RNA sequencing can simultaneously identify exonic polymorphisms and quantitate gene expression. Here we report RNA sequencing of developing maize kernels from 368 inbred lines producing 25.8 billion reads and 3.6 million single-nucleotide polymorphisms. Both the MaizeSNP50 BeadChip and the Sequenom MassArray iPLEX platforms confirm a subset of high-quality SNPs. Of these SNPs, we have mapped 931,484 to gene regions with a mean density of 40.3 SNPs per gene. The genome-wide association study identifies 16,408 expression quantitative trait loci. A two-step approach defines 95.1% of the eQTLs to a 10-kb region, and 67.7% of them include a single gene. The establishment of relationships between eQTLs and their targets reveals a large-scale gene regulatory network, which include the regulation of 31 zein and 16 key kernel genes. These results contribute to our understanding of kernel development and to the improvement of maize yield and nutritional quality.

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Variation in gene expression contributes to the diversity of phenotype. The construction of the pan-transcriptome is especially necessary for species with complex genomes, such as maize. However, knowledge of the regulation mechanisms and functional consequences of the pan-transcriptome is limited. In this study, we identified 13,382 nuclear expression presence and absence variation candidates (ePAVs, expressed in 5%~95% lines; based on the reference genome) by re-analyzing the RNA sequencing data from the kernels (15 days after pollination) of 368 maize diverse inbreds. It was estimated that only ~1% of the ePAVs are explained by DNA sequence presence and absence variations (PAV). The ePAV genes tend to be regulated by distant eQTLs when compared with non-ePAV genes (called here core expression genes, expressed in more than 95% lines). When the expression presence/absence status was used as the "genotype" to perform genome-wide association study, 56 (0.42%) ePAVs were significantly associated with 15 agronomic traits and 1,967 (14.74%) with 526 metabolic traits, measured from the mature kernels. While the above was majorly based on the reference genome, by using a modified 'assemble-then-align' strategy, 2,355 high confidence novel sequences with a total length of 1.9Mb were found absent in the current B73 reference genome (v2). Ten randomly selected novel sequences were validated with genomic PCR. A simulation analysis suggested that the pan-transcriptome of the maize whole kernel is approaching a maximum value of 63,000 genes. Two novel validated sequences annotated as NBS_LRR like genes were found to associate with flavonoid content and their homologs in rice were also found to affect flavonoids and disease-resistance. Novel sequences absent in the present reference genome might be functionally important and deserve more attentions. This study provides novel perspectives and resources to discover maize quantitative trait variations and help us to better understand the kernel regulation networks, thus enhancing maize breeding.

Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the TLR5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine.

BackgroundSince 2008, a progressive pneumonia has become prevalent in broilers and laying hens. This disease occurrs the first day after hatching and lasts more than 30 days, resulting in approximately 70% morbidity and 30% mortality in broilers. The objective of this study was to isolate and identify the pathogens that are responsible for the progressive pneumonia and establish an animal model for drug screening.Results193 serum samples were collected from 8 intensive farms from 5 provinces in China and analysed in the current research. Our clinical survey showed that 65.2% to 100% of breeding broilers, breeding layers, broilers and laying hens were seropositive for ORT antibodies. From 8 intensive farms, six ORT isolates were identified by PCR and biochemical assays, and two H9N2 viruses were isolated. Newcastle Disease Virus (NDV) and Infectious BronchitisVirus (IBV) were excluded. Typical pneumonia and airsacculitis were observed both in broilers inoculated intraperitoneally with an ORT isolate alone and in those co-infected with ORT and H9N2 virus isolates. Specifically, the survival rate was 30%, 20%, 70%, 50% and 90% in birds inoculated with ORT+H9N2 virus, ORT followed by H9N2 virus, H9N2 virus followed by ORT, and ORT or H9N2 virus alone, respectively.ConclusionsThe results of this study suggest that ORT infections of domestic poultry have been occurring frequently in China. ORT infection can induce higher economic losses and mortality if H9N2 AIV is also present. Although the isolation of ORT and H9N2 virus has been reported previously, there have been no reported co-infections of poultry with these two pathogens. This is the first report of co-infection of broilers with ORT and H9N2 virus, and this co-infection is probably associated with the outbreak of broiler airsacculitis in China, which has caused extensive economic losses.