Isoenzymatic polymorphism in Citrus spp . and Poncirus trifoliata ( L . ) Raf . ( Rutaceae )

Isoenzymatic polymorphism analysis was used to determine genetic variability among species and hybrids of Citrus pp. and one accession of Poncirus trifoliata (L.) Raf. Ten enzymatic systems aspartate aminotransferase (AAT), acid phosphatase (ACP), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), isocitrate dehydrogenase (IDH), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), diaphorase (DIA), shikimate dehydrogenase (SKD) and peroxidase (PRX) were analyzed. Twenty loci and 48 alleles were identified. Sweet orange cultivars ( C. inensis (L). Osbeck) showed the highest polymorphism with the largest number of heterozygous loci, although the alleles of those loci were the same in all cultivars, with the exception of Westin an d Lima graúda. Mandarins (C. reticulata Blanco) exhibited diverse patterns, whereas Poncirus trifoliata (L.) Raf. showed high variability with all Citrus species and hybrids. Exclusive phenotypes were observed in some enzymatic systems, and similar patterns were found among interspecific hybrids and their putative parents. Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, km 03 BR 116, Campus Universitário, 44031-460 Feira de Santana, BA, Brasil. Send correspondence to V.M.N. Fax: +55-75-224-8019. Departamento de Genética, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Bra sil. Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Cordeirópolis, SP, Brasil.


INTRODUCTION
The genus Citrus belongs to the subtribe Citrinae, tribe Citreae, subfamily Aurantioideae of the family Rutaceae (Swingle and Reece, 1967).Taxonomic relationships among members of this genus were established by Swingle and Reece (1967) and Tanaka (1954).However, these classifications differ considerably in the number of species, since Swingle and Reece recognized 16 species and Tanaka 163 species.
The Citrus genus includes the most widely producing fruit species in the world and is highly polymorphic.Several species are used as scion cultivars, such as sweet orange, mandarins, lemons and grapefruit.Many species and hybrids with related genera can also be used as rootstocks.Poncirus trifoliata (L.) Raf. has importance as a rootstock for several cultivars around the world.However, Citrus breeding programs have been hampered by factors associated with reproductive biology (sterility, incompatibility, nucellar embryony, juvenility) and scant information on the nature and mode of inheritance of economically important traits (Torres et al., 1978;Jarrel et al., 1992).
Researchers have recognized the need for genetical studies as well as identification of genetic markers as tools for clarifying taxonomic relationships and improving breeding programs in the genus (Esen and Scora, 1977;Torres et al., 1978;Gogorcena and Ortiz, 1993).Furthermore, correct identification is important for certification and registration of new cultivars.The genetic variability of Citrus and associated genera has been evaluated by morphological descriptors, which have low discriminating capacity, as well as biochemical and molecular markers (Esen and Scora, 1977;Handa et al., 1986).Isoenzymes have been extensively used as genetic markers in Citrus spp.due to their low cost and feasibility as codominant markers (Torres et al., 1978;Gogorcena et al., 1990;Durham et al., 1992;Herrero et al., 1996).

All accessions analyzed (Table
A similarity matrix was generated using the Nei unbiased genetic identity (GI) coefficient (1978, in Swofford andSelander, 1989) with the software BIOSYS 1.7 based on allelic frequencies (Swofford and Selander, 1989), and a cluster analysis using the unweighted pair-group method using arithmetic averages (UPGMA) was performed from the similarity matrix (Rohlf, 1992).

RESULTS
The number of loci and alleles in each accession varied according to the isoenzymatic system tested.Twenty loci and 48 alleles were detected from the 10 enzymatic systems analyzed (Table II).The degree of polymorphism detected (from most to least) was PRX, PGM, SKD, DIA, IDH, 6-PGD, AAT, ACP, PGI and LAP.Several accessions presented exclusive phenotypes in different enzymatic systems, and similar patterns were found among hybrids and their putative parents (Table II).The GI obtained from the allelic frequencies (Nei, 1978 in Swofford andSelander, 1989) and the UPGMA cluster analysis ranged from 1, the most related accessions, to 0.65 for the less related (Figure 1).The highest GI was observed among the different cultivars of C. sinensis (0.94 to 1.0).This group was very distinct from the other accessions.
The C. reticulata accessions did not cluster into one group.Some were more similar to accessions of other species.Carvalhaes, Clementina and Fremont accessions (C.reticulata) were grouped together with Osceola, Lee, Murcote and Orlando hybrids, that have C. reticulata as one of the progenitors.Another subgroup included some C. reticulata accessions and C. reshni, C. nobilis species and Nova tangelo.

DISCUSSION
According to Iglesias et al. (1974), isoenzymatic variability in Citrus is expected since many species and cultivars probably originated through natural hybridization, which is the route to heterozygosity in plants.
Sweet orange cultivars (C.sinensis) showed the highest level of polymorphism, with 13 or 14 heterozygous loci out of 20 (Table II), although the alleles at these loci were almost always the same.Researchers suggest that this species originated from hybridization between C. grandis (L.) Osbeck and C. reticulata Blanco (Scora, 1975(Scora, , 1988;;Esen and Scora, 1977).Although heterozygosity within these cultivars is high, the need for uniform cultivation must have resulted in the selection of a few variants, that had the de- sirable genotypic constitution, and these were multiplied through vegetative propagation (Barret and Rhodes, 1976).However, cultivars Westin and Lima graúda showed specific isoenzymatic patterns for SKD (IS) and ACP (FF/ SS), respectively, suggesting that less hybridization has occurred among these cultivars.These enzyme systems could be useful for identification.This could be important, since few studies have focused on polymorphism within cultivars of C. sinensis (Esen and Scora, 1977;Vardi, 1988;Sawasaki et al., 1992;Herrero et al., 1996).However, several cultivars of sweet orange originated from somatic mutations of seedlings or limbsport and such mutations have been difficult to detect by codominant markers, like isoenzymes.
In general, mandarins were found to be less polymorphic than sweet orange cultivars.Fourteen different heterozygous loci were observed, and the number of heterozygous loci per individual plant ranged from one to nine (Table II).The lower degree of polymorphism in this group may be due to the fact that this species originated from a cross either between two unknown C. reticulata cultivars or one C. reticulata cultivar and a different species.In all accessions, six homozygous loci and one heterozygous locus presented the same phenotype.Therefore, C. reticulata cultivars showed less polymorphism within groups than among them, suggesting some intraspecific variability from a narrow genetic base.Esen and Scora (1977) observed complete homology in amylase in Clementina and Dancy (C.reticulata), Cleópatra (C.reshni), and King (C.nobilis).In the present study seven enzymatic systems (PGI, PGM, PRX, AAT, 6-PGD, SKD and DIA) were useful in detecting differences between some of the accessions analyzed from the cultivars cited above.In fact, some phenotypes were specific, such as King (PGM = II/PP), Cleópatra and Clementina mandarins (DIA = F 1 F 1 /S 2 S 2 /S 3 S 3 , and F 1 S 1 /F 2 S 2 , respectively) (Table II).
According to Scora (1975) and Barret and Rhodes (1976), the sour orange, C. aurantium, probably originated from a cross between C. grandis (L.) Osbeck (pummelo) and C. reticulata Blanco (mandarin).In this work some alleles were common to C. aurantium and one of its probable progenitors, C. reticulata.In sour orange, specific phenotypes were detected in three enzymatic systems (PGM (II/PS), PRX (FS/II), DIA (S 1 S 1 /F 2 F 2 /F 3 S 3 )), making these three systems useful in cultivar identification.Similarly, C. limonia Osbeck showed specific phenotypes in three enzymatic systems: PRX (MM/FS), AAT (FF/FF/FS) and IDH (IS); C. sunki (Sunki) and C. reticulata cvs Cravo and Poncan could be identified by the following PRX specific patterns, MS/MM, FM/MM and MM/MM, respectively.Some enzymatic phenotypes were specific to hybrids, such as Nova tangelo, which showed the IDH specific phenotype SS.The hybrids Lee, Murcote and Orlando shared the same phenotype II/SS of 6-PGD, while Lee and Murcote had the phenotype MM of SKD.As expected, several alleles occurred in some hybrids as well as their probable progenitors.
The accession of Poncirus trifoliata showed specific phenotypes for four enzymatic systems: PGM (FF/--), IDH (FF), AAT (FF/FF/MP) and ACP (FF/--).In general, the phenotypes were very similar to those described by Torres et al. (1978Torres et al. ( , 1982)), Ballvé et al. (1991), Sawazaki et al. (1992) and Jarrel et al. (1992).Some alleles shared by P. trifoliata and other Citrus species suggest that the two genera, Citrus and Poncirus, are structurally and functionally related at the genomic level, although taxonomically distinct on a morphological level, which would explain the results of crossing these species (Torres et al., 1985;Jarrel et al., 1992).
Our data show that C. limonia, Poncirus trifoliata and C. aurantium are related species, but with a minor degree of intragroup similarity and C. aurantium is the most differentiated among them (Figure 1).It has been suggested that C. reticulata is involved in the origin of C. aurantium and C. limonia (Hodgson, 1967).The similarity observed here between the genera Poncirus and Citrus had been confirmed at the molecular level by Torres et al. (1985) and Jarrel et al. (1992), who detected high structural and functional homology between the genomes of the two genera.
In conclusion, the isoenzyme phenotype results showed a high level of heterozygosity and allows one to infer the genotype of the 31 accessions studied and the genetic similarity among them.

ACKNOWLEDGMENTS
Thanks are due to Dr. Cláudio Costa and Dr. Marcos Gimenes for their assistance.V.M.N. and C.R.L. are recipients of CNPq fellowship.Publication supported by FAPESP.

Table II -
Genotypes of cultivars of Citrus spp.and Poncirus trifoliata (L.) Raf.obtained with different enzymatic systems.