Gene-allele system of shade tolerance in southern China soybean germplasm revealed by genome-wide association study using gene-allele sequence as markers.

KEY MESSAGE: Fifty-three shade tolerance genes with 281 alleles in the SCSGP were identified directly using gene-allele sequence as markers in RTM GWAS, from which optimized crosses, evolutionary motivators, and gene-allele networks were explored. Shade tolerance is a key for optimal cultivation of soybean inter/relay-cropped with corn. To explore the shade tolerance gene-allele system in the southern China soybean germplasm, we proposed using gene-allele sequence markers (GASMs) in a restricted two-stage multi-locus model genome-wide association study (GASM-RTM-GWAS). A representative sample with 394 accessions was tested for their shade tolerance index (STI), in Nanning, China. Through whole-genome re-sequencing, 47,586 GASMs were assembled. From GASM-RTM-GWAS, 53 main-effect STI genes with 281 alleles (2-13 alleles/gene) (totally 63 genes with 308 alleles, including 38 G × E genes with 191 alleles) were identified and then organized into a gene-allele matrix composed of eight submatrices corresponding to geo-seasonal subpopulations. The population featured mild STI changes (1.69 → 1.56-1.82) and mild gene-allele changes (92.5% alleles inherited, 0% alleles excluded, 7.5% alleles emerged) from the primitive (SAIII) to the derived seven subpopulations, but large transgressive recombination potentials and optimal crosses were predicted. The 63 STI genes were annotated into six biological categories (metabolic process, catalytic activity, response to stresses, transcription and translation, signal transduction and transport and unknown functions), interacted as gene networks. From the STI gene-allele system, 38 important alleles of 22 genes were nominated for further in-depth study. GASM-RTM-GWAS performed powerful and efficient in germplasm population genetic study comparing to other procedures through facilitating direct and thorough identification of its gene-allele system, from which genome-wide breeding by design could be achieved, and evolutionary motivators and gene-allele networks could be explored.

Methylome and transcriptome analyses of soybean response to bean pyralid larvae.

BACKGROUND: Bean pyralid is one of the major leaf-feeding insects that affect soybean crops. DNA methylation can control the networks of gene expressions, and it plays an important role in responses to biotic stress. However, at present the genome-wide DNA methylation profile of the soybean resistance to bean pyralid has not been reported so far. RESULTS: Using whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq), we analyzed the highly resistant material (Gantai-2-2, HRK) and highly susceptible material (Wan82-178, HSK), under bean pyralid larvae feeding 0 h and 48 h, to clarify the molecular mechanism of the soybean resistance and explore its insect-resistant genes. We identified 2194, 6872, 39,704 and 40,018 differentially methylated regions (DMRs), as well as 497, 1594, 9596 and 9554 differentially methylated genes (DMGs) in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48 comparisons, respectively. Through the analysis of global methylation and transcription, 265 differentially expressed genes (DEGs) were negatively correlated with DMGs, there were 34, 49, 141 and 116 negatively correlated genes in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48, respectively. The MapMan cluster analysis showed that 114 negatively correlated genes were clustered in 24 pathways, such as protein biosynthesis and modification; primary metabolism; secondary metabolism; cell cycle, cell structure and component; RNA biosynthesis and processing, and so on. Moreover, CRK40; CRK62; STK; MAPK9; L-type lectin-domain containing receptor kinase VIII.2; CesA; CSI1; fimbrin-1; KIN-14B; KIN-14 N; KIN-4A; cytochrome P450 81E8; BEE1; ERF; bHLH25; bHLH79; GATA26, were likely regulatory genes involved in the soybean responses to bean pyralid larvae. Finally, 5 DMRs were further validated that the genome-wide DNA data were reliable through PS-PCR and 5 DEGs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. The results showed an excellent agreement with deep sequencing. CONCLUSIONS: Genome-wide DNA methylation profile of soybean response to bean pyralid was obtained for the first time. Several specific DMGs which participated in protein kinase, cell and organelle, flavonoid biosynthesis and transcription factor were further identified to be likely associated with soybean response to bean pyralid. Our data will provide better understanding of DNA methylation alteration and their potential role in soybean insect resistance.

Genome-wide identification of the peptide transporter family in rice and analysis of the PTR expression modulation in two near-isogenic lines with different nitrogen use efficiency.

BACKGROUND: Nitrogen (N) is a major nutrient element for crop growth. In plants, the members of the peptide transporter (PTR) gene family may involve in nitrate uptake and transport. Here, we identified PTR gene family in rice and analyzed their expression profile in near-isogenic lines. RESULTS: We identified 96, 85 and 78 PTR genes in Nipponbare, R498 and Oryza glaberrima, and the phylogenetic trees were similar in Asian cultivated rice and African cultivated rice. The number of PTR genes was higher in peanut (125) and soybean (127). The 521 PTR genes in rice, maize, sorghum, peanut, soybean and Arabidopsis could be classified into 4 groups, and their distribution was different between monocots and dicots. In Nipponbare genome, the 25 PTR genes were distributed in 5 segmental duplication regions on chromosome 1, 2, 3, 4, 5, 7, 8, 9, and 10. The PTR genes in rice have 0-11 introns and 1-12 exons, and 16 of them have the NPF (NRT1/PTR family) domain. The results of RNA-seq showed that the number of differentially expressed genes (DEGs) between NIL15 and NIL19 at three stages were 928, 1467, and 1586, respectively. Under low N conditions, the number of differentially expressed PTR genes increased significantly. The RNA-seq data was analyzed using WGCNA to predict the potential interaction between genes. We classified the genes with similar expression pattern into one module, and obtained 25 target modules. Among these modules, three modules may be involved in rice N uptake and utilization, especially the brown module, in which hub genes were annotated as protein kinase that may regulate rice N metabolism. CONCLUSIONS: In this study, we comprehensively analyzed the PTR gene family in rice. 96 PTR genes were identified in Nippobare genome and 25 of them were located on five large segmental duplication regions. The Ka/Ks ratio indicated that many PTR genes had undergone positive selection. The RNA-seq results showed that many PTR genes were involved in rice nitrogen use efficiency (NUE), and protein kinases might play an important role in this process. These results provide a fundamental basis to improve the rice NUE via molecular breeding.

Determination of the MiRNAs Related to Bean Pyralid Larvae Resistance in Soybean Using Small RNA and Transcriptome Sequencing.

Soybean is one of the most important oil crops in the world. Bean pyralid is a major leaf-feeding insect of soybean. In order to screen out the functional genes and regulatory pathways related to the resistance for bean pyralid larvae, the small RNA and transcriptome sequencing were performed based on the highly resistant material (Gantai-2-2) and highly susceptible material (Wan 82-178) of soybean. The results showed that, when comparing 48 h feeding with 0 h feeding, 55 differentially expressed miRNAs were identified in Gantai-2-2 and 58 differentially expressed miRNAs were identified in Wan82-178. When comparing Gantai-2-2 with Wan82-178, 77 differentially expressed miRNAs were identified at 0 h feeding, and 70 differentially expressed miRNAs were identified at 48 h feeding. The pathway analysis of the predicted target genes revealed that the plant hormone signal transduction, RNA transport, protein processing in the endoplasmic reticulum, zeatin biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, and isoquinoline alkaloid biosynthesis may play important roles in soybean's defense against the stress caused by bean pyralid larvae. According to conjoint analysis of the miRNA/mRNA, a total of 20 differentially expressed miRNAs were negatively correlated with 26 differentially expressed target genes. The qRT-PCR analysis verified that the small RNA sequencing results were credible. According to the analyses of the differentially expressed miRNAs, we speculated that miRNAs are more likely to play key roles in the resistance to insects. Gma-miR156q, Gma-miR166u, Gma-miR166b, Gma-miR166j-3p, Gma-miR319d, Gma-miR394a-3p, Gma-miR396e, and so on-as well as their negatively regulated differentially expressed target genes-may be involved in the regulation of soybean resistance to bean pyralid larvae. These results laid a foundation for further in-depth research regarding the action mechanisms of insect resistance.

Proteomic analysis by iTRAQ-MRM of soybean resistance to Lamprosema Indicate.

BACKGROUND: Lamprosema indicate is a major leaf feeding insect pest to soybean, which has caused serious yield losses in central and southern China. To explore the defense mechanisms of soybean resistance to Lamprosema indicate, a highly resistant line (Gantai-2-2) and a highly susceptible line (Wan 82-178) were exposed to Lamprosema indicate larval feedings for 0 h and 48 h, and the differential proteomic analyses of these two lines were carried out. RESULTS: The results showed that 31 differentially expressed proteins (DEPs) were identified in the Gantai-2-2 when comparing 48 h feeding with 0 h feeding, and 53 DEPs were identified in the Wan 82-178. 28 DEPs were identified when comparing Gantai-2-2 with Wan 82-178 at 0 h feeding. The bioinformatic analysis results showed that most of the DEPs were associated with ribosome, linoleic acid metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis, peroxisome, stilbenoid, diarylheptanoid and gingerol biosynthesis, glutathione metabolism, pant hormone signal transduction, and flavone and flavonol biosynthesis, as well as other resistance related metabolic pathways. The MRM analysis showed that the iTRAQ results were reliable. CONCLUSIONS: According to the analysis of the DEPs results, the soybean defended or resisted the Lamprosema indicate damage by the induction of a synthesis of anti-digestive proteins which inhibit the growth and development of insects, reactive oxygen species scavenging, signaling pathways, secondary metabolites synthesis, and so on.

Comparative transcriptome analysis of soybean response to bean pyralid larvae.

BACKGROUND: Soybean is one of most important oilseed crop worldwide, however, its production is often limited by many insect pests. Bean pyralid is one of the major soybean leaf-feeding insects in China. To explore the defense mechanisms of soybean resistance to bean pyralid, the comparative transcriptome sequencing was completed between the leaves infested with bean pyralid larvae and no worm of soybean (Gantai-2-2 and Wan82-178) on the Illumina HiSeq™ 2000 platform. RESULTS: In total, we identified 1744 differentially expressed genes (DEGs) in the leaves of Gantai-2-2 (1064) and Wan82-178 (680) fed by bean pyralid for 48 h, compared to 0 h. Interestingly, 315 DEGs were shared by Gantai-2-2 and Wan82-178, while 749 and 365 DEGs specifically identified in Gantai-2-2 and Wan82-178, respectively. When comparing Gantai-2-2 with Wan82-178, 605 DEGs were identified at 0 h feeding, and 468 DEGs were identified at 48 h feeding. Gene Ontology (GO) annotation analysis revealed that the DEGs were mainly involved in the metabolic process, single-organism process, cellular process, responses to stimulus, catalytic activities and binding. Pathway analysis showed that most of the DEGs were associated with the plant-pathogen interaction, phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, peroxisome, plant hormone signal transduction, terpenoid backbone biosynthesis, and so on. Finally, we used qRT-PCR to validate the expression patterns of several genes and the results showed an excellent agreement with deep sequencing. CONCLUSIONS: According to the comparative transcriptome analysis results and related literature reports, we concluded that the response to bean pyralid feeding might be related to the disturbed functions and metabolism pathways of some key DEGs, such as DEGs involved in the ROS removal system, plant hormone metabolism, intracellular signal transduction pathways, secondary metabolism, transcription factors, biotic and abiotic stresses. We speculated that these genes may have played an important role in synthesizing substances to resist insect attacks in soybean. Our results provide a valuable resource of soybean defense genes that will benefit other studies in this field.

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

BACKGROUND: Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. METHODS: Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. RESULTS: Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. CONCLUSIONS: Resistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Molecular Characteristics of Regional Chromoblastomycosis in Guangdong, China: Epidemiological, Clinical, Antifungal Susceptibility, and Serum Cytokine Profiles of 45 Cases.

Chromoblastomycosis (CBM) is a chronic disease caused by several species of dematiaceous fungi. In this study, a regional collection of 45 CBM cases was conducted in Guangdong, China, a hyper-endemic area of CBM. Epidemiology findings indicated that the mean age of cases was 61.38 ± 11.20 years, long duration ranged from 3 months to 30 years, and the gender ratio of male to female was 4.6:1. Thirteen cases (29%) declared underlying diseases. Verrucous form was the most common clinical manifestation (n = 19, 42%). Forty-five corresponding clinical strains were isolated, and 28 of them (62%) were identified as F. monophora; the remaining 17 (38%) were identified as F. nubica through ITS rDNA sequence analysis. Antifungal susceptibility tests in vitro showed low MICs in azoles (PCZ 0.015-0.25 µg/ml, VCZ 0.015-0.5 µg/ml, and ITZ 0.03-0.5 µg/ml) and TRB (0.015-1 µg/ml). Itraconazole combined with terbinafine was the main therapeutic strategy used for 31 of 45 cases, and 68% (n = 21) of them improved or were cured. Cytokine profile assays indicated upregulation of IL-4, IL-7, IL-15, IL-11, and IL-17, while downregulation of IL-1RA, MIP-1ß, IL-8, and IL-16 compared to healthy donors (p < 0.05). The abnormal cytokine profiles indicated impaired immune response to eliminate fungus in CBM cases, which probably contributed to the chronic duration of this disease. In conclusion, we investigated the molecular epidemiological, clinical, and laboratory characteristics of CBM in Guangdong, China, which may assist further clinical therapy, as well as fundamental pathogenesis studies of CBM.

Deletion C-terminal thioesterase abolishes melanin biosynthesis, affects metabolism and reduces the pathogenesis of Fonsecaea monophora.

Dematiaceous Fonsecaea monophora is one of the major pathogens of chromoblastomycosis. It has been well established that melanization is catalyzed by the type I polyketide synthase (PKS) in F. monophora. Multidomain protein Type I PKS is encoded by six genes, in which the last enzyme thioesterase (TE) catalyzes the cyclization and releases polyketide. Two PKS genes AYO21_03016 (pks1) and AYO21_10638 have been found in F. monophora and both PKS loci have the same gene arrangement but the TE domain in AYO21_10638 is truncated at 3'- end. TE may be the key enzyme to maintain the function of pks1. To test this hypothesis, we constructed a 3'-end 500 bp deletion mutant of AYO21_03016 (Δpks1-TE-C500) and its complemented strain. We profiled metabolome of this mutant and analyzed the consequences of impaired metabolism in this mutant by fungal growth in vitro and by pathogenesis in vivo. Compared with wild-type strain, we found that the mutant repressed pks1 expression and other 5 genes expression levels were reduced by more than 50%, perhaps leading to a corresponding melanin loss. The mutant also reduced sporulation and delayed germination, became vulnerable to various environmental stresses and was less resistance to macrophage or neutrophil killings in vitro, and less virulence in mice footpad model. Metabolomic analysis indicated that many metabolites were remarkably affected in Δpks1-TE-C500, in particular, an increased nicotinamide and antioxidant glutathione. In conclusion, we confirmed the crucial role of C-terminal TE in maintaining fully function of pks1 in F. monophora. Deletion of TE negatively impacts on the synthesis of melanin and metabolites that eventually affect growth and virulence of F. monophora. Any potential inhibitor of TE then could be a novel antifungal target for drug development.

Untargeted lipidomic analysis of human hippocampus for temporal lobe epilepsy with hippocampal sclerosis.

Mesial temporal lobe epilepsy (mTLE), a very common chronic neurological disorder, is frequently accompanied by neurodegeneration in the hippocampus, resulting in hippocampal sclerosis (HS); HS is the most common morphological correlate of drug resistance in mTLE patients. Incomplete knowledge of pathological changes in mTLE with HS complicates its therapy. Growing evidence indicates a role of lipid signaling pathways in epileptogenesis; thus, lipid signals emerge as potential biomarkers for the onset and evolving course of this epileptic disorder, and are potential therapeutic agents and targets. Therefore, in this study, we recruited 23 patients with medically intractable mTLE-HS and 24 non-mTLE-HS controls. We applied lipidomic analysis to identify the lipidomic profiles in the hippocampal samples of both groups. The lipidomic profiles of the hippocampus were distinctive between mTLE-HS patients and controls. We also observed that the abundance of total triglycerides showed a striking reduction in the hippocampus of mTLE-HS patients. We identified that 33 lipids were significantly differentially expressed in the hippocampus of mTLE-HS patients compared with those in the hippocampus of the controls; this might contribute to target some molecular mechanisms involved in epileptogenesis. The present study therefore reports that lipidomic changes in mTLE-HS patients may contribute to the molecular architecture of an epileptic brain.

Phaeohyphomycosis due to Exophiala spinifera greatly improved by ALA-PDT: A case report.

An 80-year-old women complained of painful erythema, papules, nodules on her right wrist for 9 months. Granuloma and inflammatory infiltrates with septate fungal hyphae were observed in pathological sections. Growth of demaciatious fungus on SDA was confirmed to be Exophiala spinifera by microscopic appearance and ITS rDNA sequencing. The diagnosis of phaeohyphomycosis due to E. spinifera was established. The patient had a poor response to oral itraconazole (200 mg/d) combining with terbinafine (250 mg/d) for 6 weeks. Then, following three courses of ALA-PDT, used as adjuvant therapy, skin lesions greatly improved and remained stable after 3 months follow-up. The case suggests that ALA-PDT is an effective adjuvant treatment option for patients with recalcitrant local subcutaneous fungal infection.

Cytokines in cerebrospinal fluid of neurosyphilis patients: Identification of Urokinase plasminogen activator using antibody microarrays.

Little is known regarding protein responses to syphilis infection in cerebrospinal fluid (CSF) of patients presenting with neurosyphilis. Protein and antibody arrays offer a new opportunity to gain insights into global protein expression profiles in these patients. Here we obtained CSF samples from 46 syphilis patients, 25 of which diagnosed as having central nervous system involvement based on clinical and laboratory findings. The CSF samples were then analyzed using a RayBioH L-Series 507 Antibody Array system designed to simultaneously analyze 507 specific cytokines. The results indicated that 41 molecules showed higher levels in patients with neurosyphilis in comparison with patients without neural involvement. For validation by single target ELISA, we selected five of them (MIP-1a, I-TAC/CXCL11, Urokinase plasminogen activator [uPA], and Oncostatin M) because they have previously been found to be involved in central nervous system (CNS) disorders. The ELISA tests confirmed that uPA levels were significantly higher in the CSF of neurosyphilis patients (109.1±7.88pg/ml) versus patients without CNS involvement (63.86±4.53pg/ml, p<0.0001). There was also a clear correlation between CSF uPA levels and CSF protein levels (p=0.0128) as well as CSF-VDRL titers (p=0.0074) used to diagnose neurosyphilis. No significant difference between the two groups of patients, however, was found in uPA levels in the serum, suggesting specific activation of the inflammatory system in the CNS but not the periphery in neurosyphilis patients. We conclude that measurements of uPA levels in CSF may be an additional parameter for diagnosing neurosyphilis.

Prevalence of Chlamydia trachomatis Genotypes in Men Who Have Sex with Men and Men Who Have Sex with Women Using Multilocus VNTR Analysis-ompA Typing in Guangzhou, China.

BACKGROUND: Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA) sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA)-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM) and men who have sex with women (MSW) attending a sexually transmitted diseases (STD) clinic in Guangzhou, China. METHODS: Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA. RESULTS: Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA. CONCLUSIONS: The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine subtypes of C. trachomatis.

Antibiotic susceptibility of Neisseria gonorrhoeae isolates from Guangzhou, China, during 2002-2011.

Antibiotic susceptibility of Neisseria gonorrhoeae in Guangzhou during 2002-2011 showed that resistance to penicillin and ciprofloxacin was high, while ceftriaxone remained effective although there was a trend towards reduced sensitivity.