Index Table of contents

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

883 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 times larger 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.

1 1 1 2 3 3 4 5

0 E5M10_126 0 E6M10_296 0 E4M10_134 0 E5M10_150 0 E4M10_340 0 E3M3_338 0 E1M3_190 0 EST4_120 2 C32_108 3 E8M14_122 4 E3M3_184 6 E6M11_130 7 E3M6_136 E8M12_180 8 E5M1_308 10 EST4_240 9 E4M4_328 10 E4M3_330 11 E5M1_100 11 E6M11_132 12 E3M3_330 13 E4M3_340 14 E1M1_260 15 E3M2_240 16 E6M10_258 18 E5M10_122 18 E8M14_118 18 E8M12_290 19 E1M2_128 22 E3M13_222 21 E6M6_102 21 E2M3_242 22 E5M10_338 23 E4M4_292 23 E5M2_332 E5M2_216 25 E3M2_260 26 E5M10_226 E5M9_246 26 E6M6_154 28 C32_110 29 E2M5_342 28 E8M12_170 32 M17_118 33 E5M2_338 35 E3M1_152 35 E8M14_336 35 E1M3_340 36 E8M12_220 39 E1M1_210 40 E3M10_124 42 E2M6_254

48 E3M3_170 50 E5M10_138 51 E6M11_158 53 E2M5_340

60 E2M1_238

66 E1M1_148

6 7 8 9 10 11 12 13

0 E5M1_220 0 E3M9_126 0 E3M6_244 0 E5M9_152 0 E4M13_174 0 E8_177 0 E2M5_330 0 E8M12_156

5 E4M2_278 8 E3M2_336 11 E4M13_134 13 E3M1_230 13 E4M8_270 14 E5M10_114 15 E4M3_174 15 E7M3_106 16 E4M13_96 17 E8M14_180 17 E3M8_160 19 E7M3_170 20 E2M1_218 21 E3M3_210 22 E4M13_340 23 E2M6_186 25 E1M2_120 27 E3M8_250 28 E8M12_216 31 E4M15_150 33 E3M13_158

38 E1M1_262 39 E5M2_198

46 M29_126

14 15 16 17 18 19 20 21

0 E6_325 0 E5M8_104 0 E4M8_170 0 E8M14_200 0 E6M9_338 0 E3M1_118 0 E2M5_358 0 E4M15_320 3 E2M5_356 4 E5M8_330 6 E6M11_290 7 E6M10_262 7 E1M1_310 9 E6M11_108 9 E4M8_110 10 E3M10_190 10 E3M8_240 13 E5M2_334 13 E4M15_190 15 E5M2_152 17 E1M1_166 18 E6M11_334 19 E3M6_340 19 E1M1_254 19 E1M1_244 22 E5M2_280 21 E1M5_338 23 E2M5_348 24 E1M2_236 26 E1M1_182 25 E3M2_334 28 E4M10_182 27 E5M9_214 30 E5M1_334 E5M10_250 30 E8M14_190 32 E7M3_336 35 E2M3_336

40 E5M8_204 42 E6M10_334

53 E7M3_190

63 E1M1_154

(A)

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22 23 24 25 26 27 28 29

0 E5M10_204 0 E5M10_326 0 E2M1_330 0 E4M1_120 0 EST2_220 0 E4M10_336 0 E3M8_146 0 E5M10_320 3 E3M13_218

9 E8M14_340

13 E3M10_310 13 E8M12_336 13 E1M1_110

18 E5M10_140 19 E8M14_332 20 E 3M6 _ 336 E6 M1 0_ 22 4 21 E3M8_130

25 E5M13_340

30 31 32 33 34 35 36 37

0 M47_134 0 E4M11_166 0 M27_138 0 E6M11_330 0 M11_145 0 M32_128 0 E6M11_100 0 E8M14_306

5 E1M2_98 5 E5M13_318

12 E5M9_282 11 E6M11_336 13 E4M11_150 13 E6M11_280 15 E5M10_188 15 E2M5_334

20 C32_104

28 E6M9_330

(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 886 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 crosses. Three significant QTLs (LOD > 3 and p < 0.001 by ANOVA) were detected for 887 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.

TS RS Sucrose Caffeine CGA Production Markers 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 *Significance at P < 0.005 **Significance at P < 0.001. Bold letters: significance at 2004 and 2005. Red letters indicate one mark associated with two traits.

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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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