Computational protocol: QTL mapping for flowering-time and photoperiod insensitivity of cotton Gossypium darwinii Watt

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[…] Genetic linkage maps were constructed from the genotypic data of polymorphic SSR and CAPs marker loci in both populations using the program JoinMap version 3.0 []. Assignment of linkage groups to the respective chromosomes was accomplished using data from CottonGen (https://www.cottongen.org/data/download/map) based on previously published molecular maps ( and Data). For a graphical representation of QTL maps and linkage groups the program MapCHART version 2.2 was used []. Regression analysis was used to identify associations between genotypic data and phenotypic traits ( and Data). Thus, two separate QTL-mapping analyses in F2 and F3 populations were performed using QTL Cartographer v2.5 [] using Kosambi [] as a mapping function, applying the algorithm of composite interval mapping (CIM). The average critical threshold LOD levels were identified by QTL Cartographer and Qgene [] at the 95% significance level after 1000 times permutation test. [...] Based on the assumption of significant gene colinearity between G. darwinii and closely related G. hirsutum (ref on phylogenetic similarity), in silico analysis of genomic regions identified by QTL were performed using whole genome sequences of G. hirsutum L. []. This was for estimation of actual physical genomic positions of loci, and for prediction of linked candidate genes/protein potentially involved in the mechanism of photoperiodic flowering. In silico PCR was carried out using the UGENE 1.20.0 [] bioinformatics software package to find the locations of 'virtual amplicons' on the cotton genome using SSR primer pairs that were associated to the mapped QTL(s). A web-based gene prediction application, AUGUSTUS 3.1.0 [], was used for identifying the genes on these chromosomes. Predicted amino acid sequences by AUGUSTUS were analyzed using Basic Local Alignment Search Tool search algorithm (www.ncbi.nlm.nih.gov/BLAST). […]

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