Fig. S1Positions of degenerate and nested degenerate PCR primers within aligned-hydroxylase sequences. The Arabidopsis, wheat, maize and rice-hydroxylase sequences were aligned using ClustalX. Only the highly conserved regions among different sequences are shown. Primers used for the nested degenerate PCR are indicated by arrows. DF, forward degenerate primer; DR, reverse degenerate primer; NDF, forward nested degenerate primer; NDR, reverse nested degenerate primer

Fig. S2Verification of the specificity of wheat HYD1 and HYD2 gene- and homeolog-specific real-time qPCR primers. Genomic DNA extracted from nullisomic-tetrasomic and ditelosomic lines of hexaploid wheat var. Chinese Spring as well as plasmid DNA of each homeolog were used as template for PCR amplifications.N2BT2D (nullisomic for 2B and tetrasomic for 2D), N2DT2A (nullisomic for 2D and tetrasomic for 2A), N5AT5D (nullisomic for 5A and tetrasomic for 5D), N4DT4A (nullisomic for 4D and tetrasomic for 4A), DT2AS (the long arm of chromosome 2A is missing), DT4BS (the long arm of chromosome 4B is missing). Kronos and UC1041 are wild type tetraploid and hexaploid wheat, respectively. Two DNA ladders, 1 Kb (left) and 100 bp (right), were loaded on each gel. When these primers were used for real-time qPCR analysis, dissociation curves were drawn for each primer pair following qPCR reactions and single peaks were observed. The qPCR products were also analyzed on agarose gels and single products at expected sizes were detected

Fig.S3Functional characterization of wheat HYD1 and HYD2 homeologs in -carotene- and -carotene-accumulating E. coli. HPLC elution profiles of a pAC-DELTA, bpAC-DELTA + pDEST17-AtBCH1, cpAC-DELTA + pDEST17-GUS, d pAC-EPSILON, e pAC-EPSILON + pDEST17-AtBCH1, f pAC-EPSILON + pDEST17-GUS,gpAC-DELTA + pDEST17-HYD-A1, hpAC-DELTA + pDEST17-HYD-B1, ipAC-DELTA + pDEST17-HYD-B1splice variant, jpAC-DELTA + pDEST17-HYD-D1, kpAC-DELTA + pDEST17-HYD-A2, lpAC-DELTA + pDEST17-HYD-B2, mpAC-DELTA + pDEST17-HYD-D2, n pAC-EPSILON + pDEST17-HYD-A1, opAC-EPSILON + pDEST17-HYD-B1, ppAC-EPSILON + pDEST17-HYD-B1splice variant, q pAC-EPSILON + pDEST17-HYD-D1, r pAC-EPSILON + pDEST17-HYD-A2, s pAC-EPSILON + pDEST17-HYD-B2, and t pAC-EPSILON + pDEST17-HYD-D2are shown. pAC-DELTA and pAC-EPSILONwere transformed with pDEST17-AtBCH1 and pDEST17-GUS for positive and negative control of -ring hydroxylation activities, respectively. u-y, Absorption spectra of peak 1, hydroxylated -carotene, peak 2, mono-hydroxylated -carotene, peak 3, lycopene, peak 4, -carotene, peak 5, -carotene

Fig. S4Carotenoid profiles of mature wheat embryos. Embryos dissected from 100 mature tetraploid (var. Kronos) and hexaploid wheat (breeding line UC1041) grains were used for carotenoid analysis. HPLC elution profiles of a Kronos andb UC1041 are shown.c and dAbsorption spectra of lutein (lut) and zeaxanthin (zea)

Table S1 Primer sequences used for RACE PCR, cDNA and genomic DNA cloning, and nested degenerate PCR

Gene / Analysis / Direction / Sequence (5’-3’)
HYD1 / 5’ RACE / Reverse / CAAATCCTAATCCGCGATCAG
Reverse
(nested) / CATATAAATAATCCTCATTGCC
3’ RACE / Forward / CGTCTACTACCGATTCGCCTG
HYD-A1 / cDNA cloning / Forward / CACCATGGCCGCTGGTCTCTCGGG
Reverse / ATAGCAACCACCAAAAAGTAAC
HYD-B1 / cDNA cloning / Forward / CACCATGGCCGCTGGTCTCTCGGG
Reverse / TCACAAGGCTCTTCTAGCTGCA
HYD-D1 / cDNA cloning / Forward / CACCATGGCGGCTGGTCTCTCCGGCTCCACT
Reverse / CGGACGCGGTGAGAGAGA
HYD-A2 / cDNA cloning / Forward / CACCATGGCCGTCGCGAGGCTGGTG
Reverse / CAAATCCTAATCCGCGATCAG
HYD-B2 / cDNA cloning / Forward / CACCATGGCCGTCGCGAGGCTGGTG
Reverse / GCACCTCGCCTTCGTTTCC
HYD-D2 / cDNA cloning / Forward / CACCATGGCCGTCGCGAGGCTGGTG
Reverse / GCACGTCGCCTCCGTTTCT
HYD-A1 / Genomic DNA / Forward / CTCCGTCCCTGTCCATTT
HYD-B1
HYD-D1 / Cloning / Reverse / CCTTCTCTACTTGTTATACAC
HYD-A2 / Genomic DNA / Forward / ATGGGGGAAGCGGTGGCCG
Cloning / Reverse / AATCCGCGATCAGAAGAAGA
HYD-B2 / Genomic DNA / Forward / CGCCTCTCGTTCCCAGAG
HYD-D2 / Cloning / Reverse / TCACACCTCTGCTTGCTGATGTAC
HYD-A1 / Chromosomal / Forward / CTCCGTCCCTGTCCATTT
Location / Reverse / ATAGCAACCACCAAAAAGTAAC
HYD-B1 / Chromosomal / Forward / ATCCTTACCGTTAGTGAGTGGCGAATT
Location / Reverse / CTGCCTGATCATTAGCTCTG
HYD-D1 / Chromosomal / Forward / TCCGTCCCTGTCCATTTATATCT
Location / Reverse / CGGACGCGGTGAGAGAGA
HYD-A2 / Chromosomal / Forward / CCTTCCAATTTCCGCTCCTTTAC
Location / Reverse / CAAATCCTAATCCGCGATCAG
HYD-B2 / Chromosomal / Forward / TGGCAAATGGAGGTATTGTA
Location / Reverse / GCAAGGCAGGTAAGAGCTGG
HYD-D2 / Chromosomal / Forward / CCACTTCCCCAAATTTCGTCG
Location / Reverse / GCAAGGCAGGTAAGAGCTGG
HYD / Degenerate PCR / Forward / GCAAGCTTGGNATGGARTTYTGGGC
(Hind III site underlined)
Reverse / GCGCTCGAGAAYTTRTCNRTRTGRTG
(Xho I site underlined)
HYD / Nested degenerate / Forward / GCGGATCCAAYGAYGTNTTYGCNAT
(BamH I site underlined)
PCR / Reverse / GCGCTCGAGAACATRTANGCCATNCC
(Xho I site underlined)

Table S2 Primer sequences used for real-time qPCR analysis and sizes of the amplicons

Gene
/homeolog / Forward (5’3’) / Reverse (5’3’) / Amplicon
(bp)
HYD1 / ACCACATGGACAAGTTCGAGG / TTGATCCTGGCGAGCTCCT / 109
HYD-A1 / ACCACATGGACAAGTTCGAGG / ATAGCAACCACCAAAAAGTAAC / 230
HYD-B1 / CACATGGACAAGTTCGAGAGC / TCACAAGGCTCTTCTAGCTGCA / 169
HYD-D1 / GAGCTCGCCAGGATCAACC / CGGACGCGGTGAGAGAGA / 341
HYD2 / AAGTTCGACAGCGTGCCATAC / TCCTGATCCTCCTCTGCACCT / 103
HYD-A2 / GCCACCGACTTACCCTTTTTC / CAAATCCTAATCCGCGATCAG / 115
HYD-B2 / CAGAAATCAGACGCCATGTAG / GCACCTCGCCTTCGTTTCC / 190
HYD-D2 / CAGAAATCAGACGCCATGCAA / GCACGTCGCCTCCGTTTCT / 190
Ta54227 / CAAATACGCCATCAGGGAGAACATC / CGCTGCCGAAACCACGAGAC / 227
Ta2291 / GCTCTCCAACAACATTGCCAAC / GCTTCTGCCTGTCACATACGC / 165
LCY-E / GTTGGTCTCATTGGCCCTGA / CAATACAGCTCTCCAGGCCG / 91

Table S3Exon and intron sizes of wheat -hydroxylase homeologs

Exon (bp) / Intron (bp)
1 / 2 / 3 / 4 / 5 / 6 / 1 / 2 / 3 / 4 / 5
HYD-A1 / 387 / 60 / 207 / 126 / 57 / 75 / 142 / 78 / 129 / 118 / 163
HYD-B1 / 387 / 60 / 207 / 126 / 57 / 75 / 122 / 82 / 109 / 109 / 152
HYD-D1 / 387 / 60 / 207 / 126 / 58 / 119 / 142 / 78 / 126 / 108 / 152
HYD-A2 / 345 / 60 / 207 / 126 / 57 / 90 / 110 / 126 / 105 / 80 / 119
HYD-B2 / 360 / 60 / 207 / 126 / 57 / 87 / 81 / 134 / 243 / 80 / 100
HYD-D2 / 354 / 60 / 207 / 126 / 57 / 90 / 115 / 122 / 251 / 77 / 108

Table S4 Results ofthe nested degenerate PCR analysis. Genomic DNA extracted from diploid wheat Ae. tauschii and hexaploid wheat breeding line UC1041 was used as template for the PCR reactions. Numbers of clones that are identical to HYD1 or HYD2 homeologs as well as non-specific PCR products are shown. The non-specific PCR products generally have less than 10% sequence identity to HYD1 or HYD2 and do not contain the conserved histidine motifs that are common to plant carotenoid -hydroxylases

Genomic DNA template / HYD1 / HYD2 / Non-specific products / Total
Ae. tauschii (DD) / 12 / 2 / 4 / 18
UC1041 (AABBDD) / 35 / 17 / 12 / 68

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