2.1 Sample collection
S. chinensis samples were collected in October, 2019, from fresh mature galls on R. chinensis , in Wufeng county (30°10′ N,110°52′ E,960 m above sea level), Hubei Province, China. S. chinensis individual for sequencing were from a single gall for PacBio sequencing, Illumina sequencing, RNA-seq and Hi-C analysis. All aphids within the gall were presumed to be clonal offspring of a single fundatrix, because allS. chinensis galls have contained only a single fundatrix and the fundatrix produces offspring in the gall via parthenogenesis. Fundatrigeniae (female) contained within a gall were moved to a petri dish after dissecting the gall. Impurities such as waxes were removed manually. Samples were separated into randomly groups with each group containing about 20 individuals. The samples were immediately frozen in liquid nitrogen for 2 hours and then stored at -80℃ f for later analysis.
In addition to the original samples, a colony was established through artificial cultivation for other genetic studies. To establish a colony, autumn migrants of S. chinensis were collected from mature galls and transferred to a nursery of the moss Plagiomnium maximoviczii , which were maintained in a greenhouse. In the following year, spring migrants (sexuparae) were collected from the mosses and cultivated in lab. Aphids were transferred to host trees after fundatrix emergence for gall induction. Aphid samples of different stages including fundatrix, fundatrigeniae, autumn migrants, overwinter nymphs, and spring migrants as well as male and female sexuales were collected separately. All aphid samples were immediately frozen in liquid nitrogen for two hours and then stored at -80℃ until further analysis.