CARYOLOGIA Vol. 51, n. 1: 19-35,1998

Pachytene chromosome morphology in Coffea L.II. C. arabica L. complement

C.A.F. PINTO-MAGLIO and N.D. DA CRUZ *Seçao de Citologia, Instituto Agronomico, Campinas - 13001-970, Sao Paulo, Brazil.

SUMMARY - A detailed morphological analysis of Coffea arabica pachytene chromosomes ispresented for the first time. The pachytene chromomeric patterns revealed that there occurs somestructural similarities in about 54% of the 22 bivalents. This fact is supported by two secondaryassociations observed, one of them involving one nucleolar bivalent. Based on these results, and otherevidences from literature, it is suggested that C. arabica is a segmental alIopolyploid with geneticallycontrolled chromosome pairing.

Key words: Coffea, pachytene karyotype, chromomere pattern, meiosis, alIopolyploidy.

INTRODUCTION

In the genus Coffea L., the most important economic species (C. arabica L.) is an exception, theonly known self-compatible polyploid of the genus, with n = 22 chromosomes (KRUG andCARVALHO 1951; CARVALHO et al. 1969). All other species are self-incompatible diploidswith n = 11 chromosomes (MENDES 1949; MEDINA and CONAGIN 1959;CONAGIN andMENDES 1961; MONACO 1972) .C. arabica origin is still undefined mainly as to the putative ancestral species envolved. It couldhave been occurred through two pathways, autopolyploidy or allopolyploidy. The last one hasbeen more accepted because the evidences in several studies (CARVALHO 1952; CARVALHOetal., 1969; BERTHOU and TROUSLOT 1977; BERTHOU et al. 1980; BERTHOU et al. 1982and BERTHOU et al., 1983) where diploid species as C. eugenioides, C. canephora, C.congensis, C. dewevrei and C. liberica have been suggested as the possible ancestrals.

This work is derived in part from a Doctoral Thesis presented by C.A.F. Pinto-Maglio atUniversity Estadual of Campinas, SP, Brazil. The financial support from Fundaçao de Amparo aPesquisa do Estado de sao Paulo (F APESP) is gratefully acknowledged.* In memory.

20 PINTO-MAGLIO and CRUZ

Though cytogenetic studies in Coffea sp. are styll very limited, it is already known that Coffeachromosomes are very small and morphologically similar to each other. BOUHARMONT (1959,1963), studying the morphology of somatic chromosomes of several Coffea species, including C.arabica, was unable to separate the karyotypes of the analysed species. Also, very poorassociation between Coffea plant morphology and lack/excess chromosomes was reported byCRUZ (1972) in aneuploid plants of C. arabica cultivar «Mundo Novo», limiting their use inbreeding programs.Somatic chromosomes of Coffea are very small (1 to 3.5 micrometers) (MENDES 1938), makingbanding a hard task. PIEROZZI (1986, 1993) had relative success in adapting C, NOR andproteolitic enzyme banding methods, to chromosomes of the diploid species C. dewevrei, C.racemosa, and C. canephora, being able to characterize them by pericentromeric and telomeric Cbands. There is no reported research on chromosome banding of C. arabica species.Pachytene chromosome analysis remains a very powerful cytogenetic tool for the Coffea genus,despite the advent of new chromosome techniques, because of the problems above mentioned.The present research was carried out to characterize C. arabica meiotic chromosomes in thepachytene stage and, as a consequence, to get basic informations about the origin and nature ofploidy in the species.

MATERIAL AND METHODS

The floral buds used in this study were collected from plants of cultivars Arabica and BourbonVermelho, both belonging to the species Coffea arabica L., available in IAC Coffea germoplasmbank. Voucher specimen numbers are UEC 14069 and IAC 25032.Those buds were fixed in 3: 1 Carnoy and the chromosome preparations were obtained bystandard propionic carmine squash method. About 200 cells of each genotype were examinedusing a phase contrast microscope. Chromosomes were drawn with camera lucida and alsophotographed. The karyotype was made from drawings, and measures for each chromosometaken from 20 to 30 drawings. The chromosomes were classified according to centromerenomenclature proposed by LEVAN et a!. (1964).

RESULTS

Chromomeric patterns of the pachytene chromosomes and measurements were obtained for the22 bivalents of the C. arabica complement and no difference was observed betweenchromomeric patterns of the chromosomes of the two cultivars studied.

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 21

A karyotype and an ideogram for the 22 pachytene bivalents, based on individual chromosomecharacteristics, are presented in Figure 1 and Figure 2, respectively.Average values of total length, arm length and arm ratio of chromosomes are summarized inTable 1, along with standard errors.Pachytene bivalents of C. arabica are well differentiated and characterized by proximal darklystained heterochromatic regions to the centromere. Some of the morphological features that wereuseful in identifying individual chromosome of the complement of this species are presentedbelow:Chromosome 1 is the longest of complement. Its median centromere is delimited by sixheterochromatic chromomeres.Chromosome 2 has the length similar to the chromosome 1; a submedian centromere is delimitedby a series of chromomeres disposed in decreasing size towards the terminal parts of a both arms.Chromosome 3 is similar to chromosome 1 and 2 in length, with a submedian centromere, but ithas much more chromomeres in both arms disposed in a decreasing way.Chromosome 4 has a submedian centromere, delimited by two large chromomeres, followed bysmaller ones. In this case, the terminal part of the short arm finishes abruptly.Chromosome 5 has the short arm terminal part similar to that of the chromosome 4, but it hasonly one large chromomere near the submedian centromere.Chromosome 6 has a submedian centromere, the main recognition feature being the presence of aconspicuous trapeziform chromomere at proximal part of the long arm.Chromosome 7 is distinguished by its short arm with five chromomeres localized from asubmedian centromere to the terminal part of this arm. It has four chromomeres at the long arm.Chromosome 8 is identified by a proeminent elliptical chromomere in the proximal part of onearm and the median localization of the centromere. Chromosome 9 has a submedian centromereand its most important characteristic is the distal end of short arm presenting a double threadaspect with terminal chromomeres.Chromosome 10 presents submedian centromere and a normal distribution of the chromomeres,with no remarkable feature.Chromosome 11 is identified by three chromomeres in the short arm and two in the long arm, allof them in the proximal region.Chromosome 12 has a submedian centromere delimited by four chromomeres of the same size,two of them at short arm e two at the long arm. Chromosome 13 has the same general aspect ofthe chromosome 7, as to the chromomere pattern. Its short arm has four chromomeres and thelong arm only three of them. The centromere is subterminal.

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 23

24 PINTO-MAGLIO and CRUZ

TABLE 1 - Coffea arabica L. pachytene chromosomes length in ìm.

Chromosome 14 is one of the three chromosomes of the complement that seemed attached to thenucleolus. This chromosome has a nucleolus organizing region with a satellite in the distal part ofthe short arm. Its chromomere pattern is formed by six chromomeres, three in each armdelimiting a little subterminal centromere.Chromosome 15 is characterized by a proeminent centromere, delimited by two heteropycnoticsegments, followed by two or three little chromomeres in both arms.Chromosome 16 has almost all chromomeres localized at the short arm. This arm, with a veryshort terminal part, has four chromomeres and the long arm has only two. The centromere issubmedian.Chromosome 17 has a submedian-subterminal centromere and very simple chromomeric pattern,localized mainly at the short arm.Chromosome 18 has a median centromere and presents only little chromomeres. One arm hasfour chromomeres and the other has a series of them. Chromosome 19 resembles chromosome 6(large chromomere in one side of the submedian centromere) .

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 25

Chromosome 20 is seldom associated with nucleolus, like chromosomes 14 and 21; itschromomere pattern is also similar to these chromosomes, with terminal satellite at short arm andthe terminalization without conspicuous landmarks at long arm.Chromosome 21 and 14 are active in nucleolus organization, the region responsible for that beinglocated at terminal position of the short arm. The chromomere pattern is very simple: twochromomeres and a terminal satellite at the nucleolus organizing region at the short arm, and twochromomeres at the long arm.Chromosome 22 has two heterochromatic segments at both side of a subterminal centromere. Atthe distal end of the short arm there is a terminal chromomere, that seems to be a nucleolarorganizer satellite, even though this chromosome has never been associated to the nucleolus. Thelong arm has a euchromatic terminalization without a well-marked end.Pairs of bivalents with similar chromomeric patterns were observed, like 2 and 3 (Fig. 3); 4-5,6-19 (Fig. 4); 12-16 (Fig. 5); nucleolar bivalents 14-21 (Fig. 5), and chromosomes 20 and 22 (Fig.6). Those similarities can only be detected when chromosomes are observed simultaneously intoone cell (Figs. 7A, 7B, chromosomes 14-21; 20-22).Three nucleolus organizing chromosomes were observed in the complement but two of them,chromosomes 14 and 21, appeared more commonly associated to the nucleolus then chromosome20 (Figs. 7 A, 7B) .Some translocations were observed (Figs. 8A, 8B), where one nucleolar chromosome wasinvolved with the chromosome 18 (Fig. 8B). Associations between pairs of bivalents, includingtwo nucleolar chromosomes, were also observed in some cells (Figs. 8C-D, 8E-F).

DISCUSSION

Morphological analysis of the pachytene chromosomes of c. arabica complement showedchromomeres located proximal to or on both sides of the centromere. So, accordingJELENKOVIC and HARRINGTON (1972), C. arabica chromosomes present a proximal patternof chromomere distribution, which is supported by other observations (PINTO-MAGLIO 1983;PINTO-MAGLIO and CRUZ 1987). Additionally, the short arm of the nucleolus-organizingchromosomes (number 14,20 e 21) are completely heterochromatic (Fig. 1).The chromomeric pattern showed little or no influence of the cellular spreading or chromosomalcontraction variations arised during the natural progression of meiosis, presenting a constancythat became the best characteristic for identification, in detriment of total length, mainly in theseparation of pairs of bivalents with similar morphology (Figs. 3-6). For example, bivalents 2

26 PINTO-MAGLIO and CRUZ

Fig. 3. - Pairs of bivalents 2 and 3 of Coffea arabica with similar morphology at pachytene phase. Head arrowsindicates centromere location. Bar = 10 ìm.

Fig. 4. - Pairs of bivalents 4 and 5, 6 and 19 of Coffea arabica with similar morphology at pachytene phase. Headarrows indicates centromere location. Bar = 10 ìm.

Fig. 5. - Pairs of bivalents 12 and 16, 14 and 21 of Coffea arabica with slmllar morpnology al pachytene. Head arrowsindicates centromere location; * = nucleolar chromosomes. Bar = 10 ìm.

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 29

.

Fig. 7. - A and B, photomicrograph and interpretation of an incomplete cell at pachytene phase of Coffea arabica . Bar= 10 ìm; nu = nucleolus

Fig. 8. - A and B, traslocations; C-D and E-F, interpretative drawings and respective photomicrographs of secondaryassociations, observed in Coffea arabica at pachytene phase. Bar = 10 ìm; nu = nucleolus; -- = centromere.

32 PINTO-MAGLIO and CRUZ

and 3 have similar total lengths and chromomeric pattern but the chromomeres of chromosome 3are bigger and more distal at the short arm (Fig. 3).Similar situations occurred with pairs of bivalents 4 and 5 and 6 and 19 (Fig. 4); 12 and 16; 14and 21 (Fig. 5) and 20 and 22 (Fig. 6). Thus, chromomeric pattern analysis showed that there aremorphological similarities between 54% of c. arabica bivalents (12 pairs, two by two) makingevident some homeology among them.Although structural similarity has been observed in 12 pairs of bivalents, only two partialsecondary associations were observed (Figs. 8C-F). So one can assume that if they were reallygenetically close, we would be able to find more partial or total secondary associations.DARLINGTON (1937), who first observed this process, reported it as an indirect effect ofpolyploidy, while THOMAS and REVELL (1946) concluded that the secondary associationswere due to heterochromatic fusion of chromosomes with enough homology and potentiality toform quadrivalents. RILEY (1960), on the other hand, believed that secondary associations werethe results of rests of attaction between homologous, at the beginning of the prophase. JENKINSand CHATTERJEE (1994) demonstrated the relationship between chromosomal structure andpairing in autotetraploids, with chromosomal sets differing in homology and chromosomalstructure. They observed that the frequency of bivalents was highly related to a major structuraldiversity among complements. Consequently, secondary associations observed in c. arabica could be reported as an additionalevidence of homeology among some chromosomes of this species.The two translocations observed, one of them in a nucleolar chromosome (Figs. 8A, 8B),suggests the possibility that the two secondary associations, also involving nucleolarchromosomes, be a consequence of those translocations, which according to many researchers,are due to altered pairing in polyploids. According to STEBBINS ( 1971) , in polyploids thestructural similarity between chromosomes not always involves a genetic correspondence. Thesame way the presence or absence of chromosome pairing not always indicate phylogeneticrelationships (DE WET and HARLAN 1972), because there are many examples whereautopolyploids do not show multivalents (polyploidization process) and allopolyploids notalways form bivalents (preferential pairing). According to JACKSON (1982), the wordsautopolyploidy, allopolyploidy and segmental allopolyploidy, created by STEBBINS (1947) toclassify the polyploids based on the behaviour of hybrids F1, represent conditions related to geneaction on chromosome pairing.There exist some features that support the hypothesis of allotetraploid origin of c. arabica,proposed by CARVALHO (1952): a) genetic analysis has not proved the existence of tetrasomicinheritance, only a disomic one; b) C. arabica marginal geographical distribution as compared tothose of the diploid species of the genus; c) some hybrids among diploid species aremorphologically

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 33

similar to C. arabica; d) high index of chromosome pairing between c. arabica and c. canephoragenomes, indicating the proximity of them.Several data obtained by other authors like pairing in di-haploids (MENDES and BACCHI 1940;BERTHAUD 1976; KAMMACHER 1980) and evidence of secondary associations andmultivalents in meiosis (KRUG and MENDES 1940; CHINNAPA 1968; GRASSIAS andKAMMACHER 1975), strongly indicated the segmental allopolyploid condition of c. arabica.Thus, C. arabica complement must have been formed by two chromosomal sets very similar,maybe from ecotypes with some degree of differentiation. According to SYLVAIN (1958), c.arabica is a highly polymorphic species at its place of origin, with several naturally ocurringecotypes.The data here obtained reinforce the opinion of CHARRIER (1978) about the relationshipsamong several Eucoffea e Mascarocoffea species with C. arabica. He concluded that if thehypothesis of allopolyploidy is true for c. arabica, it would be necessary: a) two chromosomesets more differentiated than those actually found in species of both sections above, because thespecies showed to be more related to each other than with c. arabica; b) the presence of a systemthat would favor disomic behaviour of c. arabica in case of two diploid species were involved inthe formation of this polyploid species.Based on the high morphological similarity observed among the chromosomes that form the c.arabica complement we can confirm its segmental allopolyploid condition. This morphologicalsimilarity, contrasting with low level of pairing abnormality, makes possible the hypothesis thatmaybe there exist in this species a genetic system where the pairing between homeologuechromosomes is avoided by the action of genes like Ph, found in chromosome 5Bl of Triticum(RILEY and CHAPMAN 1958; RILEY et al. 1960), Avena, Festuca, Gossypium, Nicotiana eLolium (EVANS 1987). The effect of this kind of gene system is common in segmentalallopolyploids where there are restriction of pairing between homeologue chromosomes; inautopolyploids restrictions of pairing have been reported even between homologue chromosomes(JENKINS 1987).

Acknowledgements. - The authors are grateful to Dr. Jose Alfredo Usberti Filho, for his help in the preparation of thispaper .

REFERENCES

BERTHAUD J., 1976. - Etude cytogenetique d'un haploide de c. arabica L. Cafe, Cacao, The, 20: 91-96. BERTHOUF. and TROUSLOT P., 1977. - L 'analyse du polymorphisme enzimatique dans le genre Coffea: adaptation d'unemethode d'eletroforese en serie. 9'm' Colloque Scientifique International sur le Cafe, (Abidjan), ASIC, Paris, pp. 373-383.BERTHOU F., MATHIEU C. and VEDEL F., 1982. - Variations dans I'ADN des olganites cellulaires dans lesprincipales especes de Coffea: Analyse des ADN chloroplastiques et mitochondriaux a l'aide des

34 PINTO-MAGLIO and CRUZ

enzymes de restrition. 10°mo Colloque Scientifique International sur le Cafe, (Salvador), ASIC, Paris, pp. 241-431.-, 1983. - Chloroplast and mitochondrial DNA variation as indicator of phylogenetic relationships in the genus CoffeaL. Theor. Appl. Genet., 65: 77-84.BERTHOU F., TROUSLOT P., HAMON S., VEDEL F. and QUETIER F., 1980. - Analyse en eletrophores dupolymorphism biochimique de cafeiers: variation enzimatique dans 18 popolations sauvages: variation de l'ADNmitochondrial dans les especies: C. canephora, C. eugenioides et C. arabica. Cafe, Cacao, The, 24: 313-326.BOUHARMONT J., 1959. - Recherches sur les affinites chromosomiques dans le genre CoHea. Bruxelles, InstitutNationale pour I'etude agronomique du Congo BeIge, 94p. (series scientifique n. 77).-' 1963. - Somatic chromosomes of some Coffea species. Euphytica, 12: 254-257.CARVALHO A., 1952. - Taxonomia de C. arabica L. VI. Caracteres morfol6gicos dos hapl6ides. Bragantia, 12: 201-212.CARVALHO A., FERWERDA F.P., FRAHRN-LELIVELD J.A., MEDINA D.M., MENDES A.J.T. and MONACOL.C., 1969. - Coffee: Coffea arabica L., and Coffea canephora Pierre ex Froehner. In: «Outlines of perennial cropbreeding in the tropics», ed. by F.P. Ferwerda and F. Wit, pp. 189-241, Mis. Paper 4, Landbouwhogeschool,Wageningen.CHARRIER A., 1978., - La structure genetique des cafeiers spontanes de la Region Malgache (Mascarocoffea). Leursrelations avec les cafeiers d'origine africains (Eucoffea). Office de la Recherche Scientifique et Technique Outre Mer.Memoires Orstom n. 87, pp. 221.CHINNAPPA C.C., 1968. - Interspecific hybrids of c. canephora and C. arabica. Curr. Sci., 37: 676-677, CONAGINC.H.T.M. and MENDES A.J.T., 1961. - Pesquisas citol6gicas egeneticas em tres especies de Coffea canephora Pierreex Froehner. Bragantia, 20: 787-804.CRUZ N.D. DA, 1972. - Aneupl6ides de cafe - Aspectos morfol6gicos e citol6gicos na andlise de duas progenies docafe «Mundo Novo» (Coffea arabica). Doctoral. Thesis, Escola Super. de Agricult. «Luiz de Queiroz», USP,Piracicaba, SP .DARLINGTON C.D., 1937. - Recent advances in cytology. 2nd ed., Oliver & Boyd, Edinburgh.DE WET J.M.J. and HARLAN J.R., 1972. - Chromosome pairing and phylogenetic affinities. Taxon, 21: 67-70.EVANS G.M., 1987. - The control of recombination in allopolyploids. Heredity, 59: 306-320.GRASSIAS M., and KAMMACHER P., 1975. - Observation sur la conjugaison chromosomique de Coffea arabica L.Cafe, Cacao, The, 19: 177-190.JACKSON R.C., 1982. - Polyploidy and diploidy: news perspectives on chromosome pairing and its evolutionaryimplications. Amer. J. Bot., 69: 1512-1523.JELENKOVIC G. and HARRINGTON E., 1972. - Morphology of the pachytene chromosomes in Prunus persica. Can.J. Genet. Cytol., 14: 317-324.JENKINS G., 1987. - Chromosome pairing in allopolyploids. Heredity, 59: 308.JENKINS G. and CHA TTERJEE R. , 1994. - Chromosome structure and pairing preferences in autotetraploid rye(5ecale cereale). Genome, 37: 784-793.KAMMACHER P., 1980. - Sur le comportment meiotique des dihaploides de Coffea arabica L. 9°mo ColloqueScientifique International sur le Cafe, (Londres), ASIC, Paris, pp. 717-723.KRUG C.A. and CARVALHO A., 1951. - The genetics of Coffea. Adv. Genet., 4: 127-158.KRUG C.A and MENDES A.J. T ., 1940. - Cytological observations in Coffea. IV. J. Genet., 39: 189-203. LEVAN A.,FREDGA K. and SANDEBERG A.A., 1964. - Nomenclature for centromeric position on chromosomes. Hereditas, 52:201-220.MEDINA D.M. and CONAGIN C.H.T.M., 1959. - Auto-incompatibilidade em Coffea dewevrei (De Wild et Th. Dur.).Bragantia, 18: 283-293MENDES A.J. T. and BACCHI O., 1940. - Observa!foes citol6gicas em Coffea. v. Uma variedade hapl6ide (di-hapI6ide) de C. arabica L. Jorn. Agron., 3: 183-206.MENDES A.J.T., 1983. - Os cromossomos das Rubiaceas. Bol. Inst. Agron., n° 55.MENDES C.H. T., 1949- Introdu!fiio ao estudo da autoesterilidade no genero Coffea. Bragantia, 9: 35-41. MONACOL. C., 1972. - Incompatibilidade em Coffea racemosa. Cienc. Cult., S. Paulo, 24: 150. PIEROZZI N.I., 1986. - Andlisecomparativa dos cromossomos mit6ticos de Coffea dewevrei De Wild et Th. Dur. e C. racemosa Lour. atraves detecnicas de bandamento. Master Thesis, Universidade Estadual de Campinas, Campinas, SP.

PACHYTENE CHROMOSOME MORPHOLOGY IN COFFEA ARABICA 35

- 1993. - Identifica!;iio, por metodos de bandamento dos cromossomos de Coffea (Coffea canephora Pierreex Froehner; C. dewevrei De Wild et Th. Dur.; C. racemosa Lour. Doctoral Thesis, Universidade Estadualde Campinas, Campinas, SP.PINTO-MAGLIO C.A.F., 1983. - Mor/ologia dos cromossomos nucleolares em fase de paquiteno nogenero Coffea L. Master Thesis, Universidade Estadual de Campinas, Campinas, SP.PINTO-MAGLIO C.A.F. and CRUZ N.D. DA, 1987. - Pachytene chromosome morphology in Coffea L. I.Nucleolar chromosomes. Caryologia, 40: 7-23.RrLEY R., 1960. - The secondary pairing of bivalents with genetically similar chromosomes. Nature, 135:751-752.RILEY R. and CHAPMAN V., 1958. - Genetic control of the cytologicaly diploid behavior of haxaploidwheat. Nature, 132: 713- 715.RILEY R., CHAPMAN V. and KIMBER G., 1960. - Position of the gene determining the diploid-likemeiotic behaviour of wheat. Nature, 186: 59-260.STEBBINS G.L., 1947. - Types of polyploids' their classification and significance. Adv. Genet., 1: 403429.- 1971. - Chromosomal evolution in higher plants. Edward Arnold Publ. Ltd., London.SYLVAIN P.G., 1958. - Ethiopian coffee - its significance to world coffee problems. Econ. Bot., 12: 111-139.THOMAS P.T. and REVELL S.H., 1946. - Secondary association and heterochromatin attraction in Ciceraretinum. Ann. Bot., 90: 159-164.

Received 6 May 1997; accepted 5 June 1997