Computational protocol: Linking stomatal traits and expression of slow anion channel genes HvSLAH1 and HvSLAC1 with grain yield for increasing salinity tolerance in barley

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Protocol publication

[…] The method for QTL analysis was essentially described in Xu et al. (). The DH population treated in 200 mM NaCl was used to identify QTLs controlling stomatal traits and gas exchange parameters using a map constructed with Diversity Array Technology (DArT) and Simple Sequence Repeat (SSR) markers. Using the software package MapQTL6.0 (Van Ooijen and Kyazma, ), QTLs were first analyzed by interval mapping (IM), followed by composite interval mapping (CIM). The closest marker at each putative QTL identified using interval mapping was selected as a cofactor and the selected markers were used as genetic background controls in the approximate multiple QTL model (MQM) of MapQTL6.0. Logarithm of the odds (LOD) threshold values for the presence of a QTL were estimated by performing the genome wide permutation tests implemented in MapQTL6.0 using at least 1000 permutations of the original data set for each trait, resulting in a 95% LOD threshold around 2.9. Two LOD support intervals around each QTL were established, by taking the two positions, left and right of the peak, that had LOD values of two less than the maximum (Van Ooijen and Kyazma, ), after performing restricted MQM mapping which does not use markers close to the QTL. The percentage of variance explained by each QTL (R2) was obtained using restricted MQM mapping implemented with MapQTL5.0. Graphical representation of linkage groups and QTL was carried out using MapChart 2.2 (Voorrips, ). [...] The phylogenetic tree of the SLAC/SLAH family from Arabidopsis and selected cereal crops was generated using MEGA 6 program (Tamura et al., ). The Maximum Likelihood with WAG+Freqs (F) and Gamma distribution (G) model was employed. Names of organisms and accession numbers are from the National Centre for Biological Information (NCBI). Statistical values of phylogeny were estimated by the bootstrap method and were shown at the nodes. Alignment of the barley slow anion channel like 1 (HvSLAH1) and slow anion channel 1 (HvSLAC1) with homologous proteins in selected species was conducted using the CLASTALW tool in the BioEditor software ( with 1000-bootstraps. [...] Statistical significance between control and the treatments was examined using the Student's t-test at P < 0.05 and P < 0.01. Pearson correlation analysis of all the parameters was conducted using SPSS 20 (IBM, New York, USA) and SigmaPlot 12 (Systat Software Inc., San Jose, CA, USA). […]

Pipeline specifications

Software tools MapQTL, JoinMap, MEGA, BioEdit, SPSS, SigmaPlot
Applications Miscellaneous, Phylogenetics, WGS analysis
Organisms Hordeum vulgare