Article title: A high-density genetic map of the Medicago truncatula major freezing tolerance QTL on
chromosome 6 reveals colinearity with a QTL related to freezing damage on Pisum sativum linkage group VI
Journal name: Molecular Breeding
Author names: Nadim Tayeh . Nasser Bahrman . Rosemonde Devaux . Aurélie Bluteau . Jean-Marie Prosperi .
Bruno Delbreil . Isabelle Lejeune-Hénaut
Corresponding author: Bruno Delbreil
Affiliation: Université Lille 1, UMR 1281 SADV, Bâtiment SN2, F-59655 Villeneuve d'Ascq Cedex, France
e-mail address: bruno.delbreil@univ-lille1.fr
Online Resource 2. Supplemental methods
PCR amplification
PCR reactions were performed in a final volume of 15 µL. For a single reaction, mix was composed of 1.5 µL
PCR buffer 10x (Applied Biosystems, Forster city, California, USA), 2.5 mM of MgCl2, 0.5 µM of each of the
forward and reverse primers (Sigma-Aldrich, Suffolk, UK), 0.25 mM deoxyribonucleotide triphosphates
(Invitrogen, Carlsbad, California, USA), 0.5 U of AmpliTaq DNA polymerase (Applied Biosystems) and 10 ng
of genomic DNA template. Two different thermal cycling protocols were employed using PTC-200 DNA engine
(MJ Research, Watertown, USA): i) touchdown protocol : 94°C for 4 minutes followed by 5 cycles of 94°C for
30 seconds, 60°C(-1°C/cycle) for 30 seconds, 72°C for 30 seconds and 30 other cycles of 94°C for 30 seconds,
55°C for 30 seconds, and 72°C for 30 seconds followed by a final elongation step at 72°C for 10 minutes; and ii)
three-step protocol: 94°C for 4 minutes followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds and
72°C for 30 seconds followed by a final elongation step of 10 minutes. Negative controls without DNA template
were included for all PCR reaction mixtures. PCR products were analyzed on agarose gels.
PCR product sequencing
Sequencing reactions were carried out in a final volume of 10µL, containing each 1.5 µL Ready Reaction mix
(Applied Biosystems), 1.0 µL BigDye Terminator v3.1 Sequencing Buffer (Applied Biosystems), 0.32 µM
forward or reverse primer (Sigma-Aldrich) and 10 to 40 ng of gel-purified PCR product. The following thermal
cycler conditions were applied: step 1, 96°C for 1 minute and step 2, 25 cycles of 96°C for 10 seconds, 50°C for
5 seconds and 60°C for 4 minutes. Five µL of ethylendiamintetraacetate (EDTA; 125 µM) and 60 µL of pure
ethanol were added to each PCR reaction product. After 15-minute incubation at room temperature, a
centrifugation at 3000xg for 30 minutes was realized. DNA pellets were resuspended in 20 µL of formamide Hi–
Di (Applied Biosystems) and run on a 3130xl Genetic Analyzer (Hitachi/Applied Biosystems).
Agarose gel electrophoresis
Electrophoresis were carried out on gels composed of 1.2% type D5 DNA-grade agarose (Euromedex,
Mundolsheim, France) dissolved in TBE (0.45 mM Tris, 0.45 mM Boric acid, 1 mM EDTA ; pH 8) and
containing 0,5 mg.L-1 of ethidium bromide (BioRad, Hercules, California, USA). PCR products (10 to 15µL)
were mixed with 5 µL of “loading dye” (20% Glycerol, 5 mM EDTA, 0.12%, xylene cyanole, 0.12%
bromophenol blue; pH 8) before being loaded on gel. Smart Ladder (Eurogentec, Liege, Belgium) was used as
molecular weight marker. Migration was performed in TBE at 130 volts for 1 hour. Agarose gels were visualized
by ultraviolet light using GelDoc system with Quantity One software (BioRad).
Capillary gel electrophoresis
Capillary gel electrophoresis were performed on a 3130xl Genetic Analyzer (Hitachi/Applied Biosystems) using
a 36-cm Capillary Array (Applied Biosystems), POP-7 Polymer (Applied Biosystems) and GeneScan™—500
LIZ™ (Applied Biosystems) as internal size standard. PCR reactions were conducted as described above with
the exception that at least one of the primers was labeled with FAM or HEX fluorescent dyes. For each
electrophoresis run, 1 µL of PCR product was mixed with 10 µL formamide Hi–Di (Applied Biosystems) and
0.1 µL 500LIZ. The following parameters were applied: oven temperature, 60°C; injection voltage, 1.2 Kv;
injection time, 18 seconds; and run time, 850 seconds. Data were analyzed using Genemapper software version
4.0 (Applied Biosystems).
High Resolution Melting technique (HRM)
PCR reactions and HRM analyses were performed using Rotor-Gene 6000 (Corbett Life Science, Sydney,
Australia) thermal cycler. PCR were carried out in a 15 µL final volume containing: 7.5 µl of Lightcycler 480
High Resolution Melting Master (Roche Diagnostics, Mannheim, Germany), 2.5 mM of MgCl2 (Roche
Diagnostics), 0.2 µM of each primer and 10 ng of DNA template. Amplification conditions consisted in a
denaturation step at 95°C for 5 minutes followed by 50 three-step cycles of denaturation at 95°C for 30 seconds,
annealing at 60°C or 55°C for 30 seconds and elongation at 72°C for 30 seconds and a final elongation at 72°C
for 5 minutes. High resolution melting analyses were run using the following program: 1 minute at 95°C, 1
minute at 40°C followed by temperature ramping from 65 to 93°C with 0.05°C/2 second rise. Normalization of
the melting curves and data analysis were achieved using Rotor-Gene Q Series Software version 1.7 (Qiagen,
Hilden, Germany).
Competitive Allele Specific PCR method (KASPar)
Allele-specific and common primers for KASPar genotyping were designed using a tool provided by
KBiosciences (Hoddesdon, UK). Allele-specific primers were differentially labeled using fluorescence dyes
FAM and VIC. PCR reactions were carried out in a final volume of 4 µL composed of: 2 µL KASPar 2X
Reagent Mix (KBiosciences), 0.4 mM MgCl2 (Kbiosciences), 0.165 µM of each forward primer, 0.4125 µM
reverse primer and 20 ng of template DNA. Thermal cycling was performed on a Veriti 384-Well Thermal
Cycler (Applied Biosystems) as follows: denaturation at 94°C for 15 minutes, followed by 10 cycles of 94°C for
20 seconds and 65°C (-0.8°C/cycle) for 1 minute, and 40 other cycles of 94°C for 20 seconds and 57°C for 1
minute. After amplification, fluorescence was read using LightCycler480 (Roche). Data were analyzed using the
LightCycler® 480 SW 1.5 software (Roche).
Online Resource 3. Genetic linkage map of M. truncatula chromosome 6 with increasing marker density (a to c) in Mt-FTQTL6-containing region.
Maps were constructed using genotype data from the LR3 population.
a- framework linkage map of M. truncatula chromosome 6 composed of 5 AFLP [A], 5 RAPD [R] and 4 SSR [S] markers (Avia et al. submitted);
b- chromosome 6 linkage map enriched with 13 BAC-derived markers (in blue) located on both sides of EST-SSR marker MTIC153 corresponding to Mt-FTQTL6 peak
(NT6005 and NT6044 are 8.3 cM distant);
c- high-density linkage map of Mt-FTQTL6-containing region comprising, in addition to the BAC-derived markers, 12 synteny-based markers (in red) located close to
MTIC153 (NT6048 and NT6055 are 2.1 cM distant).
Online Resource 5. Comparison of the order of BAC-derived markers on M. truncatula chromosome 6 linkage map with that of corresponding BAC clones on M. truncatula
genome assembly versions, Mt3.0 and Mt3.5.
Light to dark thick green lines refer to BAC clones in phases 1 to 3, respectively. BAC clones are named according to their respective NCBI GenBank accession numbers.
Each BAC contig is represented by a colored box, shown above or below BAC clone(s) composing it. Spaces between colored boxes refer to assembly gaps between different
contigs.
For comparison, the order and orientation of BAC contigs spanning Mt-FTQTL6 boundaries from both genome assembly versions Mt3.0 and Mt3.5 are shown. Genes or
genomic regions corresponding to BAC-derived markers are indicated in grey. Gene identifiers are different following assembly version’s respective annotation. Dotted lines
link genes/genomic regions to corresponding markers on partial linkage map of M. truncatula chromosome 6 obtained from LR3 population. Large arrows show putative
location of Mt-FTQTL6 confidence interval.
Online Resource 6. Interval mapping LOD profile (right) for M. truncatula chromosome 6 linkage map (left) using freezing tolerance data from Avia et al. (submitted).
BAC-derived markers are highlighted in blue color. Co-segregating markers were excluded because they have no effect on QTL detection. Dashed black line shows the
significance threshold (LOD = 1.8) estimated by permutation test for chromosome 6.
The position with the largest LOD on the chromosome coincides with marker MTIC153. The 95% confidence interval of Mt-FTQTL6 is located between markers NT6001
and NT6019, separated by 3.7 cM.
a
b
Online Resource 11. Agarose gel electropherograms of PCR products from different legume cultivars/accessions obtained using primers corresponding to markers
NT6058bis (a) and NT6066 (b). NT6058bis is an intron-spanning marker and NT6066 is an exon-derived marker.
Lane 1 contains the DNA ladder (SmartLadder). Lanes 2 to 24 contain PCR products from the following cultivars/accessions: Medicago truncatula 'F83005-5' (2); Medicago
truncatula 'DZA045-5' (3); Medicago sativa subsp. sativa cv. Magali (4); Medicago sativa subsp. falcata cv. Krasnokutska (5); Trifolium pratense cv. Divin (6), Trifolium
pratense cv. Pirat (7), Pisum sativum 'Champagne' (8), Pisum sativum 'Terese' (9), Lens culinaris cv. Goltandslins (10), Lens esculenta puyensis var. Anicia (11), Vicia faba
cv. Hiverna (12), Vicia faba cv. Mandoline (13), Cicer arietinum 'W6 25998' (14), Cicer arietinum 'W6 26001' (15), Vigna unguiculata (16), Phaseolus vulgaris var. vulgaris
cv. Bonita (17), Phaseolus vulgaris 'Talisman' (18), Glycine max '1177-2-18-33' (19), Glycine max '1236-4-6' (20), Lupinus albus cv. Lumen (21); Lupinus albus cv. Amiga
(22); Lupinus luteus 'PAC114' (23); Lupinus luteus cv. Sulfa (24). Lane 25 contains negative control (without DNA).
Online Resource 12. M. truncatula and P. sativum marker sequences obtained by PCR product sequencing.
Allelic variations between F83005-5 – DZA045-5 and Champagne – Terese – Eden are shown. A color code is
used for this purpose. Sequences from each species are grouped together. PCR product sequences from
polymorphic markers are listed before those from monomorphic ones.
M. truncatula [F83005-5 / DZA045-5]
>NT6003
TTTACAGGTATCTGCGGTCCCACATATATTATGCATGATATAAATGAAACAGATAGAAATACAAAG
CCATTATTATTTGTTGTACAGTTGTAATAGAAATT[T/C]TGTATTTCATAATTTAGAATTGTCAATAT
AGGATGATGAAATATTAACTAGGTTTAGTGAGAAGGGTTAGAAGTACCTCATGTAAAGGAAGGTT
CAGTCCAAGTTC[A/G]GACGAAGTTAATCGCAAATTGGCGAATCGTCGTAAATCTC
>NT6005
GATTATGAGAGAAGTTCAAATAAAAAGCATTAGAAAATGTTTCCCAAAACCAAAGTGG[T/G]ATGA
AATCTGTTATACCTGTATTAGATATGTCCAAA[T/G]AATGAAGATCCTTTTGAGTTTGAA[T/_]CCAC
GAC[G/C]GAAAGTTATGGCCGATTTGCCACGAGCTCATTCCCAATTCGTATAGCTGAAAACTAGGA
TGCCAATTGGAACCAACTTCTAAAGTGAGATTGTTCTCTGATGCATAACAATA[T/C]TGTAAACTCG
TAAGATTTGCAAGATCATCTTCCTTGACAATTCCTTGAAATAGATTTCTTTCAAGTACAAGAACCGA
CAGTTTAGAAAGTGGTCTAAGAATTTCAAATGGATTTCCACTTAATTGGTTTTTATAAAGACCAAG
AAATTGTAATGAAGAAAGATTTCCAAATGATCTA
>NT6009
CCATTCGATCAGGATGACTGGGAGCACTAT[T/G]CAAAATTAACAGCTGAAATGGGTCAGCAAGTA
CAGATTGTTGGTGATGATCTCCTTGTTACCAACCCAAAGGTATGCTTTATTAACTTCTAACTTGGGA
GTGTTCCACAAACTTGTAT[A/C]CTT[C/T]TGTGTTTTTAACATTATTCTGTCCATTATTGGCTCCTTA
GCGTGTGGAAAAAGCAATTAAGGAGAAGTCTTGCAATGCCCTTTTGTTGAAGGTAAATTTACCTTC
AGCCGTAAATTTTTATTTACTAGTG[A/G]AAGAAGAGTTCCGTCTTAATGAACTATCCTTTTTAATTC
TCTATCAAATCTGATGAGAATTTTCTTTATAACCTATTTGATAGGTGAATCAAATTGGCAGTGTAAC
TGAAAGTATTGAAGCT
>NT6012
CCTTTTTGCTCAAACAGCTCATCCAAATTCATCTTCGGAACAATAGCACGAGCCACTACAACAACA
ACAACAAAGATATTGATAAGGTGACTCACTGATATGAGAAAGTGACACAGGGATCTGAGTGATAA
ATC[A/G]GTCATGAATTCAACCTACTCAACTGACAAGTAATAATAAACAATATTCATCGATCATGAT
TACAAAAAATATAAGCACGCAGATAAACAACCAAAATTATAAACAAAGACAAGAGTAAAACACG
AACCATCAAAAACATAAGCCTGGATCTGTTCCTGTGGGTTTTGCAACTCATAA
>NT6014
AGCTCTGCATTCAAGCTGGCGTGGATATCCCGGAGTATCCATCCCGCCGAAGGACCAATCCCATAC
GAATGGTAGGGAGAAGAGTACTTGACAGAGGTGGACATCTAGAGGAGCCTAAGCCATTG[C/T]GC
ACTGCAGAGTTCTCTTCTGTCATCGACTTTGATACTTACCGAGCTTGCGAGCGATTTCCCCCTCCAC
CGTTATCAGAC[G/T]TACCTAAAATTGCTCAAGAAACTATCGATGCATATCAA[A/T]CTGTTAGAAA
GGGTGTGAGGAAGTTGATGAAGAAATACACCGTGAAAGCATGCGGTTATTGCTCGGAGGTTCATGT
GGGACCTTGGGGTCACAATGCAAAGCTTTGTGGATCATTTAAACATCAATGGAGAGATGGAAAAC
ATGGTTGGCAAGATGCTACTTTAGATGAAGTTTTACCTCCAA
>NT6034
GTTGAATGTTTTTCGTCAATTTACTTGCTTCTTTCTGATCA[T/C]GGTCATGTTTGGATAAGACGCTT
AATTAAGCACTTATAGCATAAACATTTATCGTATAAGTGTTTTTCAAA[T/A]AAGCTATTCTAGAGA
GCTTATGGAAA[C/T]AAGCTTAAA[T/A]CAATGTATGGACTTGTCATAAGCTGTTTCGATAAGTCAA
TCCAAACATGATTAGTCTAATCTAGTGTATTAGCACCATAACTAATGGTTCTATCTTTTTCTTTTTGT
TGCAGAAAAGGTCCATTTTTCAAAATC[G/T]TTCTTGCTGTTGCAATTTCTCTTTTGACATCATGCTG
T
>NT6040
GGGCAGATATTAGTATCCAGGTAAAATTGTCATTTGAAAGTGTTTTGTTGTTGTACAATGTTTTTTA
TCAGTAGGACAGGAGTCAAACTCGGTACCCCTAGAGTTTACAATACATACACACACTGCGCGCTAG
ACCCT[G/A]GTGGTGCTTTGTTGTGCAACTGATTGTTATGATGAAATTATTATTTTCTACCATTGTTT
TTATCTCTTTTATTTTACATCTAAAAAAGTTTTAAGTTTGATATACTGTCAATTTAAAACTTCAGTTA
ATTCTTTCCTTCTTTTTAAATTTCACAAAATTTAGCCGCCTGGTGATTGATAGCTTCTATAGTATTTA
TTGACTGTAGATATATCTCGTGGAT
>NT6041
TGCATGT[G/T]TTCATTTTCATTG[T/C]TATGAACACAATTCAAACTATTTTGAGGAAATAACAGAAA
TATCATGGCATTCAGAGTTTTGAATTATTTGCAAAACAACTAAACTTTATTGCATTGTTGAATGATT
GTTTCATATTAGT[A/C]TTTACTATTTAGTATGTCCGGTCTTTGTATATGTGTATCACTTTATGTAT[A/
G]GGCTT[C/T]TTAGATGACTTGAAATTCTTTTATTTAATATTTTTAAAACTTGGTGTTTCTAAAGTTT
TTGTA
>NT6044
GCACAATTTCACATCAATAGAAGATTTCAGAGGGTATGATTT[A/C]AAAACTTATAAATACATCCCT
TCCAATTTTCTCATGCAAACAATAAAGAAAAACATGTTGAATTTTTCTCTCCCCTTTATTTATTTTTA
AATTTTTCTGAATAAAAATCACAGGGCCTCTCTTCAGTATTTTACTACACACACTGACTTGGTGCAT
AGGCAGCAAGAAGCAATTAAGCAAAGGAAAGCCATAAAGAAAGGCTTGCAATCTGATAAAGACT
GGACAGGAGATGGTTT
>NT6048
ATGTTCTTTGGGTTGTATCTCTTGTACTATTGATTGCTGGGT[G/A]AGTTCCTCTCTCGAATCCTCATT
AGCAACATAACTGTTAGGGTCAAGCCTTGTTCAGCAAAGTAGAGGGAGGGAATTAATCTTAAACTA
ATATCTTGTTTAGAACTAATCATGGTTCTGTTCAGGAAGGAAAGCCAAACTGATCTGAACCATTAA
ACCAGTTTGGTAAAACTGAACTGTTAATGATT[_/CATCTCGAACCAACTGAATTTTATAAAGCTT
TGATGCACTGGTTTTGTGTTTCTGAACCTAACCTGAACTACTTTTACAGCAAACTGAACTATC
CCAAATTGCTGTTATTAAATGATTATATTAGGACTATTATGAAAACCAAAAACTTAACTGAAT
ATTGGGACTATAAAAAAATTAAAACCGAACTGCACCGAAAAGAATCAAACTGTGAAAGATAG
TTCACAAGCTGAACAAAACTGAACTCGTGAACTGCCGTCACAGT]TCAGTCTTTTC[A/C]GTGTA
TTTTGGTTTAACGGTTTAGTTTCTGGTTATAACAGTCCAACT[C/T]TGTGTTACAACAC[A/G]TGTCTT
GATAGAATACCACGTGCTAGTTAAATAAAGGAAGTAATGACATGTGAATGAACA[G/A]AACACAAT
TT[T/_]CTTAGTAATGGATAGATAGGTAGATAGATTATGTTTCATTTGTTATATCTAATC
>NT6048bis
GCTGGAATCATTCATTTGGCAGCAAACATGATCAGTCTGGTCTTCATTGGAATTCGCCTCGAACAA
CAATTTGGGTTCGGTATGTCTCTATCTTTATCATTTGTTCATTGAAGCAGTTCCTTTCAGTGTTATCC
ATTGCGGATCGTGGAAAATAGCGGCTTGTTCAAATTCCGCTACACTATAGTGACACTATAGATGCT
GTTTGACATCATTTTGTATTAAATAACGTATTGCGGAACAATA[C/G]AGACATGTTCAAATT[AA/CC
]GCTTCACTATAGCCCCGCTATTTGACAACATTGGTTCCATTTAGTGAATTTTACACAAACATAGGA
AT[CA/GG]AGAATTTTATACCTATCTGCAAGCTTATTTACCTGATGAATTTATGTGTTCAAGTTCTGC
ATTATAGTGTTGTGACCAAAA[AAA/TTT]TT[A/T]TTGGAAATTTGCAGTGAGGATAGGAATCGTTT
ACCTAGTATCTGGATTC
>NT6051
CTGCTTTTATAGGCAAGGTATAATTTTAAATAAGT[T/C]TCTGTTCATTTAGTTTGTTATTTGATTTCT
TCATTTTCTTGTTACAATCTATTTGCACATCTTCATGATTGTTTTC[A/T]TGTTTCAAAACTGAGATTC
TCGATTAGTTCTATTGTATAATGTAGTAAGCTAATTTTTTGTGTCAAATTTGTATAGTTAACCATTTT
TCTTTTGTCATTTGATTTATTATGACTTGAATCTTGAAATATTTTGATGTTCGTGTCTGATTCATAAC
AAGATTCGATTCATGACTTAAAAATAGGTCTTCGGATTCACAATT[C/T]GAAACTAGATTTGATATT
GAAAATCATATCTTATTTCCTTATTATCTCTGTATTAGGTTGGAGCAGATGAGTTTGGTTACATGTT
GGCTGA
>NT6051bis
CG[G/C]GAGTTCTTGTTTTTTCGGAATCCAAGTGCTGATATGCTTCTTCACGAATCAGAACTTGATAT
AGATCT[T/C]TTAAAGAAGGTATTTTGAATTTCATTTTTTTATTTCATGAAATGTGGTTCGAGACCTA
TGGTTTAATTTATGATTTTGATGAT[G/A]ACATTGATTTTTCAGGCTAGAATTTTCCATTATGGATCA
ATTAGTTTAATTGATGATCCATGCAAATCAGCACACATTGCTGCTATGAGG
>NT6054
TTTGAAGACTTTGCTTCTTCATGGTGGAAAAGATTCCGAATTTGAGTCAGCAATGAGAAAAAGAAT
CATGAATTATCTTAACATATCGGAAAACGATTACTTCATGGTTTTCACTGCTAATAGAACATCGGCT
TTCAAACTTGTAGCAGATTGTTACAATTTTCAAAAAAGTAGAAAGCTTTTAACAGTTTATGACCATG
AAAGTGAAGCTGTGGAAGCGATGATTAGCTCGTCCGAAAAACGAGGAGCAAAAGCT[C/A]TGTCA
GCAGAGTTTTCATGGCCAAGGCTGAGAATTCAATCAACAAAGTTGAGGAAAATGATTGTAAGTAA
GAGAAAGAAGAAGAAAAATAGGGGTCTTTTTG
>NT6055
ACCTCATGCTTTGGAGTCCTTTGATGTTGGTTATATTCAGGTACTTTTTATTTAATTAATTAAATAAT
TAATTTTTCTTGTAATAATCTATAACCCTTTGTTTAGTTTTTATCTTTTTTTCAAAGTTTTGTTAATTA
ATATGTTGGAGCTGTTTTTGTTTTGTTTGTGATGTGAAGAACAATGTTTT[G/A]TTTTTGTTTTTT[_/T]
GTGTGTTTGATAGATGAAAGGTGCAGAAAACTGCTCTTATTTGGTGATGATAACAACAAG[T/C]TGT
TCTTCTCCTAAGTTTACTACTGATAAAATCAGTATTGCTTTTGGGGATGCT
>NT6058bis
ATGAGATCTGCTTATACTTTGGTAAGTTACTCACTCATTCATATGTTTCAAAATATAATTAAGGTTT
CAATTATTTTGTTTATCACATATTACATTAGCGAAGTATCGATCGGCTTTGACGATTTGAAAAGCCT
AACTAACTACCTATATTAGTATGTTTATTTCTAAATTACGTTTTTTATCATTGTTGATTTCAAATCTG
ATCAGAGATCAAACTGATGAGACCTTCAACCGAAAAAAAAATTAAATATTTTAAAAGTAAAAAA[A
/T]A[A/T]ATATAACAAAAGGTAAGGATGAGAACCGGTTGAACCAACTGCGGCTTAACCTGGTTAAA
TCCAGTTCAACCAGAATAAGGTATAATCGTAATCAAATCAAACCGATGAACTTACTCCTTCCCGCT
TAATTGTTTGAACTAACTGGTTCGGTCTGATTTCTAAAACATTTTTTATAGTGGCTATAACCTAAGA
CATGAATGTATACATATATATGTTTTTATATATTGATAGTAATATAGGATTAATATACCA
>NT6061
AGGTTATTTGGTCCCAAAATCCAACATTTTTTCTTCAACAACCCTTTTCCCCCATGATCT[T/C]TGAT
CCGAAAAATCTTAAATAATAAACAAAAAGAAATGCACAAAGACCTATATTTGGTCTTACAAACGT
GCTTAATTTGAAAAAGCAATATTGA[G/A]TATACTTTAGCTAAAATTGCTTCAACTTATGTGCTAAC
TCCTTGGGACAAATCACTGCCCCATCTATGCA[A/G]AGACCACCTTTGGTGTGTGTGCAT
>NT6066
TGATAAGGCTTACACTATCAATTTCAAAACAAAAAGTGCTTGCAAGCTTGGAATTTCAAGATATCC
TGATTTTGT[G/A]TA[C/T]GATGCTGAAGGAGGAATAGGAACTGGTTTTGGTGCAAAGG[A/C]AACA
CAAAATTCAGATAACAA[C/T]GACATTCTTGTTTCATTTGATCTTGATACATTGTATATTCCATCGTT
AACAAGCTCTACAACAAAGTTTCTTGGATTGTCATTGCCACCATTTTTGAGGATTGATATTGTTCCA
GAAGCTTTCCAAGGAAGCATTAACCAAGAATCTGGCA
>MTIC153
ACCACCTGAGCTTA[T/C]AGGTGAAGAAACACCATTTGTCTTATTACCATGATTATGATTATTCCTC
ATCCTCAACATCTTCTTTCACTTTCACAACTATGCAACAAAAGTGGTATGAACTATGAACTATTTAG
AGAGAGAAAGAGTCAGAGAGAGA[AGAGAGA/_]GAAAGAGGGTTAGATCTCTGTGGAAACTGATT
CTATACAAAACTTGACAGAAAATTCACCGACCC
>NT6060
TCCTGGTGGGTATTGGATTCTATCTGGTCCACCAATTCACTGGAAGAGATATTGGAAAGGATGGGA
AAGAACAAAAGAAGACTTGAATGAAGAGCAAACCAAAATTGAGAATGTAGCTAAAAGCCTTTGTT
GGAATAAAATAGTGGAGAAAGGTGATATTGCAATTTGGCAGAAACCAAAAAATCATTTAGATTGC
AAATTCACTCAAAATAGGCCTTTTTGCCAAGAGCAAAATAATCCTGAA
>NT6062bis
CTACTGCAGGATATGTCAAATAACAACGGGGGAGTACAACAACAGTCTCATAGTGGACCCAATGT
AAGTGGGAATATGGCAAAGAATAATTTGGGTTTTGGGGGCCAAACTCCAACCACTAATGGAGGAG
GCTCTGCCAATGGTCAAGGAAATAATGGACCTGTTTCAAGGAGTAACAGCTTCAAAGCAGCTTCAA
ACAGTGATTCTTCTGCCGA
>NT6068
TGATGATGCATTATACGGGGACAGAAAGAAGGGAAAGAGAAGAAGCGGGAATAATCGCAGTGGT
AATATCGATTGGTGGTTAGATGGATTCAGTGGTGACCTATGGAGAGCACGCAGGAATAGTTTTGAT
TCAGCTAGTGGAGATATTCCAAAGAGTGGTGGTGTGAGTAGTACACCAAGTATGAGAGGAACCGT
TTGTTATGTTGCTCCCGAGTATGGCTATGGCGAAGATGTCA
P. sativum [Champagne / Terese / Eden]
>NT6011
TC[C/A/A]TTCAAATTCCAAGAATTCATTGACCAGAATCTCCACGGAAGATTCTTCGAATACGAGGC
GGCTAAACT[A/G/G]GCAAAAATGGCTTTGCTTTGCTCCCATGACTCTCCCTTTGA[G/A/A]AGGCCA
ACTATGGAAGCTATTGTTCAAGAACTTGCCAATTGTAGCAGTTGTCTTTGATTTATGTTTTAGTTTA
AAATACTCTTAACCATCACTAACAC[C/G/G]TAAGTTTTATGTTGTTTGGTAACTTATGATGAAAATG
TTTGTGATGGTGATGATTTTGTAATGTATGAACATGACATTGTTCTCTATGTCCTTTAACTAAAGGA
CTAGCTTTTGTG
>NT6012
AGC[G/A/A]CGAGCCACTACAACAACAACAACAA[CAA/_/_]TAGTGATAAACTGTCTCATTGGTGCG
ACGAAC[G/C/C]GACGCAGAGATCCAAGTCAGAAAAAGTCCATCAATTCAACTTTCTCAAACAACA
AAAGCAATACTAATAATAACCAGCAACATTCATCATCGGAAAAACAACCAGAATTATAAACAAAA
ACAAGATCAAAACACAAACCATCAAAAACATAAG
>NT6032
GCATAATCAATGGAAGGGACTGTTTGGTAGGTTGGATTGGCAAGGGAATTTCCCAACTTCCATCAC
CGACCCTCAACCAATGGGGAAGGTTGGAATGTGCTTCCATCCTGATCAAGATAGAATTCTTACTGT[
C/T]CGTGAATGCGCCCGATCTCAAGTAAGTTTATTACTTAAGTCACTTGTCATGCTAAAGTAGAATA
CACAAAAAGATCTTGAATGATGTTCTTTCATAGTTTGGAATTTGTGATAGGAAAATATCATCTAATT
TGGTATCATCTTTAACTTATCATCTTTTTCATCTATAAATGCAGGGCTTTCCAGACCACTATCAATTT
TCTGGTAACATCATACACAAACACAGGCAGATTGGTAACGCGGTTCCTCC
>NT6039bis
AGAAGTTAAATTGAACGTGTCTCCAGGAAAAGGTTAGTTTTTTCTCTTACTTTTTACTTTTTTAAAGT
TCTGTTTGGAT[G/C/C]AATAGCTTATTTTAAGCGCTTATAATATAAGCTCTTATGATGTATAACTTG
TTTTTGTAGCAGTTTTCACCTTGAATTGATAGGACATGCTTGAATAAATTGTTTAAACATTTAT[A/G/
G]AAGTAATTGATTATTATATAAGTGATTATGTATATAAGCTGTTACTATAACAAGAGATAAAATAA
GCTGTTTTGATTAACTATAAGTGTTTAACAGTATCACAGATATAGAAGTATTTATGTTAAGTAGATA
AGTTTAATATAAGTCAATTTAACTGTATTTTAAGTG
>NT6044
GAAGATTTCAGAGGGTATAAATAGTTTCA[C/_/_]AAATTTTGAAATAA[G/T/T]CATGTTGAGTTATA
ATCTGAGATATT[T/_/_]AATTTTTGAATAAAAATGACAGGGCATCTCT[T/A/A]GAGTATTTTACTAC
ACACACAGACTTGGTGAAAAGGCAGCAAGAAGCAATAAAGCAAAGGAAAGCAATAAAGAAAGGA
TTGCAATCTGATAAAGACTGGACAGGAGATGGTTTTG
>NT6067
CCTCTTTCACATTAAATTCACAAGGCAAGAACAATATTATTCAACATCTTCACCAAATTAGAACCA
AACCAAGGAATAGTATTGATAGTAGTGAAATTACAAGAATGCCCTCATCAAGAAAATCAGATTTTG
ATCAACATAGGCTCTCTCTTCAAATCAACAAAGAGGATGGTCTTGG[C/G/G]GATGAGGATTTTGAG
CTGCCTAGATTGTCATTTTCAAAGAGGAAAGTTGATGAGAATAGTT[T/C/C]TAAAAGTTCAAGTCA
CTATGCTAAGCAAATTGTTAAACAAGTTAAGGAAAGTGTGGTGAGTAGAAAAGTTGG
>NT6078
TCAAGGAATTGGTCAGCTACTAGACAATTACAAGCAATAGGGAACAATCTTTGTTTTCCTAAGAAT
AATGATCTTGTTGCTACAAATGGTCTTGCATTGGCTATTTATACAATGAGTTCGATTTTGTTGTTTAC
AATGTGGGCACTTGTGGCTGCAATTCCTTGCCAAGATAGGGGATTACATCTCAACTTTTCGATTCCG
CGGCAGCTATCTTGGGCTGCTCCGGTGACATT[G/A]CTCCACGAGCGGATTTCGGACGAGTCGAAG
AAGAGG
>NT6083
TTTCACAGACGAGTGAGTTATTTTCTTCCCTTTTCTCTAATCGATGAAAACAAATTTACATGCATAC
AGTATCGTGTTTTTCTGAGTTGACTTCCATTGTTTTCTTGACTCGTGTATCTGTAAAATCGAAATTAA
GTTGCATTTACTTACATACTTTTGCCGGACATGCAGAAAGAGGGTACAACAACGGCTGGCTTTGTA
TCAAGGAGTTTGCCC[G/T]GTATACATGGAGTTCAGCGACGATTC
>MTIC153
TTCAGTTCTGCAACCACCTGAGGTTATAGGTGAAGAAACACCA[C/T/T]TTGTCTTATTACTATGATT
ATGATTATTCCTCATCCTCATCATCTTCTTTCACTTTCACAGCTATGCAACAAAAGAGGTATGAACT
GAGAACTATGAGAGAGAGAGAGAGAGAG[AG/_/_]GGGGGGTTAGATCTCTGAGAACACAG
>Cabb
TATAGTAAAGAGATATGTTCGGTTTCACGTTTGGAGCACTCATAATTGATTCTCG[GTGTGTAGGA
ATGATTATACTTTTC/_]AGAATTAATTTTACTAGGATTTGTAGCTTTTGAGTTTAGACGTGCATGTT
TGTATTAACACTTGTTTTGGTGGAATTACGGTGTGCCGTTGTGATTTTAGCGAAAACTAGACTTTAG
AACTTCAATGAAATTATGGTGCTAT[A/C]GTGTTTTTTGTCGAATTCAGAGTTAATTCAACGAAGCA
AGTAGAAAGAGTGTCTTGTGGTTCCGCGGTTAGCACAATTGATTTTAGTTAAAAGTAAATTTAAGG
TAGAATGATGTATGATTGAATACATTCACGCGAAAGTGAGTTAAACAATAAATTTGTCGTGTTAAG
GCAAAA
>Ps92K09T
TGATGTTTGGTAAAATAGAGTGTG[G/A]GGATGAGAAAAGAATTCATTGATTATATTTTTGTGTTTG
ACAAGACCTTCGGTCTTGTACCTACGTACCAACATGAAAATGAGGGATCAAAACCTCGTAGTTCGT
GGTAAAAA[A/T]TTTAAGTTTGATGAATTGGTTTTAACAAGAGTTTAAATGAAAAGGCAGCAAGGG
GaCAAAAATTTGA
>NT6048
ATGTCCTATGGATTCTCTCTGTTATTCTAGTGATTGCCGGGTAAGTTCCATTCCCCATGCCTAGGAC
TATTCATTATTCAGTTCAAGAAATACATTCGAGCCAAACATGTACAGAAGTTTGGTAGAACGGAAC
CGAACTGGTATAAATTGTGATATATCTAATCACTGTATTCATTTGTTGAATTGAATGGTAGGTTATG
CACTGGCTTACTGATGCTGTTACGCGGCGAAAACGGATACGATCACTGCCATTGGTGTCACTACCC
>NT6048bis
GCTGGAATGATTCATTTGGCAGCAAACATGTTAAGTCTGGTTTTCATTGGCATTCGCCTTGAACAAC
AATTTGGTTTCGGTACGTCCCTATTTTTACAATCCATTCATTAAACGGAGAATATTAAACTTAATAG
TTTTATATTTATCTGCAAGCTTATTTACCTGACGAGTTTATGTGTTCGAGTTTGTATTATAAAGGGTT
ATGACAAACATTTTGTCGAAATATTTGCAGTGAGGATAGGAATCATTTACCTTGTATCTGGATTCGG
CGGGAGCATACTA
>NT6051bis
CGCGAGTTCTTGTTTTTCCGGAATCCAAGTGCTGATATGCTTCTTCAAGAATCAGAACTTGATAGAA
ATCTCTTAAAACAGGTATTTTGAAATTTCATTTGTCTTTGTTTCCTGACTTGTAGTTCAAGACCTATG
GTTCGAGTAATGATGTTGAATGATATATTGATTTTTCAGGCTAGAATTTTCCATTACGGTTCGATTA
GTTTAATCGATGATCCATGCAAGTCAGCACACATTGCTGCTATGA
>NT6058bis
TTGGTGGTGATGACATGAGGTCTGCTTATACTCTAGTAAGTTGCTCAATCATTTTATATATTTTTTTA
CCATATGTTATAAGCATAGAGTGTCGGCCAATTTTGCTGATGACTAATGAAAAAGTCTAACTGATT
ACCTTTGTTAGTATGTTTTTCTTTCAAGATTTTAAATCTATAAAGTAATGTTTCAAAATTTGAGTACG
AGATTTTCTAAGTTATGAATCGAATGACAAATAAATAACAATATATTTATAAATAATACTAATAAT
CTAGTTGAACCAAGTGTGGTCAAATCAATGAGACTTTCGAATTATGATTCATTGTTTGAATGAATAC
GATTGATCTAATTTTTAAAACATTATCGTAGAGGTTAAAATCCGAGACATAAATATACATATGTTTT
TATATATTTATACTAACACATGGTATGTACCAAATTAACGATGAACTTGTAGGTCATGACAGATCC
AGATGCTC
>NT6062bis
AGGGGTCCCAAGGAAATCAATCACTGCAACATCAGATGATCCAGCAGCTACTGCAAGATATGTCA
AATAACAATGGCGGGGGAGTGCAACAACAGTCTCATAGTGGACCCACCAATGGGAATAATATGGC
AAAGAATAATTTGGGTTTTGTGGGTCAAACTCCACCCACAAGCGGAGGAGGCGGCGGCTCTTCCAG
CGGTCCAGCAAATAACGGACCTGTTTCAAGGAGTAATAGCTTCAAAGCAGCTTCAAACAGTGATTC
TTCTGCCGA
>NT6064
TGATAACACTGTCCGCCTCTATGATCTGCCATCGTAAGTGGATAACTTCATATTCTTGCTCTTGTTA
GATATGTGTGTTATTCTTAAGATCTCAAATTAATGTCAGAAAATCCGGATGTGGTTCATCCATTTGC
ATGGTATTCAAAACAAGCTTGGTTTATTGTGCAAAATTGTTAGAGAAATGTTTCTTTGTTATGCCAT
CTTCTTTTGTATTTTCTGGTTTCTTGAGAGTTTGGAACTTGTTCTGAACTTTCCCTTCCATTTCTTGTG
CAGATTTGCAGAGAGAGGCAAGATATACAACAAACAAGAGGTGCGAGCAATTCAAACTGGTCCTG
GTAGCATATTCTTCACCGGCGATGGAACCGGCCTAGTGAGAGTGTGGAATTG
>NT6066
AAACAACAAAAAATCATTTCCACCATAATTGAAACTCCATCAAATGATAATAAGGCTTACACTATC
AACTTCAAGACAAAAAGTGCTTGCAAGCTTGGAATTTCAAGGTATCCTGATTTTGAATATGATGCT
GAAGGAGGAATAGGAAATGGTTTTGGTGCAAAGGCAAAAAATCAAGATGATAATGATATTATTCT
TGTTTCGTTTGATCTTGAAACATTGTATATTCCATCGTTAACAAGCTCTACTACTAAGTTTCTTGGAT
TGTCATTGCCACCATTCTTGAGGATTGATATTGTTCCAGAAGCTTTCCATGGAAGCATTAACCAAGA
ATCTGGCAA
>NT6068
GAGTGATGATACATTATATGGGGATAGAAAGAAGGAAAAGAGAAGAAGTAGGAATAATCGTGGT
AGTGTTGATTGGTGGTTAGACGGGTTCAGTGGCGAGTTATGGAGAGCGCGTAGGAATAGTTTTGAT
TCTGCTAGTGGAGACATTCCAAAGAGTGGTGGTGTGAGTAGTACACCAAGTATGAGAGGAACCATT
TGTTATGTTGCGCCTGAGTATGGCTATGGCGAAGATGTCACTGAAAAGTGTGAA
>NT6077
ACAAATGAAAGGTGTACAATTCTTGTCACTAATCTCAATCCCGATGTAAGTACATTTGACTAGAAC
TTTTTCCTTCTGGTAGAAAGTTGTCTTTTTTATTTAATTATTTGCTTTAAATTTGGTTACTTCAGAGA
ATCGATGAGGATAAACTGTTCAACTTGTTCTCCATTTATGGAAACATTGTCAGAGTTAAAATACTCC
GGAATAAACCAGATCATGCACTTGTTCAGATGGGGGATGGTTTCCAAGCCGAATTGGCAGT