Computational protocol: Genome-wide analysis of NBS-encoding disease resistance genes in Cucumis sativus and phylogenetic study of NBS-encoding genes in Cucurbitaceae crops

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Protocol publication

[…] To investigate the diversity and structure of NBS-encoding genes in cucumbers, their predicted amino acid sequences were subjected to domain and motif analyses. According to the methods of previous researchers [,], NBS-encoding genes from cucumbers were divided into three components, namely the N-terminal, NBS domain, and LRR-C-terminal regions. They were then analyzed individually using the Multiple Expectation Maximization for Motif Elicitation (MEME)/Motif Alignment and Search Tool (MAST) system (http://meme.sdsc.edu/meme/website/intro.html). Furthermore, MEME motif analyses were performed on members of TIR-NBS and CC-NBS families. Conservation of each motif among the NBS-encoding genes was performed with WebLogo version 2.8.2 (http://weblogo.berkeley.edu/) using the default settings.Gene duplication events of NBS-encoding genes were defined based on the http://criterion used by previous researchers []. NBS-encoding genes in cucumber were aligned using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and calculated by MEGA 5.0 [] for homology gene calculation. [...] To understand the phylogenetic relationship among the NBS-encoding genes in Cucurbitaceae crops, NBS-encoding RGHs from melon, bottle gourd, luffa, watermelon and squash were also identified via degenerate PCR amplification and database mining. First, PCR was performed using genomic DNA for young leaves from melon, bottle gourd, luffa, and watermelon using 3 pairs of degenerate primers. The young leaves in the second true-leaf stage were harvested, frozen immediately in liquid nitrogen, and stored at −80°C. Genomic DNA was isolated using a plant DNA extraction kit (Tiangen, China). The primers were designed by the previous researchers based on the conserved regions of P-loop and GLPL of amino acid identity among the known NBS-LRR R genes from the other plant species (Additional file ). The PCR amplifications were performed in 20 μL reaction mixtures with 1 U of LATaq DNA proof reading polymerase (TaKaRa, Kyoto, Japan), 1 × PCR buffer, 1.5 mM MgCl2, 0.5 μM each of forward/reverse primers, 0.4 mM dNTP, and 50 ng of template DNA. PCR was performed in a PTC-100 thermal cycler (MJ Research, Inc., Watertown, MA). The cycling conditions consisted of an initial denaturation performed for 3 min at 94°C, followed by 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min. These were followed by a 10 min extension step at 72°C and 10°C to terminate the reaction.The DNA fragments from the PCR were separated using 1.0% agarose gels. Fragments with the expected size (~500 bp) were excised and reclaimed from the gel and purified with a PCR purification kit (Qiagen, Germany). Subsequently, these fragments were combined with vector DNA to generate recombinant DNA molecules, and then transformed into competent Escherichia coli JM109 cells. Plasmid DNA was purified with a PCR purification kit (Qiagen, Germany). The DNA fragments were sequenced using an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA). Then, each of the acquired DNA sequences was trimmed of vector sequence contamination using VecScreen at the National Center of Biotechnology Information (NCBI). Identity and similarity searches of nucleotide and amino acid sequences were performed using BLAST at the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).Second, other RGHs in melon, watermelon, and squash were obtained from the GenBank database searches. All sequences from these species were downloaded and searched with the NBS domain of NBS-encoding sequences from A. thaliana and rice [-] as the query. The RGHs in melon were sourced from a published paper []. In addition, Arabidopsis NBS-encoding proteins, which were obtained from http://niblrrs.ucdavis.edu/At_RGenes/, were selected for phylogenetic relationship analysis. [...] Amino acid sequences of all NBS-encoding genes in the cucumber genome and RGHs from the other five Cucurbitaceae crops were aligned using Clustal X version 1.8 [], followed by manual adjustment. The conserved domains of P-loop to GLPL of these proteins and RGHs were applied to construct a phylogenetic tree using the NJ method [] and an NJ algorithm implemented in the Molecular Evolutionary Genetics Analysis software version 5.0 (MEGA 5.0) []. Bootstrapping (1000 replicates) was used to evaluate the degree of support for a particular grouping pattern in the phylogenetic tree. Branch lengths were assigned by pairwise calculations of the genetic distances, and missing data were treated by pairwise deletions of the gaps. […]

Pipeline specifications

Software tools WebLogo, BioEdit, MEGA, VecScreen, Clustal W
Applications Phylogenetics, Metagenomic sequencing analysis, Genome data visualization
Organisms Cucumis sativus, Arabidopsis thaliana
Diseases Nematode Infections, Neoplasms by Site