DefinePK

Research Objective

To analyze the resistance of Liberica coffee plants (genotypes Lim 1, Lim 2, and Bengkalis) to Coffee Leaf Rust (CLR) disease caused by Hemileia vastatrix using the functional primer CARF 005, which was previously reported for Arabica coffee.

Methodology

1. Plant Material Collection: Healthy leaves were sampled from three Liberica genotypes (Lim 1, Lim 2, Bengkalis) grown in commercial farmer fields on peatland areas in Meranti Islands, Indonesia.
2. DNA Extraction: Total genomic DNA was extracted from young leaves using a Genomic DNA Mini Kit (Plant) Geneaid protocol.
3. PCR Amplification: DNA was amplified using the CARF 005 primer pair (F: 5'-GGACATCAACACCAACCTC-3' and R: 5'-ATCCCTACCATCCACTTCAAC-3').
4. Visualization and Sequencing: PCR products were visualized on a 1% agarose gel. Products were purified and sent for bidirectional sequencing.
5. Sequence Analysis: Nucleotide sequences were analyzed using BioEdit ver 7 and BLAST against the NCBI database for similarity. Conserved domains were identified using the CDD-NCBI. Amino acid sequences were derived using Augustus and aligned using Clustal Omega. Phylogenetic relationships were constructed using MEGA X.

Methodology Flowchart

                        graph TD; A[Sample Collection: Lim 1, Lim 2, Bengkalis Leaves] --> B[Genomic DNA Extraction]; B --> C[PCR Amplification using CARF 005 Primers]; C --> D[Visualize PCR Product on Gel]; D -- ~400 bp band --> E[Purification and Bidirectional Sequencing]; E --> F[Sequence Analysis: BLAST Similarity Check]; F --> G[Conserved Domain Identification CDD]; G --> H[Amino Acid Derivation & Alignment Clustal Omega]; H --> I[Phylogenetic Analysis MEGA X]; I --> J[Conclusion on Resistance Marker Efficacy];                    

Discussion

The successful amplification and sequence similarity to known Arabica RGA sequences confirm that the DNA fragments amplified by CARF 005 are linked to the RGA region in Liberica coffee. The presence of the NB-ARC domain, which functions as a molecular switch regulating R protein conformation, supports the hypothesis that these sequences are associated with the resistance trait against H. vastatrix. This study provides the first molecular evidence using CARF 005 for CLR resistance in Liberica coffee, validating the phenotypic resistance previously reported for Lim 1 and Lim 2.

Key Findings

- The CARF 005 primers successfully amplified a DNA band of approximately 400 bp in all three Liberica genotypes (Lim 1, Lim 2, Bengkalis).
- Nucleotide sequences showed high similarity (95.89% to 98.31%) to the Coffea arabica putative disease resistance protein RGA4 sequence deposited in GeneBank (accession XM_027246611).
- Sequence analysis confirmed the presence of the NB-ARC conserved domain (Nucleotide Binding Site-ARC/APAF-1, R protein, and CED-4) and the P-loop/Walker-A domain motif (GGLGKTT) in the amplified fragments.
- Phylogenetic analysis grouped Lim 1 and Lim 2 together, separating them from Bengkalis, supporting their shared origin from Meranti Island.
- The results suggest that CARF 005 can be used to identify CLR resistance in Liberica coffee.

Conclusion

The functional marker CARF 005 is effective in amplifying RGA-linked regions in Liberica coffee genotypes. The presence of conserved domains associated with resistance genes suggests that Lim 1 and Lim 2 possess the molecular basis for resistance to Coffee Leaf Rust, supporting their use in breeding programs and germplasm conservation.

Fact Check

1. The fungal pathogen causing Coffee Leaf Rust (CLR) disease is Hemileia vastatrix. (Confirmed in Abstract/Introduction)
2. The primer pair used was CARF 005. (Confirmed in Abstract/Methods)
3. The amplified DNA band size was approximately 400 bp. (Confirmed in Results)

Mind Map