NCBI Non-redundant Protein Research Articles

Transcriptome Sequencing and Analysis for Culm Elongation of the World's Largest Bamboo (Dendrocalamus sinicus).

Dendrocalamus sinicus is the world’s largest bamboo species with strong woody culms, and known for its fast-growing culms. As an economic bamboo species, it was popularized for multi-functional applications including furniture, construction, and industrial paper pulp. To comprehensively elucidate the molecular processes involved in its culm elongation, Illumina paired-end sequencing was conducted. About 65.08 million high-quality reads were produced, and assembled into 81,744 unigenes with an average length of 723 bp. A total of 64,338 (79%) unigenes were annotated for their functions, of which, 56,587 were annotated in the NCBI non-redundant protein database and 35,262 were annotated in the Swiss-Prot database. Also, 42,508 and 21,009 annotated unigenes were allocated to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 33,920 unigenes were assigned to 128 KEGG pathways. Meanwhile, 8,553 simple sequence repeats (SSRs) and 81,534 single-nucleotide polymorphism (SNPs) were identified, respectively. Additionally, 388 transcripts encoding lignin biosynthesis were detected, among which, 27 transcripts encoding Shikimate O-hydroxycinnamoyltransferase (HCT) specifically expressed in D. sinicus when compared to other bamboo species and rice. The phylogenetic relationship between D. sinicus and other plants was analyzed, suggesting functional diversity of HCT unigenes in D. sinicus. We conjectured that HCT might lead to the high lignin content and giant culm. Given that the leaves are not yet formed and culm is covered with sheaths during culm elongation, the existence of photosynthesis of bamboo culm is usually neglected. Surprisedly, 109 transcripts encoding photosynthesis were identified, including photosystem I and II, cytochrome b6/f complex, photosynthetic electron transport and F-type ATPase, and 24 transcripts were characterized as antenna proteins that regarded as the main tool for capturing light of plants, implying stem photosynthesis plays a key role during culm elongation due to the unavailability of its leaf. By real-time quantitative PCR, the expression level of 6 unigenes was detected. The results showed the expression level of all genes accorded with the transcriptome data, which confirm the reliability of the transcriptome data. As we know, this is the first study underline the D. sinicus transcriptome, which will deepen the understanding of the molecular mechanisms of culm development. The results may help variety improvement and resource utilization of bamboos.

Transcriptomic profile of Manila clam (Ruditapes philippinarum) haemocytes in response to Perkinsus olseni infection

Ruditapes philippinarum, one of the major contributors to the global mollusc production, is susceptible to perkinsosis, a disease caused by the protozoan Perkinsus olseni (Alveolata, Perkinsidae). Understanding the molecular response of Manila clam defence cells (haemocytes) to this parasite is essential to head towards controlling this disease. Here, we assembled the Manila clam haemocyte transcriptome from healthy and P. olseni infected clams collected from wild and experimental conditions (in vivo and in vitro) using RNA-seq. The Manila clam de novo transcriptome consists of 33,079 transcripts, of which 7300 were significantly annotated (E-value <1.0E−5) with the NCBI non-redundant (nr) protein database. Among these annotated transcripts, many were functionally linked to signalling, cell proliferation, cell adhesion, immune system (e.g. immune response, immune effector process, antigen processing) and response to stress and stimuli. Our study integrates previous transcriptomic information of R. philippinarum haemocytes with present resources, including a notable amount of new transcripts with annotation description expressed after challenge with P. olseni in different infection scenarios, many of them linked to stress-related processes. A preliminary analysis of differentially expressed genes (DEGs) was performed with the sequences from the pool samples of control and challenged haemocytes obtained under similar experimental conditions. A higher number of DEGs were detected in in vitro than in vivo stimulated haemocytes, denoting the more acute and different in vitro challenges with P. olseni trophozoites, zoospores and extracellular products. Most in vitro DEGs were down-regulated and associated with immune and stress response, likely reflecting the immune-suppression occurring at the onset of infection, while DEGs in the in vivo infected haemocytes were mostly related to metabolic process. The preliminary DEG analysis on haemocyte response provides some insight into the molecular mechanism of Manila clam defence cells against perkinsosis. Statement of relevanceHigh Manila clam mortality rates have been reported due to perkinsosis caused by Perkinsus olseni, however, the molecular mechanisms of Ruditapes philippinarum response against perkinsosis are still not fully understood. Our study provided new data to enrich the R. philippinarum haemocyte transcriptome databases from previous reports, and preliminary information on haemocyte response against perkinsosis is provided.

Comparative transcriptomics reveals genes involved in metabolic and immune pathways in the digestive gland of scallop Chlamys farreri following cadmium exposure

Chlamys farreri is an economically important mollusk that can accumulate excessive amounts of cadmium (Cd). Studying the molecular mechanism of Cd accumulation in bivalves is difficult because of the lack of genome background. Transcriptomic analysis based on high-throughput RNA sequencing has been shown to be an efficient and powerful method for the discovery of relevant genes in non-model and genome reference-free organisms. Here, we constructed two cDNA libraries (control and Cd exposure groups) from the digestive gland of C. farreri and compared the transcriptomic data between them. A total of 227 673 transcripts were assembled into 105 071 unigenes, most of which shared high similarity with sequences in the NCBI non-redundant protein database. For functional classification, 24 493 unigenes were assigned to Gene Ontology terms. Additionally, EuKaryotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes analyses assigned 12 028 unigenes to 26 categories and 7 849 unigenes to five pathways, respectively. Comparative transcriptomics analysis identified 3 800 unigenes that were differentially expressed in the Cd-treated group compared with the control group. Among them, genes associated with heavy metal accumulation were screened, including metallothionein, divalent metal transporter, and metal tolerance protein. The functional genes and predicted pathways identified in our study will contribute to a better understanding of the metabolic and immune system in the digestive gland of C. farreri. In addition, the transcriptomic data will provide a comprehensive resource that may contribute to the understanding of molecular mechanisms that respond to marine pollutants in bivalves.

Whole-transcriptome analysis of differentially expressed genes in the ray florets and disc florets of Chrysanthemum morifolium.

BackgroundChrysanthemum morifolium is one of the most important global cut flower and pot plants, and has been cultivated worldwide. However, limited genomic resources are available and the molecular mechanisms involved in the two morphologically distinct floret developmental cycles in chrysanthemum remain unclear.ResultsThe transcriptomes of chrysanthemum ray florets, disc florets and leaves were sequenced using Illumina paired-end sequencing technology. In total, 16.9 G reads were assembled into 93,138 unigenes with an average length of 738 bp, of which 44,364 unigenes showed similarity to known proteins in the Swissprot or NCBI non-redundant protein databases. Additionally, 26,320, 22,304 and 13,949 unigenes were assigned to 54 gene ontology (GO) categories, 25 EuKaryotic Orthologous Groups (KOG) categories, and 280 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively.A total of 1863 differentially expressed genes (DEGs) (1210 up-regulated and 653 down-regulated) were identified between ray florets and disc florets, including genes encoding transcription factors and protein kinases. GO and KEGG pathway enrichment analyses were performed on the DEGs to identify differences in the biological processes and pathways between ray florets and disc florets. The important regulatory genes controlling flower development and flower organ determination, as well as important functional genes in the anthocyanin biosynthetic pathway, were identified, of which two leucoanthocyanidin dioxygenase-encoding genes showed specific expression in ray florets. Lastly, reverse transcription quantitative PCR was conducted to validate the DEGs identified in our study.ConclusionsComparative transcriptome analysis revealed significant differences in patterns of gene expression and signaling pathways between ray florets and disc florets in Chrysanthemum morifolium. This study provided the first step to understanding the molecular mechanism of the differential development of ray florets and disc florets in chrysanthemum, and also provided valuable genomic resources for candidate genes applicable for the breeding of novel varieties in chrysanthemum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2733-z) contains supplementary material, which is available to authorized users.

Sequencing and characterization of leaf transcriptomes of six diploid Nicotiana species.

BackgroundNicotiana belongs to the Solanaceae family that includes important crops such as tomato, potato, eggplant, and pepper. Nicotiana species are of worldwide economic importance and are important model plants for scientific research. Here we present the comparative analysis of the transcriptomes of six wild diploid Nicotiana species. Wild relatives provide an excellent study system for the analysis of the genetic basis for various traits, especially disease resistance.ResultsWhole transcriptome sequencing (RNA-seq) was performed for leaves of six diploid Nicotiana species, i.e. Nicotiana glauca, Nicotiana noctiflora, Nicotiana cordifolia, Nicotiana knightiana, Nicotiana setchellii and Nicotiana tomentosiformis. For each species, 9.0–22.3 Gb high-quality clean data were generated, and 67,073–182,046 transcripts were assembled with lengths greater than 100 bp. Over 90 % of the ORFs in each species had significant similarity with proteins in the NCBI non-redundant protein sequence (NR) database. A total of 2491 homologs were identified and used to construct a phylogenetic tree from the respective transcriptomes in Nicotiana. Bioinformatic analysis identified resistance gene analogs, major transcription factor families, and alkaloid transporter genes linked to plant defense.ConclusionsThis is the first report on the leaf transcriptomes of six wild Nicotiana species by Illumina paired-end sequencing and de novo assembly without a reference genome. These sequence resources hopefully will provide an opportunity for identifying genes involved in plant defense and several important quality traits in wild Nicotiana and will accelerate functional genomic studies and genetic improvement efforts of Nicotiana or other important Solanaceae crops in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s40709-016-0048-5) contains supplementary material, which is available to authorized users.

De novo transcriptomic analysis of the female and male adults of the blood fluke Schistosoma turkestanicum.

BackgroundSchistosoma turkestanicum is a parasite of considerable veterinary importance as an agent of animal schistosomiasis in many countries, including China. The S. turkestanicum cercariae can also infect humans, causing cercarial dermatitis in many countries and regions of the world. In spite of its significance as a pathogen of animals and humans, there is little transcriptomic and genomic data in the public databases.MethodsHerein, we performed the transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females of S. turkestanicum and de novo transcriptome assembly.ResultsApproximately 81.1 (female) and 80.5 (male) million high-quality clean reads were obtained and then 29,526 (female) and 41,346 (male) unigenes were assembled. A total of 34,624 unigenes were produced from S. turkestanicum females and males, with an average length of 878 nucleotides (nt) and N50 of 1480 nt. Of these unigenes, 25,158 (72.7 %) were annotated by blast searches against the NCBI non-redundant protein database. Among these, 21,995 (63.5 %), 22,189 (64.1 %) and 13,754 (39.7 %) of the unigenes had significant similarity in the NCBI non-redundant protein (NR), non-redundant nucleotide (NT) and Swiss-Prot databases, respectively. In addition, 3150 unigenes were identified to be expressed specifically in females and 1014 unigenes were identified to be expressed specifically in males. Interestingly, several pathways associated with gonadal development and sex maintenance were found, including the Wnt signaling pathway (103; 2 %) and progesterone-mediated oocyte maturation (77; 1.5 %).ConclusionsThe present study characterized and compared the transcriptomes of adult female and male blood fluke, S. turkestanicum. These results will not only serve as valuable resources for future functional genomics studies to understand the molecular aspects of S. turkestanicum, but also will provide essential information for ongoing whole genome sequencing efforts on this pathogenic blood fluke.

Development of Gene-Based SSR Markers in Rice Bean (Vigna umbellata L.) Based on Transcriptome Data.

Rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) is a warm season annual legume mainly grown in East Asia. Only scarce genomic resources are currently available for this legume crop species and no simple sequence repeat (SSR) markers have been specifically developed for rice bean yet. In this study, approximately 26 million high quality cDNA sequence reads were obtained from rice bean using Illumina paired-end sequencing technology and assembled into 71,929 unigenes with an average length of 986 bp. Of these unigenes, 38,840 (33.2%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases. Furthermore, 30,170 (76.3%) could be classified into gene ontology categories, 25,451 (64.4%) into Swiss-Prot categories and 21,982 (55.6%) into KOG database categories (E-value < 1.0E-5). A total of 9,301 (23.5%) were mapped onto 118 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. A total of 3,011 genic SSRs were identified as potential molecular markers. AG/CT (30.3%), AAG/CTT (8.1%) and AGAA/TTCT (20.0%) are the three main repeat motifs. A total of 300 SSR loci were randomly selected for validation by using PCR amplification. Of these loci, 23 primer pairs were polymorphic among 32 rice bean accessions. A UPGMA dendrogram revealed three major clusters among 32 rice bean accessions. The large number of SSR-containing sequences and genic SSRs in this study will be valuable for the construction of high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.

SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination.

AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation, and, in cross-sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and that protection against SIV infection by vaccination prevents enteropathogen emergence.

Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus.

BackgroundAflatoxin contamination caused by Aspergillus flavus in peanut (Arachis hypogaea) including in pre- and post-harvest stages seriously affects industry development and human health. Even though resistance to aflatoxin production in post-harvest peanut has been identified, its molecular mechanism has been poorly understood. To understand the mechanism of peanut response to aflatoxin production by A. flavus, RNA-seq was used for global transcriptome profiling of post-harvest seed of resistant (Zhonghua 6) and susceptible (Zhonghua 12) peanut genotypes under the fungus infection and aflatoxin production stress.ResultA total of 128.72 Gb of high-quality bases were generated and assembled into 128, 725 unigenes (average length 765 bp). About 62, 352 unigenes (48.43 %) were annotated in the NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Swiss-Prot, KEGG Ortholog, Protein family, Gene Ontology, or eukaryotic Ortholog Groups database and more than 93 % of the unigenes were expressed in the samples. Among obtained 30, 143 differentially expressed unigenes (DEGs), 842 potential defense-related genes, including nucleotide binding site-leucine-rich repeat proteins, polygalacturonase inhibitor proteins, leucine-rich repeat receptor-like kinases, mitogen-activated protein kinase, transcription factors, ADP-ribosylation factors, pathogenesis-related proteins and crucial factors of other defense-related pathways, might contribute to peanut response to aflatoxin production. Notably, DEGs involved in phenylpropanoid-derived compounds biosynthetic pathway were induced to higher levels in the resistant genotype than in the susceptible one. Flavonoid, stilbenoid and phenylpropanoid biosynthesis pathways were enriched only in the resistant genotype.ConclusionsThis study provided the first comprehensive analysis of transcriptome of post-harvest peanut seeds in response to aflatoxin production, and would contribute to better understanding of molecular interaction between peanut and A. flavus. The data generated in this study would be a valuable resource for genetic and genomic studies on crops resistance to aflatoxin contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0738-z) contains supplementary material, which is available to authorized users.

Transcriptome analysis of ‘Sijihua’ longan (Dimocarpus longan L.) based on next-generation sequencing technology

ABSTRACT‘Sijihua’ longan (Dimocarpus longan Lour.) blossoms throughout the year, and hence it is a useful material for studies on the mechanism of flowering. However, few genomic resources such as DNA sequences or markers are currently available for ‘Sijihua’ longan. To increase our understanding of the genetic control of important flowering characteristics in ‘Sijihua’ longan, we constructed a mixed cDNA library of ‘Sijihua’ longan using Illumina paired-end sequencing technology and obtained a total of 90,069,246 high-quality reads. Among the unigenes, 43.16% (59,588) had at least one BLASTX hit against the Arabidopsis thaliana sequence database and 42.83% (59,130) had significant similarity with proteins in the NCBI non-redundant protein database. Of these annotated unigenes, a total of 15,956 (11.56%) were mapped onto 271 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. Furthermore, 11,402 unigenes were converted into 13,236 potential simple sequence repeats (SSRs). Dinucleotide SSRs were the dominant repeat motif, with a frequency of 54.3%, followed by trinucleotide (34.6%) and tetranucleotide (4.7%) repeat motifs. AG/CT repeats (46.29%) were the most frequent motifs in the data set. We focused on gene discovery in the area of regulation of flowering, and transcripts were identified for almost every gene involved in the control of flowering. This study demonstrates that Illumina sequencing technology is a rapid and informative approach to gene discovery and molecular marker development in non-model organisms without a whole genome database. Our results provide a comprehensive sequence resource for investigating the mechanisms of flowering in ‘Sijihua’ longan, and will establish a platform for further studies on longan.

Multi-tissue transcriptomics for construction of a comprehensive gene resource for the terrestrial snail Theba pisana.

The land snail Theba pisana is native to the Mediterranean region but has become one of the most abundant invasive species worldwide. Here, we present three transcriptomes of this agriculture pest derived from three tissues: the central nervous system, hepatopancreas (digestive gland), and foot muscle. Sequencing of the three tissues produced 339,479,092 high quality reads and a global de novo assembly generated a total of 250,848 unique transcripts (unigenes). BLAST analysis mapped 52,590 unigenes to NCBI non-redundant protein databases and further functional analysis annotated 21,849 unigenes with gene ontology. We report that T. pisana transcripts have representatives in all functional classes and a comparison of differentially expressed transcripts amongst all three tissues demonstrates enormous differences in their potential metabolic activities. The genes differentially expressed include those with sequence similarity to those genes associated with multiple bacterial diseases and neurological diseases. To provide a valuable resource that will assist functional genomics study, we have implemented a user-friendly web interface, ThebaDB (http://thebadb.bioinfo-minzhao.org/). This online database allows for complex text queries, sequence searches, and data browsing by enriched functional terms and KEGG mapping.

De Novo Transcriptome Assembly of the Chinese Swamp Buffalo by RNA Sequencing and SSR Marker Discovery.

The Chinese swamp buffalo (Bubalis bubalis) is vital to the lives of small farmers and has tremendous economic importance. However, a lack of genomic information has hampered research on augmenting marker assisted breeding programs in this species. Thus, a high-throughput transcriptomic sequencing of B. bubalis was conducted to generate transcriptomic sequence dataset for gene discovery and molecular marker development. Illumina paired-end sequencing generated a total of 54,109,173 raw reads. After trimming, de novo assembly was performed, which yielded 86,017 unigenes, with an average length of 972.41 bp, an N50 of 1,505 bp, and an average GC content of 49.92%. A total of 62,337 unigenes were successfully annotated. Among the annotated unigenes, 27,025 (43.35%) and 23,232 (37.27%) unigenes showed significant similarity to known proteins in NCBI non-redundant protein and Swiss-Prot databases (E-value < 1.0E-5), respectively. Of these annotated unigenes, 14,439 and 15,813 unigenes were assigned to the Gene Ontology (GO) categories and EuKaryotic Ortholog Group (KOG) cluster, respectively. In addition, a total of 14,167 unigenes were assigned to 331 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, 17,401 simple sequence repeats (SSRs) were identified as potential molecular markers. One hundred and fifteen primer pairs were randomly selected for amplification to detect polymorphisms. The results revealed that 110 primer pairs (95.65%) yielded PCR amplicons and 69 primer pairs (60.00%) presented polymorphisms in 35 individual buffaloes. A phylogenetic analysis showed that the five swamp buffalo populations were clustered together, whereas two river buffalo breeds clustered separately. In the present study, the Illumina RNA-seq technology was utilized to perform transcriptome analysis and SSR marker discovery in the swamp buffalo without using a reference genome. Our findings will enrich the current SSR markers resources and help spearhead molecular genetic research studies on the swamp buffalo.

Transcriptome of High-Sucrose Sugarcane Variety GT35

Sugarcane (Saccharum officinarum L.) is the most important sugar crop and a major source of bio-ethanol. Sucrose accumulation is important in sugarcane, but it has not been investigated by transcriptome analysis . In this study, the transcriptome of a high-sucrose sugarcane variety, GT35, was sequenced using high-throughput Solexa technology. A total of 34,105,138 high-quality reads with an average length of 75 bp were obtained. These reads were assembled into 101,255 unigenes, with a mean length of 460 bp and an N50 length of 640 bp. BLAST searches indicated that 36,472 (36.0 %) unigenes had homologous sequences in the NCBI non-redundant protein sequences databases, and 23,811 (23.5 %) had homologous sequences in the Swiss-Prot databases. A total of 14,613 unigenes were assigned gene ontology terms, and 13,231 were assigned functional annotations and grouped into 25 functional categories. A KEGG pathway analysis of 30,756 unigenes revealed more than 30 pathways in the sugarcane transcriptome. A total of 3420 simple sequence repeats were identified in 3185 unigenes. The data in this study enriched the sugarcane transcriptome resources and will be useful for further comparison and functional genomic studies in genus Saccharum and family Poaceae, and will be useful for further detailed elucidation of sucrose accumulation in sugarcane.

High-performance computational analysis and peptide screening from databases of cyclotides from poaceae.

Cyclotides are a family of head-to-tail cyclized peptides containing three conserved disulfide bonds, in a structural scaffold also known as a cyclic cysteine knot. Due to the high degree of cysteine conservation, novel members from this peptide family can be identified in protein databases through a search through regular expression (REGEX). In this work, six novel cyclotide-like precursors from the Poaceae were identified from NCBI's non-redundant protein database by the use of REGEX. Two out of six sequences (named Zea mays L and M) showed an Asp residue in the C-terminal, which indicated that they could be cyclic. Gene expression in maize tissues was investigated, showing that the previously described cyclotide-like Z. mays J is expressed in the roots. According to molecular dynamics, the structure of Z. mays J seems to be stable, despite the putative absence of cyclization. As regards cyclotide evolution, it was hypothesized that this is an outcome from convergent evolution and/or horizontal gene transfer. The results showed that peptide screening from databases should be performed periodically in order to include novel sequences, which are deposited as the databases grow. Indeed, the advances in computational and experimental methods will together help to answer key questions and reach new horizons in defense-related peptide identification.

Development, characterization, and annotation of potential simple sequence repeats by transcriptome sequencing in pears (Pyrus pyrifolia Nakai).

Simple sequence repeats (SSRs), one of the most powerful molecular markers, can be used for DNA fingerprinting, variety identification, genetic mapping, and marker-assisted selection. Using the pear's (Pyrus pyrifolia Nakai) 75,764 unigenes (55,676,271 bp) obtained by deep transcriptome sequencing, a total of 10,622 novel SSRs were identified in 9154 unigenes, accounting for 14.02% of all unigenes. The average length and distribution of these SSRs was about 16 bp and 5.24 kb, respectively. Dinucleotide repeat motifs were the main type, with a frequency of 55.87%, followed by trinucleotides (24.45%). There were 159 kinds of repeat motifs existing in the pear transcriptome. AG/CT was the most frequent motif, accounting for 49.64%. All 9154 SSR-containing unigenes were functionally annotated using Nr (NCBI non-redundant protein database), Nt (NCBI non-redundant nucleotide database), and the Swiss-Prot database, and were classified further by Gene Ontology and Clusters of Orthologous Groups. In addition, a total of 4300 primer pairs were designed from all SSR loci obtained. Of these, 40 primers were randomly selected for PCR amplification and polyacrylamide gel (PAGE) analysis. Among the 40 primer pairs, 31 were successfully separated via PAGE. These findings also confirm that mining SSRs using next-generating sequencing technologies is a fast, effective, and reliable approach.

Transcriptome analysis and anthocyanin-related genes in red leaf lettuce.

This study aimed to analyze the transcriptome profile of red lettuce and identify the genes involved in anthocyanin accumulation. Red leaf lettuce is a popular vegetable and popular due to its high anthocyanin content. However, there is limited information available about the genes involved in anthocyanin biosynthesis in this species. In this study, transcriptomes of 15-day-old seedlings and 40-day-old red lettuce leaves were analyzed using an Illuminia HiseqTM 2500 platform. A total of 10.6 GB clean data were obtained and de novo assembled into 83,333 unigenes with an N50 of 1067. After annotation against public databases, 51,850 unigene sequences were identified, among which 46,087 were annotated in the NCBI non-redundant protein database, and 41,752 were annotated in the Swiss-Prot database. A total of 9125 unigenes were mapped into 163 pathways using the Kyoto Encyclopedia of Genes and Genomes database. Thirty-four structural genes were found to cover the main steps of the anthocyanin pathway, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. Seven MYB, three bHLH, and two WD40 genes, considered anthocyanin regulatory genes, were also identified. In addition, 3607 simple sequence repeat (SSR) markers were identified from 2916 unigenes. This research uncovered the transcriptomic characteristics of red leaf lettuce seedlings and mature plants. The identified candidate genes related to anthocyanin biosynthesis and the detected SSRs provide useful tools for future molecular breeding studies.

Discovery of genes for production of biofuels through transcriptome sequencing of Dunaliella parva

Compared with first generation plant-based biofuels, microalgae have potential advantages as feedstock for biofuel production. However, the genome sequences were insufficient in non-model microalgae with potential for biofuel production, which limited the development of genetic engineering of these microalgae. Here we described the de novo transcriptome sequencing and assembly for the halophilic green alga Dunaliella parva and identified important pathways, genes and transcription factor gene wri1 involved in biofuel production. The Illumina Hiseq 2000 platform was applied to D. parva transcriptome sequencing, which produced 26,304,060 (assembled into 60,883 contigs) and 26,797,446 (assembled into 55,236 contigs) high quality reads in samples SCH-5.0mMA (control sample) and SCH-0.5mMA (nitrogen limitation sample) respectively. Assembled contigs were subjected to Blastx search and annotated with NCBI non-redundant protein database, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes orthology identifiers. These analyses obtained the majority of genes involved in the biosynthesis and degradation of lipid and starch in D. parva. In addition, we have identified numerous differentially expressed genes, especially important transcription factor gene wri1 (GenBank KR185335) and studied differential expression of wri1 between control and nitrogen limitation sample, which provided foundation for illuminating the regulation mechanism of lipid biosynthesis induced by nitrogen limitation.

Transcriptome Analysis of Syringa oblata Lindl. Inflorescence Identifies Genes Associated with Pigment Biosynthesis and Scent Metabolism.

Syringa oblata Lindl. is a woody ornamental plant with high economic value and characteristics that include early flowering, multiple flower colors, and strong fragrance. Despite a long history of cultivation, the genetics and molecular biology of S. oblata are poorly understood. Transcriptome and expression profiling data are needed to identify genes and to better understand the biological mechanisms of floral pigments and scents in this species. Nine cDNA libraries were obtained from three replicates of three developmental stages: inflorescence with enlarged flower buds not protruded, inflorescence with corolla lobes not displayed, and inflorescence with flowers fully opened and emitting strong fragrance. Using the Illumina RNA-Seq technique, 319,425,972 clean reads were obtained and were assembled into 104,691 final unigenes (average length of 853 bp), 41.75% of which were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 36,967 were assigned to gene ontology categories and 19,956 were assigned to eukaryoticorthologous groups. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, 12,388 unigenes were sorted into 286 pathways. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at different flower stages and that were related to floral pigment biosynthesis and fragrance metabolism. This comprehensive transcriptomic analysis provides fundamental information on the genes and pathways involved in flower secondary metabolism and development in S. oblata, providing a useful database for further research on S. oblata and other plants of genus Syringa.

De Novo Assembly and Characterization of the Invasive Northern Pacific Seastar Transcriptome.

Invasive species are a major threat to global biodiversity but can also serve as valuable model systems to examine important evolutionary processes. While the ecological aspects of invasions have been well documented, the genetic basis of adaptive change during the invasion process has been hampered by a lack of genomic resources for the majority of invasive species. Here we report the first larval transcriptomic resource for the Northern Pacific Seastar, Asterias amurensis, an invasive marine predator in Australia. Approximately 117.5 million 100 base-pair (bp) paired-end reads were sequenced from a single RNA-Seq library from a pooled set of full-sibling A. amurensis bipinnaria larvae. We evaluated the efficacy of a pre-assembly error correction pipeline on subsequent de novo assembly. Error correction resulted in small but important improvements to the final assembly in terms of mapping statistics and core eukaryotic genes representation. The error-corrected de novo assembly resulted in 115,654 contigs after redundancy clustering. 41,667 assembled contigs were homologous to sequences from NCBI’s non-redundant protein and UniProt databases. We assigned Gene Ontology, KEGG Orthology, Pfam protein domain terms and predicted protein-coding sequences to > 36,000 contigs. The final transcriptome dataset generated here provides functional information for 18,319 unique proteins, comprising at least 11,355 expressed genes. Furthermore, we identified 9,739 orthologs to P. miniata proteins, evaluated our annotation pipeline and generated a list of 150 candidate genes for responses to several environmental stressors that may be important for adaptation of A. amurensis in the invasive range. Our study has produced a large set of A. amurensis RNA contigs with functional annotations that can serve as a resource for future comparisons to other echinoderm transcriptomes and gene expression studies. Our data can be used to study the genetic basis of adaptive change and other important evolutionary processes during a successful invasion.

De novo assembly and characterization of the Welsh onion (Allium fistulosum L.) transcriptome using Illumina technology.

Welsh onion (Allium fistulosum L.) has long been cultivated as a vegetable and spice for its flavor and aroma. However, transcriptomic and genomic data for A. fistulosum remain scarce. The goal of this study was to generate transcript sequences for functional genomic analyses, and identify genes potentially involved in sulfur, selenium, and vitamin metabolism. In total, 53,378,674 high-quality reads were generated, and de novo assembly resulted in 103,286 contigs and 53,837 unigenes. The average unigene length was 619 bp with an N50 of 832 bp. Similarity searches revealed that 36,155 sequences were similar to those of known proteins in public databases. Of these, 35,250 unigenes sequences were significantly similar to sequences in the NCBI non-redundant protein database and 22,804 were annotated in the Swiss-Prot database. Additionally, 13,125 and 26,660 unigenes were annotated in the Cluster of Orthologous Group and Gene Ontology databases, respectively. A total of 20,680 unigenes were classified into 128 pathways via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database. Key enzymes involved in sulfur and selenium metabolism were also identified. Additionally, our transcriptome revealed a number of unigenes encoding important enzymes involved in vitamin metabolism. We also identified 2014 simple sequence repeats in 1892 unigenes. This transcriptome analysis provides valuable information to further our understanding of the molecular mechanisms regulating the biosynthesis of organic sulfur compounds. The detected simple sequence repeats may facilitate marker-assisted selection in Welsh onion breeding experiments.