Transferability of Simple Sequence Repeats and Single ...iccri.net/download/Pelita Perkebunan/Vol 26 No 3 Desember 2010/Transferability of... · and SNP linkage information in reference

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PELITA PERKEBUNAN, Volume 26, Nomer 3, Edisi Desember 2010

Naskah diterima (received): 15 Juni 2010, disetujui (accepted): 17 Juli 2010.

1). Pusat Penelitian Kopi dan Kakao Indonesia , Jl. PB. Sudirman 90, Jember 68118, Indonesia.

2). Nestle R&D Centre, 101 Avenue Gustave Eiffel, 37097 Tours Cedex 2, France.*) Alamat penulis (Corresponding Author) : [email protected]

Pelita Perkebunan 2010, 26(3), 126—141

Transferability of Simple Sequence Repeats and Single NucleotidePolymorphisms Marker for Genetic Map Development in

Coffea canephora Pierre

Kemampuan Transfer Marka Simple Sequence Repeats dan Single NucleotidePolymorphisms Pada Pengembangan Peta Genetik Kopi Robusta

Coffea canephora Pierre

Priyono1*), Michel Rigoreau2) and Dominique Crouzillat2)

Summary

Simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP)have been developed for some crops. However the studies of transferability ofthis marker for coffee are very limited. These researches were aimed to describethe heterozygosity level of parental clones in Robusta coffee (Coffea canephora)and to evaluate the transferability of SSR and SNP markers for development ofgenetic maps in Robusta coffee. Three parental clones, namely BP 409, BP 961and Q 121 clones were used for evaluation of heterozygosity level. Three popu-lations, namely CPA (BP 961 x Q 121), CPB (BP 409 x Q 121), and CPC (BP409 x BP 961) were used for genetic mapping. SSR and SNP markers selectedfrom reference genetic map were used as molecular markers in this study. Thisresearch showed that the screening of BP 409, BP 961 and Q 121 clones SSRled to, respectively, 52%, 53% and 40%, heterozygosity. A selection of SSRand SNP linkage information in reference map of Robusta coffee allowed us toestablish the homology linkage groups among three cross pollinated (CP) maps,three backcross (BC) maps, and consensus map. Most of the markers mappedfor seven maps showed a very good collinear to references map. This resultsconfirm that SSR and SNP markers have high transferability and mapping infor-mation for Robusta coffee. This work will allow faster and more detailed investi-gations of quantitative trait loci (QTLs) and positioning of a growing number ofcandidate genes co-localized with QTLs.

Ringkasan

Marka simple sequence repeats (SSR) dan single nucleotide polymor-phisms (SNP) telah dikembangan pada beberapa tanaman, namun demikianstudi transfer marka tersebut pada kopi sangat terbatas. Penelitian inidimaksudkan untuk mempelajari tingkat heterozigositas klon dan mengevaluasitingkat transfer marker SSR dan SNP dalam pembuatan peta genetik. Tiga klon,yaitu BP 409, BP 961 dan Q 121 digunakan untuk evaluasi tingkat heterozigositas.Tiga silangan, yaitu CPA (BP 961 x Q 121), CPB (BP 409 x Q 121), dan CPC(BP 409 x BP 961) digunakan untuk pembuatan peta genetik. Marka SSR dan SNPdigunakan sebagai marker molekuler dalam penelitian ini. Hasil penelitian menunjukkan

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Transferability of SSR and SNP marker for genetic map development in Coffea canephora


bahwa dengan menggunakan marka SSR, tingkat heterozigositas kopi Robustaklon BP 409, BP 961 dan Q 121 berturut-turut 52%, 53% dan 40%. Denganmenyeleksi marka SSR dan SNP dari peta genetik referensi dapat dibuat tiga petagenetik persilangan terbuka kopi Robusta, tiga peta genetik tipe silang balik dansatu peta konsensus. Hampir semua marka SSR dan SNP pada tujuh peta genetiktersebut menempati urutan yang sama dengan peta genetik referensi. Hasil inimenunjukkan bahwa marka SSR dan SNP mempunyai tingkat transfer yang tinggipada peta genetik kopi Robusta. Hasil ini memungkinkan digunakan untuk mendeteksiQTL pada kopi Robusta serta melokalisasi posisi gen kandidat yang terpaut denganQTL.

Key words: Coffea canephora, heterozygosity, genetic map, transferable markers, simplesequence repeats, Single nucleotide polymorphisms.

INTRODUCTION

Traditional methods of plant breedinghave made a significant contribution to cropimprovement, such as Robusta coffee butthey are slow in targeting complex traits likeyield, green bean quality, drought or pest-diseases resistance. One of the main con-straints in traditional coffee breeding is thedifficulty to estimate the effect of interac-tions between the genetic and environmentfactors on phenotypic expression. The prob-lem is more complicated on quantitativecharacteristics which are poligenically con-trolled. As a perennial crop, coffee takesseveral years to be properly morphologicalevaluated. It also takes about three to fouryears after planting to produce a generation.Therefore, genetic improvement of coffeemust be continued in order to find out newsuperior planting materials to meet marketdemand. This effort, unfortunately, facesa number of constraints through conven-tional breeding methods due to lack in ge-netic markers to be used in screening andselection tools. Molecular markers wouldmake sure the resource is new, easy andconstitute a contribution for solving the aboveproblems. They allow geneticists and plantbreeders to locate and follow the numer-ous interacting genes that determine a com-plex trait. Genetic linkage maps can provide

a more direct method for selecting desir-able genes via their linkage to easily de-tectable molecular markers (Tanksley,1993).

A linkage map may be seen as a ‘roadmap’ of the chromosomes of a segregat-ing population. Linkage maps indicate theposition and relative genetic distancesbetween markers along the chromosomes.A segregating population is a mixture of geno-types recombining the genetic backgroundsof the parents. The recombinant genotypescan be used to calculate the recombinationfrequencies and the genetic distance betweenmarkers. By analyzing the marker segrega-tion, one can determine distribution and dis-tances between markers. The lower the fre-quency of recombination between two mark-ers, the closer they are on a chromosomeand reciprocally. Furthermore, recombina-tion frequencies can be converted into centiMorgans (Hartl & Jones, 2001).

To meet the great increase in coffeeproduction, biotechnology brings new andpowerful tools to plant breeders. The avai-lability of wide genetic map has become amust for the effective advance of genomicundertaking. To date, there are eight geneticmaps based on coffee genome, comprisingfour genetic maps for C. canephora(Crouzillat, person.comm.; Hendre &

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Aggarwal, 2007; Lashermes et al., 2001;Paillard et al., 1996), one genetic map forC. arabica (Pearl et al., 2004), one geneticmap for interspecific F1 (Lopez & Moncada,2006) and two genetic maps for interspecificbackcross population (Coulibaly et al., 2003;Hamon et al., 2005). Recently, Hendreet al. (2008) reported their development ofnew genomic SSR markers from Robustacoffee which showed broad cross-speciestransferability and utility in genetic studies.However no transferable marker used toconstruct different genetic map was reportedfor coffee crop. The generalized use of anincreasingly larger set of transferable markersand consensus mapping information, willallow faster and more detailed investigationof QTLs synteny among species. The purposeof this research is to create the genetic mapof three populations in C. canephora anddevelop the consensus map based on thesethree genetic maps.

MATERIAL AND METHODS

Plant material

This study involved three parental clonesnamely BP 409, BP 961, and Q 121. TheBP 409 and BP 961 clones are commercialclones in Indonesia. The mother tree ofQ 121 clone is a variety of Quillouintroduced in 1901 from Libreville, FrenchCongo to Bangelan Plantation (Malang, EastJava, Indonesia). Seeds harvested on twosurviving plants were planted and thenevaluated in this plantation. From this evalu-ation three elite clones were selected. Oneof them is Q 121 (Cramer, 1957). The choiceof these parentals was based on the priorresults of genetic diversity studies as reportedby Priyono (1998). Three controlled hybridpopulations (CP) derived from three parentalclones, namely CPA (Q 121 X BP 961), CPB(Q 121 X BP 409), and CPC (BP 409 X

BP 961) were used to create genetic maps.The numbers of progenies were 77, 93, and87 for CPA, CPB, and CPC, respectively.

SSR and SNP detectiton

The DNA extraction was performedaccording to DNeasy Plant Maxi Proto-col (www.qiagen.com). About 1 g of freshleaves of each individual (Indonesia) wasground in liquid nitrogen and used to ob-tain DNA.

For automation of the SSR detectionon the ABI PRISM® 310 capillary electro-phoresis system (Applied Biosystems), DNAfragments was labelled with specific fluo-rescent dyes. Because labelling all the prim-ers individually would prohibitively increasethe cost of genotyping projects involvingmany SSR markers, an alternative and cost-effective method was used. This techniqueemployed a two-part primer in which a stan-dard ‘tail’ corresponding to a universal M13primer was added to the 5’ end of the for-ward primer sequence. The amplificationof SSR was thus performed with three prim-ers: the forward primer with the M13 tail,the reverse non-tailed primer and thefluorescently labelled M13 primer.

PCR reactions were carried out in a fi-nal volume of 20μl. For normal reaction,PCR amplifications were conducted usingthe following procedure: 10 min 95ºC, fol-lowed by 35 cycles (30 sec 95ºC, 30 sec55ºC, 1 min 72ºC), and 7 min 72ºC. ForM13-labeled reactions, PCR amplificationswere conducted using the following proce-dure: 10 min 95ºC, followed by 30 cycles(30 sec 95ºC, 30 sec 55ºC, 30 sec 72ºC),8 cycles (30 sec 95ºC, 30 sec 5ºC, 30 sec72ºC), and then 7 min 72ºC.

Fluorescent amplification products werethen mixed with de-ionized formamide andan interval size standard (GenescanTM – 500LIZTM Applied Biosystems) in a 1:12:0.5

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volume ratio. After 5 min denaturation at95ºC, the amplification products were vi-sualized using an ABI PRISM® 310 GeneticAnalyzer (Applied Biosystems). Fragmentsizes were calculated using GENESCAN andGENOTYPER Software (Applied Bio-sys-tems), where as different alleles are repre-sented by different amplification sizes oftandem repeats.

Single nucleotide polymorphisms (SNP)are DNA sequence variations that occur whena single nucleotide of the consideredsequence is altered. PCR reactions wereconducted in 20μl (final volume) in a PCRQ7500 with the following sequence: 2 min50ºC, followed by 10 min 94ºC, and thenfollowed by 40 cycles (15 sec 95ºC, 1 min60ºC).

Molecular marker selection and geneticmap construction

Amount of SSR and SNP markers,which showed the segregation type both forF2 and BC to two parental clones for eachpopulation were selected from the 831 mark-ers of reference map of Coffea canephora,(Crouzillat, priv.comm) and supplementarySSR markers obtained from other institutesto construct the studied maps. This refer-ence map has 831 loci covering 1392 cMwith an average distance between two lociof 1.7 cM. The markers used in this refer-ence map were restriction fragments lengthpolymorphisms (RFLPs), SSR and SNPmarkers. However, only PCR-based mark-ers, i.e. SSR and SNP were selected for thepresent study.

Table 1. The origin of markers used to construct the consensus genetic map of Robusta coffee (Coffea canephora)

Tabel 1. Asal-usul marka yang digunakan untuk membuat peta genetik konsensus pada kopi Robusta (Coffea canephora)

Marka origin of consensus genetic map Asal marka pada peta genetik konsensusLinkage group

Kelompok tautanShared markerson the three CPs

Marka sama dari 3 populasi

Total numberof markers on the

consensus genetic map

Jumlah marka padapeta genetik konsensus CPA CPB CPC

A 16 21 15 6 44

B 14 28 9 12 55

C 8 17 9 4 27

D 10 16 4 7 33

E 10 15 7 2 26

F 17 35 6 7 52

G 27 19 2 7 35

H 8 20 5 5 29

I 4 5 4 5 16

J 19 20 5 4 35

K 12 16 5 5 29

Total of markersJumlah marka% to consensus

genetic map% terhadap petagenetik konsensus

135 216 71 64 381

35.4 56.7 18.6 17 100

Notes (Catatan): CPA = BP 961 x Q 121, CPB = BP 409 x Q 121, CPC = BP 409 x BP 961.

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A set of polymorphic markers mappablefor each of the cross pollinated (CP) wasselected from the C. canephora referencemap with the following requirements:

a. The markers have to be, as much as pos-sible, shared by the three populations inorder to facilitate the evaluation of mark-ers transferable in the three populations.

b. Markers have to be heterozygous forboth parents for mapping on the twoparental clones in a single run. The pref-erence was given to the markers segre-gating as F2: <ab x cd> or <ab xac>. The markers with <ab x ab> seg-regating ratio were discarded when pos-sible due to a lack of informative capa-bility.

c. Markers were selected according to theirlocation on the core map to reach a dis-tribution of about one marker every 10to 20 cM.

To evaluate the transferable ability ofSSR and SNP markers in genetic mapping,three sets of genetic maps were created:

a. Three “Cross pollinated” (CP) geneticmaps coded as CPA (BP 961 x Q 121),CPB (BP 409 x Q 121) & CPC (BP 409x BP 961) with 78, 93 and 82 progenies.

b. Three backcross (BC) genetic mapscoded as BC BP 409, BC BP 961 and BCQ 121 were developed from the com-mon parents of the three segregatingpopulations. The number of progenies foreach of these BC populations (175, 160& 171, respectively). The common het-erozygous markers segregating in eachparental clone were used to develop thesegenetic maps.

c. One consensus map was created fromthe three CP genetic maps.

All the genetic maps were performedusing JoinMap® software (Ooijen, 2006)with Kosambi’s mapping function (Kosambi,1944) on LOD threshold of 3.

RESULTS AND DISCUSSION

Genetic diversity of the parental clonesused for genetic mapping

A genetic diversity analysis was per-formed on a C. canephora collection in-cluding the three parental clones of thecontrolled hybrid populations (CP). The ge-netic diversity assessment was done usinga set of 13 SSR. The results are shown inFigure 1 and clearly indicate that the twoBP clones are belonging to the Congolesegroup and the Q 121 clone is rather partof the Conilon per Guinean hybrid group.Consequently, the two BP clones sound morerelated to each other than with Q 121 clone.This result is in agreement with previousstudy of genetic diversity using of RFLPmarkers (Priyono, 1998). Consequently, onemay expect more genetic diversity in the:BP 961 X Q 121 (CPA) and BP 409 xQ 121 (CPB) progenies than in the BP 409x BP 961 (CPC) one. Another importantgenetic factor to be considered, at least forthe genetic mapping process, is the heterozy-gosity of the three parents. In this regard,the screening of BP 409, BP 961 and Q 121clones with 757 genomic SSR led to respec-tively 52%, 53% and 40%, heterozygosity.This result suggests that the heterozygos-ity rate observed is sufficient for mappingthe three populations. Interesting to be notedthat the Q 121 clone (Conilon per Guineanhybrid group) is less heterozygous than thetwo BP clones (Congolese group).

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Six genetic maps construction

Mapping on multiple populations pro-vides several advantages over mapping basedon a single population. In particular, a largernumber of loci can be placed onto a singlemap. This is especially important when at-tempting to map specific genes of interestthat are unlikely to segregate within a singlemapping population. This also provides forgreater genomic coverage. Therefore, theconstruction of a genetic map is a key stepfor a reliable linkage analysis of importanttraits. Unfortunately, all linkage maps areunique and the product of a given popula-tion and marker set. Even when using thesame set of markers to construct differentlinkage maps, all the markers may not bepolymorphic for different populations andtherefore reliable across the genetic maps.Thus, in order to correlate the information

from one map to another, shared markersare required (Ablett et al., 2003; Flandez-Galvez et al., 2003).

Present result showed that a total of 199markers were selected from the referencemap and then transposed to construct theCPA genetic map. This genetic map covers11 linkage groups corresponding to the 11chromosomes of haploid C. canephora.Among the 199 markers, 50%, 23% and27% markers are respectively segregatingas F2, backcross to BP 409 and backcrossto Q 121. Thus, 145 markers segregate tothe first parental clone (BP 961) and154 to the second parental clone (Q 121)(Table 2). This genetic map is covering 1165cM, about 84% of the reference map length,with an average inter loci distance of6.2 cM.

-4

-3

-2

-1

0

1

2

-1,5 -1 -0,5 0 0,5 1 1,5 2

Axis 2

Axis 1

Q121

BP409

Congolese

Conillon

Guinean

BP961

-4

-3

-2

-1

0

1

2

-1,5 -1 -0,5 0 0,5 1 1,5 2

Axis 2

Axis 1

Q121

BP409

CongoleseCongolese

ConillonConillon

GuineanGuinean

BP961

Figure 1. The SSR genetic diversity of Robusta coffee (Coffea canephora) using Principal Component Analysis (PCA)among 143 accessions of Robusta coffee including the three parental clones (BP 409, BP 961 and Q 121) used forthe genetic mapping. The first and second axis explained, respectively 29.9% and 7.3% of the variance(Priyono, 2010)

Gambar 1. Keragaman kopi Robusta (Coffea canephora) berdasarkan marka SSR menggunakan uji Principal ComponentAnalysis (PCA). Uji keragaman genetik menggunakan 143 aksesi termasuk tiga klon (BP 409, BP 961 danQ 121) yang digunakan untuk pembuatan peta genetik. Varian untuk sumbu pertama dan sumbu kedua berturut-turut adalah 29,9% dan 7,3%.(Priyono, 2010)

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For the CPB genetic map a total of280 markers were selected from the ref-erence map and then transposed to con-struct the genetic map. This genetic mapalso covers of 11 linkage groups. Amongthe 280 markers, 43%, 35% and 22%markers, are segregating respectively asF2, backcross to BP 409 and backcross toQ 121. Thus, amount of 218 markerssegregate to the first parental clone (BP409) and of 181 to the second parentalclone (Q 121) (Table 2). This genetic mapcovers 1320 cM (95% of the referencemap length) with an average distancebetween loci of 4.9 cM.

Only 136 markers were transposed toconstruct the CPC genetic map. This lim-ited number of mappable marker is the con-sequence of the low genetic diversity be-tween the two BP clones and consequentlyled to a huge increase of the number ofdetected linkage groups. However it has beenpossible to align these 21 linkage groups tothe 11 groups of the reference map. Amongthe 136 markers, 63%, 20% and 17% mark-ers are segregating respectively as F2, back-cross to BP 409 and backcross to BP 961.This map includes the highest percentageof F2 <ab x ab> segregating loci with 17%compared to 1.5% for the CPA and 0% forthe CPB. This result underlines again the lowgenetic diversity between the two heterozy-gous BP clones that displays only the twoalleles (a;b). Thus, 113 markers segregateto the first parental clone (BP 409) and 109segregate to the second parental clone(BP 961) (Table 2). This genetic map covers1006 cM, about of 72% of the referencemap length with an average distance betweentwo loci of 8.9 cM. Previously, a maplength can be affected by the level ofhomology of the mapped parents such as inpapaya (Ma et al., 2004) but also by thekind of markers (Hendre et al., 2008).

To be useful in breeding programs,markers should reveal polymorphism indifferent populations derived from a widerange of parental genotypes (Langridge etal., 2001). In this study, some markerscould be mapped in a population but not ina second one. It suggests that the loci het-erozygosity was different among these popu-lations. The example of homolog map of link-age group B of the three genetic maps ofthree populations is presented in Figure 2.Due to the presence of different polymor-phic markers in these three populations, thegap regions observed on the three maps aredifferent. However, almost common markersare positioned in the same order on thesethree genetic maps.

As mentioned earlier each backcross(BC) genetic map was developed from acommon parent from two populations. Con-sequently, the number of individuals usedto construct these backcross maps could beincreased through the merging of the twoCP populations. Therefore the number ofindividuals for BC BP 409, BC BP 961,and BC Q 121 were 175 (93 from CPB and82 from CPC), 160 (78 from CPA and 82from CPC) and 171 (78 from CPA and 93from CPB), respectively.

The markers used to construct the threeBC genetic maps have to be heterozygousfor the common parent. By this approach,a total of 63 shared markers could be usedto construct the BP 409 BC genetic map.They cover 980 cM and represent 70%of the reference map length with an av-erage distance between two loci of 21.3 cM.This genetic map is composed of 17 link-age groups easily assigned to the 11 link-age groups of the reference map. The ex-ample of linkage group A of BP 409 geneticmap is presented in Figure 3.

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Notes (Catatan) : 1) Three cross pollinated map were CPA (BP 961 x Q 121), CPB (BP 409 x Q 121), and CPC (BP 409 xBP 961). 2) Three backcross map were Q 121 (BP 961 x Q 121 and BP 409 x Q 121), BP 409 (BP 409 x Q 121and BP 409 x BP 961), and CPC (BP 961 x Q 121and BP 409 x BP 961), 3) F2 type segregation loci include a/bx a/b, a/b x c/d, and a/b x a/c.(Tiga peta genetik silangan terbuka adalah CPA (BP 961 x Q 121), CPB (BP 409x Q 121), dan CPC (BP 409 x BP 961). 2) Tiga peta genetik silang baik adalah Q 121 (BP 961 x Q 121 danBP 409 x Q 121), BP 409 (BP 409 x Q 121 dan BP 409 x BP 961), dan CPC (BP 961 x Q 121 dan BP 409 xBP 961), 3) Tipe segregasi F2 lokus meliputi a/b x a/b, a/b x c/d dan a/b x a/c).

Table 2. Summary of the genetic maps of Robusta coffee (Coffea canephora) can be used for QTL study

Tabel 2. Ringkasan peta genetik kopi Robusta (Coffea canephora) yang dapat digunakan untuk analisis QTL

DescriptionUraian

Cross pollinated map1)

Peta silangan terbukaBackcross map2)

Peta silangan balik Consensus genetic mapPeta genetik konsensus

CPA CPB CPC BP 409 BP 961 Q 121

Number of individuals 78 93 82 175 160 171 -Jumlah individu

Number of loci 199 280 136 63 46 133 381Jumlah lokus

Number of loci with F2 typesegregation3) 100 119 86 - - - -Jumlah lokus dengan tipesegregasi F2

Number of loci with BC typesegregation to BP 409 - 99 27 63 - - -Jumlah lokus dengan tipesegregasi silang balikpada BP409

Number of loci with BC typesegregation to BP 961 45 - 23 - 46 - -Jumlah lokus dengan tipe segregasi silang balik padaBP961

Number of loci with BC typesegregation to Q 121 54 62 - - - 133 -Jumlah lokus dengan tipesegregasi silang balik padaQ 121

Average distance between twoloci,cM/loci 6.2 4.9 8.9 21.3 17.1 8.6 3.6Rerata jarak antara dualokus,cM/loci

Genetic map length, cM 1,165 1,320 1,006 980 547 947 1,325Panjang peta genetic, cM

Proportion of genetic maplength to reference geneticmap (%) 84 95 72 70 39 68 95Proporsi panjang peta genetikterhadap peta genetic acuan

Number of linkage groups( LOD e” 5) 11 11 21 17 14 12 11Jumlah kelompok tautan( LOD e” 5)

A total of 46 shared markers couldbe used to construct the BP 961 BC ge-netic map, covering 547 cM representingonly 39% of the reference map length. Theloci average distance was 17.1 cM. Thehighest distance between two loci was 54cM (linkage group K). Due to the low num-ber of common markers on the linkage

groups H and J, these two groups werenot constructed. This genetic map includes14 linkage groups; however they could eas-ily be assigned to nine linkage groups ofthe reference map. The example of link-age group A of BP 961 BC genetic mapis presented in Figure 3.

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LG B3_CPC

Figure 2. An example of homolog marker of the linkage group B of three CP genetic maps.

Gambar 2. Contoh kesepadanan marker peta genetik kelompok tautan B pada tiga peta genetik silanganterbuka.

Notes (Catatan): CPA (BP 961 x Q 121), CPB (BP 409 x Q 121), CPC (BP 409 x BP 961). Green, blue, and violet locicolour show that these loci heterozygote to BP 409, Q 121, and BP961 parent, respectively. Red loci colourshows that these loci heterozygote to both parent in each population (CPA (BP 961 x Q 121), CPB (B P409 xQ 121), CPC (BP 409 x BP 961). Lokus berwarna hijau, biru dan violet berturut-turut menunjukkan polasegregasi silang balik pada BP 409, Q 121, and BP 961. Lokus warna merah menunjukkan heterozigot padakedua induk).

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Finally, a total of 133 shared mark-ers could be used to construct the Q 121BC genetic map. They cover 947 cM rep-resenting 68% of the reference map lengthwith an average distance between two lociof 8.6 cM. This genetic map includes 12linkage groups easily assigned to the 11linkage groups of the reference map. Theexample of linkage group A of Q 121 BCgenetic map is presented in Figure 3.

One of the main uses of the DNAmarkers in agricultural research is the es-tablishment of linkage maps for diverse cropspecies. These maps have been utilized foridentifying chromosomal regions thatcontain genes controlling simple traits andquantitative traits using QTL analysis(Collard et al., 2005). Using largersegregating populations, the QTL detectionwill be largely improved. In our study ahigher number of markers have also beena key factor when analyzing several parentsin multi-cross experiments. It is also veryimportant for “high-resolution mapping”(at least <5 cM but ideally <1 cM), moretightly-linked markers and also todiscriminate between a single gene andseveral genes involved in a single QTL(Mohan et al., 1997).

Comparative mapping reveals a highdegree of co-linearity within species but alsobetween closely related species. This allowsthe transfer of markers between these maps.A good transferability within species wasreported in Eucalyptus sp. (Brondani et al.,2006) and Triticum sp. (Peng et al., 2000).Kuleung et al. (2004) reported the inter-specific transferability of SSR markers be-tween wheat (Triticum aestivum), rye(Secale cereale), and triticale (X TriticosecaleWittmack). 17% of wheat SSR markers weretransferable to rye, whereas 25% of ryemarkers were amplificable in wheat. 58%of wheat and 39% of rye markers could betransferred in Triticale. Although SSR mark-

ers were reported have a high transfer-ability to other coffee species (Cubryet al., 2008; Hendre et al., 2008; Poncetet al., 2004; Poncet et al., 2007), untiltoday, no report on the transferability ofgenetic map both within species and amongcoffee species is available.

Today, an important advance in ge-netic has been achieved by the construc-tion of a high density reference geneticmap of C. canephora using only co-domi-nant markers. This map is involving 831loci and covering 1392 cM. Moreover,several hundred of new SSR have beenadded to this reference genetic map andcurrently more than 1200 loci have beenmapped. Some of these markers are comingfrom collaborative work involving differentinstitutes and universities worldwide suchas: IRD and CIRAD (France), TriesteUniversity (Italy), CENICAFE (Colombia),Illionis University (USA), and Viosa Fed-eral University (Brazil) (D. Crouzillat,person.comm.). Present study showed thatsix genetic maps which developed usingSSR markers selected from this referencegenetic map showed the good homologyand high co-linearity. These results sug-gesting that this reference genetic map isreliable map.

Looking at the patterns generated bySSR, it is now claimed that, apart from ge-netic diversity, these markers are ideal forgenetic maps. In present study, all mark-ers selected from the reference map, showedthe highest success when mapped both forthe two others population (BP 961 x Q 121and BP 409 x Q 121) and three BC geneticmaps. It suggests that SSR and SNP markersare simple and fast for creating transferablemaps and for the generation of markers forhigh throughput genotyping. These trans-ferable markers can be exploited for fur-ther coffee genetic and breeding studies. Thegeneralized use of an increasingly larger set

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R1740000

AY243411CFGA0691331063616R10818

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Figure 3. An example of the homolog markers of linkage group C of the three backcross genetic maps.

Gambar 3.Contoh kesepadanan marka pada kelompok tautan C pada tiga peta genetik silang balik.

LG C_ BP 961LG C_ Q 121

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of transferable markers, will allow fasterand more precise investigations about QTLsynteny among species, QTL validationacross different genetic backgrounds, andpositioning of a growing number of candi-date genes co-localized with QTLs.

Darvasi et al. (1993) reported that thepower for QTL detection was virtually thesame for 10 cM marker spacing as for aninfinite number of markers, and only slightlydecreases for 20-50 cM spacing. If theobjective is to detect major QTLs, theaverage span between loci should be closefrom 10 cM. Several map intervals largerthan 20 cM were observed in this study.The higher the number of intervals largerthan 20 cM the lower the heterozygosity ofthe parents used to develop the map. Thesefrequencies were 0 for CPB, 7 for CPA,and 18 for CPC. These results were possiblydue to the high genetic diversity betweenthe parents BP 409 and Q 121, on thecontrary of BP 409 and BP 961 which werecloser reducing the number of mappablepolymorphic. Furthermore, the coverage andhigh density marker of 6.2 (CPA), 4.9(CPB), 8.9 (CPC), and 8.6 (BC Q 121)of these maps are satisfactory for QTL de-tection, except for CPC which has 18 in-tervals greater than 20 cM. Considering thebackcross genetic maps, an average distanceof 21.3 (BC BP 409) and 17.1 (BC BP 961)between two loci was observed. However,these maps are characterized by a high fre-quency of gaps greater than 20 cM, in thelinkage maps (21 for BC BP 409 and 12for both BC BP 961 and BC Q 121). Inthese regards these maps cannot be used forQTL detection. Therefore, it looks necessaryto pursue the construction of a linkage mapsaturated with diverse types of markersespecially for gap regions.

Consensus map

If shared markers have been incor-porated into different maps, these geneticmaps can be aligned together to producea consensus map, which provides a frame-work to align partial and complete link-age maps (Ablett et al., 2003; Karakousiset al., 2003). This map allows research-ers to rapidly and accurately select markersof chromosome regions of interest. In thisstudy, the development of the consensusmap was undertaken for localizing andcomparing the location of QTLs detectedon the six different genetic maps (Fig-ure 4). Due to this objective, the consen-sus map of C. canephorawas constructed by joining three indepen-dent CP maps. The present result showedthat all the loci previously mapped couldeasily be arranged on the 11 linkagegroups, using a LOD score threshold of5.0. Concerning the loci origin, 35.4%,56.7% and 18.6% of these markers arecoming respectively from CPB, CPA andCPC genetic maps (Table 1). Only 17 %of the loci are shared by the three CP mapsbut they are very important with regardsto the establishment of the consensus ge-netic map and for the comparison of QTLs.Seven genetic maps developed in presentstudy are summarized in Table 2. Inter-esting to be noted that all markers mappedin consensus map are homolog with threeCP maps used to develop this map. More-over, almost markers of consensus mapare colinier with the three CP maps. Onlyminor differences can be observed (Fig-ure 4). These results clearly again con-firm that SSR and SNP markers used inthis study showing a high transferabilityin genetic mapping.

In this study the consensus map is,in fact, based on the consolidation of seg-

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regating markers from three independentCP genetic maps. While the total lengthof the consensus map is close from thatof the three individual maps, the total num-ber of loci is larger resulting in a highermarker density and a reduced averageinterloci distance. It is estimated that mark-ers of the consensus map are very closefrom their true position as determined onthe three maps. All linked SSR and SNPmarkers mapped consistently on the samelinkage groups in the three CP maps andmost of the markers mapped with the sameorder along the homologous existing link-age groups. Consequently, it could beclaimed that the QTLs detected from thethree individual CP maps as well as fromthe three backcross maps, are quite pre-cisely localized on this consensus map.

Present report significantly increasesthe availability of microsatellite markersand mapping information for Robusta cof-fee. This work represents an important stepforward for Coffea comparative genomics,opening stimulating perspectives for evolu-tionary studies and molecular breeding ap-plications. The generalized use of an increas-ingly larger set of interspecific transferablemarkers and consensus mapping informa-tion, will allow faster and more detailed in-vestigations of QTL synteny among species,validation of expression-QTL across vari-able genetic backgrounds and positioning ofa growing number of candidate genes co-localized with QTLs, to be tested in asso-ciation mapping experiments.

CONCLUSIONS

Based on the SSR and SNP markersused in present study, it could be concludedthat the BP 409 and BP 961 clones belongto the Congolese group and the Q 121 cloneis rather part of the Conilon per Guineanhybrid group. These parental clones have

a high heterozygosity level, namely 52%,53% and 40% for BP 409, BP 961 andQ 121 clones, respectively. All SSR and SNPmarkers selected from the reference geneticmap of C. canephora have a high transfer-ability when used to develop three CP maps,three BC maps, and one consensus map ofthis species. Our genetic maps lead to beused for detection of quantitative trait locidetection.

Acknowladgement

The authors thank to Indonesian Cof-fee and Cocoa Research Institute, PT NestleIndonesia and Nestle R&D Centre Tours forsupporting this project.

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