Molecular diversity in Coffea canephora germplasm conserved and cultivated in Brazil

This work aimed to characterize accessions that represent the C. canephora germplasm conserved and cultivated in Brazil. A total of 130 accessions from germplasm banks of IAC (São Paulo), UFV (Minas Gerais) and also collected in plantations of the State of Espírito Santo and Rondônia were evaluated with a set of 20 new microsatellite primers. Multivariate methods were used to estimate the relationship among the accessions. High level of polymorphism and two major diversity clusters were identified. First cluster was composed by the accessions conserved in the IAC and UFV collections and the second was formed by accessions collected in areas under cultivation. Accessions from Espírito Santo and Rondônia were clear separated, composing two subclusters. Despite the great polymorphism found in Brazilian plantations, the diversity may be increased, because a new threshold in the genetic gains is expected on breeding programs with the intensification of the use of conserved germplasm.


INTRODUCTION
Coffea canephora Pierre ex.Froehner presents a wide genetic variability, with one of the widest geographic natural distribution within the subgenus Coffea (Maurin et al. 2007).Likewise most diploid species in genus Coffea, C. canephora is allogamous and presents a self-incompatibility system.
Brazil is the second largest producer of C. canephora, producing about 25% of the world yield (USDA 2012).The States of Espírito Santo and Rondônia are responsible for over 75% of the production (CONAB 2013).In that country, main C. canephora germplasm collections are placed in governmental institutions, where breeding programs are developed, i.e.: Instituto Agronômico de Campinas (IAC), in São Paulo; Universidade Federal de Viçosa (UFV), in Minas Gerais; Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural (Incaper), in Espírito Santo and Embrapa (Embrapa Rondônia), in Rondônia.The germplasm conserved in IAC and UFV are mainly composed by accessions introduced from Africa, after FAO expeditions in that continent during the last century (Silvestrini et al. 2008, Fazuoli et al. 2009).On the other hand, Incaper and Embrapa Rondônia have collected a great amount of accessions in plantations from their respective states (Ferrão et al. 2007a, Souza andSantos 2009).As a consequence, those four institutions harbor a representative sample of the germplasm conserved or grown in that country.
Brazilian C. canephora accessions were studied using phenotypic traits (Fonseca et al. 2006, Ivoglo et al. 2008, Souza and Santos 2009) and RAPD markers (Ferrão et al. 2007b, Silvestrini et al. 2008).Those studies have confirmed that there is a wide variability within the germplasm maintained in the Brazilian collections.However, despite the advantages of microsatellites -e.g.: high reproducibility, multi-allelic locus, co-dominant inheritance, high degree of polymorphism, relative abundance and good coverage of the genome (Powell et al. 1996) -there are a few works using these markers to investigate C. canephora diversity.Furthermore, there is no report of comparisons about the diversity among accessions from different institutions, providing a well representative coverage of this germplasm.FF Souza et al.Thus, this study aimed to characterize accessions representing the germplasm cultivated in Brazilian plantations and conserved in research institutions, in order to propose guide lines to management of gene banks and breeding strategies.

DNA extraction
Young and completely extended leaves were collected from each accession, frozen at -80 ºC, lyophilized, ground to become a fine powder and kept at -20 ºC until used.Genomic DNA was extracted using the method described by Diniz et al. (2005) and all DNA samples were prepared to a final concentration of 25 ηg µL-1 .

Microsatellite markers
Twenty new microsatellites were used in this study.These DNA markers were developed from non-redundant Express Sequence Tags (EST) of the Brazilian Coffee Genome Project (Table 2), in Coffee Biotechnology Lab (BIOCAFE -UFV).
Each reaction was set to a final volume of 20 µL, containing 50 ηg of genomic DNA, 0.6 unit of Taq DNA polymerase, Taq buffer 1x, 1mM of MgCl 2 , 150 µM of each dNTP and 0.1 µM of each primer.PCR amplifications were carried out using touchdown proceeding, which comprises initial denaturation at 94 ºC for 2 min, followed by 10 cycles of denaturation at 94 ºC for 0.5 min, annealing at 67ºC for 0.5 min, decreasing 1 ºC after each cycle, and extension at 72 ºC for 0.5 min.After that, another set of 30 cycles, comprising denaturation at 94 ºC for 0.5 min, annealing at 55 ºC for 0.5 min and extension at 72 ºC for 0.5 min, was accomplished followed by a final 8 min extension time at 72 ºC.Before electrophoresis, PCR products were denatured in 8 µL of denaturing dye (95% formamide) at 94 ºC for 5 min and 7 µL of sample were loaded on a standard 6% polyacrylamide gel at 50 ºC and run at a constant power of 90 W for about 2 h.Post-PCR multiplex, which involved the multi-loading of individual PCR assays (two to four SSRs per running), was performed spacing successive loads by 10 to 30 min during electrophoresis, depending on the prior information about fragment size.At last, the gel was treated with ethanol (10%) + acetic acid (1%), followed by nitric acid (1,5%); stained with silver nitrate (4%), developed with sodium carbonate (3%) and formaldehyde (0.03%); and fixed with acetic acid (5%) and dried for posterior analysis at a transilluminator apparatus.

Data analysis
The evaluation of each locus was performed considering homozygotes and heterozygotes, individuals which showed one or two alleles, respectively.The dissimilarity between the accessions was estimated based on the complement (1 -S ii') of the weighted coincidence index, using the equation: , where S ii' is the similarity between the accessions i and i'; L is the total number of loci; c j is the number of common alleles between i and i'; and p j is the weight associated to j locus, obtained by a j /A, being a j , the number of alleles in locus j and A total number of alleles.Principal Coordinate Analysis (PCoA) was performed to view the overall diversity.The dissimilarity matrix was also represented in a dendrogram based on the unweighted pair-group method using arithmetic averages (UPGMA) to establish genetic relations among accessions.Goodnessof-fit of the tree was tested comparing cophenetic value matrix, with the original dissimilarity matrix.Statistical procedures were accomplished using the software packages: Genes (Cruz 2001), Darwin 5.0 (Perrier et al. 2003) and NTSYS-pc (Rohlf 1998).

RESULTS AND DISCUSSION
Genetic relationships among the 130 accessions of C. canephora and others species of the genus Coffea were evaluated with UPGMA clustering technique (Figure 1).The cophenetic correlation was high (81.5%),and the levels of stress and distortion were low (1.4% and 11.8%, respectively), demonstrating that the dendrogram satisfactorily represents the original matrix of dissimilarities.C. arabica and Híbrido de Timor, as expected, composed an out group, confirming the efficacy of the new microsatellites to distinguish different species in genus Coffea.Accessions of germplasm collections and those sampled in plantations formed distinct cluster, but no logical subdivisions were observed inside each one.IAC and UFV accessions appeared merged on upper cluster and Rondônia and Espírito Santo accessions were also mixed in lower cluster.
The overall diversity among the 130 accessions of C. canephora was also represented in the bi-dimensional  FF Souza et al. graphics based on the principal coordinate analysis (Figure 2).The result corroborated the previous clustering observed by UPGMA, but a better layout of that diversity was found.
In the graphic, first, second and third axis exhibited, respectively, 19.1%, 5.8% and 3.3% of the total variability.Accessions were clearly separated in two major groups, in accordance their origin, i.e.: 1) accessions preserved in germplasm banks and 2) accessions collected in Brazilian plantations.Cultivated accessions were plotted at the left side of the plan, composed by the 1 st and 2 nd coordinates.A division by location was also observed in this cluster.Accessions from Rondônia and Espírito Santo occupied upper and lower halves of the plan, respectively.Nevertheless, some misclassifications were observed, i.e.: five accessions of Rondônia (RO098, RO189, RO143, RO100 and RO063) were positioned in the Espírito Santo cluster and two accessions of Espírito Santo were included in Rondônia cluster (ES02 and ES03).Accessions from UFV collection and the Robustas from IAC were positioned in the lower right side of the plan and no remarkable sub-clustering was observed.This similarity among them may indicate that UFV accessions also belong to the Robusta varietal group.Kouillou accessions from IAC occupied a slightly upper position in right side of the plan.In the graphic, composed by 1 st and 3 rd coordinates, accessions of preserved and cultivated accessions continued apart, reinforcing their genetic distance.
In Brazil, the cultivated plants of C. canephora are generically called 'Conilon', what it supposed to be a linguistic derivation of Kouillou.Nevertheless, is necessary to mention that the word 'Kouillou' was historically defined according to solely morphological criteria and may represent different populations in many countries as Ivory Coast, Benin, Gabon (Montagnon et al. 1998).Notably, only six accessions from Rondônia (RO056, RO190, RO193, RO194 and RO199) and one from Espírito Santo (ESV.3) were grouped among the accessions of IAC and UFV, which are composed by plants labeled as 'Kouillou' and 'Robusta'.Considering that only six, in a total of 73 accessions collected in plantations, share alleles with those genotypes, it is possible to infer the presence of that varietal group is still small in the most cultivated areas.Consequently, increasing the participation of that germplasm in Brazilian breeding programs would imply a lot of benefits.For instance, the use of Robusta alleles may promote the development of C. canephora clones highly resistant to leaf rust (Hemileia vastatrix Berk & Br.) and nematodes (Meloidogyne spp).Besides, those genotypes could aid to increase yield and improving beverage quality in new cultivars (Fazuoli et al. 2009, Souza andSantos 2009).Another aspect to be explored is the utilization of the heterosis resulting from intergroup crosses.Considering the genetic divergence observed between accessions of the varietal groups (Robusta and Kouillou) and the cultivated germplasm (clone from Rondônia and Espírito Santo), it is expected that their intercrosses may be advantageous.Similar strategy has been used for so long in some breeding programs around the world with remarkable success (Bouharmont et al. 1986, Leroy et al. 1997).Furthermore, 'Robusta' and 'Kouillou' are divergent heterotic groups with complementary characteristics.Robusta plants present high resistance to rust and nematodes, and give good beverage.
On the other hand, Kouillou plants are tolerant to drought and they are easier to cultivate due to the smaller size.So, these populations compose a ideal combination to use in a reciprocal recurrent selection program, as it has been already performed in Ivory Cost, since 1984 (Leroy et al. 1993, Leroy et al. 1994, Leroy et al. 1997).
The set of new microsatellites performed a suitable molecular characterization and allowed assessing an im- FF Souza et al. portant part of the diversity of C. canephora gene pool in Brazil.For the first time, representative samples of accessions from cultivated areas and germplasm collections were examined by microsatellites analysis.These markers revealed a high degree of polymorphism, which provided a satisfactory understanding of the genetic diversity among Coffea canephora accessions.Moreover, they allowed the proper grouping of different populations and varietal groups and showed to be able to resolve doubts about the accession classification.This is of great advantage, because the high intra-specific variability and the environmental effects can hinder the differentiation of populations or varietal groups based only on the phenotypic evaluation.
Despite the great polymorphism found in accessions came from areas under cultivation, the diversity may be increased.The present diversity has been enough to sup-port advances of Brazilian breeding programs, but a new threshold of genetic gains is expected with the intensification of the use of Robusta germplasm.Nowadays, Brazil plays a fundamental role in the C. canephora world production.Therefore, the establishment of new ways of germplasm interchanging with other collections around the world should be an important initiative to promote introduction of new accessions.

Figure 1 .Figure 2 .
Figure 1.Dendrogram representing the dissimilarity among 130 Coffea canephora accessions, obtained by UPGMA method, based on the weighted coincidence index estimated over the polymorphism of 20 microsatellites

Table 1 .
List of Coffea sp accessions genotyped in this study

Table 2 .
Identification, sequences of forward and reverse primer, temperature of melt and allele size for 28 microsatellites from Coffea canephora