Revealing the Diversity of Introduced Coffea canephora...

Research ArticleRevealing the Diversity of IntroducedCoffea canephora Germplasm in Ecuador Towardsa National Strategy to Improve Robusta

Rey Gastoacuten Loor Soloacuterzano1 Fabien De Bellis23 Thierry Leroy23

Luis Plaza1 Hilton Guerrero1 Cristian Subia4 Dariacuteo Calderoacuten4

Fabiaacuten Fernaacutendez4 Ivaacuten Garzoacuten5 Diana Lopez6 and Danilo Vera78

1 Instituto Nacional de Investigaciones Agropecuarias (INIAP) Programa Nacional de Cacao y Cafe (PNCC)Estacion Experimental Tropical Pichilingue (EETP) Km 5 Vıa Quevedo-El Empalme Mocache Los Rıos Ecuador2CIRAD UMR AGAP 34398 Montpellier France3AGAP Universite de Montpellier CIRAD INRA Montpellier SupAgro Montpellier France4Instituto Nacional de Investigaciones Agropecuarias (INIAP) Programa Nacional de Cacao y Cafe (PNCC)Estacion Experimental Central Amazonica (EECA) San Carlos Joya de Los Sachas Orellana Ecuador5Instituto Nacional de Investigaciones Agropecuarias (INIAP) Departamento Nacional de Biotecnologıa (DNB)Estacion Experimental Tropical Pichilingue (EETP) Km 5 Vıa Quevedo-El Empalme Mocache Los Rıos Ecuador6Instituto Nacional de Investigaciones Agropecuarias (INIAP) Departamento de ProduccionVenta de Bienes y Servicios Agropecuarios (DPVBSA) Estacion Experimental Tropical Pichilingue (EETP)Km 5 Vıa Quevedo-El Empalme Mocache Los Rıos Ecuador7Instituto Nacional de Investigaciones Agropecuarias (INIAP) Departamento Nacional de Proteccion Vegetal (DNPV)Estacion Experimental Tropical Pichilingue (EETP) Km 5 Vıa Quevedo-El Empalme Mocache Los Rıos Ecuador8Universidad Tecnica Estatal de Quevedo (UTEQ) Facultad de Ciencias Pecuarias La Marıa Km 8 Vıa Quevedo-El EmpalmeMocache Los Rıos Ecuador

Correspondence should be addressed to Rey Gaston Loor Solorzano reylooriniapgobec

Received 18 May 2017 Accepted 17 August 2017 Published 30 October 2017

Academic Editor Antonio Amorim

Copyright copy 2017 Rey Gaston Loor Solorzano et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Genetic resources of Coffea canephora have been introduced in several tropical countries with potential for crop developmentIn Ecuador the species has been cultivated since the mid-20th century However little is known about the diversity and geneticstructure of introduced germplasm This paper provides an overview of the genetic and phenotypic diversity of C canephora inEcuador and some proposals for implementing a breeding program Twelve SSR markers were used to analyze 1491 plants of Ccanephora grown in different living collections in Ecuador compared to 29 genotypes representing themain genetic and geographicdiversity groups identified within the species Results indicated that most of the genotypes introduced are of Congolese origin withaccessions from both main subgroups SG1 and SG2 Some genotypes were classed as hybrids between both subgroups Substantialphenotypic diversity was also found and correlations were observed with genetic diversity Ecuadorian Robusta coffee displayswide genetic diversity and we propose some ways of improving Robusta in Ecuador A breeding program could be based on threeoperations the choice of elite clones the introduction of newmaterial from other countries (Ivory Coast Uganda) and the creationof new hybrid material using genotypes from the different diversity groups

Hindawie Scientific World JournalVolume 2017 Article ID 1248954 12 pageshttpsdoiorg10115520171248954

2 The Scientific World Journal

1 Introduction

Coffea canephora is originated from the lowland tropicalforests of Africa which stretch from Guinea to Uganda andits cultivation is recent (end of the 19th century) Robustacoffee fields are now widely found in all lowland intertropicalregions of Africa America and Asia [1]The genetic diversityof C canephora was first described at molecular level in the1980s [1ndash7]Those studies revealed twomain diversity groupsthe Congolese and the Guinean groups (G) The Congolesegroup was subdivided into five subgroups (SG1 SG2 B Cand UW) Only a small portion of this wide diversity (iemainly SG1 and SG2) is used in current breeding programswith the exception of the recurrent breeding program in IvoryCoast which uses a larger share of this diversity except thatfrom Uganda [4 8 9] A core collection encompassing alarge share of known C canephora diversity has been recentlyproposed [10] This core collection contains genotypes fromall the known diversity groups and is an interesting startingpoint fromwhich to broaden genetic diversity inC canephorabreeding programs

In Ecuador C canephora genetic resources were firstintroduced in the mid-20th century [11 12] the origin of thisgermplasm is diverse but little information is available on itstrue geographic origin or its diversity and genetic structureThis information is considered very important for futureconservation and development conditions for a breedingprogram in the country

Ecuadorian historical records show that the first intro-ductions of C canephora genetic material came from theldquoTropical Agricultural Research and Higher Education Cen-ter (CATIE)rdquo Costa Rica in 1951 1964 1972 1977 and 1986They corresponded to the ldquoRobustardquo type (putative SG2) andall were planted at the Pichilingue Tropical Research Station(EETP) of the National Institute of Agricultural Researchof Ecuador (INIAP) with the first C canephora plantationsappearing in 1952 in Los Rıos province fromwhere they weregradually extended to several coastal provinces and towardthe north of Ecuadorian Amazonia [13]

Later in 1987 and 2006 genetic material of the ldquoConilonrdquotype (putative SG1) was imported from Brazil Additionallyunofficial sources report introductions of genetic materialimported as seeds from Vietnam and Indonesia (2009 2010)as well as Robusta from Brazil (2010) Using seeds for geneticmaterial transfers for a self-incompatible tree cannot ensureits genetic origin since crosses between genotypes fromdifferent genetic groups are likely to occur within germplasmcollections [10]

During the second half of the 20th century a nonspecificC canephora breeding program was developed However afirst group of elite material ldquoclonesrdquo was identified by INIAPin 1998 based on yield and morphological traits To datethese clonal C canephoramaterials have been recommendedfor commercial planting under the conditions of northernEcuadorian Amazonia A recent study on the phenotypiccharacterization of C canephora accessions planted in theliving genebank collection located in the EETP of theINIAP showed a high variability between and within theseaccessions [13] Consequently knowledge on the genetic

diversity of the material widely distributed in the Ecuadorianterritory could help breeders and geneticists to understandthe structure of introduced germplasm in order to design aC canephora breeding program in Ecuador

This paper will

(i) provide an overview of the genetic and pheno-typic diversity and conservation of C canephora inEcuador

(ii) address the bases for implementing a C canephorabreeding program

2 Material and Methods

21 Plant Material In all 1491 leaf samples were taken fromdifferent C canephora clonal plants collected during 20112012 and 2013 In 2011 one hundred thirty-seven leaf sampleswere collected from two fields The first field is the Dublinsa-Denisse Farm nearby Isidro Ayora in Guayas province thathas a living collection ofC canephora collected fromdifferentlocations at the Amazonian region of Ecuador The secondfield is located at the EETP research station nearby Quevedocity Los Rios province In 2012 two hundred leaf samplesfrom 12 clonal accessions and 154 leaf samples from sevenclonal accessions and a polyclonal mix were collected at theDublinsa-Denisse Farm and the EETP respectively In 2013one thousand leaf samples from 7 clones were sampled at theINIAPrsquos Amazonian Central Station (EECA)

The summary of the plant material used for genotypicanalysis across the different years is shown in Tables 1(a) and1(b) Among these 48 samples were considered as duplicatesandused to check the experimental reproducibility of the dataand accordingly homogenize the data whenever needed

22 DNA Preparation and Genotyping Genomic DNAextractions were performed according to Cubry et al [14]The 1491 accessions were genotyped with the 12 SSR markersused by Leroy et al [10] Two different methods were usedfor genotyping and allele calling in 2011 and 20122013respectively

In 2011 PCR reactions ran as described in Cubry et al[14] PCR products were analyzed by electrophoresis on a65 polyacrylamide gel using a LI-COR 4300 automatedsequencer (LI-CORBiosciencesNebraskaUSA) Size callingwas automatic and manually checked using the manufac-turerrsquos program SAGAGT

In 2012 and 2013 PCR reactions were performed accord-ing to De Bellis et al [15] In a solution A (25 120583L totalvolume) containing 25 120583L of PCR buffer (10mM Tris-HCl50mM KCl 2mM MgCl2 and 0001 glycerol) 25120583L ofdNTP (Jena Bioscience GmbH Jena Germany) 025120583L ofMgCl2 02120583L of 10 120583M forward primer with an M13 tail atthe 51015840-end (51015840-CACGACGTTGTAAAACGAC-31015840) 025 120583L of10 120583M reverse primer 025120583L of fluorescently labeled M13-tail (6-FAM NED VIC or PET from Applied BiosystemsFoster City California USA) 01 U of Taq DNA polymerase(Sigma St Louis Missouri USA) 5 120583L of template DNA(5 ng120583L) and 14 120583L of H20 The PCR conditions were asfollows an initial denaturation at 94∘C for 5min 30 cycles

The Scientific World Journal 3

Table 1 Genetic material analyzed and controls used in the genotypic analyses

(a) Analyzed plants

Year Locations Number of samples

2011Dublinsa-Denisse Farm 81

EETP 56Total 2011 137



2013 EECA 1000

(b) Controls

Diversity group Diversity subgroup Number of samplesGuinean G 11

Congolese

B 1C 3SG1 3SG2 5UW 5

Outgroup Coffea eugenioides 1

at 94∘C for 45 s 55∘C for 45 s and 72∘C for 1min and afinal extension at 72∘C for 10min PCR products were pooledin a solution B containing 2 120583L of 6-FAM 2120583L of VIC25 120583L of NED and 35 120583L of PET of Arsquos solutions From thissolution B a volume of 4 120583L was taken and added to 10 120583Lof Hi-Di formamide and 012 120583L of GeneScan 600 LIZ sizestandard and analyzed on an ABI 3500xL Genetic Analyzer(Life Technologies Carlsbad California USA) Alleles werescored usingGeneMapper v41 software (Applied BiosystemsFoster City California USA)

23 Data Analysis

231Whole of Ecuador Genetic Analyses Datawere obtainedseparately in 2011 2012 and 2013 for each sampling year fromwhich merged three matrices Standard controls of knowngenotypes and duplicates samples together with genotypingdatabase (SagaCityWeb CIRAD unpublished) were used tohelp us to poolshift alleles to ensure good coherence of thegenotyping data From the raw data were removed the 48duplicates and the samples with more than 17 of missingdata (ie two missing markers) for further analysis Thenumber of alleles detected per marker was recorded andcompared to those detected in Leroy et al [10] We computeddissimilarity matrices between individuals using a simplematching index with Darwin 60 software [16]

A first diversity tree was drawn up using data from the2011 sampling operation in Ecuador Diversity from Ecuadorwas evaluated in relation to the global diversity of the species

A global analysis using data from the three samplingyears was performed to identify unique genotypes for futureconservation and for breeding programs In all 1168 sampleswere kept for analysis based on missing values (lt8 not

available ie one marker missing) A Neighbor-Joining (NJ)tree was built on the whole data removing pairs of datawith more than 70 of missing values To take into accountthe noise due to highly repeated genotypes the max-lengthsubtree procedure was used to eliminate redundancy and toidentify the number of unique individuals without loss in thenumber of alleles A Principal Coordinates Analysis (PCoA)was used to construct a good image of the diversity betweengenotypes

Specific analyses were performed at the DUBLINSA andEECA stations to check the homogeneity and the diversity ofthe materials introduced

232 The EETP Collection Phenotypic analysis was per-formed at the EETP collection using data from 2010 to 2012The evaluation was carried out in 256 plants correspondingto 16 accessions Each accession contained different numberof individual (between 12 and 20) A phenotypic dendrogramwas performed by UPGMA clustering method using theEuclidean distance

Genotyping data were available for 146 plants Tree diver-sity was determined formolecular data and their relationshipwith phenotypic data generated by Plaza et al [13] wasstudied

Phenotypic data were measured between 2010 and 2012for all the trees planted in 2007 During this time thetraits observed were the following plant height (PH) stemdiameter (SD) number of branches (NB) number of nodesper branch (NN) and internode length (IL) Outlier datawere removed whenever found and replaced the missingvalues PCA analyses were performed using the dudipcafunction from the ade4 R package [17] Five principalcomponents were chosen after observing the screen plot of


Congolese SG2

UW

SG1

C

B

Guineans

Figure 1 Genetic diversity of 137 Ecuadorian Coffea canephora accessions within the known diversity of the species (NJ-Tree from dataproduced in 2011)

eigenvalues Correlation circle was drawn using the scorcirclefunction and the representation of individuals for the first twocomponents was drawn using the sclass function

Two types of analyses were performed on these data Aclustering tree was first built on 249 plants using the dissimi-lar Euclidian distance evaluated by average linkage for the 17traits observed across the three years Another analysis wasperformed using a Principal Components Analysis (PCA) onfive vigor traits (yield of cherry beans number of cushionflowers total of productive branches total of branches pertree and tree height) across three years observed on the 146trees analyzed for genetic diversity

3 Results

31 Whole of Ecuador Genetic Analyses The results onthe clonal trials at the EETP and Dublinsa revealed greatdiversity between ldquoclonesrdquo and also a high level of diversitywithin ldquoclonesrdquo which was more surprising The global

genetic analysis indicated the Congolese origin for all thegenotypes They were classed as SG1 or SG2 genotypes withsome hybrids between these two Congolese subgroups Thisresult can be explained by considering the origin of plantsintroduced in Ecuador the SG1 genotypes could have comefrom Brazil (Conilon genotypes are known to be from theSG1 group) and the SG2 genotypes from CATIE (from theCongo basin) Figure 1 presents the diversity tree identifyingthe genotypes from Ecuador within the global diversity of Ccanephora

A global analysis was performed in 2015 based on theresults obtained for the 1168 accessions by considering allthe different genotypic classes revealed for each year ineach plot A global diversity tree was established usingthese 1168 accessions (figure not shown) After eliminatingredundancy the 138 unique genotypes (including 29 controls)were observed in the diversity tree (figure not shown) ThePCoA analysis performed 138 unique genotypes most ofthese genotypes were included in the SG1 and SG2 diversity


Congolese SG1

Congolese SG2

SG1 versus SG2 hybrids

SG1 SG2

SG2 SG2

SG2

SG2UW UW

UWUW

C group

CC

CG

GG

GGGGG

G G G

Guinean

(Nana population)

UW

BSG1

Axis 1 268

Axis 2 121

C eugenioides

minus7 minus6 minus5 minus4 minus3 3minus2 2minus1 1

minus65

minus6

minus55

minus5

minus45

minus4

minus35

minus3

minus25

25

minus2

2

minus15

15

minus1

1

minus05

05

Figure 2 Principal coordinates analysis based on the dissimilarity matrix calculated using 12 SSR markers for 138 individual genotypes (29controls and 109 genotypes from Ecuador)

groups and six genotypes could be considered as hybridsbetween the groups (Figure 2)

Figure 3 shows the results for the genotypic analysisconsidering the ldquoclonesrdquo analyzed in 2013 at the EECAstation For each accessionclone 2 to 8 genotypic classeswere identified Accessions NP-3013 and NP-2044 only hadone genotype Some genotype classes were similar for certainaccessions genotype 1 fromNP-2024 was similar to genotype2 for NP-3056 genotype 1 fromNP-3013 was similar to geno-type 2 for NP-3018 and NP-2024 and also to genotype 3 forNP-3056 genotype 2 from NP-3072 was similar to genotype3 for NP- 4024 genotype 4 from NP-2024 was similar togenotype 6 for NP-4024 The dissimilarity tree at the EECAliving collection (Figure 4) yielded 50 genotyping classesSeveral classes grouped individuals belonged to differentaccessions

32 EETP Collection A diversity tree was constructed con-sidering the 146 plants from the 16 different origins with dataavailable for molecular diversity Twelve diverse groups were

identified within the 146 (from 154) plants analyzed in 2012(Figure 5)

On the other hand a phenotypic characterization ofthis collection was reported by Plaza et al [13] showing awide range of variation in most of the agro-morphologicaltraits evaluated for each plant The existence at this levelof phenotypic variation was found between and also withinaccessions This phenotypic result was the first to open upthe possibility of off-types in this collection despite this25 plants were selected as ldquoeliterdquo material but the mostimportant variations considered when selecting those eliteplants were plant yield and plant height [13] These 25 plants(24 analyzed) were identified by their genetic diversity basedon two analyses per origin and individually

(i) Per origin analysis COF1 p2 was different from COF1p10 COF3 p2 5 8 18 and 19 were genetically similarp7 was different COF4 p7 and 9 were similar p15 wasdifferent COF 5 p6 15 16 17 and 19 were similarNP2024 p 7 10 15 and 17 were similar NP2044 p6 16


0

UnassignedUnique genotypesGenotype 8

Genotype 7Genotype 6Genotype 5

Genotype 4 Genotype 1Genotype 3Genotype 2

NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e

Figure 3 Diagram bars showing the mixture within the 7 ldquoclonal accessionsrdquo from INIArsquos EECA experimental station For comparisonpurposes data are expressed as a percentage of plants per ldquoclonerdquo

and 17 were similar NP3018 p 8 and 19 were probablydifferent since the origin was very variable

(ii) Global genetic diversity of the 24 ldquoeliterdquo clones COF1p10 COF3 p2 5 8 18 and 19 and COF4 p7 and 9were similar COF5 p6 15 16 17 and 19 and NP2024p7 10 15 and 17 were similar NP2044 p6 16 and 17were similar COF4 p15 and COF3 p7 were similarCOF1 p2 and NP3018 p19 were almost identical theirsimilarity has to be verified

The genetic diversity of the selected clones was thereforevery low since only 5 different groups of genotypes (six ifincluding NP3018) were present in the selected clones Asregards the global diversity of the Pichilingue collectionalmost half of this diversity was not present in the selectedclones The diversity of the ConilonSG1 group was notpresent in the selected clones from this collection

33 Phenotypic Diversity The phenotypic dendrogram at theEETP C canephora collection (Figure 6) indicated 3 clustersOne cluster (group I) included 14 accessions with a highdiversity within the group The second and third groupconsisted of one isolated accession respectively

Phenotypic results were highly variable in most plantsbelonging to the same accession To determine the level of thisvariation all individuals from NP2024 accession were usedas sample The dendrogram indicated 3 clusters confirmingthe high level of variability among the individuals of thisaccession (Figure 7)

A complemented phenotypic PCA using the vigor traitswas carried out with the 12 genetic groups previously iden-tified at the EETP collection (ie Figure 5) showing a sig-nificant phenotypic diversity within of the groups (Figure 8)Group 7 was considered as presenting short internodes andgroup 9 exhibited low vigor and short internodes while group5 had rather long internodes and high vigor and group 11 wascharacterized by low vigor and long internodes

On the other hand using PCA the correlation circlebetween observed traits (Figure 9) indicated that the intern-ode length (IL) for the three years was related to axis 2 sincethe other vigor traits particularly stem diameter (SD) plantheight (PH) and the total number of nodes per tree (NNT)were related to axis 1

4 Discussion

Our analyses(i) confirmed the genetic diversity of accessions from

Ecuador covering the SG1 and SG2 subgroups ofCongolese diversity in accordance with the history ofintroductions

(ii) revealed great genetic and phenotypic diversitybetween clones but also a large number of genotypeclasses within most of the ldquoclonalrdquo accessions

(iii) suggested some ways of implementing a breedingstrategy for C canephora using the available diversity

The first point concerns the reliability of our workfor the three-year experiment The first-year analyses were


0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3

NP-2024_Other genotype






NP-2024_Other genotypeNP-2024_Other genotype




NP-2044_1







NP-3013_1NP-3013_Other genotype

NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79

Figure 4 Dissimilarity analysis of 7 ldquoclonal accessionsrdquo from the EECA station in 2013 NJ-Tree on the 63 different unique genotypes using12 SSR markers Branch support is expressed in percent of presence after 10000 bootstraps (values above 05 are displayed)

performed using LI-COR 4300 technology The followinganalyses were performed using an ABI sequencer withdifferent control plants Misidentification of alleles duringcalling and binning processes are known caveats of SSRstudies [18] To solve this issue laboratory good practiceswere implemented by including controls of known genotypesand by repeating some samples from one study to anotherThe overall results are in accordance with what was expectedand thus could be considered of good quality

It was a challenge to analyze concomitantly all the datafor the final evaluation The results could thus be consideredas a compromise between all the different data However thelow quality of some leaf material was also an element that wetook into account due to difficulties for DNA extraction andanalysis with microsatellites Some leaves samples were notcorrectly analyzed and were removed from the final analyses

In 2012 at DUBLINSA collection we detected geneticdiversity within ldquoclonesrdquo in our analyses meaning that theldquoclonesrdquo were not genetically homogeneous as they oughtto have been (data not shown) We also observed mixturesbetween genotypes in both clonal trials The CONERBO and

POLICLON genotypes which belonged to Conilon type canbe considered as plants from a mix of seeds introduced fromBrazil in the 80s these origins present high genetic diversityRegarding the EETP collection it should be noted that manyplants with different labels were quite similar from a geneticpoint of view We also had to consider that the Conilonexhibited wide diversity possibly due to its environmentalshare of diversity or due to their seed origin

In 2014 a global analysis of C canephora diversitywas carried out using a core collection approach [10]The comparison with the present study confirmed that thediversity observed within Ecuadorian accessions accountsfor about 57 of core collection diversity considering thedifferent alleles Therefore the information provided bythis study will help breeders choose the most appropriateplant(s) or accession(s) to be incorporated into their breedingprograms

Another finding was the small number of intergrouphybrids between the SG1 and SG2 diversity groups This lowlevel of hybridization can be explained by the history of theintroduced material Both introductions were composed of


G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100

Figure 5 Diversity tree for the 146 plants analyzed at the Pichilingue collection in 2012

genotypes from a single subgroup (SG2 fromCATIE and SG1from Brazil) The accessions were planted in one location(EETP) and then transferred to Manabı Santo Domingode los Tsachilas and Morona Santiago provinces mainly bycuttings Thus few hybrids can be found between groups inthe Ecuadorian collections

Nevertheless high diversity was observed within eachdiversity group (SG1 and SG2) For SG2 this diversitywas related to the large number of accessions that wereintroduced and previous studies [7] confirmed the highdiversity within this group The SG1 genotypes were mainlyintroduced by seeds for which diversity is always greaterthan the clones in this allogamous species High geneticand phenotypic diversity within this group has been recentlyestablished In the case of the Pichilingue germplasm itshould be noted that high levels of phenotypic variation werepreviously reported by Plaza et al [13] and could be relatedto the continuous pollen interchange andor possible mix ofseeds from different segregated populations

The phenotypic characterization carried out by Plaza etal [13] and our study enabled us to identify a wide rangeof variation in most of the agro-morphological traits evalu-ated per plant with significant phenotypic diversity withingenotypes In this respect it is important to keep in mindthat a phenotype is a product of genotype times environmentinteraction which we found in our results Therefore plants

may be morphologically similar but this does not necessarilyimply genetic similarity since different genetic bases canresult in similar phenotypic expression [18] and as observedin our results the same genotype can lead to substantialdifferences in phenotypic expression

In our view a combined analysis of phenotypic andmolecular marker results is crucial for a better understandingof evolutionary changes in this introduced species thiswould allow a better analysis of variation patterns withinC canephora for evaluating their future adaptive potentialin different geographical regions of Ecuador Differencesbetween phenotypic and genetic information have also beenfound in other crops [19ndash22]

Lastly this study was intended to identify diversity andways of using it to increase the production of Robusta coffeein Ecuador In Ivory Coast considering the high geneticdiversity found in C canephora a program of reciprocalrecurrent selection was conducted using the hybrid vigorobserved between genotypes of different origins [8 23 24]Based on the Ivorian experience we might propose somesteps for breeding C canephora in Ecuador with optimumuse of the existing diversity and improvedmanagement of theexistingmaterial based on the results presentedThis strategyfor the implementation of a C canephora breeding programshould also be of interest for other countries where coffeegenotypes have been introduced in recent decades


COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25

Figure 6 Phenotypic dendrogram for 16 accessions at EETP C canephora collection The data were collected between 2010 and 2012 usingvigor traits (productivity stem diameter plant height number of branches number of nodes and internode length) Branch support isexpressed in percent of presence after 10000 bootstraps (values above 02 are displayed)

As a first step we propose the following actions using thediversity existing in Ecuador

(i) To complete genotypic analyses with phenotypic datato increase knowledge on the accessions in the fieldfor their vigor productivity and stress and diseasetolerance

(ii) To reorganize the collections based on the geneticdiversity observed in our studies to avoid duplicationsand identifying unique genotypes

(iii) To implement a breeding program by selecting thebest genotypes for traits of interest (yield biotic andabiotic stress tolerance and adaptation to differentedaphoclimatic conditions) (these genotypes will beplanted as clones in a multisite trial design Attentionwill be paid to the high diversity of plants from theConilonSG1 groupThese clonal trials will enable theselection of a set of improved genotypes for farmers)

As a second step we propose the following actions byincreasing genetic diversity

(i) To introduce new genetic material from diversegroups (ie Guinean Ugandan and hybrids betweenthem) that are not present in Ecuador and test themunder Ecuadorian conditions

(ii) To establish a breeding program based on newhybrids obtained from crosses between genotypesfrom different diversity groups adapted to theedaphoclimatic conditions in Ecuador (these newhybrids will use accessions from Ecuador and intro-duced accessions to create new hybridsThis programwill use the diversity of both the SG1 and SG2 groupsas hybrids between these groups display hybrid vigorand good drought tolerance as already observedin Ivory Coast All the new hybrids will be testedunder all the conditions in Ecuador and this willlead to a new selection of elite clones or hybrids


P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20

Figure 7 Phenotypic dendrogram for 19 individuals belonging to NP-2024 accession at EETP C canephora collection The phenotypic datawere collected between 2010 and 2012 for vigor traits (productivity stem diameter plant height number of branches number of nodes andinternode length) Branch support is expressed in percent of presence after 10000 bootstraps (values above 02 are displayed)

This improved material will be distributed to farmersthrough cutting gardens (for clones) or seed gardens(for hybrids))

Over the long term hybrid selection might be theoptimum breeding method as seeds are more suitable fordistribution to growers and the nurseries are easier forfarmers to manage

5 Conclusions

The present research concluded that Ecuadorian Robustacoffee displays a wide genetic diversity between clones andalso a high level of diversity within clones This researchconfirms that most of the C canephora genotypes introducedin Ecuador are of Congolese origin containing accessionsfrom both subgroups SG1 and SG2

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Rey Gaston Loor Solorzano Fabien De Bellis and ThierryLeroy contributed equally to this work

Acknowledgments

The authors thank DUBLINSA SA the National Insti-tute of Agricultural Research (INIAP) and the Secretaryof High Education Science Technology and Innovation(SENESCYT) for financial support and Solubles InstantaneosCA for its technical assistance in the postharvest analysisThe


d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5

Figure 8 PCA for the 146 genotypes in the EETP collection for the vigor traits in 2012

PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012

Figure 9 PCA correlation circle for phenotypic vigor traits over 3 years 2010 2011 and 2012 for 146 C canephora trees in the field collectionat the EETP

authors also thank Mr Peter Biggins for revising the Englishversion

References

[1] C Montagnon T Leroy and A Yapo ldquoDiversite genotypiqueet phenotypique de quelques groupes de cafeiers (Coffeacanephora Pierre) en collectionrdquo Cafe Cacao The vol 36 pp187ndash198 1992

[2] J Berthaud Les ressources genetiques pour lrsquoamelioration descafeiers africains diploıdes Evaluation de la richesse genetiquedes populations sylvestres et de ses mecanismes organisateursrdquoConsequences pour lrsquoapplication ORSTOM Paris France 1986

[3] D Dussert P Lashermes F Anthony et al ldquoLe cafeier Coffeacanephora in P Hamon M Seguin X Perrier and JC Glasz-mannrdquo in Diversite genetique des plantes tropicales cultivees PHamon M Seguin and X Perrier and JC Glaszmann Eds pp175ndash194 CIRAD Montpellier France 1999

[4] C Montagnon ldquoOptimisation des gains genetiques dans leschema de selection recurrente reciproque de Coffea canephoraPierre [PhD thesis] PhD thesis ENSA Montpellier France2000

[5] P Musoli P Cubry P Aluka et al ldquoGenetic differentiation ofwild and cultivated populations Diversity of Coffea canephoraPierre in Ugandardquo Genome vol 52 no 7 pp 634ndash646 2009


[6] C Gomez S Dussert P Hamon S Hamon A D Kochko andV Poncet ldquoCurrent genetic differentiation of coffea canephorapierre ex a Froehn in the guineo-Congolian african zoneCumulative impact of ancient climatic changes and recenthuman activitiesrdquoBMCEvolutionary Biology vol 9 no 1 articleno 167 2009

[7] P Cubry F de Bellis D Pot P Musoli and T Leroy ldquoGlobalanalysis of Coffea canephora Pierre ex Froehner (Rubiaceae)from the Guineo-Congolese region reveals impacts from cli-matic refuges andmigration effectsrdquoGenetic Resources andCropEvolution vol 60 no 2 pp 483ndash501 2013

[8] T Leroy C Montagnon A Charrier and A B Eskes ldquoRecip-rocal recurrent selection applied to Coffea canephora Pierre ICharacterization and evaluation of breeding populations andvalue of intergroup hybridsrdquo Euphytica vol 67 no 1-2 pp 113ndash125 1993

[9] C Montagnon T Leroy and A B Eskes ldquoAmeliorationvarietale de Coffea canephora I Criteres et methodes deselectionrdquo Plantations Recherche Developpement vol 5 no 1pp 18ndash28 1998

[10] T Leroy F De Bellis H Legnate et al ldquoDeveloping corecollections to optimize the management and the exploitation ofdiversity of the coffee Coffea canephorardquo Genetica vol 142 no3 pp 185ndash199 2014

[11] INIAP (Instituto Nacional Autonomo de Investigaciones Agro-pecuarias) ldquoInforme Anual Tecnicordquo Estacion ExperimentalTropical Pichilingue Programa del Cafe Quevedo ndash Ecuador(1977)

[12] COFENAC (Consejo Cafetalero Nacional) ldquoInforme Anual dela Division Tecnicardquo Portoviejo ndash Ecuador (2004)

[13] L F Plaza R G Loor H E Guerrero and L A Duicela ldquoPhe-notypic characterization of Coffea canephora Pierre germplasmfor yield improvement in Ecuadorrdquo Espamciencia vol 6 pp 7ndash13 2015

[14] P Cubry P Musoli H Legnate et al ldquoDiversity in coffeeassessed with SSR markers Structure of the genus Coffea andperspectives for breedingrdquo Genome vol 51 no 1 pp 50ndash632008

[15] F De Bellis R Malapa V Kagy S Lebegin C Billot andJ-P Labouisse ldquoNew Development and Validation of 50 SSRMarkers in Breadfruit (Artocarpus altilis Moraceae) by Next-Generation Sequencingrdquo Applications in Plant Sciences vol 4no 8 Article ID 1600021 2016

[16] X Perrier and J P Jacquemoud-Collet ldquoDARwin softwarerdquohttpdarwinciradfrdarwin (2006)

[17] S Dray and A B Dufour ldquoThe ade4 package implementing theduality diagram for ecologistsrdquo Journal of Statistical Software vol 22 no 4 pp 1ndash20 2007

[18] E Guichoux L Lagache S Wagner et al ldquoCurrent trends inmicrosatellite genotypingrdquoMolecular Ecology Resources vol 11no 4 pp 591ndash611 2011

[19] A Mazzucato N Ficcadenti M Caioni et al ldquoGenetic diver-sity and distinctiveness in tomato (Solanum lycopersicum L)landraces The Italian case study of rsquoA pera Abruzzesersquordquo ScientiaHorticulturae vol 125 no 1 pp 55ndash62 2010

[20] J Wang S Kaur N O I Cogan et al ldquoAssessment of geneticdiversity inAustralian canola (Brassica napus L) cultivars usingSSR markersrdquo Crop amp Pasture Science vol 60 no 12 pp 1193ndash1201 2009

[21] P J Terzopoulos and P J Bebeli ldquoDNA and morphologicaldiversity of selected Greek tomato (Solanum lycopersicum L)

landracesrdquo Scientia Horticulturae vol 116 no 4 pp 354ndash3612008

[22] VVinuN Singh SVasudev et al ldquoAssessment of genetic diver-sity in Brassica juncea (Brassicaceae) genotypes using pheno-typic differences and SSRmarkersrdquoRevista de Biologıa Tropicalvol 61 no 4 pp 1919ndash1934 2013

[23] T Leroy C Montagnon A Charrier and A B Eskes ldquoRecip-rocal recurrent selection applied to Coffea canephora Pierre -II Estimation of genetic parametersrdquo Euphytica vol 74 no 1-2pp 121ndash128 1993

[24] T Leroy C Montagnon C Cilas A Yapo P Charmetant andA B Eskes ldquoReciprocal recurrent selection applied to Coffeacanephora Pierre III Genetic gains and results of first cycleintergroup crossesrdquo Euphytica vol 95 no 3 pp 347ndash354 1997

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


1 Introduction

Coffea canephora is originated from the lowland tropicalforests of Africa which stretch from Guinea to Uganda andits cultivation is recent (end of the 19th century) Robustacoffee fields are now widely found in all lowland intertropicalregions of Africa America and Asia [1]The genetic diversityof C canephora was first described at molecular level in the1980s [1ndash7]Those studies revealed twomain diversity groupsthe Congolese and the Guinean groups (G) The Congolesegroup was subdivided into five subgroups (SG1 SG2 B Cand UW) Only a small portion of this wide diversity (iemainly SG1 and SG2) is used in current breeding programswith the exception of the recurrent breeding program in IvoryCoast which uses a larger share of this diversity except thatfrom Uganda [4 8 9] A core collection encompassing alarge share of known C canephora diversity has been recentlyproposed [10] This core collection contains genotypes fromall the known diversity groups and is an interesting startingpoint fromwhich to broaden genetic diversity inC canephorabreeding programs

In Ecuador C canephora genetic resources were firstintroduced in the mid-20th century [11 12] the origin of thisgermplasm is diverse but little information is available on itstrue geographic origin or its diversity and genetic structureThis information is considered very important for futureconservation and development conditions for a breedingprogram in the country

Ecuadorian historical records show that the first intro-ductions of C canephora genetic material came from theldquoTropical Agricultural Research and Higher Education Cen-ter (CATIE)rdquo Costa Rica in 1951 1964 1972 1977 and 1986They corresponded to the ldquoRobustardquo type (putative SG2) andall were planted at the Pichilingue Tropical Research Station(EETP) of the National Institute of Agricultural Researchof Ecuador (INIAP) with the first C canephora plantationsappearing in 1952 in Los Rıos province fromwhere they weregradually extended to several coastal provinces and towardthe north of Ecuadorian Amazonia [13]

Later in 1987 and 2006 genetic material of the ldquoConilonrdquotype (putative SG1) was imported from Brazil Additionallyunofficial sources report introductions of genetic materialimported as seeds from Vietnam and Indonesia (2009 2010)as well as Robusta from Brazil (2010) Using seeds for geneticmaterial transfers for a self-incompatible tree cannot ensureits genetic origin since crosses between genotypes fromdifferent genetic groups are likely to occur within germplasmcollections [10]

During the second half of the 20th century a nonspecificC canephora breeding program was developed However afirst group of elite material ldquoclonesrdquo was identified by INIAPin 1998 based on yield and morphological traits To datethese clonal C canephoramaterials have been recommendedfor commercial planting under the conditions of northernEcuadorian Amazonia A recent study on the phenotypiccharacterization of C canephora accessions planted in theliving genebank collection located in the EETP of theINIAP showed a high variability between and within theseaccessions [13] Consequently knowledge on the genetic

diversity of the material widely distributed in the Ecuadorianterritory could help breeders and geneticists to understandthe structure of introduced germplasm in order to design aC canephora breeding program in Ecuador

This paper will

(i) provide an overview of the genetic and pheno-typic diversity and conservation of C canephora inEcuador

(ii) address the bases for implementing a C canephorabreeding program

2 Material and Methods

21 Plant Material In all 1491 leaf samples were taken fromdifferent C canephora clonal plants collected during 20112012 and 2013 In 2011 one hundred thirty-seven leaf sampleswere collected from two fields The first field is the Dublinsa-Denisse Farm nearby Isidro Ayora in Guayas province thathas a living collection ofC canephora collected fromdifferentlocations at the Amazonian region of Ecuador The secondfield is located at the EETP research station nearby Quevedocity Los Rios province In 2012 two hundred leaf samplesfrom 12 clonal accessions and 154 leaf samples from sevenclonal accessions and a polyclonal mix were collected at theDublinsa-Denisse Farm and the EETP respectively In 2013one thousand leaf samples from 7 clones were sampled at theINIAPrsquos Amazonian Central Station (EECA)

The summary of the plant material used for genotypicanalysis across the different years is shown in Tables 1(a) and1(b) Among these 48 samples were considered as duplicatesandused to check the experimental reproducibility of the dataand accordingly homogenize the data whenever needed

22 DNA Preparation and Genotyping Genomic DNAextractions were performed according to Cubry et al [14]The 1491 accessions were genotyped with the 12 SSR markersused by Leroy et al [10] Two different methods were usedfor genotyping and allele calling in 2011 and 20122013respectively

In 2011 PCR reactions ran as described in Cubry et al[14] PCR products were analyzed by electrophoresis on a65 polyacrylamide gel using a LI-COR 4300 automatedsequencer (LI-CORBiosciencesNebraskaUSA) Size callingwas automatic and manually checked using the manufac-turerrsquos program SAGAGT

In 2012 and 2013 PCR reactions were performed accord-ing to De Bellis et al [15] In a solution A (25 120583L totalvolume) containing 25 120583L of PCR buffer (10mM Tris-HCl50mM KCl 2mM MgCl2 and 0001 glycerol) 25120583L ofdNTP (Jena Bioscience GmbH Jena Germany) 025120583L ofMgCl2 02120583L of 10 120583M forward primer with an M13 tail atthe 51015840-end (51015840-CACGACGTTGTAAAACGAC-31015840) 025 120583L of10 120583M reverse primer 025120583L of fluorescently labeled M13-tail (6-FAM NED VIC or PET from Applied BiosystemsFoster City California USA) 01 U of Taq DNA polymerase(Sigma St Louis Missouri USA) 5 120583L of template DNA(5 ng120583L) and 14 120583L of H20 The PCR conditions were asfollows an initial denaturation at 94∘C for 5min 30 cycles



(a) Analyzed plants






2013 EECA 1000

(b) Controls


Congolese




23 Data Analysis










Congolese SG2

UW

SG1

C

B

Guineans




3 Results





Congolese SG1

Congolese SG2


SG1 SG2

SG2 SG2

SG2

SG2UW UW

UWUW

C group

CC

CG

GG

GGGGG

G G G

Guinean

(Nana population)

UW

BSG1

Axis 1 268

Axis 2 121

C eugenioides


minus65

minus6

minus55

minus5

minus45

minus4

minus35

minus3

minus25

25

minus2

2

minus15

15

minus1

1

minus05

05









0




NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e









4 Discussion







0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a) Analyzed plants






2013 EECA 1000

(b) Controls


Congolese




23 Data Analysis










Congolese SG2

UW

SG1

C

B

Guineans




3 Results





Congolese SG1

Congolese SG2


SG1 SG2

SG2 SG2

SG2

SG2UW UW

UWUW

C group

CC

CG

GG

GGGGG

G G G

Guinean

(Nana population)

UW

BSG1

Axis 1 268

Axis 2 121

C eugenioides


minus65

minus6

minus55

minus5

minus45

minus4

minus35

minus3

minus25

25

minus2

2

minus15

15

minus1

1

minus05

05









0




NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e









4 Discussion







0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Congolese SG2

UW

SG1

C

B

Guineans




3 Results





Congolese SG1

Congolese SG2


SG1 SG2

SG2 SG2

SG2

SG2UW UW

UWUW

C group

CC

CG

GG

GGGGG

G G G

Guinean

(Nana population)

UW

BSG1

Axis 1 268

Axis 2 121

C eugenioides


minus65

minus6

minus55

minus5

minus45

minus4

minus35

minus3

minus25

25

minus2

2

minus15

15

minus1

1

minus05

05









0




NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e









4 Discussion







0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Congolese SG1

Congolese SG2


SG1 SG2

SG2 SG2

SG2

SG2UW UW

UWUW

C group

CC

CG

GG

GGGGG

G G G

Guinean

(Nana population)

UW

BSG1

Axis 1 268

Axis 2 121

C eugenioides


minus65

minus6

minus55

minus5

minus45

minus4

minus35

minus3

minus25

25

minus2

2

minus15

15

minus1

1

minus05

05









0




NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e









4 Discussion







0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0




NP-

2024

NP-

2044

NP-

3013

NP-

3018

NP-

3056

NP-

3072

NP-

4024

01

02

03

04

05

06

07

08

09

1

Prop

ortio

n of

each

gen

otyp

e









4 Discussion







0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 01

NP-2024_1

NP-2024_2

NP-2024_4

NP-2024_3











NP-2044_1








NP-3018_1

NP-3018_2







NP-3056_1

NP-3056_2

NP-3056_3







NP-3072_1

NP-3072_2NP-3072_3


NP-4024_1NP-4024_2

NP-4024_3

NP-4024_4

NP-4024_5NP-4024_6

NP-4024_7

NP-4024_8








97

55

58

65

59

70

61

98

99

88

56

92

57

82

79









G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


G1

G2

G3

G4

G5

G6

G8

G9

G7lowast

G10lowast

G11lowast

G12lowast

0 01

100

99

94

50

88

68

95

80

67

93

74 92

70

52

100









COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


COFENAC - 001

COFENAC - 002

COFENAC - 003

COFENAC - 004

COFENAC - 005

NP-2024

NP-3018

NP-3056

NP-2044

ETP-3756-14

NP-3013

NP-4024

NP-3072

ETP-3753-13

POLICLON DE CONILON

ETP-3752-6

100

3035

90

26

45

25










P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P10

P11

P15

P7

P20

P17

P4

P16

P19

P14

P18

P21

P3

P6

P5

P8

P22

P23

P2

100

93

22

21

46

59

26

20




5 Conclusions






Acknowledgments



d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


d = 2

910

124

5

8

2

11

1

Dimension 1 409

Dim

ensio

n 2

12

5


PH_2010

SD_2010 NB_2010 NN_2010

IL_2010

PH_2011 PH_2011

SD_2011

NB_2011

NN_2011

IL_2011

PH_2012

SD_2012

NB_2012

NN_2012

IL_2012



References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Revealing the Diversity of Introduced Coffea canephora...

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