libraries constructed for Illumina sequencing. We generated 329,156,782 clean reads, integrated them, and then assembled into 236,811 unigenes averaging 595 bp long. A total of 11,992 differentially expressed genes s, of which 6119 were upregulated and 5873 downregulated, were uncovered in peloric mutant flower buds relative to normal flower buds. Kyoto Encyclopedia of Genes and Genomes enrichment assessments posited that these differentially expressed genes are associated with "Photosynthesis", "Linoleic acid metabolism", as well as "Plant hormone signal transduction" cascades. The DEGs were designated to 12 remarkably enriched GO terms, and 16 cell wall associated GO terms. The expression level of 16 determined genes were verified using RT-qPCR. Conclusions: Our gene expression data may be used to study the regulatory mechanism of flower organ development in C. geringi.

Mandarin fish (Siniperca chuatsi) is an economically important freshwater fish cultured in China. In this species, females grow faster than males. However, due to the lack of available genomic and transcriptome information, the mechanisms of sex differentiation remain poorly understood. In this study, Illumina high-throughput sequencing technology was used to sequence four cDNA libraries; the tissues examined included hypothalamus, pituitary gland, ovary, and testis. A total of 134 124 high-quality unigenes were obtained on average length of 1 361 bp and N50 of 3 312 bp. A search of all-unigene sequences against NR, NT, SwissProt, KOG, KEGG, GO, and InterPro databases resulted in 59 688 (44.50%), 76 329 (56.91%), 50 432 (37.60%), 45 741 (34.10%), 48 760 (36.35%), 5 241 (3.91%), and 46 099 (34.37%) annotations, respectively. In a comparison of ovarian and testicular libraries, 15 289 ovary-biased genes and 10 035 testis-biased genes were identified, including a series of genes related to sex determination and differentiation, such as cyp19a1a, foxl2, sox9, dmrtl, amh, and others. In addition, 49 495 SSRs and 85 899 SNPs were detected in transcriptome data. Quantitative real-time PCR results of 15 sex-related functional genes indicated that RNA-seq data was reliable. This study will contribute to a better understanding of the molecular mechanisms of sex differentiation and development in Mandarin fish.

We isolated the rye serine/threonine protein kinase (S/T PK) gene, which is responsible for intracellular signal transduction mediated by abscisic acid (ABA) synthesized following drought stress, from the huge genome, i.e., 1Cx = 7917 Mb. cDNA was synthesized from poly(A)+ RNA from ABA-treated leaves and then ligated with pUC118 via EcoRI linker to form a cDNA library. RT-PCR amplified DNA fragment of S/T PK gene was used as a probe to screen the cDNA library. One positive cDNA clone was sequenced to determine the overall 1462 bp of the S/T PK gene, which encodes the S/T PK protein consisting of deduced 363 amino acid residues including 12 catalytic subdomains. Total RNA was extracted from both ABA-treated and ABA-untreated rye Petkus leaves and then run on a 1% agarose gel denatured by formaldehyde, and the RNA was transblotted to a nylon membrane. Next, northern hybridization was conducted on the membrane using the 3′ untranslated 244 bp region as probes. The rye S/T PK gene was transcribed in ABA-treated leaves but not in untreated leaves. We have shown that rye S/T PK, localized on the 2R chromosome, is transcribed via ABA mediation, which indicates the relation to intracellular signal transduction.

Low temperatures limit crop growth, yield, and quality in cold areas, and cold tolerance is the only strategy for plants to survive in those areas. However, mulberry’s (Morus alba L.) molecular regulatory mechanisms in response to cold remain poorly understood. In this report, four cDNA libraries were built from two groups, the cold-treated group and the control group, and evaluated by RNA-Seq analysis. A total of 3593 differential expression genes (DEGs) were identified from 20,279 unigenes. Among these, 2337 up-regulated and 1256 down-regulated DEGs were identified under this standard (corrected p value 0.0). Most of these DEGs are involved in plant hormone signal transduction, MAPK signaling pathway, ubiquinone, and other terpenoid-quinone biosynthesis, nitrogen metabolism, and biosynthesis of secondary metabolites. A series of candidate genes related to cold stress were screened out and discussed based on these results. Furthermore, the expression changes of 8 genes under cold stress from RNA-Seq analysis were further validated by real-time quantitative polymerase chain reaction. This research reflects Morus alba L. transcriptome data and may help elucidate the cold response mechanisms of mulberry trees and would be useful in breeding cold-tolerant mulberry plants.

There is an increasing demand for elucidating the biosynthetic pathway of medicinal plants, which are capable of producing several metabolites with great potentials for industrial drug production. Digitalis species are important medicinal plants for the production of cardenolide compounds. Advancement on culture techniques is strictly related to our understanding of the genomic background of species. There are a limited number of genomic studies on Digitalis species. The goal of this study is to contribute to the genomic data of Digitalis ferruginea subsp. schischkinii by presenting transcriptome annotation. Digitalis ferruginea subsp. schischkinii has a limited distribution in Turkey and Transcaucasia, and has a high level of lanatoside C, an important cardenolide. In the study, we sequenced the cDNA library prepared from RNA pools of D. ferruginea subsp. schischkinii tissues treated with various stress conditions. Comprehensive bioinformatics approaches were used for de novo assembly and functional annotation of D. ferruginea subsp. schischkinii transcriptome sequence data along with TF families predictions and phylogenetic analysis. In the study, 58,369 unigenes were predicted and unigenes were annotated by analyzing the sequence data in the non-redundant (NR) protein database, the non-redundant nucleotide (NT) database, Gene Orthology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), SwissProt, and InterPro databases. This study is the first transcriptome data for D. ferruginea subsp. schischkinii.

NBS-LRR-type disease resistance gene-like cDNA, induced by salicylic acid (SA) was cloned from rye Secalecereale L. (2n = 14RR) var. Petkus, which has rust resistance genes such as Lr26, Sr31 andRr9. We designed primers based on the NBS region and performed PCR using Petkus genomic DNA as a template. Next, we TA-cloned a 532-bp DNA fragment containing five homologous amino acid sequences in the NBS region. The SA-treated rye showed strong expression of a transcript of approximately 3.5 knt in the Northern blots probed with the NBS fragment; however, no transcripts were observed with the untreated rye. We constructed a cDNA library of rye var. Petkus treated with SA, and then screened the cDNA library using the TA-cloned NBS fragments as a probe. The entire nucleotide sequence of a full length of rye NBS-LRR-containing class cDNA 3,446 bp was determined.

Sweetpotato [Ipomoea batatas (L.) Lam] is an economically important crop for fresh and processed consumption and is widely cultivated worldwide, especially in China. Various sweetpotato cultivars with different storage root colors are presently available. The purple-fleshed sweetpotato obtains its color from anthocyanin accumulation in the storage roots, which is beneficial for both plant and human health. To date, the molecular mechanism of this anthocyanin accumulation has not been studied in detail. In our study, three cDNA libraries generated from 'Xuzi8' with dark-purple flesh, 'Xuzi6' with light-purple flesh, and 'Xu28' with white flesh were sequenced utilizing an Illumina HiSeq™ 2500 platform. Corresponding totals of 28,093,466, 29,239,729 and 27,217,440 raw reads were obtained from the three libraries and assembled into 137,625 unigenes with an average length of 481 bp. Moreover, 79,203 unigenes (57.55%) were found to be annotated in several public databases, and 1285 unigenes were differentially expressed among the Xu28 vs Xuzi8, Xu28 vs Xuzi6, and Xuzi6 vs Xuzi8 libraries. After functional category enrichment analysis of differential expression genes (DEGs), 25 genes were selected as the candidate genes related to anthocyanin accumulation. Furthermore, the expression patterns of some selected DEGs were verified by quantitative real-time PCR (qRT-PCR), and the correlation between expression levels of relevant genes involved in anthocyanin biosynthesis and anthocyanin content was determined. Taken together, the results compose a transcriptomic analysis to investigate the differences in purple flesh formation in the storage roots among different sweetpotato varieties, with the notable outcome that several key genes can now be closely linked to anthocyanin biosynthesis.

Salinity and drought stress are major abiotic stresses negatively affecting the growth and productivity of alfalfa (Medicago sativa L.). Exploration of genes exhibiting superior tolerance to salinity and drought stress in alfalfa will help aid target molecular breeding and developing tolerant forages. In this study, we adopted a high-efficient yeast FOX hunting system for the identification of salinity and drought tolerant genes in alfalfa. Based on the Gateway-compatible vector system, a high-quality expression library was constructed, containing 1.3 × 107 clones with an average size of 1.44 kb. Through heterologous transformation of mixed library plasmid into salt or drought sensitive yeast mutants, monoclonal resistant strains were screened and tolerant genes were captured. Eighteen salinity-tolerance genes were obtained which were involved in several pathways, containing GRAS and zinc finger transcriptional factors, PP2A interaction module, ERVT vesicle transporter and LETM transmembrane protein. Twelve drought tolerance genes were separated, including ERF and SCL transcriptional factors, CIPK and BSK protein kinases, TGL-type ligase, cPGM and cPDL protease. The mRNA transcription levels of these tolerant genes were inducible or suppressible for response to salt or drought stress conditions following quantitative PCR detection, respectively. Furthermore, heterologous transformation of ERVT and CIPK11 can improve the salt and drought stress tolerance in Arabidopsis, which indicates the conservative function of the screening gene in yeast and Arabidopsis. Obtaining these candidate genes can provide new insights for future research with respect to plant salt and drought tolerance.

The bioaccumulation of cadmium (Cd) in crop and the subsequent food chain has aroused extensive concerns. However, the underlying molecular mechanisms of plant Cd tolerance remain to be clarified from the viewpoint of novel candidate genes. Here we described a highly efficient approach for preliminary identifying rice Cd-tolerant genes through the yeast-based cDNA library survival screening combined with high-throughput sequencing strategy. About 690 gene isoforms were identified as being Cd-tolerant candidates using this shotgun approach. Among the Cd-tolerant genes identified, several categories of genes such as BAX inhibitor (BI), NAC transcription factors and Rapid ALkalinization Factors (RALFs) were of particular interest, and their function of Cd tolerance was further validated via heterologous expression, which suggested that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves. Regarding the genes involved in ion transport, the validated Cd-tolerant heavy metal-associated domain (HMAD) isoprenylated protein HIPP42 was particularly noteworthy. Further elucidation of these genes associated with Cd tolerance in rice will benefit agricultural activities.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER− breast cancer patients.

Cytoplasmic male sterility is a well-proven mechanism for cotton hybrid production. Long non-coding RNAs belong to a class of transcriptional regulators that function in multiple biological processes. The cDNA libraries from the flower buds of the cotton CGMS, it's restorer (Rf) and maintainer lines were sequenced using high throughput NGS technique. A total of 1531 lncRNAs showed significant differential expression patterns between these three lines. Functional analysis of the co-expression network of lncRNA-mRNA using gene ontology vouchsafes that, lncRNAs play a crucial role in cytoplasmic male sterility and fertility restoration through pollen development, INO80 complex, development of anther wall tapetum, chromatin remodeling, and histone modification. Additionally, 94 lncRNAs were identified as putative precursors of 49 miRNAs. qRT-PCR affirms the concordance of expression pattern to RNA-seq data. These findings divulge the lncRNA driven miRNA-mediated regulation of gene expression profiling superintended for a better understanding of the CMS mechanisms of cotton.

Hami melon (Cucumis melo L. var. inodorus Jacq) is an important horticultural crop but susceptible to chilling injury during low temperature storage. To characterize the gene expression profiles of Gold Queen Hami melon under the cold stress and discover the key cold stress-induced genes, five cDNA libraries, C0 (control), C12 (control for 12 d), C24 (control for 24 d), L12 (cold treatment at 0.5°C for 12 d) and L24 (cold treatment at 0.5°C for 24 d), were constructed for RNA sequencing (RNA-Seq) and gene expression profiling. A total of 6479 and 7914 differentially expressed genes (DEGs) in L12 and L24 compared with C0 were respectively detected. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified the relevant biological functions and metabolic pathways of these differentially expressed genes. In addition, a diverse range of cold response candidate genes were mainly obtained from starch and sucrose metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis, such as genes encoding sucrose-phosphate synthase, α-amylase, β-amylase, sucrose synthase and glucose-6-phosphate dehydrogenase. Various transcription factors with divergent expression patterns were identified. Finally, quantitative real time polymerase chain reaction (qRT-PCR) was conducted to validate the RNA-Seq and gene differentially expression results. This study provides valuable information for further studies on cold response mechanisms and genetic improvement of cold tolerance in Gold Queen Hami melons.

There is now abundant information on genes involved in molluscan oogenesis and their associations with ovarian development. However, few studies have investigated the ovarian transcriptome of Pacific abalone (Haliotis discus hannai).The objective of this study was to identify genes related to ovarian development and maturation in Pacific abalone utilizing RNA-sequencing (RNA-seq) and to verify the genes most relevant to different stages of maturation.RNA samples from the ovarian tissues of sexually immature and mature abalone were used to construct cDNA libraries, which were paired-end sequenced on an Illumina HiSeq 2500 platform. Reads from individual samples (unigenes) were aligned to reference transcriptome databases for identification of differentially expressed genes (DEGs) between immature and mature ovarian libraries. Reverse transcription-quantitative polymerase chain reaction was used to verify the RNA-seq data.A total of 8779 unigenes were obtained from the ovaries of immature and mature abalone, with a total length of 3323,279 bp and an average length of 379 bp per gene. Gene ontology analysis assigned 5860 unigenes to biological processes, 855 to cellular components, and 1352 to molecular functions. Overall, 470 DEGs were identified, including 213 and 257 genes down-regulated and up-regulated in mature abalone, respectively. Among these, 13 relevant transcripts, including VTG1 and FZD7, were significantly highly expressed in the ovaries of mature abalone (p 0.05, fold change 2).This H. discus hannai ovary transcriptome provides molecular targets to better understand ovarian development, oogenesis, and sexual maturation, and to enhance Pacific abalone production.

Soil salinity of fields is often non-uniform. To obtain a better understanding of molecular response to non-uniform salt stress, we conducted transcriptomic analysis on the leaves and roots of alfalfa grown under 0/0, 200/200, and 0/200 mM NaCl treatments. A total of 233,742 unigenes were obtained from the assembled cDNA libraries. There were 98 and 710 unigenes identified as significantly differentially expressed genes (DEGs) in the leaves of non-uniform and uniform salt treatment, respectively. Furthermore, there were 5178 DEGs in the roots under uniform salt stress, 273 DEGs in the non-saline side and 4616 in the high-saline side roots under non-uniform salt stress. Alfalfa treated with non-uniform salinity had greater dry weight and less salt damage compared to treatment with uniform salinity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs in roots revealed that both sides of the non-uniform salinity were enriched in pathways related to “phenylpropanoid biosynthesis” and “linoleic acid metabolism”; and “MAPK signaling pathway–plant” was also indicated as a key pathway in the high-saline roots. We also combined a set of important salt-response genes and found that roots from the non-saline side developed more roots with increased water uptake by altering the expression of aquaporins and genes related to growth regulation. Moreover, the hormone signal transduction and the antioxidant pathway probably play important roles in inducing more salt-related genes and increasing resistance to non-uniform salt stress on both sides of the roots.

Heat stress has a severe impact on potato growth and tuberization process, always resulting in the decrease of tuber yield and quality. Therefore, it is of great significance for potato breeding to illuminate the mechanism of heat stress on potato and explore heat resistant genes. In this study, two cDNA libraries from normal potato leaves (20 °C day/18 °C night) and potato leaves with 3 days of heat treatment (35 °C day/28 °C night) were constructed respectively. Totally, 1420 differentially expressed genes (DEGs) were identified. The expression patterns of 12 randomly selected genes detected using droplet digital PCR agreed with the sequencing data. Gene ontology analysis showed that these DEGs were clustered into 49 different GO types, reflecting the functional diversity of the heat stress response genes. The results of KEGG pathway enrichment showed the potential biological pathways in which the DEGs were involved, indicating that these pathways may be involved in heat tolerance regulation. Most potato heat transcription factors (StHsfs) and heat shock proteins (StHsps) were not expressed efficiently based on expression profile of these DEGs. StHsp26-CP and StHsp70 were markedly increased after 3 days of heat treatment. These data will be useful for further understanding the molecular mechanisms of potato plant tolerance to heat stress and provide a basis for breeding heat-tolerance varieties.

The light–dark cycle is the primarily environmental stimulus responsible for entraining the major circadian clock located within the suprachiasmatic nucleus (SCN), which in turn coordinates various physiological processes in fish, including those in non‐SCN regions of the brain. To better understand differences in gene expression due to the light–dark cycle, we compared the transcriptomes of Pelteobagrus vachellii in non‐SCN regions of the brain in the daytime (12:00) and nighttime (24:00). The two cDNA libraries yielded 217,575,058 and 350,784,004 paired‐end clean reads, and 311,143 unique transcript fragments (unigenes) averaging 600.94 base pairs were assembled. It was possible to annotate ~7,426 (2.39%) unigenes based on homology with all database sequences. Gene expression analysis revealed that 2,406 and 1,665 annotated unigenes were up‐ and downregulated, respectively, with ≥twofold differences between daytime and nighttime. Enrichment analysis of differentially expressed genes predicted the pathways in which they operate, including development, endocrine function, adaptation to environmental changes and immunity. In summary, characterization of daytime and nighttime gene expression patterns in P. vachellii showed that the light–dark cycle coordinates gene expression in non‐SCN regions of the brain.

The basic helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors. Recent research has shown that the bHLH transcription factor DevR is involved in both sexual and asexual development as well as conidial melanin production in Aspergillus species. Our previous research also found that DevR significantly influences polysaccharide metabolism in Aspergillus oryzae. In this study, to further explore the function of DevR, its interaction proteins were screened by a yeast two-hybrid assay. An A. oryzae cDNA library was transformed into the Y187 strain by using the SMART technique and the homologous recombination method, and then hybridized with a constructed DevR bait plasmid introducing strain to obtain positive clones. Through sequencing analysis, the potential interaction proteins of DevR were determined. Among them, an AO090701000363 gene-encoding protein (named DipA), which was predicted to be a basic leucine zipper (bZIP) transcription factor, was a possible candidate. Phenotypic analysis indicated that overexpression of the AodipA may significantly suppress growth of the strain. Additionally, although no obvious change in the growth rate was found, the deletion of AodipA resulted in thicker hyphae morphology relative to the control. Comparative proteomic analysis further indicated that DipA was potentially involved in the regulation of cell wall integrity, carbon utilization, acetate catabolic process and other biological processes. Partial similarity of the phenotype to that of DevR suggested a correlation between them and implied that the DipA has a function partially similar to that of DevR.

Sogatella furcifera is an important rice pest. In order to understand the molecular basis of the sex determination in this pest, we performed de novo transcriptome sequencing of six cDNA libraries (three biological replicates) of female and male fifth-instar nymphs. Total 65,199 unigenes were obtained, with an average length of 971.5 bp and N50 length of 1708 bp. 20,287 open reading frames (ORFs) were predicted and annotated. Total 1019 differentially expressed genes with 873 upregulated and 146 downregulated were found in male compared to female. Total 164 sex-determining genes were identified, including the key sex-determining genes in fruit flies, such as Sxl, tra, dsx, etc. It implied that the sex determination mechanisms of S. furcifera may be the same as that of fruit flies. This study provided transcriptome resource as a fundamental support for future functional studies to elucidate the sex determination regulatory networks governing sexual dimorphism of S. furcifera.

In this study, we describe the further genomic characterization of several pineapple mealybug wilt-associated virus (PMWaV) members of the genus Ampelovirus, family Closteroviridae, using high-throughput sequencing (HTS). PMWaV-2 is a necessary factor in the complex etiology of mealybug wilt disease of pineapple (MWP). The complete PMWaV-1 genome was described previously, but the genomes of PMWaV-2, PMWaV-3, and putative PMWaV-4 and PMWaV-5 species lack complete characterization. To further study MWP etiology through genomic characterization of PMWaVs, a cDNA library from total RNA of pineapple plants was generated and underwent HTS. A PMWaV-4 genome of 12,932 nucleotides was assembled from the HTS data. Proteins from the PMWaV-4 genome shared high amino acid similarity to those of PMWaV-1. The RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h), and coat protein (CP) proteins of PMWaV-4 shared 88, 87, and 85% amino acid identity, respectively, to those of PMWaV-1 (AF414119) indicating PMWaV-4 is a distinct strain of PMWaV-1 but not a distinct ampelovirus species. Therefore, we suggest that PMWaV-4 be designated PMWaV-1 strain 4 (MN539275). Further, we used HTS to obtain a new isolate of PMWaV-1 (MN539276) and supplemented the available sequences of the genomes of PMWaV-2 (MN539272 and MN539273) and PMWaV-3 (MN539274) in their 5′-terminal regions.

Northern snakehead Ophiocephalus argus Cantor is an endemic freshwater species in China. This species is widely distributed in the lower Yellow and Yangtze river systems in eastern China. In the present study, six cDNA libraries of Ophiocephalus argus were sequenced using Illumina HiSeq4000 paired-end sequencing technology. 67 564 sequences from 79 500 964 paired-end reads were generated. A total of 7182 unigenes were clustered into 25 functional categories by searching against the COG database, and BLAST2GO search assigned 33 710 unigenes to 59 GO terms. From the 67 564 assembled unique sequences, 21 464 SSRs were identified in 14 997 unique sequences. Among the 7992 with compound formation (SSRs), 100 primer pairs were successfully designed for further validation, and 42 of them produced polymorphic products in Ophiocephalus argus individuals. The number of alleles per locus ranged from 5 to 19, the observed and expected heterozygosity estimates ranged from 0.178 to 0.985 and from 0.421 to 0.929, respectively. The generation of such larges cale sequence data in the present study provides a valuable resource for gene discovery and molecular marker development.

Long terminal repeats (LTR) retrotransposons have a major role in determining genome size, structure and function, thanks to their ability to transpose. We performed a meta-analysis of LTR-retrotransposon expression in roots of sunflower plantlets treated with different plant hormones, chemicals and NaCl. By using Illumina cDNA libraries, available from public repositories, we measured the number of reads matching the retrotranscriptase domains isolated from a whole genome library of retrotransposons. LTR-retrotransposons resulted in general barely expressed, except for 4 elements, all belonging to the AleII lineage, which showed high transcription levels in roots of both control and treated plants. The expression of retrotransposons in treated plants was slightly higher than in the control. Transcribed elements belonged to specific chromosomal loci and were not abundant in the genome. A few elements resulted differentially expressed depending on the treatment. Results suggest that, although most retrotransposons are not expressed, the transcription of such elements is related to their abundance, to their position in the chromosome and to their lineage.

Summary We investigated feasibility of the Full‐length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.

Background Flammulina velutipes has been recognized as a useful basidiomycete with nutritional and medicinal values. Ergosterol, one of the main sterols of F. velutipes is an important precursor of novel anticancer and anti-HIV drugs. Therefore, many studies have focused on the biosynthesis of ergosterol and have attempted to upregulate its content in multiple organisms. Great progress has been made in understanding the regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. However, this molecular mechanism in F. velutipes remains largely uncharacterized. Results In this study, nine cDNA libraries, prepared from mycelia, young fruiting bodies and mature fruiting bodies of F. velutipes (three replicate sets for each stage), were sequenced using the Illumina HiSeq™ 4000 platform, resulting in at least 6.63 Gb of clean reads from each library. We studied the changes in genes and metabolites in the ergosterol biosynthesis pathway of F. velutipes during the development of fruiting bodies. A total of 13 genes (6 upregulated and 7 downregulated) were differentially expressed during the development from mycelia to young fruiting bodies (T1), while only 1 gene (1 downregulated) was differentially expressed during the development from young fruiting bodies to mature fruiting bodies (T2). A total of 7 metabolites (3 increased and 4 reduced) were found to have changed in content during T1, and 4 metabolites (4 increased) were found to be different during T2. A conjoint analysis of the genome-wide connection network revealed that the metabolites that were more likely to be regulated were primarily in the post-squalene pathway. Conclusions This study provides useful information for understanding the regulation of ergosterol biosynthesis and the regulatory relationship between metabolites and genes in the ergosterol biosynthesis pathway during the development of fruiting bodies in F. velutipes.

With the exponential growth of sequence information stored over the last decade, including that of de novo assembled contigs from RNA-Seq experiments, quantification of chimeric sequences has become essential when assembling read data. In transcriptomics, de novo assembled chimeras can closely resemble underlying transcripts, but patterns such as those seen between co-evolving sites, or mapped read counts, become obscured. We have created a de Bruijn based de novo assembler for RNA-Seq data that utilizes a classification system to describe the complexity of underlying graphs from which contigs are created. Each contig is labelled with one of three levels, indicating whether or not ambiguous paths exist. A by-product of this is information on the range of complexity of the underlying gene families present. As a demonstration of CStones ability to assemble high-quality contigs, and to label them in this manner, both simulated and real data were used. For simulated data, ten million read pairs were generated from cDNA libraries representing four species, Drosophila melanogaster, Panthera pardus, Rattus norvegicus and Serinus canaria. These were assembled using CStone, Trinity and rnaSPAdes; the latter two being high-quality, well established, de novo assembers. For real data, two RNA-Seq datasets, each consisting of ≈30 million read pairs, representing two adult D. melanogaster whole-body samples were used. The contigs that CStone produced were comparable in quality to those of Trinity and rnaSPAdes in terms of length, sequence identity of aligned regions and the range of cDNA transcripts represented, whilst providing additional information on chimerism. Here we describe the details of CStones assembly and classification process, and propose that similar classification systems can be incorporated into other de novo assembly tools. Within a related side study, we explore the effects that chimera’s within reference sets have on the identification of differentially expression genes. CStone is available at: https://sourceforge.net/projects/cstone/., Author summary Within transcriptome reference sets, non-chimeric sequences are representations of transcribed genes, while artificially generated chimeric ones are mosaics of two or more pieces of DNA incorrectly pieced together. One area where such sets are utilized is in the quantification of gene expression patterns; where RNA-Seq reads are mapped to the sequences within, and subsequent count values reflect expression levels. Artificial chimeras can have a negative impact on count values by erroneously increasing variation in relation to the reads being mapped. Reference sets can be created from de novo assembled contigs, but chimeras can be introduced during the assembly process via the required traversal of graphs, representing gene families, constructed from the RNA-Seq data. Graph complexity determines how likely chimeras will arise. We have created CStone, a de novo assembler that utilizes a classification system to describe such complexity. Contigs created by CStone are labelled in a manner that indicates whether or not they are non-chimeric. This encourages contig dependent results to be presented with increased objectivity by maintaining the context of ambiguity associated with the assembly process. CStone has been tested extensively. Additionally, we have quantified the relationship between chimeras within reference sets and the identification of differentially expressed genes.

Rheum tanguticum (Maxim. ex Regel) Maxim. ex Balf. is a herbaceous perennial plant indigenous to China, and its root and rhizomes were usually used as an important traditional Chinese medicine. However, the genomic resources are still scarce for R. tanguticum and even for Rheum genus. Transcriptome datasets from different tissues of R. tanguticum were obtained to screen the genes related to anthraquinones biosynthesis, and five free anthraquinones were also determined. Nine cDNA libraries of roots, stems and leaves were generated, and a total of 272 million high-quality reads were assembled into 257,942 unigenes. Based on the functional annotation, A total of 227 candidate enzyme genes involved in the MVA, MEP, shikimate and polyketide pathways were identified, and several differentially expressed genes found functionally associated with anthraquinones biosynthesis showed distinct tissue-specific expression patterns. Especially, we found that the expression levels of PKS III genes might result in the content differences of free anthraquinones in different tissues of R. tanguticum. Besides, 137,400 SSR loci were identified, and 64,081 SSR primer pairs were successfully designed based on these loci. Our results not only provide cues for the genetic mechanism of anthraquinone content differences in different tissues of R. tanguticum, but also lay genomic foundation for the subsequent genetic engineering and breeding for Rheum species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01099-8.

12 Pág. Centro de Biotecnología y Genómica de Plantas, Expansins are proteins involved in cell wall metabolism that play an important role in plant growth, development, fruit ripening and abiotic stress tolerance. In the present study, we analyzed putative expansins that respond to drought stress. Five expansin genes were identified in cDNA libraries isolated from Colobanthus quitensis gown either with or without endophytic fungi under hydric stress. A differential transcript abundance was observed by qPCR analysis upon drought stress. To compare these expansin genes, and to suggest a possible mechanism of action at the molecular level, the structural model of the deduced proteins was obtained by comparative modeling methodology. The structures showed two domains and an open groove on the surface of the proteins was observed in the five structural models. The proteins were evaluated in terms of their protein-ligand interactions using four different ligands. The results suggested differences in their mode of protein-ligand interaction, in particular concerning the residues involved in the protein-ligand interaction. The presented evidence supports the participation of some members of the expansin multiprotein family in the response to drought stress in C. quitensis and suggest that the response is modulated by endophytic fungi., The Agencia Nacional de Investigación y Desarrollo (ANID, Chile) [grants REDES #190093 to L.M-Q; REDES #190078 to P.R.; FONDECYT #1211057 to P.R.] supported the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Background: The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.

Morinda officinalis How (MO) is one of the best-known traditional herbs and is widely cultivated in subtropical and tropical areas for many years, especially in southern China. Oligosaccharides are the major constituents in the roots of MO, which is well known for its therapeutic effects with anti-depression, anti-osteoporosis, memory-enhancing, ect. To date, the main gene families that regulate the biosynthetic pathway of MO oligosaccharides metabolism yet have been published. In our study, six cDNA libraries generated from six plants of MO were sequenced utilizing an Illumina HiSeq 4000 platform. Corresponding totals of more than 132.60 million clean reads were obtained from the six libraries and assembled into 25,812 unigenes with an average length of 1288 bp. Moreover, 6036 unigenes were found to be allocated to 26 pathways maps using several public databases, and 2538 differential expression genes (DEGs) were screened. Among them, 25 genes from three families were selected as the mainly candidate genes related to MO oligosaccharides biosynthesis. Then, the expression patterns of six DEGs closely related to MO oligosaccharides biosynthesis were verified by quantitative real-time PCR (qRT-PCR). Besides, the MO was clustered more closely to Coffea arabica of Rubiaceae. In summary, the transcriptomic analysis was used to investigate the differences in expression genes of oligosaccharides biosynthesis, with the notable outcome that several key gene families were closely linked to oligosaccharides biosynthesis.

The body color of Neocaridina denticulate sinensis is a compelling phenotypic trait, in which a cascade of carotenoid metabolic processes plays an important role. The study was conducted to compare the transcriptome of cephalothoraxes among three pigmentation phenotypes (red, blue, and chocolate) of N. denticulate sinensis. The purpose of this study was to explore the candidate genes associated with different colors of N. denticulate sinensis. Nine cDNA libraries in three groups were constructed from the cephalothoraxes of shrimps. After assembly, 75022 unigenes were obtained in total with an average length of 1026 bp and N50 length of 1876 bp. There were 45977, 25284, 23605, 21913 unigenes annotated in the Nr, Swissprot, KOG, and KEGG databases, respectively. Differential expression analysis revealed that there were 829, 554, and 3194 differentially expressed genes (DEGs) in RD vs BL, RD vs CH, and BL vs CH, respectively. These DEGs may play roles in the absorption, transport, and metabolism of carotenoids. We also emphasized that electron transfer across the inner mitochondrial membrane (IMM) was a key process in pigment metabolism. In addition, a total of 6328 simple sequence repeats (SSRs) were also detected in N. denticulate sinensis. The results laid a solid foundation for further research on the molecular mechanism of integument pigmentation in the crustacean and contributed to developing more attractive aquatic animals.

Made available in DSpace on 2022-04-29T08:45:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-12 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium “Candidatus Liberibacter asiaticus” (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active. Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP Departamento de Pesquisa Desenvolvimento Fundo de Defesa da Citricultura – Fundecitrus Laboratório de Interações em Insetos Departamento de Entomologia e Acarologia Escola Superior de Agricultura Luiz de Queiroz Universidade de São Paulo Instituto de Biologia Molecular y Celular de Plantas – Consejo Superior de Investigaciones Científicas Universidade Politécnica de Valencia Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP

Peucedanum praeruptorum Dunn is a perennial and one-off flowering plant of the Peucedanum genus in Umbelliferae. The cultivated P. praeruptorum Dunn usually grows nutritionally in the first year and then moves into the reproductive growth in the second year. The lignification of the roots caused by bolting leads to the quality decline of crude materials. Since most of the previous studies have dealt with coumarin biosynthesis and identification of functional genes in P. praeruptorum, the scientific connotation of the inability that the bolted P. praeruptorum cannot be used medically is still unclear. Here, we employed a transcriptome sequencing combined with coexpression analysis to unearth the regulation mechanism of key genes related to coumarin synthesis in pre- and postbolting period, and to explore the mechanisms underlying the effects of bolting on the formation and transport of coumarins between the annual and biennial plants. Six cDNA libraries were constructed, and the transcripts were sequenced and assembled by Illumina Hiseq platform. A total of 336,505 unigenes were obtained from 824,129 non-redundant spliced transcripts. Unigenes (114,488) were annotated to the NCBI nr database, 119,017 and 10,475 unigenes were aligned to Gene Ontology (GO) functional groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Differential expression analysis screened out a series of upregulated and downregulated genes related to the phenylpropanoid pathway. The heatmap clustering showed that the similar expression patterns were both observed in groups C vs. D and groups C vs. F. The WGCNA-based coexpression was performed to elucidate the module and trait relationship to unearth important genes related to the bolting process. Seven pivotal modules on the KEGG functional annotations suggested these genes were mainly enriched in the process of plant–pathogen interaction, plant hormone signal transduction, MAPK signaling pathway, α-linolenic acid metabolism, circadian rhythm, and phenylpropanoid pathway. Further analysis provided clues that the key genes of the phenylpropanoid pathway, the ABC transporters, the apoptosis-related and circadian rhythm regulatory genes may play pivotal roles in regulating bolting signaling, biosynthesis, and transportation of coumarins.

Along with being important pigments that determining the flower color in many plants, anthocyanins also perform crucial functions that attract pollinators and reduce abiotic stresses. Purple and white are two different colors of radish petals. In this study, two cDNA libraries constructed with purple and white petal plants were sequenced for transcriptome profiling. Transcriptome results implied that the expression level of the genes participating in the anthocyanin biosynthetic pathway was commonly higher in the purple petals than that in the white petals. In particular, two genes, F3′H and DFR, had a significantly higher expression pattern in the purple petals, suggesting the important roles these genes playing in radish petal coloration. BSA-seq aided-Next Generation Sequencing of two DNA pools revealed that the radish purple petal gene (RsPP) was located on chromosome 7. With additional genotyping of 617 F2 population plants, the RsPP was further confined within a region of 93.23 kb. Transcriptome and Sanger sequencing analysis further helped identify the target gene, Rs392880. Rs392880 is a homologous gene to F3′H, a key gene in the anthocyanin biosynthetic pathway. These results will aid in elucidating the molecular mechanism of plant petal coloration and developing strategies to modify flower color through genetic transformation.

Verbena bonariensis is a valuable plant for both ornament and flower border. As a major constraint, low temperature affects the growing development and survival of V. bonariensis. However, there are few systematic studies in terms of molecular mechanism on the tolerance of low temperature in V. bonariensis. In this study, Illumina sequencing technology was applied to analyze the cold resistance mechanism of plants. Six cDNA libraries were obtained from two samples of two groups, the cold-treated group and the control group. A total of 271,920 unigenes were produced from 406,641 assembled transcripts. Among these, 19,003 differentially expressed genes (DEGs) (corrected p-value 3) were obtained, including 9852 upregulated and 9151 downregulated genes. The antioxidant enzyme system, photosynthesis, plant hormone signal transduction, fatty acid metabolism, starch and sucrose metabolism pathway, and transcription factors were analyzed. Based on these results, series of candidate genes related to cold stress were screened out and discussed. The physiological indexes related to response mechanism of low temperature were tested. Eleven upregulated DEGs were validated by Quantitative Real-time PCR. In this study, we provided the transcriptome sequence resource of V. bonariensis and used these data to realize its molecular mechanism under cold stress. The results contributed to valuable clues for genetic studies and helped to screen candidate genes for cold-resistance breeding.

Sea buckthorn (Hippophae rhamnoides) seed oil is rich in omega fatty acids and various other bioactive compounds, which not only have high antioxidant activity but may also prevent cardiovascular diseases. However, the molecular mechanisms of oil biosynthesis and fatty acid accumulation in developing sea buckthorn seeds remain unknown. Altering the levels of certain miRNAs and transcription factors can affect the expression of multiple genes involved in seed development and fatty acid biosynthesis, resulting in changed seed oil contents. Considering that previous studies have focused mainly on these two regulators separately, we attempted to combine the study of miRNAs and transcription factors to construct a coregulatory network. In the present study, we generated cDNA libraries and small RNA libraries from the seeds of high-oil-content and low-oil-content sea buckthorn cultivars at four developmental stages, and then used high-throughput sequencing technology and bioinformatics methods to identify 140 differentially expressed transcription factors, 137 known miRNAs, and 250 novel miRNAs. Through target relationship prediction, 31 potential transcription factors were found to be associated with 27 miRNAs in 18 independent networks, showing the multiplicity of the regulators. In particular, 9 miRNAs and 5 transcription factors were found as hubs in the network. Furthermore, we hypothesized that 12 regulatory modules might play combinatory regulatory roles in seed development and oil accumulation. In developing seeds, the expression levels of these miRNAs and transcription factors were significantly different between the two cultivars. Taken together, these data describe the first miRNA-transcription factor coregulatory network in developing seeds of sea buckthorn. These findings will be helpful for understanding seed development and oil accumulation at the molecular level in sea buckthorn.

BackgroundMeiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors.ResultWe constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 105transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype.ConclusionSo far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis.

Norisoprenoids are produced from carotenoids under oxidative degradation mediated by carotenoid cleavage dioxygenases (CCDs) and contribute to floral and fruity notes in grape berries and wine. The diversity of CCD substrates and products has been demonstrated by in vitro recombinant proteins extracted from Escherichia coli expressing CCD genes and of in vivo proteins in an E. coli system co-expressing genes for carotenoid synthesis and cleavage. In the current study, VvCCD1 and VvCCD4b were isolated from the cDNA library of Vitis vinifera L. cv. Cabernet Sauvignon and then transformed into carotenoid-accumulating recombinant Saccharomyces cerevisiae strains. The expression of the target genes was monitored during the yeast growth period, and the accumulation of carotenoids and norisoprenoids in the recombinant strains was measured. The results indicated that both of the VvCCDs cleaved β-carotene at the 7, 8 (7', 8') position into β-cyclocitral for the first time. Additionally, the two enzymes also degraded β-carotene at the 9, 10 (9', 10') position to generate β-ionone and cleaved lycopene at the 5, 6 (5', 6') position into 6-methyl-5-hepten-2-one. These findings suggested that the VvCCDs may possess more cleavage characteristics under the eukaryotic expression system in S. cerevisiae than the prokaryotic system in E. coli, which could better explain the biochemical functions of VvCCDs in grape berries.

To acquire the eggplant transcriptome under high temperature stress, 18 cDNA libraries were constructed and sequenced. A total of 136.31 Gb of clean data was obtained, and 88.86%–92.35% of the clean reads were mapped to the eggplant reference genome. Under high temperature, the number of down-regulated genes was more than that of up-regulated genes and there were more differentially expressed genes on the 10th day after flowering than on the 15th and 20th days after flowering. On the 10th day after flowering, the key genes CHI, 3GT, F3′5′H, DFR2, ANS, and F3H in anthocyanin synthetic pathway and most ERF, WRKY, bHLH, and MYB transcription factors were all down-regulated. High temperature significantly decreased the total anthocyanin content in peels. The results showed that at the early stage of peel coloring, high temperature inhibited the expressions of key genes in anthocyanin biosynthetic pathways through the regulation of transcription factors, leading to a significant decrease in total anthocyanin content, which might reduce the peel color in eggplant.

Grass carp shares the largest portion of the aquaculture production in China, but hemorrhagic disease caused by grass carp reovirus (GCRV) often results in tremendous loss of fingerlings and yearlings, causing significant economic damages. However, it is difficult to study antiviral mechanisms in grass carp in vivo due to its large size and long reproductive cycle. Therefore, a small cyprinid species named rare minnow with high sensitivity to GCRV, is regarded as a useful model to study the mechanisms of this disease. In this study, rare minnows were infected with the type-IIGCRV (GCRV-HZ08), and pathogenesis was investigated by BGISEQ-500 transcriptome sequencing of four cDNA libraries, hepatopancreas, gills, head-kidney and spleen, and real time quantitative PCR (qRT-PCR). We obtained 51.22 Gb bases in total, and de novo assembled 107,756 unigenes with an average length of 1,441 bp. GO analysis revealed that the differentially expressed genes (DEGs) involved in the defense mechanisms were the most enriched GO terms in all four tissues. KEGG analysis revealed that the most enriched pathways were "Influenza A", "Herpes simplex infection", "Transcriptional misregulation in cancer" and "Metabolic" pathways. We also performed a comparative transcriptomic study between GCRV-infected rare minnow and grass carp data. This revealed that "IL-17 signaling pathway", "NF-kappa B signaling pathway" and "Influenza A" pathways are conserved (important) in the regulation of anti-GCRV infection in both species, and need to be further investigated. Furthermore, a total of four immune-related DEGs were selected for qRT-PCR validation, and the results confirmed the RNA-seq data. These results enhance our understanding of the antiviral responses of cyprinid fish infected by GCRV.

Tomato (Solanum lycopersicum) is one of the most widely grown and economically important vegetable crops in the world. Tomato chlorosis virus (ToCV) is one of the recently emerged viruses of tomato distributed worldwide. ToCV-tomato interaction was investigated at the molecular level for determining changes in the expression of tomato genes in response to ToCV infection in this study. A cDNA library enriched with genes induced in response to ToCV infection were constructed and 240 cDNAs were sequenced from this library. The macroarray analysis of 108 cDNAs revealed that the expression of 92 non-redundant tomato genes was induced by 1.5-fold or greater in response to ToCV infection. The majority of ToCV-induced genes identified in this study were associated with a variety of cellular functions including transcription, defense and defense signaling, metabolism, energy, transport facilitation, protein synthesis and fate and cellular biogenesis. Twenty ToCV-induced genes from different functional groups were selected and induction of 19 of these genes in response to ToCV infection was validated by RT-qPCR assay. Finally, the expression of 6 selected genes was analyzed in different stages of ToCV infection from 0 to 45 dpi. While the expression of three of these genes was only induced by ToCV infection, others were induced both by ToCV infection and wounding. The result showed that ToCV induced the basic defense response and activated the defense signaling in tomato plants at different stages of the infection. Functions of these defense related genes and their potential roles in disease development and resistance to ToCV are also discussed.

Rhizoctonia solani AG1-IA is a soil-borne necrotrophic pathogen that causes devastating rice sheath blight disease in rice-growing regions worldwide. Sclerotia play an important role in the life cycle of R. solani AG1-IA. In this study, RNA sequencing was used to investigate the transcriptomic dynamics of sclerotial development (SD) of R. solani AG1-IA. Gene ontology and pathway enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the functions and pathways of differentially expressed genes (DEGs). Six cDNA libraries were generated, and more than 300 million clean reads were obtained and assembled into 15,100 unigenes. In total, 12,575 differentially expressed genes were identified and 34.62% (4353) were significantly differentially expressed with a FDR ≤ 0.01 and |log2Ratio| ≥ 1, which were enriched into eight profiles using Short Time-series Expression Miner. Furthermore, KEGG and gene ontology analyses suggest the DEGs were significantly enriched in several biological processes and pathways, including binding and catalytic functions, biosynthesis of ribosomes, and other biological functions. Further annotation of the DEGs using the Clusters of Orthologous Groups (COG) database found most DEGs were involved in amino acid transport and metabolism, as well as energy production and conversion. Furthermore, DEGs relevant to SD of R. solani AG1-IA were involved in secondary metabolite biosynthesis, melanin biosynthesis, ubiquitin processes, autophagy, and reactive oxygen species metabolism. The gene expression profiles of 10 randomly selected DEGs were validated by quantitative real-time reverse transcription PCR and were consistent with the dynamics in transcript abundance identified by RNA sequencing. The data provide a high-resolution map of gene expression during SD, a key process contributing to the pathogenicity of this devastating pathogen. In addition, this study provides a useful resource for further studies on the genomics of R. solani AG1-IA and other Rhizoctonia species.

Macrobrachium rosenbergii is a commercially important freshwater crustacean species cultured across the world. The economic benefit of M. rosenbergii industry is closely related with gamete quality which unfortunately is unstable in artificial breeding. Improving gamete quality might be achieved through a better understanding of ovarian maturation, but the molecular mechanism is still unclear. This study presented a transcriptome analysis of eyestalk between ovary undeveloped juvenile M. rosenbergii and ovary maturing adults to identify genes involved in ovarian maturation. Six cDNA libraries were constructed with three biological replicates in each group. More than 44 billion raw reads were obtained from each sequencing library using Illumina HiSeq X Ten sequencing platform and 53,878 unigenes were generated after de novo assembly. A total of 15,792 (29.3%) unigenes were annotated by searching against the public databases including NR, Swissprot, KOG, GO and KEGG databases. The comparative transcriptome analysis identified 4815 significantly differentially expressed genes (DEGs) between two groups in which 2961 genes were up-regulated and 1854 genes were down-regulated. Sixteen DEGs were identified to be candidate genes involved in ovarian maturation including neurohormones, neurotransmitter receptor, insulin-like peptides and related receptors, steroidal hormones, methyl farnesoate (MF) related genes and so on. The function of five genes was further verified using mRNA expression detecting at different ovarian developmental stages. Enrichment analysis revealed 65 significantly changed KEGG pathways, in which 2 pathways (insulin signaling pathway and TGF-beta signaling pathway) showed obvious association with ovarian maturation. Data from this study provided fundamental information about gene expression of eyestalk at different ovarian developmental stages in M. rosenbergii, which might be helpful for revealing the regulation mechanism of ovarian maturation and then further contribute to improving gamete quality in aquaculture.

Background Carotid paragangliomas (CPGLs) are rare neuroendocrine tumors that arise from the paraganglion at the bifurcation of the carotid artery and are responsible for approximately 65% of all head and neck paragangliomas. CPGLs can occur sporadically or along with different hereditary tumor syndromes. Approximately 30 genes are known to be associated with CPGLs. However, the genetic basis behind the development of these tumors is not fully elucidated, and the molecular mechanisms underlying CPGL pathogenesis remain unclear. Methods Whole exome and transcriptome high-throughput sequencing of CPGLs was performed on an Illumina platform. Exome libraries were prepared using a Nextera Rapid Capture Exome Kit (Illumina) and were sequenced under 75 bp paired-end model. For cDNA library preparation, a TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) was used; transcriptome sequencing was carried out with 100 bp paired-end read length. Obtained data were analyzed using xseq which estimates the influence of mutations on gene expression profiles allowing to identify potential causative genes. Results We identified a total of 16 candidate genes (MYH15, CSP1, MYH3, PTGES3L, CSGALNACT2, NMD3, IFI44, GMCL1, LSP1, PPFIBP2, RBL2, MAGED1, CNIH3, STRA6, SLC6A13, and ATM) whose variants potentially influence their expression (cis-effect). The strongest cis-effect of loss-of-function variants was found in MYH15, CSP1, and MYH3, and several likely pathogenic variants in these genes associated with CPGLs were predicted. Conclusions Using the xseq probabilistic model, three novel potential causative genes, namely MYH15, CSP1, and MYH3, were identified in carotid paragangliomas.

High temperature is a common stress, which influences the growth and reproduction of plants. Maize is one of the most important crops all over the world. However, heat stress reduces significantly the yield and quality of maize. Therefore, it is important to illuminate molecular mechanism of maize response to heat stress. To estimate genes related to heat stress, we analyzed the transcriptome of maize in response to heat stress. In this study, six cDNA libraries were constructed form total RNA isolated from leaves of maize. A total of 35,209,446 and 35,205,472 clean reads were generated from CK (Control condition) and HTP (Heat stress condition) treatments, respectively. The results showed that 1857 DEGs were identified in maize after heat stress (1029 up-regulated and 828 down-regulated). KEGG pathway enrichment analysis for DEGs indicated that protein processing in endoplasmic reticulum pathways play a central role in maize response to heat stress. In addition, in the present study, 167 putative TFs were identified, which belong to various TF families (e.g., MYB, AP2-EREBP, b-ZIP, bHLH, NAC and WRKY), and may be associated with heat stress response of maize. This research may contribute to understand the molecular mechanism of maize inbred line B73 response to heat stress, which is beneficial for developing maize cultivars to improve yield and quality.

Cold stress has caused great economic loss in fish culture worldwide. Orange-spotted grouper (Epinephelus coioides) is one of the most serious lost aquatic animals in 2016 cold fronts in South China. However, the molecular mechanism of grouper's cold resistance has remained largely unknown. In the present study, HiSeq™2000 (Illumina) was used to analyze the transcriptomic profiles of the liver from grouper under control temperature (CT, 28 °C) and low temperature (LT, 13 °C). Two normalized liver cDNA libraries of CT and LT groups were created. We obtained 51,944,970 and 51,905,036 clean reads from CT and LT groups, respectively. Comparing the LT group to the CT group, a total of 5905 significantly differentially expressed genes (DEGs) were identified, including 2093 up-regulated unigenes and 3812 down-regulated unigenes. GO annotation and functional enrichment analysis indicated that all of the DEGs were classified into three categories: biological process (23 subclasses), cellular component (18 subclasses) and molecular function (13 subclasses). KEGG analysis of the DEGs showed that 2732 DEGs were annotated to 253 signaling pathways. The most highly enriched pathways were cell adhesion molecules, Staphylococcus aureus infection, PPAR signaling pathway, Vibrio cholerae infection, primary immunodeficiency, fatty acid elongation, and we found cold stress mainly affects immunity, metabolic and signal transduction. Thirteen of the DEGs were further validated by qRT-PCR. Our results provide valuable information for further analysis of the mechanisms of groupers response under cold stress.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full‑length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high‑quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P‑value

Plants growing in natural habitats are exposed to multiple environmental stresses resulting from abiotic factors such as heat, drought, and cold, which have a significant impact on cultivated potato. We have evaluated in two Solanum tuberosum varieties, Soprano and Kondor, the adaptation to different abiotic stresses as heat, cold, and drought. For this purpose plants of both varieties were stressed, and when they showed symptoms, RNA extraction was carried out and a cDNA library for each sample was constructed. The objective of this study was to detect and analyse the genes involved in the responses to abiotic stresses in potato. The assay generated transcriptome sequences from both varieties, and a total of 5.579.655 reads and 8420 putative candidate genes were generated. 4.027 of the candidate genes were polymorphic and presented a different number of patterns defined by a varying number of SNPs. Many of the generated candidate genes showed differential expression, since the candidate gene was present in the stressed plant, but not in the control plant. The application of this methodology allows us to detect numerous candidate genes or specific alleles/allele combinations, which are differentially expressed in specific samples after the application of different abiotic stresses. This will be useful to identify superior alleles which can be used in Marker Assisted Selection for resistance and tolerance to abiotic stresses.