The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant’s internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010–2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

We previously demonstrated that H9N2 subtype avian influenza viruses (AIVs) isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E) and then underwent antigenic drift from commercial vaccines, causing a country-wide outbreak during 2010–2013. In this study, H9N2 AIVs isolated from chickens during 2009–2013 were antigenically analyzed by performing hemagglutination inhibition and neutralization assays using a panel of polyclonal antibodies. Our findings confirmed the antigenic drift of recent H9N2 viruses from the commercial vaccine and showed that most of these antigenic variants form a novel HI antigenic group, F, with a few belonging to groups D and E. Slight antigenic variation was observed in group F viruses. Genetic analysis of amino acid sequences deduced from hemagglutinin (HA) gene sequences indicated that 9 of 15 mutations predominant in the 2009–2013 viruses can be mapped to known antigenic sites, which might be responsible for the novel antigenicity of group F. These antigenic changes make it necessary to modify the influenza vaccine to ensure efficient protection. A vaccine candidate, Ck/HeB/YT/10, was selected and provided significant protection against viruses from different antigenic groups in terms of reduction in virus shedding, suggesting broad cross-reactivity. Taken together, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak and now seems to be undergoing new antigenic divergence. Systematic surveillance and timely updating of vaccine strains are important for viral prevention and control in the future.

With the rapid development of sequence specific nucleases (SSNs) for genome targeting, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is now considered the most promising method for functional genetic researches, as well as genetic improvement in crop plants. However, the gene redundancy feature within the allotetraploid rapeseed genome is one of the major obstacles for simultaneous modification of different homologs in the first generation. In addition, large scale screening to identify mutated transgenic plants is very time-and labor-consuming using the conventional restriction enzyme-based approaches. In this study, a streamlined rapeseed CRISPR-Cas9 genome editing platform was developed through synthesizing a premade U6-26 driven sgRNA expression cassette and optimizing polyacrylamide gel electrophoresis (PAGE)-based screening approach. In our experiment, a sgRNA was constructed to target five rapeseed SPL3 homologous gene copies, BnSPL3-A5/BnSPL3-A4/BnSPL3-C3/BnSPL3-C4/BnSPL3-Cnn. High-throughput sequencing analysis demonstrated that the editing frequency of CRISPR/Cas9-induced mutagenesis ranged from 96.8 to 100.0% in plants with obvious heteroduplexed PAGE bands, otherwise this proportion was only 0.00–60.8%. Consistent with those molecular analyses, Bnspl3 mutants exhibited developmental delay phenotype in the first generation. In summary, our data suggest that this set of CRISPR/Cas9 platform is qualified for rapidly generating and identifying simultaneous mutagenesis of multiple gene homologs in allotetraploid rapeseed.

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum–valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.