Molecular Identification and Genetic Relationships Among Coffee Species (Coffea L) Inferred from Issr and Srap Marker Analyses

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 diccocum 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 using 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). Statistical support of the clusters was assessed ing 1 x reaction buffer (75mM Tris-HCL pH 8.8, 20 by means of 1000-bootstrap replicates. mM (NH4)2 SO4, 0.01% Tween 20), 30 ng template DNA, 200 µM dNTP mixture, 2.5 mM MgCl2, 3 µM RESULTS each of forward and reverse primer, and 1.0 U Taq DNA polymerase. The SRAP amplification program ISSR polymorphism and species identification was a 4 min initial denaturation at 96º C; 5 cycles consisting of 1 min denaturation at 94ºC, 1.15 min Out of the 20 ISSR primers initially screened, 12 primer annealing at 35ºC; and 2 min extension at primers were found to be polymorphic and produced 72ºC followed by 30 cycles consisting of 1 min dena- clear and reproducible amplification patterns. These turation at 94ºC, 1.15 min primer annealing at 50ºC; 12 primers could produce 185 distinct reproducible and 2 min extension at 72 ºC; and a final extension of bands across the 24 species with an average of 15.58 15 min at 72ºC. per primer (Table 3). The size of the amplified products ranged from 100 to 2900 bp. Of the total 185 The PCR products of both ISSR and SRAP were amplified bands, 174 (93.06%) were polymorphic, run on 2% (w/w) agarose gels containing 0.5 µg with an average of 14.5 polymorphic fragments per ethidium bromide/ml in 1X TAE buffer and then primer. All the ISSR primers except UBC-881 and visualized and photographed using the UV-transil- UBC-855 showed 100% polymorphism. The primers luminator (SYNGENE) and documented using the UBC-881 and UBC-855 showed 8.3% and 90.90% Gene Snap software program. Both ISSR and SRAP of polymorphism respectively. The average poly- 670 MANOJ KUMAR MISHRA ET AL.

Fig.1. The extent of polymorphism observed among various coffee species listed in table 1 by ISSR primer 842 Lanes M: Molecular ruler (Gene ruler 100 bp ladder plus). morphism percentage recorded with ISSR primers Out of the 12 ISSR primers, only one ISSR poly- is 93.06. The resolving power of the 12 ISSR prim- morphic primer (UBC 842) could discriminate all the ers tested ranged from 4.08 (UBC- 840) to 22.17 species independently (Fig. 1). Twelve ISSR primers (UBC-881) with an average of 8.64. Similarly, the generated 40 unique fragments of which 8, 7 and 5 polymorphism information content (PIC) of the 12 fragments are obtained in C. khasiana, C. jenkinsii ISSR primers ranged from 0.083 (UBC- 881) to 0.963 and C. arabica, respectively. Further, the ISSR primer (UBC-840) with average of 0.84. UBC-836 has amplified species diagnostic fragments in five different species (Table 4). The five Indian coffee species generated 19 unique fragments of which maximum numbers of eight unique fragments were generated by C. khasiana, whereas only one unique fragment was generated by both by C. travancorensis and C. wightiana. The genetic similarity derived from the data of the ISSR marker analysis varied from 0.27 between C. bengalensis and C. liberica to 0.89 between C. canephora and C. congensis (Ta- ble 5). Among the Indian species, C. travancorensis showed maximum genetic similarity with C. wightiana, based on the ISSR marker analysis. Further- more, Canthium diccocum, which is used as a refer- ence species, showed maximum genetic similarity (0.65) with C. racemosa.

Fig. 2 Dendrogram generated using unweighted pair group The dendrogram based on ISSR data was con- method with arithmetic average (UPGMA) analysis showing re- structed by UPGMA analysis, grouping all of the lationships among different coffee species using ISSR data. coffee species into two major clusters (Fig. 2). The Molecular based diversity analysis of coffee species 671

Fig.3 The extent of polymorphism observed among various coffee species listed in Table 1 by SRAP primer combinations Me3 and Em12 Lanes M: Molecular ruler (Gene ruler 100 bp ladder plus). first major cluster divided into two minor clusters sub-minor cluster was represented by C. eugenioides, of which the first minor cluster again divided into C. salvatrix, C. kapakata, C. arnoldiana, and C. dew- two sub-minor clusters. The first sub-minor cluster evrei var. excelsa. The second minor cluster included consisted of C. congensis, four different varieties of only the Indian species C. jenkinsii. The second major C. canephora, C. arabica and C. liberica. The second cluster divided in to two minor clusters of which the

Fig.5 Dendrogram generated using the unweighted pair group Fig.4 Dendrogram generated using the unweighted pair group method with arithmetic average (UPGMA) analysis showing re- method with arithmetic average (UPGMA) analysis showing relationships among different coffee species using ISSR and SRAP lationships among different coffee species using SRAP data. data. 672 MANOJ KUMAR MISHRA ET AL.

Table 1. Coffee species used in the study with their code, origin and conservation status Coffee Species Species code Place of origin/distribution Conservation status Coffea congensis A. Froehner C1 West Central Africa Congo Least concern C. canephora Pierre ex A. Froehner C2 West Tropical Africa Least concern C. canephora var. laurentii (De Wild.) A.Chev. C3 West Tropical Africa Least concern C. canephora var. ugandae (Cramer) A.Chev. C4 West Tropical Africa Least concern C. canephora var. quillon Philipp. C5 West Tropical Africa Least concern C. arabica cv. Kents L. C6 Ethiopia (Indian cultivar) Vulnerable C. eugenioides Moore C7 West Central Africa Least concern C. zanguebariae Lour. C8 East Tropical Africa Vulnerable C. racemosa Lour. C9 Southern Tropical Africa Near Threatened C. salvatrix Swynn.&Philipson C10 East Tropical Africa Near Threatened C. kapakata (A.Chev.) Bridson C11 West Angola Vulnerable C. stenophylla G.Don C12 West Tropical Africa Least concern C. abeokutae Cramer Ex De Wild. C13 West Tropical Africa Least concern C. liberica Bull.ex Hiern C14 West Tropical Africa Least concern C. dewevrei De Wild. & T.Durand C15 Democratic Republic of Congo Vulnerable C. arnoldiana De Wild. C16 West Central Africa Least concern C. dewevrei var. aruwimiensis (De Wild.) A.Chev. C17 West Central Africa Least concern C. dewevrei var. Excelsa (A.Chev.) C18 West Central Africa Least concern C. bengalensis Roxb.ex Schult.) C19 India endangered C. travancorensis Wight &Arn C20 India endangered C. khasiana (Korth.) Hook.f. C21 India endangered C. jenkinsii Hook.f. C22 India endangered C. wightiana Wall. Ex Wight &Arn C23 India endangered Canthium diccocum (Gaertn) C24 South Asia Threatened

Table 2. Primer sequences used for SRAP analysis of coffee species

Forward primer (5’ – 3’) Reverse primer (5’ – 3’) Me1TGAGTCCAAACCGGATA Em2 GACTGCGTACGAATTTGC Me2 TGAGTCCAAACCGGAGC Em3 GACTGCGTACGAATTGAC Me3 TGAGTCCAAACCGGAAT Em4 GACTGCGTACGAATTTGA Me4 TGAGTCCAAACCGGACC Em5 GACTGCGTACGAATTAAC Me6 TGAGTCCAAACCGGACA Em6 GACTGCGTACGAATTGCA Me9 TGAGTCCAAACCGGAGG Em7 GACTGCGTACGAATTCAA Me10 TGAGTCCAAACCGGAAA Em9 GACTGCGTACGAATTCAG Me11 TGAGTCCAAACCGGAAC Em10 GACTGCGTACGAATTCAT Em11 GACTGCGTACGAATTCTA Em12 GACTGCGTACGAATTCTC Em13 GACTGCGTACGAATTCTG Em14 GACTGCGTACGAATTCTT Em15 GACTGCGTACGAATTGAT Em16 GACTGCGTACGAATTGTC first minor cluster is again divided in to two sub-mi- sis including C. diccocum and the second sub-minor nor clusters. The first sub-minor cluster consisted of cluster included C. bengalensis, C. travancorensis and C. zanguebariae, C. racemosa, C. stenophylla, C. abe- C. wightiana whereas C. khasiana is represented in okutae, C. dewevrei and C. dewevrei var. aruwimien- the second minor cluster independently. Molecular based diversity analysis of coffee species 673

Table 3. Polymorphism obtained by RAPD, ISSR and SRAP analysis in coffee species

Primer/ primer Total Number of bands in No. of Poly- Percentage of poly- Size range (bp) RP PIC combinations bands each species morphic bands morphism ISSR UBC- 810 15 150-2000 0-9 (5.25) 15 100 10.50 0.817 UBC - 811 14 300-1800 1-8 (4.0) 14 100 8.0 0.865 UBC- 826 15 300-2200 0-9 (3.20) 15 100 6.42 0.932 UBC- 834 14 250-1900 1-8 (3.58) 14 100 7.17 0.875 UBC-835 20 200-2500 0-9 (3.5) 20 100 7.0 0.941 UBC-836 15 150-2000 0-7 (3.21) 15 100 6.42 0.921 UBC-840 16 200-1900 0-6 (2.04) 16 100 4.08 0.963 UBC-841 15 200-2000 0-10 (3.63) 15 100 7.25 0.906 UBC-842 14 200-1450 2-6 (4.08) 14 100 8.17 0.891 UBC-855 22 300-2900 2-12 (4.96) 20 90.90 9.92 0.960 UBC-880 15 150-2000 0-9 (3.33) 15 100 6.67 0.929 UBC-881 12 100-2500 11-12 (11.08) 1 8.3 22.17 0.083 Total 187 17-105 (51.86) 174 103.77 10.083 Average 15.58 1.41 – 8.75 (4.32) 14.5 93.06 8.64 0.840 SRAP Me1-Em2 12 50-2200 3-9 (6.16) 10 83.33 12.33 0.610 Me1-Em4 20 50-2900 3-10 (5.958) 19 95.0 11.92 0.848 Me1-Em6 14 250-2600 1-7 (4.13) 13 92.85 8.25 0.838 Me1-Em7 10 75-1400 2-6 (3.16) 9 90.0 6.33 0.807 Me1-Em12 23 75-1700 0-8 (4.04) 23 100 7.08 0.960 Me2-Em3 12 100-2000 1-7 (5.12) 12 100 10.25 0.716 Me2-Em4 15 150-2500 2-10 (5.70) 14 93.33 11.42 0.779 Me2-Em6 18 150-2500 2-10 (6.08) 16 88.88 12.17 0.787 Me2-Em10 12 75-3500 1-5 (3.04) 12 100 6.08 0.855 Me2-Em12 16 50-2000 1-6 (3.45) 16 100 6.02 0.879 Me3-Em3 19 75-2000 4-12 (6.91) 18 94.73 14.58 0.747 Me3-Em4 17 50-2200 3-8 (5.70) 16 94.11 11.58 0.804 Me3-Em7 12 100-3000 2-6 (3.75) 11 91.66 7.50 0.787 Me3-Em11 17 100-1500 1-6 (3.33) 16 94.11 6.67 0.961 Me3-Em12 18 200-2000 2-6 (4.0) 18 100 8.0 0.905 Me4-Em1 12 50-2200 0-6 (1.91) 12 100 3.83 0.938 Me4-Em2 18 100-2500 2-11 (6.83) 18 100 13.67 0.739 Me6-Em3 14 100-2500 2-9 (5.79) 14 100 11.58 0.726 Me6-Em5 20 100-2500 2-10 (6.46) 20 100 12.92 0.789 Me6-Em8 18 100-1500 2-10 (6.16) 18 100 12.33 0.782 Me10-Em13 19 100-2000 1-8 (5.0) 18 94.73 10.0 0.890 Total 336 37-169 (102.67) 323 204.51 17.14 Average 16.00 1.76 – 8.04 (4.88) 15.38 96.12 9.738 0.816

SRAP polymorphism and species identification erence species, C. decorum, of which 21 primer combinations are found to be highly polymorphic and Sixty SRAP primer combinations were initially produce clear amplification patterns. These 21 prim- screened against 23 coffee species along with the ref- ers could produce 336 distinct scorable bands with 674 MANOJ KUMAR MISHRA ET AL.

Тable 4. Species- diagnostic ISSR and SRAP markers in coffee species

Species ISSR marker SRAP marker C. congensis --- Me6-Em3-700, Me6-Em8- 550, Me3-Em12-400 C. canephora --- Me6-Em5-1030, Me6-Em5-75 C. canephora var. laurentii UBC 855 Me1-Em4-250, Me3-Em3- 375, Me2-Em3-500, 250 C. canephora var. ugandae Me6-Em5-1200 C. canephora var. quillon UBC-855 Me4-Em1 1350, Me6-Em3-1500, Me6-Em5-800 UBC-841 UBC-826 Me1-Em7-700, Me4-Em1 600, Me6-Em5-1500, Me10- C. arabica cv. Kents UBC-811 Em13-1200, Me3-Em11- 550, Me3-Em12- 1200 UBC-880 Me1-Em4-150, Me1-Em6-800, Me2-Em3-350, Me2-Em4-375, C. eugenioides UBC-841 Me2-Em6-375, Me3-Em7 500, 300, Me4-Em1 850, C. zanguebariae UBC-836 C. racemosa ----- Me3-Em7 600, Me1-Em4-200, Me2-Em6-550, Me3-Em4-1400, 520, Me4-Em2 UBC-835 C. salvatrix 800, Me6-Em3-500, Me6-Em5-150, Me1-Em4 240, Me2-Em12 UBC-834 1200, 550, 400, Me2-Em10 550, Me3-Em11- 1500,1100,900 C. kapakata ------Me4-Em2 1030, Me6-Em8-1250 C. stenophylla ------Me6-Em5-300, 220, Me10-Em13-800, Me3-Em11- 750 C. abeokutae ------Me3-Em12- 680 UBC-811 C. liberica Me1-Em6-900, Me1-Em7-650, Me6-Em5-200, Me2-Em10 220, UBC-880 C. dewevrei ------Me2-Em4-2000 Me2-Em6-900, Me6-Em5-1300, Me6-Em8-700, Me2-Em10 C. arnoldiana ------1000, C. dewevrei var. aruwimiensis ------Me3-Em4-1600, Me1-Em12 680, Me10-Em13-330, Me2-Em10 C. dewevrei var. excelsa UBC-836 150 UBC-811 Me1-Em6-300, Me2-Em12 400, Me10-Em13-1100, 750, 350, C. bengalensis UBC-880 Me2-Em10 750, Me3-Em12- 2000 C. travancorensis ------Me4-Em2 300, Me6-Em5-600 UBC-836 UBC-842 Me2-Em3-100, Me3-Em7 350, Me6-Em8-500, Me2-Em12 175, C. khasiana UBC-855 Me10-Em13-600, Me3-Em11- 1030 UBC-880 UBC-836 Me1-Em2-100, Me1-Em7-450, Me3-Em4-75, Me4-Em2 400, UBC-840 Me6-Em3-820, Me6-Em5-250, Me1-Em12 720, 400, , Me2- C. jenkinsii UBC-834 Em12 120, Me10-Em13-450, 170, Me2-Em10 600, Me3-Em11- UBC-855 600, 500, Me3-Em12- 1000, 650 UBC-810 Me1-Em4-550, Me2-Em12 480, Me10-Em13-270, Me3-Em12- C. wightiana UBC-836 1350 Canthium diccocum UBC-855 Me3-Em3-1450, Me4-Em1 700, Me3-Em12- 220 the number of amplified fragments ranging from 10 bands, 323 were polymorphic, with an average 15.38 (Me1-Em7) to 23 (Me1-Em12) with an average of 16 polymorphic fragments per primer combination. per primer combination (Table 3). The size of the am- The percent of polymorphism ranged from 83.33% plified products ranged from 50 to 3500 bp in differ- to a maximum of 100% with an average of 96.12% ent primer combinations. Of the total 336 amplified polymorphism. Of the total 21 primer combinations, Molecular based diversity analysis of coffee species 675

Table. 5 Similarity co-efficient of coffee species using ISSR marker (DICE)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C1 1.00 C2 0.89 1.00 C3 0.86 0.89 1.00 C4 0.83 0.85 0.83 1.00 C5 0.86 0.85 0.87 0.88 1.00 C6 0.74 0.7 0.69 0.71 0.73 1.00 C7 0.57 0.59 0.53 0.56 0.58 0.66 1.00 C8 0.39 0.39 0.37 0.39 0.36 0.37 0.54 1.00 C9 0.39 0.39 0.37 0.41 0.38 0.35 0.55 0.84 1.00 C10 0.57 0.57 0.56 0.61 0.6 0.63 0.62 0.41 0.4 1.00 C11 0.62 0.59 0.59 0.61 0.63 0.69 0.71 0.46 0.4 0.7 1.00 C12 0.52 0.49 0.45 0.55 0.48 0.44 0.65 0.57 0.6 0.41 0.48 1.00 C13 0.51 0.51 0.46 0.53 0.49 0.43 0.64 0.56 0.63 0.47 0.47 0.81 1.00 C14 0.71 0.66 0.69 0.7 0.71 0.64 0.54 0.38 0.36 0.61 0.64 0.46 0.47 1.00 C15 0.59 0.59 0.57 0.6 0.62 0.53 0.68 0.52 0.54 0.53 0.6 0.62 0.67 0.63 1.00 C16 0.57 0.57 0.57 0.59 0.57 0.64 0.54 0.4 0.39 0.64 0.71 0.42 0.45 0.65 0.53 1.00 C17 0.5 0.48 0.47 0.5 0.49 0.48 0.63 0.5 0.55 0.52 0.52 0.63 0.68 0.5 0.68 0.63 1.00 C18 0.59 0.58 0.59 0.58 0.61 0.62 0.55 0.39 0.39 0.63 0.67 0.43 0.46 0.67 0.54 0.82 0.6 1.00 C19 0.3 0.32 0.31 0.32 0.3 0.3 0.39 0.52 0.57 0.33 0.36 0.45 0.47 0.27 0.42 0.38 0.57 0.37 1.00 C20 0.41 0.43 0.41 0.41 0.4 0.42 0.48 0.58 0.58 0.4 0.4 0.52 0.54 0.38 0.43 0.41 0.6 0.45 0.62 1.00 C21 0.4 0.38 0.34 0.36 0.35 0.38 0.48 0.46 0.51 0.38 0.41 0.55 0.51 0.39 0.44 0.38 0.49 0.41 0.43 0.41 1.00 C22 0.52 0.48 0.49 0.48 0.51 0.49 0.43 0.31 0.31 0.47 0.54 0.35 0.36 0.5 0.42 0.52 0.34 0.53 0.35 0.36 0.33 1.00 C23 0.47 0.49 0.47 0.46 0.45 0.46 0.42 0.49 0.45 0.38 0.42 0.44 0.46 0.46 0.42 0.45 0.47 0.47 0.51 0.69 0.34 0.39 1.00 C24 0.45 0.47 0.41 0.47 0.44 0.39 0.55 0.59 0.65 0.39 0.42 0.56 0.61 0.4 0.51 0.42 0.55 0.39 0.57 0.46 0.51 0.39 0.44 1.00

10 primer combinations showed 100% polymor- coffee species out of which 5 Indian species gener- phism. The resolving power (RP) of 21 SRAP primer ated 30 unique fragments. Among the Indian spe- combinations ranged from 3.83 (Me4-Em1) to 14.58 cies, the maximum number of unique fragments (Me3-Em3) with an average of 9.74. Similarly, the was generated by C. jenkinsii (16) followed by C. polymorphism information content (PIC) or the khasiana (8) and the least number of unique frag- genetic diversity of 21 SRAP primer combinations ments was in C. travancorensis (1) which could be ranged from 0.61 (Me1-Em2) to 0.96 (Me1-Em12, used as a unique fingerprinting tools. The genetic Me3-Em11) with an average of 0.82. similarity derived from the data of the SRAP marker analysis varied from 0.11 between C. wightiana Only three SRAP primer pairs could discrimi- and C. congensis to 0.90 between C. canephora and nate all the species independently, although none of C. canephora var. laurentii (Table 6). Among the these primers could amplify species-specific frag- Indian species, maximum similarity was observed ments for all the 23 species (Fig. 3). 20 SRAP primer between C. travancorensis and C. wightiana and the combinations generated 75 unique fragments in 23 least genetic similarity was obtained between C. 676 MANOJ KUMAR MISHRA ET AL.

Table.6 Similarity co-efficient of Indian coffee species using SRAP marker (DICE)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C1 1.00 C2 0.86 1.00 C3 0.86 0.90 1.00 C4 0.78 0.83 0.82 1.00 C5 0.77 0.83 0.84 0.86 1.00 C6 0.71 0.74 0.77 0.76 0.79 1.00 C7 0.53 0.52 0.57 0.56 0.57 0.64 1.00 C8 0.45 0.50 0.51 0.58 0.58 0.56 0.56 1.00 C9 0.44 0.50 0.50 0.58 0.56 0.55 0.55 0.94 1.00 C10 0.45 0.47 0.51 0.52 0.53 0.54 0.54 0.62 0.60 1.00 C11 0.57 0.60 0.62 0.62 0.61 0.67 0.64 0.63 0.64 0.66 1.00 C12 0.47 0.51 0.53 0.58 0.58 0.56 0.57 0.73 0.73 0.56 0.66 1.00 C13 0.50 0.55 0.56 0.62 0.62 0.58 0.57 0.81 0.81 0.57 0.64 0.75 1.00 C14 0.64 0.66 0.67 0.64 0.63 0.60 0.55 0.40 0.39 0.50 0.58 0.43 0.42 1.00 C15 0.59 0.63 0.65 0.69 0.71 0.60 0.59 0.74 0.73 0.61 0.64 0.64 0.72 0.57 1.00 C16 0.51 0.55 0.58 0.62 0.62 0.60 0.59 0.67 0.66 0.59 0.63 0.67 0.68 0.56 0.71 1.00 C17 0.47 0.52 0.54 0.59 0.61 0.57 0.57 0.77 0.78 0.61 0.61 0.67 0.78 0.45 0.81 0.72 1.00 C18 0.57 0.59 0.62 0.65 0.67 0.62 0.59 0.70 0.69 0.61 0.68 0.68 0.71 0.57 0.73 0.79 0.76 1.00 C19 0.44 0.48 0.50 0.49 0.52 0.49 0.49 0.61 0.61 0.44 0.53 0.57 0.63 0.38 0.60 0.57 0.64 0.65 1.00 C20 0.49 0.52 0.52 0.53 0.57 0.52 0.50 0.62 0.60 0.49 0.56 0.58 0.62 0.43 0.59 0.57 0.62 0.67 0.67 1.00 C21 0.44 0.48 0.50 0.50 0.53 0.53 0.51 0.62 0.64 0.53 0.57 0.59 0.63 0.44 0.62 0.61 0.63 0.66 0.64 0.63 1.00 C22 0.41 0.43 0.48 0.48 0.51 0.49 0.49 0.57 0.56 0.42 0.46 0.50 0.52 0.40 0.57 0.50 0.59 0.57 0.50 0.59 0.57 1.00 C23 0.11 0.17 0.17 0.17 0.16 0.16 0.16 0.16 0.17 0.14 0.19 0.19 0.16 0.14 0.16 0.17 0.18 0.18 0.27 0.33 0.23 0.18 1.00 C24 0.44 0.50 0.51 0.51 0.53 0.52 0.55 0.71 0.69 0.52 0.56 0.61 0.66 0.45 0.66 0.61 0.69 0.66 0.67 0.65 0.73 0.60 0.22 1.00

khasiana and C. jenkinsii based on the SRAP mark- aruwimiensis, C. stenophylla, C. arnoldiana and C. er analysis. dewevrei var. excelsa, whereas the second group included three Indian species viz. C. bengalensis, C. The UPGMA clustering algorithm from the travancorensis, C. khasiana along with Canthium SRAP analysis grouped the 23 coffee species and C. diccocum. A strict consensus tree based on both diccocum into two major clusters (Fig. 4). The first ISSR and SRAP data were constructed (Fig. 5). This major cluster was represented by C. wightiana and dendrogram shows maximum similarity to ISSR- the second major was divided into two minor clus- based dendrogram except that the position of C. ar- ters of which the first minor cluster consisted of C. noldiana and C. dewevrei var. excelsa was taken over jenkinsii alone. The second minor cluster divided by C. zanguebariae and C. racemosa, respectively, in to two sub-minor clusters. The first sub-minor in the first major sub-cluster. The genetic similari- cluster included C. congensis, C. canephora varieties obtained using both ISSR and SRAP data has ties, C. arabica and C. liberica, whereas the second revealed the highest similarities (0.90) between C. sub-minor cluster further dived into two groups. canephora and C. canephora var. laurentii and the The first group included C. zanguebariae, C. race- lowest similarities (0.36) between C. liberica and C. mosa, C. abeokutae, C. dewevrei, C. dewevrei var. bengalensis (Table 7). Molecular based diversity analysis of coffee species 677

Table. 7 Similarity co-efficient of coffee species using combined ISSR and SRAP markers (DICE)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C1 1.00 C2 0.87 1.00 C3 0.86 0.90 1.00 C4 0.80 0.84 0.82 1.00 C5 0.81 0.84 0.85 0.87 1.00 C6 0.72 0.72 0.74 0.74 0.76 1.00 C7 0.54 0.54 0.55 0.56 0.57 0.65 1.00 C8 0.43 0.47 0.46 0.53 0.51 0.49 0.55 1.00 C9 0.43 0.46 0.46 0.54 0.51 0.48 0.55 0.92 1.00 C10 0.50 0.51 0.53 0.55 0.55 0.58 0.57 0.56 0.55 1.00 C11 0.59 0.59 0.61 0.62 0.62 0.68 0.66 0.58 0.57 0.68 1.00 C12 0.48 0.51 0.50 0.57 0.55 0.52 0.59 0.69 0.70 0.52 0.61 1.00 C13 0.50 0.54 0.53 0.60 0.59 0.53 0.59 0.75 0.77 0.54 0.59 0.77 1.00 C14 0.67 0.66 0.68 0.66 0.66 0.62 0.55 0.39 0.38 0.54 0.60 0.44 0.44 1.00 C15 0.59 0.62 0.62 0.66 0.68 0.58 0.61 0.69 0.69 0.58 0.62 0.64 0.71 0.59 1.00 C16 0.53 0.56 0.57 0.61 0.60 0.61 0.57 0.58 0.58 0.61 0.66 0.59 0.61 0.60 0.65 1.00 C17 0.48 0.50 0.51 0.56 0.57 0.54 0.59 0.70 0.73 0.58 0.58 0.66 0.76 0.47 0.78 0.68 1.00 C18 0.58 0.59 0.61 0.62 0.65 0.62 0.58 0.60 0.60 0.62 0.68 0.60 0.63 0.61 0.67 0.81 0.70 1.00 C19 0.41 0.44 0.45 0.45 0.47 0.44 0.48 0.60 0.62 0.42 0.49 0.55 0.60 0.36 0.57 0.52 0.64 0.56 1.00 C20 0.46 0.48 0.48 0.49 0.51 0.48 0.49 0.60 0.58 0.46 0.50 0.56 0.59 0.41 0.54 0.51 0.61 0.58 0.68 1.00 C21 0.43 0.44 0.43 0.44 0.46 0.46 0.49 0.56 0.59 0.48 0.50 0.57 0.58 0.41 0.56 0.51 0.58 0.56 0.57 0.54 1.00 C22 0.44 0.44 0.47 0.47 0.50 0.48 0.44 0.46 0.46 0.43 0.48 0.44 0.45 0.45 0.50 0.50 0.48 0.54 0.45 0.48 0.47 1.00 C23 0.41 0.43 0.44 0.40 0.41 0.42 0.39 0.40 0.40 0.35 0.40 0.40 0.40 0.39 0.39 0.41 0.43 0.43 0.48 0.63 0.39 0.41 1.00 C24 0.44 0.49 0.47 0.49 0.50 0.47 0.55 0.68 0.68 0.48 0.51 0.60 0.64 0.43 0.62 0.54 0.65 0.56 0.66 0.59 0.64 0.51 0.42 1.00

DISCUSSION Similar problems were experienced while developing species-specific ISSR markers in eucalyptus (Bala- Analysis of crop genetic diversity is very important saravanan et al. 2006). This could be due to the oc- for breeding and conservation programs, and molec- currence of a high divergence among the particular ular markers offer an approach to unveil the genetic species population and/or the low number of ISSR diversity among different species and cultivars based primers used in this study. In contrast to the ISSR on nucleic acid polymorphisms. In this study, both markers, SRAP is more effective and amplified spe- ISSR and SRAP marker systems were simultaneously cies specific markers in all the coffee species except used to investigate the genetic diversity among 23 C. zanguebariae, and C. dewevrei var. aruwimiensis coffee species available in the germplasm collection (Table. 4). of India. The results showed that both ISSR and SRAP markers were suitable for genetic diversity analysis Both genetic factors and selection pressure in- in coffee by amplifying several species specific diag- fluence genetic diversity (Sun and Lin 2003). Since nostic markers. Species specific ISSR primers were various coffee species have their origin in different generated in eucalyptus (Balasaravanan et al. 2006) agro-climatic zones in their native habitat, with vary- and oak (Carvalho et al. 2009). In the present study, ing selection pressure during the course of evolution, species-diagnostic ISSR markers were identified in it is therefore not surprising to find wide polymor- 12 coffee species including all the indigenous coffee phism among different coffee species. The percent- species from India except C. travancorensis (Table 4). age of polymorphic bands detected by SRAP primer 678 MANOJ KUMAR MISHRA ET AL. combinations (96.12%) was higher compared to their inclusion in the genus Coffea rather than plac- ISSR (93.06%). Similarly, the number of polymor- ing them under the genus Psilanthus or Nostolachma, phic bands detected by SRAP primer combinations as suggested by previous workers (Cross et al., 1998; (15.38) was higher than that obtained by the ISSR Lashermes et al. 1997; Andreasen et al. 1999, An- primer (14.5). This indicated that SRAP markers are dreasen and Bremer, 2003 and Davies et al. 2007). more efficient than ISSR markers in genetic diversity Further understanding of the level and partitioning analysis of coffee. of genetic variation within the species would provide an important input in designing appropriate breed- The average PIC of the SRAP primers (0.82) was ing exercises and conservation strategies. less than that of the ISSR primers (0.84), whereas the average RP of the SRAP primers (9.74) was higher REFERENCES than that of the ISSR primers (8.64). The difference Andreasen, K., and B. Bremer (2000). Combined phylogenetic in PIC and RP values among ISSR and SRAP mark- analysis in the Rubiaceae-Ixoroidae: morphology, nuclear ers is expected because both markers differ in their and chloroplast DNA data. American Journal of Botany 87, operational principle and each marker targets a dif- 1731-1748. ferent region of the genome. 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