Using RNA-seq to characterize pollen–stigma interactions for pollination studies

Insects are essential for the reproduction of pollinator-dependent crops and contribute to the pollination of 87% of wild plants and 75% of the world’s food crops. Understanding pollen flow dynamics between plants and pollinators is thus essential to manage and conserve wild plants and ensure yields are maximized in food crops. However, the determination of pollen transfer in the field is complex and laborious. We developed a field experiment in a pollinator-dependent crop and used high throughput RNA sequencing (RNA-seq) to quantify pollen flow by measuring changes in gene expression between pollination treatments across different apple (Malus domestica Borkh.) cultivars. We tested three potential molecular indicators of successful pollination and validated these results with field data by observing single and multiple visits by honey bees (Apis mellifera) to apple flowers and measured fruit set in a commercial apple orchard. The first indicator of successful outcrossing was revealed via differential gene expression in the cross-pollination treatments after 6 h. The second indicator of successful outcrossing was revealed by the expression of specific genes related to pollen tube formation and defense response at three different time intervals in the stigma and the style following cross-pollination (i.e. after 6, 24, and 48 h). Finally, genotyping variants specific to donor pollen could be detected in cross-pollination treatments, providing a third indicator of successful outcrossing. Field data indicated that one or five flower visits by honey bees were insufficient and at least 10 honey bee flower visits were required to achieve a 25% probability of fruit set under orchard conditions. By combining the genotyping data, the differential expression analysis, and the traditional fruit set field experiments, it was possible to evaluate the pollination effectiveness of honey bee visits under orchards conditions. This is the first time that pollen-stigma-style mRNA expression analysis has been conducted after a pollinator visit (honey bee) to a plant (in vivo apple flowers). This study provides evidence that mRNA sequencing can be used to address complex questions related to stigma–pollen interactions over time in pollination ecology.