Fig. 2

Screening of insertional mutants based on reverse and forward genetics. a Transformants were generated using HS211 as the parental strain and aphVIII as the insertion cassette. b1 A total of 48 transformants were grown on a 9-cm solid TAP plate and a pair of plates comprised the basic pool of 96 transformants; all plates were duplicated every 4 months for long-term storage. b2 A super pool was generated by replicating transformants from thirty 9-cm plates to two 15-cm plates. b3 Cells grown in b2 were resuspended in 200 ml TAP medium; genomic DNA was extracted and stored as super pool DNA libraries. b4 Mutants were screened by PCR using insertion cassette-specific primer, LGR06, a target gene-specific primer and 48 super-pool DNA libraries as templates. b5 PCR products were sequenced to confirm the insertion locus. b6 DNA of basic pools from the positive super pool was extracted separately. b7 The positive basic pool was identified by PCR using the specific target primer and the basic pool DNA library as templates. b8 The cells were mixed together by line and row respectively, and the positive transformant was localized by colony PCR adopting a cross-over strategy. b9 Positive transformants were recovered from the 9-cm plate for storage in b1. c1 Cells from the basic pool were transferred to nitrogen-deprived TAP (TAP-N) medium for 24 h in microtitle plates for gametogenesis. c2 Mutants with motility defects were identified under a stereomicroscope. c3 Mutants of IFT46 localization were characterized using confocal microscopy. c4 The putative insertional mutagenesis was identified using RESDA-PCR. d1 Cells in c1 are transferred into TAP-N medium for another 48 h, inducing the formation of oil droplets. d2 After Nile red staining, the mutants with fewer oil droplets were identified with fluorescence microscopy. d3 The phenotype with fewer oil droplets was confirmed under confocal microscopy. d4 The quantity of TAG in mutants identified in D3 was analyzed by thin-layer chromatography (TLC)