Assessment of genetic variability within and among coffee progenies and cultivars using RAPD markers

The RAPD technique associated with restriction digestion of genomic DNA was used to assess the genetic variability within and among nine populations of Coffea arabica, including six progenies belonging to the Sarchimor germplasm, the progeny PR 77054-40-10 (Catuaí Vermelho IAC 81 x Icatu), and two commercial cultivars (IAPAR 59 and Catuaí Vermelho IAC-81). These populations were evaluated using analysis of molecular variance (AMOVA), genetic similarity among progenies, and percentage of polymorphic loci. A total of 99 RAPD markers were evaluated of which 67 (67.67%) were polymorphic. AMOVA showed that 38.5% and 61.5% of the genetic variation was distributed among and within populations, respectively. The fixation index (FST) of the genotypes was 0.385. The mean genetic variability estimated within populations ranged from 15.58 (IAPAR 59) to 8.27 (Catuaí Vermelho IAC 81). A distinct level of genetic variability was revealed for each of the coffee progenies and varieties studied. The methodology used in this investigation was useful to determine the genetic variability within and among C. arabica L. populations providing significant information for coffee breeding.


Introduction
Coffea arabica L. is the most important species of the Coffea genus, followed by C. canephora P. Nowadays, three Brazilian varieties are grown worldwide, Mundo Novo (derived from Bourbon x Sumatra crossing), Caturra (a dwarf mutant of Bourbon), and Catuaí (derived from Caturra x Mundo Novo crossing).These cultivars are highly productive and exhibit an exceptional quality beverage (Anthony et al., 2001).The exploitation of genetic resources from the wild Coffea is essential for the development of inbred lines, which can be adapted to new production systems and also to overcome possible challenges from the world market (Carvalho et al., 1985).Nevertheless, breeding programs are limited due to the narrow genetic base of coffee, especially for pest and disease resistance improvement (Van der Vossen, 1985).Nowadays, C. canephora provides the main source for resistance genes not found in C. arabica, including leaf rust (Hemileia vastatrix), coffee berry disease (CBD) caused by Colletotrichum kahawae, and resistance to root-knot nematode (Meloidogyne sp) (Lashermes et al., 2000).
Natural and artificial hybrids derived from C. arabica x C. canephora have been intensively used in breeding programs.A good example is the Timor Hybrid, which has been exploited as a bridge to transfer rust resistance genes from C. canephora into cultivars of C. arabica (Fazuoli et al., 1996).The Timor Hybrid is an atypical tree, which was identified in a C. arabica field on the island of Timor (Bettencourt, 1973).Information about coffee germplasm introduced into Timor island suggests, on the basis of its limited fertility associated to the characteristics of disease resistance and molecular data of the original plants, that the Timor Hybrid was derived from a spontaneous interspecific cross between C. arabica and C. canephora (Lashermes et al., 2000).In 1972, the Centro de Investigações das Ferrugens do Cafeeiro (CIFC, Oeiras, Portugal) sent descendants to several producer countries that were derived from the Timor Hybrid .These plants were then crossed with C. arabica cv Villa Sarchi giving origin to the Sarchimor germplasm.
The employment of molecular markers to access the genetic variability in accessions of C. arabica has shown some difficulties due to the lack of genetic variation as observed with isozyme (Lopes, 1993) and RFLP markers (Paillard et al., 1996).Nevertheless, based on RFLP of cpDNA Lashermes et al. (1996a) suggested a recent speciation for the genus Coffea.Also, RAPD markers were an effective method for providing the genetic identification of Coffea accessions (Lashermes et al., 1996b).Diniz (2000) showed that the association of the RAPD technique with a prior digestion of the genomic DNA with restriction enzymes improved the assessment of genetic polymorphism in 40 C. arabica varieties.
In this investigation, the RAPD technique associated with a prior digestion of the DNA template was employed for the identification of the genetic variability within and among coffee populations and to estimate the percentage of polymorphic loci of the assay E9601, including six progenies of Sarchimor germplasm , the progeny PR 77054-40-10, and the cultivars IAPAR 59 and Catuaí Vermelho IAC 81.

Material and Methods
Nine coffee populations were studied.They derived from the assay E9601 performed by the Instituto Agronômico do Paraná (IAPAR), located in South Brazil.The main purpose of IAPAR in developing this assay was to search for cultivars that were resistant to orange leaf rust and adapted to the environmental, economic, and technological conditions of the plantations in Paraná State.The populations included six progenies of IAPAR 77028-11, a Sarchimor germplasm in F 5 generation, two varieties represented by IAPAR 59 and Catuaí Vermelho IAC 81, and one F 5 progeny (PR 7705-40-10) derived from Icatu x Catuaí Vermelho IAC 81.

DNA extraction, amplification, and electrophoresis
Young leaves were collected from three plants of the first and second repetitions and four plants from the third repetition with a total of 10 samples for each treatment, as described by Huff et al. (1994).Genomic DNA was isolated following the method described by Doyle and Doyle (1987), except that CTAB was replaced by MATAB (Mixed Alyltrimethylammonium Bromide, Sigma).DNA concentration was estimated using a fluorometer (DyNA Quant 200, Höefer-Pharmacia), according to the manufacturer's instructions.DNA samples of the 90 individual plants were adjusted to 10 ng/µL and used in the amplification reactions with a final volume of 15 µL containing buffer 1x (75 mM Tris-/HCl, pH 9.0, 50 mM KCl, 2.0 mM MgCl 2 , and 20 mM (NH 4 ) 2 SO 4 ), 0.2 mM each of dATP, dTTP, dCTP, and dGTP; 0.4 µM of primer (Operon Technologies), 0.9 U of Taq DNA Polymerase (Biotools), and 20 ng template DNA.For digestion, genomic DNA was incubated just before amplification, with one of the following enzymes Bam HI, Eco RI, Hae III, Hind III, and Rsa I, added directly to the RAPD reaction.DNA amplification was carried out using a PTC 100 (MJ Research) thermal cycler, programmed with 60 min at 37 °C for prior digestion of the DNA template then the thermal cycler was programmed with 3 min at 94 °C for initial DNA denaturation, followed by 48 cycles of 1 min at 94 °C, 1 min 45 s at 38 °C, and 2 min at 72 °C.The last cycle was followed by a 7 min extension at 72 °C.The samples were then stored at 4 °C until electrophoresis.Amplified products were resolved in 1.4 % agarose gels in 1x TAE (40 mM Tris-acetate, 1 mM EDTA pH 8.0) buffer, at 120 V for 3 h and stained with ethidium bromide.The RAPD profiles were visualized under UV light, and photographed using a video camera (Cohu, High Performance CCD Camera).

Statistical analysis
The markers obtained were assessed for the presence (1) or absence (0) of homologous DNA bands among the individuals of each population.The analyses were performed by using only good-quality amplified fragments.
Genetic distance and genetic identity among populations were estimated according to Nei (1978).These parameters and the percentage of polymorphic loci were calculated using the 1.31 POPGENE program (Yeh et al., 1997).A matrix of distance was obtained for each population using the NTSYS-pc software, version 2.1 (Rohlf, 2000).The dendrograms were constructed employing UPGMA (unweighted pair-group method with arithmetic averages) with the SAHN (Sequential Agglomerative, Hierarchical, and Nested Clustering) routine.
The analysis of molecular variance (AMOVA) was calculated to estimate the variation within and among populations using the RAPD polymorphic loci.The data were generated using the Arlequin 1.1 software (Schneider et al., 1997) in which the fixation index (F ST ) was also estimated.The Win AMOVA software (Excoffier et al., 1992;Excoffier, 1993) was used to obtain the square sum of variation within the populations.The bootstrap method was applied by using the DBOOT software version 1.1 (Coelho, 2001) to evaluate the reliability of the tree topology.

Results and Discussion
The RAPD technique combined with prior digestion of DNA samples improved the detection of polymorphism in C. arabica populations.Two hundred RAPD primers were tested with fifteen different restriction enzymes.Eighteen primers or primer/enzyme combinations that rendered polymorphic and reproducible fragments were then selected for DNA amplification of all samples (Table 1).The association of the selected primers with some restriction enzymes in the PCR reaction showed electrophoresis patterns different from those obtained without prior restriction as illustrated in Figure 1.This procedure allows for a straightforward analysis of the amplified products as it changes the relative intensities of the bands (Williams et al., 1990;Riede et al., 1994).Furthermore, digestion of genomic DNA may reveal a restriction site polymorphism located between the primer-annealing sites in an otherwise monomorphic band.In wheat, this method improved the level of polymorphism (Kobner, 1995) and the results were attributed to a greater efficiency of primer annealing along shorter DNA fragments, where a simplified secondary structure is unlikely to interface with the process.Kobner (1995) suggested that these differences occur because distinct annealing sites became accessible, depending on the restriction enzyme employed.Whereas Orozco-Castilho et al. (1994) and Lashermes et al. (1996b) revealed that few RAPD primers were efficient in detecting polymorphism and in generating reproducible DNA fragments in C. arabica, the use of restriction digestion of genomic DNA before amplification with RAPD primers changed the electrophoretic profiles in 40 varieties of C. arabica (Diniz, 2000).In this study, the association of restriction digestion of DNA with RAPD reaction allowed for a more efficient assessment of the existing genetic variability (Figure 1).Thus, the technique is especially suitable for the analysis of genetic differentiation and to detect loss of genetic variation in coffee populations.67.67% were polymorphic.The data were used to estimate the percentage of polymorphic loci for each population (Table 2), the differences within populations and among pairs of populations (Table 3) and for the analysis of molecular variance among populations (Table 4).The square sum of the variations within each population is illustrated in Figure 2. The dendrograms showing the associations among individuals within populations and among all individuals are displayed in Figures 3 and 4, respectively.Bootstrap analysis was used to estimate the number of bands necessary to obtain a stable association of all accessions.It was observed that approximately 70 markers were sufficient for dendrogram stability (coefficient of variation = 4.2%) and the rate of decrease was comparatively minimal beyond that (Figure 5), suggesting that 99 markers were sufficient for the analysis of coffee cultivars and progenies.

Analysis of molecular variation
The RAPD showed that 38.5% and 61.5% of the genetic variation were found among and within the populations, respectively (Table 4).Sarchimor-derived germplasm and in the PR 77054-40-10 progeny.

Analysis of genetic variability within and among the cultivars IAPAR-59 and Catuaí Vermelho IAC 81
The IAPAR 59 cultivar showed a higher percentage of polymorphic loci (42.42%) than the Catuaí Vermelho IAC 81 cultivar (26.26%) (Table 2).The genetic diversity was also higher within the cultivar IAPAR 59 cultivar than within the Catuaí cultivar (Table 3, Figures 2, 3, 4).The origin of these cultivars gives support to these observations.As outlined in Figure 6 as leaf rust resistance.The IAPAR 75163-22 progeny was then nominated IAPAR 59 and released as commercial cultivar (Sera et al., 1996).Catuaí Vermelho IAC 81, on the other hand, is a cultivar that resulted from crossing between the arabica genotypes Mundo Novo and Caturra.Genotypes derived from Timor Hybrid have shown notable genetic diversity, which appears to be approximately twice that detected in the group of cultivated and wild C. arabica accessions (Lashermes et al., 2000).Anthony et al. (2001) applied RAPD markers to the analysis of 88 wild C. arabica accessions from Ethiopia and two accessions derived from genetic populations of C. arabica var arabica and C. arabica var bourbon.According to the authors, the absence of within genetic variation strengthened the evidence of the narrow genetic base of Arabic coffee.Our results denote that much more polymorphism is present in the Sarchimor-derived germplasm than in the Arabic coffee (Catuaí Vermelho IAC 81).The analysis of genetic similarity estimated for the 10 plants of each population ranged from 0.78 to 0.94 and from 0.88 to 0.98 within the IAPAR 59 and Catuaí Vermelho IAC 81 populations, respectively.The lower genetic similarity (Table 3, Figure     genetic variation, which could be useful for breeding programs.

Progenies of Sarchimor germplasm
Besides IAPAR 59, the other progenies of the Sarchimor germplasm studied were also derived from the hybridization between Villa Sarchi (CIFC 971/10) and the Timor Hybrid (CIFC 832/2).The plant C1816 EP141 C1567, selected at IAC, was sent to IAPAR and the F 3 generation was designated as IAPAR 77028.The IAPAR LF 77028-15 progeny was then selected using the genealogical method and named, in the F 5 generation, as IPR 99.The Sarchimor progenies analyzed in this research are sister progenies of IPR 99.
The values of the genetic similarities observed within the Sarchimor-derived progenies ranged from 0.78 (within IAPAR 59) to 0.99 .The percentage of polymorphic loci and the level of within genetic variation showed that IAPAR 77028-11-1 and IAPAR 59 display similar levels of polymorphism, whereas IAPAR 77028-11-2 is more uniform (Tables 2, 3; Figure 3).The differences in genetic variability observed within the Sarchimor progenies are probably the result of both genetic segregation and selection that occurred in the origin of these progenies.The differences among the Sarchimor progenies revealed a smaller genetic difference (4.92) between IAPAR 59 and the IAPAR 77028-11-1 progeny and higher divergence (8.24) between the IAPAR 59 and the IAPAR 77028-11-6 progeny (Table 3, Figure 4).The genetic divergence between IAPAR 59 and IAPAR 77028-11-6 was accompanied by the loss of resistance that resulted in the appearance of new physiological races for the leaf rust disease.Hence, IAPAR 77028-11-6 probably carries fewer genes for leaf rust resistance in relation to the other genotypes analyzed.On the other hand, progenies that maintain genetic resistance are more related to the IAPAR 59 variety (Table 2) as for example, the progeny IAPAR 77028-11-1.The mean genetic differentiation was higher within the Sarchimor-derived progenies than within the Catuaí cultivar, probably because of the narrow genetic base of Catuai coffee.

PR 77054-40-10 progeny
The analysis of the PR 77054-40-10 progeny revealed that the values of within genetic similarity and the percentage of polymorphic loci are similar to those of the Sarchimor progenies (Tables 2, 3; Figures 2, 3).As expected, the PR 77054-40-10 progeny was more related to the cultivar Catuaí Vermelho IAC-81 than any other progeny evaluated (Figure 3).The PR 77054-40-10 progeny was obtained by selection from progenies derived by crossing between plants of the Catuaí and Icatu gene pools.The Icatu germplasm derived from interspecific hybridization between C. canephora var robusta and C. arabica var bourbon (Arabic cultivar) followed by crossing of the F 1 prog-eny with plants of the Mundo Novo (Bourbon x Sumatra) germplasm.Therefore, the genetic diversity conserved within the PR 77054-40-10 progeny (Table 3) is derived from the combination of genes from the C. arabica and the C. canephora gene pools.

Exploitation hybrids in coffee
It is acknowledged that there is a strong association between heterozigosity and heterosis and this phenomenon has led to the success of the improvement in crop yields (Duvick, 1984).Hybrid varieties have revolutionized crop production, including cross-and self-pollinated species.The usefulness of RAPD markers in the selection of hybrids with high productivity was demonstrated in rice (Xiao et al., 1996).Lanza et al. (1997) described a positive correlation between RAPD based genetic distance and singlecross hybrid grain yield of tropical maize inbred lines.In coffee, we demonstrate that the IAPAR 77028-11-4 and IAPAR 77028-11-6 progenies are the most divergent among the Sarchimor progenies (Table 2, Figure 3).Therefore, plants of these progenies may be exploited to potentialize the hybrid performance of the Sarchimor germplasm.The same approach may be used for individual plants of the six Sarchimor progenies, PR 77054-40-10 progeny, and also the IAPAR 59 and Catuaí Vermelho IAC 81 cultivars (Table 3, Figures 2, 3).Diniz (2000) demonstrated that IAPAR 59 and Mundo Novo exhibit low genetic similarity (75%).The association between genetic divergence and heterosis was demonstrated in coffee by combining the IAPAR 59 and Mundo Novo cultivars.Hybridization between these cultivars produced a heterosis of 25% (Sera, personal communication) that was possibly the result of different allele combination in the hybrid.Thus the RAPD-derived markers could be used to obtain hybrids between plants of the same progeny and between progenies or varieties of the germplasms studied.
The RAPD markers provided useful information to study genetic variability within and among C. arabica populations.The IAPAR 59 cultivar presented more genetic variation than Catuaí Vermelho IAC 81.This difference is probably the result of the hybrid origin of IAPAR 59.Furthermore, it was verified that genotypes which are more susceptible to leaf rust disease also exhibit higher genetic distance from the genotypes that maintain a high number of genes for resistance such as the IAPAR 59 cultivar.It was also evident that the level of genetic variability is not the same for the six Sarchimor progenies.The overall results of this investigation demonstrated that all genotypes studied could provide valuable material for coffee breeding.grant number 463861/00-0, and Fundação Araucária for financial support.
Figure 1 -Amplification profile of partial DNA coffee samples obtained with OPP-18 RAPD primer used alone (top) and in combination with restriction enzyme Hae III (bottom).
These data were supported by a F ST = 0.385 which measures the magnitude of genetic differentiation among populations.It has been demonstrated that the range of F ST variation for autogamous species is large, varying from 0.026 to 0.78 as shown in wild bean (Cattan-Toupance et al., 1998).Anthony et al. (2001) determined a value of G ST > 0.50 for 88 C. arabica accessions, collected by FAO in 1968 from forests and farms in Ethiopia, and two accessions derived from genetic populations of C. arabica var arabica and C. arabica var bourbon.According to Nei (1977), the G ST and the F ST of Wright (1965) are equivalent values.The genotypes studied in this research were submitted to several selection cycles, a fact that should have contributed to the reduction of F ST value (F ST = 0.385).As demonstrated by the FST value obtained in this investigation, the coffee genotypes still display high levels of genetic polymorphism.The overall genetic variation probably derived from DNA markers introgressed from the C. canephora gene pool, which are present in the 332 Silveira et al.
3) observed among Catuaí Vermelho IAC 81 plants (Arabic germplasm), when compared to IAPAR 59 plants (Sarchimor germplasm), is consequent of the narrow genetic base of the Arabic gene pool.Nevertheless, it was evident that the Catuaí genotypes studied still display enough 334 Silveira et al.

Figure 4 -
Figure 4 -Genetic relationships obtained among plants of progenies and cultivars of C. arabica on the basis of Dice's similarity coefficient.

Figure 5 -
Figure 5 -Sample variance for the Sarchimor progenies and cultivars of C. arabica, derived from a bootstrap procedure after 1000 sampling, illustrating the relationship between the mean coefficient of variation (%) and the number of bands.

Table 1 -
Primer sequences and primer/enzyme combinations used in the RAPD reaction of coffee accessions showing numbers of polymorphic and monomorphic bands.

Table 3 -
Yeh et al. (1997)of the genetic differentiation among individuals within populations and between pairs of C. arabica populations estimated according toYeh et al. (1997).

Table 4 -
Analysis of molecular variance (AMOVA) among and between the Sarchimor progenies and C. arabica cultivars.