COFFEE POLYMORPHISMS PROJECT

 

 

INTRODUCTION

 

The cultivated varieties of Coffea arabica show a very low level of genetic diversity (Bertraud and Charrier., 1980) due to autogamy and the limited number of original plants from which the main cultivars were derived.  Consequently, there are very few allelic variants, making it difficult to find polymorphisms.  Numerous techniques have been applied to study polymorphisms in C. arabica, such as RFLP (Lashermes et al., 1996a), RAPD (Orozco-Castillo, 1994, Lashermes et al., 1996b), and AFLP (Lashermes et al., 2000), and positive results have in fact been obtained.  Nonetheless, the same techniques applied to other species have provided a greater number of polymorphic loci.

Another approach to studying variability in C. arabica and identifying polymorphism is the analysis of microsatellites.  These highly polymorphic repeated sequences are very informative molecular markers because they are codominant and therefore, in contrast to the abovementioned techniques, enable the heterozygous samples to be distinguished from the homozygous.  Due to these characteristics microsatellites are powerful tools for following specific genes in assisted cross programmes.

In this section we describe the analysis by microsatellites of different crosses of Coffea arabica and of its progeny, F1 or F2.

Furthermore, we have begun a project of RFLP analysis of different varieties of C. arabica and of C. canephora for coding sequencies.  The aim of this is twofold: firstly, to find markers for the construction of a low density genetic map and secondly, to create the possibility of finding differences within the genes of Coffea arabica, that is, polymorphisms capable of marking the expressed genes.

 

 

MATERIALS AND METHODS

 

Amplification of the microsatellites

The microsatellites used for the analysis of the crosses were identified and amplified as described in Rovelli et al. (2000), beginning with two genomic libraries of C. a. Caturra enriched in di- and tri- nucleotides TG and ATC.

 

Identification of the polymorphic microsatellites

The primers which amplified the microsatellites were tested on DNA of the parent samples of three crosses; if the microsatellites presented different alleles from those of their parents, we proceeded with the analysis of the progeny.

The amplification products were analysed on sequencing gel with an ABI automatic sequencer and the length of the fragments containing microsatellites was calculated with the GENESCAN 672 (Perkin Elmer) programme.

 

Samples

The samples examined were: 1 C.a. var Caturra x C.a. var Ethiopica ET-30 cross, and 96 plants of the F2 population, originating from IRD (Montpellier, France); 2) the C.a. introgressed genotype Catimor x C.a. var Icatuá cross, and 6 plants of the F1 population, originating from IAPAR (Londrina, Brazil); 3) the C.a. introgressed genotype Sarchimor x C.a. var Ethiopica ET-6 cross and 17 plants of the F1 population, originating from CATIE (Turrialba, Costa Rica).

The DNA was extracted with a modification of the method of Murray and Thompson (1980) and Orozco-Castillo et al. (1994).

 

RFLP analysis

The fragments of DNA examined were stretches of genomic DNA amplified with primers designed on EST sequences; the EST were derived from a genomic library of radical meristems of C.a. var Bourbon red.

For the RFLP analysis we chose those fragments which provided an amplification from genomic DNA of greater length than the corresponding cDNA, and which therefore presumably contained introns.

 

 

RESULTS

 

Analysis of the crosses

The C.a. var Caturra x C.a. var Et-30 cross was analysed with 59 microsatellites.  Only five of these proved to be polymorphic in the parental samples.  The F2 population of 96 plants was analysed with 5 microsatellites to examine the distribution of the alleles.  Table 1 summarises the results:

 

Table 1

 

Microsatellite

Alleles (expressed in bp)

C.a. Caturra

C.a. Et-30

F 2

I9-3CTG

200-214

198-200-214

36 samples: 200-214

48 samples: 198-200-214

12 samples: 198-214

17-2CTG

204-215

202-215

39 samples: 204-215

32 samples: 202-204-215

25 samples: 202-215

32-2CTG

121-128

119-126

40 samples: 119-121  56:121-126

E10-3CTG

135

137

23 samples: 135

43 samples: 135-137

30 samples: 137

14-2CTG

130

128-130

96 samples: 130

 

Of the 28 microsatellites analyses in the introgressed genotype Catimor x C.a. var Icatuá 3 cross demonstrated different alleles in the parents, and therefore we analysed the 6 plants of the F1 population.  Table 2 shows the alleles, expressed in bp, relative to the three polymorphic microsatellites.

 

Table 2

 

Microsatellite

 

Alleles (expressed in bp)

Catimor

C.a. Icatuá

F1

20-6CTG

105-109

105-107-109

2 samples: 105-109

4 samples: 105-107-109

37-6CTG

119

121

6 samples: 119-121

24-4CTG

112

108

6 samples: 108-112

 

The introgressed genotype Sarchimor x C.a. var Et-6 cross was analysed with 51 microsatellites and four were polymorphic in the parents, and of these we analysed the 17 F1 plants.  The alleles of these are shown in table 3.

 

Table 3

 

microsatellite

Alleles (expressed in bp)

Sarchimor

C.a. Et-6

F1

I9-3CTG

200-214

198-200-214

10 samples: 200-214

7 samples: 198-200-214

14-2CTG

204-217

202-217

17 samples: 202-204-217

E10-3CTG

135

137

17 samples: 135-137

20-6CTG

104-106-108

104-106

17 samples: 104-106-108

 

In total 8 polymorphic microsatellites were identified.

 

For the complete list of all the polymorphic microsatellites, click  HERE

 

 

RFLP analysis

48 EST sequences were amplified, 13 of which produced a genomic amplification with a length greater than the corresponding EST.  We therefore analysed these 13 genomic loci, presuming that they also contained introns, where mutations are more likely to accumulate.  Table 4 shows the names of the genes with which the EST sequences were the most homologous in the data base:

 

Table 4

 

Clone
Homology

Clone

Homology

RM-0-L19

unknown

RM D04

thioredoxin h

RM B11

translationally controlled tumor protein (TCTP)

RM-0-I05

antimicrobial peptides precursor

RM A11

A.thaliana hypothetical protein

RM-0-E12

germin-like protein

RM C05

chlorophyll a/b-binding protein

RM B08

unknown

RMi-5-B10

60S ribosomal protein  L22

RM B10

40S ribosomal protein S23

RMi-1-E03

cysteine proteinase

RM A01

60S ribosomal protein L34

RM C11

A.thaliana hypothetical protein

 

 

Varieties of C. arabica (Ethiopica, Caturra, Mundo Novo, Laurina and a wild variety) and C. canephora were examined.

These tracts of genomic DNA were analysed with 8-11 restriction enzymes (Aci I, Alu I, Bam HI, Eco RI, Fok I, Hind III, Hinf I, Hph I, Mbo I, Mnl I, Msp I, Tsp 45 I, Tsp 509 I).  The analysis of the restriction patterns to date have not uncovered differences between the varieties of C. arabica in the size and number of bands.

Nonetheless, we could identify 5 different patterns between the two species of Coffea arabica and Coffea canephora.  Table 5 shows the size of the different bands of the two species.

 

Table 5

 

EST

ENZYME

C. canephora (in bp)

C. arabica (in bp)

RM A11

Mnl I

600

400 + 200

Rmi-5-B10

Msp I

not cut

900 + 800

RM C11

Alu I

600

500 + 100

RM C11

Mbo I

not cut

700 + 300

RM D04

Msp I

800 + 100

not cut

 

 

DISCUSSION

 

In total we identified 8 polymorphic microsatellites in varieties of C. arabica and 5 polymorphic restriction sites amongst C. arabica and C. canephora.

With regard to the microsatellites, we found between one and three alleles in the parental samples.  Where there were two or three alleles, one of them, such as the microsatellites I9-3CTG or 17-2CTG, was always present both in the parents and the progeny.  We therefore hypothesised that another locus was involved which became coamplified. Only the microsatellite 20-6CTG in the C. a. Sarchimor x C. a. Et-6 cross presented three alleles in one parent and the progeny, but two alleles in the other parent.  In this case there could be three alleles for the same locus; however, double haploid plants would be necessary to verify this.

The analysis of the microsatellites such as E10-3CTG, 37-6CTG or 24-4CTG suggests that even in this allotetraploid species the microsatellites are inherited in accordance with Mendel’s laws: when the parental samples displayed one allele in homozygosis but the parents had different alleles, then the F1 progeny displayed the classic Mendelian distribution of 1:2:1.

The microsatellites can be used to follow the traces of certain genes within specific crosses.  Indeed, the C.a. Sarchimor x C.a. Et-6 cross was produced with the intention of selecting plants resistant to nematodes.  With the identification of new polymorphic microsatellites we should be able to verify the distribution of the progeny of genes linked to resistance.

In general it is worth noting that the parental samples are heterozygotes only in 7 out of 24 loci: this demonstrates the high level of homozygosity which is found in C. arabica and which explains the high genetic uniformity of the species.

The RFLP polymorphisms to date have not provided positive results within the species C. arabica, but even polymorphisms between the two species are highly useful.  Moreover, each polymorphism found in this manner is related to differences in coding regions which could account for the phenotypic differences between the two species, as well as identifying the genes originating from C. canephora in inter-specific crosses, in which attempts are made to bring the positive qualities of resistance of C. canephora to the cultivated varieties of C. arabica.

 

 

Acknowledgments: We would like to thank Dr. T. Sera (IAPAR) for providing us with leaf samples of a cross.

 

 

BIBLIOGRAPHY

 

Bertraud J and Charrier A., Genetic Resources in Coffea. 1988 In "Coffee", Vol.4, Agronomy, R.J. Clarke and R. Macrae Eds, Elsevier Applied Science, London, pp. 1-41.

Lashermes P., Cros J., Combes M. C., Trouslot P., Anthony F., Hamon S. and Charrier A., 1996a “Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L." Theor Appl Genet 98: 626-632.

Lashermes P., Trouslot P., Anthony F., Combes M. C. and Charrier A., 1996b "Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica." Euphytica 87: 59-64.

Lashermes P., Andrzejewski S., Bertrand B., Combes M. C., Dussert S., Graziosi G., Trouslot P. and Anthony F., 2000 "Molecular analysis of introgressive breeding in coffee (Coffea arabica L.) Theor Appl Genet 100: 139-146.

Murray M. G. and Thompson W.F., 1980 “Rapid isolation of high molecular weight plant DNA" Nucleic Acis Res 8: 4321-4325.

Orozco-Castillo C., Chalmers K. J., Waugh R. and Powell, 1994 "Detection of genetic diversity and selective gene introgression in coffee using RAPD markers" Theor Appl genet 87: 934-940.

Rovelli P., Mettulio R., Anthony F., Anzueto F., Lashermes P., Graziosi G., 2000 "Microsatellites in Coffea arabica L." In "Coffee Biotechnology and Quality" Sera T., Soccol C. R., Pandey A. and Roussos S. Eds, Kluwer Academic Publishers, pp 123-133.