Thrips rearing and genomic DNA isolation

A 10th generation sibling-sibling line of F. occidentalis (Pergande) was inbred for genome homozygosity from a lab colony originating from a progenitor isolated from the Kamilo Iki valley on the island of O’hau, Hawaii [281]. Thirty-one males and females were singly paired in small 1-oz clear plastic cups with lids fitted with thrips-proof-screen, and each cup contained a small cut segment of surface-disinfested green bean pod serving as the rearing and oviposition substrate. To reduce the likelihood of parthenogenetic reproduction in subsequent generations—as unfertilized female F. occidentalis produce only male progeny—early second instar larvae (L2) developing from each mating pair were removed with a fine, water-moistened paintbrush and transferred as single pairs to individual cups with a fresh cut bean to develop to adulthood. Pairs that did not develop into male-female pairs were discarded from their lineage. By the 10th generation, four inbred lines were moved to larger colony-size, 12-oz deli cups to initiate amplification of the lines, of which one thrived to establish a healthy, reproductive colony. Pools of adult females from this colony served as the biological material for genomic DNA isolation. Genomic DNA (gDNA) was isolated from eight, 10-mg subsamples of CO₂-anesthetized females (hundreds of individuals per subsample) that were flash-frozen in liquid nitrogen, pulverized by hand with Kontes pestles (DWK Life Sciences, Thermo Fisher Scientific, Inc) then processed using the OmniPrep Genomic DNA Isolation Kit (G-Biosciences, Geno Technology, Inc., Saint Louis, MO, USA) following the manufacturer’s instructions, with the added step of 15 min room-temperature incubation of the dissolved pellet with 1 μl of LongLife RNAse (G-Biosciences) to remove residual RNA. The concentration of gDNA determined by a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc.) ranged from 48 to 89 μg of DNA. Gel electrophoresis (2% agarose gel) resolved single bands of gDNA of greater than 24 kb in size. The gDNA samples were sent to the Baylor College of Medicine Human Genome Sequencing Center (BCM-HGSC) for sequencing.