Physiological and Molecular Analysis for Development of DNA Markers in Resistance to Basal Stem Rot Disease in Oil Palm
Putranto, Riza Arief
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Basal stem rot (BSR) is a great disease concern in oil palm plantations. The cause of this disease is Ganoderma boninense, one of the fungi from the phylum Basidiomycota which can reduce oil palm productivity up to 40%. The higher generation planted in the oil palm plantations, the more widespread of the development of this disease in Ganoderma-infected areas. The response of oil palm resistance to BSR disease is very important to be discovered. The expression of genes involved in the resistance response is also a support in the superior plant material development for Ganoderma resistant in oil palm. The final objective of the study was to obtain molecular markers capable of selecting BSR disease-resistant plants in oil palm. The first experiment results “Differential expression of root specific genes of oil palm seedlings at early stage of Ganoderma boninense infection” showed two genes performed the highest transcript accumulation in susceptible treated with G. boninense. Ten out of the 16 genes belonged to Group I and the other six to Group. Seven out of the 16 genes were differentially expressed in the samples either with or without G. boninense inoculation. The seven genes may be associated with early infection responses to G. boninense. The second experiment results “Defense response changes in roots of oil palm (Elaeis guineensis Jacq.) seedlings after internal symptoms of Ganoderma boninense pat. infection” indicated that C01, C02, and C05 seedlings were susceptible, while the other three seedlings, C03, C07, and C08, were resistant based on Ganoderma Disease Index (GDI). Infection by G. boninense in the most susceptible seedlings C05 reduced fresh weight of roots (FW) by 9.0%, and lignin content by 10.9%. The most resistant seedlings C08 were reduced by only 8.4%, and 0.2% regarding their fresh weight and lignin content, respectively. BSR disease induced SA accumulation in the most susceptible C05 and decreased peroxidase (PRX) enzyme (EC 18.104.22.168) activities in root tissues of oil palm seedlings except C07 and C08 where PRX activities remained high in the 4 months after planting. Infection with G. boninense also increased glutathione S-transferase U19-like (EgGSTU19) gene expression in the root tissues of susceptible seedlings, while laccase-24 (EgLCC24) gene expression was associated with resistance against BSR disease. Based on the relative expression of twelve genes, two genes are categorized as receptors (EgWAKL5, EgMIK1), two genes as biosynthesis signal transduction compound (EgOPR5, EgACO1), five genes as defense responses (EgROMT, EgSOT12, EgLCC24, EgGLT3, EgGSTU19), and one gene as trans-resveratrol di-O-methyltransferase-like (EgRNaseIII) predicted related to BSR infection. While two other genes remain unknown (EgUnk1, EgUnk2). Ganoderma infection-induced SA accumulation and lignification in resistant accessions promote the seedlings root biomass. Oil palm seedlings have a synergistic physical, biochemical, and molecular defense mechanism to the BSR disease. The utilization of nucleotide-based molecular markers using EgLCC24 gene is able to detect resistant oil palm seedlings to G. boninense. The third experiment results “Genes expression analysis of EgUnk3, EgZFP2, and EgIPK2b in oil palm using Ct value correction and two relative quantification approaches” showed correlation regression obtained 0.28, -0.32, and 0.29 for delta Ct of EgUnk1, EgZFP2, and EgIPK2b, respectively. However, after the Ct mean have been corrected by linear regression on the EgUnk1, EgZFP2, and EgIPK2b showed the negative correlation values genes with -0.55, -0.81, and -0.29, respectively. The amplification factor (E) and efficiency value (R) using EgActin gene were 1.95 and 94.92%, respectively. Normalization of log10 on the fold change value 2-ΔΔCt and 1.95-ΔΔCt approaches using the regression coefficient showed the same results in the EgUnk1, EgZFP2, and EgIPK2b genes. Basal stem rot disease in oil palm decreased EgUnk1 and EgIPK2b expressions in the susceptible seedlings, however increased EgZFP2 gene expression in the resistant seedlings. The last experiment results “Identification of SNPs and biomarkers development of SNAP and tetra-primers ARMS-PCR on the Laccase-24 gene involved in oil palm root lignification” showed the EgLCC24 had two single nucleotide polymorphism (SNP) sites and 12 base insertion/deletion (indel) between Ganoderma resistant and susceptible plants. One base transversion from T to C at position 1128 bp did not change the protein formed, namely Asparagine (ASN), but the SNP from G to C at position 2176 bp changed Glycine (GLY) to Arginine (ARG). The insertion mutation of 12 bases at position 1529 with the first 6 bases being the repetition of the previous 2 amino acids with additions formed are Valine, Histidine, and Proline. Based on the Principal Components Analysis, eight oil palm populations are grouped into 4 quadrants. Population B is in quadrant I and II, population CS, D, E, and F is in quadrant II, population A is clustered in quadrant III, and populations CR and G are clustered in quadrant IV. Based on the dissimilarity biplot analysis using SNAP and ARMS-PCR tetra-primers, there were 3 groups of markers associated with all populations, except population A and some individuals in population B. The markers were grouped into 3 quadrants, namely primers 12 and 1128mu in quadrant I, primers 2178mu, 1128mu, 1128f1, 14, and 16 in quadrant II, while primers 70, 12, and 14 in quadrant IV. The population group in quadrant III and quadrant II did not show a specific primer for association in the oil palm population. Based on the analysis of the genetic diversity structure of the population with a determination delta of K=2, the population is grouped into 2 sub-groups, which are A and B susceptible populations in group I and CR, DR, FR, GR resistant populations in the other group.
- DT - Agriculture