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Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.

BackgroundThis study was conducted to identify epidemiological characteristics of the first documented CHIK fever outbreak in China and evaluate the effect of the preventive measures taken.Methodology/Principal FindingsFrom September 1 to October 29, 2010, China's first documented outbreak of CHIK fever occurred in the Xincun community of Wanjiang District of Dongguan city, Guangdong province; 253 case-patients were recorded, of which 129 were laboratory confirmed, with an attack rate of 1%. Before September 18th the number of CHIK fever cases remained relatively low in the Xincun community; from September 19th onwards, the number of cases increased drastically, with an outbreak peak on October 4th. Cases were distributed across nine small village groups in the Xincun community, with an attack rate of 0–12% at the village level. The household attack rates ranged between 20% and 100%. No significant difference was found in the attack rate between males and females. There was a significant difference in the attack rate in different age groups (chi-square = 18.35, p = 0.005); highest in patients aged 60 years or older and the lowest in patients aged under 10. The major clinical characteristics of patients are fever (100%), joint pain (79%) and rash (54%). Phylogenetic analysis of the E1 gene on the five earliest confirmed cases showed that the strains of CHIKV isolated from their sera were highly homologous (up to 99%) with isogeneic strains isolated in Thailand in 2009. After control measures were taken, including killing adult mosquitoes and cleaning breeding habitats of Aedes mosquitoes, the Breteau index and Mosq-ovitrap index decreased rapidly, and the outbreak ended on October 29.Conclusion/SignificanceThe infection source of the outbreak was imported. Cases showed obvious temporal, spatial, and population aggregation during the outbreak. Comprehensive control measures based on reducing the density of Aedes mosquitoes were effective in controlling the epidemic.