BACKGROUND: The hair follicle development process is regulated by sophisticated genes and signaling networks, and the hair grows from the hair follicle. The Tianzhu white yak population exhibits differences in hair length, especially on the forehead and shoulder region. However, the genetic mechanism is still unclear. Isoform sequencing (Iso-seq) technology with advantages in long reads sequencing. Hence, we combined the Iso-seq and RNA-seq methods to investigate the transcript complexity and difference between long-haired yak (LHY) and normal-haired yak (NHY).
RESULTS: The hair length measurement result showed a significant difference between LHY and NHY on the forehead and the shoulder (P-value < 0.001). The skin samples from the forehead and the shoulder of LHY and NHY were pooled for isoform sequencing (Iso-seq). We obtained numerous long transcripts, including novel isoforms, long non-coding RNA, alternative splicing events, and alternative polyadenylation events. Combined with RNA-seq data, we performed differential isoforms (DEIs) analysis between LHY and NHY. We found that some hair follicle and skin development-related DEIs, like BMP4, KRT2, IGF2R, and COL1A2 in the forehead skin; BMP1, KRT1, FGF5, COL2A1, and IGFBP5 in the shoulder skin. Enrichment analysis revealed that DEIs in both two comparable groups significantly participated in skin and hair follicle development-related pathways, like ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. The results indicated that the hair follicle development of Tianzhu white yak may influence the hair length difference. Besides, the protein-protein interaction (PPI) network of DEIs showed COL2A1 and COL3A1 exhibited a high degree of centrality, and these two genes were suggested as potential candidates for the hair length growth of Tianzhu white yak.
CONCLUSIONS: The results provided a comprehensive analysis of the transcriptome complexity and identified differential transcripts that enhance our understanding of the molecular mechanisms underlying the variation in hair length growth in Tianzhu white yak.

Moso bamboo is capable of both sexual and asexual reproduction during natural growth, resulting in four distinct types of culms: the bamboo shoot-culm, the seedling stem, the leptomorph rhizome, and a long-ignored culm-the outward-rhizome. Sometimes, when the outward rhizomes break through the soil, they continue to grow longitudinally and develop into a new individual. However, the roles of alternative transcription start sites (aTSS) or termination sites (aTTS) as well as alternative splicing (AS) have not been comprehensively studied for their development. To re-annotate the moso bamboo genome and identify genome-wide aTSS, aTTS, and AS in growing culms, we utilized single-molecule long-read sequencing technology. In total, 169,433 non-redundant isoforms and 14,840 new gene loci were identified. Among 1311 lncRNAs, most of which showed a positive correlation with their target mRNAs, one-third of these IncRNAs were preferentially expressed in winter bamboo shoots. In addition, the predominant AS type observed in moso bamboo was intron retention, while aTSS and aTTS events occurred more frequently than AS. Notably, most genes with AS events were also accompanied by aTSS and aTTS events. Outward rhizome growth in moso bamboo was associated with a significant increase in intron retention, possibly due to changes in the growth environment. As different types of moso bamboo culms grow and develop, a significant number of isoforms undergo changes in their conserved domains due to the regulation of aTSS, aTTS, and AS. As a result, these isoforms may play different roles than their original functions. These isoforms then performed different functions from their original roles, contributing to the transcriptomic complexity of moso bamboo. Overall, this study provided a comprehensive overview of the transcriptomic changes underlying different types of moso bamboo culm growth and development.

BACKGROUND: Japanese larch (Larix kaempferi) is an economically important deciduous conifer species that grows in cool-temperate forests and is endemic to Japan. Kuril larch (L. gmelinii var. japonica) is a variety of Dahurian larch that is naturally distributed in the Kuril Islands and Sakhalin. The hybrid larch (L. gmelinii var. japonica × L. kaempferi) exhibits heterosis, which manifests as rapid juvenile growth and high resistance to vole grazing. Since these superior characteristics have been valued by forestry managers, the hybrid larch is one of the most important plantation species in Hokkaido. To accelerate molecular breeding in these species, we collected and compared full-length cDNA isoforms (Iso-Seq) and RNA-Seq short-read, and merged them to construct candidate gene as reference for both Larix species. To validate the results, candidate protein-coding genes (ORFs) related to some flowering signal-related genes ​were screened from the reference sequences, and the phylogenetic relationship with closely related species was elucidated.
RESULTS: Using the isoform sequencing of PacBio RS ll and the de novo assembly of RNA-Seq short-read sequences, we identified 50,690 and 38,684 ORFs in Japanese larch and Kuril larch, respectively. BUSCO completeness values were 90.5% and 92.1% in the Japanese and Kuril larches, respectively. After comparing the collected ORFs from the two larch species, a total of 19,813 clusters, comprising 22,571 Japanese larch ORFs and 22,667 Kuril larch ORFs, were contained in the intersection of the Venn diagram. In addition, we screened several ORFs related to flowering signals (SUPPRESSER OF OVEREXPRESSION OF CO1: SOC1, LEAFY: LFY, FLOWERING Locus T: FT, CONSTANCE: CO) from both reference sequences, and very similar found in other species.
CONCLUSIONS: The collected ORFs will be useful as reference sequences for molecular breeding of Japanese and Kuril larches, and also for clarifying the evolution of the conifer genome and investigating functional genomics.

A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder (Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events (12,352) than the R group (11,452). The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.

Hucho bleekeri is a glacial relict and critically endangered fish restricted to the Yangtze River drainage in China. The lack of basic genomic information and immune characteristics will hinder the way toward protecting this species. In the present study, we conducted the first transcriptome analysis of H. bleekeri using the combination of SMRT and Illumina sequencing technology. Transcriptome sequencing generated a total of 93,330 non-redundant full-length unigenes with a mean length of 3072 bp. A total of 92,472 (99.08%) unigenes were annotated in at least one of the Nr protein, Swiss-Prot, KEGG, KOG, GO, Nt and Pfam databases. KEGG analysis showed that a total of 7240 unigenes belonging to 28 immune pathways were annotated to the immune system category. Meanwhile, differentially expressed genes between mucosa-associated tissues (skin, gill and hindgut) and systemic-immune tissues (spleen, head kidney and liver) were obtained. Importantly, genes participating in diverse immune signalling pathways and their expression profiles in H. bleekeri were discussed. In addition, a large number of long non-coding RNAs (lncRNAs) and simple sequence repeats (SSRs) were obtained in the H. bleekeri transcriptome. The present study will provide basic genomic information for H. bleekeri and for further research on analysing the characteristics of both the innate and adaptive immune systems of this critically endangered species.

BACKGROUND: Sturgeons (Acipenseriformes) are polyploid chondrostean fish that constitute an important model species for studying development and evolution in vertebrates. To better understand the mechanisms of reproduction regulation in sturgeon, this study combined PacBio isoform sequencing (Iso-Seq) with Illumina short-read RNA-seq methods to discover full-length genes involved in early gametogenesis of the Amur sturgeon, Acipenser schrenckii.
RESULTS: A total of 50.04 G subread bases were generated from two SMRT cells, and herein 164,618 nonredundant full-length transcripts (unigenes) were produced with an average length of 2782 bp from gonad tissues (three testes and four ovaries) from seven 3-year-old A. schrenckii individuals. The number of ovary-specific expressed unigenes was greater than those of testis (19,716 vs. 3028), and completely different KEGG pathways were significantly enriched between the ovary-biased and testis-biased DEUs. Importantly, 60 early gametogenesis-related genes (involving 755 unigenes) were successfully identified, and exactly 50% (30/60) genes of those showed significantly differential expression in testes and ovaries. Among these, the Amh and Gsdf with testis-biased expression, and the Foxl2 and Cyp19a with ovary-biased expression strongly suggested the important regulatory roles in spermatogenesis and oogenesis of A. schrenckii, respectively. We also found the four novel Sox9 transcript variants, which increase the numbers of regulatory genes and imply function complexity in early gametogenesis. Finally, a total of 236,672 AS events (involving 36,522 unigenes) were detected, and 10,556 putative long noncoding RNAs (lncRNAs) and 4339 predicted transcript factors (TFs) were also respectively identified, which were all significantly associated with the early gametogenesis of A. schrenckii.
CONCLUSIONS: Overall, our results provide new genetic resources of full-length transcription data and information as a genomic-level reference for sturgeon. Crucially, we explored the comprehensive genetic characteristics that differ between the testes and ovaries of A. schrenckii in the early gametogenesis stage, which could provide candidate genes and theoretical basis for further the mechanisms of reproduction regulation of sturgeon.

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed, and many new generally shorter transcripts were detected by normalization. For the same input cDNA and data yield, the normalized library recovered more total transcript isoforms and number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ∼1.25 kb and more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ∼52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk, and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ∼80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp.

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