1 Supplementary Information Appendix Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia Nestor Kippes, Juan M. Debernardi, Hans Vasquez-Gross, Bala A. Akpinar, Budak Hikmet, Kenji Kato, Shiaoman Chao, Eduard Akhunov and Jorge Dubcovsky* * correspondence to: [email protected]This file includes: SI Appendix, Methods S1 to S6 SI Appendix, Figures S1 to S14 SI Appendix, Tables S1 to S8 SI Appendix, References
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1
Supplementary Information Appendix
Identification of the VERNALIZATION 4 gene reveals the origin of
spring growth habit in ancient wheats from South Asia
Nestor Kippes, Juan M. Debernardi, Hans Vasquez-Gross, Bala A. Akpinar, Budak Hikmet,
Kenji Kato, Shiaoman Chao, Eduard Akhunov and Jorge Dubcovsky*
Figure S4. VRN-D4 protein sequence analysis. Multiple sequence alignment between the
predicted protein sequence of VRN-D4 and the publicly available VRN1 protein sequences.
Highlighted in red is the K123Q amino acid change characteristic of VRN-D4. The K-Box
domain is highlighted in light gray. T. urartu sequence was obtained from plants.ensembl.org
(scaffold60538). The complete Chinese Spring VRN-A1 (GenBank KR422423) and VRN-D4
(GenBank KR422424) sequences were obtained in this study.
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Figure S5. Conservation of amino acids affected by natural and induced mutations in VRN-D4. A multiple alignment of 268 sequences including AP1, AG, FUL, CAULIFLOWER, VRN1 and other related MAD-BOX proteins was used to generate a graphical representation of the conservation at each amino acid position. We used the WebLogo tool (weblogo.berkeley.edu) that generates a graph where the size of each amino acid letter is proportional to its frequency at that position. The region shown covers amino acids 115 to 175 (VRN-D4 coordinates) in the K-Box domain. Arrows indicate the natural amino acid change K123Q and the EMS induced amino acid change E158K. Note that the amino acids resulting from these mutations are not detected among the 268 closest available sequences for this protein domain.
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Figure S6. PCR amplification of VRN-A1 flanking genes from chromosome arm 5DS. DNA
extracted from flow-sorted 5DS chromosome arms arm (Institute of Experimental Botany,
Olomouc, Czech Republic) was used to test if genes flanking VRN-A1 on chromosome arm 5AL
(18) were also present in the 5AL segment inserted in chromosome arm 5DS. CS: Chinese
Spring, 5DS: 5DS arm DNA, TDC: Triple Dirk C. Using the same 5DS DNA sample for all
primers, only the VRN-A1 marker was amplified, indicating that PHYC, CYS and AGLG1 are not
present in 5DS. Multiple bands in CYS PCR amplification corresponds to multiple copies of
CYS genes, none of them present in the 5DS arm DNA. A BLAST search of the CS-dt5DS
database showed no genes with significant similarity to PHYC, CYS and AGLG1, confirming the
Figure S13. Fixation indexes along the 5D chromosome of T. aestivum spp. sphaerococcum.
FST values were calculated using 120 polymorphic SNPs mapped on chromosome 5D (17). The
arrow head indicates the predicted position of the centromere based on the arm location of the
SNP markers. VRN-D4 is linked to the centromere of the chromosome 5D. (The horizontal
dashed line represent the 95th percentile of the FST values on this chromosome = 0.57).
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Figure S14. Fixation indexes across T. aestivum spp. sphaerococcum chromosomes. Mean
FST values were calculated using a sliding window of 7 mapped SNPs (17) with steps of 4 SNPs
to generate smoother curves. The horizontal dashed line represent genome-specific 95th
percentile of mean FST values (A genome=0.53, B genome = 0.53, and D genome = 0.40).
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SI Appendix, Tables
Table S1. Analysis of natural and induced mutations found in VRN-D4.
Natural Induced mutations
mutations exon 4 exon 6 exon 7 exon 8
Line TDF Till-665 Till-526 Till-174 Till-481 Till278
GenBank KR422424
KR119063 KR119062 KR119061 KR119060
KR119059
nt. change A367C
G10511A G472A G538A G550A
G655A
aa. change K123Q
Splice site E158K A180T D184N
A219T
PROVEANa -3.527
- -3.292 -0.848 0.158
-0.306
PolyPhen-2b 0.999
- 0.999 0.045 0.026
0.002
SIFTc Not
tolerated -
Not
tolerated tolerated tolerated
tolerated
PSSMd 1
- -3
After
K-box
After
K-box
After
K-box
BLOSUM62 1
- 1 -1 1
-1
Nucleotide positions are calculated from the start codon in the VRN-D4 coding sequence, except for the G10511A
splicing site mutation that is indicated relative to the ATG in the genomic DNA sequence. Amino acid positions in
the protein are indicated relative to the initial methionine. The letter before the number indicates the wild type allele
and the letter after the number the mutant allele.
a PROVEAN scores were calculated at provean.jcvi.org. Scores < -2.5 are predicted to affect protein function. b Phylophen-2 scores were calculated using genetics.bwh.harvard.edu/pph2/. Values close to 0 suggest limited
effects on protein function, and values closer to 1 suggest more significant effects on protein structure and function. c SIFT was calculated at sift.jcvi.org and its scores are finally summarized into a tolerated or non-tolerated score. d Position-Specific Scoring Matrix (PSSM) scores are based on the conservation of amino acid positions among
multiple aligned proteins. Scores below zero indicate amino acid changes that are predicted to have significant
effects on the protein function. The PSSM score was calculated only for mutations within the K-box domain
(pfam01486) and the VRN-A1 protein sequence (AAW73221.1) at NCBI PSSM viewer
Table S2. List of BACs from the Chinese Spring 5DS arm library selected for sequencing. The BAC library was developed at the Institute of Experimental Botany (Czech Republic) and their nomenclature is followed. The sequence analysis confirmed that contigs CTG87 and CTG61 are connected.
No. in Figure 3 BAC Name Contig
1 TaeCsp5DShA_0045_O10 CTG87
2 TaeCsp5DShA_0085_P15 CTG87
3 TaeCsp5DShA_0056_K08 CTG87
4 TaeCsp5DShA_0076_N19 CTG87
5 TaeCsp5DShA_0038_M14 CTG87
6 TaeCsp5DShA_0036_J13 CTG87
7 TaeCsp5DShA_0082_O19 CTG61
8 TaeCsp5DShA_0043_I14 CTG61
9 TaeCsp5DShA_0035_M17 CTG61
10 TaeCsp5DShA_0058_E14 CTG61
11 TaeCsp5DShA_0086_K20 CTG61
12 TaeCsp5DShA_0039_M05 CTG61
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Table S3. Accessions sequenced for the VRN1 RIP-3 region. ‘PI’ and ‘CItr’ numbers are from GRIN identifiers, ‘UH’ numbers are from the University of Haifa wheat germplasm collection (21, 22), and ‘MG’ and ‘ IDG’ are from the University of Bologna T. dicoccum collection.
Wheat Location Identifiers
T. urartu El Beqaa, Lebanon PI 428275, PI 428280, PI 428282, PI 428283, PI 428285, PI 428286, PI 428287, PI 428288PI 428291, PI 428292, PI 428294, PI 428295, PI 428296, PI 428304, PI 428321, PI 428327, PI 538741, PI 428279, PI 538740, PI 428293, PI 428276
Urfa, Turkey PI 428222, PI 428223, PI 428240, PI 428244, PI 428251, PI 428239, PI 538733
Mardin, Turkey PI 428201, PI 428202, PI 428228
T. dicoccoides Beir‐Oren, Israel UH 28
Daliyya, Israel (south Haifa) UH 29
Diyarbakir, Turkey PI 428028, PI 428041
Gamla, Golan Heights UH 08
Iraq UH 41, UH 42
J'aba, Mid‐north Israel UH 23
Kokhav‐Hashahar UH 19
Mt. Gerizim, Israel UH 17
Rosh‐Pinna UH 09
Seryia UH 40
Golan Heights UH 11
Yabad, West Bank UH 32
Yehudiyya UH 07
T. dicoccum Armenia MG 5274
Ethiopia MG 5306
Georgia MG 5314, MG 5315
Germany MG 5398
Iran MG 5273, MG 5275, MG 5276
Italy IDG 8634
Kenia MG 3428, MG 3430
Palestine PI 355496
Russia MG 5269, MG 5270
Turkey PI 94626
T. aestivum Canada Norstar (CItr 17735), Fife (PI 283820)
Argentina Barleta (CItr 8398)
Australia Falcon (PI 292578), Golden drop (PI 92399)
Sweden Kronen (PI 278526)
United States Little club (CItr 4066), Sterling (CItr 17859), Finch (PI 628640), Alpowa (PI 566596), Louise (PI 634865), Eltan (PI 536994)
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Table S4. Geographical location of accessions postulated to carry VRN-D4 in previous studies. The presence of markers for the 5AL/5DS insertion borders, and the RIP-3VRN-D4 is indicated by a “+” and its absence by a “-”. VRN-A1 copy number variation (CNV) is described in Material and Methods. VRNA1 tandem duplication was detected as the presence/absence of a C/T double peak at the position 349 of the VRN-A1 coding region as described before (23). TDF, GABO, CS5402 and TDC were included as controls. Name of lines correspond to the previously published studies (24, 25). Names starting with ‘N’ correspond to the Institute of Cytology and Genetics (Novosibirsk, Russia).
Name Upstream
border
Downstream
border
VRN-D4
SNP A367C RIP-3 VRN-D4
CNV VRN-A1 tandem
duplication [*] Location
TDF + + A/C + 2 absent
GABO (N6827) + + A/C + 3 absent Australia
CS5402 - - A (+)1 1 absent
TDC - - A - 1 absent
CL035 - - A (+)1 1 absent China (south east)
IL193 - - A (+)1 1 absent Ethiopia
CL030 - - A - 2 present China (south east)
IL346 - - A - 2 present Afghanistan (west)
IL165 + + A/C + 3 absent Egypt
IL430 + + A/C + 2 absent Pakistan (north)
SS23 + + A/C + 3 absent Australia
IL027 + + A/C + 2 absent Afghanistan
IL047 + + A/C + 2 absent Turkey
IL114 + + A/C + 2 absent Nepal
IL154 + + A/C + 4 absent Pakistan (south)
IL163 + + A/C + 3 absent Egypt
IL213 + + A/C + 2 absent Nepal
IL216 + + A/C + 2 absent Nepal
GR007 + + A/C + 2 absent India
GR014 + + A/C + 2 absent India
GR015 + + A/C + 2 absent India
GR023 + + A/C + 2 absent India
IL164 + + A/C + 3 absent Egypt
IL175 + + A/C + 3 present Bhutan
IL176 + + A/C + 3 present Bhutan
IL008 + + A/C + 3 present Nepal
IL431 + + A/C + 4 present Pakistan (north)
GR001 + + A/C + 3 present India
N7451 + + A/C + 2 absent
N6721 + + A/C + 3 present
1 These accessions carry the RIP-3VRN-D4 SNPs in the VRN-A1 gene.
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Table S5. VRN1 and VRN-D4 spring alleles present in the T. aestivum ssp. sphaerococcum accessions used in this study.
1VRN-A1a (26)= duplication in promoter region. VRN-B1b (27) & VRN-B3 (= FT-B1) (28) = retroelement insertions in promoter. VRN-A1c, VRN-B1a and VRN-D1a (29) = intron deletions. Names starting with ‘PI’ or ‘CItr’ correspond to identification numbers of GRIN and names starting with ‘K’ to the Vavilov Institute of Plant Industry (St. Petersburg, Russia). 2 VRN-D4 tested by markers for borders of 5DS/5AL insertion, A367C SNP, and RIP-3VRN-
D4sequence. 3 Early expression of VRN-D4 in the leaves of one-week-old unvernalized plants: RT-PCR products were sequenced and only the VRN-D4 sequence was detected. NT= not tested.
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Table S6. Description of T. aestivum accessions used for the population structure analysis. PI numbers correspond to the Germplasm Resources Information Network (GRIN) identifiers (www.ars-grin.gov/npgs) and ‘K’ numbers correspond to the Novosibirsk Institute catalog numbers. T. aestivum spring and winter lines were described before (17). The last two columns indicate the identification numbers used in Fig. 5B and SI Appendix, Fig. S11.
Table S7. Average genetic diversity by genome for five T. aestivum subspecies. A total of
14,263 mapped SNPs were selected from the 16,371 polymorphic SNPs identified in the 90K iSelect Illumina SNP chip (17) to test the genetic diversity of each genome.
No. of SNPs sphaerococcum macha spelta aestivum* compactum*
A genome 5675 0.202 0.216 0.226 0.388 0.390
B genome 7344 0.243 0.189 0.215 0.388 0.382
D genome 1217 0.269 0.156 0.283 0.385 0.354
*SNPs were developed in T. aestivum ssp. aestivum. Therefore, the higher diversity observed in this subspecies and in the related T. aestivum ssp. compactum, can be influenced by ascertainment bias. These numbers were used only to adjust average diversity values per chromosome and to provide standardized diversity values.
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Table S8. PCR primers and RNA-probes used in this study.
* Lowercase letter indicates a SNP introduced to increase PCR specificity ** Primers for genomic DNA were design to be VRN-A1 specific 1, this primers amplifiy both VRN-A1 and VRN-D4 transcripts that are then differentiated by BstNI difgestion.
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