Table 1.
Primers used in this study for CDS amplification and transcript accumulation.
Table 2.
Primers used in this study for genotyping.
Fig 1.
Maximum likelihood reconstruction of the complete set of maize (Zm), sorghum (Sb) and rice (Os) PHO1 proteins along with selected PHO1 proteins of Arabidopsis (At) and canola (Br).
The moss (Physcomitrella patens) protein PpPHO1-1 is shown as an outgroup. Bootstrap support shown as a percentage at the nodes.
Table 3.
MaizePho1 genes and correspondingArabidopsis and rice orthologs.
Fig 2.
Microsynteny among Pho1;2 loci A) Annotated genes (filled boxes) in the region ofSbPho1;2 on Sorghum chromosome 4 (SbChr4) and candidate orthologs on maize (B73) chromosomes 4 (ZmChr4) and 5 (ZmChr5). Orthologous genes are connected by dashed lines. Pho1;2 genes on the three chromosomes shown in unfilled boxes. Regions shown to scale, the right hand position indicated. SbChr4 and ZmChr5 run left to right, ZmChr4 right to left. B) Pho1;2 gene models on ZmChr4, SbChr4 and ZmChr5. Exons are shown as boxes, coding regions filled, UTR unfilled. Putative anti-sense transcripts associated with ZmPho1;2a and ZmPho1;2b shown as filled boxes (NAT). Angle brackets indicate the direction of transcription. The maize syntenic paralog pair GRMZM2G164854/GRMZM5G853379 and their sorghum ortholog are also shown. Triangles indicate the position of Activator and Dissociation insertion. Refer to Table 4 for full description alleles. Shown to scale.
Fig 3.
Accumulation of Pho1;2 sense and antisense transcripts under contrasting phosphate conditions.
(A) Schematic of Pho1;2 genes indicating the position of primers (solid triangles; see Table 1). (B) Amplification of fragments corresponding to the mature sense-RNAs of ZmPho1;2a, ZmPho1;2b and SbPho1;2 from oligo-dT primed cDNA prepared from roots and shoot of 10-day-old seedlings, fertilized with Hoagland solution adjusted to 1mM (+P) or 0mM (-P) inorganic phosphate. Primer pairs used for amplification indicated by letter codes on the right-hand side of panel. (C) Amplification of fragments corresponding to putative anti-sense transcripts encoded by ZmPho1;2a, ZmPho1;2b from cDNA as Panel A. Primary PCR (left) and nested PCR performed from 1:100,000 dilution of primary reaction (right). Amplification of ZmUBQ and SbUBQ fragments and amplification from genomic DNA template (G) were used as controls. Primer pairs indicated as in (B).
Fig 4.
(A) Genetic strategy to mobilize the donor element bti31094::Ac. Individuals homozygous for the donor element, displaying the characteristic 3 Ac dosage pattern of excision from r1-sc:m3 in the triploid aleurone, were crossed as females by T43. A small proportion (~2%) of the progeny kernels were finely spotted, indicating high Ac dose as a result of replicative transposition, and were screened for potential re-insertion into the target gene. (B) Two high Ac dose kernels (white arrows) highlighted among the largely 2 Ac dose progeny of a typical test cross ear.
Fig 5.
PCR screening for novel Ac insertion in ZmPho1;2a.
(A) Schematic of Zmpho1;2a-m1 allele. Reaction 1 (588bp expected product) and reaction 2 (2496bp) are indicated. (B) PCR reactions using gene and Ac specific primers to amplify potential products from DNA pools. Pool with a star shows amplification in complementary reactions 1 and 2. (C) PCR reactions using individual DNA. Amplification from a positive individual in complementary reactions 1 and 2 indicated with a star.
Fig 6.
Recovery of Ac excision events from Zmpho1;2a-m1. (A) Schematic of primer position and fragment lengths corresponding to Zmpho1;2a-m1 (m1) and derived excision events (m1.1). (B) Amplification of apparent wild-type (Wt) and flanking products (5’, 3’) from three different individuals genotypically homozygous Zmpho1;2a-m1 on the basis of kernel phenotype and pedigree. (C) Generation of a BseYI cutting site, not present in wild-type (W22), as the result of an insertion of 8bp following excision of Ac from Zmpho1;2a-m1. (D) BseYI digestion of products amplified in (B) from three genotypically Zmpho1;2a-m1 homozygous individuals and a wild-type (W22) individual. (E) BseYI digestion of products amplified with the insertion spanning primers MS124/MS052 from a wild-type individual (W22) and an individual homozygous for the stable germinal excision allele pho1;2a’m1.1. 1Kb plus DNA ladder (Invitrogen) is loaded on the first lane in B,C and E.
Table 4.
Transposon insertion alleles of maize Pho1;2 genes.
Fig 7.
(A) Genetic strategy to re-mobilize the Ds element ZmPho1;2b-m1::Ds. Individuals carrying ZmPho1;2b-m1::Ds and the stable transposase source Ac-im were crossed as males to T43. All progeny were screened as rare kernels carrying transposed Ds were indistinguishable from other progeny on the basis of aluerone phenotype. (B) Ear carrying kernels with one copy of Ac-im in the chr7 expressed in the triploid aleurone.
Fig 8.
Generation of stable footprint alleles by Ac excision.
(A) Genetic strategy to re-mobilize to recover stable germinal excision of Ac from ZmPho1;2a-m1::Ac. An individual homozygous for ZmPho1;2a-m1::Ac was crossed as male to T43. Rare colorless kernels were selected for PCR screening. (B) Colorless kernels (white arrows) highlighted among the largely 1 Ac dose progeny of a typical test cross ear.