Abstract—Resistance gene analogues (RGAs) have been isolated from many crops such as banana, cereals, sugarcane, wheat and tomato. Cloning and characterization of RGAs offer potential in breeding for disease resistant crops through marker-assisted selection. Resistance genes from a broad range of plant species are known to share similarities in amino acid structural motifs. A pair of degenerate oligonucleotide primers designed from conserved motifs of P-loop and GLPL regions, common to many resistance genes, were used to amplify the Nucleotide-Triphosphate Binding Site (NBS) regions of RGAs from two Piper species, Piper nigrum L. cv. Semongok Aman and Piper colubrinum Link. A total of ten (10) partial RGAs fragments designated as PcRGAt5, PcRGAt6, PcRGAt7, PcRGAt8 and PcRGAt9 amplified from Piper colubrinum Link.; PnRGAt10, PnRGAt11, PnRGAt12, PnRGAt13 and PnRGAt14 amplified from Piper nigrum cv. Semongok Aman have been sequenced and characterized. The cloned partial RGAs with sequence length ranged from 498bp to 540bp encoding proteins with the size ranging from 165 amino acids to 179 amino acids. The predicted Piper RGA peptides contained the conserved motifs of Kinase-1 or the P-loop (GMGGVGKT), Kinase-2 (VLDDVW) and the hydrophobic Kinase-3 domain or GLPL. The obtained peptide sequences showed 42% to 47% similarity with published disease resistance proteins from various plant species such as Prunus persica (peach species) NBS resistance proteins, Malus baccata (apple species) and Prunus kansuensis (wild peach species) NBS-LRR-like proteins, Manihot esculenta (cassava) RCa9, RCa10.2 and RCa10.6 NBS type resistance proteins. An Un-weighted Pair Group Method with Arithmetical Averages (UPGMA) phylogenetic tree has been generated from the alignment of predicted Piper RGA peptides with the 6 published disease resistance proteins. The clustering analysis revealed that NBS-containing resistance genes comprise a large gene family in Piper species.

Index Terms—Clustering analysis, conserved motifs, disease resistant, piper nigrum L. cv. semongok aman, Piper colubrinum link., resistance gene analogues (RGAs).

L. E. Tiing is with the Research and Development Section, Malaysian Pepper Board, Sarawak, Malaysia (e-mail: lau@mpb.gov.my).
H. S. San is with the School of Engineering, Computing and Science, Swinburne University of Technology Sarawak, Malaysia (e-mail: shwang@swinburne.edu.my).
L. Eng and P. A. Det are with the Agriculture Research Centre Semongok, Department of Agriculture Sarawak, Malaysia (e-mail: lilye@sarawak.gov.my and paulusad@sarawak.gov.my).

Cite: Lau Ee Tiing, Hwang Siaw San, Lily Eng, and Paulus Amin Det, "Cloning and Characterization of Resistance Gene Analogues (RGAs) from Piper Nigrum L. cv. Semongok Aman and Piper Colubrinum Link," International Journal of Bioscience, Biochemistry and Bioinformatics vol. 2, no. 5, pp.  342-348, 2012.