SUPPORTING ONLINE MATERIALS
Association between allelic variation at the Phytoene synthase 1 (PSY-1) gene and yellow pigment content in the wheat grain
W. Zhang and J. Dubcovsky
Supporting online Table S1. Wheat germplasm used in the PSY-1 allelic diversity study. HU: Haifa University, Israel collection (population numbers), PI numbers= National Genetic Resources Program, USA. PSY-B1: “K” indicates the hybrid ‘Kofa’ allele and “U” indicates all other alleles. PSY-A1: “Ins.” indicates the repetitive element in intron four and “No Ins.” the absence of that particular insertion. All Turkish accessions from T. turgidum spp. dicoccoides are from the Diyarbakir region (putative wheat domestication center). T. aestivum lines are parents for the mapping populations of the wheat CAP project (http://maswheat.ucdavis.edu/Mapping/index.htm).
Species / Germplasm No. / Origin / PSY-B1 / PSY-A1T. turgidum / HU 41 / Iraq / U / Ins.
ssp. / HU 42 / Iraq / U / Ins.
dicoccoides / HU 43 / Iraq / U / No ins.
HU 44 / Iraq / U / Ins.
HU 05 / Israel / U / No ins.
HU 07 / Israel / U / Ins.
HU 09 / Israel / U / No ins.
HU 19 / Israel / U / No ins.
HU 29 / Israel / U / No ins.
HU 8 / Israel / U / Heterozygous
HU 11 / Israel / U / Ins.
HU 23 / Israel / U / No ins.
HU 10 / Israel / U / No ins.
HU 30 / Israel / U / No ins.
HU 33 / Israel / U / No ins.
HU 24 / Israel / U / Ins.
HU 27 / Israel / U / No ins.
HU 17 / Israel / U / No ins.
HU 28 / Israel / U / No ins.
HU 20 / Jordan / U / No ins.
HU 1 / Lebanon / U / No ins.
Table S1. Continuation
Species / Germplasm No. / Origin / PSY-B1 / PSY-A1T. turgidum / HU 32 / Palestine / U / No ins.
ssp. / HU 40 / Syria / U / No ins.
dicoccoides / PI 428017 / Turkey / U / Ins.
(continuation) / PI 428020 / Turkey / U / Ins.
PI 428028 / Turkey / - / Ins.
PI 428036 / Turkey / U / Ins.
PI 428041 / Turkey / U / Ins.
PI 428047 / Turkey / U / Ins.
PI 428055 / Turkey / U / No ins.
PI 428058 / Turkey / U / No ins.
PI 428061 / Turkey / U / No ins.
T. turgidum / PI 94627 / Asia Minor / - / Ins.
ssp. / PI 352352 / Asia Minor / U / No ins.
dicoccon / PI 355454 / Asia Minor / U / No ins.
PI 94640 / Iran / U / Ins.
PI 254180 / Iran / U / Ins.
PI 254158 / Iran / U / Ins.
PI 347230 / Iran / U / Ins.
CItr 17675 / Lebanon / U / Ins.
PI 352347 / Palestine / U / Ins.
PI 352357 / Palestine / U / Ins.
PI 352367 / Palestine / U / Ins.
PI 355496 / Palestine / U / Heterozygous
PI 355498 / Syria / U / Ins.
CItr 17676 / Turkey / U / -
PI 182743 / Turkey / U / Ins.
PI 319868 / Turkey / U / Ins.
PI 319869 / Turkey / U / Ins.
PI 352329 / Turkey / U / Ins.
PI 470737 / Turkey / U / Ins.
PI 470738 / Turkey / U / Ins.
PI 470739 / Turkey / - / Ins.
PI 606325 / Turkey / U / Ins.
PI 94626 / Turkey / U / Ins.
Table S1. Continuation
T. turgidum / Durfort / France / U / No ins.
ssp. / Exeldur / France / U / No ins.
durum / Nefer / France / U / No ins.
Neodur / France / U / No ins.
Adamello / Italy / K / No ins.
Appio / Italy / U / No ins.
Appulo / Italy / U / No ins.
Capelli ph1c / Italy / K / No ins.
Cappelli / Italy / U / No ins.
Capitti 8 / Italy / U / No ins.
Ciccio / Italy / U / No ins.
Cirillo / Italy / U / No ins.
Colosseo / Italy / U / No ins.
Duilio / Italy / U / Ins.
Karel / Italy / K / No ins.
L35 / Italy / U / No ins.
Latino / Italy / U / No ins.
Messapia / Italy / U / Ins.
Ofanto / Italy / K / No ins.
Russello / Italy / U / No ins.
San carlo / Italy / U / No ins.
Saragolla / Italy / K / -
Trinakria / Italy / K / No ins.
Valbelice / Italy / K / No ins.
Valforte / Italy / K / No ins.
Valnova / Italy / - / Ins.
Varano / Italy / K / No ins.
Zenit / Italy / U / No ins.
Vitron / Italy-Spain / U / Ins.
Aconchi 89 / Mexico / U / No ins.
Altar 84 / Mexico / U / No ins.
Mexicali 75 / Mexico / K / No ins.
Inrat 69 / Tunisia / U / No ins.
Karim / Tunisia / U / No ins.
Khiar / Tunisia / U / No ins.
Colorado / USA / K / No ins.
Desert King / USA / U / -
Table S1. Continuation
Species / Germplasm No. / Origin / PSY-B1 / PSY-A1T. turgidum / Kofa / USA / K / No ins.
ssp. / Kronos / USA / K / No ins.
durum / Langdon / USA / U / No ins.
(continuation) / Maier / USA / U / No ins.
Produra / USA / U / No ins.
Rugby / USA / U / No ins.
UC1112 / USA / K / Ins.
UC1113 / USA / U / No ins.
UC1171 / USA / U / No ins.
UC1308 / USA / U / No ins.
UC1374 / USA / U / No ins.
Westbred 881 / USA / K / No ins.
Yavaros / USA / U / Ins.
T. aestivum / Caledonia / USA / U / Ins.
Cayuga / USA / U / -
CO940610 / USA / U / Ins.
GRN*5/ND614-A / USA / - / Ins.
Harry / USA / U / Ins.
IDO444 / USA / - / Ins.
Jagger / USA / U / Ins.
Jaypee / USA / - / Ins.
Louise / USA / U / Ins.
McNeal / USA / U / Ins.
NY18/CC 40-1 / USA / U / Ins.
P91193 / USA / U / Ins.
P92201 / USA / U / Ins.
Penawawa / USA / U / No ins.
Platte / USA / U / Ins.
Rio Blanco / USA / U / Ins.
TAM 105 / USA / U / Ins.
Thatcher / USA / U / Ins.
USG3209 / USA / U / Ins.
Wesley / USA / U / -
Supporting online Fig. S1. RFLP (restriction fragment length polymorphisms) using PSY-I as a probe and DNAs from Kofa (K) and UC1113 (U) digested with restriction enzymes NcoI, NdeI, NheI, NsiI, and SacI. Southern blots were washed under very stringent conditions (0.1 X SSC buffer at 70 °C).
Supporting online Fig. S2. PSY Protein sequence alignment. Tt= Triticum turgidum ssp. durum (TtPSY-B1k= Kofa = EU096092, TtPSY-B1u= UC1113= EU096093), Ta= T. aestivum (TaPSY-A1= EU096091 and TaPSY-B1= EU096094), Lp= Lophopyrum ponticum (EU096095), Os= Oryza sativa (AAS18307.1), Zm= Zea mays (AAR08445.1). Polymorphisms in the TtPSY-B1k Kofa sequence are highlighted in yellow and those in the TaPSY-B1 sequence in red. Conserved amino acids are indicated by a *.
**
TtPSY-B1k 1 MATTVTLLLGAASSPG-----DGAARDGVQCSRMLPRRRQQRPRWVLCSLKYGCLGVGEP
TtPSY-B1u 1 MATTVTLLLGAASSPG-----DGAARDGVQCSRMLPRRRQQRPRWVLCSLKYGCLGVGEP
TaPSY-B1 1 MATTVTLLLGAASSPG-----DGAARDGVQCSRMLPRRRQQRPRWVLCSLKYGCLGVGEP
TtPSY-A1 1 MATTVTLLLGAASSPGPAAG-DGAARDGFQCSRLLPKKKQQRPRWVLCSLKYGCLGVGEP
TaPSY-A1 1 MATTVTLLLGAASSPGPAAG-DGAARDGFQCSRLLPKKKQQRPRWVLCSLKYGCLGVGEP
LpPSY-E1 1 MATTVTLLLGAVSSPGPGAGLAAGDAGHHVSLHCSRLRARKRQPWVLCSLKYGCLGVGEP
OsPSY1 1 MAAITLLRSASLPGLSDALARDAAAVQHVCSSCLPSNNKEKKRRWILCSLKYA--CLGVD
ZmPSY1 1 MAIILVRAAS--PGLS---AADSISHQGTLQCSTLLKTKRPAARRWMPCSLLG--LHPWE
***** * * * ********** ******** **
TtPSY-B1k 56 GEAGTRSAASPVYSSLTVSPGGEAAVAVVSSEQKVYDVVVKQAALLKRQLRP---QQA-A
TtPSY-B1u 56 GEAGTRSAASPVYSSLTVSPGGEAAVAVVSSEQKVYDVVVKQAALLKRQLRP---QQA-A
TaPSY-B1 56 GEAGTRSAASPVYSSLTVSPGGEAAVAVVSSEQKVYDVVLKQAALLKRQLRP---QQA-A
TtPSY-A1 60 GEAGGRSAASPVYSSLTVSPGGDAAVAVVSSEQKVYDVVVKQAALLKRQLRPSQQQQQ-A
TaPSY-A1 60 GEAGGRSAASPVYSSLTVSPGGDAAVAVVSSEQKVYDVVVKQAALLKRQLRPSQQQQQ-A
LpPSY-E1 61 GEAAGRSAASPVYSSLTVSPGGDAAVAVVSSEQKVYDVVVKQAALLKRQLRPSQQQQQQA
OsPSY1 59 PAPGEIARTSPVYSSLTVTPAGEA---VISSEQKVYDVVLKQAALLKRHLRPQP----HT
ZmPSY1 54 AGRP----SPAVYSSLAVNPAGEA---VVSSEQKVYDVVLKQAALLKRQLRTP------V
* ** ** ** **************** ***** *****************
TtPSY-B1k 112 PPAVARELDAPRGGLGEAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
TtPSY-B1u 112 PPAVARELDAPRGGLGEAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
TaPSY-B1 112 PPAVARELDAPRGGLGKAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
TtPSY-A1 119 PPAVARELDAPRGGLGEAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
TaPSY-A1 119 PPAVARELDAPRGGLGEAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
LpPSY-E1 121 PPAVARELDAPRGGLGEAYARCGEICEEYAKTFYLGTLLMTEERRRAIWAIYVWCRRTDE
OsPSY1 112 IPIVPKDLDLPRNGLKQAYHRCGEICEEYAKTFYLGTMLMTEDRRRAIWAIYVWCRRTDE
ZmPSY1 101 LDARPQDMDMPRNGLKEAYDRCGEICEEYAKTFYLGTMLMTEERRRAIWAIYVWCRRTDE
******* *** ****** ** ** *************** ******* *** ***
TtPSY-B1k 172 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
TtPSY-B1u 172 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
TaPSY-B1 172 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
TtPSY-A1 179 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
TaPSY-A1 179 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
LpPSY-E1 181 LVDGPNASHITPQALDRWERRLEDLFAGRPYDMLDAALSDTITKFPIDIQPFKDMIDGMR
OsPSY1 172 LVDGPNASHITPSALDRWEKRLDDLFTGRPYDMLDAALSDTISKSPIDIQPFRDMIEGMR
ZmPSY1 161 LVDGPNANYITPTALDRWEKRLEDLFTGRPYDMLDAALSDTISRFPIDIQPFRDMIEGMR
** * ** *************************** **** * ***** *****
TtPSY-B1k 232 TDLKKARYTNFDELYMYCYYVAGTVGLMSVPVMGIAPESKATAESVYGAALALGLANQLT
TtPSY-B1u 232 TDLKKARYKNFDELYMYCYYVAGTVGLMSVPVMGIAPESKATAESVYGAALALGLANQLT
TaPSY-B1 232 TDLKKARYKNFDELYMYCYYVAGTVGLMSVPVMGIAPESKATAESVYGAALALGLANQLT
TtPSY-A1 239 TDLKKARYKNFDELYMYCYYVAGTVGLMSVPVMGIAPDSKATAETVYGAALALGLANQLT
TaPSY-A1 239 TDLKKARYKNFDELYMYCYYVAGTVGLMSVPVMGIAPDSKATAESVYGAALALGLANQLT
LpPSY-E1 241 TDLKKARYKNFDELYMYCYYVAGTVGLMSVPVMGIAPESKATAESVYGTALALGLANQLT
OsPSY1 232 SDLRKTRYKNFDELYMYCYYVAGTVGLMSVPVMGIAPESKATTESVYSAALALGIANQLT
ZmPSY1 221 SDLRKTRYNNFDELYMYCYYVAGTVGLMSVPVMGIATESKATTESVYSAALALGIANQLT
*********************** ******** **** ** *****************
TtPSY-B1k 292 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFKGVVTDKWRKFMKRQIKRARMFFEEAE
TtPSY-B1u 292 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFKGVVTDKWRKFMKRQIKRARMFFEEAE
TaPSY-B1 292 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIVKGVVTDKWRKFMKRQIKRARMFFEEAE
TtPSY-A1 299 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFKGVVTDKWRKFMKRQIKRARMFFEEAE
TaPSY-A1 299 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFKGVVTDKWRKFMKRQIKRARMFFEEAE
LpPSY-E1 301 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFKGVVTDKWRKFMKRQIKRARMFFEEAE
OsPSY1 292 NILRDVGEDARRGRIYLPQDELAEAGLSDEDIFNGVVTNKWRSFMKRQIKRARMFFEEAE
ZmPSY1 281 NILRDVGEDARRGRIYLPQDELAQAGLSDEDIFKGVVTNRWRNFMKRQIKRARMFFEEAE
****** **************************** **** ** ******** ***
TtPSY-B1k 352 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTRRAYVGKAKKVLALPVAYGRSLL
TtPSY-B1u 352 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKAKKVLALPVAYGKSLL
TaPSY-B1 352 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKAKKVLALPVAYGKSLL
TtPSY-A1 359 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKAKKVLALPVAYGRSLL
TaPSY-A1 359 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKAKKVLALPVAYGRSLL
LpPSY-E1 361 RGVTELRKESRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKAKKVLALPVAYGRSLL
OsPSY1 352 RGVTELSQASRWPVWASLLLYRQILDEIEANDYNNFTKRTYVGKAKKLLALPVAYGRSLL
ZmPSY1 341 RGVTELSQASRWPVWASLLLYRQILDEIEANDYNNFTKRAYVGKGKKLLALPVAYGKSLL
* **** *
TtPSY-B1k 412 LPYSLRNNQT*
TtPSY-B1u 412 LPSSLRNNQT*
TaPSY-B1 412 LPSSLRNNQT*
TtPSY-A1 419 LPYSLRNNQT*
TaPSY-A1 419 LPYSLRNNQT*
LpPSY-E1 421 LPYSLRNNQT*
OsPSY1 412 MPYSLRNSQK*
ZmPSY1 401 LPCSLRNGQT*
* 281 amino acids conserved among all analyzed species.
Supporting online Fig. S3. Pedigree analysis of the PSY-B1 allele from Kofa. The presence of the PSY-B1 hybrid allele is indicated by gray rectangles and its absence by white rectangles. NA indicates not available for testing. Numbers below the variety names indicate year of release.
Supporting information for Fig. S3 (and text Fig. 4)
Origin of the unusual level of polymorphism of the PSY-B1 Kofa allele
The DNA sequence identity between the PSY-B1 alleles from Kofa and UC1113 (95.7% for the complete gene) is unusually low for orthologous genes from two different wheat varieties. Comparisons between these two sequences with the PSY-B1 sequences from Chinese Spring showed 99.7% identity with the UC1113 sequence but only 95.5% identity with the Kofa sequence, indicating that the PSY-B1 from Kofa is the unusual one. Comparison with a partial PSY-B1 sequence from Kofa (DQ642439) from an independent laboratory (Pozniak et al. 2007) confirmed that the unusual PSY-B1 sequence from Kofa was not a PCR artifact.
A more detailed analysis of Kofa and UC1113 PSY-B1 sequences showed normal levels of DNA identity between the two varieties (99.9%) within the first 1,000 bp, but significantly lower identity (93.2%) in the last 2,000 bp. The presence of some polymorphisms characteristic of the A genome prompted a more detailed analysis of the distribution of polymorphisms between the PSY-A1 and PSY-B1 genomic sequences across the Kofa allele (Fig. 4). For the first 89 polymorphisms, the Kofa sequence corresponds to the B genome in all except one polymorphism (99%). However, among the 122 distal polymorphisms the Kofa sequences were identical to the A genome variant in 85 cases (70%) and to the B genome variant in only 37 cases (30%). These results confirmed that the unusually high level of polymorphisms detected for the Kofa allele is the result of the introgression of PSY-A1 sequences in the PSY-B1 locus.
We screened a wheat germplasm collection (Supplemental Table S1) using B genome specific primers PSY1_BF3 and PSY1_BR2 (Table 1). This pair of primers amplifies a 200 bp product in Kofa and a 217 bp product from all other PSY-B1 alleles (Fig. 1E). The 200-bp allele from Kofa was not detected in any of the tetraploid durum wheat progenitors (31 accessions of T. dicoccoides, 21 accessions of T. dicoccon) or the 17 accessions of T. aestivum included in our survey, but was frequent in the US and Italian durum germplasm (31%) (Table 2 and supporting online Table S1). The absence of the Kofa allele in the T. turgidum ssp. dicoccoides and T. turgidum ssp. dicoccon accessions analyzed here supports the hypothesis that the PSY-B1 Kofa allele originated in T. turgidum ssp. durum.
Pedigree analysis showed that the CIMMYT variety Mexicali 75 (1975) and the Italian varieties Trinakria (1970) and Valforte (1980) have the PSY-B1 Kofa allele (Fig. S3). Since these different lines share Cappelli ph1c mutant as one of the ancestral progenitors (Fig. S3) we analyzed DNA from the original Cappelli line (not mutagenized) obtained from the Ente Nazionale Sementi elette (ENSE), Milan, Italy (Maccaferri et al. 2003) and from the derived Cappelli ph1c mutant line obtained from a mutagenesis study (Giorgi 1978). Since Cappelli did not show the Kofa allele but Cappelli ph1c mutant did (Fig. 1E), we concluded that the Kofa allele originated during the mutagenesis treatment used to produce the mutant population or as a result of the ph1c mutation. Sequencing of PSY-B1 from Cappelli ph1c confirmed that this allele is 100% identical to the Kofa allele.
REFERENCES. Supporting Online Materials
Giorgi B (1978) A homoeologous pairing mutant isolated in Triticum durum cv. Cappelli. Mutat Breed Newsl 11:4-5
Maccaferri M, Sanguineti MC, Donini P, Tuberosa R (2003) Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm. Theor Appl Genet 107:783-797
Pozniak CJ, Knox RE, Clarke FR, Clarke JM (2007) Identification of QTL and association of a phytoene synthase gene with endosperm colour in durum wheat. Theor Appl Genet 114:525-537
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