Computational protocol: Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat

Similar protocols

Protocol publication

[…] SDS PAGE bands (<15 kDa) of SINA and SINB were cut off from the gel and prepared for qualitative mass spectrometry analysis (http://mslab-ibb.pl/en/uslugi). A procedure includes following steps: reduction and alkylation of protein disulfide bonds, followed by tryptic digestion to obtain peptide mixture, liquid chromatography (LC) separation of a sample, MS measurement of peptides and their fragmentation spectra (tandem mass spectrometry) and searching of acquired spectra against a protein sequence database of choice (NCBI, UniProt or customer-supplied database) using Mascot search engine (http://www.matrixscience.com/), followed by validation and formatting of results. [...] Grain hardness, weight, moisture, and diameter of kernels were analyzed using the Single-Kernel Characterization System 4100 (Perten Instruments). Each sample contained 35 or 50 kernels and all parameters were calculated for each individual kernel. In total 47 individual plants from T2 and T3 generation were tested: 24 from 12 lines silenced by Pina cassette; 17 from 7 lines silenced by Pinb cassette and 6 from 3 lines transformed with the PMCG without silencing cassette. The grain protein content was determined via near-infrared transmission using a grain analyzer for the same lines (Infratec 1225).Statistical analysis was performed using Statistica software (StatSoft). The correlation coefficient between the expression level of Sin genes and the grain hardness was calculated in Microsoft Excel. […]

Pipeline specifications

Software tools Mascot Server, Statistica
Databases PINA
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Triticum aestivum
Diseases Hantavirus Pulmonary Syndrome