Arch. Biol. Sci., Belgrade, 63 (3), 667-679, 2011 DOI:10.2298/ABS1103667M MOLECULAR IDENTIFICATION AND GENETIC RELATIONSHIPS AMONG COFFEE SPECIES (COFFEA L) INFERRED FROM ISSR AND SRAP MARKER ANALYSES MANOJ KUMAR MISHRA, SANDHYARANI NISHANI and JAYARAMA Central Coffee Research Institute, Coffee Research Station P.O. Chikmagalur –577117, India Abstract – The identification and genetic relationships of 23 coffee species and one coffee-related species Canthium dic- cocum were studied using ISSR and SRAP markers. The average polymorphism information content of SRAP primers (0.81) was lower than ISSR primers (0.86), whereas the average resolving power of the SRAP primers (9.74) is higher than the ISSR primers (8.64). The genetic similarity among the species ranged from 0.30 to 0.89 using ISSR and 0.11 to 0.90 us- ing SRAP marker systems. Based on marker analysis, all twenty three coffee species were clustered into two major groups. Both the markers amplified species-specific fragments and are useful in genetic diversity analysis of coffee. Key words: Coffee species, genetic relationships, molecular identification, inter simple sequence repeats (ISSR), sequence- related amplified polymorphism (SRAP). UDC 633.73:577.2 INTRODUCTION type, floral characteristics and fruit morphology are used to characterize the various species. How- The genus Coffea L. belongs to the Rubiaceae fam- ever, developing morphological descriptors for any ily and comprises 103 species (Davis et al. 2006). particular species/cultivar has severe limitations The majority of coffee species occurs naturally in as these characteristics are influenced by environ- Africa, Madagascar and the Mascarenes, predomi- mental conditions. In contrast to the morphologi- nantly restricted to humid evergreen forest, but cal markers, DNA-based marker techniques are some species are found in seasonally dry decidu- more efficient, precise and reliable for discriminat- ous forest and/r bush land (Maurin et al. 2007). ing between closely related species and cultivars. The importance of coffee as an agricultural com- In previous studies, genetic diversity and phyloge- modity relies mainly upon two varieties, Coffea netic relationships between different coffee species arabica and C. canephora, which contribute about was carried out by using both Random Amplified 65% and 35% of total production, respectively. In Polymorphic DNA (RAPD) analysis as well as Re- spite of the commercial and social importance of striction Fragment Length Polymorphism of chlo- the genus, the genetic relationship between the ma- roplast and mitochondrial DNA (Berthou, 1983; jorities of coffee species is not extensively studied Cross et al. 1998; Lashermes et al. 1993; Lashermes and their taxonomic status is poorly understood. et al. 1996; Orozco-Castillo, et al. 1996). Recently, Understanding the genetic relationship between Davis et al. (2006) published a detailed annotated coffee species is not only important for resolving taxonomic conspectus of the genus Coffea and in- taxonomic ambiguity but also important for the ge- cluded five indigenous coffee species from India, of netic improvement program. Conventionally, mor- which C. bengalensis, C. travancorensis, C. wight- phological descriptors such as growth habit, leaf iana are placed under the genus Psilanthus and an- 667 668 MANOJ KUMAR MISHRA ET AL. other two species, C. khasiana and C. jenkinsii, are MATERIALS AND METHODS placed under the genus Nostolachma, both under Coffeeae. Based on the analysis of the internal tran- Plant materials scribed spacers (ITS) of nuclear DNA, Lashermes et al. (1997) observed limited sequence divergence Leaf material was collected from 10 individuals of between Psilanthus and Coffea and concluded that twenty three coffee species (Table 1) including five Psilanthus should not be recognized as a separate indigenous coffee species from India (C. bengalensis, genus from Coffea. Based on the comparative se- C. travancorensis, C. wightiana, C. khasiana and C. quence analysis of plastid DNA, Cross et al. (1998) jenkinsii), maintained at the germplasm plot of Cen- concurred with Lashermes et al. (1997) about the tral Coffee Research Institute, Chikmagalur, Karna- close relationship between Coffea and Psilanthus taka and Regional Coffee Research Station Thandigu- although their tree topology shows an unresolved di, Tamil Nadu. Mature seeds were also collected and relationship between two species of Psilanthus (P. grown in earthen pots under net house conditions. mannii and P. ebracteolatus) studied by them and The leaf materials were used for DNA isolation. Coffea. However, both these studies did not in- clude a representative of closely related genera for DNA extraction comparison. Further studies by Andreasen et al. (1999), Andreasen and Bremer (2000) and Davies Genomic DNA was extracted from fresh young leaves et al. (2007) showed that Psilanthus is nested within using a modified CTAB method. About 200 mg of Coffea and stressed the importance of species level leaf tissue was ground to a fine powder in liquid ni- studies to resolve the phylogenetic relationship. trogen, it was transferred to a 30 ml tube containing Maurin et al. (2007) observed that the separation of 5 ml preheated extraction buffer (2% CTAB (w/v), Coffea and Psilanthus based on morphological fea- 100mM Tris-HCL (pH 8.0), 25mM EDTA, 2M Nacl, tures is not convincing and suggested the inclusion 0.1 % beta-mercaptoethanol). The tubes were incu- of molecular data from other species of Psilanthus bated at 600 C for 1 h with occasional shaking. After to establish the relationship between these two gen- incubation, the tubes were cooled to room tempera- era. Based on the morphological features, Nostol- ture and centrifuged at 6000 rpm for 20 min. The su- achma is placed under Coffeeae (Davis et al. 2007) pernatant was transferred to a new tube and extract- although molecular data from additional species ed twice with equal volumes of chloroform-isoamyl may prove to be useful. Although various research- alcohol (24:1). The supernatant was transferred to 2 ers have studied the taxonomic and phylogenetic ml tubes, precipitated with 0.7 vol of isopropanol at relationship between various coffee species, there room temperature for 30 min., and then centrifuged are no reports yet available for the species-specif- at 8000 rpm for 20 min at 4° C. The pellet formed ic markers that are useful for distinguishing vari- after centrifugation was washed with 75% (v/v) etha- ous species. In this paper, we have used two PCR- nol for 10 min and dissolved in 60 µl of Tris-EDTA based molecular markers, Inter Simple Sequence (1-10mM). The concentration of DNA was meas- Repeats (ISSR) and Sequence Related Amplified ured using 0.8% agarose gel stained with ethidium Polymorphism (SRAP), in developing species spe- bromide as well as by UV spectrophotometry at 260 cific markers for 23 coffee species available in coffee nm and a 280 nm. The resuspended DNA was then germplasm in India. To our knowledge, both these diluted in sterile distilled water to 10ng/ µl concen- markers are used in coffee for developing species tration for use in amplification reactions. diagnostic markers for the first time. The main objectives of the study are to generate a molecu- ISSR, SRAP analysis lar database and identification tag for each species that may be useful for conservation and systematic A total of 20 ISSR primers from the University of studies as well as coffee breeding programs. British Columbia and 60 SRAP primer combina- Molecular based diversity analysis of coffee species 669 tions (8 forward primers and 14 reverse primers) PCR reactions were repeated at least twice to con- synthesized by Sigma, India, were initially screened firm the reproducibility of each PCR band. to determine the suitability of each primer for the study. Primers were selected for further analysis Data analysis based on their ability to detect clear and distinct polymorphic amplification products within the The ISSR and SRAP amplified bands were scored species of Coffea. 12 ISSR primers and 21 SRAP for the presence (1) or absence (0). The total number primer combinations that had a high level of poly- of bands, the distribution of bands across all species, morphism and the best readability were used for polymorphic bands, species-specific bands and aver- PCR amplification (Table 2). The PCR reaction age number bands per primer were calculated. The was carried out in a palm cycler (Corbett Re- value of each primer was assessed using two indices; search). PIC, which is the same as a diversity index (Botstein et al. 1980; Milbourne et al. 1997) and resolving ISSR PCR was conducted in a 20 µl reaction power (Rp) (Prevost and Wilkinson, 1999). PIC or 2 mixture containing 1x reaction buffer (10 mM Tris- DI was estimated as PIC=  (1-p i )/n, where n is the HCL pH 8.8, 50 mM KCL 0.08% Nonidet P40), 30 ng number of band positions analyzed in all the species, DNA, 200 µM dNTP mixture, 2.5 mM MgCl2, 3 µM pi is the frequency of the banding pattern. The re- ISSR primer,0.2 µl of formamide and 1.0 U Taq DNA solving power of a primer is Rp = Ib where Ib (band polymerase. The ISSR amplification conditions were: informativeness) takes the value of 1- [2x (0.5-p)] 3 min initial denaturation at 95ºC; 30 cycles consist- and p is the ratio of six species sharing the band. A ing of 1 min denaturation at 94ºC; 1.30 min primer pairwise similarity matrix was constructed using the annealing at 55ºC and 2 min extension at 72ºC and a Dice similarity coefficient (Sneath and Sokal, 1973). final 10 min extension at 72 º C. The relationship between the species was displayed as a dendrogram constructed using NTSYS –PC 2.10e SRAP analysis was performed by adapting the software (Rohlf, 1995) based on Unweighted Pair procedure described by Li and Quiros (2001) with Group Method using Arithmetic averages (UPG- minor modifications: 20 µl reaction mixture contain- MA).