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Genome resequencing uncovers genome-wide DNA polymorphisms that are useful for the development of high-density InDel markers between two barley cultivars. Discovering genomic variations and developing genetic markers are crucial for genetics studies and molecular breeding in cereal crops. Although InDels (insertions and deletions) have become popular because of their abundance and ease of detection, discovery of genome-wide DNA polymorphisms and development of InDel markers in barley have lagged behind other cereal crops such as rice, maize and wheat. In this study, we re-sequenced two barley cultivars, Golden Promise (GP, a classic British spring barley variety) and Hua30 (a Chinese spring barley variety), and mapped clean reads to the reference Morex genome, and identified in total 13,933,145 single nucleotide polymorphisms (SNPs) and 1,240,456 InDels for GP with Morex, 11,297,100 SNPs and 781,687 InDels for Hua30 with Morex, and 13,742,399 SNPs and 1,191,597 InDels for GP with Hua30. We further characterized distinct types, chromosomal distribution patterns, genome location, functional effect, and other features of these DNA polymorphisms. Additionally, we revealed the functional relevance of these identified SNPs/InDels regarding different flowering times between Hua30 and GP within 17 flowering time genes. Furthermore, we developed a series of InDel markers and validated them experimentally in 43 barley core accessions, respectively. Finally, we rebuilt population structure and phylogenetic tree of these 43 barley core accessions. Collectively, all of these genetic resources will facilitate not only the basic research but also applied research in barley.

Several QTLs and genes responsible for seed dormancy were detected and SNP candidates were shown to cause changes in seed germination. Seed dormancy is a key agricultural trait to prevent pre-harvest sprouting in crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum), and barley (Hordeum vulgare L.). However, our knowledge of seed dormancy is hampered by the complexities of studying a trait that changes over time after seed harvest, and is complicated by interactions between phytohormones, seed coat components and the environment. Here, we have conducted a genome-wide association study using a panel of 311 natural accessions of cultivated rice, examining a total of 519,158 single nucleotide polymorphisms (SNPs). Eight quantitative trait loci (QTLs) were found to associate with seed dormancy in the whole panel and five in the Japonica and Indica subpanel; expression of candidate genes within 100 kb of each QTL was examined in two published, germination-specific transcriptomic datasets. Ten candidate genes, differentially expressed within the first four days post-imbibition, were identified. Five of these genes had previously been associated with awn length, heading date, yield, and spikelet length phenotypes. Two candidates were validated using Quantitative Reverse Transcription (qRT)-PCR. In addition, previously identified genes involved in hormone signaling during germination were found to be differentially expressed between a japonica and an indica line; SNPs in the promoter of Os9BGlu33 were associated with germination index, with qRT-PCR validation. Collectively, our results are useful for future characterization of seed dormancy mechanism and crop improvement, and suggest haplotypes for further analysis that may be of use to boost PHS resistance in rice.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.

AtMYB31, a R2R3-MYB transcription factor that modulates wax biosynthesis in reproductive tissues, is involved in seed development in Arabidopsis. R2R3-MYB transcription factors play important roles in plant development; yet, the exact role of each of them remains to be resolved. Here we report that the Arabidopsis AtMYB31 is required for wax biosynthesis in epidermis of reproductive tissues, and is involved in seed development. AtMYB31 was ubiquitously expressed in both vegetative and reproductive tissues with higher expression levels in siliques and seeds, while AtMYB31 was localized to the nucleus and cytoplasm. Loss of function of AtMYB31 reduced wax accumulation in the epidermis of silique and flower tissues, disrupted seed coat epidermal wall development and mucilage production, altered seed proanthocyanidin and polyester content. AtMYB31 could direct activate expressions of several wax biosynthetic target genes. Altogether, AtMYB31, a R2R3-MYB transcription factor, regulates seed development in Arabidopsis.

Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.

The spikelet is a unique structure of inflorescence in grasses that generates one to many flowers depending on its determinate or indeterminate meristem activity. The growth patterns and number of spikelets, furthermore, define inflorescence architecture and yield. Therefore, understanding the molecular mechanisms underlying spikelet development and evolution are attractive to both biologists and breeders. Based on the progress in rice and maize, along with increasing numbers of genetic mutants and genome sequences from other grass families, the regulatory networks underpinning spikelet development are becoming clearer. This is particularly evident for domesticated traits in agriculture. This review focuses on recent progress on spikelet initiation, and spikelet and floret fertility, by comparing results from Arabidopsis with that of rice, sorghum, maize, barley, wheat, Brachypodium distachyon, and Setaria viridis. This progress may benefit genetic engineering and molecular breeding to enhance grain yield.

Cytochrome P450 monooxygenase 704B (CYP704B), a member of the CYP86 clan, was found to be needed in Arabidopsis and rice to biosynthesize precursors of sporopollenin through oxidizing fatty acids. In the present study, we cloned and characterized a CYP704B gene in Panax ginseng, named PgCYP704B1. It shared high sequence identity (98-99%) with CYP704 of Arabidopsis, Theobroma cacao, and Morus notabilis. The phylogenetic comparison of ginseng and higher plants between the members of CYP86 clan revealed that ginseng CYP704 was categorized as a group of CYP704B with dicot plants. The expression of PgCYP704B1 is low in the stem, leaf, and fruit, and high in flower buds, particularly detected in the young gametic cell and tapetum layer of the developing anther. Arabidopsis plants overexpressing PgCYP704B1 improved plant biomass such as plant height, siliques and seed number and size. A cytological observation by transverse and longitudinal semi-thin sections of the siliques cuticles revealed that the cell length increased. Furthermore a chemical analysis showed that PgCYP704B1ox lines increased their cutin monomers contents in the siliques. Our results suggest that PgCYP704B1 has a conserved role during male reproduction for fatty acid biosynthesis and its overexpression increases cutin monomers in siliques that eventually could be used for seed production.

The aperture on the pollen surface provides an exit for the emerging pollen tube. Apertures exhibit huge morphological variation across plant species-grasses, including rice, possess a complex aperture consisting of an annulus and an operculum-but little is known about how this species-specific cell-surface pattern forms. Here, we report a lectin receptor-like kinase in Oryza sativa, OsDAF1, which is essential for annulus formation and thus for fertility. OsDAF1 is evenly distributed in early microsporocytes but localizes to the distal pre-aperture site at the tetrad stage. We further reveal that the rice orthologue of a key aperture factor in Arabidopsis, OsINP1, has conserved and diversified roles in rice aperture formation. Disruption of OsINP1 prevents formation of the aperture, precluding pollen-tube germination. Furthermore, our results demonstrate that OsINP1 is required for polarization of OsDAF1 via direct protein interaction, suggesting that OsINP1 has an additional role in the formation of annulus that is absent in Arabidopsis. Our study reveals the importance of the aperture for rice grain yield and reveals mechanisms controlling pollen aperture development in cereal species.

Since January 2019, abnormal molting has been observed frequently in approximately 40-day-old Pekin ducks in China. To investigate the possible involvement of a virus, we tested the prevalence of duck circovirus (DuCV), goose hemorrhagic polyomavirus (GHPyV), and goose parvovirus (GPV) in 11 molt cases in two provinces. GPV was detected in all cases, particularly in all samples collected from the feather area. The complete genome sequences of three GPV strains were determined and found to have 52 nucleotide changes relative to GPVs associated with short beak and dwarfism syndrome of Pekin ducks. These data will enhance our understanding of GPV diversity and outcomes of GPV infection in Pekin ducks.

Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.

A novel calicivirus (strain H146) was detected in a goose and sequenced. The H146 genome consisted of two open reading frames (ORFs) with an 8-nucleotide (nt) overlap between the two ORFs, similar to what has been found in the bat sapovirus TLC58. The virus was most closely related to nacoviruses when comparing the complete genome sequence (49% identity), non-structural region (NS; 31-34% amino acid [aa] sequence identity), and major structural VP1 region (28-30% aa identity), whereas both goose calicivirus N and feline calicivirus were the closest relatives of H146 in the VP2 region (20% aa sequence identity). The levels of divergence between H146 and its closest relatives in different genomic regions are comparable to those between some members of different genera. Phylogenetic analysis based on the NS and VP1 amino acid sequences clearly demonstrated that H146 formed a separate clade. Thus, calicivirus H146 was identified as a founding member of a novel genus for which we propose the name "Sanovirus".

Panax ginseng Meyer, commonly known as ginseng, is considered one of the most important herbs with pharmaceutical values due to the presence of ginsenosides and is cultivated for its highly valued root for medicinal purposes. Recently, it has been recognized that ginseng fruit contains high contents of triterpene such as ginsenoside Re as pharmaceutical compounds. However, it is unclear how carpel, the female reproductive tissue of flowers, is formed during the three-year-old growth before fruit is formed in ginseng plants. Here, we report P. ginseng carpel development at the cytological level, starting from the initial stage of ovule development to seed development. The carpel of P. ginseng is composed of two free stigmas, two free styles, and one epigynous bilocular ovary containing one ovule in each locule. Based on our cytological study, we propose that the female reproductive development in P. ginseng can be classified into seven stages: early phase of ovule development, megasporogenesis, megagametogenesis, pre-fertilization, fertilization, post-fertilization, and seed development. We also describe the correlation of the female and male gametophyte development and compare morphological differences in carpel development between ginseng and other higher plants. One unique feature for ginseng seed development is that it takes 40 days for the embryo to develop to the early torpedo stage and that the embryo is small relative to the seed size, which could be a feature of taxonomic importance. This study will provide an integral tool for the study of the reproductive development and breeding of P. ginseng.

Background Tembusu virus (TMUV) usually affects adult ducks, causing a severe drop of egg production. It has also been shown to be pathogenic in commercial Pekin ducklings below 7 weeks of age. Here, we report a TMUV-caused neurological disease in young egg-type ducklings and the pathogenicity of the egg-type duck-origin TMUV isolates in meat-type Pekin ducklings. Results The disease occurred in 25 to 40-day-old Jinding ducklings in China, and was characterized by paralysis. Gross lesions were lacking and microscopic lesions appeared chiefly in brain and spleen. Inoculation in embryonated duck eggs resulted in isolation of TMUV Y and GL. The clinical signs and microscopic lesions observed in the spontaneously infected egg-type ducks were repeated in Pekin ducklings by experimental infection. Notably, both Y and GL strains caused 100% mortality in the case of 2-day-old inoculation by intracerebral route. High mortalities (80 and 70%) also occurred following infection of the Y virus at 2 days of age by intramuscular route and at 9 days of age by intracerebral route. Conclusions These findings demonstrate that the egg-type duck-origin TMUVs exhibit high pathogenicity in Pekin ducklings, and that the severity of the disease in ducklings is dependent on the infection route and the age of birds at the time of infection. The availability of the highly pathogenic TMUV strains provides a useful material with which to begin investigations into the molecular basis of TMUV pathogenicity in ducks.

Two Arabidopsis ABC transporters, ABCG1 and ABCG16, are expressed in the tapetal layer, specifically after postmeiotic microspore release, and play important roles in pollen surface development. The male gametophytic cells of terrestrial plants, the pollen grains, travel far before fertilization, and thus require strong protective layers, which take the form of a pollen coat and a pollen wall. The protective surface structures are generated by the tapetum, the tissue surrounding the developing gametophytes. Many ABC transporters, including Arabidopsis thaliana ABCG1 and ABCG16, have been shown to play essential roles in the development of such protective layers. However, the details of the mechanism of their function remain to be clarified. In this study, we show that ABCG1 and ABCG16 are localized at the plasma membrane of tapetal cells, specifically after postmeiotic microspore release, and play critical roles in the postmeiotic stages of male gametophyte development. Consistent with this stage-specific expression, the abcg1 abcg16 double knockout mutant exhibited defects in pollen development after postmeiotic microspore release; their microspores lacked intact nexine and intine layers, exhibited defects in pollen mitosis I, displayed ectopic deposits of arabinogalactan proteins, failed to complete cytokinesis, and lacked sperm cells. Interestingly, the double mutant exhibited abnormalities in the internal structures of tapetal cells, too; the storage organelles of tapetal cells, tapetosomes and elaioplasts, were morphologically altered. Thus, this work reveals that the lack of ABCG1 and ABCG16 at the tapetal cell membrane causes a broad range of defects in pollen, as well as in tapetal cells themselves. Furthermore, these results suggest that normal pollen surface development is necessary for normal development of the pollen cytoplasm.

The transgenic maize (Zea mays L.) line BVLA430101 overexpressing Aspergillus niger phyA2 was approved in 2009 to be planted in a given area in China. However, the flanking sequences and event-specific qualitative/quantitative PCR detection methods for this transgenic event have not been reported. In this study, we characterized the molecular features of the exogenous integration in BVLA430101 and developed event-specific qualitative/quantitative PCR detection methods. Using genome walking, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and conventional PCR assays, we revealed that one intact copy of phytase construct was integrated in the maize genome chromosome 3, which is followed by a fragment of the transformation vector containing partial sequence of pH2b promoter. The defined 3′ flanking sequence was 1644 bp in length, based on which the event-specific qualitative and quantitative PCR assays for BVLA430101 were developed. The limit of detection (LOD) of the qualitative PCR assay was 10 haploid genome copies, and the limits of detection and quantification (LOD and LOQ) of the quantitative PCR were 10 and 10 copies of maize haploid genome, respectively. In-house validation of the developed event-specific quantitative PCR method with three practical maize samples showed that the quantified biases between the test and true values ranged between 2.60 and 7.52 %. These results indicated that the developed event-specific qualitative and quantitative PCR methods based on the newly characterized 3′ flanking sequences can be used successfully for the identification and quantification of transgenic maize BVLA430101 and its derived products.

Ginseng (Panax ginseng), a valued medicinal herb, is a slow-growing plant that flowers after 3 years of growth with the formation of a solitary terminal umbel inflorescence. However, little is known about cytological events during ginseng reproduction, such as the development of the male organ, the stamen. To better understand the mechanism controlling ginseng male reproductive development, here, we investigated the inflorescence and flower structure of ginseng. Moreover, we performed cytological analysis of anther morphogenesis and showed the common and specialized cytological events including the formation of four concentric cell layers surrounding male reproductive cells followed by subsequent cell differentiation and degeneration of tapetal cells, as well as the formation of mature pollen grains via meiosis and mitosis during ginseng anther development. Particularly, our transverse section and microscopic observations showed that the ginseng tapetal layer exhibits obvious nonsynchronous cell division evidenced by the observation of one or two tapetal layers frequently observed in one anther lobe, suggesting the unique control of cell division. To facilitate the future study on ginseng male reproduction, we grouped the anther development into 10 developmental stages according to the characterized cytological events.

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Metabolites in maize kernels are associated not only with nutritional value but also physiological properties such as maturation, desiccation, and germination. However, comprehensive information concerning the metabolome of maize kernels is limited. In this study, we identified 210 metabolites in mature kernels of 14 representative maize lines using a non-targeted metabolomic profiling approach. Further statistical analysis revealed that 75 metabolites were significantly variable among those tested lines, and certain metabolites out of the detected 210 metabolites played critical roles in distinguishing one line from another. Additionally, metabolite–metabolite correlation analysis dissected key regulatory elements or pathways involved in metabolism of lipids, amino acids and carbohydrates. Furthermore, an integrated metabolic map constructed with transcriptomic, proteomic and metabolic data uncovered characteristic regulatory mechanisms of maize kernel metabolism. Altogether, this work provides new insights into the maize kernel metabolome that would be useful for metabolic engineering and/or molecular breeding to improve maize kernel quality and yield.

The tapetum of the anther locule encloses the male reproductive cells and plays a supportive role for normal pollen development. However, the underlying mechanism remains less understood. Previously, we identified a complete recessive male sterile mutant, post-meiotic deficient anther1 (pda1), with abnormal postmeiotic tapetal development. In this study we comprehensively characterized pda1. Chemical analysis uncovered that pda1 anther had significant lower levels of cutin monomers and cuticular waxes. PDA1 gene encodes an ATP-binding cassette (ABC) half-transporter, namely OsABCG15, which is conserved from algae to higher plants. In situ RNA hybridization assay showed that PDA1 is strongly expressed in tapetal cells, and weakly in microspores during the anther development. Additionally, the expression of two pollen exine biosynthetic genes CYP704B2 and CYP703A3 was dramatically reduced in pda1 mutant anthers. Altogether, these observations suggest that the tapetum-expressed ABC transporter PDA1 plays a crucial role in secreting lipidic precursors from the tapetum to developing microspores and the anther epidermis.

Seed development dedicates to reserve synthesis and accumulation and uncovering its genetic and biochemical mechanisms has been a major research focus. Although proteomic and transcriptomic analyses revealed dynamic changes of genes and enzymes involved, the information regarding concomitant metabolic changes is missing. Here we investigated the dynamic metabolic changes along the rice grain development of two japonica and two indica cultivars using non-targeted metabolomics approach, in which we successfully identified 214 metabolites. Statistical analyses revealed both cultivar and developmental stage dependent metabolic changes in rice grains. Generally, the stage specific metabolic kinetics corresponded well to the physiological status of the developing grains and metabolic changes in developing rice grain are similar to those of dicot Arabidopsis and tomato at reserve accumulation stage but are different from those of dicots at seed desiccation stage. The remarkable difference in metabolite abundances between japonica and indica rice grain was observed at the reserve accumulation stage. Metabolite-metabolite correlation analysis uncovered potential new pathways for several metabolites. Taken together, this study uncovered both conserved and diverse development associated metabolic kinetics of rice grains, which facilitates further study to explore fundamental questions regarding the evolution of seed metabolic capabilities as well as their potential applications in crop improvement.

We conducted experiments to culture Pycnoporus sp. SYBC-L3 in a medium comprising an industrial waste (dye-containing textile effluent) and a lignocellulosic waste (Phragmites australis) that achieved laccase production while having the color removed from the wastewater. Our experimental results showed that the fungus grew well in liquid submerged cultivation with the diluted textile effluent as the sole culture medium, but relatively low extracellular laccase activity (1.8 U/mL) was produced. Addition of the lignocellulosic biomass enhanced laccase production and color removal. The highest laccase activity was found to be 6.5 U/mL in the presence of Phragmites australis stem. Under this condition, 70 % color removal occurred in the culture medium. This study provided an alternative novel scheme to remove color in textile wastewater while having an economic value added by producing laccase.

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

A novel manganese peroxidase of Rhizoctonia sp. SYBC-M3 (R-MnP) was purified by (NH4)2SO4 fractionation, DEAE-cellulose-32 column chromatography, and Sephadex G100 column chromatography. The molecular mass of R-MnP was determined to be approximately 40.4 kDa by SDS-PAGE. The optimum temperature and pH for R-MnP were 55°C and 4.5, respectively. R-MnP was highly stability when the temperature was below 50°C. R-MnP could retain about 60% of its activity when the pH was between 4 and 6.5. However, R-MnP activity was inhibited by Fe3+, Cu2+, and Co3+ as well as increased by Zn2+ and Ca2+. R-MnP demonstrated oxidation of DMP, ABTS, veratryl alcohol, and guaiacol. The K m values of RMnP for H2O2 and Mn2+ were 25.3 and 53.9 μmol/L, respectively.

Maximum catalase production by mesophilic bacterium Serratia marcescens SYBC-01 was obtained by an optimization of culture medium and conditions. A novel cold-adapted catalase from the strain was purified and characterized. The Cat-2 without peroxidase activity was a homodimer with a molecular mass of 154 kDa, consisting of two identical subunits of about 70 kDa. Its apparent Km and Vmax value were 29.7 mM and 80,925 U/mg of protein, respectively. The Cat-2 exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. The enzyme was most active at approximately 20°C and had 73.8% activity at 0°C. After incubation at 60°C for 60 min, the enzyme still maintained 75% of its initial activity. The Cat-2 displayed relatively higher thermostability compared to that of other cold-adapted and some mesophilic catalases.

Estimation of the homoplasmy of transgene in chloroplast is a necessary step in chloroplast transformation. This task is usually achieved by Southern blot analysis, which is laborious, time-consuming, requires large amounts of plant materials and needs hazardous radioisotopes in some cases. To develop a fast, sensitive, stable and effective technique for determining the homoplasmy of transgene in chloroplast, one real-time PCR system based on TaqMan probe technique was developed for evaluating the homoplasmy degree. In the real-time PCR system, one assay targets the exogenous faeG gene, and another one the tobacco chloroplast reference gene, maturase gene within the trnK intron (matK). The homoplasmy of the transgene was determined by the comparison of copy number of faeG and matK. The analyzed results of 17 transplastomic tobacco lines using the developed real-time PCR system were consistent with those from Southern blot analysis, indicating that the real-time PCR method is suitable for estimating the homoplasmy degree of transplastomic plant with the advantages of high efficiency and throughput, low cost and saving time.

To develop effective alternatives for detecting genetically modified organisms (GMOs), we reported one optimized visual loop-mediated isothermal amplification (LAMP) method for the detection of exogenous DNA targets from two GM soybean events in this study. This isothermal amplification can be performed within 40 min without polymerase chain reaction (PCR) equipment and the derived LAMP products can be directly observed by naked eye employing SybrGreen I dye instead of conventional gel electrophoresis analysis. The limits of detection of these established visual LAMP assays were about 4 copies of haploid soybean genomic DNA, and which were much higher than those of reported conventional PCR assays. Furthermore, the high specificity of LAMP assays was determined. All the results demonstrated that the developed visual LAMP assays are convenient, cost-efficient, and rapid for on spot detection of GMOs.

Although AGAMOUS-LIKE6 (AGL6) MADS-box genes are ancient with wide distributions in gymnosperms and angiosperms, their functions remain poorly understood. Here, we show the biological role of the AGL6-like gene, OsMADS6, in specifying floral organ and meristem identities in rice (Oryza sativa L.). OsMADS6 was strongly expressed in the floral meristem at early stages. Subsequently, OsMADS6 transcripts were mainly detectable in paleas, lodicules, carpels and the integument of ovule, as well as in the receptacle. Compared to wild type plants, osmads6 mutants displayed altered palea identity, extra glume-like or mosaic organs, abnormal carpel development and loss of floral meristem determinacy. Strikingly, mutation of a SEPALLATA (SEP)-like gene, OsMADS1 (LHS1), enhanced the defect of osmads6 flowers, and no inner floral organs or glume-like structures were observed in whorls 2 and 3 of osmads1-z osmads6-1 flowers. Furthermore, the osmads1-z osmads6-1 double mutants developed severely indeterminate floral meristems. Our finding, therefore, suggests that the ancient OsMADS6 gene is able to specify "floral state" by determining floral organ and meristem identities in monocot crop rice together with OsMADS1.

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml(-1), which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0 degrees C but also a high thermostable enzyme. The half-lives at 60, 70 and 80 degrees C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.

Effective analysis methods for genetically modified organisms (GMOs) using reliable reference molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the reference molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new reference molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new reference molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the reference material derived from plant raw materials.

Male reproductive development is a complex biological process which includes the formation of the stamen with differentiated anther tissues, in which microspores/pollens are generated, then anther dehiscence and subsequently pollination. Stamen specification and anther development involve a number of extraordinary events such as meristem transition, cell division and differentiation, cell to cell communication, etc., which need the cooperative interaction of sporophytic and gametophytic genes. The advent of various tools for rice functional gene identification, such as complete genome sequence, genome-wide microarrays, collections of mutants, has greatly facilitated our understanding of mechanisms of rice stamen specification and anther development. Male sterile lines are critical for hybrid rice breeding, therefore understanding these processes will not only contribute greatly to the basic knowledge of crop developmental biology, but also to the development of new varieties for hybrid rice breeding in the future.

The nucleotide sequences of the complete VP1, VP0, VP3, and partial 3D regions of seven duck hepatitis virus (DHV) serotype 1 (DHV-1) strains isolated in China between 2001 and 2007 and one DHV-1 strain originally obtained from ATCC were determined and compared with previously available DHV sequences in GenBank. Phylogenetic analysis on the basis of VP1 sequences demonstrated three distinct genetic groups. There was an excellent concordance among the genetic groups assigned based on the complete VP0, VP3, and the partial 3D regions. In view of the growing importance of molecular techniques in diagnosis, we propose that the three genetic groups should be termed DHV types A, B, and C. All DHV-1 strains grouped in genotype A, whereas the new serotype strains isolated in Taiwan and the new serotype strains isolated in South Korea clustered into genotypes B and C, respectively, suggesting a potential genetic correlates of serotype. In pairwise comparisons of complete VP1, VP0, and VP3 nucleotide and amino acid sequences and the partial 3D nucleotide sequence, DHVs of the same genotype were clearly distinguished from those of heterologous genotypes. Analysis of the amino acid sequences of the three capsid proteins demonstrated the presence of conserved elements that form the eight-stranded beta-barrel structures, as well as intervening domains that vary in sequence between strains of different genotypes as seen in other picorna viruses.

Six molecularly imprinted polymers (MIPs) of erythromycin (ERY) were prepared by noncovalent bulk polymerization using methacrylic acid (MAA) as the functional monomer. On the basis of binding analysis, the MIPs with 1:2 optimum ratio of template to MAA were selected for subsequent scanning electron microscopy and Brunauer-Emmett-Teller analyses, which indicated that the MIPs had more convergent porous structures than the nonimprinted polymers. The equilibrium binding experiments showed that the binding sites of MIPs were heterogeneous, with two dissociation constants of 0.005 and 0.63 mg mL(-1), respectively. Furthermore, the performance of the MIPs as solid-phase extraction (SPE) sorbents was evaluated, and the selectivity analysis showed that the MIPs could recognize ERY with moderate cross-reactivity for other macrolides. The overall investigation of molecularly imprinted SPE for cleanup and enrichment of the ERY in pig muscle and tap water confirmed the feasibility of utilizing the MIPs obtained as specific SPE sorbents for ERY extraction in real samples. [figure: see text]

A rice mutant with Japonica 9522 cultivar background Oryza sativa extraordinary glume 1 (Oseg 1) was identified from the M 2 mutant pool mutagenized by 60Co γ-ray. Compared with wild type plants, Oseg 1 developed longer empty glumes and rudimentary glumes. In some Oseg 1 mutants, the number of stamens of flowers was reduced and leaf-like lodicules occurred, and excessive lemma/palea-like organ could be observed in some mutant spikelets. This indicated that OsEG1 could regulate the edevelopment of rudimentary glumes, empty glumes, lemma/palea, lodicules, and stamens. Genetic analysis indicated that Oseg 1 came from a single recessive genetic locus. To clone OsEG1 gene, F 2 population was constructed by a cross between Oseg 1 (Japonica) and Guangluai4 (Indica). Using map-based cloning approach, OsEG1 was mapped on chromosome 4, between INDEL marker OS407 and WHM0466 with genetic distance of 2.0 cm and 1.0 cm, respectively. These results are useful for further cloning and functional analysis of the OsEG1 gene.

To understand the expansion of multicopy microRNA (miRNA) families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miR160 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicate that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.

A rice male-sterile mutantOsMS-L ofjaponica cultivar 9522 background, was obtained in M4 population treated with60Co γ-Ray. Genetic analysis indicated that the male-sterile phenotype was controlled by a single recessive gene. Results of tissue section showed that at microspore stage,OsMS-L tapetum was retarded. Then tapetal cells expanded and microspores degenerated. No matured pollens were observed inOsMS-L anther locus. To mapOsMS-L locus, an F2 population was constructed from the cross between theOsMS-L (japonica) and LongTeFu B(indica). Firstly, theOsMS-L locus was roughly mapped between two SSR markers, RM109 and RM7562 on chromosome 2. And then eleven polymorphic markers were developed for further fine fine-mapping. At last theOsMS-L locus was mapped between the two InDel markers, Lhs10 and Lhs6 with genetic distance of 0.4 cM, respectively. The region was delimited to 133 kb. All these results were useful for further cloning and functional analysis ofOsMS-L.

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T0) were calculated by comparing threshold cycle (CT) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsCT value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.

Ethylene responsive factors (ERFs) are important in regulating plant pathogen resistance, abiotic stress tolerance and plant development. Recent studies have greatly enlarged the ERF protein family and revealed more important roles of ERFs in plants. Here, we report our finding of a tomato ERF protein TSRF1, which is transcriptionally up-regulated by ethylene, salicylic acid, or Ralstonia solanacearum strain BJ1057 infection. Biochemical analysis indicates that TSRF1 specifically interacts in vitro with the GCC box, an element present in the promoters of many pathogenesis-related (PR) genes. Further investigation evidences that TSRF1 activates in vivo the expression of reporter beta-glucuronidase gene controlled by GCC box. More importantly, overexpressing TSRF1 in tobacco and tomato constitutively activates the expression of PR genes, and subsequently enhancing transgenic plant resistance to the bacterial wilt caused by Ralstonia solanacearum strain BJ1057. Therefore our investigation not only extends the functions of ERF proteins in plant resistance to R. solanacearum, but also provides further clues to understanding the mechanism of host regulatory proteins in response to the infection of pathogens.

The spikelet is the basal unit of inflorescence in grasses, and its formation is crucial for reproductive success and cereal yield. Here, we report a previously unknown role of the plant hormone jasmonic acid (JA) in determining rice (Oryza sativa) spikelet morphogenesis. The extra glume 1 (eg1) and eg2 mutants exhibit altered spikelet morphology with changed floral organ identity and number, as well as defective floral meristem determinacy. We show that EG1 is a plastid-targeted lipase that participates in JA biosynthesis, and EG2/OsJAZ1 is a JA signalling repressor that interacts with a putative JA receptor, OsCOI1b, to trigger OsJAZ1's degradation during spikelet development. OsJAZ1 also interacts with OsMYC2, a transcription factor in the JA signalling pathway, and represses OsMYC2's role in activating OsMADS1, an E-class gene crucial to the spikelet development. This work discovers a key regulatory mechanism of grass spikelet development and suggests that the role of JA in reproduction has diversified during the flowering plant evolution.

Background Assessment of potential allergenicity of protein is necessary whenever transgenic proteins are introduced into the food chain. Bioinformatics approaches in allergen prediction have evolved appreciably in recent years to increase sophistication and performance. However, what are the critical features for protein's allergenicity have been not fully investigated yet. Results We presented a more comprehensive model in 128 features space for allergenic proteins prediction by integrating various properties of proteins, such as biochemical and physicochemical properties, sequential features and subcellular locations. The overall accuracy in the cross-validation reached 93.42% to 100% with our new method. Maximum Relevance Minimum Redundancy (mRMR) method and Incremental Feature Selection (IFS) procedure were applied to obtain which features are essential for allergenicity. Results of the performance comparisons showed the superior of our method to the existing methods used widely. More importantly, it was observed that the features of subcellular locations and amino acid composition played major roles in determining the allergenicity of proteins, particularly extracellular/cell surface and vacuole of the subcellular locations for wheat and soybean. To facilitate the allergen prediction, we implemented our computational method in a web application, which can be available at http://gmobl.sjtu.edu.cn/PREAL/index.php. Conclusions Our new approach could improve the accuracy of allergen prediction. And the findings may provide novel insights for the mechanism of allergies.

Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events.

Background Allergy involves a series of complex reactions and factors that contribute to the development of the disease and triggering of the symptoms, including rhinitis, asthma, atopic eczema, skin sensitivity, even acute and fatal anaphylactic shock. Prediction and evaluation of the potential allergenicity is of importance for safety evaluation of foods and other environment factors. Although several computational approaches for assessing the potential allergenicity of proteins have been developed, their performance and relative merits and shortcomings have not been compared systematically. Results To evaluate and improve the existing methods for allergen prediction, we collected an up-to-date definitive dataset consisting of 989 known allergens and massive putative non-allergens. The three most widely used allergen computational prediction approaches including sequence-, motif- and SVM-based (Support Vector Machine) methods were systematically compared using the defined parameters and we found that SVM-based method outperformed the other two methods with higher accuracy and specificity. The sequence-based method with the criteria defined by FAO/WHO (FAO: Food and Agriculture Organization of the United Nations; WHO: World Health Organization) has higher sensitivity of over 98%, but having a low specificity. The advantage of motif-based method is the ability to visualize the key motif within the allergen. Notably, the performances of the sequence-based method defined by FAO/WHO and motif eliciting strategy could be improved by the optimization of parameters. To facilitate the allergen prediction, we integrated these three methods in a web-based application proAP, which provides the global search of the known allergens and a powerful tool for allergen predication. Flexible parameter setting and batch prediction were also implemented. The proAP can be accessed at http://gmobl.sjtu.edu.cn/proAP/main.html. Conclusions This study comprehensively evaluated sequence-, motif- and SVM-based computational prediction approaches for allergens and optimized their parameters to obtain better performance. These findings may provide helpful guidance for the researchers in allergen-prediction. Furthermore, we integrated these methods into a web application proAP, greatly facilitating users to do customizable allergen search and prediction.

Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.

Background ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites. Results Here, we present SIPeS (S ite I dentification from P aired-e nd S equencing), a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS), which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS. Conclusions When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.