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Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 () and 1.95 to 2.14 (), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392 bp fragment using cDNA and 1.5 kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.

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Coffea canephora Pierre ex Frohener is popularly known as robusta coffee contributes to about 39% of the total world coffee production. Coffea canephora a native to West Africa was introduced to India as early as 1900 AD. However adequate information regarding the diversity and genetic structure of C. canephora germplasm available in India is not yet available. In the present study, sequence related amplified polymorphism (SRAP) and start codon targeted (SCoT) molecular markers were employed to access the genetic diversity and relationships of 58 C. canephora germplasm accessions available in the Indian gene bank. Forty-nine SRAP and thirty-one SCoT primers amplified 632 and 331 fragments respectively, of which 507 (80.22%) and 225 (67.97%) were polymorphic. The average polymorphism information content (SRAP, 0.48; SCoT, 0.37) and resolving power (SRAP, 15.60; SCoT,14.84) revealed high genetic diversity among the accessions The SRAP markers were found more informative with regards to the amount of diversity detected which is evident from effective multiplex ratio (SRAP = 8.59, SCoT = 5.61) and marker index (SRAP = 4.60, SCoT = 2.58). The neighbor-joining clustering revealed that 58 accessions were grouped into four major clusters which were also supported by Principal coordinate analysis. An admixture model-based clustering method in STRUCTURE grouped all the accessions in four subpopulations (K = 4) as similar to NJ clustering. Our study demonstrated the suitability of SRAP and SCoT markers for coffee genetic diversity and discovered thirty-one diverse genotypes in the germplasm that could be integrated into the C. canephora genetic improvement program.

Start codon targeted polymorphism (SCoT), a novel and gene-targeted marker, has recently become the marker of choice in genetic diversity studies. In the present study, 31 SCoT primers were tested for their suitability in the genetic analysis of 21 coffee genotypes representing 18 species. A total of 647 distinct PCR amplified fragments were produced with a mean of 20.9 fragments per primer and 80.80% of which were polymorphic. The polymorphic information content of SCoT primers ranged from 0.16 to 0.86, with a mean value of 0.63. Resolving power ranged from 6.19 to 28.29, with a mean value of 20.2. Species-specific unique PCR amplified fragments were identified for 16 species, which could be used as genetic fingerprints. The genetic similarity among various coffee species calculated using the Dice similarity coefficient ranged between 0.60 and 0.89. The dendrogram constructed using the unweighted pair group of arithmetic means (UPGMA) clustered the 21 coffee genotypes into two major groups. The study revealed that Coffea jenkinsii, an indigenous species from India, showed the highest similarity with C. arabica, which is of Ethiopian origin. The results proved the suitability of SCoT markers in genetic analysis of coffee genotypes.

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