Computational protocol: Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza)

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Protocol publication

[…] Rice seeds were obtained from germplasm collections of the United States Department of Agriculture (USDA), the International Rice Research Institute (IRRI) or from Dr. Beng Kah Song (Monash University Malaysia), who kindly provided Malaysian weedy rice samples. Plants were grown to the seedling stage (10–20 cm), and young leaf tissue was collected for DNA extraction. Genomic DNA was extracted using Qiagen DNeasy kits or a modified CTAB extraction protocol []. DNA quality and quantity were determined using 0.8 % agarose gel electrophoresis and ethidium bromide staining. In total, 282 plants were included in the analysis, representing six AA genome Oryza species: O. barthii (N = 9), O. glaberrima (N = 9), O. glumaepatula (N = 5), O. meridionalis (N = 6), O. rufipogon (N = 40), and O. sativa (N = 114 crop varieties, 99 weed accessions) (Additional file : Table S1).The PSTOL1 presence/absence polymorphism was scored using three sets of PCR primers spanning different portions of the gene, which consists of a single exon of 975 bp (Additional file : Table S2). PCR amplifications followed standard protocols [] and were optimized before large scale screening. Plants that yielded a negative PCR result at least twice for all three primer pairs were scored as absent for the PSTOL1 gene. If any of the three primer pairs individually yielded a product while the other two primer pairs were negative, the amplicon was sequenced; however, there were no cases where the resulting product had sequence similarity to PSTOL1, and these plants were therefore scored as negative. PCR cleanup was carried out using Wizard PCR cleanup kits. All amplicons were direct Sanger sequenced in both directions following previously described protocols [].PSTOL1 sequences were contiged, aligned and checked for quality using Phred/Phrap and BioLign version 4.0.6.2 (Tom Hall, http://en.bio-soft.net/dna/BioLign.html). Only high quality base calls (quality score of 30 or better) were retained. Alignments were trimmed to the start and stop codon for the PSTOL1 gene. Evidence for heterozygous SNPs (double peaks) was screened by eye and found to be absent in the dataset, consistent with the highly selfing nature of both cultivated and weedy rice.A Maximum Likelihood tree was constructed using MEGA version 5 [] with complete deletion of gaps and 10,000 bootstrap replicates. Tree construction employed a GTR plus gamma mutation model, which was selected using Akaike information criteria as implemented in Modeltest []. DnaSP version 5 [] was used to calculate summary statistics for genetic diversity, including numbers of segregating sites, average pairwise nucleotide diversity at silent sites (π) with Jukes-Cantor correction, and Watterson’s estimator of θ at silent sites. Neutral-equilibrium models were tested by estimating Tajima’s D amd Fu’s FS statistics in DnaSP with 10,000 coalescent simulations to assess significance. […]

Pipeline specifications

Software tools BioLign, MEGA, ModelTest-NG, DnaSP
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Oryza sativa