Pyramiding of Genes Conferring Resistance to Coffee Berry Disease Using Marker Assisted Selection
Gimase, James Mwita
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The epidemics of Coffee Berry Disease (CBD), caused by Colletotrichum kahawae, destroy up to 80% of the crop on susceptible varieties if not controlled. CBD control using fungicides increases production cost and environmental pollution, hence the need for resistant varieties. The conventional development of varieties takes 25-30 years, a period that can be reduced using DNA markers. Resistance to CBD is conferred by three genes; R, T and k from varieties Rume Sudan (RS), Hibrido de Timor (HDT) and K7 respectively. The T gene was mapped and adopted in marker-assisted selection (MAS) and there is a need for mapping of the other genes for selection efficiency. The objectives of this study were: to evaluate the suitability of the RSxSL28 F2 genotypes for mapping of a dominant gene, to identify the DNA marker for R gene in RS, to identify genotypes within R11 and Batian with T and R genes using MAS and to pyramid the three genes for CBD resistance in one population. Resistance to CBD of the F2 genotypes from RSxSL28 was evaluated by hypocotyl inoculation test. Genomic DNA was extracted using standard protocols. DNA analysis was carried out using Genotyping by sequencing (GBS) and the filtered sequence reads aligned to the C. canephora reference genome where 1170 Single Nucleotide Polymorphism (SNP) markers were obtained within the 11 coffee chromosomes (Chr). The population structure was determined by Principal Component Analysis (PCA) and marker-based kinship. Genome-Wide Association Study (GWAS) was carried out using the Compressed Mixed Linear Model (CMLM) while QTL analysis was carried out at LOD ≥ 2.5. The DNA markers for R and T genes were used to identify crosses within varieties R11 and Batian with multiple resistance to CBD, which were further utilized to develop a population with the three CBD resistant genes by crossing them to K7. The analysis of variance revealed significant variations among the F2 genotypes and their parents. The PCA and kinship coefficient matrix revealed that 84 F2 genotypes, whose Chi-Square (χ²) test fitted the 3:1 (χ² = 0 and, P≤0.05) Mendelian ratio of segregation for a dominant gene, were ideal for GWAS and QTL mapping. Two SNP markers, 100025973|F|0-59:T>C-59:T>C and 100034991|F|0-44:C>T-44:C>T, were significantly associated with CBD resistance in Chr 1 and 2 respectively, at threshold -log10(P) value > 2.0 on GWAS. The markers were confirmed by QTL mapping, where the first marker was at a distance of 3 centi Morgans (cM) while the second was 12.5 cM from the right and left genes respectively, accounting for 12.5% and 11% of the total phenotypic variation. The marker sequences were associated with proteins for plant defense mechanisms to disease pathogens. Eleven coffee genotypes were confirmed with multiple resistance conferred by T and R-genes. Three out of the eight genotypes were used to develop a pyramiding population with T, R and k genes. In this study, the 84 F2 genotypes are suitable for genetic mapping and are recommended for mapping genes for other diseases, segregating between the two parents. The Two SNP markers associated with CBD resistance in RS are recommended for use in MAS. The 11 crosses confirmed with multiple resistance to CBD are recommended for further distribution to growers since their resistance is broad-based and thus durable while the pyramiding population developed with T, R and k genes is recommended for subsequent selfing to fix the k gene and release as varieties with durable resistance. The study also recommends the mapping of the k gene in K7 for efficiency in selection for multiple resistance to CBD.