Sorghum seeds showing “candystripe” pericarp phenotype of the y-cs allele.

Transposition of Candystripe1 element. Southern blot analysis was done on pairs of candystripe and red revertant sibling plants (Materials and Methods). Results obtained from three pairs (nos. 12, 14, and 20) are presented. Genomic DNA from parental lines Tx2737 (white pericarp, lane T) and CS8110419 (candystripe, lane CS) and three pairs of candystripe (C) and red revertant (R) siblings was digested with BamHI, gel-fractionated, and blotted. The blot was hybridized with Cs1 probe 28E, a 1.1-kb subfragment of the F1 fragment (Fig. 2B). The 8.0-kb polymorphic BamHI band present in the parental candystripe line (CS) and sibling candystripe plants (C) is shown by an arrow. This band is absent from homozygous (genotype YY) red revertants 14-R and 20-R. Transposition of Cs1 to a new genomic site is demonstrated by the presence of a new Cs1-hybridizing band, indicated by an asterisk in these red revertants. Note that red revertant 12-R shows the presence of the 8.0-kb y-cs band as well as a second band in the genome; progeny analysis confirmed that this revertant is of a heterozygous Y/y-cs genotype.

Structure of Candystripe1 transposon. Line diagram showing the structural features of the Cs1 element. Arrow heads at the end represent the 20-bp TIR sequences. Subterminal ends are enlarged to show the arrangement of the 12-bp repeat motifs. Predicted genes 1, 2, 3, and 4 are indicated with exons as black boxes and introns as diagonal lines joining the exons. Other sequence elements are indicated.

Molecular characterization of the Y-cs allele. (A) Detection of RFLP. Genomic DNA samples from red revertant (R) and candystripe (C) plants were digested with BamHI and hybridized to the indicated probe fragments. Probe 8A is a genomic DNA fragment of the maize p1 gene containing exons 1 and 2, which encode part of the MYB DNA-binding domain (37). Probes F3, F2, and F1 are restriction fragments of the y-cs clone λCS-16 (B). The approximately 8.0-kb polymorphic band present in candystripe DNA that hybridizes to all the probe fragments is indicated with an asterisk. The 5.0-kb band (arrow) present in red revertant DNA hybridizes to the maize p1-Myb probe 8A and sorghum Y gene intron2 probe F3. Nonpolymorphic bands hybridizing to 8A probe may represent other Myb-homologous genes in sorghum. Molecular sizes are shown in kb. (B) Partial restriction map of the y-cs allele (not to scale). The Y gene exons are represented as black boxes. The Candystripe1 (Cs1) element present in the second intron of the Y gene is indicated by a bold line. Outwardly oriented arrow heads represent 20-bp TIR sequences found at the borders of the Cs1 element. Positions of DNA fragments 8A, 12, F1, F2, F3, and 28E used as hybridization probes are indicated below the y-cs map. Restriction enzyme sites shown are B, BamHI; E, EcoRI; H, HindIII; and X, XhoI. The y-cs map was constructed from overlapping λ clones (Lower). Arrows marked as P-1 and P-2 indicate positions of primer 1 and primer 2, respectively, used for the PCR amplification.

Homologs of Candystripe1 in sorghum races and grasses. Cs1-homologous sequences in diverse sorghum lines (A) and grass species (B) were identified through Southern blot analysis. Gel blots prepared from BamHI-digested leaf DNA were hybridized to the Candystripe1 probe fragment 28E (Fig. 2B). Plant introduction (PI) numbers and country of collection of diverse sorghum races used in A are: 1, Bicolor (PI147837, Ethiopia); 2, Caudatum (PI267459, India); 3, Caudatum (PI276837, Ethiopia); 4, Caudatum (PI570719, Sudan); 5, Durra (PI534132, Ethiopia); 6, Durra (PI248317, India); 7, Guinea (PI534070, Nigeria); 8, Guinea-Caudatum (PI533871, Nigeria); 9, Kafir-Caudatum (PI152621, Sudan). Sorghum breeding lines used are CS, CS8110419 (candystripe); and RR, red revertant. Grass species included in B are Sg, Sudangrass; Tr, Tripsacum; Te, Teosinte; M, Maize; W, Wheat; R, Rice; Su, Sugarcane; and O, Oats.

Multiple sequence alignment of a Cs1 predicted protein. Sequence alignment of 50 aa regions of proteins encoded by gene 2 of Cs1 (position 344–393), TNP1 of Tam1 (position 191–240; accession no. S23817), SOR-BAC, a hypothetical protein from sorghum (position 215–264; accession no. AAD27567), and TNPA protein of En-1 (position 293–342; accession no. AAA66268). Conserved residues are highlighted in black, and similar residues are shown in gray.