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| Status |
Public on Jul 25, 2023 |
| Title |
Tpd maternal BSA sterile bulk 5 |
| Sample type |
SRA |
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| Source name |
leaf
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| Organism |
Zea mays subsp. mays x Zea mays subsp. mexicana |
| Characteristics |
tissue: leaf
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 |
High molecular weight (HMW) genomic DNA was used as input for all Nanopore and bulk Illumina sequencing experiments. For extraction, bulked seedlings were dark treated for 1 week prior to tissue collection. Four grams of frozen tissue was ground under liquid N2 and pre-washed twice with 1.0M sorbital. The tissue was then transferred to 20ml pre-warmed lysis buffer (100mM Tris-HCl pH 9.0, 2% w/v CTAB, 1.4M NaCl, 20mM EDTA, 2% PVP-10, 1% 2-mercaptoethanol, 0.1% sarkosyl, 100ug/mL proteinase K), mixed gently, and incubated for 1 hour at 65˚C. Organic extraction in phase-lock tubes was performed using 1 vol phenol:chloroform:isoamyl alcohol (25:24:1) followed by 1 vol chloroform:isoamyl alcohol. DNA was precipitated by adding 0.1 vol 3M NaOAc pH 5.2 followed by 0.7 vol isopropanol. HMW DNA was hooked out with a pasteur pipette and washed with 70% EtOH, air dried for 2 minutes, and resuspended in 200ul Tris-Cl pH 8.5 (EB). The solution was treated with 2ul 20mg/ml RNase A at 37˚C for 20 minutes followed by 2ul 50mg/ml proteinase K at 50˚C for 30 minutes. 194ul EB, 100ul NaCl, and 2ul 0.5M EDTA were added and organic extractions were performed as before. DNA was precipitated with 1.7 vol EtOH, hooked out of solution with a pasteur pipette, washed with 70% EtOH, and resuspended in 50ul EB.
HMW DNA from separately maintained Tpd1;Tpd2 lineages (BC5S3, BC8S3) and bulk segregation analysis (BSA) pools were extracted as detailed above. Libraries were prepared using the Illumina TruSeq DNA PCR-Free kit (Illumina, cat#20015962) with 2ug of DNA input.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
bsa.vcf.gz
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| Data processing |
library strategy: WGS
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. Reference and alternate allele frequencies at each marker were calculated and average signal was consolidated into 100kb bins. ∆SNPindex was then calculated for each bin in a sliding window.
Assembly: Zm-W22-REFERENCE-NRGENE-2.0; Zm-TPD
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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) |
| GSE234921 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference [WGS] |
| GSE234925 |
Teosinte Pollen Drive guides maize domestication and evolution by RNA interference |
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| Relations |
| BioSample |
SAMN35739927 |
| SRA |
SRX20696976 |
| 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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