Resource For Identification Of Genes Research Articles

Full-length agave transcriptome reveals candidate glycosyltransferase genes involved in hemicellulose biosynthesis

Agave species are typical crassulacean acid metabolism (CAM) plants commonly cultivated to produce beverages, fibers, and medicines. To date, few studies have examined hemicellulose biosynthesis in Agave H11648, which is the primary cultivar used for fiber production. We conducted PacBio sequencing to obtain full-length transcriptome of five agave tissues: leaves, shoots, roots, flowers, and fruits. A total of 41,807 genes were generated, with a mean length of 2394 bp and an annotation rate of 97.12 % using public databases. We identified 42 glycosyltransferase genes related to hemicellulose biosynthesis, including mixed-linkage glucan (1), glucomannan (5), xyloglucan (16), and xylan (20). Their expression patterns were examined during leaf development and fungal infection, together with hemicellulose content. The results revealed four candidate glycosyltransferase genes involved in xyloglucan and xylan biosynthesis, including glucan synthase (CSLC), xylosyl transferase (XXT), xylan glucuronyltransferase (GUX), and xylan α-1,3-arabinosyltransferase (XAT). These genes can be potential targets for manipulating xyloglucan and xylan traits in agaves, and can also be used as candidate enzymatic tools for enzyme engineering. We have provided the first full-length transcriptome of agave, which will be a useful resource for gene identification and characterization in agave species. We also elucidated the hemicellulose biosynthesis machinery, which will benefit future studies on hemicellulose traits in agave.

A novel Synthetic phenotype association study approach reveals the landscape of association for genomic variants and phenotypes

IntroductionGenome-Wide Association Studies (GWAS) identify tagging variants in the genome that are statistically associated with the phenotype because of their linkage disequilibrium (LD) relationship with the causative mutation (CM). When both low-density genotyped accession panels with phenotypes and resequenced data accession panels are available, tagging variants can assist with post-GWAS challenges in CM discovery. ObjectivesOur objective was to identify additional GWAS evaluation criteria to assess correspondence between genomic variants and phenotypes, as well as enable deeper analysis of the localized landscape of association. MethodsWe used genomic variant positions as Synthetic phenotypes in GWAS that we named “Synthetic phenotype association study” (SPAS). The extreme case of SPAS is what we call an “Inverse GWAS” where we used CM positions of cloned soybean genes. We developed and validated the Accuracy concept as a measure of the correspondence between variant positions and phenotypes. ResultsThe SPAS approach demonstrated that the genotype status of an associated variant used as a Synthetic phenotype enabled us to explore the relationships between tagging variants and CMs, and further, that utilizing CMs as Synthetic phenotypes in Inverse GWAS illuminated the landscape of association. We implemented the Accuracy calculation for a curated accession panel to an online Accuracy calculation tool (AccuTool) as a resource for gene identification in soybean. We demonstrated our concepts on three examples of soybean cloned genes. As a result of our findings, we devised an enhanced “GWAS to Genes” analysis (Synthetic phenotype to CM strategy, SP2CM). Using SP2CM, we identified a CM for a novel gene. ConclusionThe SP2CM strategy utilizing Synthetic phenotypes and the Accuracy calculation of correspondence provides crucial information to assist researchers in CM discovery. The impact of this work is a more effective evaluation of landscapes of GWAS associations.

Extensive transcriptome changes during seasonal leaf senescence in field-grown black cottonwood (Populus trichocarpa Nisqually-1)

To better understand the molecular control of leaf senescence, we examined transcriptome changes during seasonal leaf senescence in Populus trichocarpa Nisqually-1, the Populus reference genome, growing in its natural habitat. Using monthly (from May to October) transcriptomes for three years (2009, 2015, and 2016), we identified 17,974 differentially expressed genes (DEGs; false discovery rate <0.05; log-fold change cutoff = 0) from 36,007 expressed Populus gene models. A total of 14,415 DEGs were directly related to transitions between four major developmental phases – growth, senescence initiation, reorganization, and senescence termination. These DEGs were significantly (p < 0.05) enriched in 279 gene ontology (GO) terms, including those related to photosynthesis, metabolic process, catalytic activity, protein phosphorylation, kinase activity, pollination, and transport. Also, there were 881 differentially expressed transcription factor (TF) genes from 54 TF families, notably bHLH, MYB, ERF, MYB-related, NAC, and WRKY. We also examined 28 DEGs known as alternative splicing (AS) factors that regulate AS process, and found evidence for a reduced level of AS activity during leaf senescence. Furthermore, we were able to identify a number of promoter sequence motifs associated with leaf senescence. This work provides a comprehensive resource for identification of genes involved in seasonal leaf senescence in trees, and informs efforts to explore the conservation and divergence of molecular mechanisms underlying leaf senescence between annual and perennial species.

Genome-Wide Identification of Arabidopsis LBD29 Target Genes Reveals the Molecular Events behind Auxin-Induced Cell Reprogramming during Callus Formation.

Auxin-induced callus formation represents an important cell reprogramming process during in vitro regeneration of plants, in which the pericycle or pericycle-like cells within plant organs are reprogrammed into the pluripotent cell mass termed callus that is generally required for subsequent regeneration of root or shoot. However, the molecular events behind cell reprogramming during auxin-induced callus formation are largely elusive. We previously identified that auxin-induced LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors act as the master regulators to trigger auxin-induced callus formation. Here, by ChIP-seq (chromatin immunoprecipitation-based sequencing) and RNA sequencing approaches, we identified the potential LBD29 target genes at the genome-wide level and outlined the molecular events of LBD-triggered cell reprogramming during callus formation. We showed that LBD29 preferentially bound to the G-box (CACGTG) and TGGGC[C/T] motifs and potentially targeted >350 genes, among which the genes related to methylation, reactive oxygen species (ROS) metabolism, cell wall hydrolysis and lipid metabolism were rapidly activated, while most of the light-responsive genes were suppressed by LBD29. Further examination of a few representative genes validated that they were targeted by LBD29 and participated in the regulation of cell reprogramming during callus formation. Our data not only outline a framework of the early molecular events behind auxin-induced cell reprogramming of callus formation, but also provide a valuable resource for identification of genes that regulate cell fate switch during in vitro regeneration of plants.

Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses.

The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.

Construction of a Spinach Bacterial Artificial Chromosome (BAC) Library as a Resource for Gene Identification and Marker Development

Spinach (Spinacae oleracae) is a nutritious vegetable and an economically important crop worldwide. Spinach is threatened by a number of diseases, and the most significant of which is downy mildew, caused by Peronospora farinosa f. sp. spinaciae (Pfs). The spinach locus RPF1 confers resistance to 11 of the 15 known races of Pfs. To study the RPF1 locus in depth, a BAC library was constructed from DNA of a single spinach plant that was homozygous at the RPF1 locus. The BAC library contained 73,728 clones, with an average insert size of 183 kb, which represented approximately 13 spinach genome equivalents (989 Mb). The ends of 1920 BAC clones were sequenced from both directions, and 3405 BAC end sequences (BESs) ranging from 100 to 935 bp were obtained. About 38.59 % of the BESs contained repetitive sequences, including 363 copies of retrotransposable elements and 32 copies of transposable elements. Less than 3 % of the clones originated from chloroplasts or mitochondria. Various categories of protein-encoding genes, including transcription factors, stress responsive genes, and reproduction-associated genes, were identified. Putative disease resistance genes containing nucleotide binding site-leucine-rich repeat (NBS-LRR) domains were also identified, one of which was closely linked to the RPF1 locus. Simple sequence repeat (SSR) motifs were identified from 154 BESs, including various di-, tri-, tetra-, and penta-nucleotide repeats. One hundred pairs of SSR primers and 24 retrotransposon primers were designed from BESs, all of which successfully amplified spinach DNA. This spinach BAC library will provide a useful resource for spinach genetic studies and breeding efforts.

5′-Cap selection methods and their application in full-length cDNA library construction and transcription start site profiling

With the accomplishment of the genome draft sequences, identification of functional elements in genome has become an urgent task. Full-length cDNAs provide an important resource for gene identification and their precise structural feature determination. It also provides a basis for genomic element definition. As many regulatory elements are around transcription start sites (TSSs), precise localization of TSSs in the genome becomes a critical step for identifying the associated core promoters. Massive parallel snapshot of TSSs at a particular time under a specific experimental condition makes it possible to globally analyze important regulatory elements around TSSs and further construct transcriptional regulatory networks. In this paper, we first reviewed two important full-length cDNA cloning techniques: cap-trapper technique and oligo-capping technique. Then, we introduced deepCAGE, a cap-trapper and deep sequencing-based TSS profiling technique, and its applications in the research of transcriptional regulation.

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1st to 5th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.

Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

Bacterial artificial chromosome (BAC) libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa), using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

The immune gene repertoire of an important viral reservoir, the Australian black flying fox

BackgroundBats are the natural reservoir host for a range of emerging and re-emerging viruses, including SARS-like coronaviruses, Ebola viruses, henipaviruses and Rabies viruses. However, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. Massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. Although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. In this study, we describe the immune transcriptome of the Australian flying fox, Pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity.ResultsTowards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from P. alecto. We identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. A small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts.ConclusionsThis study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. In addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection.

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.

Construction and Evaluation of Directionally Cloned cDNA Libraries from Lactating and Non-lactating Mammary Gland of River Buffalo (Bubalus bubalis): A Resource for Gene Identification in Bubaline Genome

Mukesh, M., Kataria, R.S., Kumar, V., Pandey, D., Sodhi, M., Ahlawat, S.P.S., Sobti, R.C. and Mishra, B.P. 2008. Construction and evaluation of directionally cloned cDNA libraries from lactating and non-lactating mammary gland of river buffalo (Bubalus bubalis): A resource for gene identification in bubaline genome. J. Appl. Anim. Res., 33: 81–84. Directionally cloned cDNA libraries from lactating (Lac-MG) and non-lactating (NLac-MG) buffalo mammary gland were constructed and characterized. The total number of independent primary clones were: 8.0 × 104 and 1.1 × 105 for Lac-MG and Nlac-MG cDNA libraries, respectively, with >90% of the clones having cDNA inserts. The occurrence of large cDNA insert length (average 0.90 Kb: range 0.45–2.5 Kb in Lac-MG and average 0.57 Kb: range 0.32–1.8 Kb in NLac-MG cDNA libraries) was indicative of the good quality status of both the libraries. The single-pass sequencing of randomly selected cDNA clones found several full-length cDNA sequences with complete 5′ and 3′ UTR. The initial sequence analysis showed several clones harboring milk protein, ribosomal and regulatory gene transcripts like ubiquitin, mammary derived growth inhibitor, lipoprotein lipase etc. The two cDNA libraries can be utilized as gene resources for cataloguing and annotation of mammary gland transcripts/ESTs in river buffaloes.

Penaeus monodon gene discovery project: The generation of an EST collection and establishment of a database

A large-scale expressed sequence tag (EST) sequencing project was undertaken for the purpose of gene discovery in the black tiger shrimp Penaeus monodon. Initially, 15 cDNA libraries were constructed from different tissues (eyestalk, hepatopancrease, haematopoietic tissue, haemocyte, lymphoid organ, and ovary) of shrimp, reared under normal or stress conditions, to identify tissue-specific genes and genes responding to infection and heat stress. A total of 10,100 clones were analyzed by single-pass sequencing from the 5′ end. Clustering and assembling of these ESTs resulted in a total of 4845 unique sequences with 917 overlapping contigs and 3928 singletons. The redundancy of each cDNA library ranged from 13.4% to 61.3% with an overall redundancy of 61.1%. About half of these ESTs (2365 clones, 48.8%) showed significant homology (BLASTX, e-values < 10 − 4 ) to known genes. A high proportion of P. monodon ESTs was most similar to the predicted protein sequences from various organisms, e.g. Homo sapiens (9%), Mus musculus (7%) , Drosophila (6%), Gallus sp.(6%), and Anopheles (5%). Only 6% showed the highest similarity to other known genes from shrimp due to the limited sequence entries of the species in the public database. Several tissue-specific transcripts were identified as well as the candidate genes that may be implicated in the immune response. In addition, bioinformatic mining of microsatellites from the P. monodon ESTs identified 997 unique microsatellite containing ESTs in which 74 loci resided within the genes of known functions. Consequently, the P. monodon EST database was established. The EST sequence data and the BLAST results were stored and made available through a web-accessible database ( http://pmodon.biotec.or.th). This EST database provides a useful resource for gene identification and functional genomic studies of shrimp.

REGANOR

With >1,000 prokaryotic genome sequencing projects ongoing or already finished, comprehensive comparative analysis of the gene content of these genomes has become viable. To allow for a meaningful comparative analysis, gene prediction of the various genomes should be as accurate as possible. It is clear that improving the state of genome annotation requires automated gene identification methods to cope with the influence of artifacts, such as genomic GC content. There is currently still room for improvement in the state of annotations. We present a web server and a database of high-quality gene predictions. The web server is a resource for gene identification in prokaryote genome sequences. It implements our previously described, accurate gene finding method REGANOR. We also provide novel gene predictions for 241 complete, or almost complete, prokaryotic genomes. We demonstrate how this resource can easily be utilised to identify promising candidates for currently missing genes from genome annotations with several examples. All data sets are available online. The gene finding server is accessible via https://www.cebitec.uni-bielefeld.de/groups/brf/software/reganor/cgi-bin/reganor_upload.cgi. The server software is available with the GenDB genome annotation system (version 2.2.1 onwards) under the GNU general public license. The software can be downloaded from https://sourceforge.net/projects/gendb/. More information on installing GenDB and REGANOR and the system requirements can be found on the GenDB project page http://www.cebitec.uni-bielefeld.de/groups/brf/software/wiki/GenDBWiki/AdministratorDocumentation/GenDBInstallation

Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis.

The oocyte is a key regulator of ovarian folliculogenesis and early embryonic development. However, the composition of the oocyte transcriptome and identities and functions of key oocyte-specific genes involved in the above processes are relatively unknown. Using a PCR-based cDNA amplification method (SMART technology), we constructed a bovine oocyte cDNA library. Analysis of 230 expressed sequence tags (ESTs) from this library identified 102 unique sequences. Although some correspond to housekeeping genes (e.g., ribosomal protein L15) and some represent genes previously known to be expressed in oocytes and other tissues, most encode for genes whose expression in mammalian oocytes has not been reported previously (e.g., cocaine- and amphetamine-regulated transcript) or genes of unknown function. Sixteen did not show significant sequence similarity to any entries in the GenBank database and were classified as novel. Using over 2,000 unsequenced, randomly selected cDNA clones from the library, we constructed an oocyte microarray and performed experiments to identify genes preferentially expressed in fetal ovary (an enriched source of oocytes) relative to somatic tissues. Eleven clones were identified by microarray analysis with consistently higher expression in fetal ovaries (collected from animals at days 210-260 of gestation) compared with spleen and liver. DNA sequence analysis of these clones revealed that two correspond to JY-1, a novel bovine oocyte-specific gene. The remaining nine clones represent five identified genes and one additional completely novel gene. Increased abundance of mRNA in fetal ovary for five of the six genes identified was confirmed by real-time PCR. Results demonstrate the potential utility of these unique resources for identification of oocyte-expressed genes potentially important for reproductive function.

Delineation of a minimal region of deletion at 6q16.3 in follicular lymphoma and construction of a bacterial artificial chromosome contig spanning a 6‐megabase region of 6q16–q21

Regional deletions of 6q are frequent karyotypic alterations in malignant lymphoma and are associated with an adverse clinical outcome. One such region of recurrent deletion is 6q16-q21; however, the specific genes affected have not been identified. Our objective in this study was to identify cases with deletion of 6q16-q21 in follicular lymphoma and to define a minimal region of deletion. A physical map of 6q16.2-q21 was constructed using map information from both sequence-based and bacterial artificial chromosome (BAC) fingerprint-based maps. Forty-three BAC clones spanning a 6-Mb region of 6q16.2-q21 were identified and obtained from the RP-11 library. Selected BACs were fluorescence-labeled and hybridized to a series of 34 follicular lymphomas with a regional 6q deletion detected by G banding. Twenty-four cases with deletion of the 6q16.3 region were detected. A minimal deletion of 2.3 Mb was defined. Our study has identified a limited region of deletion of 6q16.3 that may implicate four known genes in follicular lymphoma and possibly in other cancers. A BAC contig spanning a 6-Mb region has been anchored to the 6q16.2-q21 region. This map represents a useful resource for gene identification in this region, not only in lymphoma but also in other neoplasms with 6q alterations.