Identification of zygotic and nucellar tangerine seedlings (Citrus spp.) using RAPD

Identification of zygotic and nucellar tangerine seedlings (Citrus spp.) using RAPD

Marinês Bastianel¹, Sérgio F. Schwarz¹, Helvécio Della Coleta Filho², Linda Lee Lin³, Marcos Machado² and Otto C. Koller¹

¹ Departamento de Horticultura e Silvicultura, Faculdade de Agronomia, Universidade Federal do Rio Grande Sul, Caixa Postal 776, 91501-970 Porto Alegre, RS, Brasil. Send correspondence to S.F.S.

² Centro de Citricultura Sylvio Moreira, IAC, Cordeirópolis, SP, Brasil.

³ Centro de Ciências Agrárias, UFSCAR, Araras, SP, Brasil.

ABSTRACT

The randomly amplified polymorphic DNA (RAPD) technique was used to distinguish nucellar and zygotic seedlings resulting from crosses between the‘ Montenegrina’ (

Citrus deliciosa Tenore) and ‘King’ (

C. nobilis Loureiro) tangerines. The aim of the present study was to develop tangerine varieties with a reduced number of seeds and organoleptic characteristics similar to the‘ Montenegrina’ tangerine. Embryos were isolated from seeds, cultivated

in vitro, and acclimated in a greenhouse. Four random primers were used to identify 54 plants of sexual origin from a total of 202 individuals. The degree of polymorphism of each primer was reflected in the number of zygotic plants obtained per primer. Cluster analysis of parents and progeny separated the individuals into distinct groups with a maximum genetic dissimilarity of 20%. INTRODUCTION The tangerine ‘Montenegrina’ (

Citrus deliciosa Tenore) is a highly acceptable fresh fruit because of its pleasant flavor. Its coloring, physical and chemical traits are enhanced by the climate and soil of Rio Grande do Sul, Brazil. These characteristics along with its late maturing season - it is ripe when similar fruits are scarce on the market - make it an excellent alternative crop, because of the better prices achieved. However, its large number of seeds has precluded export to Europe, which demands seedless fruits of good appearance and flavor. Crossing diploids (2x) of tetraploids (4x) to produce triploids (3x) has become a useful method for producing seedless citrus varieties (Soost and Cameron, 1985). One of the main problems found in citrus breeding programs is undesirable nucellar polyembryogenesis. Many polyembryonic tangerine varieties such as ‘Montenegrina’ and ‘King’ have been used in breeding programs. Consequently, the demand for methods to separate nucellar from zygotic embryos has increased.

Zygotic and nucellar embryos could be differentiated with the development of isozyme techniques (Torres et al., 1978; Soost et al., 1980; Soost and Torres, 1981). Although there are about 18 known enzymatic systems for citrus (Ashari et al., 1989), the accuracy of enzymatic analysis is influenced by the choice of enzyme and the type and age of the tissue analyzed (Roose, 1988; Asíns et al., 1995). In preliminary isozyme tests, polymorphism was not found among the tangerines‘ Montenegrina’ and ‘King’. Recently, molecular markers have been able to analyze DNA directly, without any influence from the environment or tissue age (Tansksley et al., 1989). Among these, random amplified polymorphic DNA markers (RAPD) have been widely used in citrus because of their assumed phenotypic neutrality and their ability to quickly and easily reveal a large number of markers. The technique has been used mainly for genotype typification, phylogenetic studies, mapping and mutant identification (Luro et al., 1995; Cai et al., 1994; Deng et al., 1995). The RAPD technique does not need previous information about the targeted DNA and shows great polymorphism (Ferreira and Grattapaglia, 1995).

The objective of the present study was to identify ‘Montenegrina’ and‘ King’ tangerine hybrids using the RAPD technique. The results would be used to develop a breeding program for tangerine varieties with organoleptic characteristics similar to ’Montenegrina’, eventually with fewer seeds.

MATERIAL AND METHODS

In vitro embryo culture

The parent plants used in this study came from the citrus collection at the Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul, Eldorado do Sul, RS. Controlled pollinations were carried out in September 1993 using‘ Montenegrina’ tangerines as the female parent and ‘King’ as the male.

The resulting seeds from the crosses were extracted and washed in running water to remove mucus. They were treated with 70% alcohol for 2 min, 2% sodium hypochlorite for 20 min and washed three times in sterilized water (Pescador, 1993). The embryos were removed with tweezers and a histological needle under a magnifying glass, placed in MS culture medium (Murashige and Skoog) and cultivated in a growth chamber for 45 to 60 days, until seedling formation was complete. These were placed on carbonized rice husks and turf substrate and acclimatized in a greenhouse.

Extraction of genomic DNA

Approximately 50 mg of leaves, dried in a drying oven for 24 h at 40

^oC, was used for DNA extraction. Total DNA was extracted using Murray and Thompson’s (1980) methodology and quantified by agarose gel electrophoresis according to Sambrook

et al. (1989).

Amplification

Four out of 12 10-bp random primers revealed polymorphisms between the parents. These primers were OPI11 (5’-ACATGCCGTG-3’) OPH15 (5’- AATGGCGCAG-3’), P140 (5’-AGGTCACTGA-3’) and P141 (5’-GGGGTTGACC-3’).

Amplification reactions were prepared in a volume of 12.3 ml, containing 1.0 unit of Taq polymerase (Cembiot, RS, Brazil); 200 mM each of dATP, dTTP, dCTP and dGTP (Boehringer Mannheim); 1.3

ml of 10x buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 2.0 mM MgCl

₂ and 0.05% gelatine); 15 ng of primer; 15 ng genomic DNA, and 15 ml mineral oil was added to prevent evaporation. Amplification reactions were carried out in a MJ Research Inc. thermocycler programmed for 36 cycles of 1 min at 92

^oC, 1 min at 36

^oC, and 2 min at 72

^oC, followed by a final cycle of 10 min at 72

^oC.

The amplified and separated fragments were visualized in 1.4% agarose gel stained with 0.5

mg/ml ethidium bromide and photographed under ultraviolet light with Polaroid 667 film.

Data analysis

Presence (1) and absence (0) of intensely stained products were recorded and used in the analyses. Ten nucellar individuals, together with the zygotes and the parents (‘Montenegrina’ and ‘King’), were analyzed using numerical and multivariate analyses (NTSYS-PC) 1.7 Version (Rohlf, 1992). A similarity matrix was generated using SM (simple matching) coefficients and a dendogram constructed using the UPGMA method (unweighted pair group method). RESULTS AND DISCUSSION Controlled pollination of 30 ‘Montenegrina’ flowers using ‘King’ pollen resulted in eight fruits and a total of 80 seeds. Two hundred and seventy individuals were obtained from embryo culture of these seeds. Loss through contamination, physical damage or other causes was about 25%. Some studies have obtained up to 6.15 seedlings per seed (Machuka

et al., 1993), but the mean of the‘ Montenegrina’ and ‘King’ cross in this study was 3.37. This was probably associated with the degree of polyembryogenesis of the species used. Frost and Soost (1967) reported that some cultivars have numerous embryos per seed, whereas others have few extraneous embryos.

Figure 1 (A,B) shows RAPD amplification patterns obtained through comparisons of some F1 individuals with their parents using OPI11 primer (Figure 1A). Zygotic individuals (lines 69, 72a and 80) were identified by the absence of 2000-bp fragments, which are absent in the male parent ‘King’, but present in the female parent ‘Montenegrina’. Figure 1B shows RAPD patterns obtained with primer P141. F1 zygotes were identified by the absence of 1400-bp fragments (lines 24 and 26), which are present in the female parent, but absent in the male parent. Furthermore, 840-bp fragments (lines 21, 16 and 29), which are present in both parents, did not amplify.

Figure 1
- Visualization of RAPD reaction of individuals from crosses of tangerines ‘Montenegrina’ (Citrus deliciosa) and ‘King’ (C. nobilis). A, Primer OPI11; B, Primer P141.

The primers used (OPI11, OPH15 , P141 and P140) amplified 31 well-resolved products, 12 of which were polymorphic. Different levels of polymorphism were obtained with each primer used. Primer OPI11 showed one polymorphic product, whereas three polymorphic products were obtained with primer P140, and four polymorphic products with primers OPH15 and P141. These different polymorphism levels influenced the number of zygotic individuals identified by each primer, since the more polymorphic primers revealed a greater number of zygotic progeny (Table I). Thus, a wider selection of polymorphic primers may increase the chances of identifying zygotes, as different regions of amplification could be recognized in both parents and progeny.

Primer No. of polymorphic fragments No. of identified zygotes* OPI11 1 22 P140 3 27 OPH15 4 33 P141 4 38

Primer	No. of polymorphic fragments	No. of identified zygotes*
OPI11	1	22
P140	3	27
OPH15	4	33
P141	4	38

Table I - Degree of polymorphism generated per primer and the number of zygotes identified among progeny of ‘Montenegrina’ (Citrus deliciosa Tenore) and‘ King’ (C. nobilis Loureiro) tangerines.

^*

Some zygotes were identified by more than one primer.

Among the 202 plants, 54 F1 hybrids (26.7%) were identified with only four primers. Frost and Soost (1967) showed zygote frequencies of 78.7% and 14.02% for‘ King’ and ‘Willowleaf’ tangerines, respectively, using pollen from Poncirus trifoliata. Soost et al. (1980) found a zygote frequency of about 85% using‘ King’ as the female parent and pollen from ‘Parson Special’ tangerine. Several studies have shown that zygote frequency does not exceed 15% depending on the species and, in some cases, only nucellar individuals are obtained (Hirai et al., 1986; Cameron and Soost, 1980; Roose and Traugh, 1988). The difference among the values obtained here and those in the literature may be attributed to pollination efficiency, loss of zygotic individuals during culture, and plant acclimatization. Environmental and genetic factors, in addition to nutritional and varietal factors, may also have affected zygote frequency (Khan and Roose, 1988; Roose and Traugh, 1988).

Data clustering (Figure 2) grouped hybrid and nucellar individuals and the parents. Only 10 nucellar individuals were presented, because all nucellar plants showed 100% similarity. Two large groups based on the markers were found. One group was formed by individuals similar to the male parent (‘King’ - K), and the other was formed by individuals similar to the female parent (‘Montenegrina’ - M). The latter was associated with a group of nucellar progeny. Maximum dissimilarity between hybrid individuals was 20%. Medina Filho et al. (1993) using isozymes showed the occurrence of twins and triplets in progeny from crosses involving different Citrus species and P. trifoliata with the following frequencies: monozygotic twins (1.5%), monozygotic triplets (0.15%) and dizygotic twins (0.315%). In the present study, the hybrids which make up the subgroups with 100% similarity came from different seeds. Thus, the possibility of twins was eliminated. According to the RAPD technique principle, trials with larger numbers of primers with these sub-groups could lead to differentiation of these individuals.

Figure 2
- Similarity among individuals from crosses of tangerines ‘Montenegrina’ (Citrus deliciosa) and ‘King’ (C. nobilis) generated by the SM (simple matching) coefficients using UPGMA grouping.

The RAPD technique was efficient in the identification of hybrids from crosses of‘ Montenegrina’ and ‘King’, with the identification of 54 hybrids from a progeny of 202 individuals. Plants obtained through in vitro cultures of previously separated embryos probably increase survival and development of zygote embryos. This does not normally happen when the seed is placed to germinate directly in a substrate, which indirectly contributed to the zygote frequency obtained in this experiment.

ACKNOWLEDGMENTS We are grateful to FINEP, FAPERGS, PROPESP/ UFRGS and RHAE/CNPq for financial support and to the Sylvio Moreira Citrus Center/IAC for the use of their biotechnology laboratory. Publication supported by FAPESP. RESUMO A técnica RAPD (random amplified polymorphic DNA) foi utilizada para distinguir plântulas nucelares e zigóticas resultantes do cruzamento entre as tangerineiras ‘Montenegrina’ (

Citrus deliciosa Tenore) e ‘King’ (

C. nobilis Loureiro). Este cruzamento foi realizado objetivando a obtenção de variedades de tangerineiras com características organolépticas de fruto semelhantes à tangerina ‘Montenegrina’ e menor número de sementes. Embriões foram isolados das sementes e cultivados

in vitro e aclimatizados em casa de vegetação. Utilizando-se de 4 primers de seqüência randômica foram identificadas 54 plantas de origem sexual de um total de 202 indivíduos. O grau de polimorfismo de cada primer refletiu no número de plantas zigóticas obtidas por primer, sendo o total de zigóticos identificados pela soma das informações geradas pelos 4 primers. Análise de agrupamento com os parentais e a progênie separou os indivíduos em grupos distintos com uma dissimilaridade genética máxima de 20%. REFERENCES

Ashari, S.,

Aspinall, D. and

Sedgley, M. (1989). Identification and investigation of relationships of mandarin types using isozyme analysis.

Sci. Hortic.

40: 305-315.

Asíns, M.J., Herrero, R. and Navarro, L. (1995). Factors affecting Citrus tree isozyme-gene expression. Theor. Appl. Genet. 90: 892-898.

Cai, Q., Guy, C.L. and Moore, G.A. (1994). Extension of the linkage map in citrus using random amplified polymorphic DNA and RFLP mapping of cold-acclimation-responsive loci. Theor. Appl. Genet. 89: 606-614.

Cameron, J.W. and Soost, R.K. (1980). Mono- and poly- embryony among tetraploid citrus hybrids. HortScience 15: 730-731.

Deng, Z.N., Gentile, A., Nicolosi, E., Domina, F., Vardi, A. and Tribulato, E. (1995). Identification of in vivo and in vitro lemon mutants by RAPD markers. J. Hortic. Sci. 70: 117-125.

Ferreira, M.E. and Grattapaglia, D. (1995). Introdução ao Uso de Marcadores RAPD e RFLP em Análise Genética. Embrapa - Cenargem, Brasília, pp. 220.

Frost, H.B. and Soost, R.K. (1967). Seed reproduction: development of gametes and embryos. In: The Citrus Industry (Reuther, W., Batchelor, L.D. and Webber, H.J., eds). University of California, Berkeley, CA, 290-324.

Hirai, M., Kozaki, I. and Kajiura, I. (1986). The rate of spontaneous inbreeding of trifoliate orange and some characteristics of the inbred seedlings. Jpn. J. Breed. 36: 138-146.

Khan, I.A and Roose, M.L. (1988). Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of Trifoliate Orange. J. Am. Soc. Hort. Sci. 113: 105-110.

Luro, F., Bové, F.L., Bové, J.M. and Ollitrault, P. (1995). DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in Citrus. HortScience 30: 1063-1067.

Machuka, J.S., Waithaka, K. and Gopalan, H.N.B. (1993). Embryo culture and gel electrophoretic identification of nucellar and zygotic seedlings of Citrus limon (L.) and Citrus sinensis (L.). Discovery and Innovation 5: 75-80.

Medina Filho, H.P., Bordignon, R., Ballve, R.M.L. and Siqueira, J.S. (1993). Genetic proof of the occurrence of mono and dizygotic hybrid twins in citrus rootstocks. Rev. Bras. Genet. 16: 703-711.

Murray, H.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.

Pescador, R. (1993). Cultivo de embriões de larangeira Cipó (Citrus Sinensis Osb.) in vitro e uso de padrões enzimáticos na identificação dos" seedlings" zigóticos e nucelares. Master’s thesis, UFRGS, Porto Alegre.

Rohlf, F.J. (1992). NTSYS-PC Numerical Taxonomy and Multivariate Analyses System (version 1.7). State Univer sity of New York, New York.

Roose, M.J. (1988). Isozymes and DNA restriction fragment length polymorphisms in citrus breeding and systematics. Proc. Int. Soc. Citricult. 1: 155-165.

Roose, M.L. and Traugh, S.N. (1988). Identification and performance of Citrus trees on nucellar and zygotic rootstocks. J. Am. Soc. Hort. Sci. 113: 100-105.

Sambrook J., Fritsch, E.F. and Maniats, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.

Soost, R.K. and Cameron, J.W. (1985). ‘Melogold’, a triploid pommelo-grapefruit hybrid. HortScience 20: 1134-1135.

Soost, R.K. and Torres, A.M. (1981). Leaf isozymes as genetic markers in Citrus. Proc. Int. Soc. Citricult. 1: 7-10.

Soost, R.K., Williams, T.E. and Torres, A.M. (1980). Identification of nucellar and zygotic seedlings of citrus with leaf isozymes. HortScience 15: 728-729.

Tansksley, S.D., Young, N.D., Paterson, A.H. and Bonierbale, M.W. (1989). RFLP mapping in plant breeding: new tools for an old science. Biotechnology 7: 257-264.

Torres, A.M., Soost, R.K. and Diedenhofen, U. (1978). Leaf isozymes as genetic markers in citrus. Am. J. Bot. 65: 869-881.

(Received July 16, 1996)

References

• Ashari, S, Aspinall, D and Sedgley, M (1989). Identification and investigation of relationships of mandarin types using isozyme analysis. Sci. Hortic.40: 305-315.
• Asíns, M.J, Herrero, R and Navarro, L (1995). Factors affecting Citrus tree isozyme-gene expression. Theor. Appl. Genet 90: 892-898.
• Cai, Q, Guy, C.L and Moore, G.A (1994). Extension of the linkage map in citrus using random amplified polymorphic DNA and RFLP mapping of cold-acclimation-responsive loci. Theor. Appl. Genet 89: 606-614.
• Cameron, J.W and Soost, R.K (1980). Mono- and poly- embryony among tetraploid citrus hybrids. HortScience 15: 730-731.
• Ferreira, M.E. and Grattapaglia, D. (1995). Introduçăo ao Uso de Marcadores RAPD e RFLP em Análise Genética Embrapa - Cenargem, Brasília, pp. 220.
• Frost, H.B. and Soost, R.K. (1967). Seed reproduction: development of gametes and embryos. In: The Citrus Industry (Reuther, W., Batchelor, L.D. and Webber, H.J., eds). University of California, Berkeley, CA, 290-324.
• Hirai, M, Kozaki, I and Kajiura, I (1986). The rate of spontaneous inbreeding of trifoliate orange and some characteristics of the inbred seedlings. Jpn. J. Breed.36: 138-146.
• Khan, I.A and Roose, M.L. (1988). Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of Trifoliate Orange J. Am. Soc. Hort. Sci. 113: 105-110.
• Luro, F, Bové, F.L, Bové, J.M and Ollitrault, P. (1995). DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in Citrus. HortScience 30: 1063-1067.
• Machuka, J.S, Waithaka, K and Gopalan, H.N.B (1993). Embryo culture and gel electrophoretic identification of nucellar and zygotic seedlings of Citrus limon (L.) and Citrus sinensis (L.). Discovery and Innovation 5: 75-80.
• Medina Filho, H.P., Bordignon, R., Ballve, R.M.L. and Siqueira, J.S. (1993). Genetic proof of the occurrence of mono and dizygotic hybrid twins in citrus rootstocks. Rev. Bras. Genet. 16: 703-711.
• Murray, H.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.
• Pescador, R. (1993). Cultivo de embriőes de larangeira Cipó (Citrus Sinensis Osb.) in vitro e uso de padrőes enzimáticos na identificaçăo dos" seedlings" zigóticos e nucelares. Masters thesis, UFRGS, Porto Alegre.
• Sambrook J, Fritsch, E.F and Maniats, T (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.
• Soost, R.K and Cameron, J.W (1985). Melogold, a triploid pommelo-grapefruit hybrid. HortScience 20: 1134-1135.
• Soost, R.K and Torres, A.M (1981). Leaf isozymes as genetic markers in Citrus Proc.Int. Soc. Citricult. 1: 7-10.
• Soost, R.K, Williams, T.E. and Torres, A.M (1980). Identification of nucellar and zygotic seedlings of citrus with leaf isozymes. HortScience 15: 728-729.
• Tansksley, S.D., Young, N.D., Paterson, A.H. and Bonierbale, M.W. (1989). RFLP mapping in plant breeding: new tools for an old science. Biotechnology 7: 257-264.
• Torres, A.M, Soost, R.K and Diedenhofen, U (1978). Leaf isozymes as genetic markers in citrus. Am. J. Bot. 65: 869-881.

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  16 July 1996