LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly.

Background: Completing a genome is an important goal of genome assembly. However, many assemblies, including reference assemblies, are unfinished and have a number of gaps. Long reads obtained from third-generation sequencing (TGS) platforms can help close these gaps and improve assembly contiguity. However, current gap-closure approaches using long reads require extensive runtime and high memory usage. Thus, a fast and memory-efficient approach using long reads is needed to obtain complete genomes. Findings: We developed LR_Gapcloser to rapidly and efficiently close the gaps in genome assembly. This tool utilizes long reads generated from TGS sequencing platforms. Tested on de novo assembled gaps, repeat-derived gaps, and real gaps, LR_Gapcloser closed a higher number of gaps faster and with a lower error rate and a much lower memory usage than two existing, state-of-the art tools. This tool utilized raw reads to fill more gaps than when using error-corrected reads. It is applicable to gaps in the assemblies by different approaches and from large and complex genomes. After performing gap-closure using this tool, the contig N50 size of the human CHM1 genome was improved from 143 kb to 19 Mb, a 132-fold increase. We also closed the gaps in the Triticum urartu genome, a large genome rich in repeats; the contig N50 size was increased by 40%. Further, we evaluated the contiguity and correctness of six hybrid assembly strategies by combining the optimal TGS-based and next-generation sequencing-based assemblers with LR_Gapcloser. A proposed and optimal hybrid strategy generated a new human CHM1 genome assembly with marked contiguity. The contig N50 value was greater than 28 Mb, which is larger than previous non-reference assemblies of the diploid human genome. Conclusions: LR_Gapcloser is a fast and efficient tool that can be used to close gaps and improve the contiguity of genome assemblies. A proposed hybrid assembly including this tool promises reference-grade assemblies. The software is available at http://www.fishbrowser.org/software/LR_Gapcloser/.

[Response of grain filling characteristics of maize to sowing date.] / 玉米籽粒灌浆特性对播期的响应.

To study the effect of different sowing dates on the process of grain filling characteristics, Jingke 528 (JK528) and Zhengdan 958 (ZD958) were used in the field experiment with two sowing dates (10th April and 14th May). The results showed that the 100-grain dry mass and yield of JK528 and ZD958 with the early spring sowing were significantly higher than that with the spring sowing date, which increased by 6.8% and 10.1%, 17.8% and 9.2%, respectively. The maximum grain filling rate (Gmax) and the average grain filling rate (Gave) were higher with the early spring sowing than that with the spring sowing date, while the active grain filling period (P) showed an opposite trend. The 100-grain mass and yield of JK528 were significantly improved by 7.4 g and 1189.6 kg·hm-2 compared to ZD958, with the increase of 21.6% and 10.8%, respectively. The P of ZD958 was higher than that of JK528, while Wmax, Gmax, Gave and Tmax of JK528 were higher than that of ZD958. The average grain filling rate of JK528 during early and mid grain filling stages was higher than that of late grain filling stages, which was also higher than that of ZD958 at the same sowing date. Furthermore, correlation analysis revealed that the mean temperature and the accumulated temperature were significantly positively correlated with the grain dry matter accumulation. Therefore, the full use of hot-ray resources and the increase of the mean grain-filling rate could improve maize grain yield. The higher grain filling rate during early and mid grain filling stages in JK528 could compensate for the short active grain filling period, leading to higher yields under different sowing dates.

[Effects of different accumulated temperature on photosynthetic performances of spring maize varieties during grain-filling period].

Taking cold-resistant maize variety Fengdan 3 and cold-sensitive maize variety Zhengdan 958 as test materials, field experiments were conducted in I, II, and III accumulated temperature zones in Heilongjiang Province of Northeast China to study the effects of different accumulated temperature on the photosynthetic performances of different types of cold-resistant spring maize varieties during their grain-filling period. In the three accumulated temperature zones, the tasseling and maturing periods of Fengdan 3 and Zhengdan 958 were prolonged in the order of I > II > and III, and the grain bulk density decreased in the same order. The RuBPCase and PEPCase activities of Fengdan 3 and Zhengdan 958 leaves had different temperature sensitivity. For Fengdan 3, its leaf RuBPCase and PEPCase activities were high in early grain filling period (0-20 days after anthesis), and the variety could ripen in temperature-limited region. For Zhengdan 958, its leaf RuBPCase and PEPCase activities were high within 0-10 days and 40-60 days after anthesis but not sensitive to the active accumulated temperature during 10-40 days after anthesis, and the variety could not ripen in temperature-limited region. The photosynthetic rates of the two varieties were significantly positively correlated with the active accumulated temperature during 0-10 days and 30-40 days after anthesis. The effects of the accumulated temperature in the three zones on the photosynthetic performances were significant at both early and later grain filling stages. For the same varieties, the higher the active accumulated temperature in grain filling period, the higher the grain yield. Zhengdan 958 had higher yield than Fengdan 3.

The first complete mitochondrial genome from Bostrychus genus (Bostrychus sinensis) and partitioned Bayesian analysis of Eleotridae fish phylogeny.

To understand the phylogenetic position of Bostrychus sinensis in Eleotridae and the phylogenetic relationships of the family, we determined the nucleotide sequence of the mitochondrial (mt) genome of Bostrychus sinensis. It is the first complete mitochondrial genome sequence of Bostrychus genus. The entire mtDNA sequence was 16508 bp in length with a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region. The mitochondrial genome of B. sinensis had common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures. Phylogenetic hypotheses within Eleotridae fish have been controversial at the genus level. We used the mitochondrial cytochrome b (cytb) gene sequence to examine phylogenetic relationships of Eleotridae by using partitioned Bayesian method. When the specific models and parameter estimates were presumed for partitioning the total data, the harmonic mean -lnL was improved. The phylogenetic analysis supported the monophyly of Hypseleotris and Gobiomorphs. In addition, the Bostrychus were most closely related to Ophiocara, and the Philypnodon is also the sister to Microphlypnus, based on the current datasets. Further, extensive taxonomic sampling and more molecular information are needed to confirm the phylogenetic relationships in Eleotridae.

[Effects of PASP-KT-NAA on the grain-filling of maize in different accumulated temperature zones of Hilongjiang Province, Norheast China].

Taking the two maize varieties Zhengdan 958 and Fengdan 3 grown on the three accumulated temperature zones (I, II and III) in Heilongjiang Provice as test materials, a field investigation was made in 2010 and 2011 to study the effects of PASP-KT-NAA (PKN), a compound of exogenous plant growth regulators, on the grain filling and yield of the varieties under different environmental temperatures. From zone I to III, the air temperature at the grain filling stage had a decreasing trend, with the average minimum temperature being 12.16, 11.40, and 9.56, respectively. The effective accumulated temperature at the mid-ate amt sae stage of grain filling was too low to be sufficient for grain filling, which severely affected the grain filling process. Applying N, P and K promoted the dry matter accumulation of maize grain and the grain filling rate in the three zones, delayed the peak time (Tmax) of the grain filling rate of Fengdan 3 but advanced that of Zhengdan 958, promoted the growth capacity at peak time of grain filling rate and the maximum grain filling rate of the two varieties, and shortened their active grain filling period. Applying N, P, and K increased the grain yield of the two varieties in the three zones obviously, and, as compared with those in zones I and II , the grain yields of Zhengdan 958 and Fengdan 3 in zone III were increased by 8.2% and 5.1% , and 3.4% and 0.8% , respectively. Therefore, applying N, P and K could help maize utilizing the limited accumulation temperature, improve the grain filling rate, decrease the grain water content, and ultimately, increase the maize yield.

[Characteristics and phylogenetic analysis of mitochondrial genome in the gobies].

The vast number of species, small size and high variation of morphology make the morphological identification and classification of gobies very difficult. In this study, the complete mitochondrial genome (mitogenome) of 26 species of gobies was analyzed, aiming at accumulating the molecular information on the identification, classification and molecular evolution of gobies. The results showed that the gene composition and arrangement of mitogenome of gobies are similar to most vertebrates. Due to various degrees of repetitive sequences in the control region, the mitogenome of 26 gobies exhibits a great variation in length. The A+T content of the mitogenome is greater than 50% and the lowest frequency is for G among the four bases. Thirty-seven coding gene sequences were used to calculate the average Kimura 2-parameter genetic distance of 26 species of gobies. Acanthogobius hasta and A. ommaturus, Glossogobius olivaceus and G circumspectus were synonyms, respectively. By comparing the control region sequences of 26 gobies, the terminal associated sequences, central conserved sequence block and conserved sequence block were identified, respectively. Thirty-six coding gene sequences of 26 gobies were used to construct the phylogenetic tree and the results were different from the traditional morphological classification. The five subfamilies of Gobiidae were obviously evolved: Amblyopinae, Oxudercinae and Sicydiinae were clustered into a group and then formed a sister group with Gobionellinae; the fishes of Gobiinae had distant relationship with the four subfamilies and formed a group alone. Molecular clock analysis estimated that gobies probably originated in the late Eocene to Oligocene time and further evolved into modern characteristic gobies in the Miocene.

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11.

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.

The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae): genome characterization and phylogenetic considerations.

The complete mitochondrial genome sequence of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) was determined and phylogenetic analysis was conducted to elucidate the evolutionary relationship of the marbled rockfish with other Sebastinae species. This mitochondrial genome, consisting of 17301 bp, is highly similar to that of most other vertebrates, containing the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one putative control region. Most of the genes are encoded on the H-strand, while the ND6 and seven tRNA genes (for Gln, Ala, Asn, Tyr, Ser (UCA), Glu, and Pro) are encoded on the L-strand. The reading frame of two pairs of genes overlapped on the same strand (the ATPase 8 and 6 genes overlapped by ten nucleotides; ND4L and ND4 genes overlapped by seven nucleotides). The possibly nonfunctional light-strand replication origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNA(Cys) gene. An extent termination-associated sequence (ETAS) and conserved sequence blocks (CSB) were identified in the control region, except for CSB-1; unusual long tandem repeats were found at the 3' end of the control region. Phylogenetic analyses supported the view that Sebastinae comprises four genera (Sebates, Hozukius, Helicolenus, and Sebasticus).

Molecular characterization of miiuy croaker CC chemokine gene and its expression following Vibrio anguillarum injection.

A CC chemokine gene was isolated from miiuy croaker (Miichthys miiuy) by expressed sequence tag analysis. The Mimi-CC cDNA contains an open reading frame of 429 nucleotides encoding 142 amino acid residues. The deduced Mimi-CC possesses the typical arrangement of four cysteines as found in other known CC chemokines (C³¹, C³², C56, and C7°). It shares 15.3%-37.4% identity to CC chemokines of mammal and teleost. Phylogenetic analysis showed that miiuy croaker was most closely related to Atlantic cod. Genomic analysis revealed that Mimi-CC gene consists of four exons and three introns, which is not typical of CC chemokines but resembles that of CXC chemokines. Real-time quantitative RT-PCR demonstrated that Mimi-CC is constitutively expressed in most tissues including lymphoid organs, and the highest expression of Mimi-CC transcripts in normal tissues was observed in muscle. Challenge of miiuy croaker with Vibrio anguillarum resulted in significant changes in the expression of CC chemokine transcripts in four tissues, especially in kidney and spleen.

Sequence and expression analysis of cathepsin S gene in the miiuy croaker Miichthys miiuy.

Cathepsin S (CTSS) is a lysosomal cysteine endopeptides of the papain family. In the present study, CTSS gene was isolated from miiuy croaker (Miichthys miiuy) by expressed sequence tag analysis. The complete miiuy croaker CTSS cDNA comprised 1,454 nucleotides, including 56 bp at the 5'-UTR and 394 bp at the 3'-UTR. The open reading frame is 1,017 bp, and it encodes 338 amino acid residues with a calculated molecular weight of 37.1 kDa. BLAST analysis revealed that miiuy croaker cathepsin S shared high similarity with other known cathepsin S, and it showed significant homology with that of Japanese flounder, the degree of conservation of the miiuy croaker CTSS nucleotide sequence in comparison with other teleost species ranged from 63.0 to 81.7%. The N-linked glycosylation sites and catalytic sites are conserved in miiuy croaker CTSS. The CTSS transcript was expressed in all tissues examined; high levels of transcripts of CTSS were detected liver, muscle, and fin. These results provide important information for further exploring the roles of cathepsin S in antigen processing.

Growth differences and differential expression analysis of pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) between the sexes in half-smooth tongue sole Cynoglossus semilaevis.

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months (P<0.05) and at 18 onths the mean body weight of the females (771.0±44.3 g) was as much as 4.9 times higher than that of males (130.6±6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females (P<0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.

Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene, after infection by Vibrio anguillarum.

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. MHC gene family contains two main subgroups of immunologically active molecules. In order to study the molecular function and genomic characteristic of class I gene in teleost, the full lengths of MHC class Iα cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). Seven exons and six introns were identified in miiuy croaker class Iα gene. This genomic structural feature of miiuy croaker is similar to that present in some fishes such as Japanese flounder and Atlantic salmon, but different from that present in some other fishes such as half-smooth tongue sole and channel catfish. The deduced amino acid sequence of class Iα gene had 25.9-54.1% identity with those of mammal and teleost. Real-time quantitative RT-PCR demonstrated that the MHC class Iα gene was ubiquitously expressed in 10 normal tissues; expression levels of MHC Iα gene were found first upregulated and then downregulated throughout the pathogenic bacteria infection process in spleen and kidney.

The complete mitochondrial genome of bighead croaker, Collichthys niveatus (Perciformes, Sciaenidae): structure of control region and phylogenetic considerations.

Sciaenidae is a diverse, commercially important family. To understand the phylogenetic position of Collichthys niveatus in this family, we present its complete mitochondrial genome sequence. The genome is 16469 bp in length and contains 37 mitochondrial genes (13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes) and a control region (CR) as in other bony fishes. Further sequencing for the complete control region was performed on Collichthys lucida. Although the conserved sequence domains such as extend termination associated sequence (ETAS) and conserved sequence block domains (CSB-1, CSB-2 and CSB-3) are recognized in the control region of the two congeneric species, the typical central conserved blocks (CSB-F, CSB-E and CSB-D) could not be detected, while they are found in Miichthys miiuy and Cynoscion acoupa of Sciaenidae and other Percoidei fishes. Phylogenetic analyses do not support the monophyly of Pseudosciaeniae, which is against with the morphological results. C. niveatus is most closely related to Larimichthys polyactis, and Collichthys and Larimichthys may be merged into one genus, based on the current datasets.

Molecular cloning, genomic structure and expression analysis of major histocompatibility complex class Iα gene of half-smooth tongue sole (Cynoglossus semilaevis).

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. The full length of MHC class Iα cDNA was cloned from half-smooth tongue sole by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR), genomic organization and expression of MHC Iα were examined to study the function of MHC gene in fish. The domain structure feature and antigen-binding motifs of other teleost and mammals MHC are conserved in the half-smooth tongue sole MHC Iα gene. The deduced amino acid sequence of half-smooth tongue sole MHC Iα (GenBank accession no. FJ372720) had 12.1-61.8% identity with those of human and other fish. Eight exons and seven introns were identified in MHC Iα gene. Real-time quantitative PCR demonstrated that MHC Iα gene was ubiquitously expressed in normal tissues, while that in Vibrio anguillarum infected fish was significantly increased in intestines and decreased in spleen and liver from 24 to 72 h after infection, followed by a recovery to normal level after 96 h.

Allelic variation, balancing selection and positive selected sites detected from MHC class Iα gene of olive flounder.

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and pattern of evolution in MHC genes has been investigated in many mammals, however, much less is known in teleost. In the present study, we have investigated complete MHC Iα gene consists of 7 exons and 6 introns in Olive flounder (Paralichthys olivaceus). Genetic variation in the MHC class Iα gene was also tested in flounder. In 32 individuals, a total of 62 alleles were detected from exon 2 of MHC class Iα gene. The rate of non-synonymous substitutions (d ( N )) occurred at a significantly higher frequency than that of synonymous substitutions (d ( S )) in PBR and non-PBR, suggesting that balancing selection for maintaining polymorphisms at the MHC Iα locus. Many positive selection sites were found to act very intensively on antigen binding sites. Our founding suggests a snapshot in an evolutionary process of MHC Iα gene evolution of the P. olivaceus.

Identification of immune genes of the miiuy croaker (Miichthys miiuy) by sequencing and bioinformatic analysis of ESTs.

Miiuy croaker (Miichthys miiuy) is an economically important fish in China. However, genomic research on this species is still in its infancy, and genomic resources are largely unavailable. In order to isolate functional genes involved in immunity, a normalized cDNA library was constructed from the spleen of the miiuy croaker. A total of 5053 ESTs from the library were sequenced and compared with sequences in the GenBank database. The 4609 high-quality ESTs were assembled into 3221 unigenes. Based on sequence similarities, 193 immune genes were identified such as major histocompatibility complex, cytokines and cytokine receptors, adhesive proteins, stress proteins, transcription factors for immune response, immunoglobulin and coagulation factors. Our study thus provides both a detailed annotation of immune genes in miiuy croaker and a collection of novel transcripts of Fc receptor-like 5 in teleost for the first time.