Study on the differential gene expression of elm leaves fed on by Tetraneura akinire Sasaki Abstract Background To study the essential molecular mechanism of gall formation is very important. Objective To investigate the differential gene expression in leaves fed on by Tetraneura akinire Sasaki and to provide a basis for the better understanding of the essential molecular mechanism of gall formation. Methods The infected leaves of the elm were divided into three periods: initial formation period (T2), growth and differentiation period (T3), and cracking period (T4). The untouched leaves were used as the control (T1). RNA-Seq was performed, and the high-quality sequences were mapped to the reference genome and the elm gene database to obtain the gene expression profiles. The expression level of each gene was calculated by the RPKM method. A combination of FDR ≤ 0.01 and the absolute value of |log2 ratio (T/CK)| ≥ 2 was used as the threshold to determine the significance of gene expression. Finally, GO and pathway enrichment analyses were used to identify the significantly enriched functional classification and metabolic pathways in DEGs. Results The results revealed that approximately 244 mRNAs were detected between T1 and T2, including 192 up-regulated and 52 down-regulated mRNAs; approximately 175 mRNAs were detected between T1 and T3, including 145 up-regulated and 30 down-regulated mRNAs; and approximately 372 mRNAs were detected between T1 and T4, including 360 up-regulated and 12 down-regulated mRNAs. Approximately 34 differentially expressed genes were identified by Venn analysis. Comparing the three infection periods to the control, there were 28 up-regulated and six down-regulated mRNAs. Additionally, 562 genes were used for cluster analysis, which revealed that the gene expression in T2 and T3 changed greatly. Genes related to cell proliferation and respiration, such as microtubulin and 6-phosphoric acid fructose kinase were mainly up-regulated during the T2 period. Genes encoding lipoxygenase, glutathione-S-transferase, superoxide dismutase and protease inhibitor were up-regulated during T2 and T3. Genes encoding lignocellulose synthase were up-regulated during T4, which suggests the reinforcement of the cell wall to improve the resistance to the damage of the Tetraneura akinire Sasaki. Conclusions The results showed that the feeding of Tetraneura akinire Sasaki caused the differential expression of elm genes and influenced cellular energy metabolism. These changes in physiological response and gene expression of the elm compose the physiological and molecular basis of the gall formation and may improve the resistance of elm to Tetraneura akinire Sasaki.

Genome-wide association analysis of salt tolerance QTLs with SNP markers in maize ( Zea mays L.) Abstract Background Salt-tolerant breeding of maize has great significance to the development and utilization of saline–alkaline soil and the maintenance of grain security. Genome-wide association study (GWAS) has been widely used in maize genetics and breeding. Objective To discover new salt-tolerant genes in maize by association analysis, which can provide technical supports for the innovation and genetic improvement of salt-tolerant germplasm resources in maize. Methods Totally 150 maize inbred lines were genotyped with a high-density chip. GWAS was carried out to identify the significant single nucleotide polymorphisms (SNPs) which were associated with maize salt tolerance. Totally 34,972 SNPs with high quality and diversity were selected from 56,110 SNP markers, which were distributed on 10 chromosomes of maize. The GLM algorithm in TASSEL5.2 was used to analyze the five traits related to salt tolerance. Results Using a strict LOD threshold of 4.5, totally 7 SNP loci were identified, which were significantly correlated with plant height change rate and fresh weight change rate. The high density fingerprints of 150 inbred lines were clustered by TASSEL5.2 software to construct genetic clustering map to estimate the genetic distance and the subgroups. The 150 maize inbred lines were divided into two groups: SS group and NSS group, and the SNP loci of the salt-tolerant index showed difference in chromosome distribution. Based on previous studies, we screened 8 candidate genes for salt tolerance in maize and four of them were further validated by real-time quantitative PCR. Conclusion Totally 7 SNP loci and 8 candidate genes related to salt tolerance in maize were identified, which will be of special value in molecular breeding of salt-tolerant maize.

Influence of single nucleotide polymorphisms (SNPs) in genetic susceptibility towards periprosthetic osteolysis Abstract Wear debris-induced inflammatory osteolysis remains a significant limiting factor for implant replacement surgeries. Hence, a comprehensive understanding of the complex network of cellular and molecular signals leading to these inflammatory responses is required. Both macrophages and monocytes have a critical role in the instigation of the inflammatory reaction to wear debris but differ in the extent to which they induce cytokine expression in patients. Lately, single nucleotide polymorphisms (SNPs) have been associated with genetic susceptibility among individual patients with implant failure. Studies have shown that SNPs in key pro-inflammatory cytokines and their receptors are associated with osteolytic susceptibility. Likewise, SNPs within several genes involved in the regulation of bone turnover have also been found to be associated with wear debris induced osteolysis. It is presumed that SNP variance might play a decisive role in the activation and signaling of macrophages, osteoblasts, chondrocytes, fibroblasts and other cells involved in inflammatory bone loss. Understanding the extent to which SNPs exist among genes that are responsible for inflammatory bone loss may provide potential targets for developing future therapeutic interventions. Herein, we attempt to summarize the various susceptible genes with possible SNP variance that could contribute to the severity of periprosthetic osteolysis in patients with implants.

Physiological responses and small RNAs changes in maize under nitrogen deficiency and resupply Abstract Background Maize is an important crop in the world, nitrogen stress severely reduces maize yield. Although a large number of studies have identified the expression changes of microRNAs (miRNAs) under N stress in several species, the miRNAs expression patterns of N-deficient plants under N resupply remain unclear. Objective The primary objective of this study was to identify miRNAs in response to nitrogen stress and understand relevant physiological changes in nitrogen-deficient maize after nitrogen resupply. Methods Physiological parameters were measured to study relevant physiological changes under different nitrogen conditions. Small RNA sequencing and qRT-PCR analysis were performed to understand the response of miRNAs under different nitrogen conditions. Results The content of chlorophyll, soluble protein and nitrate nitrogen decreased than CK by 0.52, 0.49 and 0.82 times after N deficiency treatment and increased than ND by 0.52, 1.36 and 0.65 times after N resupply, respectively. Conversely, the activity of superoxide dismutase (SOD) and peroxidase (POD) increased by 0.67 and 1.64 times than CK after N deficiency, respectively, and decreased by 0.09 and 0.35 times than ND after N resupply. A total of 226 known miRNAs were identified by sRNA sequencing; 106 miRNAs were differentially expressed between the control and N-deficient groups, and 103 were differentially expressed between the N-deficient and N-resupply groups (P < 0.05). Real-time quantitative PCR (qPCR) was used to further validate and analyze the expression of the identified miRNAs. A total of 1609 target genes were identified by target prediction, and some differentially expressed miRNAs were predicted to target transcription factors and functional proteins. Gene Ontology (GO) analysis was used to determine the biological function of these targets and revealed that some miRNAs, such as miR169, miR1214, miR2199, miR398, miR408 and miR827 might be involved in nitrogen metabolism regulation. Conclusion Our study comprehensively provides important information on miRNA functions and molecular mechanisms in response to N stress. These findings may assist to improve nitrogen availability in plants.

Association analysis of polymorphism in the NR6A1 gene with the lumbar vertebrae number traits in sheep Abstract Introduction The vertebral number is an economically significant trait, which is associated with body length and carcass traits. Nuclear Receptor Subfamily 6, Group A, Member 1 (NR6A1) is a member of the nuclear receptor superfamily and it plays an important role in the early development of embryos. Objectives The NR6A1 gene was considered as an important candidate for influence vertebrae number, while the potential associations between this gene and the number of lumbar vertebrae traits of sheep have not been explored. Methods In this study, we detected the genetic variants of NR6A1 gene and analyzed the associations of the polymorphisms with lumbar number traits in 130 Kazakh sheep. We use single-strand conformation polymorphism (SSCP) technique to detect single nucleotide polymorphism (SNP) of NR6A1 gene, and the association of the genotype and lumbar number variation was analyzed by independent Chi-square test. Results We detect SNP of NR6A1 gene by PCR-SSCP technique, and polymorphisms were only found in the coding region of exon-6 and exon-8 of NR6A1 gene. In order to investigate the connection between the SNP locus and lumbar number traits in sheep, we conducted a Chi-square test for independence for exon-6 and exon-8 of NR6A1 gene, respectively. Association analysis revealed significant associations between the SNP (rs414302710: A >C) in the exon-8 of NR6A1 gene with the number of lumbar vertebrae (P < 0.01). Conclusion Our study indicated that this SNP (rs414302710: A>C) locus of exon-8 of NR6A1 gene in sheep possible influence the number of lumbar vertebrae, which has the potential to be applied in selective breeding of sheep.

Phylogeographic studies on two shore crab species from East Asia: similar but different stories Abstract Background The genetic structure of marine organisms in the East Asian region has long been a subject of interest. Two grapsid crab species, Hemigrapsus penicillatus (De Haan, 1835) and Hemigrapsus sanguineus (De Haan, 1835), are commonly found in the rocky intertidal zones around this region. They are known to spread via larval migration, which makes them an appropriate model species for observing the genetic structure of East Asian intertidal invertebrate animals. Objective We investigated the genetic structure of the East Asian crabs H. penicillatus and H. sanguineus. Methods We collected specimens of H. penicillatus from seven locations (42 individuals) and of H. sanguineus from ten locations (58 individuals) in Korea, Japan, and Taiwan. We investigated and compared the genetic diversity and structure of populations of these species using mitochondrial cytochrome oxidase subunit I (COI) sequences. Results Our results show that both species are genetically structured between South Korea and Japan, and that the Taiwan population forms a cluster that is separate from those of the other countries. Populations of H. penicillatus contain less genetic diversity than those of H. sanguineus. Conclusion These results suggest that there is a genetic structure between the two species at present in East Asia.

Comparative transcript profiling and cytological observation of the newly bred recessive genic male sterility non-heading Chinese cabbage ( Brassica rapa ssp. chinensis) line WS24-3A Abstract Background WS24-3A is a newly bred non-heading Chinese cabbage genic male-sterile line, in which sterility is controlled by a recessive gene, designated as Bra2ms. WS24-3A has been used for hybrid breeding. Objective To reveal the underlying molecular mechanisms responsible for the sterility of WS24-3A. Methods Cytological observation of the process of sterile/fertile anther development was performed to determine the tissue and stage in which sterility occurs. Phenotyping and transcriptomic analyses were performed to identify differentially expressed genes (DEGs) between sterile and fertile flower buds at different stages. Results Cytological analysis revealed no tetrads at stage 7 or at later stages of anther development, and the degradation of callose was delayed. Abnormal meiocytes were surrounded by sustaining callose that degenerated gradually in WS24-3A. Comparative transcript profiling identified 3282 DEGs during three anther developmental stages, namely, pre-meiotic anther, meiotic anther, and anthers with single-celled pollen stage. The difference in DEG percentage between up-regulated and down-regulated at meiotic anther stage was obviously larger than at the other two stages; further, most DEGs are important for male meiosis, callose synthesis and dissolution, and tapetum development. Ten DEGs were found to be involved in anther and pollen development, which were analyzed by quantitative PCR. Conclusion Bra2ms affected gene expression in meiocytes and associated with callose synthesis, degradation and tapetum development. Our results provide clues to elucidate the molecular mechanism of genic male sterility in non-heading Chinese cabbage.

(Cyto)genomic and epigenetic characterization of BICR 10 cell line and three new established primary human head and neck squamous cell carcinoma cultures Abstract Background Head and neck squamous cell carcinoma cell lines are useful preclinical models to understand the molecular processes underlying the development of such tumors, and to establish targeted therapies. Objective We performed a comprehensive (cyto)genomic and epigenetic characterization of three new established primary human head and neck squamous cell carcinoma cultures and an established, yet undercharacterized cell line: BICR 10. Methods Karyotyping, multiplex fluorescence in situ hybridization, array comparative genomic hybridization and methylation-specific multiplex ligation-dependent probe amplification were applied. Results The three primary cultures turned out to be a near-triploid and BICR 10 near-diploid. Banding and molecular cytogenetic analysis revealed non-random numerical and structural aberrations. The most common rearrangements identified in BICR 10 cell line were non-complex derivatives of reciprocal translocations, in which the breakpoints often appeared in centromeric/near-centromeric regions. In the 3 primary cell cultures the most common rearrangements observed were iso- and derivatives chromosomes derived from translocations. Overall, gains of 7p, 8q and losses at 3p, 8p, 9p, 18q and Xp were present in all four studied samples. Among the analyzed genes, BICR 10 cell line exhibited enhanced methylation of gene promoter; however, in all studied samples PAX5, WT1 and GATA5 were methylated. Conclusion The here reported comprehensive characterization of BICR 10 cell line and the new established cultures enriches the resources available for head and neck cancer research, especially for testing therapeutic agents.

Comparison of library construction kits for mRNA sequencing in the Illumina platform Abstract Background The emergence of next-generation sequencing (NGS) technologies has made a tremendous contribution to the deciphering and significance of transcriptome analysis in biological fields. Since the advent of NGS technology in 2007, Illumina, Inc. has provided one of the most widely used sequencing platforms for NGS analysis. Objective Although reagents and protocols provided by Illumina are adequately performed in transcriptome sequencing, recently, alternative reagents and protocols which are relatively cost effective are accessible. However, the kits derived from various manufacturers have advantages and disadvantages when researchers carry out the transcriptome library construction. Methods We compared them using a variety of protocols to produce Illumina-compatible libraries based on transcriptome. Three different mRNA sequencing kits were selected for this study: TruSeq® RNA Sample Preparation V2 (Illumina, Inc., USA), Universal Plus mRNA-Seq (NuGEN, Ltd., UK), and NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England BioLabs, Ltd., USA). We compared them focusing on cost, experimental time, and data output. Results The quality and quantity of sequencing data obtained through the NGS technique were strongly influenced by the type of the sequencing library kits. It suggests that for transcriptome studies, researchers should select a suitable library construction kit according to the goal and resources of experiments. Conclusion The present work will help researchers to choose the right sequencing library construction kit for transcriptome analyses.

Analysis of longissimus muscle quality characteristics and associations with DNA methylation status in cattle Abstract Background As cattle represent one of the most important livestock species for meat production, control of muscle development in regards to quality is an important research focus. Objectives In this study, the phenotypic quality traits and its associations with DNA methylation levels of the longissimus muscle in two cattle breeds were studied. Methods The pH value, water loss rate, fat and protein and fatty acid content were measured in three beef cattle breeds of longissimus mucle; The longissimus mucle was analyzed by MethylRAD-seq and RNA-seq. The differentially methylated and differentially expressed related genes were subjected to BSP. Results Methylation status of longissimus mucle was analyzed by MethylRAD-seq. Compared with Simmental, there were 39 differentially methylated and expressed genes in muscle of Yunling cattle, and 123 differentially methylated and expressed genes in Wenshan muscle. A combined analysis of MethylRAD-seq and RNA-seq results revealed differential methylation and expression level of 18 genes between Simmental and Wenshan cattle, and 14 genes between Simmental and Yunling cattle. In addition, 28 genes were differentially methylated between Wenshan and Yunling cattle. Results of promoter methylation analysis of ACAD11, FADS6 and FASN showed that the overall degree of DNA methylation of FADS6 and FASN was negatively correlated with their expression levels. Methylation level of FASN in Simmental was greater than Yunling and Wenshan. The degree of methylation at the FADS6 CpG4 site was significantly higher in Simmental than that in Yunling. The levels of methylation at the CpG7 locus of the Simmental and Yunling breeds were greater than Wenshan cattle. A negative correlation was detected between the methylation levels and the expression of FASN CpG1, CpG2, CpG3, CpG5, CpG7, and CpG10. Conclusion The functional and molecular regulatory mechanism of the genes related to meat quality can be revealed systematically from aspects of the genetic and epigenetic regulation. These studies will help to further explore the molecular mechanisms and phenotypic differences that regulate growth and quality of different breeds of cattle.