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| Status |
Public on Jul 25, 2023 |
| Title |
Tpd single pollen drive 277 |
| Sample type |
SRA |
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| Source name |
pollen
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| Organism |
Zea mays subsp. mays x Zea mays subsp. mexicana |
| Characteristics |
tissue: pollen
genotype: Tpd1+/-;Tpd2+/-
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| Growth protocol |
The TPD lineage traces to teosinte mexicana collected near Copándaro, Michoacán, Mexico in December 1993. Gamete a, plant 4 of collection 107 was used in an initial outcross to the Mid-western US dent inbred W22 and subsequently backcrossed. Tpd1;Tpd2 (BC8S3) homozygous lines were used for whole-genome sequencing and de novo genome assembly. All additional experiments were performed using Tpd1/tpd1; Tpd2/tpd2 (BC11-BC13) plants or populations derived from maternal segregation of these lines. The lbl1-rgd1 and dcl2-mu1 alleles were backcrossed to W22 ≥ 4 times. All genetic experiments used segregating wild-type progeny as experimental controls. Plants were grown under greenhouse and field conditions. In the greenhouse, starter flats were transplanted to pots containing divot mix and a single scoop of Osmocote 19-6-12. Once established, pots were on an irrigation drip of 20-20-20 at 0.8 EC (200 PPM Nitrogen) with a weekend water flush. Marathon was added as a drench and biweekly sprays of insecticides were applied as needed. Long day (6:00 AM until 10:00 PM) conditions were maintained at a 28˚C set point with cooling to 24˚C at night.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Pollen grains from Tpd1/tpd1; Tpd2/tpd2 plants were suspended in ice-cold PBS on a microscope slide under a dissecting scope. Individual plump, viable pollen grains were deposited into the 0.2mL wells of a 96-well plate using a p20 pipette. Lysis and whole genome amplification were performed using the REPLI-g single-cell kit (Qiagen, cat#150345) with the following modifications; 1/4th the specified volume of amplification mix was deposited in each well and isothermal amplification was limited to 5 hours. All steps prior to amplification were performed in a UV-decontaminated PCR hood. WGA products were cleaned up using a Genomic DNA Clean & Concentrator kit (Zymo Research, cat#D4067) and yields were quantified using with the QuantiFluor dsDNA system (Promega, cat#E2670) in a 96-well microplate format.
Libraries were prepared using the TruSeq Nano DNA High Throughput kit (Illumina, cat#20015965) with 200ng input. Samples were sequenced on a NextSeq500 platform using 2x150bp high output run.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
single-pollen-tpd.drive.vcf.gz
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| Data processing |
library strategy: DNA-seq
Adapter trimming was performed with Cutadapt v3.1 and read quality was assessed using FastQC v0.11.9. Paired-end reads were aligned to the to the W22 reference genome with BWA-MEM v0.7.17. Alignments were filtered by mapping quality (mapQ≥30) and PCR duplicates were removed using Samtools v1.10. SNP calling was performed using Freebayes v1.3.2. Putative SNP calls were filtered by quality, depth, and allele frequency (AF=1) to obtain a high-confidence mexicana marker set that was subsequently validated against the TPD assembly. For BSA analysis, SNP calls were filtered against the gold-standard TPD marker set.
All calls at validated marker sites were extracted and encoded in a sparse matrix format (rows = markers, columns = samples) and encoded (1 = alt allele, -1 = ref allele, 0 = missing). To assess mexicana introgression in individual pollen grains, mean SNP signal was calculated in 100kb bins across the genome. A sliding window (1Mb window, 200kb step) was applied in order to smooth the data and identify regions with mexicana SNP density. To identify genomic intervals overrepresented in surviving Tpd pollen grains, aggregate allele frequency was calculated across all pollen grains at each marker site.
Assembly: Zm-W22-REFERENCE-NRGENE-2.0; Zm-TPD-1.0
Supplementary files format and content: single-pollen-tpd.fertile.vcf.gz
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| Submission date |
Jun 14, 2023 |
| Last update date |
Jul 25, 2023 |
| Contact name |
Robert A Martienssen |
| E-mail(s) |
martiens@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Department |
Delbruck Bldg.
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| Lab |
Martienssen
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| Street address |
1 Bungtown Rd
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL33489 |
| Series (2) |
| GSE234920 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference [dna_single_pollen] |
| GSE234925 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference |
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| Relations |
| BioSample |
SAMN35738429 |
| SRA |
SRX20683458 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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