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  • SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124

    NATURE PLANTS | www.nature.com/natureplants 1

    1    

    A trehalose-6-phosphate phosphatase enhances anaerobic germination

    tolerance in rice

    Tobias Kretzschmar, Margaret Anne F. Pelayo, Kurniawan R. Trijatmiko, Lourd F. Gabunada,

    Rejbana Alam, Rosario Jimenez, Merlyn S. Mendioro, Inez H. Slamet-Loedin,

    Nese Sreenivasulu, Julia Bailey-Serres, Abdelbagi M. Ismail, David J. Mackill,

    Endang M. Septiningsih

    Supplementary Information

    http://dx.doi.org/10.1038/nplants.2015.124

  • 2 NATURE PLANTS | www.nature.com/natureplants

    SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124

    2    

    Supplementary Methods

    Near isogenic line (NIL) development to fine map the qAG-9-2  

    NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected

    BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao

    Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with

    background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the

    original mapping populations were used to confirm the presence of the QTL. Selected BC4F4

    recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers

    RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with

    overlapping introgressions. Genotyping was performed to identify new recombinants, which

    were then phenotyped and further genotyped along with the informative recombinants identified

    in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine

    different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-

    20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to

    IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina

    Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services

    Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on

    chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-

    (Supplementary Fig. 8).

    3    

    Survival rate experiment

    Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted

    following an established protocol6. For the QTL confirmation, 16 trays per replication were used

    to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a

    total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing

    for two replicates. Randomization for all entries including the parental controls was performed

    with Alpha Plus design. Germination was also performed in air on moistened paper towels.

    Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with

    a similar set up.

    De novo-assembly of qAG-9-2 from KHO

    A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the

    Arizona Genomics Institute (AGI). Established methods were used for BAC library

    construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC

    library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).

    BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and

    hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).

    TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360

    region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI

    fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the

    KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb

    fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the

    DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as

    http://dx.doi.org/10.1038/nplants.2015.124

  • NATURE PLANTS | www.nature.com/natureplants 3

    SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124

    2    

    Supplementary Methods

    Near isogenic line (NIL) development to fine map the qAG-9-2  

    NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected

    BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao

    Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with

    background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the

    original mapping populations were used to confirm the presence of the QTL. Selected BC4F4

    recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers

    RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with

    overlapping introgressions. Genotyping was performed to identify new recombinants, which

    were then phenotyped and further genotyped along with the informative recombinants identified

    in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine

    different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-

    20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to

    IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina

    Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services

    Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on

    chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-

    (Supplementary Fig. 8).

    3    

    Survival rate experiment

    Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted

    following an established protocol6. For the QTL confirmation, 16 trays per replication were used

    to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a

    total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing

    for two replicates. Randomization for all entries including the parental controls was performed

    with Alpha Plus design. Germination was also performed in air on moistened paper towels.

    Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with

    a similar set up.

    De novo-assembly of qAG-9-2 from KHO

    A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the

    Arizona Genomics Institute (AGI). Established methods were used for BAC library

    construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC

    library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).

    BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and

    hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).

    TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360

    region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI

    fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the

    KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb

    fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the

    DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as

    http://dx.doi.org/10.1038/nplants.2015.124

  • 4 NATURE PLANTS | www.nature.com/natureplants

    SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124

    4    

    previously described42, followed by visualization with a STORM PhosphorImager “ATMI”.

    Positive BAC clones were gathered from the arrayed library and confirmed by secondary

    hybridization and PCR. One BAC clone (BAC4), positive for both probes, was sequenced on a

    Ion Personal Genome Machine (PGM) (Life Technologies) resulting in a total of 310,000 reads

    with an average length of 255 bp. Reads were trimmed, filtered for BAC vector sequence and the

    rice genomic sequence was assembled using DNASTAR 11 SeqMan NGen (Lasergene). The

    assembly covered chromosome 9 from LOC_Os09g20340 to LOC_Os09g20390. As the region

    LOC_Os09g20390 to LOC_Os09g20400, not covered by BAC4, this portion of the qAG-9-2

    region was assembled via cloning and Sanger-based sequencing of overlapping amplicons.

    Fragments were amplified with primer pairs AMP5.4 to AMP5.8 (#77-86), AG400_B (#87-88),

    and AG1-400 (#89-90). Resulting products were cloned into the pGEM-T Easy (Promega) and

    sequenced by a service provider (Macrogen) with their in-house M13_F and M13_R primers and

    amplicon-specific primers spaced at a distance of ~500 bp along the expected sequence (primers

    #91-115) and sequences assembled using DNASTAR

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