Index Table of contents
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Construction of a Genetic Map Based on an Interspecific F2
Population between Coffea arabica and Coffea canephora and its
Usefulness for Quality Related Traits
R.H.G PRIOLLI1, L.C.S. RAMOS2, D. POT3,4, M. MOLLER2, P.B. GALLO2,
M.M. PASTINA1, A.A.F. GARCIA1, P.Y. YAMAMOTO2, S.D. LANNES3,
L.P. FERREIRA3, M.B.S. SCHOLZ3, P. MAZZAFERA5, L.F.P. PEREIRA6,
C.A. COLOMBO2
1ESALQ/USP, Departamento de Genética, Piracicaba (SP), Brazil
2IAC (Instituto Agronômico de Campinas), CPD de Recursos Genéticos Vegetais,
Campinas (SP), Brazil
3IAPAR (Instituto Agronômico do Paraná), Londrina (PR) Brazil
4Cirad, UMR DAP, Montpellier, France
5UNICAMP, Instituto de Biologia, Campinas (SP), Brazil
6 Embrapa Café, Brasília, DF, Brazil
SUMMARY
Genetic maps based on molecular markers have been developed in a large set of plants and this strategy has proven its efficiency towards the identification of tools for marker assisted selection. In the present study, AFLP and SSR markers were used to build a genetic map of an
interspecific F2 population between Coffea arabica and Coffea canephora. It was identified
349 AFLP markers and 50 SSR alleles segregating in 90 F2 plants from forty four AFLP
combinations and 19 SSR loci. For the map construction, only single dose markers
segregating 3:1 in the F2 were considered (248 AFLP markers and 27 SSR alleles, standing
for to 68.9% of the polymorphic markers). The genetic map was build and one hundred and sixty nine markers were mapped, corresponding to 155 AFLP markers and 14 SSR loci. Thirty seven linkage groups corresponding to a total map length of 1011 cM were obtained, with an average distance between the markers of 5.98 cM and an average of 4.58 markers per linkage group. Forty-six marker trait associations were found; of which, nineteen were associated with sugar content, eight for caffeine, eight for CGA, one for caffeine and CGA and ten for total production per plant. Only four single markers associations were detected at both years of determinations. The single markers analysis for QTL detection allowed us to obtain previous information of putative QTL association for coffee quality and productivity. Additional markers are being added to this working linkage map for more complete coverage of the coffee genome.
INTRODUCTION
C. arabica L. the only self-fertile tetraploid species (2n = 4x = 44) of the Coffea genus, is
characterized by low genetic diversity which has been attributed to its allotetraploid origin, its reproductive biology and its domestication history. In contrast, diploid species from the coffea genus (2n = 2X = 22) are alogamous and are highly diverse at the phenotypic and molecular levels. These species form valuable gene reservoirs for different breeding purposes (Carvalho 1988).
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Transfer of desirable genes from C. canephora to C. arabica varieties through interspecific crosses is one of the breeding strategies used for coffee improvement. C.arabica x
C.canephora hybrids, resulting from the hybridization between C. arabica and colchicine
doubled C. canephora have been cited as reasonably fertile (Berthaud, 1978; Owuor and Van der Wossen, 1981). They are also particularly favorable to intergenomic recombination and gene introgressions (Lashermes et al., 2000; Herrera et al., 2002; Priolli et al., 2008).
Molecular markers are being used successfully in many crops to assist directed germplasm improvement. Marker-assisted selection allows screening of large numbers of trees for a gene of interest at early stage and reduces the number of backcrosses required to select elite genotypes (Lashermes et al., 1999).
In coffee, a saturated Coffea canephora genetic was built by Lashermes et al. (2001) and
others partial genetic maps were obtained for interspecific crosses involving diploid species (C. pseudozanguebarie x C. liberica (Ky et al., 2000) e C. canephora x C. heterocalyx
(Coulibaly et al.2003)). Identification of quantitative trait loci allowed the localization of two
markers that flanked a fructification time genomic region (Akaffou et al. 2003) and three markers associated with pollen viability (Coulibaly et al., 2003), which could be used for early marker-assisted selection.
In C. arabica, according to its low polymorphism level associated with its tetraploid, the strategy consists on the construction of partial genetic maps (Pearl et al., 2004; Teixeira-Cabral et al., 2004) with posterior integration of the partial genetic maps.
In the present study, AFLP and SSR markers were used to build a genetic map of an F2
interspecic population between C. arabica and C.canephora. In addition, association between segregating markers and quality related traits were analyzed.
MATERIAL AND METHODS Plant material
The F1 tetraploid hybrid between Coffea arabica L. var. Bourbon Vermelho and Coffea
canephora var. Robusta 4x, an artificial tetraploid obtained by Mendes (1947), has, since
1996, been advanced to F2 by selfing three F1 clones from the same plant. The F2 segregating
population was grown in a field trial at a site near the municipality of Mococa (latitude 21º 28' S, longitude 47º 01' W and altitude 665 m) in São Paulo State Brazilian state received treatment with inorganic fertilizer, and weed and pest control and all other treatments recommended for growing coffee under Brazilian conditions (Thomaziello et al., 1996).
Sample preparation and field data
Leaves and fruits from each F2 were harvested two successive years (2004 and 2005). Leaves were collected from the third and fourth leaf pairs from different sides of the tree canopy. Fruits were harvested at mature stage for analysis of caffeine, chlorogenic acids and sugar contents.
Seeds were manually removed from the pericarp, dried at 70 °C for two weeks and then finely ground with a blade grinder or with a pestle and mortar. Caffeine and chlorogenic acids were extracted according to Priolli et al. (2008). Total and reducing sugars were extracted according to Rogers et al. (1999) and quantified using Somogyi and Nelson reagent. Sucrose
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content was estimated by the substraction of reducing sugars content from total sugar contents. Production was also evaluated (Kg fruits / Plant).
Molecular marker assay
Total genomic DNA was extracted from freeze-dried leaves of the parental, F1 hybrid and F2
genotypes as described by Ky et al. (2000). Nineteen microsatellite loci, previously identified as polymorphic between C. arabica and C. canephora, were analyzed using PCR. Some of these microsatellite loci (Table 1) have been mapped in C. canephora (Lashermes et al, 2001) and other were obtained by Combes et al (2000). The specific primer pairs, amplification conditions, radioactive labelling and polyacrylamide gel electrophoresis were as reported elsewhere (Priolli et al., 2008). The amplified fragment length polymorphism (AFLP) procedure was performed as previously reported (Vos et al., 1995). Briefly, 500 ng of genomic DNA was digested with the restriction enzymes EcoRI and MseI. Restriction fragments were then ligated with double-strand EcoRI and MseI adapters. A selective pre-amplification was performed using the appropriate primers (named E and M, respectively) without selective nucleotide at the 3′ end (ie E+0/M+0). The reaction mixture was diluted 1/10 and 3 µL was used for the final amplification with two primers, each containing three selective nucleotides (Table 1).
Data analysis
Only markers polymorphic between the parents and present in the F1 were considered. This
strategy was applied whatever the marker used. Consequently, both SSRs and AFLPs were considered here as dominant markers. Segregation distortion from the 3:1 expected ratio for single dose markers was analyzed by the chi-square test. Bonferoni correction was applied to control type I error for multiple tests. Map construction was carried out using Joinmap version 3.0 (Stam 1993). Linkage groups were established using two-point analysis with LOD threshold values of 4 and recombination fraction of 0.50. The Kosambi function was used for converting recombination fractions into map distances. Associations between markers and the analyzed traits (total sugar, reducing sugar, sucrose, caffeine, chlorogenic acids contents and production) were analyzed by one-way ANOVA. Significant association were considered for
P value lower than 0.001 and suggestive associations were considered for P value between
0.001 and 0.005.
RESULTS AND DISCUSSION Linkage map
Fifty alleles of SSR markers and 349 AFLP markers were polymorphic in F2 populations.
Chi-square analysis revealed 275 markers (69%) fitted a 3:1 ratio. The remaining 99 (33%) showed segregation distortion within the population. One hundred and sixty nine markers were mapped, corresponding to 155 AFLP markers and 14 SSR alleles. 66% of the mapped markers came from the Coffea arabica parent, 30% from the Coffea canephora 4x and 4% from both parent. These results suggest that the divergence between the two ancestral
genomes of Coffea arabica (i.e C. canephora and Coffea eugenioides) is two timeslarger than
the one between the two haplotypes of the C. canephora genotype used as a parent to create
the F1 hybrid. Similar results were observed by Pearl et al (2004) who found in a pseudo F2
population derived from a cross between the cultivars of C. arabica , 68% of AFLP markers were from cv. Catimor, 30% from cv. Mokka hybrid, and 2% were codominant.
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Thirty seven linkage groups corresponding to a total map length of 1011 cM were obtained, with an average distance between the markers of 5.98 cM and 4.58 markers per linkage group (Figure 1). Twenty linkage groups include only 2 markers.
(A) E5M10_126 0 E3M13_222 22 1 E6M10_296 0 E4M3_340 13 E5M10_226 E5M9_246 26 C32_110 28 E3M3_170 48 E6M11_158 51 E2M5_340 53 E1M1_148 66 1 E4M10_134 0 E4M3_330 10 E1M2_128 19 E2M5_342 29 E2M6_254 42 1 E5M10_150 0 C32_108 2 E6M11_130 6 E3M6_136 E8M12_180 7 EST4_240 10 E6M11_132 11 E3M2_240 15 E5M10_122 18 E6M6_102 21 E4M4_292 23 E8M12_170 28 E3M1_152 35 E5M10_138 50 2 E4M10_340 0 E4M4_328 9 E3M3_330 12 E2M3_242 21 E3M2_260 25 E6M6_154 26 E5M2_338 33 E1M1_210 39 E2M1_238 60 3 E3M3_338 0 E8M14_122 3 E8M14_118 18 3 E1M3_190 0 E5M1_308 8 E5M1_100 11 E1M1_260 14 E6M10_258 16 E8M12_290 18 E5M2_332 E5M2_216 23 M17_118 32 E8M14_336 35 E8M12_220 36 4 EST4_120 0 E3M3_184 4 E5M10_338 22 E1M3_340 35 E3M10_124 40 5 E5M10_126 0 E3M13_222 22 1 E6M10_296 0 E4M3_340 13 E5M10_226 E5M9_246 26 C32_110 28 E3M3_170 48 E6M11_158 51 E2M5_340 53 E1M1_148 66 1 E4M10_134 0 E4M3_330 10 E1M2_128 19 E2M5_342 29 E2M6_254 42 1 E5M10_150 0 C32_108 2 E6M11_130 6 E3M6_136 E8M12_180 7 EST4_240 10 E6M11_132 11 E3M2_240 15 E5M10_122 18 E6M6_102 21 E4M4_292 23 E8M12_170 28 E3M1_152 35 E5M10_138 50 2 E4M10_340 0 E4M4_328 9 E3M3_330 12 E2M3_242 21 E3M2_260 25 E6M6_154 26 E5M2_338 33 E1M1_210 39 E2M1_238 60 3 E3M3_338 0 E8M14_122 3 E8M14_118 18 3 E1M3_190 0 E5M1_308 8 E5M1_100 11 E1M1_260 14 E6M10_258 16 E8M12_290 18 E5M2_332 E5M2_216 23 M17_118 32 E8M14_336 35 E8M12_220 36 4 EST4_120 0 E3M3_184 4 E5M10_338 22 E1M3_340 35 E3M10_124 40 5 E5M1_220 0 E3M2_336 8 E4M3_174 15 E1M2_120 25 6 E3M9_126 0 E3M1_230 13 E3M8_250 27 7 E3M6_244 0 E7M3_106 15 8 E5M9_152 0 E4M13_340 22 9 E4M 13_174 0 E4M 13_96 16 10 E8_177 0 E8M14_180 17 11 E2M5_330 0 E2M1_218 20 12 E8M 12_156 0 E4M 2_278 5 E4M 13_134 11 E4M 8_270 13 E5M 10_114 14 E3M 8_160 17 E7M 3_170 19 E3M 3_210 21 E2M 6_186 23 E8M 12_216 28 E4M 15_150 31 E3M 13_158 33 E1M 1_262 38 E5M 2_198 39 M29_126 46 13 E5M1_220 0 E3M2_336 8 E4M3_174 15 E1M2_120 25 6 E3M9_126 0 E3M1_230 13 E3M8_250 27 7 E3M6_244 0 E7M3_106 15 8 E5M9_152 0 E4M13_340 22 9 E4M 13_174 0 E4M 13_96 16 10 E8_177 0 E8M14_180 17 11 E2M5_330 0 E2M1_218 20 12 E8M 12_156 0 E4M 2_278 5 E4M 13_134 11 E4M 8_270 13 E5M 10_114 14 E3M 8_160 17 E7M 3_170 19 E3M 3_210 21 E2M 6_186 23 E8M 12_216 28 E4M 15_150 31 E3M 13_158 33 E1M 1_262 38 E5M 2_198 39 M29_126 46 13 E6_325 0 E5M8_330 4 E6M11_108 9 E1M1_166 17 E1M2_236 24 E5M1_334 E5M10_250 30 E2M3_336 35 E6M10_334 42 E7M3_190 53 E1M1_154 63 14 E5M8_104 0 E2M5_356 3 E6M10_262 7 E3M10_190 10 E3M6_340 19 E4M10_182 28 15 E4M8_170 0 E1M1_310 7 E1M1_254 19 E2M5_348 23 E1M1_182 26 E5M9_214 27 16 E8M 14_200 0 E8M 14_190 30 17 E6M9_338 0 E3M8_240 10 E6M11_334 18 E5M2_280 22 E3M2_334 25 E7M3_336 32 E5M8_204 40 18 E3M1_118 0 E1M1_244 19 19 E2M5_358 0 E6M11_290 6 E4M8_110 9 E5M2_334 13 E5M2_152 15 20 E4M15_320 0 E4M15_190 13 E1M5_338 21 21 E6_325 0 E5M8_330 4 E6M11_108 9 E1M1_166 17 E1M2_236 24 E5M1_334 E5M10_250 30 E2M3_336 35 E6M10_334 42 E7M3_190 53 E1M1_154 63 14 E5M8_104 0 E2M5_356 3 E6M10_262 7 E3M10_190 10 E3M6_340 19 E4M10_182 28 15 E4M8_170 0 E1M1_310 7 E1M1_254 19 E2M5_348 23 E1M1_182 26 E5M9_214 27 16 E8M 14_200 0 E8M 14_190 30 17 E6M9_338 0 E3M8_240 10 E6M11_334 18 E5M2_280 22 E3M2_334 25 E7M3_336 32 E5M8_204 40 18 E3M1_118 0 E1M1_244 19 19 E2M5_358 0 E6M11_290 6 E4M8_110 9 E5M2_334 13 E5M2_152 15 20 E4M15_320 0 E4M15_190 13 E1M5_338 21 21
886 (B)
Figure 1.Genetic linkage map constructed from C.arabica x C.canephora containing 155 AFLP markers and 14 SSR markers. Mapping distances are represented in centiMorgans (cM) and markers codes are provided on the right side of each linkage group.
Segregation analyses of restriction fragment length polymorphism (RFLP) loci-markers have indicated tetrasomic inheritance resulting from the pairing of homologous chromosomes in meiosis of first generation C. arabica x C. canephora 4x hybrids (Lashermes et al., 2000). In
two BC1F1 populations, (C. arabica × C. canephora 4x) × C. arabica, segregations and
co-segregation of RFLP and microsatellite loci-markers conformed to the expected ratio assuming random chromosome segregation and the absence of selection (Herrera et al., 2002).
In a study using SSR loci, the hybrid F1 showed that the ratios of the gametes genotype did
not differ significantly from those expected assuming random associations and tetrasomic inheritance (Priolli et al., 2008).
In C. arabica, according to its low polymorphism level associated with its tetraploid, the strategy consists on the construction of partial genetic maps with posterior integration of the partial genetic maps. A linkage map of arabica coffee was constructed from 288 AFLP primer combinations resulting in 16 major linkage groups containing 4-21 markers, and 15 small linkage groups consisting of 2-3 linked markers. The total length of the map was 1,802.8 cM, with an average distance of 10.2 cM between adjacent markers (Pearl et al., 2004). In a backcross population of the C. arabica, a partial genetic map was constructed with 82 RAPD loci and covered the estimated length of 540.6cM, average distance of 7.3 cM between
adjacent markers in a total of eight linkage groups (Teixeira-Cabral et al 2004). In C.
canephora, a genetic map of 1402 cM with 15 linkage groups was reported using 47 RFLP
and 100 RAPD markers (Paillard et al., 1996). Eleven linkage groups that putatively correspond to the 11 gametic chromosomes of C. canephora were identified from 162 loci (97 AFLP, 11 RAPD, 18 microsatellite, and 36 RFLP) in a total map length of 1041 cM and
E5M10_204 0 E5M10_140 18 22 E5M 10_326 0 E3M 10_310 13 23 E2M1_330 0 E8M14_340 9 24 E4M1_120 0 E3M13_218 3 25 EST2_220 0 E8M12_336 13 26 E4M10_336 0 E1M1_110 13 E3M6_336 E6M10_224 20 27 E3M 8_146 0 E3M 8_130 21 28 E5M10_320 0 E8M14_332 19 E5M13_340 25 29 E5M10_204 0 E5M10_140 18 22 E5M 10_326 0 E3M 10_310 13 23 E2M1_330 0 E8M14_340 9 24 E4M1_120 0 E3M13_218 3 25 EST2_220 0 E8M12_336 13 26 E4M10_336 0 E1M1_110 13 E3M6_336 E6M10_224 20 27 E3M 8_146 0 E3M 8_130 21 28 E5M10_320 0 E8M14_332 19 E5M13_340 25 29 M47_134 0 E6M9_330 28 30 E4M11_166 0 E4M11_150 13 31 M27_138 0 C32_104 20 32 E6M11_330 0 E6M11_280 13 33 M11_145 0 E5M10_188 15 34 M32_128 0 E5M9_282 12 35 E6M11_100 0 E1M2_98 5 E6M11_336 11 E2M5_334 15 36 E8M14_306 0 E5M13_318 5 37 M47_134 0 E6M9_330 28 30 E4M11_166 0 E4M11_150 13 31 M27_138 0 C32_104 20 32 E6M11_330 0 E6M11_280 13 33 M11_145 0 E5M10_188 15 34 M32_128 0 E5M9_282 12 35 E6M11_100 0 E1M2_98 5 E6M11_336 11 E2M5_334 15 36 E8M14_306 0 E5M13_318 5 37
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average distance of 6.5 cM. (Lashermes et al., 2001). Genetic maps for interspecific diploid
crosses were also obtained for C. pseudozanguebarie x C. liberica (Ky et al., 2000) and C.
canephora x C. heterocalyx (Coulibaly et al., 2003) leading to the identification of 14 linkage
groups covering 1,144 cM and 15 linkage groups with 1,360 cM respectively.
Table 1. List of SSR loci and selective AFLP primers (EcoRI+3 and MseI+3) with their codes presents in C.arabica X C.canephora genetic map.
Loci SSR Code Primer AFLP Code Primer AFLP Code
17-2CTG M17 ACC E1 CAA M7
32-2CTG C32 ACT E2 CAG M8
C2-2CATC C2 AAC E3 CGA M9
E6-3CTG E6 ACA E4 CGT M10
E8-3CTG E8 AAG E5 CGG M11
EST1 EST1 AGC E6 CCC M12
EST2 EST2 AGG E7 CGC M13
EST4 EST4 ACG E8 CCG M14
M11 M11 CAC M1 CCA M15 M25 M25 CTG M2 M27 M27 CTT M3 M29 M29 CTC M4 M32 M32 CAT M5 M47 M47 CTA M6
Single marker analysis
Single marker analysis to detect associations between phenotypic traits including total sugar, reducing sugar, sucrose, caffeine, chlorogenic acids contents and production and molecular markers were performed. Two threshold levels corresponding to significant association (P value < 0.001) and suggestive level (0.001 < P value < 0.005) were considered (Table 2).
Overall, 46 marker trait associations were found corresponding to seven from SSR makers
and 39 from AFLP markers. The percentage of the phenotypic variance explained by each marker ranged from 10.62 (E3M1_118 marker) to 20.69% (E3M3_170 marker). In relation to biochemical contents, ten marker-trait associations were found for total sugar, seven to reducing sugar, eleven to sucrose, nine to caffeine and nine to CGA. For field production data, ten markers presenting suggestive and significant effect were detected. Nine markers were associated to total sugar and sucrose but only two of these markers presented significant
or suggestive effects in the two years analysed (E3M8_250 and E5M1_308). Marker
E4M3_330 also presented association with caffeine content in the two successive years, the
same effect stability was observed for E3M8_146 in relation with the production level. These
markers can be viewed as consistent markers, with potential to be applied in a further marker assisted selection.
In a few cases the same markers presented association with two distinct traits. Such results were observed for marker E2M5_342 for caffeine and GCA contents. The same observation was done for markers that presented associations with total sugars and sucrose contents. However such association was expected as sucrose content was derived from the total sugar and reducing sugar contents.
Some QTLs in Coffea were detected using segregating population, derived from interspecific
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pollen viability in a backcrossed progenies originating from a cross between Coffea
canephora and Coffea heterocalyx (Coulibaly et al., 2003).
Table 2. Single marker-test for total and reducing sugar (TS and RS), sucrose, caffeine, chlorogenic acids (CGA), and production at years 2004 and 2005, with their effects in %
of the phenotypic variability.
2004 2005 2004 2005 2004 2005 2004 2005 2004 2005 2004 2005 E3M8_250* 11.23 12.36 11.16 12.24 E5M1_308** 16.76 16.97 16.39 16.79 E5M2_216** 15.21 15.53 E5M2_332* 12.00 11.93 E5M8_330* 10.96 11.07 E3M1_118* 10.62 11.01 E3M1_230* 12.33 12.31 E5M1_100** 14.16 13.80 EST1_110* 13.96 14.38 E8_179* 11.11 E5M9_290** 14.55 E6M6_272** 12.92 E7M3_126* 12.79 M47_151** 16.57 E4M4_292** 13.01 E6M9_108* 10.64 M11_45** 14.96 E3M6_244* 11.16 E6_325* 11.01 E4M3_330* 11.36 11.69 E6M8_120** 16.74 E8M12_156* 13.44 E8M14_180** 13.78 E3M10_120** 16.31 E3M1_260* 11.96 E5M1_270* 12.04 E8M14_118* 12.17 E2M5_342** 17.77 11.28 E1M1_166* 10.84 E7M3_120** 16.99 E2M5_350** 14.17 E3M3_338* 11.29 E4M4_328** 16.36 E5M1_326** 14.13 E8M14_118** 14.46 E8M14_122** 16.86 E2M3_336* 12.20 E2M5_340** 16.51 E3M3_170** 20.69 E3M8_146** 13.99 13.34 E3M8_320** 18.35 E6M11_158** 15.36 E6M9_336* 11.29 EST4_240** 18.23 C32_110** 12.95 E3M6_244* 11.34
Markers TS RS Sucrose Caffeine CGA Production
*Significance at P < 0.005
**Significance at P < 0.001. Bold letters: significance at 2004 and 2005.
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Only one QTL was identified for fructification time using a one-way ANOVA with a significance level of P < 0.001 in a cross between Coffea pseudozanguebariae X C. liberica var. Dewevrei . The QTL was located on linkage group E defined by Ky et al. (2000), between the AFLP marker ACCCTT1 and the RFLP marker G13 The ACCCTT1 marker explained 64% of the fructification variance (Akaffou et al., 2003).
The single markers analysis allowed us to obtain preliminary information of putative QTL for biochemical components involved in the quality of coffee beverage an their relation with production. However, others QTL detection approaches such as interval mapping (Lander and Botstein, 1989) and composite interval mapping (Zeng, 1994), both, having more power to detect single QTL marker association are planned to be applied to our data set in a short-term. REFERENCES
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